endothelial growth factor." *Am J Physiol Cell Physiol* 280(6): C1375-86. **6**

### **Platelet and Liver Regeneration**

Nobuhiro Ohkohchi, Soichiro Murata and Kazuhiro Takahashi

*Department of Surgery, University of Tsukuba Japan* 

#### **1. Introduction**

108 Tissue Regeneration – From Basic Biology to Clinical Application

Zachary, I. (2001). "Signaling mechanisms mediating vascular protective actions of vascular

Platelets are the smallest structures in circulating blood and have a convex disc construction with an equatorial diameter of 2-3 microns and have no nucleus. They are derived from megakaryocytes in the bone marrow. Following their normal life span of 8-10 days, they are removed from the circulation when passing through the spleen. Platelets have three types of secretory granules, i.e., alpha-granules, dense-granules, and lysosomal granules in the cytosol (Fig. 1). Each granule contains a specific mix of soluble factors, such as plateletderived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), serotonin, adenosine diphosphate (ADP), adenosine tri-phosphate (ATP), epidermal growth factor (EGF), and transforming growth factor beta (TGF-beta) (Blair et al., 2009; McNicol & Israels., 1999; Polasek et al., 2005). After being activated by physiological substances such as thrombin, collagen, thromboxane A2 (TXA2), epinephrine, and platelet-activating factor (PAF), or by nonphysiological substances such as divalent cationophores and phorobol esters, platelets release these biologically active substances that exert various effects depending on the specific context (Holmsen, 1989; Suzuki H et al., 1992; Broos et al., 2011)

The main physiological role for circulating platelets is hemostasis when a vessel is injured (Holmsen, 1989). This process involves rapid adhesion of the platelets to the exposed subendothelium followed by platelet aggregation which culminates in the formation of a platelet plug that temporarily seals off the injured vessel walls. As they undergo this process, platelet activation leads to exocytosis of granular substances, release of newly synthesized mediators, and discharge of membrane-bond trans-cellular signaling molecules (Holmsen, 1989; Broos et al., 2011). Numbers of the various kinds of mechanisms facilitate platelet participation in other physiological or pathological process including inflammation (McNicol et al., 2008), malignancy (Mehta, 1984; Nash et al., 2002), immune response (Elzey et al., 2005; Sowa et al., 2009; Klinger & Jelkmann 2002; Sprague et al., 2008), wound healing (Mazzucco et al., 2010; Ranzato et al., 2009; Rozman & Bolta., 2007; Yamaguchi et al., 2010), and tissue regeneration (Radice et al., 2010; Dugrillon et al., 2002; Hartmann et al., 2010; de Vos et al., 2010; Rodeo et al., 2010).

Platelets have been reported to accumulate in the liver under some kinds of pathologic conditions, such as ischemia/reperfusion injury (Khandoga et al., 2003, 2006; Pak et al., 2010), liver cirrhosis (Zaldivar et al., 2010), cholestatic liver (Laschke et al., 2008) and viral

Platelet and Liver Regeneration 111

a cooperative effect with liver sinusoidal endothelial cells (LSEC), and 3) a collaborative

Liver regeneration occurs by proliferation of all of the existing mature cellular populations including hepatocytes, biliary epithelial cells, LSEC, Kupffer cells, and hepatic stellate cells. Of these, hepatocytes are the first cells to proliferate (Malik et al., 2002); they usually replicate once or twice following partial hepatectomy and return to the quiescent state. The kinetics of cell proliferation differ between species, the peak of DNA synthesis in hepatocytes usually being at 24 hours in rats but at 36 hours in mice (Michalopoulos & DeFrances., 1997; Michalopoulos, 2010; Fausto et al, 1995, 2000). Intercellular interactions mediated by many growth factors and cytokines, including HGF, tumor necrosis factor-alpha (TNF-apha), interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta), EGF etc. appear to play important role in this process. Each growth factor leads subsequent activation of downstream transcription cascades that drive transition of the quiescent hepatocytes into the cell cycle and ensure progression beyond the restriction point in the G1 phase of the cycle. Several transcription factors are involved, and nuclear factor-kappa B (NF-KB) (Tewari et al., 1992; Cressman et al., 1994; FitzGerld et al., 1995), activator protein 1 (Ap-1) (Stepniak et al., 2006), CCAAT/enhancer binding protein-beta (C/EBPbeta) (Wang et al., 2008), extracellular signalregulated kinase 1/2 (ERK 1/2) (Borowiak et al., 2004; Bard-Chapeau et al., 2006; Factor et al., 2010), signal transducer and activator of transcription 3 (STAT3) (Cressman et al., 1995; Li et al., 2002; Moh et al., 2007), and phosphatidylinositol-3-kinase (PI3K)/Akt (Jackson et al., 2008; Haga et al., 2005; Nechemia-Arbely et al., 2011) are representatives. Among these transcription factors and corresponding signaling transductions, the TNF-alpha/NF-KB, IL-6/STAT3, and PI3K/Akt pathways are considered the three major cascades through which platelets exert

The TNF-alpha/NF-KB pathway is activated within 30 minutes of partial hepatectomy and the activation usually lasts no longer than 4-5 hours (Michalopoulos & DeFrances, 1997). NF-KB is found in almost every cell including hepatocytes and non-paranchymal cells. It is a heterodimer composed of two subunits, p65 and p50, which are assembled in the cytosol (Solt & May, 2008). It is inactivated by Inhibitor of NF-KB (IKB) which binds to the p65 subunit. After being stimulated by TNF-alpha, NF-kB is activated by the removal of IkB from the p65 subunit; it migrates to the cell nucleus, where it binds to the promoter of

STAT3 is activated more slowly; it becomes detectable 1 to 2 hours after partial hepatectomy and lasts about 4-6 hours (Michalopoulos & DeFrances, 1997). IL-6 binding causes dimerization of the corresponding receptor and the activation of intracellular tyrosine kinase which phosphorylates gp130 and creates a docking site fof STAT3 (Heinrich et al., 1998). STAT3 is phosphorylated and translocates to the nucleus where it promotes the expression of cyclin-D1 and p21 to control the progression of the cell cycle (Turkson & Jove, 2000; Terui et al., 2005). It has been reported that hepatocytic mitosis of STAT3-knockout mice was significantly suppressed after partial hepatectomy in liver regeneration (Haga et al., 2009). The absence of STAT3 in hepatocytes exacerbates liver fibrosis during cholestasis

cyclin-D1, which regulates G0/G1-to-S-phase transition (Hinz et al., 1999).

**2. Growth factors, cytokines, and signal transduction related to platelets'** 

effect with Kupffer cells.

**effect on liver regeneration** 

their effects on liver regeneration (Fig. 2).

(Shigekawa et al., 2011).

hepatitis (Lang et al., 2008). Furthermore, platelets flow out slow, with rolling and adhesion in the liver sinusoids, under stressed situations such as ischemia/reperfusion injury (Nakano et al., 2008). Previous works on such conditions have focused on platelets as producers of inflammatory cytokines and therefore being pro-inflammatory (Pereboom et al., 2008). However, recent clinical and basic studies have revealed other ways in which they affect liver biology and pathology.

Fig. 1. Platelet ultrastructure. Transmission electron microscopic representation of a human platelets; the microtubules (MT), open cannalicular system (OCS), dense tubular system (DTS), mitochondria (M), alpha-granules (αG), dense granules (DG), and glycogen particles (Gly) are indicated. This electromicrograph is produced by kind permission of Dr. Hidenori Suzuki, Division of Morphological and Biomolecular Research, Graduate School of Medicine, Nippon Medical School.

In clinical studies, Marubashi et al. reported that there was a positive correlation between graft size and post-transplant thrombocytosis (Marubashi et al., 2006). Alkozai et al. described that a peri-operative low platelet count after partial hepatectomy was a predictor of delayed postoperative recovery of liver function and was associated with an increased risk of post-operative mortality (Alkozai et al., 2010). Kim et al. stated that total amount of platelet transfusion was positively associated with graft regeneration (Kim et al., 2010). In basic research, Lesurtel et al. showed that platelet-derived serotonin mediated liver regeneration in mice (Lesurtel et al., 2006). Nocito et al. demonstrated that platelets and platelet-derived serotonin promoted tissue repair after normothermic hepatic ischemia in mice (Nocito et al., 2007). In addition, we have obtained several types of evidence for platelets promoting liver regeneration using different experimental models of liver dysfunction in small and large animals.

In this chapter, we describe our evidence for platelets in promoting liver regeneration. Furthermore, we explain three different mechanisms i.e., 1) a direct effect on hepatocytes, 2)

hepatitis (Lang et al., 2008). Furthermore, platelets flow out slow, with rolling and adhesion in the liver sinusoids, under stressed situations such as ischemia/reperfusion injury (Nakano et al., 2008). Previous works on such conditions have focused on platelets as producers of inflammatory cytokines and therefore being pro-inflammatory (Pereboom et al., 2008). However, recent clinical and basic studies have revealed other ways in which they

Fig. 1. Platelet ultrastructure. Transmission electron microscopic representation of a human platelets; the microtubules (MT), open cannalicular system (OCS), dense tubular system (DTS), mitochondria (M), alpha-granules (αG), dense granules (DG), and glycogen particles (Gly) are indicated. This electromicrograph is produced by kind permission of Dr. Hidenori

In clinical studies, Marubashi et al. reported that there was a positive correlation between graft size and post-transplant thrombocytosis (Marubashi et al., 2006). Alkozai et al. described that a peri-operative low platelet count after partial hepatectomy was a predictor of delayed postoperative recovery of liver function and was associated with an increased risk of post-operative mortality (Alkozai et al., 2010). Kim et al. stated that total amount of platelet transfusion was positively associated with graft regeneration (Kim et al., 2010). In basic research, Lesurtel et al. showed that platelet-derived serotonin mediated liver regeneration in mice (Lesurtel et al., 2006). Nocito et al. demonstrated that platelets and platelet-derived serotonin promoted tissue repair after normothermic hepatic ischemia in mice (Nocito et al., 2007). In addition, we have obtained several types of evidence for platelets promoting liver regeneration using different experimental models of liver

In this chapter, we describe our evidence for platelets in promoting liver regeneration. Furthermore, we explain three different mechanisms i.e., 1) a direct effect on hepatocytes, 2)

Suzuki, Division of Morphological and Biomolecular Research, Graduate School of

affect liver biology and pathology.

Medicine, Nippon Medical School.

dysfunction in small and large animals.

a cooperative effect with liver sinusoidal endothelial cells (LSEC), and 3) a collaborative effect with Kupffer cells.

#### **2. Growth factors, cytokines, and signal transduction related to platelets' effect on liver regeneration**

Liver regeneration occurs by proliferation of all of the existing mature cellular populations including hepatocytes, biliary epithelial cells, LSEC, Kupffer cells, and hepatic stellate cells. Of these, hepatocytes are the first cells to proliferate (Malik et al., 2002); they usually replicate once or twice following partial hepatectomy and return to the quiescent state. The kinetics of cell proliferation differ between species, the peak of DNA synthesis in hepatocytes usually being at 24 hours in rats but at 36 hours in mice (Michalopoulos & DeFrances., 1997; Michalopoulos, 2010; Fausto et al, 1995, 2000). Intercellular interactions mediated by many growth factors and cytokines, including HGF, tumor necrosis factor-alpha (TNF-apha), interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta), EGF etc. appear to play important role in this process. Each growth factor leads subsequent activation of downstream transcription cascades that drive transition of the quiescent hepatocytes into the cell cycle and ensure progression beyond the restriction point in the G1 phase of the cycle. Several transcription factors are involved, and nuclear factor-kappa B (NF-KB) (Tewari et al., 1992; Cressman et al., 1994; FitzGerld et al., 1995), activator protein 1 (Ap-1) (Stepniak et al., 2006), CCAAT/enhancer binding protein-beta (C/EBPbeta) (Wang et al., 2008), extracellular signalregulated kinase 1/2 (ERK 1/2) (Borowiak et al., 2004; Bard-Chapeau et al., 2006; Factor et al., 2010), signal transducer and activator of transcription 3 (STAT3) (Cressman et al., 1995; Li et al., 2002; Moh et al., 2007), and phosphatidylinositol-3-kinase (PI3K)/Akt (Jackson et al., 2008; Haga et al., 2005; Nechemia-Arbely et al., 2011) are representatives. Among these transcription factors and corresponding signaling transductions, the TNF-alpha/NF-KB, IL-6/STAT3, and PI3K/Akt pathways are considered the three major cascades through which platelets exert their effects on liver regeneration (Fig. 2).

The TNF-alpha/NF-KB pathway is activated within 30 minutes of partial hepatectomy and the activation usually lasts no longer than 4-5 hours (Michalopoulos & DeFrances, 1997). NF-KB is found in almost every cell including hepatocytes and non-paranchymal cells. It is a heterodimer composed of two subunits, p65 and p50, which are assembled in the cytosol (Solt & May, 2008). It is inactivated by Inhibitor of NF-KB (IKB) which binds to the p65 subunit. After being stimulated by TNF-alpha, NF-kB is activated by the removal of IkB from the p65 subunit; it migrates to the cell nucleus, where it binds to the promoter of cyclin-D1, which regulates G0/G1-to-S-phase transition (Hinz et al., 1999).

STAT3 is activated more slowly; it becomes detectable 1 to 2 hours after partial hepatectomy and lasts about 4-6 hours (Michalopoulos & DeFrances, 1997). IL-6 binding causes dimerization of the corresponding receptor and the activation of intracellular tyrosine kinase which phosphorylates gp130 and creates a docking site fof STAT3 (Heinrich et al., 1998). STAT3 is phosphorylated and translocates to the nucleus where it promotes the expression of cyclin-D1 and p21 to control the progression of the cell cycle (Turkson & Jove, 2000; Terui et al., 2005). It has been reported that hepatocytic mitosis of STAT3-knockout mice was significantly suppressed after partial hepatectomy in liver regeneration (Haga et al., 2009). The absence of STAT3 in hepatocytes exacerbates liver fibrosis during cholestasis (Shigekawa et al., 2011).

Platelet and Liver Regeneration 113

thrombocytosis, mice were injected TPO. Anti-mouse platelet monoclonal antibody (Pm-1) was administrated to induce thrombocytopenia. Liver regeneration, cytokine and signaling pathways in the three groups were compared. Differences of platelet accumulation in the liver by using immunohistochemical staining technique were also

The liver/body weight ratios in the thrombocytotic group and normal group were significantly higher than in the thrombocytopenic group, 48 hours after partial hepatectomy. In the thrombocytotic group, the liver/body weight ratio 48 hours after partial hepatectomy was significantly higher than that in normal group (Fig. 3A). The hepatocyte Ki-67 labeling index and hepatocyte mitotic index 48 hours after partial hepatectomy in the thrombocytotic group was obviously higher than that of normal and thrombocytopenic groups (Fig. 3B). Furthermore, the hepatocyte proliferating cell nuclear antigen (PCNA) labeling index 48 hours after partial hepatectomy in the thrombocytopenic group was remarkably lower than

(A)

observed.

that in normal and thrombocytotic groups.

The PI3K/Akt pathway is activated immediately after partial hepatectomy (Murata et al., 2007). The pathway is initiated by the activation of the receptor tyrosine kinases or receptors coupled with G proteins by HGF, IL-6, TNF-alpha, TGF-beta and many other signaling molecules (Osawa et al., 2002; Okano et al., 2003; Tulasne & Foveau, 2008; Kato et al., 2009; Nechemia-Arbely et al., 2011). Met is a tyrosine kinase receptors on the surface of hepatocytes tha binds HGF (Bottaro et al., 1991; Tulasne & Foveau, 2008). HGF/c-met signaling activates PI3K which recruits Akt to the site of membranes, and subsequently phosphorylates Akt (Fresno et al., 2004). Glycogen synthase kinase 3-beta (GSK3-beta) acts downstream of Akt and plays a critical role in liver regeneration by regulating cell growth along with other downstream Akt factors, such as mTOR and 70S6K (Faridi et al., 2003; Latronico et al., 2004; Haga et al., 2005). Phosphorylation of Akt results in activation of GSK3-beta by phosphorylation at serine-9, resulting in accumulation of beta-catenin and cyclin-D1 in the nucleus, which induce DNA synthesis and cellular mitosis of hepatocytes (Gotoh et al., 2003; Chen et al., 2005).

Fig. 2. Cytokines and growth factors for liver regeneration.

#### **3. Effect of platelets on liver regeneration**

Our first study was focused on liver regeneration under thrombocytotic conditions induced by thrombopoietin (TPO) (Murata et al., 2007). A 70% partial hepatectomy was carried out and mice were subsequently divided into three groups as follows; untreated mice as normal group, a thrombocytotic group, and a thrombocytopenic group. To induce

The PI3K/Akt pathway is activated immediately after partial hepatectomy (Murata et al., 2007). The pathway is initiated by the activation of the receptor tyrosine kinases or receptors coupled with G proteins by HGF, IL-6, TNF-alpha, TGF-beta and many other signaling molecules (Osawa et al., 2002; Okano et al., 2003; Tulasne & Foveau, 2008; Kato et al., 2009; Nechemia-Arbely et al., 2011). Met is a tyrosine kinase receptors on the surface of hepatocytes tha binds HGF (Bottaro et al., 1991; Tulasne & Foveau, 2008). HGF/c-met signaling activates PI3K which recruits Akt to the site of membranes, and subsequently phosphorylates Akt (Fresno et al., 2004). Glycogen synthase kinase 3-beta (GSK3-beta) acts downstream of Akt and plays a critical role in liver regeneration by regulating cell growth along with other downstream Akt factors, such as mTOR and 70S6K (Faridi et al., 2003; Latronico et al., 2004; Haga et al., 2005). Phosphorylation of Akt results in activation of GSK3-beta by phosphorylation at serine-9, resulting in accumulation of beta-catenin and cyclin-D1 in the nucleus, which induce DNA synthesis and cellular mitosis of hepatocytes

(Gotoh et al., 2003; Chen et al., 2005).

Fig. 2. Cytokines and growth factors for liver regeneration.

Our first study was focused on liver regeneration under thrombocytotic conditions induced by thrombopoietin (TPO) (Murata et al., 2007). A 70% partial hepatectomy was carried out and mice were subsequently divided into three groups as follows; untreated mice as normal group, a thrombocytotic group, and a thrombocytopenic group. To induce

**3. Effect of platelets on liver regeneration** 

thrombocytosis, mice were injected TPO. Anti-mouse platelet monoclonal antibody (Pm-1) was administrated to induce thrombocytopenia. Liver regeneration, cytokine and signaling pathways in the three groups were compared. Differences of platelet accumulation in the liver by using immunohistochemical staining technique were also observed.

The liver/body weight ratios in the thrombocytotic group and normal group were significantly higher than in the thrombocytopenic group, 48 hours after partial hepatectomy. In the thrombocytotic group, the liver/body weight ratio 48 hours after partial hepatectomy was significantly higher than that in normal group (Fig. 3A). The hepatocyte Ki-67 labeling index and hepatocyte mitotic index 48 hours after partial hepatectomy in the thrombocytotic group was obviously higher than that of normal and thrombocytopenic groups (Fig. 3B). Furthermore, the hepatocyte proliferating cell nuclear antigen (PCNA) labeling index 48 hours after partial hepatectomy in the thrombocytopenic group was remarkably lower than that in normal and thrombocytotic groups.

Platelet and Liver Regeneration 115

Fig. 4. Effect of platelet increment or reduction on Akt, STAT3, and ERK 1/2 after partial hepatectomy. (Reproduced from Murata et al., 2007, World J Surg. with permission).

contact with hepatocytes in the thrombocytotic group (Fig. 5B).

hepatocytes, by translocating into the space of Disse.

regeneration.

Platelet accumulation in the liver was investigated in all groups before and 5 minutes after partial hepatectomy. Platelets accumulated in the residual liver within 5 minutes after partial hepatectomy and a two-fold increase in platelet levels was observed in the normal and thrombocytotic groups (Fig. 5A). On the other hand, in thrombocytotic group, platelets in the residual liver increased remarkably compared with normal and thrombocytopenic groups 5 minutes after partial hepatectomy. However, no increment was observed in thrombocytopenic group. In addition, under transmission electron microscopy, platelets translocated from the liver sinusoidal space into the space of Disse, and they had direct

These results suggest that platelets accumulate in the liver within a few minutes of partial hepatectomy and cause rapid hepatocyte proliferation through direct contact with

Taken together, the results described above demonstrate that platelets affect liver regeneration in the acute phase after partial hepatectomy and suggested that the PI3K/Akt pathway is the main signaling pathway involved in platelet mediated liver

The following study was done to investigate the role of platelets in liver regeneration using a thrombocytosis model in mice after 90% partial hepatectomy (Myronovych et al. 2008). All mice in the normal group died within 30 hours, predominantly between 20 and 30 hours. In contrast, the survival rate at 30 hours and at one week after partial hepatectomy was 54.5% and 27.3%, respectively in thrombocytotic group (Fig. 6A). Phosphorylation of Akt and STAT3 started earlier and stronger in thrombocytotic group than normal group. Serum albumin levels decreased in both groups after partial hepatectomy, but more rapidly in normal group, and there was a significant difference with higher levels being detected at 24 hours post-hepatectomy in the livers of the thrombocytotic group (Fig 6B). Serum

(B)

Fig. 3. Effect of thrombocytosis on liver regeneration after 70% of partial hepatectomy. (A) Liver/body weight ratio before and 48 hours after partial hepatectomy (PH) in thrombocytotic , normal and thrombocytopenic groups. Data were expressed as mean ± SD. \*p < 0.05, \*\*p < 0.01 versus thrombocytopenic group. #p < 0.05 normal group versus thrombocytotic group. (B) Ki-67 labeling index 48 hours after partial hepatectomy in thrombocytotic, normal and thrombocytopenic groups. Representative immunohistochemical images are shown. Data were expressed as mean ± SD. \*p < 0.05 versus thrombocytopenic group. (Reproduced from Murata et al., 2007, World J Surg with permission.)

HGF and PDGF expression in the liver tissue in thrombocytotic group were significantly higher than in the normal and thrombocytopenic groups. Akt was strongly phosphorylated in the thrombocytotic group compared with the thrombocytopenic group. Activation of Akt in the thrombocytotic group started 5 minutes after partial hepatectomy and persisted for 2 hours. On the other hand, although activation of Akt was seen from 5 minutes after partial hepatectomy in the normal group, activation reduced in 2 hours after partial hepatectomy. In the thrombocytopenic group, Akt was not activated during the first 6 hours after partial hepatectomy. There was no difference in activation in ERK 1/2 and STAT3 among the three groups after partial hepatectomy (Fig. 4).

(B)

thrombocytotic , normal and thrombocytopenic groups. Data were expressed as mean ± SD.

HGF and PDGF expression in the liver tissue in thrombocytotic group were significantly higher than in the normal and thrombocytopenic groups. Akt was strongly phosphorylated in the thrombocytotic group compared with the thrombocytopenic group. Activation of Akt in the thrombocytotic group started 5 minutes after partial hepatectomy and persisted for 2 hours. On the other hand, although activation of Akt was seen from 5 minutes after partial hepatectomy in the normal group, activation reduced in 2 hours after partial hepatectomy. In the thrombocytopenic group, Akt was not activated during the first 6 hours after partial hepatectomy. There was no difference in activation in ERK 1/2 and STAT3 among the three

Fig. 3. Effect of thrombocytosis on liver regeneration after 70% of partial hepatectomy. (A) Liver/body weight ratio before and 48 hours after partial hepatectomy (PH) in

\*p < 0.05, \*\*p < 0.01 versus thrombocytopenic group. #p < 0.05 normal group versus thrombocytotic group. (B) Ki-67 labeling index 48 hours after partial hepatectomy in

immunohistochemical images are shown. Data were expressed as mean ± SD. \*p < 0.05 versus thrombocytopenic group. (Reproduced from Murata et al., 2007, World J Surg with

thrombocytotic, normal and thrombocytopenic groups. Representative

permission.)

groups after partial hepatectomy (Fig. 4).

Fig. 4. Effect of platelet increment or reduction on Akt, STAT3, and ERK 1/2 after partial hepatectomy. (Reproduced from Murata et al., 2007, World J Surg. with permission).

Platelet accumulation in the liver was investigated in all groups before and 5 minutes after partial hepatectomy. Platelets accumulated in the residual liver within 5 minutes after partial hepatectomy and a two-fold increase in platelet levels was observed in the normal and thrombocytotic groups (Fig. 5A). On the other hand, in thrombocytotic group, platelets in the residual liver increased remarkably compared with normal and thrombocytopenic groups 5 minutes after partial hepatectomy. However, no increment was observed in thrombocytopenic group. In addition, under transmission electron microscopy, platelets translocated from the liver sinusoidal space into the space of Disse, and they had direct contact with hepatocytes in the thrombocytotic group (Fig. 5B).

These results suggest that platelets accumulate in the liver within a few minutes of partial hepatectomy and cause rapid hepatocyte proliferation through direct contact with hepatocytes, by translocating into the space of Disse.

Taken together, the results described above demonstrate that platelets affect liver regeneration in the acute phase after partial hepatectomy and suggested that the PI3K/Akt pathway is the main signaling pathway involved in platelet mediated liver regeneration.

The following study was done to investigate the role of platelets in liver regeneration using a thrombocytosis model in mice after 90% partial hepatectomy (Myronovych et al. 2008). All mice in the normal group died within 30 hours, predominantly between 20 and 30 hours. In contrast, the survival rate at 30 hours and at one week after partial hepatectomy was 54.5% and 27.3%, respectively in thrombocytotic group (Fig. 6A). Phosphorylation of Akt and STAT3 started earlier and stronger in thrombocytotic group than normal group. Serum albumin levels decreased in both groups after partial hepatectomy, but more rapidly in normal group, and there was a significant difference with higher levels being detected at 24 hours post-hepatectomy in the livers of the thrombocytotic group (Fig 6B). Serum

Platelet and Liver Regeneration 117

cholesterol levels were higher in thrombocytotic group at all time points with a significant difference at 24 hours after partial hepatectomy (Fig. 6C). In our measurement of insulin-like growth factor binding protein (IGFBP-1) by real-time PCR, the peak value of IGFBP-1 expression was reached sooner in the thrombocytotic group than in the normal group after

The findings described above indicated that liver regeneration occurs even in 90% hepatectomized mice under conditions of thrombocytosis. Platelets contribute to cell cycle progression and metabolic pathways, and maintain liver function after the extended

We also evaluated the effect of TPO on liver regeneration after partial hepatectomy and on fibrosis under conditions of liver cirrhosis in rats (Murata et al., 2008). Rats were divided into three groups as follows; a normal group without any treatment, a liver cirrhosis (LC) group, and an LC group with a single administration of TPO (LC+TPO). 70% of partial hepatectomy was performed and liver regeneration and anti-fibrotic effects

In the LC group, the platelet count in the blood was significantly lower than that in the normal group. In the LC+TPO group, platelet count increased 2-fold higher than that in the normal group (Fig. 7A). The hepatocyte PCNA labeling index 24 hours after partial hepatectomy in the LC group was significantly lower than that in the normal group; the PCNA labeling index in the LC+TPO group was significantly higher than that in the LC group and the same level as that in normal group (Fig. 7B). HGF concentration in liver tissue in the LC+TPO group at the time of partial hepatectomy was clearly higher than that in the normal group. IGF-1 concentration in the liver tissue in LC+TPO group was significantly higher than that in normal group. Fibrotic change around the portal regions in the LC group was more prominent than that in the normal group. In contrast, fibrotic change decreased

These results described above indicated that a single administration of TPO in cirrhotic liver induces the remarkable increment of the platelets and then improves liver regeneration and

We examined whether the TPO itself or increased platelets have a hepatocyte-proliferative effect and anti-fibrotic effect on the fibrotic liver. We injected anti-platelet serum (APS) into LC+TPO group (LC+TPO+APS). The platelet count of LC+TPO+APS group decreased remarkably compared with LC and LC+TPO groups (Fig. 8A). PCNA labeling index 24 hours after partial hepatectomy was markedly lower in LC+TPO+APS group than that in LC and LC+TPO groups (Fig. 8B). Furthermore, liver fibrotic area before partial hepatectomy increased significantly in LC+TPO+APS group compared with LC+TPO group

These results clearly indicate that acceleration of liver regeneration and anti-fibrotic effects

We investigated whether exogenous platelets have the similar encouraging effect on liver regeneration. Platelet-rich plasma (PRP) was infused via the portal vein after 70% partial

of TPO administration are induced by increment of platelets, not by TPO itself.

partial hepatectomy and decreased moderately afterwards.

hepatectomy.

were compared.

(Fig. 8C).

remarkably in the LC+TPO group (Fig. 7C)

fibrosis of cirrhotic liver after 70% of partial hepatectomy.

(B)

Fig. 5. Immunohistochemistry and Transmission electron microscopic photograph of the residual liver. (A) Immunohistochemistry of liver frozen section. Red; platelets. Platelets are stained by Pm-1 antibody. Representative images 5 minutes after partial hepatectomy (PH) are shown. Platelets were counted before and 5 minutes after partial hepatectomy in thrombocytopenic, normal, and thrombocytotic groups. Data were expressed as mean ± SD. \*p < 0.05 versus before partial hepatectomy. Original magnification X 400. (B) Transmission electron microscopic image of partial hepatectomy in the residual liver 5 minutes after partial hepatectomy in thrombocytotic group. Arrow indicates platelet translocations into the space of Disse through the porosity of a flattened process in a sinusoidal endothelial cell (SEC). Original magnification X 7500. (Reproduced from Murata et al., 2007, World J Surg with permission.)

(A)

(B) Fig. 5. Immunohistochemistry and Transmission electron microscopic photograph of the residual liver. (A) Immunohistochemistry of liver frozen section. Red; platelets. Platelets are stained by Pm-1 antibody. Representative images 5 minutes after partial hepatectomy (PH) are shown. Platelets were counted before and 5 minutes after partial hepatectomy in thrombocytopenic, normal, and thrombocytotic groups. Data were expressed as mean ± SD. \*p < 0.05 versus before partial hepatectomy. Original magnification X 400. (B) Transmission electron microscopic image of partial hepatectomy in the residual liver 5 minutes after partial hepatectomy in thrombocytotic group. Arrow indicates platelet translocations into the space of Disse through the porosity of a flattened process in a sinusoidal endothelial cell (SEC). Original magnification X 7500. (Reproduced from

Murata et al., 2007, World J Surg with permission.)

cholesterol levels were higher in thrombocytotic group at all time points with a significant difference at 24 hours after partial hepatectomy (Fig. 6C). In our measurement of insulin-like growth factor binding protein (IGFBP-1) by real-time PCR, the peak value of IGFBP-1 expression was reached sooner in the thrombocytotic group than in the normal group after partial hepatectomy and decreased moderately afterwards.

The findings described above indicated that liver regeneration occurs even in 90% hepatectomized mice under conditions of thrombocytosis. Platelets contribute to cell cycle progression and metabolic pathways, and maintain liver function after the extended hepatectomy.

We also evaluated the effect of TPO on liver regeneration after partial hepatectomy and on fibrosis under conditions of liver cirrhosis in rats (Murata et al., 2008). Rats were divided into three groups as follows; a normal group without any treatment, a liver cirrhosis (LC) group, and an LC group with a single administration of TPO (LC+TPO). 70% of partial hepatectomy was performed and liver regeneration and anti-fibrotic effects were compared.

In the LC group, the platelet count in the blood was significantly lower than that in the normal group. In the LC+TPO group, platelet count increased 2-fold higher than that in the normal group (Fig. 7A). The hepatocyte PCNA labeling index 24 hours after partial hepatectomy in the LC group was significantly lower than that in the normal group; the PCNA labeling index in the LC+TPO group was significantly higher than that in the LC group and the same level as that in normal group (Fig. 7B). HGF concentration in liver tissue in the LC+TPO group at the time of partial hepatectomy was clearly higher than that in the normal group. IGF-1 concentration in the liver tissue in LC+TPO group was significantly higher than that in normal group. Fibrotic change around the portal regions in the LC group was more prominent than that in the normal group. In contrast, fibrotic change decreased remarkably in the LC+TPO group (Fig. 7C)

These results described above indicated that a single administration of TPO in cirrhotic liver induces the remarkable increment of the platelets and then improves liver regeneration and fibrosis of cirrhotic liver after 70% of partial hepatectomy.

We examined whether the TPO itself or increased platelets have a hepatocyte-proliferative effect and anti-fibrotic effect on the fibrotic liver. We injected anti-platelet serum (APS) into LC+TPO group (LC+TPO+APS). The platelet count of LC+TPO+APS group decreased remarkably compared with LC and LC+TPO groups (Fig. 8A). PCNA labeling index 24 hours after partial hepatectomy was markedly lower in LC+TPO+APS group than that in LC and LC+TPO groups (Fig. 8B). Furthermore, liver fibrotic area before partial hepatectomy increased significantly in LC+TPO+APS group compared with LC+TPO group (Fig. 8C).

These results clearly indicate that acceleration of liver regeneration and anti-fibrotic effects of TPO administration are induced by increment of platelets, not by TPO itself.

We investigated whether exogenous platelets have the similar encouraging effect on liver regeneration. Platelet-rich plasma (PRP) was infused via the portal vein after 70% partial

Platelet and Liver Regeneration 119

(A)

(B)

(C)

Fig. 7. Effect of TPO on platelet count, liver regeneration and fibrosis. (A) Platelet count before partial hepatectomy in normal, LC, and LC+TPO groups. Data were expressed as mean ± SD. \*p < 0.05 versus normal group. (B) The hepatocyte PCNA labeling index before and 24 hours after partial hepatectomy (PH) in normal, LC, and LC+TPO groups. Data were

hepatectomy. \*p < 0.05 versus normal group 24 hours after partial hepatectomy. \$p < 0.05

(C) Fibrotic change in the liver in normal, LC, and LC+TPO groups. Representative image in each group. Sirius red staining of liver sections. Original magnification × 200. (Reproduced

expressed as mean ± SD. #p < 0.05, ##p < 0.01 versus normal group before partial

versus LC group 24 hours after partial hepatectomy.

from Murata et al., 2008, Ann Surg with permission.)

Fig. 6. Effect of thrombocytosis on survival and metabolic pathways after 90% of partial hepatectomy (PH). (A) Survival rate with Kaplan-Meier method. \*p < 0.05 versus normal group. (B) Change in serum albumin concentration. Data were expressed as mean ± SD. \*p < 0.05 versus normal group. (C) Change in serum total cholesterol concentration. Data were expressed as mean ± SD. \*p < 0.05 versus normal group.

(Reproduced from Myronovych et al., 2008, J Hepatol with permission.)

(A)

(B)

(C)

Fig. 6. Effect of thrombocytosis on survival and metabolic pathways after 90% of partial hepatectomy (PH). (A) Survival rate with Kaplan-Meier method. \*p < 0.05 versus normal group. (B) Change in serum albumin concentration. Data were expressed as mean ± SD. \*p < 0.05 versus normal group. (C) Change in serum total cholesterol concentration. Data

were expressed as mean ± SD. \*p < 0.05 versus normal group.

(Reproduced from Myronovych et al., 2008, J Hepatol with permission.)

Fig. 7. Effect of TPO on platelet count, liver regeneration and fibrosis. (A) Platelet count before partial hepatectomy in normal, LC, and LC+TPO groups. Data were expressed as mean ± SD. \*p < 0.05 versus normal group. (B) The hepatocyte PCNA labeling index before and 24 hours after partial hepatectomy (PH) in normal, LC, and LC+TPO groups. Data were expressed as mean ± SD. #p < 0.05, ##p < 0.01 versus normal group before partial hepatectomy. \*p < 0.05 versus normal group 24 hours after partial hepatectomy. \$p < 0.05 versus LC group 24 hours after partial hepatectomy.

(C)

(C) Fibrotic change in the liver in normal, LC, and LC+TPO groups. Representative image in each group. Sirius red staining of liver sections. Original magnification × 200. (Reproduced from Murata et al., 2008, Ann Surg with permission.)

Platelet and Liver Regeneration 121

(A)

(B)

(C)

Fig. 8. Effect of platelets on liver regeneration and fibrosis of the liver. (A) Platelet count before partial hepatectomy (PH). (B) PCNA labeling index before and 24 hours after partial hepatectomy. (C) Fibrotic area of the liver in LC, LC+TPO, and LC+TPO+APS groups. Data were expressed as mean ± SD. #p < 0.05, ##p < 0.01 versus LC group. \*p < 0.05 versus LC+TPO group. (Reproduced from Murata et al., 2008, Ann Surg with permission.)

hepatectomy and residual liver regeneration was evaluated in rats (Matsuo et al., 2011). To clarify the mechanisms by which platelet promote liver regeneration, we also analyzed the dynamics of platelets infused in the liver before and after partial hepatectomy using an intravital microscope (IVM).

The liver/body weight ratio 24 hours after partial hepatectomy was significantly higher in PRP transfused group (PRP+) than in the normal saline administered group (PRP-). The hepatocyte Ki-67 labeling index was significantly higher in the PRP+ group than that in the PRP- group. Akt and ERK 1/2 became phosphorylated earlier in the PRP+ group than in the PRP- group, whereas phosphorylation of STAT3 did not apparently differ between the two groups. Under IVM, although platelets flowed fast and few of them rolled and adhered in the liver sinusoids before partial hepatectomy, a significant proportion of platelets accumulated in the liver sinusoids and flowed slowly with adhesion and rolling after partial hepatectomy. The findings in this experiment indicate that exogenous platelets also promote liver regeneration.

Since the anatomy of porcine liver is similar to that of the human, and porcine liver is useful for mimicking human liver surgery, we evaluated the effect of platelets in anti-liver damage and liver regeneration using pigs (Hisakura et al., 2010). To induce thrombocytosis, pigs received TPO administration (TPO+) or were performed splenectomy (Sp+). Pigs underwent 80% partial hepatectomy and were assigned to either TPO-, TPO+, Sp-, or Sp+ groups; liver damage, histological findings including necrotic changes, ballooning, cholestasis, and liver regeneration were compared among these groups. Serum aspartate aminotransferase levels in the TPO+ group were significantly lower than that in the TPO- group on day 2 after partial hepatectomy. Serum alanine aminotransferase levels in the Sp+ group were significantly lower than that in the Spgroup on day 2 after partial hepatectomy. Serum alkaline phosphatase levels in the TPO+ and the Sp+ groups were significantly lower than those in the TPO- and the Sp- group at 6 hours and on day 2 after partial hepatectomy. Histological analysis for cholestasis, ballooning, and hepatocyte necrosis was carried out by using a scoring system (Table. 1). Although cholestasis, ballooning, and hepatocyte necrosis were observed in zone 2 in TPO- and Sp- groups, structure was mostly preserved in TPO+ and Sp+ group (Fig. 9). On the other hand, the liver/body weight ratio and the hepatocyte PCNA labeling index showed no significant difference among the groups on day 2 and 7 after partial hepatectomy.

Under transmission microscopy, the sinusoidal endothelial linings were destroyed and detached into sinusoidal space with the enlargement of the spaces of Disse and the cytoplasm of sinusoidal endothelial cells was swollen with secondary lysosomes 2 hours after partial hepatectomy in TPO- or Sp- group (Fig. 10). In contrast, the structure of the endothelial lining was well preserved in TPO+ and Sp+ group.

Although there was no direct evidence of platelets in promoting liver regeneration in this experiment, the results indicated that increase in the number of platelets protect sinusoidal linings from disturbance and prevent acute liver damage after extended hepatectomy.

hepatectomy and residual liver regeneration was evaluated in rats (Matsuo et al., 2011). To clarify the mechanisms by which platelet promote liver regeneration, we also analyzed the dynamics of platelets infused in the liver before and after partial hepatectomy using an

The liver/body weight ratio 24 hours after partial hepatectomy was significantly higher in PRP transfused group (PRP+) than in the normal saline administered group (PRP-). The hepatocyte Ki-67 labeling index was significantly higher in the PRP+ group than that in the PRP- group. Akt and ERK 1/2 became phosphorylated earlier in the PRP+ group than in the PRP- group, whereas phosphorylation of STAT3 did not apparently differ between the two groups. Under IVM, although platelets flowed fast and few of them rolled and adhered in the liver sinusoids before partial hepatectomy, a significant proportion of platelets accumulated in the liver sinusoids and flowed slowly with adhesion and rolling after partial hepatectomy. The findings in this experiment indicate that exogenous platelets also promote

Since the anatomy of porcine liver is similar to that of the human, and porcine liver is useful for mimicking human liver surgery, we evaluated the effect of platelets in anti-liver damage and liver regeneration using pigs (Hisakura et al., 2010). To induce thrombocytosis, pigs received TPO administration (TPO+) or were performed splenectomy (Sp+). Pigs underwent 80% partial hepatectomy and were assigned to either TPO-, TPO+, Sp-, or Sp+ groups; liver damage, histological findings including necrotic changes, ballooning, cholestasis, and liver regeneration were compared among these groups. Serum aspartate aminotransferase levels in the TPO+ group were significantly lower than that in the TPO- group on day 2 after partial hepatectomy. Serum alanine aminotransferase levels in the Sp+ group were significantly lower than that in the Spgroup on day 2 after partial hepatectomy. Serum alkaline phosphatase levels in the TPO+ and the Sp+ groups were significantly lower than those in the TPO- and the Sp- group at 6 hours and on day 2 after partial hepatectomy. Histological analysis for cholestasis, ballooning, and hepatocyte necrosis was carried out by using a scoring system (Table. 1). Although cholestasis, ballooning, and hepatocyte necrosis were observed in zone 2 in TPO- and Sp- groups, structure was mostly preserved in TPO+ and Sp+ group (Fig. 9). On the other hand, the liver/body weight ratio and the hepatocyte PCNA labeling index showed no significant difference among the groups on day 2 and 7 after partial

Under transmission microscopy, the sinusoidal endothelial linings were destroyed and detached into sinusoidal space with the enlargement of the spaces of Disse and the cytoplasm of sinusoidal endothelial cells was swollen with secondary lysosomes 2 hours after partial hepatectomy in TPO- or Sp- group (Fig. 10). In contrast, the structure of the

Although there was no direct evidence of platelets in promoting liver regeneration in this experiment, the results indicated that increase in the number of platelets protect sinusoidal linings from disturbance and prevent acute liver damage after extended

endothelial lining was well preserved in TPO+ and Sp+ group.

intravital microscope (IVM).

liver regeneration.

hepatectomy.

hepatectomy.

Fig. 8. Effect of platelets on liver regeneration and fibrosis of the liver. (A) Platelet count before partial hepatectomy (PH). (B) PCNA labeling index before and 24 hours after partial hepatectomy. (C) Fibrotic area of the liver in LC, LC+TPO, and LC+TPO+APS groups. Data were expressed as mean ± SD. #p < 0.05, ##p < 0.01 versus LC group. \*p < 0.05 versus LC+TPO group. (Reproduced from Murata et al., 2008, Ann Surg with permission.)

Platelet and Liver Regeneration 123

Table 1. Scoring system of cholestasis, ballooning, and hepatocyte necrosis. (Reproduced

Fig. 10. Transmission electron microscopic findings 2 hours after partial hepatectomy. Magnification × 6000. In TPO- (A), and Sp- groups (B), the sinusoidal endothelial lining was destroyed and detached into the sinusoidal space with enlargement of the space of Disse (arrows), and the cytoplasm of sinusoidal endothelial cells were swollen with secondary lysosomes (arrow head). In contrast, in TPO+ (C), and Sp+ groups (D), the sinusoidal endothelial cells were well preserved. (Reproduced from Hisakura et al., 2010, *J Hepatobiliary* 

*Pancreat Sci* with permission.)

from Hisakura et al., 2010, *J Hepatobiliary Pancreat Sci* with permission.)

Fig. 9. Semiquantitative scoring for cholestasis, ballooning, and hepatocyte necrosis in TPO-, TPO+, Sp-, and Sp+ groups. Data are expressed as means ± SD. \*p < 0.05 versus TPO- group or Sp- group. (Reproduced from Hisakura et al, 2010, J Hepatobiliary Pancreat Sci with permission.)

Fig. 9. Semiquantitative scoring for cholestasis, ballooning, and hepatocyte necrosis in TPO-, TPO+, Sp-, and Sp+ groups. Data are expressed as means ± SD. \*p < 0.05 versus TPO- group or Sp- group. (Reproduced from Hisakura et al, 2010, J Hepatobiliary Pancreat Sci with

permission.)


Table 1. Scoring system of cholestasis, ballooning, and hepatocyte necrosis. (Reproduced from Hisakura et al., 2010, *J Hepatobiliary Pancreat Sci* with permission.)

Fig. 10. Transmission electron microscopic findings 2 hours after partial hepatectomy. Magnification × 6000. In TPO- (A), and Sp- groups (B), the sinusoidal endothelial lining was destroyed and detached into the sinusoidal space with enlargement of the space of Disse (arrows), and the cytoplasm of sinusoidal endothelial cells were swollen with secondary lysosomes (arrow head). In contrast, in TPO+ (C), and Sp+ groups (D), the sinusoidal endothelial cells were well preserved. (Reproduced from Hisakura et al., 2010, *J Hepatobiliary Pancreat Sci* with permission.)

Platelet and Liver Regeneration 125

same level as that of platelet- group. Moreover, this effect in the upper-mix group was of the same level as in the co-mix group. These results indicate that, upon direct contact with hepatocytes, platelets released soluble factors that induce hepatocyte proliferation. A proliferative effect was also observed in the thrombin-stimulated group, despite there being

To clarify which component of platelets had an effect on hepatocyte proliferation, the mitogenic activity of the whole disrupted platelets, the soluble fraction, and the membrane fraction were evaluated. The whole disrupted platelets and the soluble fraction had significant proliferative effects, whereas the membrane fraction did not have the effect. To determine which element of the platelet soluble factor exerted the proliferative effect, platelet extracts were gel-excluded into 18 fractions, and mitogenic activity of each fraction was evaluated on BrdU assay. Mitogenic activity was strongly induced in the fraction of HGF, VEGF, and IGF-1 (Fig. 12A). In addition, when hepatocyte signals were analyzed in response to growth factors, HGF, IGF-1, and VEGF strongly activated the Akt and the ERK1/2 pathways, whereas PDGF and serotonin did not induced activation (Fig. 12B). For further confirmation of the platelets soluble factors, IGF-1 and HGF inhabitation using anti-IGF-1 and anti-HGF antibodies significantly inhibited hepatocyte

The results of this examination indicated that the direct contact between platelets and hepatocytes triggered the release of soluble factors from the platelets such as IGF-1 and

We assessed the direct effect of platelets using Kupffer cell depletion model (Murata et al., 2008). Liposome-encapsulated dichloromethylene diphosphonate (Cl2-MDP) was used for the depletion of Kupffer cells. Mice were divided into four groups as follows: mice without any treatment (normal), mice with Kupffer cell depletion (KD), mice with thrombocytosis caused by injection of thrombopoietin (thrombocytosis), and mice undergoing Kupffer cell depletion and thrombocytosis by injection of thrombopoietin (TKD). Each group of mice underwent 70% partial hepatectomy, and liver regeneration, cytokine and growth factors expression, and phosphorylation of Akt were assayed in the

The liver/body weight ratio in KD group was significantly lower than that in normal group 48 hours after partial hepatectomy. The liver/body weight ratio in TKD group was almost the same as that in normal group. In thrombocytotic group, the liver/body weight ratio was significantly higher than that in normal group (Fig. 13 A). The hepatocyte mitotic index of the KD group 48 hours after partial hepatectomy was significantly lower than that in normal group. And, the hepatocyte mitotic index in the TKD group was almost the same as that in normal group. Furthermore, the hepatocyte mitotic index in thrombocytotic group was significantly higher than those in other groups. Moreover, the hepatocyte PCNA labeling index 48 hours after partial hepatectomy in the KD group was significantly lower compared with normal group. And, in the TKD group, it was significantly higher than that in KD group and the same as that in thrombocytotic and

no direct contact between platelets and hepatocytes (Fig. 11).

HGF, which caused a proliferative effect on the hepatocytes.

proliferation.

groups.

normal groups (Fig. 13 B).

#### **4. Mechanisms of direct effect of platelets on liver regeneration**

Up to the beginning of the 21st century, there was no report regarding the effect of platelets on liver regeneration. Two studies were reported in which platelets promoted liver regeneration (Murata et al., 2004; Lesurtel et al., 2006). We reported that platelets accumulate in the liver and translocate actively to the space of Disse through fenestrae of LSECs after partial hepatectomy, which enables platelets to contact directly with hepatocytes (Murata et al., 2007). To clarify the role of the direct contact of platelets with hepatocytes, we investigated by using co-culturing chamber systems where platelets and hepatocytes were separated by a permeable membrane (Matsuo et al., 2008). To elucidate characteristics of the direct contact, four groups of separated co-culture were prepared as follows: a without platelet group (platelet-), a mixed co-culture group (co-mix), a separated co-culture group (co-sep), a group with mixed cells (the upper mix group: upper-mix), and the thrombinstimulated group (thrombin stimulated) were prepared (Fig. 11). TLR2 cells, the murine immortalized primary hepatocytes, in the lower chamber were counted 72 hours after incubation. In the upper-mix group, platelets induced significant proliferation of hepatocytes in the lower chamber, whereas the proliferation in co-sep group was almost the

Fig. 11. Co-culture system to elucidate the characteristics of direct contact. Without platelet group (platelet-): neither hepatocytes nor platelets were seeded in the upper chamber. Separated co-cultured group (co-sep): platelets were seeded in the upper chamber. Upper mix group (upper-mix): hepatocytes were seeded in the upper chamber and overlayered with platelets. Co-mixed group (co-mix): platelets and hepatocytes were seeded in the lower chamber. Thrombin stimulated group (thrombin-stimulated): platelets in the upper chamber were stimulated with thrombin to release soluble factors such as cytokines and growth factors. Hepatocytes in the lower chamber are counted after 72 hours of incubation. Data are expressed as means SD. \*p < 0.05 versus platelet-. (Reproduced from Matsuo et al., *J Surg Res* with permission.)

Up to the beginning of the 21st century, there was no report regarding the effect of platelets on liver regeneration. Two studies were reported in which platelets promoted liver regeneration (Murata et al., 2004; Lesurtel et al., 2006). We reported that platelets accumulate in the liver and translocate actively to the space of Disse through fenestrae of LSECs after partial hepatectomy, which enables platelets to contact directly with hepatocytes (Murata et al., 2007). To clarify the role of the direct contact of platelets with hepatocytes, we investigated by using co-culturing chamber systems where platelets and hepatocytes were separated by a permeable membrane (Matsuo et al., 2008). To elucidate characteristics of the direct contact, four groups of separated co-culture were prepared as follows: a without platelet group (platelet-), a mixed co-culture group (co-mix), a separated co-culture group (co-sep), a group with mixed cells (the upper mix group: upper-mix), and the thrombinstimulated group (thrombin stimulated) were prepared (Fig. 11). TLR2 cells, the murine immortalized primary hepatocytes, in the lower chamber were counted 72 hours after incubation. In the upper-mix group, platelets induced significant proliferation of hepatocytes in the lower chamber, whereas the proliferation in co-sep group was almost the

**4. Mechanisms of direct effect of platelets on liver regeneration** 

Fig. 11. Co-culture system to elucidate the characteristics of direct contact.

of incubation. Data are expressed as means SD. \*p < 0.05 versus platelet-.

(Reproduced from Matsuo et al., *J Surg Res* with permission.)

Without platelet group (platelet-): neither hepatocytes nor platelets were seeded in the upper chamber. Separated co-cultured group (co-sep): platelets were seeded in the upper chamber. Upper mix group (upper-mix): hepatocytes were seeded in the upper chamber and overlayered with platelets. Co-mixed group (co-mix): platelets and hepatocytes were seeded in the lower chamber. Thrombin stimulated group (thrombin-stimulated): platelets in the upper chamber were stimulated with thrombin to release soluble factors such as cytokines and growth factors. Hepatocytes in the lower chamber are counted after 72 hours same level as that of platelet- group. Moreover, this effect in the upper-mix group was of the same level as in the co-mix group. These results indicate that, upon direct contact with hepatocytes, platelets released soluble factors that induce hepatocyte proliferation. A proliferative effect was also observed in the thrombin-stimulated group, despite there being no direct contact between platelets and hepatocytes (Fig. 11).

To clarify which component of platelets had an effect on hepatocyte proliferation, the mitogenic activity of the whole disrupted platelets, the soluble fraction, and the membrane fraction were evaluated. The whole disrupted platelets and the soluble fraction had significant proliferative effects, whereas the membrane fraction did not have the effect. To determine which element of the platelet soluble factor exerted the proliferative effect, platelet extracts were gel-excluded into 18 fractions, and mitogenic activity of each fraction was evaluated on BrdU assay. Mitogenic activity was strongly induced in the fraction of HGF, VEGF, and IGF-1 (Fig. 12A). In addition, when hepatocyte signals were analyzed in response to growth factors, HGF, IGF-1, and VEGF strongly activated the Akt and the ERK1/2 pathways, whereas PDGF and serotonin did not induced activation (Fig. 12B). For further confirmation of the platelets soluble factors, IGF-1 and HGF inhabitation using anti-IGF-1 and anti-HGF antibodies significantly inhibited hepatocyte proliferation.

The results of this examination indicated that the direct contact between platelets and hepatocytes triggered the release of soluble factors from the platelets such as IGF-1 and HGF, which caused a proliferative effect on the hepatocytes.

We assessed the direct effect of platelets using Kupffer cell depletion model (Murata et al., 2008). Liposome-encapsulated dichloromethylene diphosphonate (Cl2-MDP) was used for the depletion of Kupffer cells. Mice were divided into four groups as follows: mice without any treatment (normal), mice with Kupffer cell depletion (KD), mice with thrombocytosis caused by injection of thrombopoietin (thrombocytosis), and mice undergoing Kupffer cell depletion and thrombocytosis by injection of thrombopoietin (TKD). Each group of mice underwent 70% partial hepatectomy, and liver regeneration, cytokine and growth factors expression, and phosphorylation of Akt were assayed in the groups.

The liver/body weight ratio in KD group was significantly lower than that in normal group 48 hours after partial hepatectomy. The liver/body weight ratio in TKD group was almost the same as that in normal group. In thrombocytotic group, the liver/body weight ratio was significantly higher than that in normal group (Fig. 13 A). The hepatocyte mitotic index of the KD group 48 hours after partial hepatectomy was significantly lower than that in normal group. And, the hepatocyte mitotic index in the TKD group was almost the same as that in normal group. Furthermore, the hepatocyte mitotic index in thrombocytotic group was significantly higher than those in other groups. Moreover, the hepatocyte PCNA labeling index 48 hours after partial hepatectomy in the KD group was significantly lower compared with normal group. And, in the TKD group, it was significantly higher than that in KD group and the same as that in thrombocytotic and normal groups (Fig. 13 B).

Platelet and Liver Regeneration 127

(A)

(B)

(A) Liver/body weight ratio 2 and 48 hours after partial hepatectomy. Data are expressed as means ± SD. \*p < 0.05 versus normal group, \$p < 0.05 versus thrombocytosis; #p < 0.05 versus TKD group. (B) The hepatocyte PCNA labeling index 48 hours after partial hepatectomy. Data are expressed as means ± SD. \*p < 0.05 versus normal group.

The liver content of TNF-alpha, HGF, and IGF-1 was assessed in normal, KD, and TKD groups. TNF-alpha expression increased and reached the peak 2 hours after partial

Fig. 13. Liver regeneration indexes under Kupffer cell depletion and thrombocytosis.

(Reproduced from Murata et al., 2008, World J Surg with permission.)

(B)

Fig. 12. Gel exclusion chromatography of platelet extracts and mitotic activities. (A) The platelet extracts were gel filtrated on Superdex G200 gel. The solid line shows the resulting absorbance profile at 280 nm. The broken line shows the mitogenic activity of each fraction. Fraction 1 and 2 were nonspecifically macroaggregated proteins. Significant mitogenic activity was observed in fraction 1 ,2, 5-7, and 14-17. On western blotting, fractions 4-6 were rich in HGF, fraction 5-7 were rich in VEGF, fractions 7-9 were rich in PDGF, and fraction 14-17 were rich in IGF-1. Data were expressed as means ± SD of each experiments. (B) Cellular signals of hepatocytes stimulated by platelets and PDGF, HGF, PDGF, IGF-1, VEGF, and Serotonin (5-HT). (Reproduced from Matsuo et al., 2008, J Surg Res with permission.)

(A)

(B)

(A) The platelet extracts were gel filtrated on Superdex G200 gel. The solid line shows the resulting absorbance profile at 280 nm. The broken line shows the mitogenic activity of each fraction. Fraction 1 and 2 were nonspecifically macroaggregated proteins. Significant mitogenic activity was observed in fraction 1 ,2, 5-7, and 14-17. On western blotting, fractions 4-6 were rich in HGF, fraction 5-7 were rich in VEGF, fractions 7-9 were rich in PDGF, and fraction 14-17 were rich in IGF-1. Data were expressed as means ± SD of each experiments. (B) Cellular signals of hepatocytes stimulated by platelets and PDGF, HGF, PDGF, IGF-1, VEGF, and Serotonin (5-HT). (Reproduced from Matsuo et al., 2008, J Surg Res

Fig. 12. Gel exclusion chromatography of platelet extracts and mitotic activities.

with permission.)

Fig. 13. Liver regeneration indexes under Kupffer cell depletion and thrombocytosis. (A) Liver/body weight ratio 2 and 48 hours after partial hepatectomy. Data are expressed as means ± SD. \*p < 0.05 versus normal group, \$p < 0.05 versus thrombocytosis; #p < 0.05 versus TKD group. (B) The hepatocyte PCNA labeling index 48 hours after partial hepatectomy. Data are expressed as means ± SD. \*p < 0.05 versus normal group. (Reproduced from Murata et al., 2008, World J Surg with permission.)

The liver content of TNF-alpha, HGF, and IGF-1 was assessed in normal, KD, and TKD groups. TNF-alpha expression increased and reached the peak 2 hours after partial

Platelet and Liver Regeneration 129

Fig. 14. Scheme for liver regeneration promoted directly by platelets.

of mitosis.

p21.

regards to liver regeneration.

Platelets translocate to the space of Disse and release growth factors such as IGF-1 and HGF through direct contact with hepatocytes. The growth factors subsequently induce initiation

as nitric oxide and endothelin (Wisse et al., 1996; Vollmar & Menger, 2009; Ping et al., 2006). IL-6 produced by LSECs and Kupffer cells is one of the important components of early signaling pathways in liver regeneration, and it activates the acute phase of protein synthesis by hepatocytes as part of the overall inflammatory response (Gauldie et al., 1992; Michalopoulos & DeFrances, 1997). After hepatectomy, plasma IL-6 concentration is reported to increase from 6 hours to a peak by 24 hours (Rai et al., 1996; Badia et al., 1998). IL-6 binds to its receptor on hepatocytes, which subsequently leads to phosphorylation of STAT3 monomers (Fausto et al., 2006). STAT3 homodimerizes and translocates to the nucleus, where it stimulates transcription of a number target genes such as cyclin-D1 and

The relationship between platelets and LSECs has been well-documented in ischemia/reperfusion injury models. Rolling and adhering of leukocytes on LSECs with subsequent interaction with platelets is the important pathogenesis of ischemia/reperfusion injury (Montalvo-Jave et al., 2008; Croner et al., 2006, Pak et al., 2010). It was also reported that transient interaction, i.e., rolling, and permanent adhesion of platelets to the postischemic hepatic endothelium stimulate platelet activation and expression of endothelial adhesion molecules (Massberg et al., 1998; Khandoga et al., 2003). There have not, however, been any prior studies focused on the relationship between human platelets and LSECs with

To clarify the role of platelets in liver regeneration in relation to LSECs, we used coculturing chamber systems where platelets and LSECs could be separated by a permeable

hepatectomy in normal group, whereas it remained low in KD and TKD groups. HGF concentration in the liver tissue in TKD group at the time of partial hepatectomy was significantly higher than that in normal group, and it persisted 6 hours after partial hepatectomy. At the same time, IGF-1 concentration in the liver tissue in KD and TKD groups at the time of partial hepatectomy was significantly lower than that in normal group, and IGF-1 concentration in the TKD groups was higher than that in the KD group. Furthermore, Akt was strongly phosphorylated in normal group compared with the KD group. In the TKD group, phosphorylation of Akt was started at the time of PH and lasted until 120 minutes after PH, and it was almost the same level as it was in normal group.

Platelet accumulation 2 hours after partial hepatectomy was investigated in each group. Platelet accumulation in the KD group demonstrated a significant decrease compared with the normal group. Moreover, platelet accumulation in the TKD group showed a significantly higher level than that in the KD group, and it was almost the same level as that in the normal group. In the thrombocytotic group, platelet accumulation increased significantly compared with the normal group. Transmission electron microscopy demonstrated that in the thrombocytotic group, platelets translocated from the liver sinusoidal space to the space of Disse and were in direct contact with hepatocytes at 5 minutes after hepatectomy.

These results clearly demonstrate that platelets promote liver regeneration under conditions of Kupffer cell depletion. Increase of platelets recruited platelets in the liver tissue and elevated and HGF concentrations in the liver, which activated downstream signaling transduction and hepatocyte mitosis.

In conclusion, our previous studies clarified the direct effect of platelets in promoting liver regeneration. The mechanism is explained as follows; after partial hepatectomy, platelets accumulate in the liver, they translocate to the space of Disse and release growth factors such as IGF-1 and HGF through direct contact with hepatocytes. The growth factors stimulate initiation of hepatocyte mitosis, which eventually promote liver regeneration. Especially in human, since it was reported that human platelets do not contain a significant amount of HGF (Nakamura et al., 1989), IGF-1 is the most important mediator for liver regeneration, which is contained in human platelets (Fig. 14).

#### **5. The effect with liver sinusoidal endothelial cells**

LSECs comprise 70% of the sinusoidal cells (Knook & Sleyster, 1976; Smedsrod et al., 1990). By construction of a thin and continuous layer, the sinusoidal endothelium forms the structural barrier, separating the hepatic parenchyma from blood constituents passing through the liver. Unlike other vascular endothelial cells, LSECs have large cytoplasmic gaps without basal membranes. These enable maximal contact between circulating blood and hepatocytes to help exchange various soluble macromolecules and nano-particles such as lipoproteins and endocytosis (Braet & Wisse, 2002). LSECs are involved in liver regeneration as well as Kupffer cells and hepatic stellate cells, and they are known to produce immunoregulatory and pro-inflammatory cytokines including HGF, interleukin-1 (IL-1), IL-6, and interferon. In addition, they synthesize eicosanoids, particularly TXA2, prostaglandin E2, as well as synthesizing important regulators of vascular tone, such

hepatectomy in normal group, whereas it remained low in KD and TKD groups. HGF concentration in the liver tissue in TKD group at the time of partial hepatectomy was significantly higher than that in normal group, and it persisted 6 hours after partial hepatectomy. At the same time, IGF-1 concentration in the liver tissue in KD and TKD groups at the time of partial hepatectomy was significantly lower than that in normal group, and IGF-1 concentration in the TKD groups was higher than that in the KD group. Furthermore, Akt was strongly phosphorylated in normal group compared with the KD group. In the TKD group, phosphorylation of Akt was started at the time of PH and lasted until 120 minutes after PH, and it was almost the same level as it was in

Platelet accumulation 2 hours after partial hepatectomy was investigated in each group. Platelet accumulation in the KD group demonstrated a significant decrease compared with the normal group. Moreover, platelet accumulation in the TKD group showed a significantly higher level than that in the KD group, and it was almost the same level as that in the normal group. In the thrombocytotic group, platelet accumulation increased significantly compared with the normal group. Transmission electron microscopy demonstrated that in the thrombocytotic group, platelets translocated from the liver sinusoidal space to the space of Disse and were in direct contact with hepatocytes at 5

These results clearly demonstrate that platelets promote liver regeneration under conditions of Kupffer cell depletion. Increase of platelets recruited platelets in the liver tissue and elevated and HGF concentrations in the liver, which activated downstream signaling

In conclusion, our previous studies clarified the direct effect of platelets in promoting liver regeneration. The mechanism is explained as follows; after partial hepatectomy, platelets accumulate in the liver, they translocate to the space of Disse and release growth factors such as IGF-1 and HGF through direct contact with hepatocytes. The growth factors stimulate initiation of hepatocyte mitosis, which eventually promote liver regeneration. Especially in human, since it was reported that human platelets do not contain a significant amount of HGF (Nakamura et al., 1989), IGF-1 is the most important mediator for liver

LSECs comprise 70% of the sinusoidal cells (Knook & Sleyster, 1976; Smedsrod et al., 1990). By construction of a thin and continuous layer, the sinusoidal endothelium forms the structural barrier, separating the hepatic parenchyma from blood constituents passing through the liver. Unlike other vascular endothelial cells, LSECs have large cytoplasmic gaps without basal membranes. These enable maximal contact between circulating blood and hepatocytes to help exchange various soluble macromolecules and nano-particles such as lipoproteins and endocytosis (Braet & Wisse, 2002). LSECs are involved in liver regeneration as well as Kupffer cells and hepatic stellate cells, and they are known to produce immunoregulatory and pro-inflammatory cytokines including HGF, interleukin-1 (IL-1), IL-6, and interferon. In addition, they synthesize eicosanoids, particularly TXA2, prostaglandin E2, as well as synthesizing important regulators of vascular tone, such

normal group.

minutes after hepatectomy.

transduction and hepatocyte mitosis.

regeneration, which is contained in human platelets (Fig. 14).

**5. The effect with liver sinusoidal endothelial cells** 

Fig. 14. Scheme for liver regeneration promoted directly by platelets. Platelets translocate to the space of Disse and release growth factors such as IGF-1 and HGF through direct contact with hepatocytes. The growth factors subsequently induce initiation of mitosis.

as nitric oxide and endothelin (Wisse et al., 1996; Vollmar & Menger, 2009; Ping et al., 2006). IL-6 produced by LSECs and Kupffer cells is one of the important components of early signaling pathways in liver regeneration, and it activates the acute phase of protein synthesis by hepatocytes as part of the overall inflammatory response (Gauldie et al., 1992; Michalopoulos & DeFrances, 1997). After hepatectomy, plasma IL-6 concentration is reported to increase from 6 hours to a peak by 24 hours (Rai et al., 1996; Badia et al., 1998). IL-6 binds to its receptor on hepatocytes, which subsequently leads to phosphorylation of STAT3 monomers (Fausto et al., 2006). STAT3 homodimerizes and translocates to the nucleus, where it stimulates transcription of a number target genes such as cyclin-D1 and p21.

The relationship between platelets and LSECs has been well-documented in ischemia/reperfusion injury models. Rolling and adhering of leukocytes on LSECs with subsequent interaction with platelets is the important pathogenesis of ischemia/reperfusion injury (Montalvo-Jave et al., 2008; Croner et al., 2006, Pak et al., 2010). It was also reported that transient interaction, i.e., rolling, and permanent adhesion of platelets to the postischemic hepatic endothelium stimulate platelet activation and expression of endothelial adhesion molecules (Massberg et al., 1998; Khandoga et al., 2003). There have not, however, been any prior studies focused on the relationship between human platelets and LSECs with regards to liver regeneration.

To clarify the role of platelets in liver regeneration in relation to LSECs, we used coculturing chamber systems where platelets and LSECs could be separated by a permeable

Platelet and Liver Regeneration 131

(A)

(B) Fig. 15. Assay of IL-6 and VEGF in the supernatant of cultured LSECs after the addition of platelets, and the necessity of contact with platelets for excretion of IL-6 from LSECs. (A) The amounts of IL-6 and VEGF in the supernatant of LSECs were measured 0, 6, and 24 hours after the addition of platelets. Data are expressed as means ± SD. \*p < 0.05 versus platelet– group. (B) To investigate the necessity of direct contact between platelets and LSECs, LSECs were cultured for 6 hours with platelets mixed (platelet+mixed) or platelets separated (platelet + separated), and the excretion of IL-6 from LSECs was measured. Data are expressed as means ± SD. \*p < 0.05 versus platelet+separated group. (Reproduced from

Kawasaki et al., 2010, *J Hepatol* with permission.)

membrane (Kawasaki et al., 2010). We used TMNK-1 cells (immortalized human LSECs), instead of primary LSECs, since their utility and efficiency was confirmed in the previous basic research (Matsumura et al., 2004).

Proliferation of LSECs and concentrations of IL-6 and VEGF in the supernatant were significantly higher in the group in which LSECs were co-cultured with human platelets (platelet+ or platelet+ mixed) than they were in the group in which LSECs were cultured without human platelets (platelet-) (Fig. 15A,B). However, when the platelets and LSECs were cultured separately (platelet+separated), no significant increase of IL-6 was observed (Fig. 15B). These results indicated that human platelets increase proliferation of LSECs and induced IL-6 release from LSECs and that the direct contact between platelets and LSECs is required for the production of IL-6. BrdU uptake of the primary hepatocytes in the group administered with the supernatant co-cultured with platelets and LSECs was significantly higher than that in the group administered with the supernatant cultured without platelets. When a specific antagonist for sphingosine 1-phosphate (S1P) 2 receptors were added to LSECs and cocultured with platelets, the concentration of IL-6 showed significant decrease (Fig. 16A). On the contrary, the concentration of IL-6 was clearly increased in the group administered with S1P compared with those without S1P (Fig. 16B). These results revealed that S1P in platelets played important roles in liver regeneration by release of IL-6 from LSECs.

S1P is generally expressed in human plasma. It belongs to the class of lipid mediators and has been shown to regulate diverse biological processes, including proliferation, survival migration, or cytoskeletal reorganization (Yatomi et al., 2000; Xia & Wadham., 2011). S1P is produced from platelets and interacts with endothelial cells under the conditions of critical platelet-endothelial interactions, i.e., thrombosis, angiogenesis, and atherosclerosis (Yatomi et al., 2000). It was reported that the biological effect of S1P is partially mediated by endothelial nitric oxide synthetic activation and subsequent nitric oxide formation; extracellular S1P could contribute to sinusoidal protection and remodeling in alcoholic liver injury (Zheng et al., 2006). However, it was also described that S1P in human hepatic myofibroblast has an anti-mitogenic effect by increasing expression of TGF-β (Ikeda et al., 2003). As described above, S1P has various kinds of biophysical effects.

From the results of our experiment, the promotive effect of platelets on liver regeneration could be explained by follows; the direct contact between platelets and LSECs induce S1P release from platelets, which subsequently induce excretion of IL-6 from LSECs. LSECderived IL-6 promotes DNA synthesis of hepatocytes through STAT3 pathway (Fig. 17).

#### **6. The role of Kupffer cells on liver regeneration**

Kupffer cells are the principal constituents of the non-paranchymal cells of the liver (Malik et al., 2002). They locate within the lumen of the liver sinusoids, and are adherent to the LSECs. Kupffer cells play a role as macrophages against bacteria, bacterial endotoxins and microbial debris derived from gastrointestinal tract (Bilzer at al., 2006). Kupffer cells have been postulated to play a key role in liver regeneration after partial hepatectomy, and they could produce important biologically-active mediators that have both stimulatory and inhibitory influence on hepatocyte proliferation after hepatectomy (Boulton et al., 1998). Except for a report stating augmentation of the early phase of liver regeneration with Kupffer cell depletion (Meijer et al., 2000), depletion of Kupffer cells is basically well-known

membrane (Kawasaki et al., 2010). We used TMNK-1 cells (immortalized human LSECs), instead of primary LSECs, since their utility and efficiency was confirmed in the previous

Proliferation of LSECs and concentrations of IL-6 and VEGF in the supernatant were significantly higher in the group in which LSECs were co-cultured with human platelets (platelet+ or platelet+ mixed) than they were in the group in which LSECs were cultured without human platelets (platelet-) (Fig. 15A,B). However, when the platelets and LSECs were cultured separately (platelet+separated), no significant increase of IL-6 was observed (Fig. 15B). These results indicated that human platelets increase proliferation of LSECs and induced IL-6 release from LSECs and that the direct contact between platelets and LSECs is required for the production of IL-6. BrdU uptake of the primary hepatocytes in the group administered with the supernatant co-cultured with platelets and LSECs was significantly higher than that in the group administered with the supernatant cultured without platelets. When a specific antagonist for sphingosine 1-phosphate (S1P) 2 receptors were added to LSECs and cocultured with platelets, the concentration of IL-6 showed significant decrease (Fig. 16A). On the contrary, the concentration of IL-6 was clearly increased in the group administered with S1P compared with those without S1P (Fig. 16B). These results revealed that S1P in platelets

S1P is generally expressed in human plasma. It belongs to the class of lipid mediators and has been shown to regulate diverse biological processes, including proliferation, survival migration, or cytoskeletal reorganization (Yatomi et al., 2000; Xia & Wadham., 2011). S1P is produced from platelets and interacts with endothelial cells under the conditions of critical platelet-endothelial interactions, i.e., thrombosis, angiogenesis, and atherosclerosis (Yatomi et al., 2000). It was reported that the biological effect of S1P is partially mediated by endothelial nitric oxide synthetic activation and subsequent nitric oxide formation; extracellular S1P could contribute to sinusoidal protection and remodeling in alcoholic liver injury (Zheng et al., 2006). However, it was also described that S1P in human hepatic myofibroblast has an anti-mitogenic effect by increasing expression of TGF-β (Ikeda et al.,

From the results of our experiment, the promotive effect of platelets on liver regeneration could be explained by follows; the direct contact between platelets and LSECs induce S1P release from platelets, which subsequently induce excretion of IL-6 from LSECs. LSECderived IL-6 promotes DNA synthesis of hepatocytes through STAT3 pathway (Fig. 17).

Kupffer cells are the principal constituents of the non-paranchymal cells of the liver (Malik et al., 2002). They locate within the lumen of the liver sinusoids, and are adherent to the LSECs. Kupffer cells play a role as macrophages against bacteria, bacterial endotoxins and microbial debris derived from gastrointestinal tract (Bilzer at al., 2006). Kupffer cells have been postulated to play a key role in liver regeneration after partial hepatectomy, and they could produce important biologically-active mediators that have both stimulatory and inhibitory influence on hepatocyte proliferation after hepatectomy (Boulton et al., 1998). Except for a report stating augmentation of the early phase of liver regeneration with Kupffer cell depletion (Meijer et al., 2000), depletion of Kupffer cells is basically well-known

played important roles in liver regeneration by release of IL-6 from LSECs.

2003). As described above, S1P has various kinds of biophysical effects.

**6. The role of Kupffer cells on liver regeneration** 

basic research (Matsumura et al., 2004).

Fig. 15. Assay of IL-6 and VEGF in the supernatant of cultured LSECs after the addition of platelets, and the necessity of contact with platelets for excretion of IL-6 from LSECs. (A) The amounts of IL-6 and VEGF in the supernatant of LSECs were measured 0, 6, and 24 hours after the addition of platelets. Data are expressed as means ± SD. \*p < 0.05 versus platelet– group. (B) To investigate the necessity of direct contact between platelets and LSECs, LSECs were cultured for 6 hours with platelets mixed (platelet+mixed) or platelets separated (platelet + separated), and the excretion of IL-6 from LSECs was measured. Data are expressed as means ± SD. \*p < 0.05 versus platelet+separated group. (Reproduced from Kawasaki et al., 2010, *J Hepatol* with permission.)

Platelet and Liver Regeneration 133

to exert an inhibitory influence on liver regeneration by alteration of hepatic cytokine expression (Takeishi et al, 1999). A critical early event following partial hepatectomy is the increase in plasma levels of TNF-alpha. In support of this view, an experiment using antibody against TNF-alpha has demonstrated significant reduction of hepatocyte proliferation (Akerman et al., 1992). Mice lacking TNF receptor-1 were shown to demonstrate severe impairment in liver regeneration (Yamada et al., 1998). Activation of the TNF receptor increases hepatic level of the NF-KB in both hepatocytes and nonparanchymal cells, and it is followed by production and release of IL-6 from Kupffer cells. Kupffer cells are assumed to be one of the most important sources of both TNF-alpha and IL-6 (Kahn et al., 1994; Decker., 1998). This is supported by the report that Kupffer celldepleted mice failed to increase TNF-alpha, and IL-6 levels were equivalent to the level of

The relationship between platelet and Kupffer cells has been also well-documented in ischemia/reperfusion injury models. Platelets act in concert with the activated Kupffer cells and leukocytes, and a triangular interaction between these cells has been demonstrated as the main mechanism of the injury (Vollmar & Menger, 2009; Sindram et al., 2001). It was reported that when rats with depletion of Kupffer cells were subjected to ischemia and reperfusion, platelet adhesion in sinusoids was suppressed and, as consequence, attenuation of sinusoidal perfusion failure and endothelial damage were seen (Nakano et al., 2008). It is also reported that Kupffer cells produce PAF, which is a potent phospholipid mediator of platelet aggregation (Karidis et al., 2006). PAF is also believed to play important roles in the acute liver injury with ischemia/reperfusion (Karidis et al., 2006; Toledo-Pereyra & Suzuki, 1994), liver graft dysfunction (Hashikura et al., 1994), and post-operative liver failure after extended hepatectomy (Mizuno et al., 2001). As shown above, the role of platelets in relation

Kupffer cell-competent mice after partial hepatectomy (Abshagen et al., 2007).

to Kupffer cells have been described mainly with inflammatory injuries of the liver.

in the liver.

Nakamura et al. described a different character of Kupffer cell function associated with platelets. They reported that in response to LPS, IL-1, and TNF-alpha, platelets accumulated in the liver and a large number of platelets were found in the space of Disse (Endo et al., 1992, 1993; Nakamura et al., 1998). They also observed that platelets in the liver sinusoids were mostly surrounded by well-developed cell processes of Kupffer cells without being phagocytosed (Nakamura et al., 1998). However, depletion of Kupffer cells resulted in abolition of hepatic accumulation and migration of platelets (Nakamura et al., 1998). Although the precise mechanism was not clear, these reports indicated that cellular interaction between platelets and Kupffer cells plays an important role in platelet behavior

Previously, we reported that even under condition of Kupffer cell depletion, platelets accumulated in the liver in the thrombocytotic state and promoted liver regeneration by direct contact with hepatocytes through their migration from the liver sinusoidal space to the space of Disse (Murata et al., 2008). In our recent study using SCID mice with human platelet transfusion, we demonstrated that concentrations of mouse-derived TNF-alpha and IL-6 in the liver tissue after 70% of partial hepatectomy were significantly higher in the mice with platelet transfusion than in the mice without transfusion. These results may indicate that platelet transfusion enhances secretory activity of Kupffer cells after hepatectomy. Furthermore, in the mice with platelet transfusion, significant accumulation and activation of platelets transfused

Fig. 16. Effects of inhibitor of S1P and S1P on excretion of IL-6 from LSECs (A) Excretion of IL-6 from LSECs was evaluated using a specific antagonist for S1P2 receptors. LSECs were cultured with platelets for 6 hours, and the amount of IL-6 in the supernatant of LSECs was measured. Data are expressed as means ± SD. \*p < 0.05 versus inhibitor- group. (B) To determine whether S1P had an effect on excretion of IL-6 from LSECs, LSECs were cultured with S1P for 6 hours, and the amount of IL-6 in the supernatant of TMNK-1 cells was measured. Data are expressed as means ± SD. \*p < 0.05 versus S1Pgroup. (Reproduced from Kawasaki et al., 2010, *J Hepatol* with permission.)

Fig. 17. Scheme for liver regeneration promoted by LSECs and platelets. The direct contact between platelets and LSECs triggers excretion of S1P from platelets, which subsequently causes excretion of IL-6 from LSECs. IL-6 from LSECs promotes DNA synthesis of hepatocytes.

 (A) (B) Fig. 16. Effects of inhibitor of S1P and S1P on excretion of IL-6 from LSECs

group. (Reproduced from Kawasaki et al., 2010, *J Hepatol* with permission.)

Fig. 17. Scheme for liver regeneration promoted by LSECs and platelets.

synthesis of hepatocytes.

The direct contact between platelets and LSECs triggers excretion of S1P from platelets, which subsequently causes excretion of IL-6 from LSECs. IL-6 from LSECs promotes DNA

(A) Excretion of IL-6 from LSECs was evaluated using a specific antagonist for S1P2 receptors. LSECs were cultured with platelets for 6 hours, and the amount of IL-6 in the supernatant of LSECs was measured. Data are expressed as means ± SD. \*p < 0.05 versus inhibitor- group. (B) To determine whether S1P had an effect on excretion of IL-6 from LSECs, LSECs were cultured with S1P for 6 hours, and the amount of IL-6 in the supernatant of TMNK-1 cells was measured. Data are expressed as means ± SD. \*p < 0.05 versus S1P-

to exert an inhibitory influence on liver regeneration by alteration of hepatic cytokine expression (Takeishi et al, 1999). A critical early event following partial hepatectomy is the increase in plasma levels of TNF-alpha. In support of this view, an experiment using antibody against TNF-alpha has demonstrated significant reduction of hepatocyte proliferation (Akerman et al., 1992). Mice lacking TNF receptor-1 were shown to demonstrate severe impairment in liver regeneration (Yamada et al., 1998). Activation of the TNF receptor increases hepatic level of the NF-KB in both hepatocytes and nonparanchymal cells, and it is followed by production and release of IL-6 from Kupffer cells. Kupffer cells are assumed to be one of the most important sources of both TNF-alpha and IL-6 (Kahn et al., 1994; Decker., 1998). This is supported by the report that Kupffer celldepleted mice failed to increase TNF-alpha, and IL-6 levels were equivalent to the level of Kupffer cell-competent mice after partial hepatectomy (Abshagen et al., 2007).

The relationship between platelet and Kupffer cells has been also well-documented in ischemia/reperfusion injury models. Platelets act in concert with the activated Kupffer cells and leukocytes, and a triangular interaction between these cells has been demonstrated as the main mechanism of the injury (Vollmar & Menger, 2009; Sindram et al., 2001). It was reported that when rats with depletion of Kupffer cells were subjected to ischemia and reperfusion, platelet adhesion in sinusoids was suppressed and, as consequence, attenuation of sinusoidal perfusion failure and endothelial damage were seen (Nakano et al., 2008). It is also reported that Kupffer cells produce PAF, which is a potent phospholipid mediator of platelet aggregation (Karidis et al., 2006). PAF is also believed to play important roles in the acute liver injury with ischemia/reperfusion (Karidis et al., 2006; Toledo-Pereyra & Suzuki, 1994), liver graft dysfunction (Hashikura et al., 1994), and post-operative liver failure after extended hepatectomy (Mizuno et al., 2001). As shown above, the role of platelets in relation to Kupffer cells have been described mainly with inflammatory injuries of the liver.

Nakamura et al. described a different character of Kupffer cell function associated with platelets. They reported that in response to LPS, IL-1, and TNF-alpha, platelets accumulated in the liver and a large number of platelets were found in the space of Disse (Endo et al., 1992, 1993; Nakamura et al., 1998). They also observed that platelets in the liver sinusoids were mostly surrounded by well-developed cell processes of Kupffer cells without being phagocytosed (Nakamura et al., 1998). However, depletion of Kupffer cells resulted in abolition of hepatic accumulation and migration of platelets (Nakamura et al., 1998). Although the precise mechanism was not clear, these reports indicated that cellular interaction between platelets and Kupffer cells plays an important role in platelet behavior in the liver.

Previously, we reported that even under condition of Kupffer cell depletion, platelets accumulated in the liver in the thrombocytotic state and promoted liver regeneration by direct contact with hepatocytes through their migration from the liver sinusoidal space to the space of Disse (Murata et al., 2008). In our recent study using SCID mice with human platelet transfusion, we demonstrated that concentrations of mouse-derived TNF-alpha and IL-6 in the liver tissue after 70% of partial hepatectomy were significantly higher in the mice with platelet transfusion than in the mice without transfusion. These results may indicate that platelet transfusion enhances secretory activity of Kupffer cells after hepatectomy. Furthermore, in the mice with platelet transfusion, significant accumulation and activation of platelets transfused

Platelet and Liver Regeneration 135

beginning to be utilized in clinical settings; the importance and effects of platelets will become more apparent in the near future. With several lines of evidence showing platelets to be effective in anti-fibrosis (Watanabe et al., 2008; Kodama et al., 2010), anti-apoptosis (Hisakura et al., 2011), and liver regeneration, platelet therapy would open a new avenue to develop novel strategies for the treatments of liver diseases. Through these researches, we believe that platelet therapy could offer a therapeutic strategy for liver regeneration after

The authors thank Dr. N. Kobayashi, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, for providing the sinusoidal endothelial cell line-TMNK-1. The authors also thank Dr. N. Yanai, University of Tohoku, for providing the hepatocyte

Abshagen, K.; Eipel, C.; Kalff, JC.; Menger, MD. & Vollmar, B. (2007). Loss of NF-kappaB

Akerman, P.; Cote, P.; Yang, SQ.; McClain, C.; Nelson, S.; Bagby, GJ. & Diehl, AM. (1992).

hepatectomy. *Am J Physiol,* Vol.263, No.4Pt1, (October 1992), pp. 579-585. Alkozai, EM.; Nijsten, MW.; de Jong, KP.; de Boer, MT.; Peeters, PM.; Slooff, MJ.; Porte, RJ.

Badia, JM.; Ayton, LC.; Evans, TJ.; Carpenter, AJ.; Nawfal, G.; Kinderman, H.; Zografos, G.;

Bard-Chapeau, EA.; Yuan, J.; Droin, N.; Long, S.; Zhang, EE.; Nguyen, TV. & Feng, GS.

hepatoprotection. *Mol Cell Biol,* Vol.26, No.12, (June 2006), pp. 4664-4674. Bilzer, M.; Roggel, F. & Gerbes, AL. (2006). Role of Kupffer cells in host defense and liver

Blair, P. & Flaumenhaft, R. (2009). Platelet alpha-granules: basic biology and clinical

Bode, AP. & Fischer, TH. (2007). Lyophilized platelets: fifty years in the making. *Artif Cells* 

Borowiak, M.; Garratt, AN.; Wüstefeld, T.; Strehle, M.; Trautwein, C. & Birchmeier, C.

Bottaro, DP.; Rubin, JS.; Faletto, DL.; Chan, AM.; Kmiecik, TE.; Vande Woude, GF. &

(2004). Met provides essential signals for liver regeneration. *Proc Natl Acad Sci U S* 

Aaronson, SA. (1991). Identification of the hepatocyte growth factor receptor as the

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activation in Kupffer cell-depleted mice impairs liver regeneration after partial hepatectomy. *Am J Physiol Gastrointest Liver Physiol,* Vol.292, No.6 (June 2007),

Antibodies to tumor necrosis factor-alpha inhibit liver regeneration after partial

& Lisman, T. (2010). Immediate postoperative low platelet count is associated with delayed liver function recovery after partial liver resection. *Ann Surg,* Vol.251,

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extended hepatectomy, liver injuries or small grafts in liver transplantation.

**8. Acknowledgement** 

pp.1570-1577.

pp. 185-190.

No.2, (February 2010), pp. 300-306.

cell line-TLR2.

**9. References** 

were observed in the liver after hepatectomy. Although a few platelets transfused were adhering to the Kupffer cells in the mice without hepatectomy, the majority of platelets adhered to the surface of Kupffer cells in the mice with hepatectomy. It is insufficient to conclude only from these findings, however, it was assumed that platelets promote liver regeneration by interactions with Kupffer cells after hepatectomy. In other words, after hepatectomy, Kupffer cells induce accumulation and activation of platelets in the liver by direct adhering, and function of Kupffer cells are enhanced by the accumulated platelets. As described above, liver regeneration is promoted by the direct effect of growth factors released from platelets and by the paracrine effect of Kupffer cells enhanced by platelets (Fig. 18).

Fig. 18. Scheme for liver regeneration promoted by Kupffer cells

After partial hepatectomy, the activated Kupffer cells induce accumulation and activation of platelets through direct adhering. By the direct effect of growth factors released from platelets, and by the paracrine effect of Kupffer cells enhanced by platelets, liver regeneration is promoted.

#### **7. Conclusion**

In this chapter we have described our previous reports of platelets in promoting liver regeneration and the three different mechanisms by which platelete promote liver regeneration, i.e., 1) the direct effect on hepatocytes, 2) the cooperative effect with LSECs, and 3) the collaborative effect with Kupffer cells. Platelets are blood components that contain various kinds of biologically-active growth factors and cytokines. Nowadays artificial platelets (Bode & Fischer, 2007; Okamura et al., 2009), TPO formulae (Rhodes & Stasi, 2010), and freeze-dried platelets (Hoshi et al., 2007) are being developed and are

were observed in the liver after hepatectomy. Although a few platelets transfused were adhering to the Kupffer cells in the mice without hepatectomy, the majority of platelets adhered to the surface of Kupffer cells in the mice with hepatectomy. It is insufficient to conclude only from these findings, however, it was assumed that platelets promote liver regeneration by interactions with Kupffer cells after hepatectomy. In other words, after hepatectomy, Kupffer cells induce accumulation and activation of platelets in the liver by direct adhering, and function of Kupffer cells are enhanced by the accumulated platelets. As described above, liver regeneration is promoted by the direct effect of growth factors released from platelets and by the paracrine effect of Kupffer cells enhanced by platelets (Fig. 18).

Fig. 18. Scheme for liver regeneration promoted by Kupffer cells

regeneration is promoted.

**7. Conclusion** 

After partial hepatectomy, the activated Kupffer cells induce accumulation and activation of platelets through direct adhering. By the direct effect of growth factors released from platelets, and by the paracrine effect of Kupffer cells enhanced by platelets, liver

In this chapter we have described our previous reports of platelets in promoting liver regeneration and the three different mechanisms by which platelete promote liver regeneration, i.e., 1) the direct effect on hepatocytes, 2) the cooperative effect with LSECs, and 3) the collaborative effect with Kupffer cells. Platelets are blood components that contain various kinds of biologically-active growth factors and cytokines. Nowadays artificial platelets (Bode & Fischer, 2007; Okamura et al., 2009), TPO formulae (Rhodes & Stasi, 2010), and freeze-dried platelets (Hoshi et al., 2007) are being developed and are beginning to be utilized in clinical settings; the importance and effects of platelets will become more apparent in the near future. With several lines of evidence showing platelets to be effective in anti-fibrosis (Watanabe et al., 2008; Kodama et al., 2010), anti-apoptosis (Hisakura et al., 2011), and liver regeneration, platelet therapy would open a new avenue to develop novel strategies for the treatments of liver diseases. Through these researches, we believe that platelet therapy could offer a therapeutic strategy for liver regeneration after extended hepatectomy, liver injuries or small grafts in liver transplantation.

#### **8. Acknowledgement**

The authors thank Dr. N. Kobayashi, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, for providing the sinusoidal endothelial cell line-TMNK-1. The authors also thank Dr. N. Yanai, University of Tohoku, for providing the hepatocyte cell line-TLR2.

#### **9. References**


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**7** 

 *Russia* 

Eleonora Grigoryan

**Shared Triggering Mechanisms of Retinal** 

*Kol'tzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow,* 

Neural retina (NR) of the eye of vertebrates is underlined by retinal pigmented epithelium (RPE). NR↔RPE interconnection is critical for development, regeneration and function of both compartments of the retina. A disturbance of the normal quantitative correlation of RPE cells and photoreceptors, their structural and functional integrity unavoidably breaks visual cycle and induces retinal pathology. The majority of retinal diseases - inherent, age-

In the lab NR↔RPE disintegration can be achieved under some experimental conditions, such as NR separation and explantation, elimination of photoreceptors by bright light, chemical or mechanical damage of RPE and photoreceptors, and etc. A usage of experimental models in studies on the retina of lower and higher vertebrates endows a lot for understanding of cellular and molecular mechanisms of retinal pathology, on one part,

In tailed amphibian (Urodela) complete removal of NR or NR artificial detachment leads to RPE cell transdifferentiation that two months later results in regeneration of functioning retina (Chiba & Mitashov, 2007; Grigoryan & Mitashov, 1979; Hasegawa, 1958; Mitashov, 1997; Stroeva & Mitashov, 1983). In mammals RPE-based NR regeneration has not been reported. It is well known that NR detachment causes serious complications and blinding diseases despite of switching on some protective mechanisms for NR rescue (Fisher et al., 2005; Fisher & Lewis, 2010a, b; Pasto, 1998). This review represents an attempt to study early cellular and molecular mechanisms triggering NR regeneration in amphibians and NR

RPE of all vertebrates being localized between choroidal coat and NR has a big range of very important functions. RPE protects NR photoreceptors against overabundant light, participate in visual cycle, releases growth factors, regulates ion balance, transports nutrients, etc. (Strauss, 2005). In development RPE and NR have a common origin and both derived from the neuroepithelium of the optic cup. The latter delaminates into two layers, NR and RPE. Differentiation of these two tissues is a result of the expression of complex molecular network that is recently named the "*oculome"* (Lachke & Maas, 2010)*.* In the

related or systemic, links with a disturbance of NR↔RPE relationship.

and natural mechanisms of its rescue, on the other*.* 

rescue/pathology in mammals.

**1. Introduction** 

**Regeneration in Lower Vertebrates and** 

**Retinal Rescue in Higher Ones** 


### **Shared Triggering Mechanisms of Retinal Regeneration in Lower Vertebrates and Retinal Rescue in Higher Ones**

Eleonora Grigoryan

*Kol'tzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia* 

#### **1. Introduction**

144 Tissue Regeneration – From Basic Biology to Clinical Application

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Sato, N. (2006). Sphingosine 1-phosphate protects rat liver sinusoidal endothelial cells from ethanol-induced apoptosis: Role of intracellular calcium and nitric oxide.

> Neural retina (NR) of the eye of vertebrates is underlined by retinal pigmented epithelium (RPE). NR↔RPE interconnection is critical for development, regeneration and function of both compartments of the retina. A disturbance of the normal quantitative correlation of RPE cells and photoreceptors, their structural and functional integrity unavoidably breaks visual cycle and induces retinal pathology. The majority of retinal diseases - inherent, agerelated or systemic, links with a disturbance of NR↔RPE relationship.

> In the lab NR↔RPE disintegration can be achieved under some experimental conditions, such as NR separation and explantation, elimination of photoreceptors by bright light, chemical or mechanical damage of RPE and photoreceptors, and etc. A usage of experimental models in studies on the retina of lower and higher vertebrates endows a lot for understanding of cellular and molecular mechanisms of retinal pathology, on one part, and natural mechanisms of its rescue, on the other*.*

> In tailed amphibian (Urodela) complete removal of NR or NR artificial detachment leads to RPE cell transdifferentiation that two months later results in regeneration of functioning retina (Chiba & Mitashov, 2007; Grigoryan & Mitashov, 1979; Hasegawa, 1958; Mitashov, 1997; Stroeva & Mitashov, 1983). In mammals RPE-based NR regeneration has not been reported. It is well known that NR detachment causes serious complications and blinding diseases despite of switching on some protective mechanisms for NR rescue (Fisher et al., 2005; Fisher & Lewis, 2010a, b; Pasto, 1998). This review represents an attempt to study early cellular and molecular mechanisms triggering NR regeneration in amphibians and NR rescue/pathology in mammals.

> RPE of all vertebrates being localized between choroidal coat and NR has a big range of very important functions. RPE protects NR photoreceptors against overabundant light, participate in visual cycle, releases growth factors, regulates ion balance, transports nutrients, etc. (Strauss, 2005). In development RPE and NR have a common origin and both derived from the neuroepithelium of the optic cup. The latter delaminates into two layers, NR and RPE. Differentiation of these two tissues is a result of the expression of complex molecular network that is recently named the "*oculome"* (Lachke & Maas, 2010)*.* In the

Shared Triggering Mechanisms of Retinal Regeneration

**2.1 A disturbance of retinal cell contacts and behavior** 

photoreceptor degeneration due to constant light (Uehara et al., 2001).

cells and to form new additional NR (Grigoryan & Mitashov, 1985) (fig. 2).

underwent a phenotypic change resulted in the formation of macrophages.

**2. Results and discussion** 

in Lower Vertebrates and Retinal Rescue in Higher Ones 147

One of the earliest events caused by NR↔RPE separation is the loss of the adhesion and communication between two tissues. Adhesion of NR to RPE is provided by interaction of the RPE apical processes with the outer segments of the photoreceptors. An adhesion force between NR and RPE is ensured by constant elimination of water from the subretinal space. The latter remains closely tight in the normal retina and is essential for retinal functions and visual processing (Ghazi & Green, 2003; Marmor, 1993). The interface between RPE and NR is the interphotoreceptor matrix (IPM) that serves for chemical cross-talk between two tissues for their coordinated function. IPM consists of ECM components but a disruption of them often causes NR detachment. For instance, when IPM chondroitin 6-sulfate proteoglycan is perturbed *in vivo* by intravitreal injections of xyloside (a sugar inhibited chondroitin sulfate proteoglycan synthesis), shallow NR detachment could be observed. This suggests that adhesion between NR and RPE is dependent on continuous presence and synthesis of IPM proteoglycans (Lazarus & Hageman, 1992). Moreover, there are evidences that IPM molecules responsible for adhesion have a neuroprotective effect as well. For instance in the rat galectin-3, participating in RPE↔NR adhesion, inhibits apoptosis through the bcl-2 or cysteine protease pathways and, in contrast, intravitreous injection of anti-galectin-3 antibody accelerates

IPM disruption, subsequent weakening or even loss of RPE lateral contacts and attachment to NR and Bruch membrane, induces a change of RPE cell behavior. In parallel, after NR detachment when part of photoreceptors degenerate a large amount of their debris overwhelm the phagocytic ability of the RPE cells. As a response for altered conditions RPE manifests their multipotential capacity. After retinal removal in newts RPE cells stop to synthesize melanin and increase their proliferative activity (Grigoryan & Mitashov, 1979). In the same animals after NR detachment RPE also can display the unique capacity to transdifferentiate into retinal

Another of known differentiation potencies of urodelean RPE is the transformation to mobile cells with many of the characteristics of macrophages. The observation was made long time ago in the course of NR regeneration after retinal removal or optic nerve cutting in the newt (Keefe 1973) and in the case of NR experimental detachment in mammals (Johnson & Faulds, 1977). In those early works it was shown that after NR removal in Urodela some RPE cells withdrawn from the layer, moved along vitreal direction and phagocytosed retinal cell remnants (figure 3a, b). Due to this ability they were named "melanophages" (Keefe, 1973). Something comparable was carrying out in the rodent models of NR detachment (figure 3 c, d). When NR was separated from RPE in the rat and rabbit, epithelium

The morphology of RPE cell conversion to macrophages was well described by electron microscopy (Johnson & Faulds, 1977). Nowadays using organotypic 3D culturing of the posterior sector (RPE+choroids+sclera) of the rat eye we found that RPE also gave rise to macrophages: double nuclei cells, morphologically different from typical monocytes, and expressing macrophage-specific antigenes (Grigoryan et al., 2007; Novikova et al., 2010b). Alternatively or additionally, the RPE of mammals can proliferate and then participate in the formation of multilaminar layer of cells with characteristics of mesenchimal ones in

development as well as in the adult state both retinal tissues are in precise, well coordinated interconnection*.* On the outer side of the eye RPE is separated from vascular (choroid) and scleral coat by Bruch "membrane" that is formed by basal membranes of RPE and choroidal endothelium (fig.1).

Fig. 1. Retinal pigmented epithelium interplay with other tissues of the eye back wall. Schematic representation, modified from: Strauss, 2005

In this review we concentrated on the early cellular and molecular events induced by disturbance of NR↔RPE interconnections. We should point out that even the very early changes induced by NR↔RPE separation are complex and effect not only on RPE and NR but also other tissues of the eye back wall. Inner part of NR: interneurons, Müller glial cells and ganglion cells, although somewhat later, undergo changes in their function and behavior as a response for alterations in the outer NR. In this review we do not consider these, secondary in time course, processes.

Initiation of retinal regeneration in Urodela and its rescue in mammals as well as progress of following events are represented by comprehensive processes, in which different molecular families and signaling pathways participate. These molecules introduced by NR as well as adjoining tissues, and, in particular by RPE, play important, controlling, and regulative roles. The study of the complexity of intercellular and intertissue communications requires approaches using animals whose RPE↔NR disintegration does not lead to retinal loss but, in contrast, induces epimorphic regeneration. These approaches allow us to understand what kind of molecular changes are introduced by RPE and NR and what subsequent cellular and molecular events they are able to induce. As we'll see the changes initiating retinal regeneration in lower vertebrates and retinal rescue in higher ones have a high degree of universality. This gives us a hope for an existing preservation of some regenerative responses in evolutionary row and not complete block of them on the top, in human.

### **2. Results and discussion**

146 Tissue Regeneration – From Basic Biology to Clinical Application

development as well as in the adult state both retinal tissues are in precise, well coordinated interconnection*.* On the outer side of the eye RPE is separated from vascular (choroid) and scleral coat by Bruch "membrane" that is formed by basal membranes of RPE and choroidal

Fig. 1. Retinal pigmented epithelium interplay with other tissues of the eye back wall.

In this review we concentrated on the early cellular and molecular events induced by disturbance of NR↔RPE interconnections. We should point out that even the very early changes induced by NR↔RPE separation are complex and effect not only on RPE and NR but also other tissues of the eye back wall. Inner part of NR: interneurons, Müller glial cells and ganglion cells, although somewhat later, undergo changes in their function and behavior as a response for alterations in the outer NR. In this review we do not consider

Initiation of retinal regeneration in Urodela and its rescue in mammals as well as progress of following events are represented by comprehensive processes, in which different molecular families and signaling pathways participate. These molecules introduced by NR as well as adjoining tissues, and, in particular by RPE, play important, controlling, and regulative roles. The study of the complexity of intercellular and intertissue communications requires approaches using animals whose RPE↔NR disintegration does not lead to retinal loss but, in contrast, induces epimorphic regeneration. These approaches allow us to understand what kind of molecular changes are introduced by RPE and NR and what subsequent cellular and molecular events they are able to induce. As we'll see the changes initiating retinal regeneration in lower vertebrates and retinal rescue in higher ones have a high degree of universality. This gives us a hope for an existing preservation of some regenerative responses in evolutionary row and not complete block of them on the top, in

Schematic representation, modified from: Strauss, 2005

these, secondary in time course, processes.

human.

endothelium (fig.1).

#### **2.1 A disturbance of retinal cell contacts and behavior**

One of the earliest events caused by NR↔RPE separation is the loss of the adhesion and communication between two tissues. Adhesion of NR to RPE is provided by interaction of the RPE apical processes with the outer segments of the photoreceptors. An adhesion force between NR and RPE is ensured by constant elimination of water from the subretinal space. The latter remains closely tight in the normal retina and is essential for retinal functions and visual processing (Ghazi & Green, 2003; Marmor, 1993). The interface between RPE and NR is the interphotoreceptor matrix (IPM) that serves for chemical cross-talk between two tissues for their coordinated function. IPM consists of ECM components but a disruption of them often causes NR detachment. For instance, when IPM chondroitin 6-sulfate proteoglycan is perturbed *in vivo* by intravitreal injections of xyloside (a sugar inhibited chondroitin sulfate proteoglycan synthesis), shallow NR detachment could be observed. This suggests that adhesion between NR and RPE is dependent on continuous presence and synthesis of IPM proteoglycans (Lazarus & Hageman, 1992). Moreover, there are evidences that IPM molecules responsible for adhesion have a neuroprotective effect as well. For instance in the rat galectin-3, participating in RPE↔NR adhesion, inhibits apoptosis through the bcl-2 or cysteine protease pathways and, in contrast, intravitreous injection of anti-galectin-3 antibody accelerates photoreceptor degeneration due to constant light (Uehara et al., 2001).

IPM disruption, subsequent weakening or even loss of RPE lateral contacts and attachment to NR and Bruch membrane, induces a change of RPE cell behavior. In parallel, after NR detachment when part of photoreceptors degenerate a large amount of their debris overwhelm the phagocytic ability of the RPE cells. As a response for altered conditions RPE manifests their multipotential capacity. After retinal removal in newts RPE cells stop to synthesize melanin and increase their proliferative activity (Grigoryan & Mitashov, 1979). In the same animals after NR detachment RPE also can display the unique capacity to transdifferentiate into retinal cells and to form new additional NR (Grigoryan & Mitashov, 1985) (fig. 2).

Another of known differentiation potencies of urodelean RPE is the transformation to mobile cells with many of the characteristics of macrophages. The observation was made long time ago in the course of NR regeneration after retinal removal or optic nerve cutting in the newt (Keefe 1973) and in the case of NR experimental detachment in mammals (Johnson & Faulds, 1977). In those early works it was shown that after NR removal in Urodela some RPE cells withdrawn from the layer, moved along vitreal direction and phagocytosed retinal cell remnants (figure 3a, b). Due to this ability they were named "melanophages" (Keefe, 1973). Something comparable was carrying out in the rodent models of NR detachment (figure 3 c, d). When NR was separated from RPE in the rat and rabbit, epithelium underwent a phenotypic change resulted in the formation of macrophages.

The morphology of RPE cell conversion to macrophages was well described by electron microscopy (Johnson & Faulds, 1977). Nowadays using organotypic 3D culturing of the posterior sector (RPE+choroids+sclera) of the rat eye we found that RPE also gave rise to macrophages: double nuclei cells, morphologically different from typical monocytes, and expressing macrophage-specific antigenes (Grigoryan et al., 2007; Novikova et al., 2010b). Alternatively or additionally, the RPE of mammals can proliferate and then participate in the formation of multilaminar layer of cells with characteristics of mesenchimal ones in

Shared Triggering Mechanisms of Retinal Regeneration

lower and higher vertebrates.

progress of eye diseases.

Nathans, 2005).

**2.2 Visual cycle disturbance and apoptosis** 

in Lower Vertebrates and Retinal Rescue in Higher Ones 149

Pigmented macrophage-like cells of RPE origin may be seen in a wide variety of pathological processes affecting the interface between NR and RPE. As we mentioned above a stimulus to cellular activity in the RPE is a loss of the milieu stabilizing RPE differentiation. Such an influence produced by adjacent tissues now finds explanations in terms of molecular biology. Various molecules, as like as: Na-K-ATPase, soluble component of the IPM interphotoreceptor retinoid binding protein (IRBP) (Duffy et al., 1993; Gonzalez-Fernandez, 2003), mucin-type components of IPM for the retinal adhesion, and the serumtype ones for the transport of metabolites (Uehara et al., 1991), are suggested to play a role in the maintenance of intact photoreceptor-RPE complex. In other words, in outer retina cellto-cell and tissue-to-tissue contacts are participants of the maintenance of retinal integrity and destruction of which leads to NR cell death and RPE cell type transformation both in

Another event taking place soon after RPE↔NR separation is a disturbance of visual cycle. In the norm the latter represents a complex of biochemical reactions for regeneration of visual chromophore 11-*cis*-retinal from all-*trans*-retinol. It is well known that visual cycle is based on the renewal of photoreceptor outer segment disks. In this process disks are newly built from the base of the segments, at the connecting cilium and then, at the tips of the outer segments they are shed from the photoreceptors. Shed disks are phagocytosed by the RPE cells where they are digested. *Retinal* undergoes the RPE-specific part of visual cycle and then is redelivered as *11-cis-retinal* to photoreceptors (Bok, 1993). This interaction is essential for maintaining not only the visual function, but also structural integrity of photoreceptors. The processes of disk shedding, phagocytosis, and chromophore renewal must be tightly coordinated between RPE and NR photoreceptors. After disturbance of RPE↔NR interconnection caused by different reasons - from inherent to acquired pathology - the visual cycle gets unavoidably disturbed and brings a part into initiation and following

Recent molecular biology studies of NR detachment suggest an essential inhibition of genes coding visual cycle proteins. "Genomic response" (Rattner et al., 2008) after NR detachment was studied in comprehensive analysis of changes in transcript abundance in the murine RPE. In that work all RPE transcripts coding visual cycle components (Rpe65, Lrat, Cralbp, Rdh5, Rdh10, and Rbp1) showed down-regulation. In parallel, an increase of a small set of transcripts for secreted proteins and cell surface receptors were registered. In accordance with Rattner and co-workers (2008) the decrease in RPE transcripts coding for the visual cycle proteins could be a protective strategy of retinal cells in conditions of detachment when slowing the light-dependent cycling of retinoids takes place. In this case the damage response of the RPE showed similarity to that described for the NR. In particular, a fraction of certain transcripts (e.g., *Cebpd*, *Osmr*, *Serpin a3n*) is induced in both tissues (Rattner &

Meanwhile, there are numerous data suggested a decrease of the expression of particular components of visual cycle. Thus, in human IRBP (interphotoreceptor retinoid- binding protein) principal for transport of visual cycle proteins is down-regulated after NR detachment associated with diabetes retinopathy (Garcia-Ramirez et al., 2009). When we compared RPE in the intact eye and RPE soon after NR removal in Urodela we found by

connective tissue. The process of RPE transition to myofibroblasts is an attribute of well known ocular fibrotic disease, proliferative vitreoretinopathy (Saika et al., 2008).

Fig. 2. Growth of additional neural retina (2) derived from RPE after detachment of the initial one (1) in the adult newt.

Fig. 3. Detachment of the neural retina in the newt (a,b) and albino rat (c,d) eye. In both cases RPE cells (arrows) withdraw from the layer and migrate in the vitreous direction.

connective tissue. The process of RPE transition to myofibroblasts is an attribute of well

known ocular fibrotic disease, proliferative vitreoretinopathy (Saika et al., 2008).

Fig. 2. Growth of additional neural retina (2) derived from RPE after detachment of the

 Fig. 3. Detachment of the neural retina in the newt (a,b) and albino rat (c,d) eye. In both cases RPE cells (arrows) withdraw from the layer and migrate in the vitreous direction.

initial one (1) in the adult newt.

Pigmented macrophage-like cells of RPE origin may be seen in a wide variety of pathological processes affecting the interface between NR and RPE. As we mentioned above a stimulus to cellular activity in the RPE is a loss of the milieu stabilizing RPE differentiation. Such an influence produced by adjacent tissues now finds explanations in terms of molecular biology. Various molecules, as like as: Na-K-ATPase, soluble component of the IPM interphotoreceptor retinoid binding protein (IRBP) (Duffy et al., 1993; Gonzalez-Fernandez, 2003), mucin-type components of IPM for the retinal adhesion, and the serumtype ones for the transport of metabolites (Uehara et al., 1991), are suggested to play a role in the maintenance of intact photoreceptor-RPE complex. In other words, in outer retina cellto-cell and tissue-to-tissue contacts are participants of the maintenance of retinal integrity and destruction of which leads to NR cell death and RPE cell type transformation both in lower and higher vertebrates.

#### **2.2 Visual cycle disturbance and apoptosis**

Another event taking place soon after RPE↔NR separation is a disturbance of visual cycle. In the norm the latter represents a complex of biochemical reactions for regeneration of visual chromophore 11-*cis*-retinal from all-*trans*-retinol. It is well known that visual cycle is based on the renewal of photoreceptor outer segment disks. In this process disks are newly built from the base of the segments, at the connecting cilium and then, at the tips of the outer segments they are shed from the photoreceptors. Shed disks are phagocytosed by the RPE cells where they are digested. *Retinal* undergoes the RPE-specific part of visual cycle and then is redelivered as *11-cis-retinal* to photoreceptors (Bok, 1993). This interaction is essential for maintaining not only the visual function, but also structural integrity of photoreceptors. The processes of disk shedding, phagocytosis, and chromophore renewal must be tightly coordinated between RPE and NR photoreceptors. After disturbance of RPE↔NR interconnection caused by different reasons - from inherent to acquired pathology - the visual cycle gets unavoidably disturbed and brings a part into initiation and following progress of eye diseases.

Recent molecular biology studies of NR detachment suggest an essential inhibition of genes coding visual cycle proteins. "Genomic response" (Rattner et al., 2008) after NR detachment was studied in comprehensive analysis of changes in transcript abundance in the murine RPE. In that work all RPE transcripts coding visual cycle components (Rpe65, Lrat, Cralbp, Rdh5, Rdh10, and Rbp1) showed down-regulation. In parallel, an increase of a small set of transcripts for secreted proteins and cell surface receptors were registered. In accordance with Rattner and co-workers (2008) the decrease in RPE transcripts coding for the visual cycle proteins could be a protective strategy of retinal cells in conditions of detachment when slowing the light-dependent cycling of retinoids takes place. In this case the damage response of the RPE showed similarity to that described for the NR. In particular, a fraction of certain transcripts (e.g., *Cebpd*, *Osmr*, *Serpin a3n*) is induced in both tissues (Rattner & Nathans, 2005).

Meanwhile, there are numerous data suggested a decrease of the expression of particular components of visual cycle. Thus, in human IRBP (interphotoreceptor retinoid- binding protein) principal for transport of visual cycle proteins is down-regulated after NR detachment associated with diabetes retinopathy (Garcia-Ramirez et al., 2009). When we compared RPE in the intact eye and RPE soon after NR removal in Urodela we found by

Shared Triggering Mechanisms of Retinal Regeneration

**2.3 Changes in vascular and immune systems** 

initiated just after NR↔RPE separation (Moriya et al., 1986).

the key role of which in NR regeneration and rescue is discussed below.

**2.4 Expression of growth factors and major signaling pathways** 

specifically ECM changes (see below).

In mammals similar changes in vascular and immune systems can be induced experimentally or come out from eye diseases. For instance, there are data obtained by proteomic analysis of subretinal fluid and vitreous body of patients suffered with retinopathy of different kind and NR detachment, in particular. Authors found an increase of the content of fibrinogenic and inflammatory associated proteins for all types of pathology (Shimata et al., 2008). There are data suggested tPA (tissue plasminogen activator) may be involved in remodeling of the extracellular milieu during eye development (Collinge et al., 2005). tPA was found at the apical interface between the developing RPE and NR and then began to down-regulate once the photoreceptors have differentiated. Therefore, tPA as well as other components of the fibrinolytic system can be involved in regulation of the processes subsequent to retinal tissue disintegration, and

Anatomic and functional relationship between NR, RPE and RPE underlying tissues (Bruch membrane, choroidal coat) is consistent with the idea that signals pass between tissues for

in Lower Vertebrates and Retinal Rescue in Higher Ones 151

cellular responses to destroyed RPE↔NR communication in the retina. However, it's necessary to note that NR detachment from the RPE does not lead to immediate death of the cells and retinal apoptosis is a secondary event conditioned by a number of processes

In the eye of vertebrates, RPE and NR represent a structural unit that acts only in case when two tissues are interactive. Other tissues of the eye back wall, namely Bruch membrane and choroidal coat underlying RPE, also endow retinal integrity and function (figure 1). It is known that vascular occlusion, thrombus formation, accumulation of fibrinopeptides and inflammatory associated cells and proteins all are participants of many eye diseases accompanying RPE↔NR separation. In our experiments on Urodela when eye surgery was applied for retinal removal the occlusion of choroidal small vessels, an inflammation, and a decrease of a tension of the eye back wall tissues represented the very first events that brought later to RPE cell-type conversion, cell proliferation, and withdrawal from the layer (Grigoryan & Mitashov, 1979). Recently it was shown that in newts the thrombin (a participant of hemostasis and other immediate responses to any damage) pretends to be a regulator of iris cell transdifferentiation (Imokawa & Brockes, 2003; Imokawa et al., 2004). It is known that thrombin derives from prothrombin when activated by coagulation factors and, in particular, by transmembrane protein TF (tissue factor). In the work of Godwin and co-authors (2010) it was found that TF expression correlates topologically and in the time course with lens regeneration. TF and other molecules responsible for clot formation are pretending now to be initiators of tissue regeneration in lower vertebrates. In urodelean amphibians the role of complement system was proposed also in limb and lens regeneration (Kimura et al., 2003) and recently in the chick in retinal regeneration (Haynes et al., 2010). It is not inconceivable that comparable mechanism participates in triggering of retinal rescue in mammals. On the other hand, activated leucocytes associated with TF/thrombin/fibrin system can be also important participants in the initiation of NR epimorphic regeneration in amphibians and NR rescue in mammals. It is proposed (Song et al., 2010) that soon after damage they can be a source of FGF,

PCR a considerable decrease of RPE65 transcript abundance (Avdonin et al., 2008). Other results strongly suggest that RPE65 in the RPE-derived cells of early retinal regenerate in Urodela is the only reminder after protein degradation or discharge (Chiba et al., 2006).

When RPE and NR are disintegrated, there is not only a decrease of visual cycle protein synthesis but also protein translocation into the inner compartments of photoreceptors. We observed the phenomenon not at once in experiments on the model of NR detachment in the newt *in vivo* or in isolated NR under conditions *in vitro.* In those experiments we used recoverin (the protein involved in calcium-dependent regulation of rhodopsin phosphorylation) as a marker protein of photoreceptors (Grigoryan, 2007; Grigoryan et al., 2009; Krasnov et al., 2003; Novikova et al., 2010a). In both cases: *in vivo* after NR detachment and *in vitro* newt retina retained the ability to express recoverin but its immunoreactivity was displaced from the segments to perikarya and even axons of photoreceptors. As suggested by Liljekvist-Larsson et al*.* (2003) who observed the same phenomenon in cultured retina of newborn rats, the synthesis of recoverin in the cytoplasm of retinal cells continues, but the transport of its newly synthesized molecules within the cell is impaired. It seems in vertebrates a decrease of expression of visual protein genes and a change of synthesized protein location in RPE and photoreceptors is the characteristic closely related with RPE↔NR separation.

One more event in cohort of those induced by experimental NR separation is an activation of mitochondrial DNA synthesis in retinal photoreceptors. Recently we showed that in the rat NR isolated from RPE and cultivated "whole amount" in 3D conditions *in vitro* the intensive incorporation of DNA synthesis precursor (BrdU) is localized in photoreceptor inner segments - cell compartments extremely rich with mitochondria (Novikova et al., 2010b). We believe that increased synthetic activity of mitochondrial DNA in photoreceptors is an attempt of photoreceptor cells to rescue and avoid apoptosis. It should be also noted that there are some other mechanisms, for instance protein degradation, taking a part in the decrease of the expression or content of visual cycle proteins in RPE and NR after loss of these tissue integrity.

A suppression of biochemical machinery of visual cycle in RPE and NR undoubtedly affects their vitality and differentiation stability. Disturbance of visual cycle and/or other RPE↔NR co-reactions lead to photoreceptor cell apoptosis. This can be a result of accumulation of photochemically active molecules and ROS which in the absence of regular visual cycle trigger an apoptotic cascade. Apoptosis includes a formation of AP-1 complex (transcription factor) and up-regulation of genes coding apoptotic enzymes – caspases (Reme et al., 2003). An evaluation of the possible induction of RPE cell apoptosis by transforming growth factor-beta (TGF-beta) was undertaken by Esser and colleagues (1997). Proapoptotic effect of TGF-beta was well demonstrated in cultured human RPE cells by electron microscopy, in situ DNA end labeling, comet assay, and a photometric enzyme immunoassay for histone associated DNA fragments. In Urodela the occurrence of apoptosis following ablation of the retina was examined by an in situ technique for detecting DNA fragmentation. It was shown that apoptosis occurs not only just after retinal removal but at the initial phase of NR regeneration as well. Authors of the work (Kaneko et al., 1999) came to a conclusion that the apoptosis is closely related to the phenomena of retinal regeneration in Urodela. Therefore, at the early time subsequent to NR↔RPE separation, both in Urodela and in mammals, the programming cell death is one more of

PCR a considerable decrease of RPE65 transcript abundance (Avdonin et al., 2008). Other results strongly suggest that RPE65 in the RPE-derived cells of early retinal regenerate in Urodela is the only reminder after protein degradation or discharge (Chiba et al., 2006).

When RPE and NR are disintegrated, there is not only a decrease of visual cycle protein synthesis but also protein translocation into the inner compartments of photoreceptors. We observed the phenomenon not at once in experiments on the model of NR detachment in the newt *in vivo* or in isolated NR under conditions *in vitro.* In those experiments we used recoverin (the protein involved in calcium-dependent regulation of rhodopsin phosphorylation) as a marker protein of photoreceptors (Grigoryan, 2007; Grigoryan et al., 2009; Krasnov et al., 2003; Novikova et al., 2010a). In both cases: *in vivo* after NR detachment and *in vitro* newt retina retained the ability to express recoverin but its immunoreactivity was displaced from the segments to perikarya and even axons of photoreceptors. As suggested by Liljekvist-Larsson et al*.* (2003) who observed the same phenomenon in cultured retina of newborn rats, the synthesis of recoverin in the cytoplasm of retinal cells continues, but the transport of its newly synthesized molecules within the cell is impaired. It seems in vertebrates a decrease of expression of visual protein genes and a change of synthesized protein location in RPE and photoreceptors is the characteristic closely related

One more event in cohort of those induced by experimental NR separation is an activation of mitochondrial DNA synthesis in retinal photoreceptors. Recently we showed that in the rat NR isolated from RPE and cultivated "whole amount" in 3D conditions *in vitro* the intensive incorporation of DNA synthesis precursor (BrdU) is localized in photoreceptor inner segments - cell compartments extremely rich with mitochondria (Novikova et al., 2010b). We believe that increased synthetic activity of mitochondrial DNA in photoreceptors is an attempt of photoreceptor cells to rescue and avoid apoptosis. It should be also noted that there are some other mechanisms, for instance protein degradation, taking a part in the decrease of the expression or content of visual cycle proteins in RPE and NR after loss of

A suppression of biochemical machinery of visual cycle in RPE and NR undoubtedly affects their vitality and differentiation stability. Disturbance of visual cycle and/or other RPE↔NR co-reactions lead to photoreceptor cell apoptosis. This can be a result of accumulation of photochemically active molecules and ROS which in the absence of regular visual cycle trigger an apoptotic cascade. Apoptosis includes a formation of AP-1 complex (transcription factor) and up-regulation of genes coding apoptotic enzymes – caspases (Reme et al., 2003). An evaluation of the possible induction of RPE cell apoptosis by transforming growth factor-beta (TGF-beta) was undertaken by Esser and colleagues (1997). Proapoptotic effect of TGF-beta was well demonstrated in cultured human RPE cells by electron microscopy, in situ DNA end labeling, comet assay, and a photometric enzyme immunoassay for histone associated DNA fragments. In Urodela the occurrence of apoptosis following ablation of the retina was examined by an in situ technique for detecting DNA fragmentation. It was shown that apoptosis occurs not only just after retinal removal but at the initial phase of NR regeneration as well. Authors of the work (Kaneko et al., 1999) came to a conclusion that the apoptosis is closely related to the phenomena of retinal regeneration in Urodela. Therefore, at the early time subsequent to NR↔RPE separation, both in Urodela and in mammals, the programming cell death is one more of

with RPE↔NR separation.

these tissue integrity.

cellular responses to destroyed RPE↔NR communication in the retina. However, it's necessary to note that NR detachment from the RPE does not lead to immediate death of the cells and retinal apoptosis is a secondary event conditioned by a number of processes initiated just after NR↔RPE separation (Moriya et al., 1986).

#### **2.3 Changes in vascular and immune systems**

In the eye of vertebrates, RPE and NR represent a structural unit that acts only in case when two tissues are interactive. Other tissues of the eye back wall, namely Bruch membrane and choroidal coat underlying RPE, also endow retinal integrity and function (figure 1). It is known that vascular occlusion, thrombus formation, accumulation of fibrinopeptides and inflammatory associated cells and proteins all are participants of many eye diseases accompanying RPE↔NR separation. In our experiments on Urodela when eye surgery was applied for retinal removal the occlusion of choroidal small vessels, an inflammation, and a decrease of a tension of the eye back wall tissues represented the very first events that brought later to RPE cell-type conversion, cell proliferation, and withdrawal from the layer (Grigoryan & Mitashov, 1979). Recently it was shown that in newts the thrombin (a participant of hemostasis and other immediate responses to any damage) pretends to be a regulator of iris cell transdifferentiation (Imokawa & Brockes, 2003; Imokawa et al., 2004). It is known that thrombin derives from prothrombin when activated by coagulation factors and, in particular, by transmembrane protein TF (tissue factor). In the work of Godwin and co-authors (2010) it was found that TF expression correlates topologically and in the time course with lens regeneration. TF and other molecules responsible for clot formation are pretending now to be initiators of tissue regeneration in lower vertebrates. In urodelean amphibians the role of complement system was proposed also in limb and lens regeneration (Kimura et al., 2003) and recently in the chick in retinal regeneration (Haynes et al., 2010). It is not inconceivable that comparable mechanism participates in triggering of retinal rescue in mammals. On the other hand, activated leucocytes associated with TF/thrombin/fibrin system can be also important participants in the initiation of NR epimorphic regeneration in amphibians and NR rescue in mammals. It is proposed (Song et al., 2010) that soon after damage they can be a source of FGF, the key role of which in NR regeneration and rescue is discussed below.

In mammals similar changes in vascular and immune systems can be induced experimentally or come out from eye diseases. For instance, there are data obtained by proteomic analysis of subretinal fluid and vitreous body of patients suffered with retinopathy of different kind and NR detachment, in particular. Authors found an increase of the content of fibrinogenic and inflammatory associated proteins for all types of pathology (Shimata et al., 2008). There are data suggested tPA (tissue plasminogen activator) may be involved in remodeling of the extracellular milieu during eye development (Collinge et al., 2005). tPA was found at the apical interface between the developing RPE and NR and then began to down-regulate once the photoreceptors have differentiated. Therefore, tPA as well as other components of the fibrinolytic system can be involved in regulation of the processes subsequent to retinal tissue disintegration, and specifically ECM changes (see below).

#### **2.4 Expression of growth factors and major signaling pathways**

Anatomic and functional relationship between NR, RPE and RPE underlying tissues (Bruch membrane, choroidal coat) is consistent with the idea that signals pass between tissues for

Shared Triggering Mechanisms of Retinal Regeneration

embryonic chicken (Sakami et al., 2008).

phenotype, cell proliferation and apoptosis take place.

proliferation.

in Lower Vertebrates and Retinal Rescue in Higher Ones 153

increase in FGF2 protein level was demonstrated by ELISA in RPE cell supernatants after incubation with BDNF or exposure to intense light or oxidizing agents. These data indicate that in RPE cells FGF2 is modulated by stress and by agents that provide protection from stress (Hackett et al., 1997). In addition, it was found that FGF2 immunoreactivity in the interphotoreceptor matrix tends to increase during first 24 hours after retinal detachment in the rat. It is proposed that the interphotoreceptor matrix has its own endogenous local source(s) of FGF2 (Ozaki et al., 2000). Therefore, it is possible to consider that in both cases, at the initiation of NR regeneration in amphibia and NR rescue in mammals, FGF2 signaling pathway participate in neuroprotection and regulation of RPE cell differentiation and

Other signaling pathways, as like as IGF-1, CNTF, and TGFβ represent also a part of the molecular network, regulating RPE and NR cell behavior after separation of these tissues in mammals. However, for today there are only few data on their activity in NR regeneration in Urodela. There is the evidence that IGF-1 (as like as FGF2) can accelerate proliferation and proneuronal differentiation of amphibian RPE under *in vitro* conditions (Yoshii et al., 2007). Meanwhile, proapoptotic growth factor TGFβ more likely plays prohibitive role in RPE cell type conversion. Activin, a TGF-β family signaling protein has been shown to contribute to the loss in competence of the RPE to regenerate retina. Sakami and co-authors (2008) have found that additing of activin blocked regeneration from the RPE, even when the cells were competent. Conversely, a small molecule inhibitor of the activin/TGF-β/nodal receptors could delay and reverse the developmental restriction in FGF-stimulated NR regeneration in

Earlier it was shown that TGFβ inhibits proliferation at the vitreoretinal interface after NR detachment in human (Esser et al., 1997). Nowadays the study of the role of TGFβ is carried out on the model of retinal detachment in experiments using mice null for Smad3, TGFβ functional cooperator, a key signaling intermediate downstream of TGFβ and activin receptors. Obtained results showed that Smad3 is essential for the epithelial-mesenchymal transition of RPE cells induced by NR detachment. *De novo* accumulation of fibrous tissue derived from multilayered RPE cells was seen in experimental NR detachment in eyes of wild type, but not in Smad3-null mice (Saika et al., 2004). Activation of several signaling pathways, particularly TGFβ /Smad, was also fixed by Zacks and coworkers (2006). Soon after NR detachment the interleukin-6/STAT, TGFβ-Smad, and stress response pathway (aryl hydrocarbon receptor) – all were transcriptionally and translationally upregulated, suggesting that retina produces survival factors after detachment and that there is a possible cross-talk between up-regulated pathways (Zacks et al., 2006). In sum, knowing of signaling pathways with proliferative and anti-proliferative as well as pro-apoptotic and antiapoptotic effects is very important, because in both, retinal epimorphic regeneration in amphibian and proliferative retinopathy after detachment in mammals, changes of RPE cell

**2.5 Up-regulation of heat shock proteins and immediate-early response genes** 

RPE↔NR disintegration results in the early activation of stress-response genes and specific signaling pathways which may enable retinal cells to survive at the most acute period of time. During NR detachment/regeneration in Urodela and detachment in mammals, heat shock proteins (HSPs) are involved in fast regenerative responses. Our preliminary

coordinated processes in the eye back wall. The RPE secretes a variety of growth factors that support photoreceptor survival and ensure a structural basis for optimal circulation and nutrients' supply (Campochiaro, 1993). One of the signal molecules released by RPE is PEDF (pigment epithelium-derived factor) that plays a broad spectrum of developmental and neuroprotective roles (Tombran-Tink et al., 1995). In particular, it was shown that PEDF can act as an antiangiogenic factor that inhibits endothelial cell proliferation in the choriocapillaris. VEGF is another vasoactive factor of RPE known as preventing endothelial cell apoptosis (Saint-Geniez et al., 2009). In a healthy eye, PEDF and VEGF are secreted at opposite sides of the RPE cell. At the apical side PEDF acts on neurons and photoreceptors but the majority of VEGF is secreted to the basal side where it acts on the choroidal endothelium. It was found that the balance between PEDF and VEGF is disturbed in the early course of retinopathy. Thus, in subretinal fluid of patients suffered with early PVR the concentrations of both factors essentially increase changing a normal balance where PEDF counteracts to angiogenic potential of VEGF (Dieudonné et al., 2007). It is important to note that, as a rule, NR↔RPE disintegration is accompanying by oxidative stress that, in turn, induces a decrease of PEDF correct level (Ohno-Matsui et al., 2001).

In response to retinal damage or injury RPE also secretes the row of neuroprotective factors including those of FGF, CNTF, IGF, and TGF families, all of which are included in the regulative network of the eye and retina (for review: Strauss, 2005). For instance, many extracellular stimuli have been proposed to induce an increasing of VEGF secretion. This signaling exploits growth factors such as IGF-I that can contribute to a pathway in which photoreceptors can stimulate VEGF secretion by RPE cells. Fibroblast growth factor basic (FGF2) is one of several agents that elicits most profound effects in RPE and NR cells. Since 90s the role of FGF2 in RPE transdifferentiation and NR regeneration after RPE↔NR separation in adult amphibians and bird embryos is received the intensive study (Araki, 2007; Mitsuda et al., 2005; Park & Hollenberg, 1993). In accordance with the data including our own, FGF2 and FGF2R coupled with that of transcription factor Pax6 control urodelean RPE cell dedifferentiation and proliferation after NR removal (Avdonin, 2010; Chiba & Mitashov, 2007). FGF-FGFR-MEK cascade and Pax6 up-regulation depended on changes of the cell-ECM and/or cell-cell interaction are supposed important for realization of the first steps of NR regeneration (Avdonin, 2010; Susaki & Chiba, 2007). In the *in vitro – in vivo* like systems it was shown that cells of isolated RPE could be induced to faster dedifferentiation by additing of FGF2 to culture medium (Ikegami et al., 2001; Novikova et al. 2010b).

In mammals, soon after NR detachment FGF2 gene up-regulation also takes place in parallel with high expression of FGF receptors (FGFR) (Hackett et al., 1997; Ozaki et al., 2000). When the retina is perturbed, significant changes occur in the expression of FGFR1 by photoreceptors: FGFR1 immunoreactivity increases rapidly (in 24 hours after injury) and steadily (Ozaki et al., 2000). That appears to be accompanied by similar increase of FGF2 in the IPM. Ozaki and co-workers suggest that this describes a paracrine mechanism: FGF2 is released or activated after retinal injury and then binds to FGFR1 on photoreceptor target cells. The latter, in turn, initiates an intracellular cascade that "protects" the cells from further damage.

The study of the effect of light, various types of stress, neurotrophic factors, and cytokines on FGF2 levels in human RPE cultured *in vitro* showed that many agents of photoreceptor protection (for instance, BDNF, CNTF, IL-1β) can up regulate FGF2 mRNA in RPE cells. An

coordinated processes in the eye back wall. The RPE secretes a variety of growth factors that support photoreceptor survival and ensure a structural basis for optimal circulation and nutrients' supply (Campochiaro, 1993). One of the signal molecules released by RPE is PEDF (pigment epithelium-derived factor) that plays a broad spectrum of developmental and neuroprotective roles (Tombran-Tink et al., 1995). In particular, it was shown that PEDF can act as an antiangiogenic factor that inhibits endothelial cell proliferation in the choriocapillaris. VEGF is another vasoactive factor of RPE known as preventing endothelial cell apoptosis (Saint-Geniez et al., 2009). In a healthy eye, PEDF and VEGF are secreted at opposite sides of the RPE cell. At the apical side PEDF acts on neurons and photoreceptors but the majority of VEGF is secreted to the basal side where it acts on the choroidal endothelium. It was found that the balance between PEDF and VEGF is disturbed in the early course of retinopathy. Thus, in subretinal fluid of patients suffered with early PVR the concentrations of both factors essentially increase changing a normal balance where PEDF counteracts to angiogenic potential of VEGF (Dieudonné et al., 2007). It is important to note that, as a rule, NR↔RPE disintegration is accompanying by oxidative stress that, in turn,

In response to retinal damage or injury RPE also secretes the row of neuroprotective factors including those of FGF, CNTF, IGF, and TGF families, all of which are included in the regulative network of the eye and retina (for review: Strauss, 2005). For instance, many extracellular stimuli have been proposed to induce an increasing of VEGF secretion. This signaling exploits growth factors such as IGF-I that can contribute to a pathway in which photoreceptors can stimulate VEGF secretion by RPE cells. Fibroblast growth factor basic (FGF2) is one of several agents that elicits most profound effects in RPE and NR cells. Since 90s the role of FGF2 in RPE transdifferentiation and NR regeneration after RPE↔NR separation in adult amphibians and bird embryos is received the intensive study (Araki, 2007; Mitsuda et al., 2005; Park & Hollenberg, 1993). In accordance with the data including our own, FGF2 and FGF2R coupled with that of transcription factor Pax6 control urodelean RPE cell dedifferentiation and proliferation after NR removal (Avdonin, 2010; Chiba & Mitashov, 2007). FGF-FGFR-MEK cascade and Pax6 up-regulation depended on changes of the cell-ECM and/or cell-cell interaction are supposed important for realization of the first steps of NR regeneration (Avdonin, 2010; Susaki & Chiba, 2007). In the *in vitro – in vivo* like systems it was shown that cells of isolated RPE could be induced to faster dedifferentiation

by additing of FGF2 to culture medium (Ikegami et al., 2001; Novikova et al. 2010b).

further damage.

In mammals, soon after NR detachment FGF2 gene up-regulation also takes place in parallel with high expression of FGF receptors (FGFR) (Hackett et al., 1997; Ozaki et al., 2000). When the retina is perturbed, significant changes occur in the expression of FGFR1 by photoreceptors: FGFR1 immunoreactivity increases rapidly (in 24 hours after injury) and steadily (Ozaki et al., 2000). That appears to be accompanied by similar increase of FGF2 in the IPM. Ozaki and co-workers suggest that this describes a paracrine mechanism: FGF2 is released or activated after retinal injury and then binds to FGFR1 on photoreceptor target cells. The latter, in turn, initiates an intracellular cascade that "protects" the cells from

The study of the effect of light, various types of stress, neurotrophic factors, and cytokines on FGF2 levels in human RPE cultured *in vitro* showed that many agents of photoreceptor protection (for instance, BDNF, CNTF, IL-1β) can up regulate FGF2 mRNA in RPE cells. An

induces a decrease of PEDF correct level (Ohno-Matsui et al., 2001).

increase in FGF2 protein level was demonstrated by ELISA in RPE cell supernatants after incubation with BDNF or exposure to intense light or oxidizing agents. These data indicate that in RPE cells FGF2 is modulated by stress and by agents that provide protection from stress (Hackett et al., 1997). In addition, it was found that FGF2 immunoreactivity in the interphotoreceptor matrix tends to increase during first 24 hours after retinal detachment in the rat. It is proposed that the interphotoreceptor matrix has its own endogenous local source(s) of FGF2 (Ozaki et al., 2000). Therefore, it is possible to consider that in both cases, at the initiation of NR regeneration in amphibia and NR rescue in mammals, FGF2 signaling pathway participate in neuroprotection and regulation of RPE cell differentiation and proliferation.

Other signaling pathways, as like as IGF-1, CNTF, and TGFβ represent also a part of the molecular network, regulating RPE and NR cell behavior after separation of these tissues in mammals. However, for today there are only few data on their activity in NR regeneration in Urodela. There is the evidence that IGF-1 (as like as FGF2) can accelerate proliferation and proneuronal differentiation of amphibian RPE under *in vitro* conditions (Yoshii et al., 2007). Meanwhile, proapoptotic growth factor TGFβ more likely plays prohibitive role in RPE cell type conversion. Activin, a TGF-β family signaling protein has been shown to contribute to the loss in competence of the RPE to regenerate retina. Sakami and co-authors (2008) have found that additing of activin blocked regeneration from the RPE, even when the cells were competent. Conversely, a small molecule inhibitor of the activin/TGF-β/nodal receptors could delay and reverse the developmental restriction in FGF-stimulated NR regeneration in embryonic chicken (Sakami et al., 2008).

Earlier it was shown that TGFβ inhibits proliferation at the vitreoretinal interface after NR detachment in human (Esser et al., 1997). Nowadays the study of the role of TGFβ is carried out on the model of retinal detachment in experiments using mice null for Smad3, TGFβ functional cooperator, a key signaling intermediate downstream of TGFβ and activin receptors. Obtained results showed that Smad3 is essential for the epithelial-mesenchymal transition of RPE cells induced by NR detachment. *De novo* accumulation of fibrous tissue derived from multilayered RPE cells was seen in experimental NR detachment in eyes of wild type, but not in Smad3-null mice (Saika et al., 2004). Activation of several signaling pathways, particularly TGFβ /Smad, was also fixed by Zacks and coworkers (2006). Soon after NR detachment the interleukin-6/STAT, TGFβ-Smad, and stress response pathway (aryl hydrocarbon receptor) – all were transcriptionally and translationally upregulated, suggesting that retina produces survival factors after detachment and that there is a possible cross-talk between up-regulated pathways (Zacks et al., 2006). In sum, knowing of signaling pathways with proliferative and anti-proliferative as well as pro-apoptotic and antiapoptotic effects is very important, because in both, retinal epimorphic regeneration in amphibian and proliferative retinopathy after detachment in mammals, changes of RPE cell phenotype, cell proliferation and apoptosis take place.

#### **2.5 Up-regulation of heat shock proteins and immediate-early response genes**

RPE↔NR disintegration results in the early activation of stress-response genes and specific signaling pathways which may enable retinal cells to survive at the most acute period of time. During NR detachment/regeneration in Urodela and detachment in mammals, heat shock proteins (HSPs) are involved in fast regenerative responses. Our preliminary

Shared Triggering Mechanisms of Retinal Regeneration

Disturbance of retinal cell contacts and change of RPE cell behavior

Visual cycle disturbance and cell apoptosis

Changes in vascular and immune systems in the

Participation of growth factors and major signaling pathways

Up-regulation of heat shock proteins and immediate-early response

extracellular matrix and RPE cytoskeleton.

genes

Remodeling of

eye back wall

Kind of events Prior NR epimorphic

in Lower Vertebrates and Retinal Rescue in Higher Ones 155

weakening of RPE cell lateral

RRE cell withdrawal from

high proliferative activity of

apoptosis of small set of RPE and photoreceptor cells

Possible involvement of fibrinolytic (TF, thrombin) and complement systems in triggering of RPE cell transdifferentiation

HSP70,90 proteins; *c-Myc* 

Redistribution of fibronectin, laminin stimulating effect on

transition of RPE cells: shift of specific intermediate filaments (cytokeratins →

Table 1. A comparison of known NR detachment-induced cellular and molecular events preceding retinal regeneration in Urodelean amphibians and retinal rescue in mammals

RPE conversion, epithelial–neuronal

neurofilaments).

gene

RPE cell conversion to macrophagal and proneuronal phenotypes

Blockage of melanin synthesis in RPE cells, down regulation of visual

cycle proteins,

Prior NR rescue

IPM disruption Weakening of RPE cell lateral contacts,

the layer,

RPE cells,

cycle proteins,

cells

and TGFβ

RPE cells,

FGF2, IGF1, TGFβ (activin) PEDF, VEGF, FGF1,2,

RRE cell withdrawal from

low proliferative activity of

fibroblast-like phenotypes

Up regulation of secreted proteins in RPE cells, down regulation of visual

apoptosis of small set of RPE and photoreceptor

Possible involvement of fibrinogenic (tPA), inflammatory associated proteins, and activated lymphocytes at the first stage of NR rescue

CNTF, BDNF, IGF1, IL-1β,

HSPs, *c-Fos* and *c-Jun* genes, AP1-complex

Role of laminin and integrins in modulation of

epithelial-mesenchymal transition of RPE cells: change in composition of specific intermediate filaments (cytokeratins, vimentin, GFAP).

RPE cell conversion to macrophagal and

regeneration

contacts,

the layer,

RPE cells,

IPM disruption,

(unpublished) results show an accumulation and co-distribution of HSP70, 90 and FGF2 in the NR soon after its detachment in the newt. In the experiments we observed well correlated changes in the intensity of HSPs and FGF2 expression and in the localization of these proteins in the retina. These data preliminary show that besides well known role of HSPs in the protection of newly synthesizing proteins from degradation a regulative link between HSPs and FGF may play a role in triggering of early retinal cell death/survival events. It is interesting also when infected with MC29, a myc expressing virus, the RPE cells in developing eye can be induced to transdifferentiate to neuroretinal epithelium. Beside genes whose work is involved in regulating neuronal differentiation myc also induced a transient expression of Mitf, well-known regulator of the pigmented differentiation (Beche-Belsot et al., 2001). HSPs, growth factors, and mitogen-activated protein kinase (MAPK) signaling are capable of immediate-early response gene up-regulation in different systems. Retinal detachment in the rat results in early up-regulation of genes, coding HSPs, FGF, early emergency genes (c-Fos and c-Jun), and transcription factor AP-1 complex (Faktorovich et al., 1992; Geller et al., 2001). Authors hypothesize that NR detachment causes the rapid release of FGF2 from intra- and/or extra-cellular stores, leading to the activation of FGFR1 and ERK, and proximate induction of c-Fos and c-Jun protein expression in RPE. Up-regulation of these intracellular components linked with FGF expression [HSPs → FGF2→FGFR → ERK&MAPK (MEK) pathway → c-fos&c-jun (AP-I) →] pretends to be an important early step on the way to RPE cell type transformation, migration and proliferation in amphibian and mammals. It is likely that increased AP-1 expression besides entering to apoptosis can regulate a variety of genetic and cellular responses induced by NR↔RPE separation.

#### **2.6 Remodeling of extracellular matrix (ECM) and RPE cytoskeleton**

It is quite possible that RPE↔NR separation associated changes of cytoskeleton are involved in regulation of HSPs and AP-1 complex in RPE and NR cells. Our early studies showed fast down-regulation of epithelium-specific intermediate filament expression and up-regulation of pan-neuronal one in RPE soon after NR removal in the newt (Grigoryan & Anton, 1993, 1995; Grigoryan, 1995). Keratins of the cytoskeletal intermediate filaments have been identified immunohistochemically in RPE of the adult newt retina. In conditions of NR surgical removal or complete detachment the expression of keratins markedly decreased. Similar observation has been made immediately after dissociation of the RPE cells isolated from nonoperated newt eyes. The results obtained provide an evidence for the inhibition of cytokeratin expression just after destabilization of RPE cell phenotype. *In vivo* in RPE disappeared cytokeratins were replaced by NF-200 neurofilament proteins that testified an existence of the mechanism responsible for gradual change of cytoskeleton in modified RPE in amphibians.

Changes suggested cytoskeleton rearrangement were also registered in mammalian animal models simulating RPE epithelial-mesenchymal transition specific for NR detachment. It was found that RPE cells lost their initial phenotype, dedifferentiated and acquired mesenchymal migratory morphology and cytoskeleton proteins (Casaroli–Marano et al., 1999). Recently thrombin (see above) pretends to play a promoting role in actin stress fiber formation, an important determinant in eye diseases involving transformation and migration of RPE cells (Ruiz-Loredo et al., 2011). On the other hand cytoskeleton changes in

(unpublished) results show an accumulation and co-distribution of HSP70, 90 and FGF2 in the NR soon after its detachment in the newt. In the experiments we observed well correlated changes in the intensity of HSPs and FGF2 expression and in the localization of these proteins in the retina. These data preliminary show that besides well known role of HSPs in the protection of newly synthesizing proteins from degradation a regulative link between HSPs and FGF may play a role in triggering of early retinal cell death/survival events. It is interesting also when infected with MC29, a myc expressing virus, the RPE cells in developing eye can be induced to transdifferentiate to neuroretinal epithelium. Beside genes whose work is involved in regulating neuronal differentiation myc also induced a transient expression of Mitf, well-known regulator of the pigmented differentiation (Beche-Belsot et al., 2001). HSPs, growth factors, and mitogen-activated protein kinase (MAPK) signaling are capable of immediate-early response gene up-regulation in different systems. Retinal detachment in the rat results in early up-regulation of genes, coding HSPs, FGF, early emergency genes (c-Fos and c-Jun), and transcription factor AP-1 complex (Faktorovich et al., 1992; Geller et al., 2001). Authors hypothesize that NR detachment causes the rapid release of FGF2 from intra- and/or extra-cellular stores, leading to the activation of FGFR1 and ERK, and proximate induction of c-Fos and c-Jun protein expression in RPE. Up-regulation of these intracellular components linked with FGF expression [HSPs → FGF2→FGFR → ERK&MAPK (MEK) pathway → c-fos&c-jun (AP-I) →] pretends to be an important early step on the way to RPE cell type transformation, migration and proliferation in amphibian and mammals. It is likely that increased AP-1 expression besides entering to apoptosis can regulate a variety of genetic and cellular

responses induced by NR↔RPE separation.

in amphibians.

**2.6 Remodeling of extracellular matrix (ECM) and RPE cytoskeleton** 

It is quite possible that RPE↔NR separation associated changes of cytoskeleton are involved in regulation of HSPs and AP-1 complex in RPE and NR cells. Our early studies showed fast down-regulation of epithelium-specific intermediate filament expression and up-regulation of pan-neuronal one in RPE soon after NR removal in the newt (Grigoryan & Anton, 1993, 1995; Grigoryan, 1995). Keratins of the cytoskeletal intermediate filaments have been identified immunohistochemically in RPE of the adult newt retina. In conditions of NR surgical removal or complete detachment the expression of keratins markedly decreased. Similar observation has been made immediately after dissociation of the RPE cells isolated from nonoperated newt eyes. The results obtained provide an evidence for the inhibition of cytokeratin expression just after destabilization of RPE cell phenotype. *In vivo* in RPE disappeared cytokeratins were replaced by NF-200 neurofilament proteins that testified an existence of the mechanism responsible for gradual change of cytoskeleton in modified RPE

Changes suggested cytoskeleton rearrangement were also registered in mammalian animal models simulating RPE epithelial-mesenchymal transition specific for NR detachment. It was found that RPE cells lost their initial phenotype, dedifferentiated and acquired mesenchymal migratory morphology and cytoskeleton proteins (Casaroli–Marano et al., 1999). Recently thrombin (see above) pretends to play a promoting role in actin stress fiber formation, an important determinant in eye diseases involving transformation and migration of RPE cells (Ruiz-Loredo et al., 2011). On the other hand cytoskeleton changes in


Table 1. A comparison of known NR detachment-induced cellular and molecular events preceding retinal regeneration in Urodelean amphibians and retinal rescue in mammals

Shared Triggering Mechanisms of Retinal Regeneration

in Lower Vertebrates and Retinal Rescue in Higher Ones 157

Fig. 4. Main shared processes taking place in the eye back wall soon after neural retina

A comparison of initial mechanisms triggering retinal regeneration and retinal rescue/pathology seems efficient. It could be very helpful for understanding why quite similar intrinsic protective responses, occurring at early stages of NR regeneration in amphibians and NR detachment in mammals, give such contrast final results. In regards of this, being based on the accumulated data we can make several suppositions. The first one is the difference in the level of RPE cell differentiation. In Urodela it has some developmental traits whose expression in permissive conditions *in vivo* leads to acquiring of neuronal phenotype. Contrast to amphibians, in adult mammals changes of RPE differentiation *in vivo* imply the epithelial-mesenchymal transition and cell transformation into migrated macrophages, the processes resembling an inflammation and scarring. The second assumption is a difference in the external molecular network, its signals, and cross-talks which regulate RPE cell differentiation in amphibians and mammals. The search of key factors which distinguish detachment induced signaling for amphibian RPE from that for mammalian and human RPE is rather difficult though also necessary step for future work. Finally and more likely, both: RPE cell intrinsic competence (including epigenetic features) and molecular regulation by microsurrounding are different in lower

detachment in amphibians (NR epimorphic regeneration) and mammals (NR rescue/pathology). ONL – outer nuclear layer, R – rod, C – cone photoreceptors.

RPE reflect an alteration of cell micro-surrounding. The latter, in turn, is a response for mechanical and chemical changes which are produced inevitably by RPE↔NR separation. Earlier we showed a decrease of fibronectin in Bruch membrane and its redistribution in RPE after NR detachment in the newt (Grigoryan et al., 1990). Similar results were obtained by Ortiz and co-authors (1992): at the beginning of RPE cell transdifferentiation in the eye of the adult newt, fibronectin was the first to appear in the cell border of the newforming neuroepithelium. A dependence of RPE phenotype on changes of ECM was also observed by Reh and co-authors in *in vitro* experiments (1987). They reported that RPE transdifferentiation is profoundly influenced by the substrate on which the cells are cultured. RPE cells plated on laminin-containing substrates frequently were conversed into neurons. Recently some data suggest that interaction of laminins and integrins in Bruch membrane leads to differential behavior of RPE cells in mammals (Aisenbrey et al., 2006).

Degradation of ECM is one more important stimulus for the initiation of RPE cell migration and phenotype transformation. Metalloproteases are known molecules for ECM changes and, vice versa, metalloprotease inhibitors (TIMPs) are factors that stabilize ECM. Mechanical trauma induced by NR↔RPE separation is associated with an increased activity of proteolytic enzymes. To ascertain whether RPE cells release proteases due to mechanical stress special tests *in vitro* were performed by Kain and Reuter (1995). In traction conditions created *in vitro* RPE might release proteases to cut intercellular adhesions in order to escape mechanical strain. Authors suggest that release of proteases from RPE may be involved in the pathology of traction detachment, facilitating the disconnection between RPE and photoreceptor outer segments. In human RPE cultured *in vitro* stromelysin which degrades important constituents of the ECM was found (Schönfeld, 1997). Therefore, the action of lysosomal proteases may change the surrounding that, in turn, can induce further detachment of RPE cells from the basement membrane and initiate RPE proliferation and dedifferentiation under conditions of RPE↔NR separation.

#### **3. Conclusion**

In the review we summarized our own and literature data on the early cellular and molecular events taking place after separation of neural retina (NR) from the retinal pigmented epithelium (RPE) in the eyes of vertebrates (Table 1). In amphibians RPE↔NR disintegration leads to the formation of the new NR by means of RPE cell transdifferentiation into retinal cells, while in mammals NR detachment triggers a retinal pathology. A comparison of these two opposite phenomena unexpectedly reveals a similarity of early cell and molecular processes induced by RPE↔NR separation (figure 4). In both cases alterations of RPE cell contacts, changes in cytoskeleton and ECM composition as well as perturbations in blood circulation and immune system can be found. These alterations lead to RPE cell type destabilization, phenotypic transformation, cell withdrawal from the layer, and migration. In parallel, down-regulation of the expression of visual cycle molecules takes place. In contrast heat shock proteins, FGF signaling, immediate-early response genes, and AP-1 complex demonstrate up-regulation. In all animals and in human these, NR detachment associated events represent a limited, universal range of retinal cell responses to the stress. Among them RPE dedifferentiation, proliferation and migration seems most important for both, subsequent NR epimorphic regeneration in amphibians and a progress of detachment induced eye diseases in mammals.

RPE reflect an alteration of cell micro-surrounding. The latter, in turn, is a response for mechanical and chemical changes which are produced inevitably by RPE↔NR separation. Earlier we showed a decrease of fibronectin in Bruch membrane and its redistribution in RPE after NR detachment in the newt (Grigoryan et al., 1990). Similar results were obtained by Ortiz and co-authors (1992): at the beginning of RPE cell transdifferentiation in the eye of the adult newt, fibronectin was the first to appear in the cell border of the newforming neuroepithelium. A dependence of RPE phenotype on changes of ECM was also observed by Reh and co-authors in *in vitro* experiments (1987). They reported that RPE transdifferentiation is profoundly influenced by the substrate on which the cells are cultured. RPE cells plated on laminin-containing substrates frequently were conversed into neurons. Recently some data suggest that interaction of laminins and integrins in Bruch membrane leads to differential

Degradation of ECM is one more important stimulus for the initiation of RPE cell migration and phenotype transformation. Metalloproteases are known molecules for ECM changes and, vice versa, metalloprotease inhibitors (TIMPs) are factors that stabilize ECM. Mechanical trauma induced by NR↔RPE separation is associated with an increased activity of proteolytic enzymes. To ascertain whether RPE cells release proteases due to mechanical stress special tests *in vitro* were performed by Kain and Reuter (1995). In traction conditions created *in vitro* RPE might release proteases to cut intercellular adhesions in order to escape mechanical strain. Authors suggest that release of proteases from RPE may be involved in the pathology of traction detachment, facilitating the disconnection between RPE and photoreceptor outer segments. In human RPE cultured *in vitro* stromelysin which degrades important constituents of the ECM was found (Schönfeld, 1997). Therefore, the action of lysosomal proteases may change the surrounding that, in turn, can induce further detachment of RPE cells from the basement membrane and initiate RPE proliferation and

In the review we summarized our own and literature data on the early cellular and molecular events taking place after separation of neural retina (NR) from the retinal pigmented epithelium (RPE) in the eyes of vertebrates (Table 1). In amphibians RPE↔NR disintegration leads to the formation of the new NR by means of RPE cell transdifferentiation into retinal cells, while in mammals NR detachment triggers a retinal pathology. A comparison of these two opposite phenomena unexpectedly reveals a similarity of early cell and molecular processes induced by RPE↔NR separation (figure 4). In both cases alterations of RPE cell contacts, changes in cytoskeleton and ECM composition as well as perturbations in blood circulation and immune system can be found. These alterations lead to RPE cell type destabilization, phenotypic transformation, cell withdrawal from the layer, and migration. In parallel, down-regulation of the expression of visual cycle molecules takes place. In contrast heat shock proteins, FGF signaling, immediate-early response genes, and AP-1 complex demonstrate up-regulation. In all animals and in human these, NR detachment associated events represent a limited, universal range of retinal cell responses to the stress. Among them RPE dedifferentiation, proliferation and migration seems most important for both, subsequent NR epimorphic regeneration in amphibians and a progress

behavior of RPE cells in mammals (Aisenbrey et al., 2006).

dedifferentiation under conditions of RPE↔NR separation.

of detachment induced eye diseases in mammals.

**3. Conclusion** 

Fig. 4. Main shared processes taking place in the eye back wall soon after neural retina detachment in amphibians (NR epimorphic regeneration) and mammals (NR rescue/pathology). ONL – outer nuclear layer, R – rod, C – cone photoreceptors.

A comparison of initial mechanisms triggering retinal regeneration and retinal rescue/pathology seems efficient. It could be very helpful for understanding why quite similar intrinsic protective responses, occurring at early stages of NR regeneration in amphibians and NR detachment in mammals, give such contrast final results. In regards of this, being based on the accumulated data we can make several suppositions. The first one is the difference in the level of RPE cell differentiation. In Urodela it has some developmental traits whose expression in permissive conditions *in vivo* leads to acquiring of neuronal phenotype. Contrast to amphibians, in adult mammals changes of RPE differentiation *in vivo* imply the epithelial-mesenchymal transition and cell transformation into migrated macrophages, the processes resembling an inflammation and scarring. The second assumption is a difference in the external molecular network, its signals, and cross-talks which regulate RPE cell differentiation in amphibians and mammals. The search of key factors which distinguish detachment induced signaling for amphibian RPE from that for mammalian and human RPE is rather difficult though also necessary step for future work. Finally and more likely, both: RPE cell intrinsic competence (including epigenetic features) and molecular regulation by microsurrounding are different in lower

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#### **4. Acknowledgment**

This study was supported by the Russian Foundation for Basic Research, project no. 11-04- 00125-a (for E.N. Grigoryan).

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extracellular matrix components during lens and neural retina regeneration in the adult newt, *Experimental Eye Research,* Vol.54, No.6, pp. 861-870, ISSN 0014-

receptor 1 (flg) by rat photoreceptor cells after injury. *Investigative Ophthalmology* 

evidence for endothelin signaling from photoreceptors to glia, *Journal of* 

retinal pigment epithelium to light damage and retinal detachment, *Journal of* 

retinal degenerations and how? *Documenta Ophthalmologica,* Vol. 106, No.1, pp. 25-

transdifferentiate to neurons by laminin, *Nature*, Vol. 330, pp. 68-71, ISSN 0028-

formation in RPE through Rho/ROCK-mediated MLC phosphorylation, *Journal of* 

Ooshima, A.; Yoo J.; Flanders, K. & Roberts, A. (2004). Smad3 is required for dedifferentiation of retinal pigment epithelium following retinal detachment in


**Part 2** 

**Application of Stem Cells** 

Zacks, D.; Han, Y.; Zeng, Y. & Swaroop, A. (2006). Activation of signaling pathways and stress-response genes in an experimental model of retinal detachment, *Investigative Ophthalmology and Visual Science*, Vol. 47, No.4, pp.1691-1695, ISSN 1552-5783

**Part 2** 

**Application of Stem Cells** 

164 Tissue Regeneration – From Basic Biology to Clinical Application

Zacks, D.; Han, Y.; Zeng, Y. & Swaroop, A. (2006). Activation of signaling pathways and

1552-5783

stress-response genes in an experimental model of retinal detachment, *Investigative Ophthalmology and Visual Science*, Vol. 47, No.4, pp.1691-1695, ISSN

**8** 

*1,4USA 2Philippines 3Germany* 

**The Therapeutic Potential of Stimulating** 

The past decade has seen a fast and extensive development of various therapies and treatment protocols based on Adult Stem Cells (ASC) and their application to various diseases. While some of these treatment protocols have been well documented in the scientific literature and used in well controlled clinical set ups, others have been developed and are being used by a growing numbers of clinics throughout the world, without thorough documentation though nevertheless with good clinical care and with the reports

Despite the wide variety of methods, the general procedure guiding these various protocols follows a series of common steps. The first step is the isolation of stem cells from a source. For the purpose of banking or clinical application, stem cells can be isolated from a variety of sources including umbilical cord (Can and Balci, 2011; Zhang et al., 2011), adipose tissuederived stem cells (Insausti et al., 2011; Zachar et al., 2011), peripheral blood stem cells (Kolbe et al., 2010; Hofmann et al., 2009), amniotic and placental stem cells (Klein and Fauza, 2011; Tsagias et al., 2011), dental pulp stem cells (Gronthos et al., 2011; Tirino et al., 2011), olfactory stem cells (Chen et al., 2006; Viktorov et al., 2008), and even human limbal

The second step is proliferation. This is not a necessary step with regard to stem cell function, however the small number of stem cells present in one umbilical cord, one placenta, one blood sample, one liposuction or one dental pulp makes clinical application difficult without the ability to expand the harvested stem cells. Methods to expand embryonic stem cells have been developed more than a decade ago, however it is only a few years ago that methods to significantly expand ASC have been developed, leading to an expansion of the stem cell banking market and greater clinical application (Ivanovic et al.,

**1. Introduction** 

of very compelling results.

epithelial stem cells (Vasania et al., 2011).

2011; Dos Santos et al., 2011; Pineault et al., 2011).

**Endogenous Stem Cell Mobilization** 

Christian Drapeau1, George Eufemio2, Paola Mazzoni1,

*2Cardinal Santos Medical Center, Medical Arts Building, Manila,* 

Gerhard D. Roth3 and Susan Strandberg4 *1Stemtech International, San Clemente, CA,* 

*3Neurology and Psychiatry Center, Ostfildern,* 

*4Stem Cell Center, Spokane, WA* 

### **The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization**

Christian Drapeau1, George Eufemio2, Paola Mazzoni1, Gerhard D. Roth3 and Susan Strandberg4 *1Stemtech International, San Clemente, CA, 2Cardinal Santos Medical Center, Medical Arts Building, Manila, 3Neurology and Psychiatry Center, Ostfildern, 4Stem Cell Center, Spokane, WA 1,4USA 2Philippines 3Germany* 

#### **1. Introduction**

The past decade has seen a fast and extensive development of various therapies and treatment protocols based on Adult Stem Cells (ASC) and their application to various diseases. While some of these treatment protocols have been well documented in the scientific literature and used in well controlled clinical set ups, others have been developed and are being used by a growing numbers of clinics throughout the world, without thorough documentation though nevertheless with good clinical care and with the reports of very compelling results.

Despite the wide variety of methods, the general procedure guiding these various protocols follows a series of common steps. The first step is the isolation of stem cells from a source. For the purpose of banking or clinical application, stem cells can be isolated from a variety of sources including umbilical cord (Can and Balci, 2011; Zhang et al., 2011), adipose tissuederived stem cells (Insausti et al., 2011; Zachar et al., 2011), peripheral blood stem cells (Kolbe et al., 2010; Hofmann et al., 2009), amniotic and placental stem cells (Klein and Fauza, 2011; Tsagias et al., 2011), dental pulp stem cells (Gronthos et al., 2011; Tirino et al., 2011), olfactory stem cells (Chen et al., 2006; Viktorov et al., 2008), and even human limbal epithelial stem cells (Vasania et al., 2011).

The second step is proliferation. This is not a necessary step with regard to stem cell function, however the small number of stem cells present in one umbilical cord, one placenta, one blood sample, one liposuction or one dental pulp makes clinical application difficult without the ability to expand the harvested stem cells. Methods to expand embryonic stem cells have been developed more than a decade ago, however it is only a few years ago that methods to significantly expand ASC have been developed, leading to an expansion of the stem cell banking market and greater clinical application (Ivanovic et al., 2011; Dos Santos et al., 2011; Pineault et al., 2011).

The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization 169

For ESCM to have any clinical relevance, the demonstration must be made that one of the natural roles of stem cells in the body is to participate to tissue repair in cases of injury or degenerative diseases. Therefore, the clinical relevance of mobilizing endogenous bone marrow-derived stem cells (BMSC) would be to increase the number of circulating stem cells available to migrate into affected tissues and contribute to tissue repair. For this phenomenon to be natural: 1) the body must have a mechanism that triggers BMSC mobilization after an injury; 2) BMSC must traffic in the blood and be recruited by the injured tissue; 3) in the injured tissue BMSC must proliferate and 4) a mechanism must

The most common compound known to naturally stimulate BMSC mobilization is Granulocyte-Colony Stimulating Factor (G-CSF). Discovered in 1985 by Welte et al., G-CSF is a cytokine secreted by various tissues that stimulates the proliferation, differentiation and function of neutrophil precursors and mature neutrophils. But G-CSF was also shown to stimulate BMSC mobilization (Petit et al., 2003; Cottler-Fox et al., 2003), making it a common tool in protocols of stem cell apheresis for the purpose of cryopreservation and stem cell

Given the vital importance of the heart and the fact that cardiovascular diseases are a leading cause of death in the world, much of the clinical stem cell research has focused its efforts on the role of stem cells in cardiac repair taking place after acute myocardial infarction (AMI). A number of studies have revealed the sequence of events taking place after AMI. A few hours after AMI, the cardiac tissue releases or causes to release G-CSF (Leone et al., 2006). As its concentration slowly increases in the bloodstream, G-CSF triggers the release of stem cells from the bone marrow, increasing the number of PBSC which peaks at around 4-7 days after AMI (Shintani et al., 2001; Leone et al., 2006). It is worth mentioning that the serum level of G-CSF and the number of PBSC are also increased in cases of chronic angina (Leone et al., 2006). Similar stem cell mobilization and increase in PBSC have been

Other chemokines such as interleukine-8 (IL-8), Stromal-Derived Factor-1 (SDF-1), Stem Cell Factor (SCF), Groß, and vascular endothelial factor (VEGF) have been shown to trigger BMSC mobilization (King et al., 2001; Lapidot & Petit, 2002; Fukuda et al., 2007; Lapid et al., 2009). Contrary to G-CSF and SCF, which lead to a slow increase in the number of PBSC over a period of a few days, other cytokines such as IL-8 lead to a significant increase in the

As has been described with the heart following AMI, a stroke also triggers the release of cytokines that induce the mobilization of BMSC and their migration into the brain. For example, the number of PBSC in stroke patients nearly tripled within 7 days after the stroke (Hennemann et al., 2008; Paczkowska et al., 2005). In one study, the magnitude of stem cell release was actually correlated with the functional recovery of the patients (Dunac et al, 2007). Interestingly, the number of circulating stem cells did not increase in patients who received thrombolysis therapy immediately after their stroke. Therefore, it appears that it is

exist to trigger the differentiation of BMSC into cells of that tissue.

documented following skeletal muscle injury (Stout et al., 2007).

transplant (Gordon et al., 2008; Croop et al., 2001).

number of PBSC within hours (Fibbe et al., 1999).

**2. The repair system of the body** 

**2.1 Signaling for mobilization** 

The third step is pre-conditioning or treatment to trigger commitment of the stem cells into a specific cellular lineage. For example, stem cells can be led to differentiate into dopamineproducing neuron by an exposure to a cocktail containing sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), and basic fibroblast growth factor (bFGF) (Trzaska and Rameshwar, 2011; Wang et al., 2011) or into neurons responding to multiple neurotransmitters by a simple exposure to retinoic acid and other growth factors (Greco et al., 2008). Likewise, stem cells can be guided to differentiate in cardiomyocytes by exposure to a cocktail containing transforming growth factor-beta(1), bone morphogenetic protein-4, activin A, vascular endothelial growth factor (VEGF), insulin-like growth factor-1, fibroblast growth factor-2, Epidermal growth factor (EGF), and interleukin-6 (Behfar et al., 2010; Behfar et al., 2008). Pre-conditioning with these cytokines can also enhance the formation of gap junction and improve therapeutic efficacy (Hahn et al., 2008). Exposure of stem cells to a cocktail containing insulin, transferrin, selenium and the GLP-1 (glucagon-like peptide-1) analogue exendin-4 leads to the formation of insulin-producing pancreatic cells (Docherty, 2009; Chandra et al., 2009). Various cocktails have been shown to trigger the differentiation of mesenchymal stem cells in into a wide variety of cell types (Snykers et al., 2011; Arufe et al., 2009; Keilhoff et al., 2006). Nevertheless, this pre-conditioning step is not essential since ASC will naturally differentiate into the cell type with which they find themselves, upon contact with cellular debris or cell marker specific to that cell type. For example, as they migrate into the heart, stem cells can be triggered to differentiate into cardiomyocytes (Orlic et al., 2001), or into keratinocytes and skin appendages, insulin-producing pancreatic cells or hepatocytes as they respectively migrate in a skin wound (Zhang and Fu, 2008), the pancreas (Hasegawa et al., 2007) or the liver (Theise et al., 2000).

The third and final step is the injection of stem cells into the target organ, in the main artery leading to the target organ or in the bloodstream from where a number of them will migrate on their own to the affected organ. In the case of a heart attack for example, stem cells can be injected in coronary artery (Wollert et al., 2004) or directly in the border zone of the infarct (Stamm et al., 2003; Tse et al., 2003). Treatment efficacy can vary significantly with the various methods of injection. For the treatment of acute myocardial infarction, injection in the border zone of the infarct seems to yield the best results, followed by intracoronary and intravenous injection, respectively (Karra and Wu, 2008). For the treatment of spinal cord injury, injection directly in the lesion or in the cerebrospinal fluid seems far superior to intravenous injection (Lima et al., 2010), yet very compelling cases have been documented following intravenous stem cell injection or simple bone marrow stem cell mobilization (see Section 4). For the treatment of diabetes however, intravenous injection seems to yield better results than stem cells transplantation directly into the pancreas (Hasegawa et al., 2007).

In this multistep procedure in which each step can be accomplished according to a wide variety of protocols and methods, it remains that peripheral blood stem cells (PBSC) can, without expansion, pre-conditioning or injection reach various target organs and participate to the process of tissue repair. This observation has led a number of researchers to look at the therapeutic potential of simply stimulating Endogenous Bone Marrow Stem Cell Mobilization (ESCM). This chapter will look in detail into the clinical and therapeutic potential of ESCM by describing its physiological basis, by reviewing the existing literature on the clinical application of ESCM and by presenting a few clinical cases.

#### **2. The repair system of the body**

168 Tissue Regeneration – From Basic Biology to Clinical Application

The third step is pre-conditioning or treatment to trigger commitment of the stem cells into a specific cellular lineage. For example, stem cells can be led to differentiate into dopamineproducing neuron by an exposure to a cocktail containing sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), and basic fibroblast growth factor (bFGF) (Trzaska and Rameshwar, 2011; Wang et al., 2011) or into neurons responding to multiple neurotransmitters by a simple exposure to retinoic acid and other growth factors (Greco et al., 2008). Likewise, stem cells can be guided to differentiate in cardiomyocytes by exposure to a cocktail containing transforming growth factor-beta(1), bone morphogenetic protein-4, activin A, vascular endothelial growth factor (VEGF), insulin-like growth factor-1, fibroblast growth factor-2, Epidermal growth factor (EGF), and interleukin-6 (Behfar et al., 2010; Behfar et al., 2008). Pre-conditioning with these cytokines can also enhance the formation of gap junction and improve therapeutic efficacy (Hahn et al., 2008). Exposure of stem cells to a cocktail containing insulin, transferrin, selenium and the GLP-1 (glucagon-like peptide-1) analogue exendin-4 leads to the formation of insulin-producing pancreatic cells (Docherty, 2009; Chandra et al., 2009). Various cocktails have been shown to trigger the differentiation of mesenchymal stem cells in into a wide variety of cell types (Snykers et al., 2011; Arufe et al., 2009; Keilhoff et al., 2006). Nevertheless, this pre-conditioning step is not essential since ASC will naturally differentiate into the cell type with which they find themselves, upon contact with cellular debris or cell marker specific to that cell type. For example, as they migrate into the heart, stem cells can be triggered to differentiate into cardiomyocytes (Orlic et al., 2001), or into keratinocytes and skin appendages, insulin-producing pancreatic cells or hepatocytes as they respectively migrate in a skin wound (Zhang and Fu, 2008), the pancreas (Hasegawa

The third and final step is the injection of stem cells into the target organ, in the main artery leading to the target organ or in the bloodstream from where a number of them will migrate on their own to the affected organ. In the case of a heart attack for example, stem cells can be injected in coronary artery (Wollert et al., 2004) or directly in the border zone of the infarct (Stamm et al., 2003; Tse et al., 2003). Treatment efficacy can vary significantly with the various methods of injection. For the treatment of acute myocardial infarction, injection in the border zone of the infarct seems to yield the best results, followed by intracoronary and intravenous injection, respectively (Karra and Wu, 2008). For the treatment of spinal cord injury, injection directly in the lesion or in the cerebrospinal fluid seems far superior to intravenous injection (Lima et al., 2010), yet very compelling cases have been documented following intravenous stem cell injection or simple bone marrow stem cell mobilization (see Section 4). For the treatment of diabetes however, intravenous injection seems to yield better results than stem cells transplantation directly into the pancreas (Hasegawa et al., 2007).

In this multistep procedure in which each step can be accomplished according to a wide variety of protocols and methods, it remains that peripheral blood stem cells (PBSC) can, without expansion, pre-conditioning or injection reach various target organs and participate to the process of tissue repair. This observation has led a number of researchers to look at the therapeutic potential of simply stimulating Endogenous Bone Marrow Stem Cell Mobilization (ESCM). This chapter will look in detail into the clinical and therapeutic potential of ESCM by describing its physiological basis, by reviewing the existing literature

on the clinical application of ESCM and by presenting a few clinical cases.

et al., 2007) or the liver (Theise et al., 2000).

For ESCM to have any clinical relevance, the demonstration must be made that one of the natural roles of stem cells in the body is to participate to tissue repair in cases of injury or degenerative diseases. Therefore, the clinical relevance of mobilizing endogenous bone marrow-derived stem cells (BMSC) would be to increase the number of circulating stem cells available to migrate into affected tissues and contribute to tissue repair. For this phenomenon to be natural: 1) the body must have a mechanism that triggers BMSC mobilization after an injury; 2) BMSC must traffic in the blood and be recruited by the injured tissue; 3) in the injured tissue BMSC must proliferate and 4) a mechanism must exist to trigger the differentiation of BMSC into cells of that tissue.

#### **2.1 Signaling for mobilization**

The most common compound known to naturally stimulate BMSC mobilization is Granulocyte-Colony Stimulating Factor (G-CSF). Discovered in 1985 by Welte et al., G-CSF is a cytokine secreted by various tissues that stimulates the proliferation, differentiation and function of neutrophil precursors and mature neutrophils. But G-CSF was also shown to stimulate BMSC mobilization (Petit et al., 2003; Cottler-Fox et al., 2003), making it a common tool in protocols of stem cell apheresis for the purpose of cryopreservation and stem cell transplant (Gordon et al., 2008; Croop et al., 2001).

Given the vital importance of the heart and the fact that cardiovascular diseases are a leading cause of death in the world, much of the clinical stem cell research has focused its efforts on the role of stem cells in cardiac repair taking place after acute myocardial infarction (AMI). A number of studies have revealed the sequence of events taking place after AMI. A few hours after AMI, the cardiac tissue releases or causes to release G-CSF (Leone et al., 2006). As its concentration slowly increases in the bloodstream, G-CSF triggers the release of stem cells from the bone marrow, increasing the number of PBSC which peaks at around 4-7 days after AMI (Shintani et al., 2001; Leone et al., 2006). It is worth mentioning that the serum level of G-CSF and the number of PBSC are also increased in cases of chronic angina (Leone et al., 2006). Similar stem cell mobilization and increase in PBSC have been documented following skeletal muscle injury (Stout et al., 2007).

Other chemokines such as interleukine-8 (IL-8), Stromal-Derived Factor-1 (SDF-1), Stem Cell Factor (SCF), Groß, and vascular endothelial factor (VEGF) have been shown to trigger BMSC mobilization (King et al., 2001; Lapidot & Petit, 2002; Fukuda et al., 2007; Lapid et al., 2009). Contrary to G-CSF and SCF, which lead to a slow increase in the number of PBSC over a period of a few days, other cytokines such as IL-8 lead to a significant increase in the number of PBSC within hours (Fibbe et al., 1999).

As has been described with the heart following AMI, a stroke also triggers the release of cytokines that induce the mobilization of BMSC and their migration into the brain. For example, the number of PBSC in stroke patients nearly tripled within 7 days after the stroke (Hennemann et al., 2008; Paczkowska et al., 2005). In one study, the magnitude of stem cell release was actually correlated with the functional recovery of the patients (Dunac et al, 2007). Interestingly, the number of circulating stem cells did not increase in patients who received thrombolysis therapy immediately after their stroke. Therefore, it appears that it is

The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization 171

In brief, the sudden drop in blood pressure taking place at the postcapillary venule triggers turbulence whose shear force mechanically activates L-selectin which in turn triggers the externalization of CXCR4, making PBSC more responsive to signals coming from tissues. If the tissue is in need of repair, it is secreting SDF-1 as well as other compounds such as stem cell factor (SCF) and hepatocyte growth factor (HGH) (Kucia et al., 2004; , Neuss et al., 2004) that diffuse locally to the capillaries. Binding of SDF-1 and SCF to their specific receptors (e.g. CXCR4 and c-Kit) leads to the expression of adhesion molecules at the surface of stem cells (Voermans et al., 2000; Peled et al., 1999; Peled et al., 2000). Through a complex interaction of microvilli at the surface of both the capillary and PBSC, the stem cells initiate the process of tethering and then arrest on the capillary endothelium (Middleton et al., 1997;

Following firm attachment, SDF-1 and HGH continue to bind to their respective receptors CXCR4 and c-met at the surface of SC, which triggers the release and activation of matrix metalloproteinases (MMPs) which digest the endothelial lining, allowing for the extravasation of PBSC (Mannello et al., 2006; Janowska-Wieczorek et al., 2000; Neuss et al.,

When studying the migration behavior of SCs to a wide array of chemokines, SCs were found to migrate only toward SDF-1 (Wright et al., 2002; Jo et al., 2000). The migration to SDF-1 is a polarized phenomenon toward the chemokine source (chemotactic) and not a simple random motion (chemokinetic), as SCs only migrated in a gradient of SDF-1 and not

SDF-1 is normally secreted, to some extent, by cardiomyocytes (Askari et al., 2003), skeletal muscles (Ratajczak et al., 2003), liver (Hatch et al., 2002; Kollet et al., 2003), brain (Bagri et al., 2002; Lazarini et al., 2003; Zou et al., 1998), and kidney (Schrader et al., 2002). However, its secretion increases during tissue damage such as AMI (Wojakowski et al., 2004; Abbott et al., 2004), ischemia (Takahashi et al., 1999, Iwaguro et al., 2002), toxic liver damage (Kollet et al., 2003; Swenson et al., 2008; Hatch et al., 2002), and excessive bleeding (Ratajczak et al., 2004).

In the tissue, the process of migration toward the site of injury relies on the interaction between CD44 and its ligand hyaluronic acid (HYA). HYA is one of a family of polysaccharides known as glycosaminoglycans (GAGS), typically found in the connective tissues of vertebrates. Alongside other proteins such as collagen, elastin, fibrillin, fibronectin and laminin, GAGS and HYA constitute the extracellular matrix (ECM) of most tissues. Studies in rats have shown that half of the HYA found in the body is in the skin, while 25% is found in joints and bones together (Reed et al., 1988). The rest is distributed somewhat equally in muscles and viscera (Clarris and Fraser,1968; Comper and Laurent, 1978) where the highest concentrations are found in connective tissue that form the ECM of most tissues. After extravasation, CXCR4 on the surface of SC and SDF-1 secreted by the effected tissue continue their interaction which, in the tissue, leads to the formation of pseudopodia (see Figure 1) toward the source of SDF-1 secretion. As SDF-1 binds to CXCR4 in the tissue, CD44 adhesion molecules are externalized at the tip of the pseudopodia, leading to adhesion to HYA pathways within the tissue. In the tissue, the binding of CD44 to HYA is transient, as CD44 molecules shed soon after binding to HYA (Friedl et al., 1995), thus enabling pseudopodia detachment from the ECM. CD44 can also be cleaved by specific enzymes whose secretion is enhanced by SDF-1 (Heissig et al., 2002; Okamoto et al., 1999;

2002).

2004; Ries et al., 2007).

when SDF-1 is uniformly distributed in the media.

the lingering injury that slowly leads to the mobilization of stem cells from the bone marrow.

Finally, injuries to the skin and bones were also shown to trigger mobilization of BMSC and their migration into the injured tissue. For example, within 24 hours of a severe burn, a rapid increase of up to 9-fold in the number of PBSC has been observed in the blood of burn patients (Fox et al., 2008). Furthermore, the size of the area of the body affected by the burns strongly correlated with the magnitude of the mobilization. The affected skin also released cytokines such as SDF-1 and VEGF, which are involved in the migration of PBSC to the skin and their differentiation into blood vessels, respectively (Mansilla et al., 2006). In one study (Lee et al., 2008), the number of PBSC peaked around 3 days after a bone fracture and rapidly returned to basal level within a few days. These results were confirmed in another study in which stem cells were shown to migrate to the fracture site and to promote neovascularization. The formation of new vessels was shown to peak at the fracture site 7 days after the fracture, which corresponds to the early phase of ossification of the fracture line (Matsumoto et al., 2008). Therefore BMSC mobilization was documented to naturally follow an injury or even be associated with chronic conditions.

The natural process by which stem cells are mobilized from the bone marrow is still not fully understood. Contrary to most tissues in which SDF-1 is secreted consequent to an injury or a degenerative condition, in the bone marrow SDF-1 is constitutively produced and released, and binding of SDF-1 to its exclusive receptor CXCR4 leads to the externalization of adhesion molecules, namely integrins, which allow for the adherence of stem cells to the bone marrow matrix. The binding of SDF-1 to CXCR4 is referred to as the SDF-1/CXCR4 axis. The general understanding is that disruption of the SDF-1/CXCR4 axis reduces the expression of adhesion molecules, leading to a reduction in the adherence of stem cells to the bone marrow matrix and the consequent mobilization of stem cells. Various compounds known to trigger stem cell mobilization all affect the SDF-1/CXCR4 axis in various ways.

For example, G-CSF disrupts the SDF-1/CXCR4 axis by activating a series of proteolytic enzymes including elastase, cathepsin G, and various matrix metalloproteinases (MMP2 and MMP9) that inactivate SDF-1 (Mannello et al., 2006; Jin et al., 2006; Carion et al., 2003). AMD3100 is a newly developed BMSC mobilizer and it acts by blocking CXCR4, disrupting the SDF-1/CXCR4 axis (Broxmeyer et al., 2005). A blocker of L-selectin was recently isolated from the cyanophyta *Aphanizomenon flos-aquae* and shown to trigger BMSC mobilization (Jensen et al., 2007). Inhibition of L-selectin leads to a down-regulation of CXCR4 expression, partially disrupting the SDF-1/CXCR4 axis. The mobilization mechanism of IL-8, SCF, VEGF and Groß are not well understood.

Therefore, the human body has a mechanism to naturally mobilize BMSC which can then traffic to various areas of the body and contribute to tissue regeneration and repair.

#### **2.2 Extravasation & recruitment**

Recruitment is the process by which PBSC are recruited by a specific tissue signaling for repair. The process of PBSC recruitment in a tissue takes place predominantly at the level the postcapillary venule, in a manner similar to neutrophils (Henschler et al., 2008).

the lingering injury that slowly leads to the mobilization of stem cells from the bone

Finally, injuries to the skin and bones were also shown to trigger mobilization of BMSC and their migration into the injured tissue. For example, within 24 hours of a severe burn, a rapid increase of up to 9-fold in the number of PBSC has been observed in the blood of burn patients (Fox et al., 2008). Furthermore, the size of the area of the body affected by the burns strongly correlated with the magnitude of the mobilization. The affected skin also released cytokines such as SDF-1 and VEGF, which are involved in the migration of PBSC to the skin and their differentiation into blood vessels, respectively (Mansilla et al., 2006). In one study (Lee et al., 2008), the number of PBSC peaked around 3 days after a bone fracture and rapidly returned to basal level within a few days. These results were confirmed in another study in which stem cells were shown to migrate to the fracture site and to promote neovascularization. The formation of new vessels was shown to peak at the fracture site 7 days after the fracture, which corresponds to the early phase of ossification of the fracture line (Matsumoto et al., 2008). Therefore BMSC mobilization was documented to naturally

The natural process by which stem cells are mobilized from the bone marrow is still not fully understood. Contrary to most tissues in which SDF-1 is secreted consequent to an injury or a degenerative condition, in the bone marrow SDF-1 is constitutively produced and released, and binding of SDF-1 to its exclusive receptor CXCR4 leads to the externalization of adhesion molecules, namely integrins, which allow for the adherence of stem cells to the bone marrow matrix. The binding of SDF-1 to CXCR4 is referred to as the SDF-1/CXCR4 axis. The general understanding is that disruption of the SDF-1/CXCR4 axis reduces the expression of adhesion molecules, leading to a reduction in the adherence of stem cells to the bone marrow matrix and the consequent mobilization of stem cells. Various compounds known to trigger stem cell mobilization all affect the SDF-1/CXCR4 axis in

For example, G-CSF disrupts the SDF-1/CXCR4 axis by activating a series of proteolytic enzymes including elastase, cathepsin G, and various matrix metalloproteinases (MMP2 and MMP9) that inactivate SDF-1 (Mannello et al., 2006; Jin et al., 2006; Carion et al., 2003). AMD3100 is a newly developed BMSC mobilizer and it acts by blocking CXCR4, disrupting the SDF-1/CXCR4 axis (Broxmeyer et al., 2005). A blocker of L-selectin was recently isolated from the cyanophyta *Aphanizomenon flos-aquae* and shown to trigger BMSC mobilization (Jensen et al., 2007). Inhibition of L-selectin leads to a down-regulation of CXCR4 expression, partially disrupting the SDF-1/CXCR4 axis. The mobilization mechanism of IL-8, SCF,

Therefore, the human body has a mechanism to naturally mobilize BMSC which can then

Recruitment is the process by which PBSC are recruited by a specific tissue signaling for repair. The process of PBSC recruitment in a tissue takes place predominantly at the level

traffic to various areas of the body and contribute to tissue regeneration and repair.

the postcapillary venule, in a manner similar to neutrophils (Henschler et al., 2008).

follow an injury or even be associated with chronic conditions.

marrow.

various ways.

VEGF and Groß are not well understood.

**2.2 Extravasation & recruitment** 

In brief, the sudden drop in blood pressure taking place at the postcapillary venule triggers turbulence whose shear force mechanically activates L-selectin which in turn triggers the externalization of CXCR4, making PBSC more responsive to signals coming from tissues. If the tissue is in need of repair, it is secreting SDF-1 as well as other compounds such as stem cell factor (SCF) and hepatocyte growth factor (HGH) (Kucia et al., 2004; , Neuss et al., 2004) that diffuse locally to the capillaries. Binding of SDF-1 and SCF to their specific receptors (e.g. CXCR4 and c-Kit) leads to the expression of adhesion molecules at the surface of stem cells (Voermans et al., 2000; Peled et al., 1999; Peled et al., 2000). Through a complex interaction of microvilli at the surface of both the capillary and PBSC, the stem cells initiate the process of tethering and then arrest on the capillary endothelium (Middleton et al., 1997; 2002).

Following firm attachment, SDF-1 and HGH continue to bind to their respective receptors CXCR4 and c-met at the surface of SC, which triggers the release and activation of matrix metalloproteinases (MMPs) which digest the endothelial lining, allowing for the extravasation of PBSC (Mannello et al., 2006; Janowska-Wieczorek et al., 2000; Neuss et al., 2004; Ries et al., 2007).

When studying the migration behavior of SCs to a wide array of chemokines, SCs were found to migrate only toward SDF-1 (Wright et al., 2002; Jo et al., 2000). The migration to SDF-1 is a polarized phenomenon toward the chemokine source (chemotactic) and not a simple random motion (chemokinetic), as SCs only migrated in a gradient of SDF-1 and not when SDF-1 is uniformly distributed in the media.

SDF-1 is normally secreted, to some extent, by cardiomyocytes (Askari et al., 2003), skeletal muscles (Ratajczak et al., 2003), liver (Hatch et al., 2002; Kollet et al., 2003), brain (Bagri et al., 2002; Lazarini et al., 2003; Zou et al., 1998), and kidney (Schrader et al., 2002). However, its secretion increases during tissue damage such as AMI (Wojakowski et al., 2004; Abbott et al., 2004), ischemia (Takahashi et al., 1999, Iwaguro et al., 2002), toxic liver damage (Kollet et al., 2003; Swenson et al., 2008; Hatch et al., 2002), and excessive bleeding (Ratajczak et al., 2004).

In the tissue, the process of migration toward the site of injury relies on the interaction between CD44 and its ligand hyaluronic acid (HYA). HYA is one of a family of polysaccharides known as glycosaminoglycans (GAGS), typically found in the connective tissues of vertebrates. Alongside other proteins such as collagen, elastin, fibrillin, fibronectin and laminin, GAGS and HYA constitute the extracellular matrix (ECM) of most tissues. Studies in rats have shown that half of the HYA found in the body is in the skin, while 25% is found in joints and bones together (Reed et al., 1988). The rest is distributed somewhat equally in muscles and viscera (Clarris and Fraser,1968; Comper and Laurent, 1978) where the highest concentrations are found in connective tissue that form the ECM of most tissues.

After extravasation, CXCR4 on the surface of SC and SDF-1 secreted by the effected tissue continue their interaction which, in the tissue, leads to the formation of pseudopodia (see Figure 1) toward the source of SDF-1 secretion. As SDF-1 binds to CXCR4 in the tissue, CD44 adhesion molecules are externalized at the tip of the pseudopodia, leading to adhesion to HYA pathways within the tissue. In the tissue, the binding of CD44 to HYA is transient, as CD44 molecules shed soon after binding to HYA (Friedl et al., 1995), thus enabling pseudopodia detachment from the ECM. CD44 can also be cleaved by specific enzymes whose secretion is enhanced by SDF-1 (Heissig et al., 2002; Okamoto et al., 1999;

The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization 173

al., 2002) to 15% of cardiomyocytes were Y-chromosome positive (Quaini et al., 2002; Laflamme et al., 2002). In one patient who died of cardiac rejection, 29% of the cardiomyocytes contained the Y chromosome in ''hot spots'' of high cardiac repair. In male patients who received lung transplants from female donors, recipient-derived BMSCs cells were detected as bronchial epithelial cells, type II pneumocytes, and seromucous glands in the transplanted lungs of all tested patients. In patients suffering from chronic damage, up to 24% of bronchial epithelial cells carried the Y-chromosome, indicating ongoing repair of

In one study investigating this process of tissue repair by BMSC, irradiated mice were transplanted with GFP-positive SC before being injected in the right tibialis muscle with a large dose of cardiotoxin, which led to a loss of mobility within a few days. Yet, after eight weeks the injured muscle showed massive regeneration, with the right tibialis muscle significantly reconstructed with GFP-positive myocytes. By contrast, the contralateral noninjured leg showed very small incorporation of GFP-positive myocytes (Drapeau et al., 2010). Similar observations were made by Abedi et al. (2004), using smaller injections of cardiotoxin. Four weeks after the injection of cardiotoxin in the right leg, the area of the injection contained 1-2% of GFP-positive muscle cells while the left leg showed no

Similar observations were made in models of skin injury (Abedi et al., 2004). In a similar protocol, irradiated mice were transplanted with GFP-positive stem cells. BMSCs were then mobilized during five days using G-CSF. On the fourth day, mice were subjected to punch biopsies on their flank. The area of the injury was rebiopsied and sutured 48 hours and 1 month after the injury, in order to assess the incorporation of GFP-positive cells in the healing skin. While analysis at 48 hours showed significant infiltration by GFP-positive undifferentiated stem cells in the deep layer of the skin (hypodermis), after 4 weeks there was a large number of GFP-positive tissue cells in the dermis composing the structure of the healed skin, such as keratinocytes, sebaceous glands, blood vessels, and some rare muscle fibers and hair follicles. In the control animals that received G-CSF but no punch biopsy, none of these skin structures were positive for GFP, indicating that the few SC that had

In other studies looking at the incorporation of BMSCs in injured tissues, directed migration was demonstrated in the gut after section of an intestinal segment, (Hayakawa et al., 2003) in the heart after AMI (Orlic et al., 2001a; Fukuhara et al., 2004) or induced cardiomyopathy, (Hisashi et al., 2004) in the brain after stroke, (Sanchez-Ramos et al., 2002; Hoehn et al., 2002) and in the liver after drug-induced liver damage (Abedi et al., 2004). Taken altogether these studies clearly establish that BMSCs primarily migrate to areas subjected to injury, damage

When stem cells reach the site of an injury they must proliferate and expand, as there are not enough PBSC to accomplish full repair of any significant injury or degenerative process.

Several chemokines such as SDF-1 have been reported to enhance stem cell proliferation (Bonavia et al., 2003). The direct effect of SDF-1 on cell proliferation and survival is not well understood, but SDF-1 has been found to stimulate the proliferation and survival of stem

damaged tissue by the recipient's own stem cells (Kleeberger et al., 2003).

fluorescence at all.

or simple degeneration.

**2.3 Proliferation** 

migrated in the skin had done so randomly.

Janowska-Wieczorek et al., 1999). Through this process, stem cells can migrate within the tissue toward the site of injury.

The fact that PBSC primarily migrate to organs affected by an injury or a degenerative process has been documented in several studies. For example, in the cases of male recipients of liver transplants from female donors, biopsies performed 4-13 months after transplantation contained a significant number of Y-chromosome positive hepatocytes (16% to 43%). In one patient who suffered from hepatitis C after liver transplant and died 4.5 months after transplantation, up to 43% of the transplanted liver was made of Ychromosome positive hepatocytes in comparison with 5% after 4.5 months in a woman with a sound liver who received a bone marrow transplant from a man (Theise et al., 2000). Investigations carried out on archival autopsy and biopsy liver specimens obtained from two women who received a bone marrow transplant from male donors for the treatment of leukemia revealed that 5 to 10% of the liver had been replaced by donor derived BMSCs after 4.5 and 13 months, respectively (Theise et al., 2000).

Fig. 1. **CD44 is localized to the leading edge of polarized human stem cells migrating toward SDF-1.** Cord blood-derived CD34+ cells were plated on HA coverslips and allowed to adhere for 30 minutes before recording cell movement. The position of SDF-1 source is indicated by arrowheads. (A-C) Phase contrast microscopy of untreated cells (A), cells stimulated with polarized source of SDF-1 (B), and cells treated with anti-CD44 mAb F10-44- 2 and stimulated with polarized source of SDF-1 (C). (D-F) Cells treated as above were fixed 5 and 30 minutes after exposure to polarized source of SDF-1 and indirectly immunolabeled with antihuman CD44 mAb (red) and anti-CXCR4 mAb (green). An arrow is pointing to the fine CD44-positive protrusions at the direction of SDF-1. Bars = 5µm. (Taken from Avigdor et al., 2004)

Similar observations were made on men who received cardiac transplants from female donors. Analyses of tissue samples from biopsies revealed that an average of 0.1% (Muller et al., 2002) to 15% of cardiomyocytes were Y-chromosome positive (Quaini et al., 2002; Laflamme et al., 2002). In one patient who died of cardiac rejection, 29% of the cardiomyocytes contained the Y chromosome in ''hot spots'' of high cardiac repair. In male patients who received lung transplants from female donors, recipient-derived BMSCs cells were detected as bronchial epithelial cells, type II pneumocytes, and seromucous glands in the transplanted lungs of all tested patients. In patients suffering from chronic damage, up to 24% of bronchial epithelial cells carried the Y-chromosome, indicating ongoing repair of damaged tissue by the recipient's own stem cells (Kleeberger et al., 2003).

In one study investigating this process of tissue repair by BMSC, irradiated mice were transplanted with GFP-positive SC before being injected in the right tibialis muscle with a large dose of cardiotoxin, which led to a loss of mobility within a few days. Yet, after eight weeks the injured muscle showed massive regeneration, with the right tibialis muscle significantly reconstructed with GFP-positive myocytes. By contrast, the contralateral noninjured leg showed very small incorporation of GFP-positive myocytes (Drapeau et al., 2010). Similar observations were made by Abedi et al. (2004), using smaller injections of cardiotoxin. Four weeks after the injection of cardiotoxin in the right leg, the area of the injection contained 1-2% of GFP-positive muscle cells while the left leg showed no fluorescence at all.

Similar observations were made in models of skin injury (Abedi et al., 2004). In a similar protocol, irradiated mice were transplanted with GFP-positive stem cells. BMSCs were then mobilized during five days using G-CSF. On the fourth day, mice were subjected to punch biopsies on their flank. The area of the injury was rebiopsied and sutured 48 hours and 1 month after the injury, in order to assess the incorporation of GFP-positive cells in the healing skin. While analysis at 48 hours showed significant infiltration by GFP-positive undifferentiated stem cells in the deep layer of the skin (hypodermis), after 4 weeks there was a large number of GFP-positive tissue cells in the dermis composing the structure of the healed skin, such as keratinocytes, sebaceous glands, blood vessels, and some rare muscle fibers and hair follicles. In the control animals that received G-CSF but no punch biopsy, none of these skin structures were positive for GFP, indicating that the few SC that had migrated in the skin had done so randomly.

In other studies looking at the incorporation of BMSCs in injured tissues, directed migration was demonstrated in the gut after section of an intestinal segment, (Hayakawa et al., 2003) in the heart after AMI (Orlic et al., 2001a; Fukuhara et al., 2004) or induced cardiomyopathy, (Hisashi et al., 2004) in the brain after stroke, (Sanchez-Ramos et al., 2002; Hoehn et al., 2002) and in the liver after drug-induced liver damage (Abedi et al., 2004). Taken altogether these studies clearly establish that BMSCs primarily migrate to areas subjected to injury, damage or simple degeneration.

#### **2.3 Proliferation**

172 Tissue Regeneration – From Basic Biology to Clinical Application

Janowska-Wieczorek et al., 1999). Through this process, stem cells can migrate within the

The fact that PBSC primarily migrate to organs affected by an injury or a degenerative process has been documented in several studies. For example, in the cases of male recipients of liver transplants from female donors, biopsies performed 4-13 months after transplantation contained a significant number of Y-chromosome positive hepatocytes (16% to 43%). In one patient who suffered from hepatitis C after liver transplant and died 4.5 months after transplantation, up to 43% of the transplanted liver was made of Ychromosome positive hepatocytes in comparison with 5% after 4.5 months in a woman with a sound liver who received a bone marrow transplant from a man (Theise et al., 2000). Investigations carried out on archival autopsy and biopsy liver specimens obtained from two women who received a bone marrow transplant from male donors for the treatment of leukemia revealed that 5 to 10% of the liver had been replaced by donor derived BMSCs

Fig. 1. **CD44 is localized to the leading edge of polarized human stem cells migrating toward SDF-1.** Cord blood-derived CD34+ cells were plated on HA coverslips and allowed to adhere for 30 minutes before recording cell movement. The position of SDF-1 source is indicated by arrowheads. (A-C) Phase contrast microscopy of untreated cells (A), cells stimulated with polarized source of SDF-1 (B), and cells treated with anti-CD44 mAb F10-44- 2 and stimulated with polarized source of SDF-1 (C). (D-F) Cells treated as above were fixed 5 and 30 minutes after exposure to polarized source of SDF-1 and indirectly immunolabeled with antihuman CD44 mAb (red) and anti-CXCR4 mAb (green). An arrow is pointing to the

Similar observations were made on men who received cardiac transplants from female donors. Analyses of tissue samples from biopsies revealed that an average of 0.1% (Muller et

fine CD44-positive protrusions at the direction of SDF-1. Bars = 5µm.

(Taken from Avigdor et al., 2004)

tissue toward the site of injury.

after 4.5 and 13 months, respectively (Theise et al., 2000).

When stem cells reach the site of an injury they must proliferate and expand, as there are not enough PBSC to accomplish full repair of any significant injury or degenerative process.

Several chemokines such as SDF-1 have been reported to enhance stem cell proliferation (Bonavia et al., 2003). The direct effect of SDF-1 on cell proliferation and survival is not well understood, but SDF-1 has been found to stimulate the proliferation and survival of stem

The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization 175

This process was beautifully put in evidence by the study of Jang et al. (2004) where hematopoietic stem cells (HSC) were co-cultured with either normal or damaged liver tissue separated by a semi-permeable membrane with 0.4 µm pores. Using immunofluorescence to detect markers specific to either HSC (CD45) or hepatocytes (albumin), the authors could follow the transformation of the HSC population. When HSCs were cultured alone for 8 hours, they only expressed CD45 and no albumin. However, when HSCs were exposed to injured liver tissue, they rapidly became positive for albumin. Over time, the population of cells positive for CD45 began to decrease as the population positive for albumin began to increase. Albumin-positive cells were seen as early as 8 hours and constituted 3.0% of the cell population at 48 hours. The conversion was minimal and delayed when HSC were exposed to undamaged liver (control for injury). Therefore the presence of an injury appears to be an important factor in the process of SC differentiation into a specific type of somatic

The authors further investigated the phenomenon of differentiation by tracking the presence of various markers found in developing fetal liver cells, such as αFP, and in mature hepatocytes, such as CK18, albumin, fibrinogen, and transferrin. They found that αFP was expressed only at 8 hours and was lost thereafter. On the other hand, the expression of CK18, albumin, fibrinogen and transferrin each increased over time. While at time 0 the HSCs expressed only CD45, after as little as 8 hours all liver-specific markers were positive. So during the differentiation process, the SC seemed to take a route similar to the development of hepatocytes in the developing fetus, leading to mature hepatocytes within less than 48 hours. This retracing of fetal development has also being documented in

In this study, differentiation did not involve cellular fusion, as the new liver cells only contained one set of chromosomes. The differentiation was necessarily triggered by signaling molecules produced by the damaged tissue. It has been suggested that MMPs produced by damaged tissue could be playing an important role in SC differentiation by digesting specific ECM components that would then diffuse and get into contact with SCs. Binding of such compounds to specific receptors would activate internal messengers that would trigger the process of differentiation by activating specific genes, as suggested by the high level of mRNA found in differentiating cells. (Mannello et al., 2006; Chavey et al., 2003) Gap junction intercellular communication (GJIC) and tunneling nanotubules (TNTs) could constitute other mechanisms playing a significant role in SC differentiation, by direct cell-tocell contact (Behfar et al., 2010). The diameter of one gap junction is around 2 nm and the molecular size cut-off level is around 1-2 kDa, which is sufficient for the intercellular exchange of ions, nucleotides and even small proteins (Neijssen et al., 2005). GJIC is known to play a crucial role in modulating several cellular functions, to the point that impaired or lack of GJIC has been associated with severe diseases (Dasgupta et al., 1999). It has been suggested that GJIC could play an important role in SC differentiation (Loewenstein and Rose, 1992). Likewise, recent studies indicated that two cells can also exchange information via TNTs (Rustom et al., 2004). TNTs would form a cytoplasma bridge between two cells that could be large enough to allow the transport of large molecules or even whole cell organelles. Such information could play a role in the finalizing process of differentiation, though much work needs to be done in this field before we obtain a better understanding of

cells.

cardiopoiesis (Behfar et al., 2008).

the role of GJIC and TNTs in stem cell differentiation.

cells under certain experimental conditions (Broxmeyer et al., 2003). In tissues, SDF-1 appears to act as a "cellular survival factor" (Hwang et al., 2006). Insulin-like growth factor (IGF-1), when coupled with epidermal growth factor (EGF) or fibroblast growth factor-2 (FGF-2), was shown to support the proliferation and survival of neural (Arsenijevic et al., 2001) and muscle stem cells (Deasy et al., 2002). Extracellular nucleotides were also shown to support the proliferation of brain stem cell (Mishra et al., 2006).

#### **2.4 Differentiation**

The ability of adult stem cells to differentiate into various cell types has been well documented, though the mechanism behind such transformation is still not well understood.

As reported by Krause et al. (2001), 11 months after injection of male stem cells in female mice, Y-chromosome bearing cells were found in various tissues including the liver, muscle, skin, lung, and intestine. It has been well demonstrated that BMSC can differentiate into a wide variety of cell types including myocytes (Ferrari et al, 1998), hepatocytes (Lee et al., 2004), epithelial cells (Krause et al., 2001), neurons (Mezey et al., 2003; Sanchez-Rarnos et al., 2000; Woodbury et al., 2000), retinal cells (Tomita et al., 2002), endothelial cells and cardiomyocytes (Jackson et al., 2001; Orlic et al, 2001), gastrointestinal epithelium (Krause et al., 2001; Okamoto et al., 2002), pancreatic endocrine cells (Ianus et al., 2003), bone and cartilage (Pereira et al., 1995; 1998).

Although little work has been done in this field and many questions remain to be answered, two possible mechanisms have been proposed for SC differentiation.

One proposed mechanism is cellular fusion, which takes place when two cells fuse together to become one cell. A few studies have suggested that SC have the ability to fuse with somatic cells, rescuing the target cell (Tarada et al., 2002; Vassilopoulos et al., 2003; Spees et al., 2003). Although there is clear evidence that this phenomenon did take place in a few experiments and that it may take place naturally in certain tissue such as the heart (Nygren et al., 2004), it is unlikely to be a significant physiological phenomenon (Wurmser and Gage, 2002). For example, while the process of fusion involves the interaction of one single SC with one somatic cell, therefore a ratio of 1:1, the extent of SC-mediated tissue repair that has been documented in numerous studies, involving various tissues, far exceeds the actual number of SCs migrating in the tissue. Furthermore, the process of cellular fusion results in a cell that is tetraploid. Although this has been observed in a few in vitro studies, it has been a very rare observation in vivo. In fact, even in vitro, relatively harsh conditions had to be used in order to obtain cellular fusion. Finally, cellular fusion would imply the merging of two different cellular membranes, a process that in itself is rigged with challenges, as cells are designed not to fuse.

So, although cellular fusion could possibly naturally take place in the body, it is unlikely to contribute significantly to the process of repair that has been documented with ASCs. The other most likely mechanism is differentiation through contact with cellular components when the affected tissue is locally subjected to the action of various matrix metalloproteinases (MMPs) or differentiation induced by cytokines released by neighboring cells.

cells under certain experimental conditions (Broxmeyer et al., 2003). In tissues, SDF-1 appears to act as a "cellular survival factor" (Hwang et al., 2006). Insulin-like growth factor (IGF-1), when coupled with epidermal growth factor (EGF) or fibroblast growth factor-2 (FGF-2), was shown to support the proliferation and survival of neural (Arsenijevic et al., 2001) and muscle stem cells (Deasy et al., 2002). Extracellular nucleotides were also shown to

The ability of adult stem cells to differentiate into various cell types has been well documented, though the mechanism behind such transformation is still not well

As reported by Krause et al. (2001), 11 months after injection of male stem cells in female mice, Y-chromosome bearing cells were found in various tissues including the liver, muscle, skin, lung, and intestine. It has been well demonstrated that BMSC can differentiate into a wide variety of cell types including myocytes (Ferrari et al, 1998), hepatocytes (Lee et al., 2004), epithelial cells (Krause et al., 2001), neurons (Mezey et al., 2003; Sanchez-Rarnos et al., 2000; Woodbury et al., 2000), retinal cells (Tomita et al., 2002), endothelial cells and cardiomyocytes (Jackson et al., 2001; Orlic et al, 2001), gastrointestinal epithelium (Krause et al., 2001; Okamoto et al., 2002), pancreatic endocrine cells (Ianus et al., 2003), bone and

Although little work has been done in this field and many questions remain to be answered,

One proposed mechanism is cellular fusion, which takes place when two cells fuse together to become one cell. A few studies have suggested that SC have the ability to fuse with somatic cells, rescuing the target cell (Tarada et al., 2002; Vassilopoulos et al., 2003; Spees et al., 2003). Although there is clear evidence that this phenomenon did take place in a few experiments and that it may take place naturally in certain tissue such as the heart (Nygren et al., 2004), it is unlikely to be a significant physiological phenomenon (Wurmser and Gage, 2002). For example, while the process of fusion involves the interaction of one single SC with one somatic cell, therefore a ratio of 1:1, the extent of SC-mediated tissue repair that has been documented in numerous studies, involving various tissues, far exceeds the actual number of SCs migrating in the tissue. Furthermore, the process of cellular fusion results in a cell that is tetraploid. Although this has been observed in a few in vitro studies, it has been a very rare observation in vivo. In fact, even in vitro, relatively harsh conditions had to be used in order to obtain cellular fusion. Finally, cellular fusion would imply the merging of two different cellular membranes, a process that in itself is rigged with challenges, as cells

So, although cellular fusion could possibly naturally take place in the body, it is unlikely to contribute significantly to the process of repair that has been documented with ASCs. The other most likely mechanism is differentiation through contact with cellular components when the affected tissue is locally subjected to the action of various matrix metalloproteinases (MMPs) or differentiation induced by cytokines released by neighboring

two possible mechanisms have been proposed for SC differentiation.

support the proliferation of brain stem cell (Mishra et al., 2006).

**2.4 Differentiation** 

cartilage (Pereira et al., 1995; 1998).

are designed not to fuse.

cells.

understood.

This process was beautifully put in evidence by the study of Jang et al. (2004) where hematopoietic stem cells (HSC) were co-cultured with either normal or damaged liver tissue separated by a semi-permeable membrane with 0.4 µm pores. Using immunofluorescence to detect markers specific to either HSC (CD45) or hepatocytes (albumin), the authors could follow the transformation of the HSC population. When HSCs were cultured alone for 8 hours, they only expressed CD45 and no albumin. However, when HSCs were exposed to injured liver tissue, they rapidly became positive for albumin. Over time, the population of cells positive for CD45 began to decrease as the population positive for albumin began to increase. Albumin-positive cells were seen as early as 8 hours and constituted 3.0% of the cell population at 48 hours. The conversion was minimal and delayed when HSC were exposed to undamaged liver (control for injury). Therefore the presence of an injury appears to be an important factor in the process of SC differentiation into a specific type of somatic cells.

The authors further investigated the phenomenon of differentiation by tracking the presence of various markers found in developing fetal liver cells, such as αFP, and in mature hepatocytes, such as CK18, albumin, fibrinogen, and transferrin. They found that αFP was expressed only at 8 hours and was lost thereafter. On the other hand, the expression of CK18, albumin, fibrinogen and transferrin each increased over time. While at time 0 the HSCs expressed only CD45, after as little as 8 hours all liver-specific markers were positive. So during the differentiation process, the SC seemed to take a route similar to the development of hepatocytes in the developing fetus, leading to mature hepatocytes within less than 48 hours. This retracing of fetal development has also being documented in cardiopoiesis (Behfar et al., 2008).

In this study, differentiation did not involve cellular fusion, as the new liver cells only contained one set of chromosomes. The differentiation was necessarily triggered by signaling molecules produced by the damaged tissue. It has been suggested that MMPs produced by damaged tissue could be playing an important role in SC differentiation by digesting specific ECM components that would then diffuse and get into contact with SCs. Binding of such compounds to specific receptors would activate internal messengers that would trigger the process of differentiation by activating specific genes, as suggested by the high level of mRNA found in differentiating cells. (Mannello et al., 2006; Chavey et al., 2003)

Gap junction intercellular communication (GJIC) and tunneling nanotubules (TNTs) could constitute other mechanisms playing a significant role in SC differentiation, by direct cell-tocell contact (Behfar et al., 2010). The diameter of one gap junction is around 2 nm and the molecular size cut-off level is around 1-2 kDa, which is sufficient for the intercellular exchange of ions, nucleotides and even small proteins (Neijssen et al., 2005). GJIC is known to play a crucial role in modulating several cellular functions, to the point that impaired or lack of GJIC has been associated with severe diseases (Dasgupta et al., 1999). It has been suggested that GJIC could play an important role in SC differentiation (Loewenstein and Rose, 1992). Likewise, recent studies indicated that two cells can also exchange information via TNTs (Rustom et al., 2004). TNTs would form a cytoplasma bridge between two cells that could be large enough to allow the transport of large molecules or even whole cell organelles. Such information could play a role in the finalizing process of differentiation, though much work needs to be done in this field before we obtain a better understanding of the role of GJIC and TNTs in stem cell differentiation.

The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization 177

Similar data has been obtained by Vasa et al. (2001) who documented that a higher number of endothelial progenitor cells (EPC) was associated with greater cardiovascular health. In fact, hypoxia has been associated with the secretion of SDF-1 and VEGF by the ischemic tissue (Schioppa et al., 2003; Bachelder et al., 2002). Circulating EPCs therefore are attracted to the ischemic heart by the action of SDF-1 and the migrating EPCs contribute to the formation of new blood vessels upon the action of VEGF (Lee et al., 2007). Therefore, a higher number of EPCs or PBSC helps maintain optimal blood flow and a strong cardiac

The link between disease formation and the number of circulating stem cells is not limited to the heart. Similar observations have been made with muscular dystrophy where the rate of progression of the disease has been linked to the number of circulating stem cells and for which the number of PBSC is now considered one of the most important predictors of the disease progression (Marchesi et al., 2008). Likewise, the progression of pulmonary arterial hypertension (Diller et al., 2008; Junhui et al., 2008), arthritis (Herbrig et al., 2006; Grisar et al., 2005), atherosclerosis (Zhu et al., 2006 ), lupus erythematosus (Westerweel et al., 2007; Moonen et al., 2007), kidney failure (Choi et al., 2004; Herbrig et al., 2004; Eizawa et al., 2003), migraine (Lee et al., 2008), erectile dysfunction (Baumhakel et al., 2007) and other diseases have all been linked to a reduction in the number of PBSC. Recently, a direct relationship has been established between the number of PBSC and the development of diabetes, linking impaired fasting glucose, impaired glucose tolerance, and insulindependent diabetes mellitus to progressively lower levels of PBSCs (Fadini et al., 2010).

If the number of PBSC constitutes such a key parameter in the process of SC-mediated tissue repair, therefore increasing the number of PBSC could constitute a therapeutic approach. Following is the description of a number of clinical trials investigating the clinical potential

The heart has been traditionally seen as having little regenerative capabilities after birth, although many recent studies have challenged this view. Evidence clearly suggests that there is a low level of constant regeneration of cardiac cells (Soonpaa and Field, 1998; Bergmann et al., 2009; Quaini et al., 2002), and the number of dividing cells can increase by up to 10-fold in chronic heart disease or after AMI (Kajstura et al., 1998; Beltrami et al., 2001). Yet, this level of proliferation seems insufficient to rescue the cardiac muscle after AMI (Schwartz and Kornowski, 2003) and survivors of heart attacks are left with reduced

A number of studies in animals using G-CSF and SCF have shown that ESCM can lead to significant cardiac repair after AMI. Injection of SCF and G-CSF for 8 days after inducing AMI significantly increased the number of PBSCs, which led to the migration of PBSCs into the myocardium (Orlic et al., 2001b). Twenty-seven days after AMI, a band of newly formed cardiac tissue occupied more than 75% of the infarcted region of the ventricule and newly formed blood vessels were supplying the infarcted tissue. The blood vessels were surrounded by smooth muscles and microscopic observations revealed the presence of red blood cells, indicating that the newly formed arterioles integrated structurally with the remaining functional vasculature. By comparison, in control animals the ventricular wall

tissue.

of ESCM in various diseases or injuries.

quality of life and little prospect for improvement.

**3.1 Acute myocardial infarction** 

#### **2.5 Paracrine effect**

Finally, a number of studies investigating the effect of BMSC on various diagnostic entities have revealed that oftentimes the extent of the benefits observed cannot be accounted for by the sheer number of BMSC that have differentiated into somatic cells, even when there is clear evidence of tissue regeneration mediated by newly formed cells. For example, irradiated female mice transplanted with transgenic GFP+ BMSC showed much better recovery from experimental stroke after G-CSF-induced BMSC mobilization when compared to non-mobilized control mice (Kawada et al., 2006). Both motor and cognitive functions were improved by BMSC mobilization, and the treatment also reduced infarct size. However, using bromodeoxyuridine (BrdU), it was observed that a significant number of the newly formed brain cells did not derive from BMSC but rather from local neural stem cells. Similar results were seen with spinal cord lesion and muscle injury (Kinnaird et al., 2004; Osada et al., 2010). The regenerative effect is believed to be triggered by the secretion of growth factors and paracrine signaling by BMSC (Uccelli et al., 2011).

The paracrine effect has been best put in evidence in the heart after AMI. Delivery of BMSC to ischemic cardiac tissue has led to a significant increase in the concentration of IL-10, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and other cytokines in the cardiac tissue, which contributed to neovascularization and reduction of infarct size (Kamihata et al., 2001; Burchfield et al., 2008; Gnecchi et al., 2005; Mirotsou et al., 2011). Condition media in which BMSC were exposed to hypoxia proved to be cytoprotective to cardiomyocytes and was able to reduce infarct size (Gnecchi et al., 2006).

In all, a growing body of evidence supports the hypothesis that as BMSC migrate into tissues, aside from differentiating into cells of the target tissue, they also exert their regenerative effect at least in part -and maybe even to a large extent- through the secretion of paracrine signaling compounds.

#### **3. ESCM as a treatment approach**

Given the fact that SDF-1 is secreted by various organs and tissues upon injury or degeneration, it would follow that the number of PBSCs should be an important parameter in the overall effectiveness of SC-mediated tissue repair and regeneration. A higher number of PBSC would mean more SC available to respond to SDF-1 signaling and migrate into tissues. In this regard, Tomoda et al. (2003) reported that after a heart attack individuals with a higher number of PBSC showed greater recovery of cardiac functions after 6 months when compared to people having fewer PBSC at the time of the cardiac event.

In a prospective study using more than 500 individuals, Werner et al. (2005, 2007) put in evidence that the number of PBSC is a critical parameter in the role of SCs in tissue repair. The authors quantified the baseline number of PBSC in 519 individuals (average 66.6±10.8 years old) at risk for cardiovascular problems, and monitored their condition for one year. A first major cardiovascular event occurred in 214 patients. After adjustment for age, sex, vascular risk factors, and other relevant variables, increased levels of PBSC were associated with reduced risk of death from cardiovascular causes, lesser risk of a first major cardiovascular event, greater revascularization and lesser frequency of hospitalization.

Similar data has been obtained by Vasa et al. (2001) who documented that a higher number of endothelial progenitor cells (EPC) was associated with greater cardiovascular health. In fact, hypoxia has been associated with the secretion of SDF-1 and VEGF by the ischemic tissue (Schioppa et al., 2003; Bachelder et al., 2002). Circulating EPCs therefore are attracted to the ischemic heart by the action of SDF-1 and the migrating EPCs contribute to the formation of new blood vessels upon the action of VEGF (Lee et al., 2007). Therefore, a higher number of EPCs or PBSC helps maintain optimal blood flow and a strong cardiac tissue.

The link between disease formation and the number of circulating stem cells is not limited to the heart. Similar observations have been made with muscular dystrophy where the rate of progression of the disease has been linked to the number of circulating stem cells and for which the number of PBSC is now considered one of the most important predictors of the disease progression (Marchesi et al., 2008). Likewise, the progression of pulmonary arterial hypertension (Diller et al., 2008; Junhui et al., 2008), arthritis (Herbrig et al., 2006; Grisar et al., 2005), atherosclerosis (Zhu et al., 2006 ), lupus erythematosus (Westerweel et al., 2007; Moonen et al., 2007), kidney failure (Choi et al., 2004; Herbrig et al., 2004; Eizawa et al., 2003), migraine (Lee et al., 2008), erectile dysfunction (Baumhakel et al., 2007) and other diseases have all been linked to a reduction in the number of PBSC. Recently, a direct relationship has been established between the number of PBSC and the development of diabetes, linking impaired fasting glucose, impaired glucose tolerance, and insulindependent diabetes mellitus to progressively lower levels of PBSCs (Fadini et al., 2010).

If the number of PBSC constitutes such a key parameter in the process of SC-mediated tissue repair, therefore increasing the number of PBSC could constitute a therapeutic approach. Following is the description of a number of clinical trials investigating the clinical potential of ESCM in various diseases or injuries.

#### **3.1 Acute myocardial infarction**

176 Tissue Regeneration – From Basic Biology to Clinical Application

Finally, a number of studies investigating the effect of BMSC on various diagnostic entities have revealed that oftentimes the extent of the benefits observed cannot be accounted for by the sheer number of BMSC that have differentiated into somatic cells, even when there is clear evidence of tissue regeneration mediated by newly formed cells. For example, irradiated female mice transplanted with transgenic GFP+ BMSC showed much better recovery from experimental stroke after G-CSF-induced BMSC mobilization when compared to non-mobilized control mice (Kawada et al., 2006). Both motor and cognitive functions were improved by BMSC mobilization, and the treatment also reduced infarct size. However, using bromodeoxyuridine (BrdU), it was observed that a significant number of the newly formed brain cells did not derive from BMSC but rather from local neural stem cells. Similar results were seen with spinal cord lesion and muscle injury (Kinnaird et al., 2004; Osada et al., 2010). The regenerative effect is believed to be triggered by the secretion

The paracrine effect has been best put in evidence in the heart after AMI. Delivery of BMSC to ischemic cardiac tissue has led to a significant increase in the concentration of IL-10, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and other cytokines in the cardiac tissue, which contributed to neovascularization and reduction of infarct size (Kamihata et al., 2001; Burchfield et al., 2008; Gnecchi et al., 2005; Mirotsou et al., 2011). Condition media in which BMSC were exposed to hypoxia proved to be cytoprotective to cardiomyocytes and

In all, a growing body of evidence supports the hypothesis that as BMSC migrate into tissues, aside from differentiating into cells of the target tissue, they also exert their regenerative effect at least in part -and maybe even to a large extent- through the secretion

Given the fact that SDF-1 is secreted by various organs and tissues upon injury or degeneration, it would follow that the number of PBSCs should be an important parameter in the overall effectiveness of SC-mediated tissue repair and regeneration. A higher number of PBSC would mean more SC available to respond to SDF-1 signaling and migrate into tissues. In this regard, Tomoda et al. (2003) reported that after a heart attack individuals with a higher number of PBSC showed greater recovery of cardiac functions after 6 months

In a prospective study using more than 500 individuals, Werner et al. (2005, 2007) put in evidence that the number of PBSC is a critical parameter in the role of SCs in tissue repair. The authors quantified the baseline number of PBSC in 519 individuals (average 66.6±10.8 years old) at risk for cardiovascular problems, and monitored their condition for one year. A first major cardiovascular event occurred in 214 patients. After adjustment for age, sex, vascular risk factors, and other relevant variables, increased levels of PBSC were associated with reduced risk of death from cardiovascular causes, lesser risk of a first major cardiovascular event, greater revascularization and lesser frequency of hospitalization.

when compared to people having fewer PBSC at the time of the cardiac event.

of growth factors and paracrine signaling by BMSC (Uccelli et al., 2011).

was able to reduce infarct size (Gnecchi et al., 2006).

of paracrine signaling compounds.

**3. ESCM as a treatment approach** 

**2.5 Paracrine effect** 

The heart has been traditionally seen as having little regenerative capabilities after birth, although many recent studies have challenged this view. Evidence clearly suggests that there is a low level of constant regeneration of cardiac cells (Soonpaa and Field, 1998; Bergmann et al., 2009; Quaini et al., 2002), and the number of dividing cells can increase by up to 10-fold in chronic heart disease or after AMI (Kajstura et al., 1998; Beltrami et al., 2001). Yet, this level of proliferation seems insufficient to rescue the cardiac muscle after AMI (Schwartz and Kornowski, 2003) and survivors of heart attacks are left with reduced quality of life and little prospect for improvement.

A number of studies in animals using G-CSF and SCF have shown that ESCM can lead to significant cardiac repair after AMI. Injection of SCF and G-CSF for 8 days after inducing AMI significantly increased the number of PBSCs, which led to the migration of PBSCs into the myocardium (Orlic et al., 2001b). Twenty-seven days after AMI, a band of newly formed cardiac tissue occupied more than 75% of the infarcted region of the ventricule and newly formed blood vessels were supplying the infarcted tissue. The blood vessels were surrounded by smooth muscles and microscopic observations revealed the presence of red blood cells, indicating that the newly formed arterioles integrated structurally with the remaining functional vasculature. By comparison, in control animals the ventricular wall

The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization 179

Many studies have shown that extensive neuronal death in the brain after a stroke triggers the migration of neural stem cells to the site of injury, followed by their proliferation and differentiation into neurons and glial cells (Peterson, 2002; Fallon et al., 2000; Arvidsson et al., 2002; Nakatomi et al., 2002; Schmidt and Reymann 2002). However, this natural process does not appear to be sufficient to produce significant functional recovery (Yamamoto et al.,

As with the heart after AMI, stroke has been associated with BMSC mobilization. Studies have shown that the number of PBSC in stroke patients can increase up to 3-fold within 7 days after the stroke (Hennemann et al., 2008; Paczkowska et al., 2005). In one study, the magnitude of BMSC mobilization was correlated with the patients' functional recovery

When rats were injected with rat (Chen et al., 2001; Pavlichenko et al., 2008; Willing et al., 2003) or human SC (Li et al. 2002) after an induced stroke, significant motor and cognitive improvements were observed. Although a significant number of BM-derived cells could be identified as newly formed neurons and glial cells in the stroke foci, they accounted for only a small percentage of the total number of newly formed brain cells. Most of the newly formed brain cells are believed to be derived from neural SC upon the action of paracrines secreted by the migrating SC. Similar results were obtained using human umbilical cord stem cells (HUCSCs) where intravenous injection of HUCSCs 24 hours after a stroke greatly improved functional recovery (Chen et al., 2001). Injection of HUCSCs 7 days after the stroke still led to significant functional recovery, though the extent of the recovery was less

Mobilization of BMSCs induced by G-CSF was shown in a number of studies to improve the outcome of a stroke. For example, when tested 14 and 28 days after a stroke, animals treated with G-CSF showed much greater body coordination than control animals (Shyu et al., 2004). When the brains were analyzed using imaging, the infarcted area was much smaller in the treated group (61 mm3) when compared to the control group (176 mm3). All these benefits were greatly reduced when the animals were pre-treated with a blocker of CXCR4, indicating that the observed effects were dependent upon the migration of BMSCs into the

Similar results have been reported by other scientific teams (Six et al., 2003; Kawada et al., 2006). For example, after repopulating the bone marrow with GFP-positive stem cells, G-CSF-induced BMSCs mobilization immediately and roughly 2 weeks after inducing a stroke dramatically improved motor and cognitive performances four weeks after the stroke, as measured by using the Morris water maze (Kawada et al., 2006). In this study, while all the mice in the treated group reached the platform within 40 seconds, none of the control mice reached the submerged platform within the allotted 120 seconds. As in the study by Shyu et al. (2004), the infarct size was much smaller in the treated animals when compared to control. Using BrdU it was observed that the number of new brain cells found in the infarcted area was much higher in the treated group than in the control group. Yet very few of the new brain cells were GFP-positive, supporting the view that as they migrate in the brain BMSCs secrete growth factors that support the proliferation and differentiation of neural stem cells (Yoo et al., 2008). BMSCs also support neovascularization, which further

**3.2 Stroke** 

2001; Magavi and Macklis, 2002).

than with treatment at 24 hours.

brain.

(Dunac et al, 2007).

was filled with scar tissue covering the entire area of the infarct and no new blood vessels could be seen.

In summary, while only 17% (9 of 52) of the untreated animals survived AMI, showing severe signs of cardiomyopathy and compromised blood circulation, up to 73% (11 of 15) of the animals treated with G-CSF survived with significantly improved cardiac function and restored blood circulation. After 27 days, ejection fraction was 114% greater in the treated group and other parameters such as end-diastolic pressure, systolic pressure, and other parameters of cardiovascular function were all improved in treated versus non-treated mice.

Injection of G-CSF, however, can have significant negative effect in humans if done at large dose for more than 5-6 days (Bensinger et al., 1996; Shimoda et al., 1993). At lower dose and with shorter treatment duration, human trials have so far delivered mitigated results, though the approach remains promising. While some groups did report very promising results (Ince et al., 2005; Sesti et al., 2005), others reported no effect at all (Ellis et al., 2006; Zohlnhofer et al., 2007; Ripa and Kastrup, 2008) . A comprehensive review of the various studies however reveals that each study used slightly different protocols with regard to the time of treatment after AMI (from hours to 3 months), as well as the intensity and duration of the treatment, suggesting that ESCM could indeed hold great promise once the most effective treatment protocol has been developed (Abdel-Latif et al., 2008).

For example, Wojakowski et al. (2006) reported in 43 cardiac patients that if the patients were treated early after AMI (<12 hours) with G-CSF, the number of PBSCs following BMSC mobilization correlated with the extent of cardiac repair. In another study, G-CSF was injected within 5 days post-AMI in 41 patients at high risk for unfavorable left ventricular remodeling. Five months after G-CSF treatment, ejection fraction had improved 12.5% compared to no improvement in the control group (Leone et al., 2007). The improvements in cardiac function appeared to be linked to the prevention of left ventricular remodeling.

A meta-analysis reviewing the effectiveness of BMSC mobilization for the treatment of AMI included 7 studies and a total of 364 patients. The analysis concluded that treatment with G-CSF can improve LV ejection fraction if the treatment is administered early after the heart attack (Kang et al., 2007). However, in spite of the improvements in ejection fraction, other general parameters of cardiovascular health such as ventricular arrhythmia, rehospitalization for heart failure, and the composite of other cardiovascular events (i.e., death from heart attack, recurrent heart attack, and stroke), were not significantly different in the G-CSF treatment groups compared with the control groups. Similar results were reported by another meta-analysis that included eight studies and 385 patients (Abdel-Latif et al., 2008).

So, it remains unclear whether the simple mobilization of BMSCs can constitute an effective treatment for AMI. While some studies have yielded promising results, others suggest no benefits at all. However, positive results obtained in some studies should not be denied on the basis of the negative results obtained in others. Reconciliation of all this data and the development of an effective treatment protocol will most probably come through the determination of optimal treatment parameters: 1) intensity of ESCM, 2) duration of the treatment, 3) time after AMI, 4) number of treatments received over time, and 5) other yet unidentified parameters. Compounds other than G-CSF might also be discovered that could provide more consistent results (Broxmeyer et al., 2005; DeClercq, 2005).

#### **3.2 Stroke**

178 Tissue Regeneration – From Basic Biology to Clinical Application

was filled with scar tissue covering the entire area of the infarct and no new blood vessels

In summary, while only 17% (9 of 52) of the untreated animals survived AMI, showing severe signs of cardiomyopathy and compromised blood circulation, up to 73% (11 of 15) of the animals treated with G-CSF survived with significantly improved cardiac function and restored blood circulation. After 27 days, ejection fraction was 114% greater in the treated group and other parameters such as end-diastolic pressure, systolic pressure, and other parameters of cardiovascular function were all improved in treated versus non-treated mice. Injection of G-CSF, however, can have significant negative effect in humans if done at large dose for more than 5-6 days (Bensinger et al., 1996; Shimoda et al., 1993). At lower dose and with shorter treatment duration, human trials have so far delivered mitigated results, though the approach remains promising. While some groups did report very promising results (Ince et al., 2005; Sesti et al., 2005), others reported no effect at all (Ellis et al., 2006; Zohlnhofer et al., 2007; Ripa and Kastrup, 2008) . A comprehensive review of the various studies however reveals that each study used slightly different protocols with regard to the time of treatment after AMI (from hours to 3 months), as well as the intensity and duration of the treatment, suggesting that ESCM could indeed hold great promise once the most

For example, Wojakowski et al. (2006) reported in 43 cardiac patients that if the patients were treated early after AMI (<12 hours) with G-CSF, the number of PBSCs following BMSC mobilization correlated with the extent of cardiac repair. In another study, G-CSF was injected within 5 days post-AMI in 41 patients at high risk for unfavorable left ventricular remodeling. Five months after G-CSF treatment, ejection fraction had improved 12.5% compared to no improvement in the control group (Leone et al., 2007). The improvements in cardiac function appeared to be linked to the prevention of left ventricular remodeling.

A meta-analysis reviewing the effectiveness of BMSC mobilization for the treatment of AMI included 7 studies and a total of 364 patients. The analysis concluded that treatment with G-CSF can improve LV ejection fraction if the treatment is administered early after the heart attack (Kang et al., 2007). However, in spite of the improvements in ejection fraction, other general parameters of cardiovascular health such as ventricular arrhythmia, rehospitalization for heart failure, and the composite of other cardiovascular events (i.e., death from heart attack, recurrent heart attack, and stroke), were not significantly different in the G-CSF treatment groups compared with the control groups. Similar results were reported by another meta-analysis that included eight studies and 385 patients (Abdel-Latif

So, it remains unclear whether the simple mobilization of BMSCs can constitute an effective treatment for AMI. While some studies have yielded promising results, others suggest no benefits at all. However, positive results obtained in some studies should not be denied on the basis of the negative results obtained in others. Reconciliation of all this data and the development of an effective treatment protocol will most probably come through the determination of optimal treatment parameters: 1) intensity of ESCM, 2) duration of the treatment, 3) time after AMI, 4) number of treatments received over time, and 5) other yet unidentified parameters. Compounds other than G-CSF might also be discovered that could

provide more consistent results (Broxmeyer et al., 2005; DeClercq, 2005).

effective treatment protocol has been developed (Abdel-Latif et al., 2008).

could be seen.

et al., 2008).

Many studies have shown that extensive neuronal death in the brain after a stroke triggers the migration of neural stem cells to the site of injury, followed by their proliferation and differentiation into neurons and glial cells (Peterson, 2002; Fallon et al., 2000; Arvidsson et al., 2002; Nakatomi et al., 2002; Schmidt and Reymann 2002). However, this natural process does not appear to be sufficient to produce significant functional recovery (Yamamoto et al., 2001; Magavi and Macklis, 2002).

As with the heart after AMI, stroke has been associated with BMSC mobilization. Studies have shown that the number of PBSC in stroke patients can increase up to 3-fold within 7 days after the stroke (Hennemann et al., 2008; Paczkowska et al., 2005). In one study, the magnitude of BMSC mobilization was correlated with the patients' functional recovery (Dunac et al, 2007).

When rats were injected with rat (Chen et al., 2001; Pavlichenko et al., 2008; Willing et al., 2003) or human SC (Li et al. 2002) after an induced stroke, significant motor and cognitive improvements were observed. Although a significant number of BM-derived cells could be identified as newly formed neurons and glial cells in the stroke foci, they accounted for only a small percentage of the total number of newly formed brain cells. Most of the newly formed brain cells are believed to be derived from neural SC upon the action of paracrines secreted by the migrating SC. Similar results were obtained using human umbilical cord stem cells (HUCSCs) where intravenous injection of HUCSCs 24 hours after a stroke greatly improved functional recovery (Chen et al., 2001). Injection of HUCSCs 7 days after the stroke still led to significant functional recovery, though the extent of the recovery was less than with treatment at 24 hours.

Mobilization of BMSCs induced by G-CSF was shown in a number of studies to improve the outcome of a stroke. For example, when tested 14 and 28 days after a stroke, animals treated with G-CSF showed much greater body coordination than control animals (Shyu et al., 2004). When the brains were analyzed using imaging, the infarcted area was much smaller in the treated group (61 mm3) when compared to the control group (176 mm3). All these benefits were greatly reduced when the animals were pre-treated with a blocker of CXCR4, indicating that the observed effects were dependent upon the migration of BMSCs into the brain.

Similar results have been reported by other scientific teams (Six et al., 2003; Kawada et al., 2006). For example, after repopulating the bone marrow with GFP-positive stem cells, G-CSF-induced BMSCs mobilization immediately and roughly 2 weeks after inducing a stroke dramatically improved motor and cognitive performances four weeks after the stroke, as measured by using the Morris water maze (Kawada et al., 2006). In this study, while all the mice in the treated group reached the platform within 40 seconds, none of the control mice reached the submerged platform within the allotted 120 seconds. As in the study by Shyu et al. (2004), the infarct size was much smaller in the treated animals when compared to control. Using BrdU it was observed that the number of new brain cells found in the infarcted area was much higher in the treated group than in the control group. Yet very few of the new brain cells were GFP-positive, supporting the view that as they migrate in the brain BMSCs secrete growth factors that support the proliferation and differentiation of neural stem cells (Yoo et al., 2008). BMSCs also support neovascularization, which further

The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization 181

cells were also positive for insulin, for insulin RNA, and for Y-chromosome, demonstrating that they originated from the transplanted BMSCs. These cells showed functional characteristics typical of normal pancreatic ß-cells, such as fluctuations of intracellular calcium upon exposure to various concentrations of glucose. Within the time frame of that study (4–6 weeks), 1.7–3% of BM–derived GFP-positive cells were detected in the pancreatic islets. In a similar study, BMSCs were also shown to participate into the development of new blood vessels, further supporting the regeneration of the pancreatic tissue (Mathews et

Then, using a protocol similar to that used by Ianus et al., Hasegawa et al. (2007) further demonstrated that mobilization of BMSCs was not only effective but essential for pancreatic regeneration. Hasegawa et al. induced diabetes by injection of streptozotocin (STZ) in lethally irradiated female mice followed by infusion of BMSC from GFP transgenic mice. Infusion of BMSCs led to the incorporation of GFP-positive BMSCs into islets of Langerhans in the pancreatic tissue, partially restoring pancreatic islet number and size, and improving STZ-induced hyperglycemia. However, when the same experiment was done while simply infusing the pancreas with BMSCs, without preirradiation, no improvement was obtained. Furthermore, when the experiment was repeated with full BMSC transplant in a model of mice with impaired ability to mobilize stem cells, no benefits were obtained. Therefore, natural mobilization of BMSCs from the bone marrow appears essential for the regeneration

Fig. 3. **Pancreatic islets of STZ-treated mice receiving subsequent bone marrow transplant** 

antibodies. *Green* indicates insulin-positive and *red* glucagon-positive cells. Pancreases from normoglycemic control mouse (a), hyperglycemic control mouse (b), and STZ-treated mouse receiving BMT (c). BMT improved STZ-induced hyperglycemia. (Taken from Hasegawa et

In one recent study in humans, ESCM showed great promise in the treatment of diabetes. The study selected individuals recently diagnosed for diabetes and the treatment consisted of both stem cell mobilization and autologous stem cell transplant. The patients first received injections of G-CSF in order to harvest PBSC, followed later by autologous stem cell thansplant and, 5 days post-transplant, a second round of G-CSF treatment. The endpoints monitored in the study were overall morbidity along with temporal changes in exogenous insulin requirements. Before the treatment, all patient required daily insulin injection. By the end of the study, 14 of the 15 patients had experienced insulin-free episodes ranging

**(BMT)**. Double immunostaining of pancreases with anti-insulin and anti-glucagon

between 1 and 35 months (mean 16.2 months) (Voltarelli et al., 2007).

al., 2004; Gao et al., 2008).

al., 2007)

of pancreatic function after inducing diabetes with STZ.

contributes to the regeneration of the brain tissue (Lee et al., 2005; Hess et al., 2002; Kan et al., 2005).

Although much of this work needs to be reproduced in humans, ESCM for the treatment of stroke appears promising and would constitute a safe approach to the treatment of stroke.

#### **3.3 Diabetes**

The ability of BMSCs to leave the bone marrow, migrate to the pancreas and become insulinproducing cells was beautifully shown by Ianus et al. (2003). In brief, female mice were lethally irradiated and then transplanted with male BMSCs that express, using a CRE-LoxP system, GFP if the insulin gene is actively transcribed. When analyzed 4-6 weeks after the transplantation, GFP-positive cells were found in the pancreas (Figure 2). The GFP-positive

Fig. 2. **FISH and immunofluorescence marking of BM-derived insulin-producing cells.**  Immunofluorescence and FISH of isolated, dispersed pancreatic islet cells after transplantation of lethally irradiated female mice with male BMSCs that express, using a CRE-LoxP system, GFP if the insulin gene is actively transcribed. a) Bright-field phase, b) GFP imaging note slight autofluorescence of control isolated islet cells; c) Immunostaining with rhodamine X–labeled secondary antibody for insulin; d) FISH for Y chromosome (in yellow) and nucleus stain with DAPI (blue). Y chromosome is present only in GFP-positive cells. Scale bar, 5 µm; X630. (Taken from Ianus et al., 2003)

contributes to the regeneration of the brain tissue (Lee et al., 2005; Hess et al., 2002; Kan et

Although much of this work needs to be reproduced in humans, ESCM for the treatment of stroke appears promising and would constitute a safe approach to the treatment of

The ability of BMSCs to leave the bone marrow, migrate to the pancreas and become insulinproducing cells was beautifully shown by Ianus et al. (2003). In brief, female mice were lethally irradiated and then transplanted with male BMSCs that express, using a CRE-LoxP system, GFP if the insulin gene is actively transcribed. When analyzed 4-6 weeks after the transplantation, GFP-positive cells were found in the pancreas (Figure 2). The GFP-positive

Fig. 2. **FISH and immunofluorescence marking of BM-derived insulin-producing cells.** 

transplantation of lethally irradiated female mice with male BMSCs that express, using a CRE-LoxP system, GFP if the insulin gene is actively transcribed. a) Bright-field phase, b) GFP imaging note slight autofluorescence of control isolated islet cells; c) Immunostaining with rhodamine X–labeled secondary antibody for insulin; d) FISH for Y chromosome (in yellow) and nucleus stain with DAPI (blue). Y chromosome is present only in GFP-positive

Immunofluorescence and FISH of isolated, dispersed pancreatic islet cells after

cells. Scale bar, 5 µm; X630. (Taken from Ianus et al., 2003)

al., 2005).

stroke.

**3.3 Diabetes** 

cells were also positive for insulin, for insulin RNA, and for Y-chromosome, demonstrating that they originated from the transplanted BMSCs. These cells showed functional characteristics typical of normal pancreatic ß-cells, such as fluctuations of intracellular calcium upon exposure to various concentrations of glucose. Within the time frame of that study (4–6 weeks), 1.7–3% of BM–derived GFP-positive cells were detected in the pancreatic islets. In a similar study, BMSCs were also shown to participate into the development of new blood vessels, further supporting the regeneration of the pancreatic tissue (Mathews et al., 2004; Gao et al., 2008).

Then, using a protocol similar to that used by Ianus et al., Hasegawa et al. (2007) further demonstrated that mobilization of BMSCs was not only effective but essential for pancreatic regeneration. Hasegawa et al. induced diabetes by injection of streptozotocin (STZ) in lethally irradiated female mice followed by infusion of BMSC from GFP transgenic mice. Infusion of BMSCs led to the incorporation of GFP-positive BMSCs into islets of Langerhans in the pancreatic tissue, partially restoring pancreatic islet number and size, and improving STZ-induced hyperglycemia. However, when the same experiment was done while simply infusing the pancreas with BMSCs, without preirradiation, no improvement was obtained. Furthermore, when the experiment was repeated with full BMSC transplant in a model of mice with impaired ability to mobilize stem cells, no benefits were obtained. Therefore, natural mobilization of BMSCs from the bone marrow appears essential for the regeneration of pancreatic function after inducing diabetes with STZ.

Fig. 3. **Pancreatic islets of STZ-treated mice receiving subsequent bone marrow transplant (BMT)**. Double immunostaining of pancreases with anti-insulin and anti-glucagon antibodies. *Green* indicates insulin-positive and *red* glucagon-positive cells. Pancreases from normoglycemic control mouse (a), hyperglycemic control mouse (b), and STZ-treated mouse receiving BMT (c). BMT improved STZ-induced hyperglycemia. (Taken from Hasegawa et al., 2007)

In one recent study in humans, ESCM showed great promise in the treatment of diabetes. The study selected individuals recently diagnosed for diabetes and the treatment consisted of both stem cell mobilization and autologous stem cell transplant. The patients first received injections of G-CSF in order to harvest PBSC, followed later by autologous stem cell thansplant and, 5 days post-transplant, a second round of G-CSF treatment. The endpoints monitored in the study were overall morbidity along with temporal changes in exogenous insulin requirements. Before the treatment, all patient required daily insulin injection. By the end of the study, 14 of the 15 patients had experienced insulin-free episodes ranging between 1 and 35 months (mean 16.2 months) (Voltarelli et al., 2007).

The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization 183

Investigation of the clinical potential of ESCM has been limited, largely due to the significant risk associated with the use of G-CSF, the main stem cell mobilizer used in clinical trial, for extended periods of time (Bensinger et al., 1996; Shimoda et al., 1993). Recently, a new stem cell mobilizer (StemEnhance®; SE) has been developed that triggers a much milder increase in the number of PBSC, but its safety allows for a sustained oral daily consumption over long periods of time, allowing for safe daily ESCM (Jensen et al., 2007). In brief, SE is an extract from the cyanophyta *Aphanizomenon flos-aquae* that concentrates a protein with an estimated molecular weight of 160–180 kDa, which was shown to be a selective L-selectin blocker. Oral consumption of 1 gram of SE was been shown to trigger an average 25% increase in the number of PBSC within 60 minutes after consumption. The magnitude of the mobilization induced by SE is much smaller than that triggered by G-CSF, however its safety allows for continuous use and therefore offers a novel approach in the study of ESCM. To test its therapeutic potential, SE was used in a number of preliminary

MC is a 62 year old male with a 17 year history of Parkinson's Disease. MC was initially diagnosed with Parkinson's type resting pill-rolling tremor affecting his left hand and then progressing to the limbs on left side of his body. Over the course of five years the tremors increased gradually affecting the right side as well. At ten years into the disease process, MC was no longer able to practice as an attorney, mobility was affected and limitations

In 2009, before the treatment of SE, the left side tremors had significantly worsened, symptoms of stiffness and bradykinesia which introduced a shuffling gait and poor balance made it unsafe for HM to ambulate without the use of a cane. At the time MC began consuming SE he was unable to dress himself, put on his watch, write, feed himself or drive his car. After 45 days on the product, consuming 1 gram of SE, three times per day, MC showed significant improvement with decreased tremors, less stiffening and bradykinesia; at this time MC was able to get around without the use of his cane. After 60 days on the product, judging that the benefits had plateaued, MC discontinued the use of SE. Forty - five days later, signs of tremor and bradykinesia returned and MC was seeking medical attention, once again. MC resumed SE with a dose of 2 grams, three times per day and within six weeks showed much improvement with less tremors, stiffness and bradykinesia. MC was able to participate in activities of daily living, such as dressing and feeding himself, he was also able to tie his own tie, put on his watch, and once again, he could ambulate without using a cane. To date MC has been on SE for two years, he has returned to driving his car without limitations, he is independent with all activities of daily living and he also

Another patient, MT, is a 52 year old woman with an early onset of Parkinson at age 36, with tremor as the primary symptom. Early treatment consisted of Pergolid, then Budipin up to 30 mg three times a day. However due to QTc increase, Budipin was later reduced to 10 mg three times a day. The patient was also treated with Methydopamin 62.5 mg four times a day. With this treatment, MT's main problem was the experience of fluctuations and

**4. Clinical application (SE)** 

**4.1 Parkinson** 

clinical trials involving a number of diagnostic entities.

increased compromising his personal and professional abilities.

participates in some level of professional activity.

In this study the patients benefited from two instances of ESCM and one instance of autologous SC transplant. It is not possible to determine what were the respective contributions of the ESCM and SC injection, however it is likely that the mobilizations by themselves significantly contributed to the benefits experienced. While the first mobilization lasted several days and the second mobilization lasted about one week, there was only one instance of stem cell injection.

Diabetes is an interesting disease to study the link between disease progression and the number of circulating PBSCs, as it follows a series of relatively well defined stages with regard to carbohydrate metabolism status, namely normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and newly diagnosed diabetes mellitus (DM). Fadini et al. (2010) quantified the number of circulating CD34+ cells by flow cytometry in 425 individuals divided among these four stages of disease progression. The data showed a clear trend of decreased number of PBSCs with disease progression through IFG, IGT and DM (Figure 4). The number of circulating PBSCs was significantly lower in the IGT and DM groups when compared to the NGT group. The reduction in the number of PBSCs can either be a consequence of higher blood glucose levels that might affect the ability of stem cells to mobilize from the bone marrow or a causal factor in the development of DM whereby a reduced number of circulating PBSCs reduces the ability of the pancreas to renew itself over the years, or both. This supports the view previously suggested that diabetes could be a stem cell disease (Fadini et al., 2009).

Fig. 4. Variation of circulating CD34+ cells and diabetes. **V**ariation of circulating CD34+ cells in patients grouped according to carbohydrate metabolism, namely normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT) or diabetes (DM) duration, as appropriate. The mean value of patients with NGT was taken to represent the zero point. Bars indicate 95% CIs of means. \* Values significantly different when compared to NGT. (Taken from Fadini et al., 2009)

### **4. Clinical application (SE)**

182 Tissue Regeneration – From Basic Biology to Clinical Application

In this study the patients benefited from two instances of ESCM and one instance of autologous SC transplant. It is not possible to determine what were the respective contributions of the ESCM and SC injection, however it is likely that the mobilizations by themselves significantly contributed to the benefits experienced. While the first mobilization lasted several days and the second mobilization lasted about one week, there was only one

Diabetes is an interesting disease to study the link between disease progression and the number of circulating PBSCs, as it follows a series of relatively well defined stages with regard to carbohydrate metabolism status, namely normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and newly diagnosed diabetes mellitus (DM). Fadini et al. (2010) quantified the number of circulating CD34+ cells by flow cytometry in 425 individuals divided among these four stages of disease progression. The data showed a clear trend of decreased number of PBSCs with disease progression through IFG, IGT and DM (Figure 4). The number of circulating PBSCs was significantly lower in the IGT and DM groups when compared to the NGT group. The reduction in the number of PBSCs can either be a consequence of higher blood glucose levels that might affect the ability of stem cells to mobilize from the bone marrow or a causal factor in the development of DM whereby a reduced number of circulating PBSCs reduces the ability of the pancreas to renew itself over the years, or both. This supports the view

previously suggested that diabetes could be a stem cell disease (Fadini et al., 2009).

Fig. 4. Variation of circulating CD34+ cells and diabetes. **V**ariation of circulating CD34+ cells

in patients grouped according to carbohydrate metabolism, namely normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT) or diabetes (DM) duration, as appropriate. The mean value of patients with NGT was taken to represent the zero point. Bars indicate 95% CIs of means. \* Values significantly different

when compared to NGT. (Taken from Fadini et al., 2009)

instance of stem cell injection.

Investigation of the clinical potential of ESCM has been limited, largely due to the significant risk associated with the use of G-CSF, the main stem cell mobilizer used in clinical trial, for extended periods of time (Bensinger et al., 1996; Shimoda et al., 1993). Recently, a new stem cell mobilizer (StemEnhance®; SE) has been developed that triggers a much milder increase in the number of PBSC, but its safety allows for a sustained oral daily consumption over long periods of time, allowing for safe daily ESCM (Jensen et al., 2007).

In brief, SE is an extract from the cyanophyta *Aphanizomenon flos-aquae* that concentrates a protein with an estimated molecular weight of 160–180 kDa, which was shown to be a selective L-selectin blocker. Oral consumption of 1 gram of SE was been shown to trigger an average 25% increase in the number of PBSC within 60 minutes after consumption. The magnitude of the mobilization induced by SE is much smaller than that triggered by G-CSF, however its safety allows for continuous use and therefore offers a novel approach in the study of ESCM. To test its therapeutic potential, SE was used in a number of preliminary clinical trials involving a number of diagnostic entities.

#### **4.1 Parkinson**

MC is a 62 year old male with a 17 year history of Parkinson's Disease. MC was initially diagnosed with Parkinson's type resting pill-rolling tremor affecting his left hand and then progressing to the limbs on left side of his body. Over the course of five years the tremors increased gradually affecting the right side as well. At ten years into the disease process, MC was no longer able to practice as an attorney, mobility was affected and limitations increased compromising his personal and professional abilities.

In 2009, before the treatment of SE, the left side tremors had significantly worsened, symptoms of stiffness and bradykinesia which introduced a shuffling gait and poor balance made it unsafe for HM to ambulate without the use of a cane. At the time MC began consuming SE he was unable to dress himself, put on his watch, write, feed himself or drive his car. After 45 days on the product, consuming 1 gram of SE, three times per day, MC showed significant improvement with decreased tremors, less stiffening and bradykinesia; at this time MC was able to get around without the use of his cane. After 60 days on the product, judging that the benefits had plateaued, MC discontinued the use of SE. Forty - five days later, signs of tremor and bradykinesia returned and MC was seeking medical attention, once again. MC resumed SE with a dose of 2 grams, three times per day and within six weeks showed much improvement with less tremors, stiffness and bradykinesia. MC was able to participate in activities of daily living, such as dressing and feeding himself, he was also able to tie his own tie, put on his watch, and once again, he could ambulate without using a cane. To date MC has been on SE for two years, he has returned to driving his car without limitations, he is independent with all activities of daily living and he also participates in some level of professional activity.

Another patient, MT, is a 52 year old woman with an early onset of Parkinson at age 36, with tremor as the primary symptom. Early treatment consisted of Pergolid, then Budipin up to 30 mg three times a day. However due to QTc increase, Budipin was later reduced to 10 mg three times a day. The patient was also treated with Methydopamin 62.5 mg four times a day. With this treatment, MT's main problem was the experience of fluctuations and

The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization 185

Perindopril 4 mg daily, Elantan 20 mg twice per day, Simvostatin 20 mg daily, Adelat xl 30 mg daily, and Aspirin 1 per day. JP was then put on a list for possible heart transplant.

Three weeks after discharge JP began consuming SE, 1 gram three times per day. After 3 months of taking the product, he received a call for a possible heart transplant and returned to see his cardiologist for evaluation and comprehensive testing. The cardiologist reported that JP was making a remarkable recovery, the heart transplant surgery was postponed. Four months later JP was re-evaluated and found to have made a complete recovery. JP has since been returning at 6 month intervals for follow up evaluation, and to date he has been stable with no further coronary incidents. The evaluations and most recent ECG conclude that JP has normal heart function. Presently, JP shares that he is not on any medications, blood pressure is 126/65 mm Hg, he continues to take SE and experiences good quality of

NA is a 47 year old Colombian woman who was diagnosed at 18 year of age with deforming rheumatoid arthritis. Over the years she managed her condition with the use of Prednisone, Methotrexate, Sulfasalazina, and Diclofenac. Four years ago NA was diagnosed with diabetes mellitus with glycemia reaching 308 mg/dL and assumed treatment with Euglucon 5 mg twice a day along with NPH insulin. About one year ago, due to her arthritic condition she became wheelchair bound and required assistance for bathing and dressing. At that time her medical records show levels of C-Reactive Protein (CRP) of 96.2 mg/L, Erythrocytes sedimentation rate (ESR) of 81 mm/H, Platelet count of 535 K/ul, fasting glucose of 147 mg/dL and HgbA1c (or Glycosylated Hemoglobin) of 8.07. A few months after becoming wheelchair bound NA began consuming 1 gram of SE once a day. After one month she subjectively reported a reduction in pain and inflammation, while 3 months later her mobility had improved to the point that she could bathe and dress alone. After 6 months she began using a walker, then switched to a cane and one year later she was walking unaided. Her last medical records indicate a level of CRP of 2.1 mg/L, ESR of 34 mm/H, Platelet count of 485 K/ul. NA has also discontinued the use of any diabetes medication; her records indicate a fasting glucose of 106 mg/dL with a glycemia not exceeding 120 mg/dL and

At the time of writing this report, NA does not take any anti-inflammatory drug, undergoes a remarkable improvement in her quality of life, keeping daily consumption for 15 consecutive months of 1 gram of SE, bone support supplementation of 500 mg Calcitriol (1,25-dihydroxycholecalciferol) every month, milk of magnesia and an annual dose of

In September 2008, GE, a 78 year old male surgical oncologist who was otherwise in good health, had a stroke. The MRI/MRA revealed an acute infarct involving the right lentiform nucleus, moderately extensive chronic small vessel ischemic changes, chronic lacunar infarct involving the right ventromedial thalamus, and intracranial atherosclerotic vascular disease. The stroke left GE with aphasia and a reduced ability to perform any physical activity. October 2009, 13 months after the stroke, GE began taking SE; GE consumed 1 gram twice

life.

HgbA1c of 5.62.

Zoledronic Acid.

**4.5 Cerebrovascular accident (Stroke)** 

**4.4 Diabetes and rheumatoid arthritis** 

the beginning of ON-OFF syndrome. One year ago MT began consuming StemEnhance 1 gram three times a day. Today MT's experiences only minor tremor accompanied with some dyskinetic syndrome and sometimes propulsion. Her quality of life has increased and she is much more socially active.

#### **4.2 Traumatic spinal cord injury**

A preliminary trial with 8 individuals with spinal cord injuries was performed in a community center in Hawaii. The cases all involved paraplegia and various degrees of quadriplegia. Of the 8 cases, 4 dropped due to various circumstances unrelated to the consumption of SE. Of the 4 remaining participants, 2 had repetitive periods of hospitalization that made their consumption of SE irregular. Of the two remaining participants, one experienced mild though significant improvement in mobility while the other participant experienced significant improvements in mobility.

The latter participant, VS, had a serious car accident 17 years prior to SE consumption and was left with traumatic brain injury that affected her speech and a significant spinal cord lesion. At the beginning of the trial, VS was able to lift her right leg approximately 15 centimeter from her chair with no lateral movement, and showed a total absence of movement in her left leg. She could move her arms, hands and fingers though the movements were very slow with little dexterity and precision. She had some ability to move in her bed but was unable to turn herself without assistance. No data was available to document peripheral sensory perception of the lower limbs or nerve conductivity. After 6 months of daily consumption of 3 grams of SE three times a day, VS could lift her right leg more than 30 centimeters from her chair, with lateral movement outside of her chair. She could also lift her left leg off the chair and laterally outside of the chair. VS had less control over the movement of her left leg, though the magnitude of the movements was comparable to the movements seen with the right leg. After 10 months, VS was able to rotate herself in her bed unassisted and from a supine position she could lift both legs to a 90 degree angle and sit in her bed unassisted. Over the period of the trial her upper limbs also improved in dexterity and her speech showed mild though significant improvement. VS comes from a disadvantage socio-economic environment and did not have access to physical therapy beyond the first few years of her injury, she therefore developed leg and feet deformities that prevented her from bearing weight and possibly resuming physical therapy.

#### **4.3 Coronary Artery Disease**

JP is a 60 year old South African male who experienced a heart attack at 51 years of age. After diagnosis of Coronary Artery Disease was made a stent placement was performed. Dietary and lifestyle changes were implemented immediately by JP following the hospitalization. Unfortunately, 3 years later, JP suffered four additional heart attacks. During the hospitalization, the angiogram revealed the right and left coronary arteries were obstructed 100% and 40%, respectively, thus determining that JP was not a good candidate for bypass surgery. At the time of the last hospitalization, JP had decreased energy and experienced "stable" angina with any exertion, and his overall quality of life was greatly compromised. JP was put on a medication regimen which consisted of Atenolol 50mg daily, Perindopril 4 mg daily, Elantan 20 mg twice per day, Simvostatin 20 mg daily, Adelat xl 30 mg daily, and Aspirin 1 per day. JP was then put on a list for possible heart transplant.

Three weeks after discharge JP began consuming SE, 1 gram three times per day. After 3 months of taking the product, he received a call for a possible heart transplant and returned to see his cardiologist for evaluation and comprehensive testing. The cardiologist reported that JP was making a remarkable recovery, the heart transplant surgery was postponed. Four months later JP was re-evaluated and found to have made a complete recovery. JP has since been returning at 6 month intervals for follow up evaluation, and to date he has been stable with no further coronary incidents. The evaluations and most recent ECG conclude that JP has normal heart function. Presently, JP shares that he is not on any medications, blood pressure is 126/65 mm Hg, he continues to take SE and experiences good quality of life.

#### **4.4 Diabetes and rheumatoid arthritis**

184 Tissue Regeneration – From Basic Biology to Clinical Application

the beginning of ON-OFF syndrome. One year ago MT began consuming StemEnhance 1 gram three times a day. Today MT's experiences only minor tremor accompanied with some dyskinetic syndrome and sometimes propulsion. Her quality of life has increased and she is

A preliminary trial with 8 individuals with spinal cord injuries was performed in a community center in Hawaii. The cases all involved paraplegia and various degrees of quadriplegia. Of the 8 cases, 4 dropped due to various circumstances unrelated to the consumption of SE. Of the 4 remaining participants, 2 had repetitive periods of hospitalization that made their consumption of SE irregular. Of the two remaining participants, one experienced mild though significant improvement in mobility while the

The latter participant, VS, had a serious car accident 17 years prior to SE consumption and was left with traumatic brain injury that affected her speech and a significant spinal cord lesion. At the beginning of the trial, VS was able to lift her right leg approximately 15 centimeter from her chair with no lateral movement, and showed a total absence of movement in her left leg. She could move her arms, hands and fingers though the movements were very slow with little dexterity and precision. She had some ability to move in her bed but was unable to turn herself without assistance. No data was available to document peripheral sensory perception of the lower limbs or nerve conductivity. After 6 months of daily consumption of 3 grams of SE three times a day, VS could lift her right leg more than 30 centimeters from her chair, with lateral movement outside of her chair. She could also lift her left leg off the chair and laterally outside of the chair. VS had less control over the movement of her left leg, though the magnitude of the movements was comparable to the movements seen with the right leg. After 10 months, VS was able to rotate herself in her bed unassisted and from a supine position she could lift both legs to a 90 degree angle and sit in her bed unassisted. Over the period of the trial her upper limbs also improved in dexterity and her speech showed mild though significant improvement. VS comes from a disadvantage socio-economic environment and did not have access to physical therapy beyond the first few years of her injury, she therefore developed leg and feet deformities that prevented her from bearing weight and possibly resuming physical

JP is a 60 year old South African male who experienced a heart attack at 51 years of age. After diagnosis of Coronary Artery Disease was made a stent placement was performed. Dietary and lifestyle changes were implemented immediately by JP following the hospitalization. Unfortunately, 3 years later, JP suffered four additional heart attacks. During the hospitalization, the angiogram revealed the right and left coronary arteries were obstructed 100% and 40%, respectively, thus determining that JP was not a good candidate for bypass surgery. At the time of the last hospitalization, JP had decreased energy and experienced "stable" angina with any exertion, and his overall quality of life was greatly compromised. JP was put on a medication regimen which consisted of Atenolol 50mg daily,

other participant experienced significant improvements in mobility.

much more socially active.

therapy.

**4.3 Coronary Artery Disease** 

**4.2 Traumatic spinal cord injury** 

NA is a 47 year old Colombian woman who was diagnosed at 18 year of age with deforming rheumatoid arthritis. Over the years she managed her condition with the use of Prednisone, Methotrexate, Sulfasalazina, and Diclofenac. Four years ago NA was diagnosed with diabetes mellitus with glycemia reaching 308 mg/dL and assumed treatment with Euglucon 5 mg twice a day along with NPH insulin. About one year ago, due to her arthritic condition she became wheelchair bound and required assistance for bathing and dressing. At that time her medical records show levels of C-Reactive Protein (CRP) of 96.2 mg/L, Erythrocytes sedimentation rate (ESR) of 81 mm/H, Platelet count of 535 K/ul, fasting glucose of 147 mg/dL and HgbA1c (or Glycosylated Hemoglobin) of 8.07. A few months after becoming wheelchair bound NA began consuming 1 gram of SE once a day. After one month she subjectively reported a reduction in pain and inflammation, while 3 months later her mobility had improved to the point that she could bathe and dress alone. After 6 months she began using a walker, then switched to a cane and one year later she was walking unaided. Her last medical records indicate a level of CRP of 2.1 mg/L, ESR of 34 mm/H, Platelet count of 485 K/ul. NA has also discontinued the use of any diabetes medication; her records indicate a fasting glucose of 106 mg/dL with a glycemia not exceeding 120 mg/dL and HgbA1c of 5.62.

At the time of writing this report, NA does not take any anti-inflammatory drug, undergoes a remarkable improvement in her quality of life, keeping daily consumption for 15 consecutive months of 1 gram of SE, bone support supplementation of 500 mg Calcitriol (1,25-dihydroxycholecalciferol) every month, milk of magnesia and an annual dose of Zoledronic Acid.

#### **4.5 Cerebrovascular accident (Stroke)**

In September 2008, GE, a 78 year old male surgical oncologist who was otherwise in good health, had a stroke. The MRI/MRA revealed an acute infarct involving the right lentiform nucleus, moderately extensive chronic small vessel ischemic changes, chronic lacunar infarct involving the right ventromedial thalamus, and intracranial atherosclerotic vascular disease. The stroke left GE with aphasia and a reduced ability to perform any physical activity. October 2009, 13 months after the stroke, GE began taking SE; GE consumed 1 gram twice

The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization 187

proliferation and differentiation of tissue stem cells. Much work remains to be done to clearly elucidate the mechanisms of action behind the benefits of ECSM in various diagnostic entities, however from a clinical standpoint it remains that regardless of the mechanism of action, ESCM appears to be a valuable approach to increase the quality of life

Various stem cell mobilizers have been documented in the scientific literature and many of them have been associated with side effects that prevent the application in humans of what has been documented as effective in various animal models. Such mobilizers include G-CSF, Stem Cell Factor, interleukin-8 and plerixafor (Lemery et al., 2011), which have all been associated with side effects going from diarrhea, nausea, pain and numbness to pericarditis and thrombosis. In spite of the potential benefits, such side effects have largely prevented the use of such compounds for ESCM in humans, and the lack of safe stem cell mobilizers largely explains the limited interest so far in this therapeutic approach. The main challenge in further investigating the therapeutic potential of ESCM remains therefore the development of safe stem cell mobilizers. In the meanwhile, SE appears to be a valuable tool

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**6. References** 

per day and after 8 weeks on the product GE noticed improvement with his speech and experienced more energy with improved balance. In April 2010 a repeat MRI/MRA showed no evidence of an acute infarct. GE's aphasia was completely resolved at this point and his overall mobility was improved allowing him to perform all activities of daily living. In January of 2011 a repeat MRI/MRA of the anterior and posterior cerebral arteries demonstrated no evidence of hemodynamically significant stenosis, and revealed normal vertebrobasilar arteries with no evidence of intracranial aneurysm or vascular malformation.

To date, GE has resumed playing tennis at age 81, walks around the mall in the neighborhood with other senior citizens and has returned to his professional activity as a surgeon.

#### **4.6 Kidney failure**

The whole paragraph should read: "GW is 36 months old; he was born with a malfunctioning valve in the urethra, compromising the flow of urine at birth. At ten days old he had surgery to repair the urethra, however, the damage to the kidneys and the bladder were already evident, caused from the retention of urine at birth. Following the surgical procedure, scar tissue was observed to the wall of the bladder and one of the kidneys did not respond, eventually that kidney quit working and began to atrophy. A scar was also left on his hand caused from an IV being ripped out after surgery. Before SE was introduced, GW had stopped growing at 15 months of age and multiple health issues were leading him toward a kidney transplant. Furthermore, GW had been on antibiotics for one year due to multiple infections. His body was not eliminating fluid as it should, as evidenced by the worsening of edema resulting in the use of diuretics. GW had purple feet caused from compromised circulation, dark spots were observed on his back, his eyes were blood shot and his overall presentation was pale in appearance. GW's activity was nothing normal for a 1 ½ year old child; his energy level was very depleted and he was unable to play like a normal toddler.

GW was 21 months old when SE was introduced at 250 mg per day. After 3 days on SE his mother reported that his eyes were crystal clear and he was observed running through the house full of energy. The dark spots on his back began to fade after one week of SE consumption. Red color was seen on GW cheeks for the first time since he was born. After 75 days on SE, GW had grown 6cm taller and the shrinking kidney at birth had measured 0.8cm larger. GW is off all pharmaceutical medications to date and his bladder and kidney are back to normal functioning to their fullest capacity, the scar to his hand is completely gone.

#### **5. Conclusion**

The benefits of ESCM on various degenerative conditions have been documented in several animal models and in humans. In some cases, BMSC clearly migrate into tissues and directly contribute to the formation of new functional somatic cells of the target tissue. However in other cases, especially diseases affecting the heart and central nervous system, a primary mechanism of action appears to be the secretion of paracrines that stimulate the proliferation and differentiation of tissue stem cells. Much work remains to be done to clearly elucidate the mechanisms of action behind the benefits of ECSM in various diagnostic entities, however from a clinical standpoint it remains that regardless of the mechanism of action, ESCM appears to be a valuable approach to increase the quality of life of patients affected by various degenerative diseases.

Various stem cell mobilizers have been documented in the scientific literature and many of them have been associated with side effects that prevent the application in humans of what has been documented as effective in various animal models. Such mobilizers include G-CSF, Stem Cell Factor, interleukin-8 and plerixafor (Lemery et al., 2011), which have all been associated with side effects going from diarrhea, nausea, pain and numbness to pericarditis and thrombosis. In spite of the potential benefits, such side effects have largely prevented the use of such compounds for ESCM in humans, and the lack of safe stem cell mobilizers largely explains the limited interest so far in this therapeutic approach. The main challenge in further investigating the therapeutic potential of ESCM remains therefore the development of safe stem cell mobilizers. In the meanwhile, SE appears to be a valuable tool to study the clinical benefits of ESCM.

#### **6. References**

186 Tissue Regeneration – From Basic Biology to Clinical Application

per day and after 8 weeks on the product GE noticed improvement with his speech and experienced more energy with improved balance. In April 2010 a repeat MRI/MRA showed no evidence of an acute infarct. GE's aphasia was completely resolved at this point and his overall mobility was improved allowing him to perform all activities of daily living. In January of 2011 a repeat MRI/MRA of the anterior and posterior cerebral arteries demonstrated no evidence of hemodynamically significant stenosis, and revealed normal vertebrobasilar arteries with no evidence of intracranial aneurysm or vascular

To date, GE has resumed playing tennis at age 81, walks around the mall in the neighborhood with other senior citizens and has returned to his professional activity as a

The whole paragraph should read: "GW is 36 months old; he was born with a malfunctioning valve in the urethra, compromising the flow of urine at birth. At ten days old he had surgery to repair the urethra, however, the damage to the kidneys and the bladder were already evident, caused from the retention of urine at birth. Following the surgical procedure, scar tissue was observed to the wall of the bladder and one of the kidneys did not respond, eventually that kidney quit working and began to atrophy. A scar was also left on his hand caused from an IV being ripped out after surgery. Before SE was introduced, GW had stopped growing at 15 months of age and multiple health issues were leading him toward a kidney transplant. Furthermore, GW had been on antibiotics for one year due to multiple infections. His body was not eliminating fluid as it should, as evidenced by the worsening of edema resulting in the use of diuretics. GW had purple feet caused from compromised circulation, dark spots were observed on his back, his eyes were blood shot and his overall presentation was pale in appearance. GW's activity was nothing normal for a 1 ½ year old child; his energy level was very depleted and he was unable to

GW was 21 months old when SE was introduced at 250 mg per day. After 3 days on SE his mother reported that his eyes were crystal clear and he was observed running through the house full of energy. The dark spots on his back began to fade after one week of SE consumption. Red color was seen on GW cheeks for the first time since he was born. After 75 days on SE, GW had grown 6cm taller and the shrinking kidney at birth had measured 0.8cm larger. GW is off all pharmaceutical medications to date and his bladder and kidney are back to normal functioning to their fullest capacity, the scar to his hand is completely

The benefits of ESCM on various degenerative conditions have been documented in several animal models and in humans. In some cases, BMSC clearly migrate into tissues and directly contribute to the formation of new functional somatic cells of the target tissue. However in other cases, especially diseases affecting the heart and central nervous system, a primary mechanism of action appears to be the secretion of paracrines that stimulate the

malformation.

**4.6 Kidney failure** 

play like a normal toddler.

gone.

**5. Conclusion** 

surgeon.


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**9** 

*USA* 

**Spermatogonial Stem Cells:** 

*University of Illinois at Urbana-Champaign, Urbana, IL* 

*University of Florida, Gainesville, FL* 

**An Alternate Source of Pluripotent** 

**Stem Cells for Regenerative Medicine** 

Liz Simon1, Marie-Claude Hofmann2 and Paul S. Cooke3 *1Department of Biomedical Sciences,College of Veterinary Medicine, Nursing and Allied Health, Tuskegee University, Tuskegee, AL* 

*2Department ofComparative Biosciences, College of Veterinary Medicine,* 

*3Department of Physiological Sciences, College of Veterinary Medicine,* 

There is immense scientific and medical interest in stem cells as a potential source material for regenerative medicine to replace or restore lost, damaged, or aging cells, tissues or organs (Mason and Dunnill 2008). The ideal stem cell candidate for regenerative medicine is a pluripotent stem cell that is easily obtainable, has a stable developmental potential even after prolonged culture, forms derivatives of all three embryonic germ layers from the progeny of a single cell and generates teratomas after injection into immunosuppressed mice (Mason and Dunnill 2008). Stem cell-based therapy has the potential to offer important new treatment options for insulin-dependent diabetes, Parkinson's disease, cardiovascular, renal, musculoskeletal and retinal diseases and spinal cord diseases and trauma, among others. A critical question in this field is to establish which stem cells could be efficiently

In 1981, Martin and Evans achieved a milestone in stem cell biology with the derivation of mouse embryonic stem cells (ESCs; Evans and Kaufman 1981). The subsequent derivation of pluripotent human ESCs in 1998 by James Thomson (Thomson et al. 1998) and pluripotent stem cells from human primordial germ cells (Shamblott et al. 1998) ushered in a revolution in the field of regenerative medicine and tissue engineering. However, the destruction of human embryos to obtain ESCs and the need for therapeutic cloning to use them optimally made their clinical application highly controversial. In addition to ethical, legal and moral issues, ESCs have inherent limitations that must be overcome before their clinical use. One prime concern is the potential tumorigenicity of these cells in vivo. Although efforts to eliminate this possibility are underway, it remains a serious issue (Blum and Benvenisty 2008; Fujikawa et al. 2005; Strulovici et al. 2007; Wu, Boyd, and Wood 2007). Other concerns are immune rejection (Drukker et al. 2002; Wobus and Boheler 2005), genetic instability and incomplete epigenetic reprogramming (Wobus and Boheler 2005). These concerns have

**1. Introduction** 

used clinically.


### **Spermatogonial Stem Cells: An Alternate Source of Pluripotent Stem Cells for Regenerative Medicine**

Liz Simon1, Marie-Claude Hofmann2 and Paul S. Cooke3 *1Department of Biomedical Sciences,College of Veterinary Medicine, Nursing and Allied Health, Tuskegee University, Tuskegee, AL 2Department ofComparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 3Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL* 

*USA* 

#### **1. Introduction**

202 Tissue Regeneration – From Basic Biology to Clinical Application

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599

There is immense scientific and medical interest in stem cells as a potential source material for regenerative medicine to replace or restore lost, damaged, or aging cells, tissues or organs (Mason and Dunnill 2008). The ideal stem cell candidate for regenerative medicine is a pluripotent stem cell that is easily obtainable, has a stable developmental potential even after prolonged culture, forms derivatives of all three embryonic germ layers from the progeny of a single cell and generates teratomas after injection into immunosuppressed mice (Mason and Dunnill 2008). Stem cell-based therapy has the potential to offer important new treatment options for insulin-dependent diabetes, Parkinson's disease, cardiovascular, renal, musculoskeletal and retinal diseases and spinal cord diseases and trauma, among others. A critical question in this field is to establish which stem cells could be efficiently used clinically.

In 1981, Martin and Evans achieved a milestone in stem cell biology with the derivation of mouse embryonic stem cells (ESCs; Evans and Kaufman 1981). The subsequent derivation of pluripotent human ESCs in 1998 by James Thomson (Thomson et al. 1998) and pluripotent stem cells from human primordial germ cells (Shamblott et al. 1998) ushered in a revolution in the field of regenerative medicine and tissue engineering. However, the destruction of human embryos to obtain ESCs and the need for therapeutic cloning to use them optimally made their clinical application highly controversial. In addition to ethical, legal and moral issues, ESCs have inherent limitations that must be overcome before their clinical use. One prime concern is the potential tumorigenicity of these cells in vivo. Although efforts to eliminate this possibility are underway, it remains a serious issue (Blum and Benvenisty 2008; Fujikawa et al. 2005; Strulovici et al. 2007; Wu, Boyd, and Wood 2007). Other concerns are immune rejection (Drukker et al. 2002; Wobus and Boheler 2005), genetic instability and incomplete epigenetic reprogramming (Wobus and Boheler 2005). These concerns have

Spermatogonial Stem Cells:

may have significant clinical use.

**3. Spermatogonial Stem Cells** 

source of cells for regenerative therapy.

**3.1 Development and differentiation potential of SSCs** 

An Alternate Source of Pluripotent Stem Cells for Regenerative Medicine 205

(Mason and Manzotti 2010). The initial research and monetary investment to develop stemcell based therapy is high. However, since the treatment involves transplantation of cells or tissues that can function normally for extended periods of time or even potentially for the remainder of a patient's life, there is negligible need for ongoing treatment, rendering this

The limitations and problems associated with iPSCs suggest that they may not offer the optimal solution for all aspects of regenerative medicine, raising the question of whether other stem cells may be preferable in some or potentially all regenerative medicine applications. Other stem cells that have received widespread attention in this regard include hematopoietic (HSC), mesenchymal and fetal cord blood stem cells. An adult allogenic stem cell source with developmental potential similar to ESCs and iPSCs would potentially be the

Another stem cell that has been studied extensively for years and has recently been shown to have potential in regenerative medicine is the spermatogonial stem cell (SSC). This chapter focuses on recent developments in this field and the advantages and limitations of SSC use in regenerative medicine. We also briefly discuss other multipotent stem cells that

In mammalian testes, SSCs are found along the basement membrane of seminiferous tubules. They produce the spermatogenic lineage, ensuring lifelong fertility of the individual. Like other stem cells, SSCs are undifferentiated and capable of self-renewal. While in the testicular microenvironment, they differentiate only into one specialized cell lineage, spermatozoa. However, when SSCs are isolated from the testis and placed into a different environment, they acquire or manifest pluripotency and differentiate into tissues belonging to all three embryonic germ layers (Kanatsu-Shinohara et al. 2008; Golestaneh et al. 2009; Simon et al. 2009; Ning et al. 2010). This property makes SSCs a powerful potential

In the embryo, the germ line arises from primordial germ cells (PGCs). PGCs are initially identifiable as a small cluster of cells in the proximal epiblast near the extra-embryonic ectoderm. This lineage differentiates under the influence of bone morphogenetic proteins (BMPs) and diverges from the somatic lineage in the late embryonic and early fetal stage (Lawson et al. 1999; Ying et al. 2000). Due to their extra-embryonal origin, they are not classified as belonging to a particular embryonic germ layer. Therefore, they are not subjected to many of the differentiation signals other stem cells receive (Lawson and Hage 1994; Simon, Hess, and Cooke 2010); this may allow them to remain more undifferentiated than other stem cells. Following their initial development, PGCs subsequently migrate into the mesoderm, the endoderm (hindgut) and across the dorsal mesentery to reach the developing gonads at about 4-5 weeks of gestation in humans and 11-13 day post-coitum in rodents (Culty 2009). In the testis, PGCs then become mitotically quiescent until birth, and are called gonocytes. Shortly after birth, gonocytes resume mitosis and migrate to the basement membrane of seminiferous tubules, where they form SSCs and remain

modality of treatment potentially cost-effective (Mason and Dunnill 2008).

most promising clinical approach if an optimal starting cell was found.

sparked interest within the scientific community in finding alternate sources of pluripotent/multipotent cells that have similar can as ESCs, but potentially circumvent the problems associated with these cells.

The derivation of induced pluripotent stem cells (iPSCs) through the introduction of a small combination of transcription factors into terminally differentiated cells has raised the possibility of producing an alternate pluripotent stem cell source for use in regenerative medicine. Researchers at Kyoto University, Japan, were first to identify conditions that allowed mouse skin fibroblasts to be induced into pluripotency (Takahashi and Yamanaka 2006) and a year later human skin fibroblasts were also induced to pluripotency using this same approach (Takahashi, Okita et al. 2007; Takahashi, Tanabe et al. 2007; Yu et al. 2007). The iPSC technology has immense potential for clinical therapy as these cells avoid some ethical and moral issues associated with ESCs. The technology involves producing critical levels of proteins needed for pluripotency through introducing the genes into the target cells exogenously using viral vectors (Takahashi, Tanabe et al. 2007; Takahashi and Yamanaka 2006; Yu et al. 2007), adenoviral vectors (Stadtfeld et al. 2008; Okita et al. 2008) or non-viral plasmids (Okita et al. 2008) or introducing the actual recombinant proteins themselves into cells (Zhou et al. 2009). Although initially there was concern over use of viral vectors integrating into the human genome and potentially inducing neoplastic changes, the use of alternate methods to deliver the pluripotency genes appears to circumvent that problem. Nonetheless, because there is genetic manipulation involved (Stadtfeld et al. 2010; Stadtfeld and Hochedlinger 2010) and an increased risk of tumorigenicity (Nakagawa et al. 2008; Ben-David and Benvenisty 2011; Kooreman and Wu 2010) extensive research has to be conducted before iPSCs are used clinically. Since iPSCs can be derived from patientspecific cells, one potential benefit of these cells was a low or no risk of immune rejection. However, a recent finding suggests that this might not be true: In contrast to derivatives of ESCs, abnormal gene expression in iPSCs introduced into syngeneic mice in vivo induced T-cell-dependent immune responses, leading to immune infiltration and ultimately rejection of these cells (Zhao et al. 2011). This raises concerns for use of these cells in regenerative medicine. As with any new technology, there are a number of other hurdles to overcome before using iPSCs clinically, such as refractoriness of many adult cells to reprogramming, transient epigenetic memory of donor cells (Kim et al. 2010; Polo et al. 2010) and possible non-recurrent mutations. Despite these concerns, the tremendous progress and obvious potential of iPSCs over the past few years has overshadowed work on other potentially pluripotent stem cells, although some of these cells may ultimately offer therapeutic potential equal to that of iPSCs.

#### **2. Progress in regenerative medicine**

In 2010, two ESC-based human clinical trials were approved by the U. S. Food and Drug Administration, one initiated by Geron Corporation and the other by Advanced Cell Technology. Though the clinical trials are underway, there is concern that introducing stem cells that have not transformed into specialized cells into patients may pose the risk of teratoma formation. However, remarkable progress has been made in cell-based regenerative medicine over the past decade. The cell-therapy based industry now has an annual revenue of over a billion dollars and it is projected to rise to \$5.1 billion by 2014 (Mason and Manzotti 2010). The initial research and monetary investment to develop stemcell based therapy is high. However, since the treatment involves transplantation of cells or tissues that can function normally for extended periods of time or even potentially for the remainder of a patient's life, there is negligible need for ongoing treatment, rendering this modality of treatment potentially cost-effective (Mason and Dunnill 2008).

The limitations and problems associated with iPSCs suggest that they may not offer the optimal solution for all aspects of regenerative medicine, raising the question of whether other stem cells may be preferable in some or potentially all regenerative medicine applications. Other stem cells that have received widespread attention in this regard include hematopoietic (HSC), mesenchymal and fetal cord blood stem cells. An adult allogenic stem cell source with developmental potential similar to ESCs and iPSCs would potentially be the most promising clinical approach if an optimal starting cell was found.

Another stem cell that has been studied extensively for years and has recently been shown to have potential in regenerative medicine is the spermatogonial stem cell (SSC). This chapter focuses on recent developments in this field and the advantages and limitations of SSC use in regenerative medicine. We also briefly discuss other multipotent stem cells that may have significant clinical use.

### **3. Spermatogonial Stem Cells**

204 Tissue Regeneration – From Basic Biology to Clinical Application

sparked interest within the scientific community in finding alternate sources of pluripotent/multipotent cells that have similar can as ESCs, but potentially circumvent the

The derivation of induced pluripotent stem cells (iPSCs) through the introduction of a small combination of transcription factors into terminally differentiated cells has raised the possibility of producing an alternate pluripotent stem cell source for use in regenerative medicine. Researchers at Kyoto University, Japan, were first to identify conditions that allowed mouse skin fibroblasts to be induced into pluripotency (Takahashi and Yamanaka 2006) and a year later human skin fibroblasts were also induced to pluripotency using this same approach (Takahashi, Okita et al. 2007; Takahashi, Tanabe et al. 2007; Yu et al. 2007). The iPSC technology has immense potential for clinical therapy as these cells avoid some ethical and moral issues associated with ESCs. The technology involves producing critical levels of proteins needed for pluripotency through introducing the genes into the target cells exogenously using viral vectors (Takahashi, Tanabe et al. 2007; Takahashi and Yamanaka 2006; Yu et al. 2007), adenoviral vectors (Stadtfeld et al. 2008; Okita et al. 2008) or non-viral plasmids (Okita et al. 2008) or introducing the actual recombinant proteins themselves into cells (Zhou et al. 2009). Although initially there was concern over use of viral vectors integrating into the human genome and potentially inducing neoplastic changes, the use of alternate methods to deliver the pluripotency genes appears to circumvent that problem. Nonetheless, because there is genetic manipulation involved (Stadtfeld et al. 2010; Stadtfeld and Hochedlinger 2010) and an increased risk of tumorigenicity (Nakagawa et al. 2008; Ben-David and Benvenisty 2011; Kooreman and Wu 2010) extensive research has to be conducted before iPSCs are used clinically. Since iPSCs can be derived from patientspecific cells, one potential benefit of these cells was a low or no risk of immune rejection. However, a recent finding suggests that this might not be true: In contrast to derivatives of ESCs, abnormal gene expression in iPSCs introduced into syngeneic mice in vivo induced T-cell-dependent immune responses, leading to immune infiltration and ultimately rejection of these cells (Zhao et al. 2011). This raises concerns for use of these cells in regenerative medicine. As with any new technology, there are a number of other hurdles to overcome before using iPSCs clinically, such as refractoriness of many adult cells to reprogramming, transient epigenetic memory of donor cells (Kim et al. 2010; Polo et al. 2010) and possible non-recurrent mutations. Despite these concerns, the tremendous progress and obvious potential of iPSCs over the past few years has overshadowed work on other potentially pluripotent stem cells, although some of these cells may ultimately

In 2010, two ESC-based human clinical trials were approved by the U. S. Food and Drug Administration, one initiated by Geron Corporation and the other by Advanced Cell Technology. Though the clinical trials are underway, there is concern that introducing stem cells that have not transformed into specialized cells into patients may pose the risk of teratoma formation. However, remarkable progress has been made in cell-based regenerative medicine over the past decade. The cell-therapy based industry now has an annual revenue of over a billion dollars and it is projected to rise to \$5.1 billion by 2014

problems associated with these cells.

offer therapeutic potential equal to that of iPSCs.

**2. Progress in regenerative medicine** 

In mammalian testes, SSCs are found along the basement membrane of seminiferous tubules. They produce the spermatogenic lineage, ensuring lifelong fertility of the individual. Like other stem cells, SSCs are undifferentiated and capable of self-renewal. While in the testicular microenvironment, they differentiate only into one specialized cell lineage, spermatozoa. However, when SSCs are isolated from the testis and placed into a different environment, they acquire or manifest pluripotency and differentiate into tissues belonging to all three embryonic germ layers (Kanatsu-Shinohara et al. 2008; Golestaneh et al. 2009; Simon et al. 2009; Ning et al. 2010). This property makes SSCs a powerful potential source of cells for regenerative therapy.

#### **3.1 Development and differentiation potential of SSCs**

In the embryo, the germ line arises from primordial germ cells (PGCs). PGCs are initially identifiable as a small cluster of cells in the proximal epiblast near the extra-embryonic ectoderm. This lineage differentiates under the influence of bone morphogenetic proteins (BMPs) and diverges from the somatic lineage in the late embryonic and early fetal stage (Lawson et al. 1999; Ying et al. 2000). Due to their extra-embryonal origin, they are not classified as belonging to a particular embryonic germ layer. Therefore, they are not subjected to many of the differentiation signals other stem cells receive (Lawson and Hage 1994; Simon, Hess, and Cooke 2010); this may allow them to remain more undifferentiated than other stem cells. Following their initial development, PGCs subsequently migrate into the mesoderm, the endoderm (hindgut) and across the dorsal mesentery to reach the developing gonads at about 4-5 weeks of gestation in humans and 11-13 day post-coitum in rodents (Culty 2009). In the testis, PGCs then become mitotically quiescent until birth, and are called gonocytes. Shortly after birth, gonocytes resume mitosis and migrate to the basement membrane of seminiferous tubules, where they form SSCs and remain

Spermatogonial Stem Cells:

introduction in iPSCs.

**3.3 Spermatogonial stem cells are pluripotent** 

hypomethylation of pluripotency marker genes (Zechner et al. 2009)

**4. Direct differentiation of spermatogonial stem cells** 

**4.1 Epithelial-mesenchymal interactions** 

Simon, Hess, and Cooke 2010).

In recent years, several research groups reported methodologies for isolation and culture of human SSCs and also demonstrated that these cells were pluripotent/multipotent (Conrad et al. 2008; Golestaneh et al. 2009; Kossack et al. 2009; Dym et al. 2009; Izadyar et al. 2011). SSCs isolated from human testicular tissues and cultured for a week or more spontaneously produced ESC-like small colonies, which were then transferred into ESC media and cultured for about 4 weeks to get sufficient numbers of ESC-like colonies and these ESC-like cells could then be differentiated into specific cell types. These results indicate that human SSCs have the potential to be used as an alternate source of pluripotent stem cells in regenerative cell therapy, without the ethical concerns of ESC and the concerns involving exogenous gene

Epithelial-mesenchymal interactions are critical for organogenesis in many organs such as lung, prostate, mammary gland, liver, pancreas and salivary glands (Grobstein 1953). Mesenchyme, which is undifferentiated connective tissue, signals to the epithelium to induce epithelial morphogenesis and cytodifferentiation in a wide variety of organs. The central role of epithelial-mesenchymal interactions in organ development was postulated initially by Pander and later experimentally demonstrated by Spemann and Saunders (in

An Alternate Source of Pluripotent Stem Cells for Regenerative Medicine 207

In the testicular microenvironment, SSCs produce only the spermatogenic lineage, and the assumption was that this was their sole potential developmental fate. However, the iconoclastic finding of Kanatsu-Shinohara et al. (Kanatsu-Shinohara et al. 2004) demonstrated that neonatal murine SSCs produced ESC-like cells when isolated from the testis and grown for extended periods in ESC culture conditions. Although initial work was done with neonatal SSCs (Kanatsu-Shinohara et al. 2004; Kanatsu-Shinohara et al. 2008; Simon et al. 2009), subsequent work showed that adult murine SSCs (Glaser et al. 2008; Izadyar et al. 2008; Seandel et al. 2007; Guan et al. 2006) grown for 4-7 weeks in vitro also produced a low frequency of ESC-like colonies. These ESC-like cells can give rise to cell types derived from all three embryonic germ layers, and also produce teratomas when injected subcutaneously into nude mice. In addition, these ESC-like cells contribute to embryonic development when injected into blastocysts. ESC-like cells derived from SSCs have been termed multipotent germline stem cells (mGSCs) and these cells have been differentiated into many cell types. For example, two separate groups demonstrated that mGSCs could differentiate into mature cardiac and endothelial cells and that these cardiac cells were contractile and had electric potentials and ion channels (Baba et al. 2007; Guan et al. 2007). mGSCs derived from adult mouse SSCs could be differentiated into functional neurons and glia (Glaser et al. 2008; Streckfuss-Bomeke et al. 2009). Pluripotent stem cells were derived from adult mGSCs that not only could differentiate into a variety of cell types both in vivo and in vitro, but also showed germline transmission to the next generation when injected into blastocysts (Ko et al. 2009). Moreover, mGSCs have pluripotency characteristics similar to ESCs such as telomerase activity, telomere length and

throughout life. Thus, the embryological origin of SSCs is unique. This may facilitate their potential for differentiation into cell types of different germ cell layers and underlines their clinical potential for regenerative medicine. Furthermore, teratomas occur exclusively in gonads (Stevens 1964) and are of germ cell origin, and gene expression in early germ cells is very similar to ESCs (Zwaka and Thomson 2005; Simon, Hess, and Cooke 2010) , which emphasizes the broad developmental potential of SSCs. SSCs express genes such as POU domain class 5, transcription factor 1 (Pou5f1; Huang et al. 2009; Bhartiya et al.), Lin 28 (Zheng et al. 2009), undifferentiated embryonic cell transcription factor 1 (UTF-1) and Zinc finger protein 42 (Rex-1; Kristensen et al. 2008), which impart pluripotency. However, expression of another pluripotency gene, Nanog, is repressed in the testis by transformation related protein 53 (TRP53) and phosphatase and tensin homolog (PTEN). Both proteins belong to a critical signaling pathway preventing SSCs from being pluripotent while in the testis (Kuijk et al. 2009). Overall, this gene expression pattern suggests that SSCs have a gene profile similar to ESCs and thus are more undifferentiated than other adult stem cells.

#### **3.2 Current methods of isolation and propagation of SSCs with emphasis on human SSC isolation**

Spermatogonial stem cell constitute only 0.03% of the total germ cell population in rodent and human testis (Tegelenbosch and de Rooij 1993). Thus, their small numbers and the lack of specific markers are the main hurdles to their characterization. Nonetheless, significant progress has recently been made in the isolation and propagation of cells with SSCs properties from rodent (Dym et al. 2009; Guan et al. 2009; Kanatsu-Shinohara, Takehashi, and Shinohara 2008; Kubota, Avarbock, and Brinster 2004; Oatley and Brinster 2006, 2008; Ogawa et al. 2003; Hofmann et al. 2005; Kanatsu-Shinohara et al. 2010) and human testes (Conrad et al. 2008; Glaser et al. 2008; Golestaneh et al. 2009; Kossack et al. 2009; Zovoilis et al. 2008; Izadyar et al. 2011; He et al. 2009). Human testicular tissue is currently obtained from testicular biopsies (Izadyar et al. 2011; Kossack et al. 2009), orchiectomies (Izadyar et al. 2011) and organ donors (Golestaneh et al. 2009). Testicular biopsies of approximately one gram can yield sufficient number of human SSCs for most clinical applications. Although our knowledge of mouse and human SSCs phenotype is still limited, studies suggest that human SSCs express proteins such as cluster of differentiation antigens 49f, 90 and 133 (CD49f, CD90, and CD133, respectively), glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRA1), G protein-coupled receptor 125 (GPR125), melanoma antigen family A 4 (MAGE4), promyelocytic leukemia zinc finger (PLZF) and stage-specific embryonic antigen-4 (SSEA-4; Costoya et al. 2004; Conrad et al. 2008; He et al. 2009; Izadyar et al. 2011). Using these markers in magnetic- or fluorescent-activated cell sorting, SSCs can be isolated with a high degree of purity (Gassei et al. 2009; Izadyar et al. 2011; Kokkinaki et al. 2009; Simon et al. 2010). Although there have been no definitive culture conditions for propagation of either mouse or human SSCs, culture systems established by different groups seem to be conducive for their propagation. At least in rodents, glial cell line-derived neurotrophic factor (GDNF) was found to be essential to maintain SSCs in an undifferentiated state in vivo and in vitro (Tyagi et al. 2009; Hofmann 2008; Sariola and Immonen 2008; Oatley, Avarbock, and Brinster 2007; Oatley et al. 2006; Naughton et al. 2006; Kubota, Avarbock, and Brinster 2004; Meng et al. 2000).

#### **3.3 Spermatogonial stem cells are pluripotent**

206 Tissue Regeneration – From Basic Biology to Clinical Application

throughout life. Thus, the embryological origin of SSCs is unique. This may facilitate their potential for differentiation into cell types of different germ cell layers and underlines their clinical potential for regenerative medicine. Furthermore, teratomas occur exclusively in gonads (Stevens 1964) and are of germ cell origin, and gene expression in early germ cells is very similar to ESCs (Zwaka and Thomson 2005; Simon, Hess, and Cooke 2010) , which emphasizes the broad developmental potential of SSCs. SSCs express genes such as POU domain class 5, transcription factor 1 (Pou5f1; Huang et al. 2009; Bhartiya et al.), Lin 28 (Zheng et al. 2009), undifferentiated embryonic cell transcription factor 1 (UTF-1) and Zinc finger protein 42 (Rex-1; Kristensen et al. 2008), which impart pluripotency. However, expression of another pluripotency gene, Nanog, is repressed in the testis by transformation related protein 53 (TRP53) and phosphatase and tensin homolog (PTEN). Both proteins belong to a critical signaling pathway preventing SSCs from being pluripotent while in the testis (Kuijk et al. 2009). Overall, this gene expression pattern suggests that SSCs have a gene profile similar to ESCs and thus are more

**3.2 Current methods of isolation and propagation of SSCs with emphasis on human** 

Spermatogonial stem cell constitute only 0.03% of the total germ cell population in rodent and human testis (Tegelenbosch and de Rooij 1993). Thus, their small numbers and the lack of specific markers are the main hurdles to their characterization. Nonetheless, significant progress has recently been made in the isolation and propagation of cells with SSCs properties from rodent (Dym et al. 2009; Guan et al. 2009; Kanatsu-Shinohara, Takehashi, and Shinohara 2008; Kubota, Avarbock, and Brinster 2004; Oatley and Brinster 2006, 2008; Ogawa et al. 2003; Hofmann et al. 2005; Kanatsu-Shinohara et al. 2010) and human testes (Conrad et al. 2008; Glaser et al. 2008; Golestaneh et al. 2009; Kossack et al. 2009; Zovoilis et al. 2008; Izadyar et al. 2011; He et al. 2009). Human testicular tissue is currently obtained from testicular biopsies (Izadyar et al. 2011; Kossack et al. 2009), orchiectomies (Izadyar et al. 2011) and organ donors (Golestaneh et al. 2009). Testicular biopsies of approximately one gram can yield sufficient number of human SSCs for most clinical applications. Although our knowledge of mouse and human SSCs phenotype is still limited, studies suggest that human SSCs express proteins such as cluster of differentiation antigens 49f, 90 and 133 (CD49f, CD90, and CD133, respectively), glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRA1), G protein-coupled receptor 125 (GPR125), melanoma antigen family A 4 (MAGE4), promyelocytic leukemia zinc finger (PLZF) and stage-specific embryonic antigen-4 (SSEA-4; Costoya et al. 2004; Conrad et al. 2008; He et al. 2009; Izadyar et al. 2011). Using these markers in magnetic- or fluorescent-activated cell sorting, SSCs can be isolated with a high degree of purity (Gassei et al. 2009; Izadyar et al. 2011; Kokkinaki et al. 2009; Simon et al. 2010). Although there have been no definitive culture conditions for propagation of either mouse or human SSCs, culture systems established by different groups seem to be conducive for their propagation. At least in rodents, glial cell line-derived neurotrophic factor (GDNF) was found to be essential to maintain SSCs in an undifferentiated state in vivo and in vitro (Tyagi et al. 2009; Hofmann 2008; Sariola and Immonen 2008; Oatley, Avarbock, and Brinster 2007; Oatley et al. 2006; Naughton et al. 2006;

undifferentiated than other adult stem cells.

Kubota, Avarbock, and Brinster 2004; Meng et al. 2000).

**SSC isolation** 

In the testicular microenvironment, SSCs produce only the spermatogenic lineage, and the assumption was that this was their sole potential developmental fate. However, the iconoclastic finding of Kanatsu-Shinohara et al. (Kanatsu-Shinohara et al. 2004) demonstrated that neonatal murine SSCs produced ESC-like cells when isolated from the testis and grown for extended periods in ESC culture conditions. Although initial work was done with neonatal SSCs (Kanatsu-Shinohara et al. 2004; Kanatsu-Shinohara et al. 2008; Simon et al. 2009), subsequent work showed that adult murine SSCs (Glaser et al. 2008; Izadyar et al. 2008; Seandel et al. 2007; Guan et al. 2006) grown for 4-7 weeks in vitro also produced a low frequency of ESC-like colonies. These ESC-like cells can give rise to cell types derived from all three embryonic germ layers, and also produce teratomas when injected subcutaneously into nude mice. In addition, these ESC-like cells contribute to embryonic development when injected into blastocysts. ESC-like cells derived from SSCs have been termed multipotent germline stem cells (mGSCs) and these cells have been differentiated into many cell types. For example, two separate groups demonstrated that mGSCs could differentiate into mature cardiac and endothelial cells and that these cardiac cells were contractile and had electric potentials and ion channels (Baba et al. 2007; Guan et al. 2007). mGSCs derived from adult mouse SSCs could be differentiated into functional neurons and glia (Glaser et al. 2008; Streckfuss-Bomeke et al. 2009). Pluripotent stem cells were derived from adult mGSCs that not only could differentiate into a variety of cell types both in vivo and in vitro, but also showed germline transmission to the next generation when injected into blastocysts (Ko et al. 2009). Moreover, mGSCs have pluripotency characteristics similar to ESCs such as telomerase activity, telomere length and hypomethylation of pluripotency marker genes (Zechner et al. 2009)

In recent years, several research groups reported methodologies for isolation and culture of human SSCs and also demonstrated that these cells were pluripotent/multipotent (Conrad et al. 2008; Golestaneh et al. 2009; Kossack et al. 2009; Dym et al. 2009; Izadyar et al. 2011). SSCs isolated from human testicular tissues and cultured for a week or more spontaneously produced ESC-like small colonies, which were then transferred into ESC media and cultured for about 4 weeks to get sufficient numbers of ESC-like colonies and these ESC-like cells could then be differentiated into specific cell types. These results indicate that human SSCs have the potential to be used as an alternate source of pluripotent stem cells in regenerative cell therapy, without the ethical concerns of ESC and the concerns involving exogenous gene introduction in iPSCs.

#### **4. Direct differentiation of spermatogonial stem cells**

#### **4.1 Epithelial-mesenchymal interactions**

Epithelial-mesenchymal interactions are critical for organogenesis in many organs such as lung, prostate, mammary gland, liver, pancreas and salivary glands (Grobstein 1953). Mesenchyme, which is undifferentiated connective tissue, signals to the epithelium to induce epithelial morphogenesis and cytodifferentiation in a wide variety of organs. The central role of epithelial-mesenchymal interactions in organ development was postulated initially by Pander and later experimentally demonstrated by Spemann and Saunders (in Simon, Hess, and Cooke 2010).

Spermatogonial Stem Cells:

differentiate into various derivatives.

CK8.

An Alternate Source of Pluripotent Stem Cells for Regenerative Medicine 209

appropriate mesenchyme and grafted in vivo (Fig. 1). To track cell lineages derived from SSCs in tissue recombinations and verify that these cells were undergoing differentiation, wt transgenic C57BL/6 mice expressing enhanced green fluorescent protein (GFP) ubiquitously were used. For example, UGM derived from wild-type mice (wt-UGM) were recombined with SSCs derived from mice expressing GFP (G-SSC) and grafted under the renal capsule of syngeneic male hosts. After 4 weeks of growth, the epithelium in these, wt-UGM + G-SSC, tissue recombinants expressed NKX3.1, a prostatic epithelial marker and androgen receptor but not germ cell nuclear antigen 1 (GCNA1), a germ cell marker. The tissue recombinants had an epithelium that stained intensely for GFP (Fig. 1A), indicating that it was of SSC origin, while stromal cells lacked GFP staining. Similarly uterine mesenchyme (UtM) from mice expressing GFP (G-UtM) recombined with SSCs derived from wt-mice (wt-SSC) differentiated into uterine epithelium that expressed cytokeratin 8 (CK8), estrogen receptor–alpha and progesterone receptor. In these G-UtM + wt-SSC tissue recombinants, stromal cells strongly expressed GFP, while epithelium did not, indicating that the epithelium was of SSC origin (Fig. 1B). This approach provides a method to directly differentiate SSCs into specific cell types from all three embryonic germ layers without the extended culture period needed in vitro to produce ESC-like cells that can subsequently

Fig. 1. Differentiation of SSCs into prostatic and uterine epithelium. A) Urogenital sinus mesenchyme (UGM) from wild-type (wt) mice was recombined with SSCs derived from mice that expressed enhanced green fluorescent protein (G) ubiquitously and was grafted under the renal capsule of syngeneic male hosts. After 4 weeks of in vivo growth, the wt-UGM + G-SSC tissue recombinants formed prostatic epithelium (E) that was of SSC origin expressing GFP and androgen receptor (AR, red nuclei). B) Uterine mesenchyme (UtM) from GFP mice was recombined with SSCs derived from wt mice and grafted under the renal capsule of syngeneic female hosts. After 4 weeks of growth, the G-UtM + wt-SSC tissue recombinants formed uterine epithelium (E) that was of SSC origin expressing

cytokeratin 8 (CK8). Stromal cells (S) express GFP but E does not. SSCs do not express AR or

When SSCs were mixed with mammary epithelial cells and grafted into the mammary fat pad in vivo, SSCs differentiated into mammary epithelial cells, but the stem cells alone could not be differentiated into mammary cells (Boulanger et al. 2007). This suggests that the

Classical tissue recombination experiments conducted by Cunha and coworkers with reproductive tissue demonstrated that the mesenchyme dictates the fate of the epithelium in various reproductive tissues. The urogenital sinus is an ambisexual fetal organ that gives rise to the prostate in males and a portion of the vagina in females. Under the influence of androgen, urogenital sinus mesenchyme (UGM) induces urogenital sinus epithelium (UGE) to differentiate into prostatic epithelium and UGM also regulates epithelial ductal morphogenesis and cytodifferentiation in prostate. UGM also instructively induces prostatic morphogenesis in other epithelia (Cunha et al. 1983; Cunha, Lung, and Reese 1980; Cunha, Sekkingstad, and Meloy 1983). For example, UGM instructively induces adult bladder epithelium to form prostatic epithelium in tissue recombinants in vivo (Cunha et al. 1983). Similarly, neonatal uterine mesenchyme instructively induces neonatal vaginal epithelium to form uterine epithelium (Cunha 1976).

#### **4.1.1 Potential signaling pathways involved in epithelial-mesenchymal interactions**

Although mesenchymal paracrine signaling is essential for determining epithelial fate, the specific signaling pathway(s) involved in this phenomenon remain unclear. Some possible signaling molecules are Wnt7a (Miller and Sassoon 1998), hepatocyte growth factor (HGF) and fibroblast growth factor (FGF) family molecule(s) in the uterus (Chen, Spencer, and Bazer 2000, 2000), HGF and FGF family molecule(s) for lung organogenesis (Ohmichi et al. 1998) (for review see Kumar et al. 2005,) and FGF and Wnt and Hedgehog signaling in the prostate (for review see Thomson, Cunha, and Marker 2008; Cunha, Cooke, and Kurita 2004; Lin and Wang, 2010; Taylor et al. 2009).

#### **4.1.2 Mesenchyme dictates the fate of stem cell differentiation**

The demonstrations that epithelial-mesenchymal interactions are essential for organogenesis also triggered interest in using this approach in regenerative therapy. ESCs were differentiated into prostatic epithelium by recombining mouse UGM and human ESCs and growing these tissue recombinations in vivo (Taylor et al. 2006). Using a similar methodology (Oottamasathien et al. 2007; Anumanthan et al. 2008), human ESCs or bone marrow-derived mesenchymal stem cells have been differentiated into bladder epithelium by exposing these cells to the inductive influence of bladder mesenchyme in a tissue recombinant. More recently, Taylor et al. demonstrated that stroma could induce adult stem cells to express dual phenotypes (Taylor et al. 2009). Prostatic stroma induced putative mammary epithelial stem cells to generate glandular epithelia expressing both prostatic and mammary markers. These results demonstrate that the mesenchyme can instructively direct the differentiation of ESCs or other stem cells into a specific cell fate.

#### **4.2 Spermatogonial stem cells differentiate into tissues of all three embryonic germ layers in response to instructive inducers**

Based on demonstrations of the importance of the stem cell niche (Tyagi et al. 2009; de Rooij 2009; Hess et al. 2006; Simon et al. 2010; Oatley, Racicot, and Oatley 2010), the pluripotential nature of SSCs and the instructive potential of various mesenchymes, we postulated and subsequently demonstrated that neonatal mouse SSCs could directly differentiate into prostatic, uterine and skin epithelium (Simon et al. 2009) when recombined with the

Classical tissue recombination experiments conducted by Cunha and coworkers with reproductive tissue demonstrated that the mesenchyme dictates the fate of the epithelium in various reproductive tissues. The urogenital sinus is an ambisexual fetal organ that gives rise to the prostate in males and a portion of the vagina in females. Under the influence of androgen, urogenital sinus mesenchyme (UGM) induces urogenital sinus epithelium (UGE) to differentiate into prostatic epithelium and UGM also regulates epithelial ductal morphogenesis and cytodifferentiation in prostate. UGM also instructively induces prostatic morphogenesis in other epithelia (Cunha et al. 1983; Cunha, Lung, and Reese 1980; Cunha, Sekkingstad, and Meloy 1983). For example, UGM instructively induces adult bladder epithelium to form prostatic epithelium in tissue recombinants in vivo (Cunha et al. 1983). Similarly, neonatal uterine mesenchyme instructively induces neonatal vaginal epithelium

**4.1.1 Potential signaling pathways involved in epithelial-mesenchymal interactions**  Although mesenchymal paracrine signaling is essential for determining epithelial fate, the specific signaling pathway(s) involved in this phenomenon remain unclear. Some possible signaling molecules are Wnt7a (Miller and Sassoon 1998), hepatocyte growth factor (HGF) and fibroblast growth factor (FGF) family molecule(s) in the uterus (Chen, Spencer, and Bazer 2000, 2000), HGF and FGF family molecule(s) for lung organogenesis (Ohmichi et al. 1998) (for review see Kumar et al. 2005,) and FGF and Wnt and Hedgehog signaling in the prostate (for review see Thomson, Cunha, and Marker 2008; Cunha, Cooke, and Kurita 2004;

The demonstrations that epithelial-mesenchymal interactions are essential for organogenesis also triggered interest in using this approach in regenerative therapy. ESCs were differentiated into prostatic epithelium by recombining mouse UGM and human ESCs and growing these tissue recombinations in vivo (Taylor et al. 2006). Using a similar methodology (Oottamasathien et al. 2007; Anumanthan et al. 2008), human ESCs or bone marrow-derived mesenchymal stem cells have been differentiated into bladder epithelium by exposing these cells to the inductive influence of bladder mesenchyme in a tissue recombinant. More recently, Taylor et al. demonstrated that stroma could induce adult stem cells to express dual phenotypes (Taylor et al. 2009). Prostatic stroma induced putative mammary epithelial stem cells to generate glandular epithelia expressing both prostatic and mammary markers. These results demonstrate that the mesenchyme can instructively direct

**4.2 Spermatogonial stem cells differentiate into tissues of all three embryonic germ** 

Based on demonstrations of the importance of the stem cell niche (Tyagi et al. 2009; de Rooij 2009; Hess et al. 2006; Simon et al. 2010; Oatley, Racicot, and Oatley 2010), the pluripotential nature of SSCs and the instructive potential of various mesenchymes, we postulated and subsequently demonstrated that neonatal mouse SSCs could directly differentiate into prostatic, uterine and skin epithelium (Simon et al. 2009) when recombined with the

to form uterine epithelium (Cunha 1976).

Lin and Wang, 2010; Taylor et al. 2009).

**layers in response to instructive inducers** 

**4.1.2 Mesenchyme dictates the fate of stem cell differentiation** 

the differentiation of ESCs or other stem cells into a specific cell fate.

appropriate mesenchyme and grafted in vivo (Fig. 1). To track cell lineages derived from SSCs in tissue recombinations and verify that these cells were undergoing differentiation, wt transgenic C57BL/6 mice expressing enhanced green fluorescent protein (GFP) ubiquitously were used. For example, UGM derived from wild-type mice (wt-UGM) were recombined with SSCs derived from mice expressing GFP (G-SSC) and grafted under the renal capsule of syngeneic male hosts. After 4 weeks of growth, the epithelium in these, wt-UGM + G-SSC, tissue recombinants expressed NKX3.1, a prostatic epithelial marker and androgen receptor but not germ cell nuclear antigen 1 (GCNA1), a germ cell marker. The tissue recombinants had an epithelium that stained intensely for GFP (Fig. 1A), indicating that it was of SSC origin, while stromal cells lacked GFP staining. Similarly uterine mesenchyme (UtM) from mice expressing GFP (G-UtM) recombined with SSCs derived from wt-mice (wt-SSC) differentiated into uterine epithelium that expressed cytokeratin 8 (CK8), estrogen receptor–alpha and progesterone receptor. In these G-UtM + wt-SSC tissue recombinants, stromal cells strongly expressed GFP, while epithelium did not, indicating that the epithelium was of SSC origin (Fig. 1B). This approach provides a method to directly differentiate SSCs into specific cell types from all three embryonic germ layers without the extended culture period needed in vitro to produce ESC-like cells that can subsequently differentiate into various derivatives.

Fig. 1. Differentiation of SSCs into prostatic and uterine epithelium. A) Urogenital sinus mesenchyme (UGM) from wild-type (wt) mice was recombined with SSCs derived from mice that expressed enhanced green fluorescent protein (G) ubiquitously and was grafted under the renal capsule of syngeneic male hosts. After 4 weeks of in vivo growth, the wt-UGM + G-SSC tissue recombinants formed prostatic epithelium (E) that was of SSC origin expressing GFP and androgen receptor (AR, red nuclei). B) Uterine mesenchyme (UtM) from GFP mice was recombined with SSCs derived from wt mice and grafted under the renal capsule of syngeneic female hosts. After 4 weeks of growth, the G-UtM + wt-SSC tissue recombinants formed uterine epithelium (E) that was of SSC origin expressing cytokeratin 8 (CK8). Stromal cells (S) express GFP but E does not. SSCs do not express AR or CK8.

When SSCs were mixed with mammary epithelial cells and grafted into the mammary fat pad in vivo, SSCs differentiated into mammary epithelial cells, but the stem cells alone could not be differentiated into mammary cells (Boulanger et al. 2007). This suggests that the

Spermatogonial Stem Cells:

**sources** 

An Alternate Source of Pluripotent Stem Cells for Regenerative Medicine 211

Fig. 2. Potential use of spermatogonial stem cells (SSCs) in regenerative medicine.

**5. Advantages and limitations of SSCs over other pluripotent stem cell** 

SSCs, as well as other adult derived stem cells, may be safer to use therapeutically than ESCs or iPSCs. Since SSCs are more differentiated than ESCs, they are less likely to induce teratomas (Kossack et al. 2009). However, the risk of malignant transformation cannot be totally rejected since SSCs are relatively more undifferentiated than other adult stem cells. This type of problem is illustrated by the formation of brain tumors from donor-derived cells in patients who received fetal stem cells for treatment of ataxia-telangiectasia (Amariglio et al. 2009), emphasizing that tumorigenicity is the biggest impediment for the use of pluripotent stem cells in cell therapy. Another obstacle is the immunogenicity of SSCs and potential risk of rejection of the cells (Dressel et al. 2009). But immune rejection of autologous or allogenic stem cell transplants can be minimized by routine immunosuppression treatments as is used for organ transplantation. Another possible alternative is genetic manipulation of stem cells and elimination of genes responsible for

and differentiate into cells of all three embryonic germ layers.

immune rejection (Wobus and Boheler 2005).

Spermatogonia in the murine seminiferous tubule expressing PLZF, a SSC marker. SSCs are isolated from the testis using magnetic- or fluorescent-activated cell sorting. Some of the possible methods of differentiating SSCs into specific cell types are 1) Long-term culture of SSCs, selection for cells that form embryonic stem cell-like colonies and subsequent culture and differentiation of these pluripotent cells into specific cell types. 2) Recombination of SSCs with instructive inducers to directly differentiate SSCs into specific epithelial cell types; 3) Direct injection to a specific site of injury or specific microenvironment. Since SSCs are unspecialized, they could home and respond to signals in the new microenvironment

inductive potential of the adult mammary fat pad might not be sufficient for directing the differentiation of SSCs into mammary epithelial cells. This is a potential hurdle for the use of this methodology, as the inductive mesenchyme for most of the organs is present during fetal life, and as they transition to form adult stromal cells they might lose this ability to instructively induce other epithelia. However, a recent study demonstrated direct differentiation of SSCs into hematopoietic cells (Ning et al., 2010). When SSCs were injected into the bone marrow of adult female mice, these mice had Y chromosome positive-cells that had phenotypical and functional characteristics of hematopoietic cells both in vivo and in vitro, emphasizing both the pluripotential nature of SSCs and that the microenvironment even in adult organs plays a decisive role in directing the differentiation of SSCs.

As discussed earlier, our understanding of the mechanistic basis of epithelial-mesenchymal interactions is fragmentary. Fetal mesenchymes are potent instructive inducers, and one of the challenges for using this methodology will be to determine whether or not adult stroma is capable of similar instructive inductions. Once we are able to dissect out the molecular mechanisms used by mesenchyme to instructively induce epithelial morphogenesis, it will be feasible to expose SSCs to the inductive signals produced by these tissues in vitro, on artificial scaffolds or some other arrangement. Another major limitation of this methodology is that it is inapplicable to tissues that do not involve epithelial-mesenchymal interactions during development. However, the direct differentiation of SSCs into hematopoietic cells (Ning et al., 2010) discussed above suggests that SSCs will differentiate into a specific tissue type when exposed to an appropriate microenvironment, even in the absence of epithelialmesenchymal interactions. Thus further research is necessary to determine both the full developmental potential of SSCs and the most appropriate methodology for inducing specific cell types, but SSCs appear to have great potential in this regard.

#### **4.3 Mechanism of differentiation of spermatogonial stem cells into other cell types**

The mechanism of differentiation of SSCs into other cell types under the influence of instructive inducers is poorly understood. One possibility is that in response to an inductive mesenchyme, the SSCs de-differentiate into ESC-like cells (as has been reported in vitro), and then subsequently differentiate into a new epithelia. However, our preliminary studies indicate that SSCs may not undergo a de-differentiation step in the presence of an inductive mesenchyme, but instead may differentiate directly from SSCs to another epithelial type without going through an intermediate ESC-like cell stage. Shinohara and coworkers (Kanatsu-Shinohara et al. 2008) demonstrated that a single spermatogonial stem cell could produce an embryonic stem-like line that was multipotent and germline stem cells that were committed to spermatogenesis, indicating that all SSCs may be capable of becoming pluripotent. This is supported by other studies (Ko et al. 2009). Conversely, Izadyar et al. suggested that there are two distinct populations of SSCs, one that is OCT4+ and c-KIT- that gives rise to multipotent cells and another that is OCT4+ and c-KIT+ that gives rise to the spermatogenic lineage (Izadyar et al. 2008). A definitive elucidation of how SSCs differentiate into other tissue types, as well as definitively establishing whether all SSCs or a specific subpopulation can be converted into other tissues, will be an essential prerequisite for successful use of this approach in a clinical setting. Nonetheless, SSCs can be differentiated into specific cell types using different approaches and be a potential source for pluripotent cells for stem cell-based therapy (Fig. 2).

inductive potential of the adult mammary fat pad might not be sufficient for directing the differentiation of SSCs into mammary epithelial cells. This is a potential hurdle for the use of this methodology, as the inductive mesenchyme for most of the organs is present during fetal life, and as they transition to form adult stromal cells they might lose this ability to instructively induce other epithelia. However, a recent study demonstrated direct differentiation of SSCs into hematopoietic cells (Ning et al., 2010). When SSCs were injected into the bone marrow of adult female mice, these mice had Y chromosome positive-cells that had phenotypical and functional characteristics of hematopoietic cells both in vivo and in vitro, emphasizing both the pluripotential nature of SSCs and that the microenvironment

As discussed earlier, our understanding of the mechanistic basis of epithelial-mesenchymal interactions is fragmentary. Fetal mesenchymes are potent instructive inducers, and one of the challenges for using this methodology will be to determine whether or not adult stroma is capable of similar instructive inductions. Once we are able to dissect out the molecular mechanisms used by mesenchyme to instructively induce epithelial morphogenesis, it will be feasible to expose SSCs to the inductive signals produced by these tissues in vitro, on artificial scaffolds or some other arrangement. Another major limitation of this methodology is that it is inapplicable to tissues that do not involve epithelial-mesenchymal interactions during development. However, the direct differentiation of SSCs into hematopoietic cells (Ning et al., 2010) discussed above suggests that SSCs will differentiate into a specific tissue type when exposed to an appropriate microenvironment, even in the absence of epithelialmesenchymal interactions. Thus further research is necessary to determine both the full developmental potential of SSCs and the most appropriate methodology for inducing

even in adult organs plays a decisive role in directing the differentiation of SSCs.

specific cell types, but SSCs appear to have great potential in this regard.

pluripotent cells for stem cell-based therapy (Fig. 2).

**4.3 Mechanism of differentiation of spermatogonial stem cells into other cell types**  The mechanism of differentiation of SSCs into other cell types under the influence of instructive inducers is poorly understood. One possibility is that in response to an inductive mesenchyme, the SSCs de-differentiate into ESC-like cells (as has been reported in vitro), and then subsequently differentiate into a new epithelia. However, our preliminary studies indicate that SSCs may not undergo a de-differentiation step in the presence of an inductive mesenchyme, but instead may differentiate directly from SSCs to another epithelial type without going through an intermediate ESC-like cell stage. Shinohara and coworkers (Kanatsu-Shinohara et al. 2008) demonstrated that a single spermatogonial stem cell could produce an embryonic stem-like line that was multipotent and germline stem cells that were committed to spermatogenesis, indicating that all SSCs may be capable of becoming pluripotent. This is supported by other studies (Ko et al. 2009). Conversely, Izadyar et al. suggested that there are two distinct populations of SSCs, one that is OCT4+ and c-KIT- that gives rise to multipotent cells and another that is OCT4+ and c-KIT+ that gives rise to the spermatogenic lineage (Izadyar et al. 2008). A definitive elucidation of how SSCs differentiate into other tissue types, as well as definitively establishing whether all SSCs or a specific subpopulation can be converted into other tissues, will be an essential prerequisite for successful use of this approach in a clinical setting. Nonetheless, SSCs can be differentiated into specific cell types using different approaches and be a potential source for

Fig. 2. Potential use of spermatogonial stem cells (SSCs) in regenerative medicine. Spermatogonia in the murine seminiferous tubule expressing PLZF, a SSC marker. SSCs are isolated from the testis using magnetic- or fluorescent-activated cell sorting. Some of the possible methods of differentiating SSCs into specific cell types are 1) Long-term culture of SSCs, selection for cells that form embryonic stem cell-like colonies and subsequent culture and differentiation of these pluripotent cells into specific cell types. 2) Recombination of SSCs with instructive inducers to directly differentiate SSCs into specific epithelial cell types; 3) Direct injection to a specific site of injury or specific microenvironment. Since SSCs are unspecialized, they could home and respond to signals in the new microenvironment and differentiate into cells of all three embryonic germ layers.

#### **5. Advantages and limitations of SSCs over other pluripotent stem cell sources**

SSCs, as well as other adult derived stem cells, may be safer to use therapeutically than ESCs or iPSCs. Since SSCs are more differentiated than ESCs, they are less likely to induce teratomas (Kossack et al. 2009). However, the risk of malignant transformation cannot be totally rejected since SSCs are relatively more undifferentiated than other adult stem cells. This type of problem is illustrated by the formation of brain tumors from donor-derived cells in patients who received fetal stem cells for treatment of ataxia-telangiectasia (Amariglio et al. 2009), emphasizing that tumorigenicity is the biggest impediment for the use of pluripotent stem cells in cell therapy. Another obstacle is the immunogenicity of SSCs and potential risk of rejection of the cells (Dressel et al. 2009). But immune rejection of autologous or allogenic stem cell transplants can be minimized by routine immunosuppression treatments as is used for organ transplantation. Another possible alternative is genetic manipulation of stem cells and elimination of genes responsible for immune rejection (Wobus and Boheler 2005).

Spermatogonial Stem Cells:

application in regenerative medicine.

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urothelium. *J Urol* 180 (4 Suppl):1778-83.

cells. *Stem Cells* 25 (6):1375-83.

**7. Conclusions** 

**8. References** 

An Alternate Source of Pluripotent Stem Cells for Regenerative Medicine 213

Among pluripotent/multipotent stem cells (Table 1), spermatogonial stem cells have great potential and some unique advantages. Despite their promise, numerous hurdles must be overcome before clinical use of SSCs. The small population of SSCs in the testis and the difficulty in propagating and maintaining them in culture is one major hurdle. The methodology proposed here is promising but extensive work is needed before its

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#### **6. Alternate sources of stem cells for use in regenerative medicine**

The use of stem cells in regenerative medicine began when bone marrow cells were transplanted to treat acute leukemia (Thomas et al. 1959), and presently HSC therapy is the only stem cell therapy widely used clinically (Helmy et al. 2010). There is growing evidence that HSCs are plastic—and can give rise to tissues other than those of the blood system, e.g., liver cells (Lagasse et al. 2000). Another readily available source is mesenchymal stem cells that can be isolated from adult tissue, fetal tissue and umbilical cord blood. These cells can differentiate into osteoblasts, chondrocytes and adipocytes (Friedenstein et al. 1974; Pittenger et al. 1999) neurons (Cho et al. 2005), astrocytes (Kopen, Prockop, and Phinney 1999) and hepatocyte-like cells (Petersen et al. 1999). The use of adipose stem cells as a source for cell therapy is increasing rapidly as methods of isolation and culture are standardized; subcutaneous depots are easily accessible, replenishable and are often abundant. Adult stem cells derived from adipose tissues can differentiate in vitro into many cell types including adipocyte, chondrocyte, endothelial, epithelial, hematopoietic support, hepatocyte, neuronal, myogenic, and osteoblast lineages (Gimble and Guilak 2003; Halvorsen et al. 2001; Safford et al. 2002; Zuk et al. 2001). Fetal stem cells are self-renewing cells located in various types of fetal tissue, including umbilical cord blood, umbilical cord matrix, fetal blood and the amniotic membrane (Reinisch and Strunk 2009; Jager et al. 2009; Zeddou et al., 2010). Umbilical cord blood contain multiple populations of stem cells that can be effective in treating many diseases such as hematological malignancies, hemoglobinopathies, metabolic disorders and the greatest advantage of these cells is decreased immune rejection (Liao et al., 2011).


Table 1. Potential advantages and limitations of stem cells in regenerative medicine

#### **7. Conclusions**

212 Tissue Regeneration – From Basic Biology to Clinical Application

The use of stem cells in regenerative medicine began when bone marrow cells were transplanted to treat acute leukemia (Thomas et al. 1959), and presently HSC therapy is the only stem cell therapy widely used clinically (Helmy et al. 2010). There is growing evidence that HSCs are plastic—and can give rise to tissues other than those of the blood system, e.g., liver cells (Lagasse et al. 2000). Another readily available source is mesenchymal stem cells that can be isolated from adult tissue, fetal tissue and umbilical cord blood. These cells can differentiate into osteoblasts, chondrocytes and adipocytes (Friedenstein et al. 1974; Pittenger et al. 1999) neurons (Cho et al. 2005), astrocytes (Kopen, Prockop, and Phinney 1999) and hepatocyte-like cells (Petersen et al. 1999). The use of adipose stem cells as a source for cell therapy is increasing rapidly as methods of isolation and culture are standardized; subcutaneous depots are easily accessible, replenishable and are often abundant. Adult stem cells derived from adipose tissues can differentiate in vitro into many cell types including adipocyte, chondrocyte, endothelial, epithelial, hematopoietic support, hepatocyte, neuronal, myogenic, and osteoblast lineages (Gimble and Guilak 2003; Halvorsen et al. 2001; Safford et al. 2002; Zuk et al. 2001). Fetal stem cells are self-renewing cells located in various types of fetal tissue, including umbilical cord blood, umbilical cord matrix, fetal blood and the amniotic membrane (Reinisch and Strunk 2009; Jager et al. 2009; Zeddou et al., 2010). Umbilical cord blood contain multiple populations of stem cells that can be effective in treating many diseases such as hematological malignancies, hemoglobinopathies, metabolic disorders and the greatest advantage of these cells is

**6. Alternate sources of stem cells for use in regenerative medicine** 

Stem Cells Advantages Major limitations

Indefinite self-renewal

Initial source of cells are easy

No ethical or moral concerns Indefinite self-renewal

No ethical or moral concerns Relatively easy to obtain Less tumorigenic potential

Relatively easy to obtain Minimal immunorejection

Table 1. Potential advantages and limitations of stem cells in regenerative medicine

during autologous transplantation

Easy to obtain Reduced risk of tumorigenicity

Ethical concerns Tumorigenicity Therapeutic cloning

Tumorigenicity Genetic instability Use of viral vectors to introduce genes

Introduction of exogenous

Relatively small numbers Difficult to maintain in

Restricted differentiation and self-renewal potential

Restricted differentiation and self-renewal potential

involved

genes

cultures

potential

to obtain

potential

Pluripotent

decreased immune rejection (Liao et al., 2011).

Embryonic stem cells Pluripotent

Induced pluripotent cells Pluripotent

Spermatogonial stem

Fetal stem cells (Fetal cord blood, umbilical

mesenchymal, adipose)

cord tissue)

Adult stem cells (hematopoietic,

cells

Among pluripotent/multipotent stem cells (Table 1), spermatogonial stem cells have great potential and some unique advantages. Despite their promise, numerous hurdles must be overcome before clinical use of SSCs. The small population of SSCs in the testis and the difficulty in propagating and maintaining them in culture is one major hurdle. The methodology proposed here is promising but extensive work is needed before its application in regenerative medicine.

#### **8. References**


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**10** 

*Japan* 

**Therapeutic Application of Allogeneic Fetal Membrane-Derived Mesenchymal Stem Cell** 

**Transplantation in Regenerative Medicine** 

In 1968, Friedenstein et al. isolated clonogeneic spindle-shaped cells from bone marrow (BM) in monolayer cultures, which they called colony-forming-unit fibroblasts (Friedenstein et al., 1974). These cells showed the ability to self-renew and to differentiate toward a mesodermal lineage as adipocytes, chondrocytes, osteocytes and connective stromal cells. Several studies reported that BM-derived multipotential stromal precursor cells can also differentiate into lineages such as ectodermal cells and endodermal cells (Kopen et al., 1999; Pittenger et al., 1999). For this reason, BM-derived stromal cells were first considered to be stem cells by Caplan and were named mesenchymal stem cells (MSCs) (Caplan, 1991). The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposed the following minimal criteria for defining human MSCs: (1) MSCs must be plastic-adherent when maintained under standard culture conditions, (2) MSCs must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR surface molecules, (3) MSCs must differentiate into osteoblasts, adipocytes and chondroblasts *in vitro* (Dominici et al., 2006; Sensebe et al., 2010)*.*  MSCs have been obtained from adipose tissue, cord blood and many other tissues, and can differentiate into a variety of cells, including adipocytes, osteocytes, chondrocytes, endothelial cells and myocytes (Campagnoli et al., 2001; Kim et al., 2006; Zuk et al., 2001). MSCs secrete a variety of angiogenic, antiapoptotic and mitogenic factors, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) (Kinnaird et al., 2004; Nagaya et al., 2005). Among MSCs derived from various tissues, BM-derived MSCs (BM-MSCs) are widely used in the field of stem cell transplantation. We previously reported that autologous BM-MSC transplantation induced therapeutic angiogenesis in a rat model of hind-limb ischemia and improved cardiac function in rat models of dilated cardiomyopathy and acute autoimmune myocarditis (Iwase et al., 2005; Nagaya et al., 2005; Ohnishi et al., 2007). However, there are several limitations to using an autologous cell source for cell transplantation, such as the

**1. Introduction** 

Shin Ishikane1, Hiroshi Hosoda1 and Tomoaki Ikeda1,2 *1Department of Regenerative Medicine and Tissue Engineering, National Cerebral and Cardiovascular Center Research Institute,* 

> *2Department of Perinatology and Gynecology, National Cerebral and Cardiovascular Center,*


### **Therapeutic Application of Allogeneic Fetal Membrane-Derived Mesenchymal Stem Cell Transplantation in Regenerative Medicine**

Shin Ishikane1, Hiroshi Hosoda1 and Tomoaki Ikeda1,2 *1Department of Regenerative Medicine and Tissue Engineering, National Cerebral and Cardiovascular Center Research Institute, 2Department of Perinatology and Gynecology, National Cerebral and Cardiovascular Center, Japan* 

#### **1. Introduction**

220 Tissue Regeneration – From Basic Biology to Clinical Application

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and M. H. Hedrick. 2001. Multilineage cells from human adipose tissue:

In 1968, Friedenstein et al. isolated clonogeneic spindle-shaped cells from bone marrow (BM) in monolayer cultures, which they called colony-forming-unit fibroblasts (Friedenstein et al., 1974). These cells showed the ability to self-renew and to differentiate toward a mesodermal lineage as adipocytes, chondrocytes, osteocytes and connective stromal cells. Several studies reported that BM-derived multipotential stromal precursor cells can also differentiate into lineages such as ectodermal cells and endodermal cells (Kopen et al., 1999; Pittenger et al., 1999). For this reason, BM-derived stromal cells were first considered to be stem cells by Caplan and were named mesenchymal stem cells (MSCs) (Caplan, 1991). The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposed the following minimal criteria for defining human MSCs: (1) MSCs must be plastic-adherent when maintained under standard culture conditions, (2) MSCs must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR surface molecules, (3) MSCs must differentiate into osteoblasts, adipocytes and chondroblasts *in vitro* (Dominici et al., 2006; Sensebe et al., 2010)*.* 

MSCs have been obtained from adipose tissue, cord blood and many other tissues, and can differentiate into a variety of cells, including adipocytes, osteocytes, chondrocytes, endothelial cells and myocytes (Campagnoli et al., 2001; Kim et al., 2006; Zuk et al., 2001). MSCs secrete a variety of angiogenic, antiapoptotic and mitogenic factors, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) (Kinnaird et al., 2004; Nagaya et al., 2005). Among MSCs derived from various tissues, BM-derived MSCs (BM-MSCs) are widely used in the field of stem cell transplantation. We previously reported that autologous BM-MSC transplantation induced therapeutic angiogenesis in a rat model of hind-limb ischemia and improved cardiac function in rat models of dilated cardiomyopathy and acute autoimmune myocarditis (Iwase et al., 2005; Nagaya et al., 2005; Ohnishi et al., 2007). However, there are several limitations to using an autologous cell source for cell transplantation, such as the

Therapeutic Application of Allogeneic Fetal Membrane-Derived

Mesenchymal Stem Cell Transplantation in Regenerative Medicine 223

MSCs expressed compounds characteristic of several angiogenesis-related genes, including VEGF-C, platelet-derived growth factor-B, angiopoietins, chemokines and interleukins. These results show that FM-MSCs have properties similar to those of BM-MSCs and suggest that transplantation of FM-MSCs may induce therapeutic angiogenesis in cases of ischemic disease.

Fig. 1. Characterization of FM-MSCs and BM-MSCs: (A) Morphology of FM-MSCs and BM-MSCs derived from Lewis rats. In the early passages, FM- and BM-MSC derived cells appeared microscopically heterogeneous. After several passages, these cells formed a morphologically homogenous population of fibroblast-like cells, which was similar to BM-MSCs. Scale bars: 100 m. (B) Multipotency of FM-MSCs and BM-MSCs. Differentiation into adipocytes was observed by oil red O. Differentiation into osteocytes was observed by alizarin red S. Differentiation into chondrocytes was observed by safranin O. Scale bars: 50 m. (C) Flow cytometric analysis of FM-MSCs and BM-MSCs at passage 3. Closed areas indicate staining with a specific antibody, whereas open areas represent staining with

isotype control antibodies.

invasiveness of the cell collection procedure, inadequate numbers of cells and donor-site morbidity, and the functionality of precursor cells in patients with cardiovascular risk factors has been questioned. The frequency and differentiation capacity of BM-MSCs decrease with age (D'Ippolito et al., 1999; Mareschi et al., 2006). An alternative source of MSCs that could provide large quantities of cells would be advantageous. One way to circumvent these limitations could be to use allogeneic MSCs. If allogeneic MSCs could be isolated from healthy young donors, and if they had a therapeutic effect similar to that of autologous MSCs, they would be considered a superior new cell source because it would be possible to overcome the problems noted above, and wider clinical applications of cell therapy would become available. Therefore, we focused on fetal membranes (FMs), which are generally discarded as medical waste after delivery, as an alternative source of autologous MSCs. Several studies reported that human FMs contain multipotent cells similar to BM-MSCs and are easy to expand (Alviano et al., 2007; Int Anker et al., 2004; Portmann-Lanz et al., 2006). If FM-MSCs could be used in allogeneic transplantation, FMs would be a useful source of cells for transplantation and regenerative medicine.

In this review, we compare the cellular characteristics and utilization of FM-MSCs with those of BM-MSCs and discuss the potential of allogeneic FM-MSC transplantation therapy in the tissue regeneration (Ishikane et al. 2008, 2010).

#### **2. Fetal membrane-derived mesenchymal stem cells**

The two FMs, the amnion and the chorion, marginate outward from the basal surface of the placenta and encase the amniotic fluid in which the fetus is suspended during pregnancy. The FMs facilitate gas and waste exchange and play a critical role as defense barriers, in maintenance of pregnancy and in parturition (Bourne, 1962). Human FMs, which are generally discarded as medical waste after delivery, were recently shown to be rich sources of MSCs. Because fetal tissues are routinely discarded postpartum, FMs are inexpensive and easy to obtain and their availability is virtually limitless, avoiding the need for mass tissue banking. Human amnion membrane-derived MSCs (hAM-MSCs) were isolated for the first time from second and third trimester AMs by In't Anker et al., who demonstrated their potential for differentiation into osteogenic and adipogenic cells (In't Anker et al., 2004). Later, Portmann-Lanz et al. demonstrated their capacity for differentiation into chondrogenic, myogenic and neurogenic lines (Portmann-Lanz et al., 2006). In 2007, Alviano et al. reported that hAM-MSCs are superior in proliferation and differentiation potential to adult hBM-MSCs, providing the first evidence of the angiogenic potential of hAM-MSCs (Alviano et al., 2007). A large quantity of MSCs was isolated from hFMs by serial passaging them prior to senescence at about 15 passages (Kim et al., 2007; Soncini et al., 2007). The availability of a fetal tissue that is usually discarded without any ethical conflict and the high yield in stem cell recovery make FMs a truly exciting alternative source that offers new prospects for expanding the range of clinical applications for stem cells.

In our study, FM-MSCs derived from Lewis rats did not express the hematopoietic or endothelial surface markers CD11b/c, CD31, CD34 and CD45, but stained positive for CD29, CD73 and CD90 (Ishikane et al., 2008). These rat FM-MSCs differentiated into adipocytes, osteocytes and chondrocytes (Figure 1). In culture medium, FM-MSCs secreted the angiogenic factors, VEGF and HGF. In an angiogenic gene polymerase chain reaction array analysis, FM-

invasiveness of the cell collection procedure, inadequate numbers of cells and donor-site morbidity, and the functionality of precursor cells in patients with cardiovascular risk factors has been questioned. The frequency and differentiation capacity of BM-MSCs decrease with age (D'Ippolito et al., 1999; Mareschi et al., 2006). An alternative source of MSCs that could provide large quantities of cells would be advantageous. One way to circumvent these limitations could be to use allogeneic MSCs. If allogeneic MSCs could be isolated from healthy young donors, and if they had a therapeutic effect similar to that of autologous MSCs, they would be considered a superior new cell source because it would be possible to overcome the problems noted above, and wider clinical applications of cell therapy would become available. Therefore, we focused on fetal membranes (FMs), which are generally discarded as medical waste after delivery, as an alternative source of autologous MSCs. Several studies reported that human FMs contain multipotent cells similar to BM-MSCs and are easy to expand (Alviano et al., 2007; Int Anker et al., 2004; Portmann-Lanz et al., 2006). If FM-MSCs could be used in allogeneic transplantation, FMs

would be a useful source of cells for transplantation and regenerative medicine.

in the tissue regeneration (Ishikane et al. 2008, 2010).

applications for stem cells.

**2. Fetal membrane-derived mesenchymal stem cells** 

In this review, we compare the cellular characteristics and utilization of FM-MSCs with those of BM-MSCs and discuss the potential of allogeneic FM-MSC transplantation therapy

The two FMs, the amnion and the chorion, marginate outward from the basal surface of the placenta and encase the amniotic fluid in which the fetus is suspended during pregnancy. The FMs facilitate gas and waste exchange and play a critical role as defense barriers, in maintenance of pregnancy and in parturition (Bourne, 1962). Human FMs, which are generally discarded as medical waste after delivery, were recently shown to be rich sources of MSCs. Because fetal tissues are routinely discarded postpartum, FMs are inexpensive and easy to obtain and their availability is virtually limitless, avoiding the need for mass tissue banking. Human amnion membrane-derived MSCs (hAM-MSCs) were isolated for the first time from second and third trimester AMs by In't Anker et al., who demonstrated their potential for differentiation into osteogenic and adipogenic cells (In't Anker et al., 2004). Later, Portmann-Lanz et al. demonstrated their capacity for differentiation into chondrogenic, myogenic and neurogenic lines (Portmann-Lanz et al., 2006). In 2007, Alviano et al. reported that hAM-MSCs are superior in proliferation and differentiation potential to adult hBM-MSCs, providing the first evidence of the angiogenic potential of hAM-MSCs (Alviano et al., 2007). A large quantity of MSCs was isolated from hFMs by serial passaging them prior to senescence at about 15 passages (Kim et al., 2007; Soncini et al., 2007). The availability of a fetal tissue that is usually discarded without any ethical conflict and the high yield in stem cell recovery make FMs a truly exciting alternative source that offers new prospects for expanding the range of clinical

In our study, FM-MSCs derived from Lewis rats did not express the hematopoietic or endothelial surface markers CD11b/c, CD31, CD34 and CD45, but stained positive for CD29, CD73 and CD90 (Ishikane et al., 2008). These rat FM-MSCs differentiated into adipocytes, osteocytes and chondrocytes (Figure 1). In culture medium, FM-MSCs secreted the angiogenic factors, VEGF and HGF. In an angiogenic gene polymerase chain reaction array analysis, FM-

MSCs expressed compounds characteristic of several angiogenesis-related genes, including VEGF-C, platelet-derived growth factor-B, angiopoietins, chemokines and interleukins. These results show that FM-MSCs have properties similar to those of BM-MSCs and suggest that transplantation of FM-MSCs may induce therapeutic angiogenesis in cases of ischemic disease.

Fig. 1. Characterization of FM-MSCs and BM-MSCs: (A) Morphology of FM-MSCs and BM-MSCs derived from Lewis rats. In the early passages, FM- and BM-MSC derived cells appeared microscopically heterogeneous. After several passages, these cells formed a morphologically homogenous population of fibroblast-like cells, which was similar to BM-MSCs. Scale bars: 100 m. (B) Multipotency of FM-MSCs and BM-MSCs. Differentiation into adipocytes was observed by oil red O. Differentiation into osteocytes was observed by alizarin red S. Differentiation into chondrocytes was observed by safranin O. Scale bars: 50 m. (C) Flow cytometric analysis of FM-MSCs and BM-MSCs at passage 3. Closed areas indicate staining with a specific antibody, whereas open areas represent staining with isotype control antibodies.

Therapeutic Application of Allogeneic Fetal Membrane-Derived

(Amado et al., 2005; Hare et al., 2009; Le Blanc et al., 2008).

allogeneic lymphocyte co-cultures.

in a mixed lymphocyte culture (MLC) test (Di Nicola et al., 2002).

Mesenchymal Stem Cell Transplantation in Regenerative Medicine 225

2007). Allogeneic BM-MSC transplantation has been used in several preclinical and clinical studies, in which allogeneic MSCs were not rejected in the absence of immunosuppression

The use of BM-MSCs not only avoids allogeneic rejection but also may confer immunosuppressive effects. Several studies demonstrated that MSCs modulate the function of T cells, major executors of the adaptive immune response (Krampera et al., 2003; Le Blanc et al., 2003). Di Nicola et al. showed that BM-MSCs strongly suppressed T cell proliferation

In our study of rats, FM-MSCs had immunological properties similar to those of BM-MSCs. In an MLC test with haplotype-mismatched allogeneic cells, FM-MSCs did not provoke alloreactive lymphocyte proliferation. Interleukin (IL)-2 plays a role in the activation and proliferation of T cells. IL-2 concentrations in supernatants of FM-MSC and allogeneic lymphocyte co-cultures and in the MLC were lower than those in lymphocyte and

To investigate T cell alloreactivity to transplanted allogeneic FM-MSCs, FM-MSCs, BM-MSCs or splenic lymphocytes obtained from GFP-transgenic Lewis rats were injected into the hind-limb tissue of MHC-mismatched August–Copenhagen Irish (ACI) rats. One week after cell injection, slight T cell infiltration was observed at the injection site of allogeneic FM-MSC-injected hind-limb muscles, but the degree of infiltration was less marked than that after allogeneic splenic lymphocyte transplantation and was equivalent to that induced by allogeneic BM-MSCs. Use of non-autologous cells for transplant also requires that one consider the possibility of graft rejection. Although most clinical applications of FM-MSC transplantation apply to allogeneic transplantation, our results suggest that FM-MSCs evade

T cell alloreactivity and may be successfully transplanted across MHC barriers.

**stem cell transplantation in a hind-limb ischemia model** 

after ischemic events (Moon et al., 2006; Nakagami et al., 2005).

allogeneic BM-MSC transplantation.

**2.2 Therapeutic angiogenesis in allogeneic fetal membrane-derived mesenchymal** 

Therapeutic angiogenesis, a strategy to treat tissue ischemia by promoting the proliferation of collateral vessels, has emerged as one of the most promising therapies developed to date (Carmeliet, 2003). In a rat model of hind-limb ischemia, autologous BM-MSC transplantation enhanced angiogenesis and peripheral blood flow in the ischemic limb, and these cells were incorporated into sites of angiogenesis after tissue ischemia (Iwase et al., 2005). MSC transplantation was shown to be a promising approach for restoring tissue vascularization

In a previous study, we demonstrated that allogeneic transplantation of FM-MSCs induced angiogenesis in a rat model of hind-limb ischemia (Ishikane et al., 2008). One day after left common iliac artery resection, FM-MSCs obtained from Lewis rats were transplanted into the ischemic thigh muscle of MHC-mismatched ACI rats with hind-limb ischemia (5 105 cellsanimal). The blood perfusion of the ischemic limb and the capillary density of the ischemic muscle were increased 2 and 3 weeks, respectively, after allogeneic FM-MSC transplantation (Figure 2). It is noteworthy that the therapeutic gain was similar to that of


Table 1. Comparison of the characteristics of FM-MSCs and BM-MSCs observed in our studies. Abbreviations: BM-MSC, bone marrow-derived mesenchymal stem cell; FM-MSC, fetal membrane-derived mesenchymal stem cell; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; MSC, mesenchymal stem cell; MHC, major histocompatibility complex; VEGF, vascular endothelial growth factor.

#### **2.1 Immunomodulatory effect of fetal membrane-derived mesenchymal stem cells**

MSCs have received renewed interest, particularly for their use in transplantation medicine. Although the main driving force responsible for interest in the regenerative capacity of MSCs in the past was their presumptive plasticity, their ability to modulate the immune response is now attracting greater interest. MSCs are positive for major histocompatibility complex (MHC) class I but negative for MHC class II and for costimulatory factors such as CD40, CD80 and CD86, and are therefore considered nonimmunogenic (Chamberlain et al.

Invasive

CD34, CD45, CD73,

MHC class I, MHC class II

In acute myocarditis: induced

Smooth muscle cells: very low Myocardium: very low

Vascular endothelial cells:

very low or none

Low

CD90,

Osteogenic Chondrogenic

induced

Low

Evade

Suppress

Suppress

VEGF, HGF, IGF-1, adrenomedullin

Immunophenotype CD11, CD29, CD31,

Angiogenesis In hind limb ischemia:

In vitro multipotency Adipogenic

**FM-MSC Characteristic BM-MSC** 

procedure

Placenta Donor tissue Adult bone marrow

secretion

In vivo differentiation

effect)

infiltration

Table 1. Comparison of the characteristics of FM-MSCs and BM-MSCs observed in our studies. Abbreviations: BM-MSC, bone marrow-derived mesenchymal stem cell; FM-MSC, fetal membrane-derived mesenchymal stem cell; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; MSC, mesenchymal stem cell; MHC, major histocompatibility

**2.1 Immunomodulatory effect of fetal membrane-derived mesenchymal stem cells** 

MSCs have received renewed interest, particularly for their use in transplantation medicine. Although the main driving force responsible for interest in the regenerative capacity of MSCs in the past was their presumptive plasticity, their ability to modulate the immune response is now attracting greater interest. MSCs are positive for major histocompatibility complex (MHC) class I but negative for MHC class II and for costimulatory factors such as CD40, CD80 and CD86, and are therefore considered nonimmunogenic (Chamberlain et al.

Suppress Fibrosis Suppress

transplanted cells

activation (rejection)

(immunomodulatory

cells

Noninvasive MSC harvest

VEGF, HGF Growth factor

Low Engraftment of

Evade Alloreactive T cell

Suppress Inflammatory cell

complex; VEGF, vascular endothelial growth factor.

Suppress CD4T cell activation

CD11, CD29, CD31, CD34,

In hind limb ischemia: induced In acute myocarditis: not

Vascular endothelial cells: none

Myocardium: none

CD45, CD73, CD90, MHC class I, MHC class II

Adipogenic Osteogenic Chondrogenic

High Number of obtained

2007). Allogeneic BM-MSC transplantation has been used in several preclinical and clinical studies, in which allogeneic MSCs were not rejected in the absence of immunosuppression (Amado et al., 2005; Hare et al., 2009; Le Blanc et al., 2008).

The use of BM-MSCs not only avoids allogeneic rejection but also may confer immunosuppressive effects. Several studies demonstrated that MSCs modulate the function of T cells, major executors of the adaptive immune response (Krampera et al., 2003; Le Blanc et al., 2003). Di Nicola et al. showed that BM-MSCs strongly suppressed T cell proliferation in a mixed lymphocyte culture (MLC) test (Di Nicola et al., 2002).

In our study of rats, FM-MSCs had immunological properties similar to those of BM-MSCs. In an MLC test with haplotype-mismatched allogeneic cells, FM-MSCs did not provoke alloreactive lymphocyte proliferation. Interleukin (IL)-2 plays a role in the activation and proliferation of T cells. IL-2 concentrations in supernatants of FM-MSC and allogeneic lymphocyte co-cultures and in the MLC were lower than those in lymphocyte and allogeneic lymphocyte co-cultures.

To investigate T cell alloreactivity to transplanted allogeneic FM-MSCs, FM-MSCs, BM-MSCs or splenic lymphocytes obtained from GFP-transgenic Lewis rats were injected into the hind-limb tissue of MHC-mismatched August–Copenhagen Irish (ACI) rats. One week after cell injection, slight T cell infiltration was observed at the injection site of allogeneic FM-MSC-injected hind-limb muscles, but the degree of infiltration was less marked than that after allogeneic splenic lymphocyte transplantation and was equivalent to that induced by allogeneic BM-MSCs. Use of non-autologous cells for transplant also requires that one consider the possibility of graft rejection. Although most clinical applications of FM-MSC transplantation apply to allogeneic transplantation, our results suggest that FM-MSCs evade T cell alloreactivity and may be successfully transplanted across MHC barriers.

#### **2.2 Therapeutic angiogenesis in allogeneic fetal membrane-derived mesenchymal stem cell transplantation in a hind-limb ischemia model**

Therapeutic angiogenesis, a strategy to treat tissue ischemia by promoting the proliferation of collateral vessels, has emerged as one of the most promising therapies developed to date (Carmeliet, 2003). In a rat model of hind-limb ischemia, autologous BM-MSC transplantation enhanced angiogenesis and peripheral blood flow in the ischemic limb, and these cells were incorporated into sites of angiogenesis after tissue ischemia (Iwase et al., 2005). MSC transplantation was shown to be a promising approach for restoring tissue vascularization after ischemic events (Moon et al., 2006; Nakagami et al., 2005).

In a previous study, we demonstrated that allogeneic transplantation of FM-MSCs induced angiogenesis in a rat model of hind-limb ischemia (Ishikane et al., 2008). One day after left common iliac artery resection, FM-MSCs obtained from Lewis rats were transplanted into the ischemic thigh muscle of MHC-mismatched ACI rats with hind-limb ischemia (5 105 cellsanimal). The blood perfusion of the ischemic limb and the capillary density of the ischemic muscle were increased 2 and 3 weeks, respectively, after allogeneic FM-MSC transplantation (Figure 2). It is noteworthy that the therapeutic gain was similar to that of allogeneic BM-MSC transplantation.

Therapeutic Application of Allogeneic Fetal Membrane-Derived

severe peripheral vascular disease.

allogeneic FM-MSCs *in vitro*.

2010).

Mesenchymal Stem Cell Transplantation in Regenerative Medicine 227

secreting large amounts of humoral factors involved in angiogenesis, such as VEGF and HGF (Kinnaird et al., 2004; Nagaya et al., 2005). VEGF is one of the more powerful angiogenic cytokines and can also mobilize endothelial progenitor cells (EPCs) from BM and inhibit EPC apoptosis (Asahara et al., 1999). HGF plays important roles in tissue regeneration, morphogenesis and angiogenesis (Zarnegar and Michalopoulos, 1995). HGF is thought to stimulate endothelial cell proliferation and to induce angiogenesis, and is a key signaling factor that promotes infiltration of circulating stem cells from the peripheral circulation to an ischemic area (Morishita et al., 1999; Weimar et al., 1998). Further studies are needed to improve the availability of transplanted MSCs for engraftment, but allogeneic FM-MSC transplantation could provide a new therapeutic strategy for the treatment of

**2.3 Immunomodulatory effect of allogeneic fetal membrane-derived mesenchymal** 

Several studies reported that MSCs have immunomodulatory effects mediated by secretion of soluble factors such as prostaglandin E2, indoleamine 2,3-dioxygenase, IL-6, IL-10, heme oxygenase-1 and galectin (Aggarwal and Pittenger, 2005; Chabannes et al., 2007; Meisel et al., 2004; Sioud et al., 2011). Based on the immunomodulatory property of MSCs, allogeneic FM-MSC transplantation may be an attractive treatment for autoimmune myocarditis.

Experimental autoimmune myocarditis (EAM) is induced by injecting porcine cardiac myosin in Lewis rats. Allogeneic FM-MSCs obtained from MHC-mismatched ACI rats (5 105 cellsanimal) were transplanted intravenously into EAM rats 1 week after myosin injection. Two weeks after transplantation, the intravenous allogeneic transplantation of FM-MSCs reduced fibrosis, edema, necrosis, granulation and eosinophil infiltration in hearts exhibiting EAM and significantly attenuated infiltration of inflammatory cells (CD68 positive monocytes and macrophages) and MCP-1 expression in the myocardium (Figure 4A and B). Hemodynamic and echocardiographic tests showed a significant improvement in cardiac function as a result of allogeneic FM-MSC transplantation (Ishikane et al., 2010). The extent of the improvement ranged from 30% to 60 according to various indices of the level of dysfunction, which is equivalent to that observed in our previous study on autologous BM-MSC transplantation in EAM (Ohnishi et al., 2007). Allogeneic transplantation of FM-MSCs significantly reduced infiltration of T cells (CD3-positive cells) into EAM hearts (Figure 4C). In a T lymphocyte proliferation assay, splenic T lymphocytes collected from allogeneic FM-MSC-transplanted EAM rats had a reduced proliferative response to myosin compared with the response of splenic T lymphocytes from untransplanted EAM rats. In addition, proliferation of activated T lymphocytes was suppressed by co-culture with

Okada et al. reported that Th2-type cytokine expression in EAM was increased by HGF, whereas Th1-type cytokine expression was suppressed by intramyocardial transplantation of autologous BM-MSCs (Okada et al., 2007). An increase in HGF expression may reduce the severity of EAM by suppressing the Th1 response. Van Linthout et al. reported that MSCs improved murine acute coxsackievirus B3-induced myocarditis via their immunomodulatory properties in a nitric oxide-dependent manner (Van Linthout et al.,

**stem cell transplantation in an autoimmune myocarditis model** 

Fig. 2. Comparison of angiogenesis after allogeneic FM-MSC and BM-MSC transplantation in rats with hindlimb ischemia

(A) Representative examples of serial A laser doppler perfusion image (LDPI). Blood perfusion of the ischemic hindlimb was markedly increased in the allogeneic FM-MSCs and BM-MSCs transplanted group 3 weeks after cell injection (red to orange). (B) Quantitative analysis of hindlimb blood perfusion. LDPI index was significantly higher in the allogeneic FM-MSCs and BM-MSCs transplanted groups than in the phosphate-buffer saline (PBS) treated control group 3 weeks after cell injection. The LDPI index was determined as the ratio of ischemic to nonischemic hindlimb blood perfusion. Data are mean ± S.E.M. \**P* 0.05 FM-MSC vs. PBS; †*P* 0.05 BM-MSC vs. PBS.

The allogeneic FM- and BM-MSCs in the ischemic hind-limb tissue survived for 3 weeks after transplantation, but the number of engrafted cells decreased significantly in both cases (Figure 3). In a previous trial, intramuscularly transplanted allogeneic BM-MSCs were observed 6 months after transplantation (Dai et al., 2005). In other studies, the number of engrafted autologous and allogeneic MSCs gradually decreased, and MSCs were absent after several weeks (Fouillard et al., 2007; Kraitchman et al., 2005; Shake et al., 2002). Muller-Ehmsen et al. reported the observed transplanted MSC loss was predominantly caused by cell death rather than migration of cells to other organs (Muller-Ehmsen et al., 2006).

To investigate differentiation of transplanted FM-MSCs into blood vessel endothelial cells, we performed immunofluorescent staining of MSC-transplanted ischemic hind-limb sections. GFP-positive transplanted FM-MSCs and BM-MSCs and lectin-positive endothelial cells were observed in hind-limb tissue, but GFPlectin double-positive cells were not observed. Some studies reported that transplanted BM-MSCs directly differentiated into the vascular endothelial cells and vascular smooth muscles in ischemic models (Al-Khaldi et al., 2003; Moon et al., 2006). However, recent studies demonstrated that the direct contribution of grafted MSCs is minimal or even absent, and that paracrine actions are of major importance in mediating their regenerative effects (Aranguren et al., 2008; Au et al., 2008; Muller-Ehmsen et al., 2006). MSCs were considered to induce neovascularization by

Fig. 2. Comparison of angiogenesis after allogeneic FM-MSC and BM-MSC transplantation

The allogeneic FM- and BM-MSCs in the ischemic hind-limb tissue survived for 3 weeks after transplantation, but the number of engrafted cells decreased significantly in both cases (Figure 3). In a previous trial, intramuscularly transplanted allogeneic BM-MSCs were observed 6 months after transplantation (Dai et al., 2005). In other studies, the number of engrafted autologous and allogeneic MSCs gradually decreased, and MSCs were absent after several weeks (Fouillard et al., 2007; Kraitchman et al., 2005; Shake et al., 2002). Muller-Ehmsen et al. reported the observed transplanted MSC loss was predominantly caused by

To investigate differentiation of transplanted FM-MSCs into blood vessel endothelial cells, we performed immunofluorescent staining of MSC-transplanted ischemic hind-limb sections. GFP-positive transplanted FM-MSCs and BM-MSCs and lectin-positive endothelial cells were observed in hind-limb tissue, but GFPlectin double-positive cells were not observed. Some studies reported that transplanted BM-MSCs directly differentiated into the vascular endothelial cells and vascular smooth muscles in ischemic models (Al-Khaldi et al., 2003; Moon et al., 2006). However, recent studies demonstrated that the direct contribution of grafted MSCs is minimal or even absent, and that paracrine actions are of major importance in mediating their regenerative effects (Aranguren et al., 2008; Au et al., 2008; Muller-Ehmsen et al., 2006). MSCs were considered to induce neovascularization by

cell death rather than migration of cells to other organs (Muller-Ehmsen et al., 2006).

(A) Representative examples of serial A laser doppler perfusion image (LDPI). Blood perfusion of the ischemic hindlimb was markedly increased in the allogeneic FM-MSCs and BM-MSCs transplanted group 3 weeks after cell injection (red to orange). (B) Quantitative analysis of hindlimb blood perfusion. LDPI index was significantly higher in the allogeneic FM-MSCs and BM-MSCs transplanted groups than in the phosphate-buffer saline (PBS) treated control group 3 weeks after cell injection. The LDPI index was determined as the ratio of ischemic to nonischemic hindlimb blood perfusion. Data are mean ± S.E.M. \**P* 0.05

in rats with hindlimb ischemia

FM-MSC vs. PBS; †*P* 0.05 BM-MSC vs. PBS.

secreting large amounts of humoral factors involved in angiogenesis, such as VEGF and HGF (Kinnaird et al., 2004; Nagaya et al., 2005). VEGF is one of the more powerful angiogenic cytokines and can also mobilize endothelial progenitor cells (EPCs) from BM and inhibit EPC apoptosis (Asahara et al., 1999). HGF plays important roles in tissue regeneration, morphogenesis and angiogenesis (Zarnegar and Michalopoulos, 1995). HGF is thought to stimulate endothelial cell proliferation and to induce angiogenesis, and is a key signaling factor that promotes infiltration of circulating stem cells from the peripheral circulation to an ischemic area (Morishita et al., 1999; Weimar et al., 1998). Further studies are needed to improve the availability of transplanted MSCs for engraftment, but allogeneic FM-MSC transplantation could provide a new therapeutic strategy for the treatment of severe peripheral vascular disease.

#### **2.3 Immunomodulatory effect of allogeneic fetal membrane-derived mesenchymal stem cell transplantation in an autoimmune myocarditis model**

Several studies reported that MSCs have immunomodulatory effects mediated by secretion of soluble factors such as prostaglandin E2, indoleamine 2,3-dioxygenase, IL-6, IL-10, heme oxygenase-1 and galectin (Aggarwal and Pittenger, 2005; Chabannes et al., 2007; Meisel et al., 2004; Sioud et al., 2011). Based on the immunomodulatory property of MSCs, allogeneic FM-MSC transplantation may be an attractive treatment for autoimmune myocarditis.

Experimental autoimmune myocarditis (EAM) is induced by injecting porcine cardiac myosin in Lewis rats. Allogeneic FM-MSCs obtained from MHC-mismatched ACI rats (5 105 cellsanimal) were transplanted intravenously into EAM rats 1 week after myosin injection. Two weeks after transplantation, the intravenous allogeneic transplantation of FM-MSCs reduced fibrosis, edema, necrosis, granulation and eosinophil infiltration in hearts exhibiting EAM and significantly attenuated infiltration of inflammatory cells (CD68 positive monocytes and macrophages) and MCP-1 expression in the myocardium (Figure 4A and B). Hemodynamic and echocardiographic tests showed a significant improvement in cardiac function as a result of allogeneic FM-MSC transplantation (Ishikane et al., 2010). The extent of the improvement ranged from 30% to 60 according to various indices of the level of dysfunction, which is equivalent to that observed in our previous study on autologous BM-MSC transplantation in EAM (Ohnishi et al., 2007). Allogeneic transplantation of FM-MSCs significantly reduced infiltration of T cells (CD3-positive cells) into EAM hearts (Figure 4C). In a T lymphocyte proliferation assay, splenic T lymphocytes collected from allogeneic FM-MSC-transplanted EAM rats had a reduced proliferative response to myosin compared with the response of splenic T lymphocytes from untransplanted EAM rats. In addition, proliferation of activated T lymphocytes was suppressed by co-culture with allogeneic FM-MSCs *in vitro*.

Okada et al. reported that Th2-type cytokine expression in EAM was increased by HGF, whereas Th1-type cytokine expression was suppressed by intramyocardial transplantation of autologous BM-MSCs (Okada et al., 2007). An increase in HGF expression may reduce the severity of EAM by suppressing the Th1 response. Van Linthout et al. reported that MSCs improved murine acute coxsackievirus B3-induced myocarditis via their immunomodulatory properties in a nitric oxide-dependent manner (Van Linthout et al., 2010).

Therapeutic Application of Allogeneic Fetal Membrane-Derived

Mesenchymal Stem Cell Transplantation in Regenerative Medicine 229

Fig. 4. Histopathological changes in autoimmune myocarditis at 2 weeks after

decreased in the allogeneic FM-MSCs transplanted group. (B) CD68-positive

transplantation induced by transplantation of allogeneic FM-MSCs. (A) Myocardial sections showed markedly less inflammation in the allogeneic FM-MSCs transplanted group than in the untransplanted myocarditis group. Insets are transverse sections of the myocardium. The semiquantitative histological grade of edema and eosinophil infiltration were markedly

macrophage/monocyte infiltration, and (C) CD3-positive T cell infiltration were markedly reduced by allogeneic FM-MSC transplantation. Scale bars = 50 m. Data are expressed as mean ± SEM. \**P* < 0.05 vs. the sham group; †*P* < 0.05 vs. the untreated myocarditis group.

Fig. 3. Engraftment of allogeneic FM-MSCs and BM-MSCs injected into ischemic hindlimb muscles. (A) Representative sections show that GFP-positive allogeneic FM-MSCs and BM-MSCs were present in the hindlimb muscles of rats with hindlimb ischemia 1 and 3 weeks after cell injection (brown stain; black arrows). Scale bars: 50 m. (B) Quantitative analysis

allogeneic BM-MSCs were observed in ischemic hindlimbs 1 week after cell injection. Three weeks after cell injection, a few GFP-positive allogeneic FM-MSCs and BM-MSCs were

demonstrated that comparable numbers of GFP-positive allogeneic FM-MSCs and

observed. Data are mean ± S.E.M.

Fig. 4. Histopathological changes in autoimmune myocarditis at 2 weeks after transplantation induced by transplantation of allogeneic FM-MSCs. (A) Myocardial sections showed markedly less inflammation in the allogeneic FM-MSCs transplanted group than in the untransplanted myocarditis group. Insets are transverse sections of the myocardium. The semiquantitative histological grade of edema and eosinophil infiltration were markedly decreased in the allogeneic FM-MSCs transplanted group. (B) CD68-positive macrophage/monocyte infiltration, and (C) CD3-positive T cell infiltration were markedly reduced by allogeneic FM-MSC transplantation. Scale bars = 50 m. Data are expressed as mean ± SEM. \**P* < 0.05 vs. the sham group; †*P* < 0.05 vs. the untreated myocarditis group.

Therapeutic Application of Allogeneic Fetal Membrane-Derived

and autoimmune myocarditis.

**5. Acknowledgments** 

EGFP)1Ys.

**6. References** 

0006-4971

pp.204-209, ISSN 0003-4975

(February 2007), pp.11, ISSN 1471-213X

Mesenchymal Stem Cell Transplantation in Regenerative Medicine 231

alloreactive T lymphocyte proliferation, and allogeneic FM-MSC transplantation induced therapeutic angiogenesis in a rat model of hind-limb ischemia. The angiogenic effects may be induced in a paracrine manner rather than via vascular differentiation of the transplanted MSCs. It is expected that allogeneic FM-MSC transplantation will be an effective therapy for autoimmune myocarditis with rapidly progressive heart failure. The beneficial effects of allogeneic FM-MSC transplantation are mainly attributable to suppression of T lymphocyte activation and anti-inflammatory effects. FM are potentially promising cell source for clinical use; they are medical waste material, are abundantly available from maternity wards. The unlimited availability of term gestational tissue, large number of cell that can be isolated from FM without invasive procedures, minimal ethical and legal barriers associated with their usage and immune tolerance make these cells highly attractive for stem cell based regenerative and reparative medicine and tissue engineering. Meanwhile, the risk of tumor formation from transplanting allogeneic FM-MSC into patients remains undetermined, and long-term follow-up studies are needed to clarify safety. Although further experiments are needed to adapt the current results for clinical application, we predict that allogeneic FM-MSC transplantation therapy will become a treatment for severe peripheral vascular disease

Our studies were supported by a Research Grant for Cardiovascular Disease (18C-1) and Human Genome Tissue Engineering 009 from the Ministry of Health, Labor and Welfare. We are grateful to Dr Kenichi Yamahara, Dr Makoto Kodama, Dr Hatsue Ishibashi-Ueda, Dr Shunsuke Ohnishi and Dr Noritoshi Nagaya for their support of our studies. We are thankful to the National BioResource Project for the Rat in Japan (http://www.anim.med.kyoto-u.ac.jp/NBR/) for providing rat strain LEW-TgN(CAG-

Aggarwal, S., & Pittenger, M. F. (2005). Human mesenchymal stem cells modulate allogeneic

Al-Khaldi, A.; Al-Sabti, H.; Galipeau, J., & Lachapelle, K. (2003). Therapeutic angiogenesis

Alviano, F.; Fossati, V.; Marchionni, C.; Arpinati, M.; Bonsi, L.; Franchina, M.; Lanzoni, G.;

Amado, L. C.; Saliaris, A. P.; Schuleri, K. H.; St John, M.; Xie, J. S.; Cattaneo, S.; Durand, D. J.;

immune cell responses. *Blood*, Vol.105, No.4, (October 2004), pp.1815-1822, ISSN

using autologous bone marrow stromal cells: improved blood flow in a chronic limb ischemia model. *Annals of Thoracic Surgery*, Vol.75, No.1, (January 2003),

Cantoni, S.; Cavallini, C.; Bianchi, F.; Tazzari, P. L.; Pasquinelli, G.; Foroni, L.; Ventura, C.; Grossi, A., & Bagnara, G. P. (2007). Term Amniotic membrane is a high throughput source for multipotent Mesenchymal Stem Cells with the ability to differentiate into endothelial cells in vitro. *BMC Developmental Biology*, Vol.7,

Fitton, T.; Kuang, J. Q.; Stewart, G.; Lehrke, S.; Baumgartner, W. W.; Martin, B. J.; Heldman, A. W., & Hare, J. M. (2005). Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. *Proceedings of the* 

Allogeneic transplantation of FM-MSCs may be an attractive therapy for the treatment of autoimmune myocarditis. Further studies are needed to elucidate the therapeutic mechanisms.

#### **3. Potential of mesenchymal stem cell sheet transplantation therapy**

As discussed above, MSC transplantation has attractive possibilities as a tool for cell transplantation therapy. However, further experiments are needed to develop data obtained with MSCs for application to humans because evidence of an ameliorating effect on angiogenesis and cardiac function is not necessarily sufficient to warrant clinical use. To date, intramuscular and intravenous injections have been used for cell transplantation therapy, but the engraftment rate of MSCs transplanted via these routes was very low (Ishikane et al. 2008, 2010). Although intramuscularly transplanted allogeneic FM-MSCs survived in ischemic hindlimb tissue for 3 weeks after transplantation, the number of engrafted cells decreased significantly. In EAM, some of the intravenously transplanted MSCs were found in the lung, heart, spleen and liver 1 week after transplantation, but these engrafted cells could not be detected 4 weeks after transplantation. Most homing and engraftment studies demonstrated little, if any, long-term (1 week) engraftment of MSCs after systemic administration (Parekkadan and Milwid, 2010). Studies showed that the majority of administered MSCs (80) immediately accumulate in the lung and are cleared with a half-life of 24 h. Although intravenous cell transplantation is very convenient, it is not suitable for transplantation of large numbers of cells. Thus, a more effective transplantation route is needed to enhance angiogenesis and cardiac functional improvement in MSC transplantation.

Recently, cell sheet engineering received attention as a method for heart tissue repair. Okano et al. developed engineered cell sheets containing scaffoldless tissue using temperatureresponsive culture dishes (Yamada et al., 1990). These cell sheets enable cell-to-cell connections and maintain the presence of adhesion proteins. The cell sheets preserve extracellular matrix proteins deposited on the basal surface of the cultured cells. These adhesive proteins play an important role in enhancing attachment between stacked cell sheets and between cell sheets and the myocardial surface, thereby enabling stable fixation of the cell sheet constructs to the target tissues. The cell sheets can readily be transferred and grafted to scarred myocardium without additives or suturing. Memon et al. demonstrated that layered skeletal myoblast sheets transplanted to infarcted rat hearts enhanced left ventricular contraction, reduced fibrosis and prevented left ventricular dilation (Memon et al., 2005). Kondoh et al. showed that in hamsters with dilated cardiomyopathy, myoblast sheet graft implantation improved cardiac performance and prolonged life expectancy in association with a reduction in myocardial fibrosis (Kondoh et al., 2006). In our study on rats, adipose tissue-derived MSC sheets improved cardiac function in damaged hearts, with reversal of cardiac wall thinning and prolonged survival after myocardial infarction (Miyahara et al., 2006). These cell sheets enable transplantation of many more cells than with intramuscular or intravenous needle injection. MSC sheet transplantation is expected to increase the number of engrafted cells and to enhance paracrine signaling.

#### **4. Conclusion**

This review shows the potential of allogeneic transplantation of FM-MSCs for the treatment of peripheral vascular disease and autoimmune myocarditis. FM-MSCs did not elicit alloreactive T lymphocyte proliferation, and allogeneic FM-MSC transplantation induced therapeutic angiogenesis in a rat model of hind-limb ischemia. The angiogenic effects may be induced in a paracrine manner rather than via vascular differentiation of the transplanted MSCs. It is expected that allogeneic FM-MSC transplantation will be an effective therapy for autoimmune myocarditis with rapidly progressive heart failure. The beneficial effects of allogeneic FM-MSC transplantation are mainly attributable to suppression of T lymphocyte activation and anti-inflammatory effects. FM are potentially promising cell source for clinical use; they are medical waste material, are abundantly available from maternity wards. The unlimited availability of term gestational tissue, large number of cell that can be isolated from FM without invasive procedures, minimal ethical and legal barriers associated with their usage and immune tolerance make these cells highly attractive for stem cell based regenerative and reparative medicine and tissue engineering. Meanwhile, the risk of tumor formation from transplanting allogeneic FM-MSC into patients remains undetermined, and long-term follow-up studies are needed to clarify safety. Although further experiments are needed to adapt the current results for clinical application, we predict that allogeneic FM-MSC transplantation therapy will become a treatment for severe peripheral vascular disease and autoimmune myocarditis.

#### **5. Acknowledgments**

230 Tissue Regeneration – From Basic Biology to Clinical Application

Allogeneic transplantation of FM-MSCs may be an attractive therapy for the treatment of autoimmune myocarditis. Further studies are needed to elucidate the therapeutic

As discussed above, MSC transplantation has attractive possibilities as a tool for cell transplantation therapy. However, further experiments are needed to develop data obtained with MSCs for application to humans because evidence of an ameliorating effect on angiogenesis and cardiac function is not necessarily sufficient to warrant clinical use. To date, intramuscular and intravenous injections have been used for cell transplantation therapy, but the engraftment rate of MSCs transplanted via these routes was very low (Ishikane et al. 2008, 2010). Although intramuscularly transplanted allogeneic FM-MSCs survived in ischemic hindlimb tissue for 3 weeks after transplantation, the number of engrafted cells decreased significantly. In EAM, some of the intravenously transplanted MSCs were found in the lung, heart, spleen and liver 1 week after transplantation, but these engrafted cells could not be detected 4 weeks after transplantation. Most homing and engraftment studies demonstrated little, if any, long-term (1 week) engraftment of MSCs after systemic administration (Parekkadan and Milwid, 2010). Studies showed that the majority of administered MSCs (80) immediately accumulate in the lung and are cleared with a half-life of 24 h. Although intravenous cell transplantation is very convenient, it is not suitable for transplantation of large numbers of cells. Thus, a more effective transplantation route is needed to enhance

Recently, cell sheet engineering received attention as a method for heart tissue repair. Okano et al. developed engineered cell sheets containing scaffoldless tissue using temperatureresponsive culture dishes (Yamada et al., 1990). These cell sheets enable cell-to-cell connections and maintain the presence of adhesion proteins. The cell sheets preserve extracellular matrix proteins deposited on the basal surface of the cultured cells. These adhesive proteins play an important role in enhancing attachment between stacked cell sheets and between cell sheets and the myocardial surface, thereby enabling stable fixation of the cell sheet constructs to the target tissues. The cell sheets can readily be transferred and grafted to scarred myocardium without additives or suturing. Memon et al. demonstrated that layered skeletal myoblast sheets transplanted to infarcted rat hearts enhanced left ventricular contraction, reduced fibrosis and prevented left ventricular dilation (Memon et al., 2005). Kondoh et al. showed that in hamsters with dilated cardiomyopathy, myoblast sheet graft implantation improved cardiac performance and prolonged life expectancy in association with a reduction in myocardial fibrosis (Kondoh et al., 2006). In our study on rats, adipose tissue-derived MSC sheets improved cardiac function in damaged hearts, with reversal of cardiac wall thinning and prolonged survival after myocardial infarction (Miyahara et al., 2006). These cell sheets enable transplantation of many more cells than with intramuscular or intravenous needle injection. MSC sheet transplantation is expected to

**3. Potential of mesenchymal stem cell sheet transplantation therapy** 

angiogenesis and cardiac functional improvement in MSC transplantation.

increase the number of engrafted cells and to enhance paracrine signaling.

This review shows the potential of allogeneic transplantation of FM-MSCs for the treatment of peripheral vascular disease and autoimmune myocarditis. FM-MSCs did not elicit

mechanisms.

**4. Conclusion** 

Our studies were supported by a Research Grant for Cardiovascular Disease (18C-1) and Human Genome Tissue Engineering 009 from the Ministry of Health, Labor and Welfare. We are grateful to Dr Kenichi Yamahara, Dr Makoto Kodama, Dr Hatsue Ishibashi-Ueda, Dr Shunsuke Ohnishi and Dr Noritoshi Nagaya for their support of our studies. We are thankful to the National BioResource Project for the Rat in Japan (http://www.anim.med.kyoto-u.ac.jp/NBR/) for providing rat strain LEW-TgN(CAG-EGFP)1Ys.

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**11** 

*USA* 

Arshak R. Alexanian

*Milwaukee, Wisconsin,* 

**Mesenchymal Stem Cells in CNS Regeneration** 

**1.1 Mesenchymal stem cells as an ideal source of cells for regenerative medicine** 

rejection still remains one of the important issues for regenerative medicine.

manipulated more easily (Niibe et al., 2011).

During the last two decades, stem cells have become recognized as a promising tool for various biomedical applications including disease modeling, drug development, and cell replacement therapies. However, identification of the reliable sources of stem cells that can be easily harvested, expanded on a large enough scale, and carry no risk of immune

Mesenchymal stem cells (MSCs) are promising tools for cell therapy (Zeidan-Chulia & Noda, 2009) by autologous and allogeneic transplantation for two significant reasons. Firstly, MSC can easily be isolated and expanded from different adult and postnatal tissues, such as BM (Prockop, 1997), peripheral blood (Kuznetsov et al., 2001), muscle (J. Y. Lee et al., 2000), vasculature (Brighton et al., 1992), skin (Mizuno & Glowacki, 1996), adipose tissue (Zuk et al., 2001) and umbilical cord (O. K. Lee et al., 2004). Secondly, MSCs can differentiate into multiple cell types of mesodermal, endodermal, and epidermal origin such as bone (Pereira et al., 1995), cartilage (Pereira et al., 1998), fat (Umezawa et al., 1991), muscle (Ferrari et al., 1998), cardiomyocytes (Makino et al., 1999), and neurons (Kohyama et al., 2001). Such a surprising high plasticity of MSCs might be explained by the expression of a variety of gene families in undifferentiated MSCs. Several recent studies have shown that MSCs express several embryonic stem cell markers (pluripotent markers) such as Oct4, Nanog, alkaline phosphatase and SSEA-4, and SOX2 (Park & Patel, 2010; Pierantozzi et al., 2011; Riekstina et al., 2009). It also has been demonstrated that the translational and transcriptional machinery in MSCs responsible for the expression of multiple genes typical of several derivatives of three germ layers are not silenced, rather operating at the low level (Blondheim et al., 2006; Tondreau et al., 2008). Most importantly, at appropriate environmental conditions in vitro and in vivo MSCs can upregulate the expression of these genes and exhibit several characteristics of mature cells of different tissues such as heart (Choi, Kurtz, & Stamm, 2011; Hattan et al., 2005; Makino et al., 1999), lever (Stock et al., 2010) and central nervous system (Alexanian, 2010). While it still many controversy concerning transdifferentiation of MSCs these recent data suggest that MSCs could be ideal autologous source of easily reprogrammable cells. Harboring such a high plasticity these cells, in contrast to adult and other tissue specific stem cells and progenitors, could be

**1. Introduction** 

*Medical College of Wisconsin, Neuroscience Research Laboratories, Department of Neurosurgery, VA Medical Center - Research 151,* 


### **Mesenchymal Stem Cells in CNS Regeneration**

#### Arshak R. Alexanian

*Medical College of Wisconsin, Neuroscience Research Laboratories, Department of Neurosurgery, VA Medical Center - Research 151, Milwaukee, Wisconsin, USA* 

#### **1. Introduction**

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#### **1.1 Mesenchymal stem cells as an ideal source of cells for regenerative medicine**

During the last two decades, stem cells have become recognized as a promising tool for various biomedical applications including disease modeling, drug development, and cell replacement therapies. However, identification of the reliable sources of stem cells that can be easily harvested, expanded on a large enough scale, and carry no risk of immune rejection still remains one of the important issues for regenerative medicine.

Mesenchymal stem cells (MSCs) are promising tools for cell therapy (Zeidan-Chulia & Noda, 2009) by autologous and allogeneic transplantation for two significant reasons. Firstly, MSC can easily be isolated and expanded from different adult and postnatal tissues, such as BM (Prockop, 1997), peripheral blood (Kuznetsov et al., 2001), muscle (J. Y. Lee et al., 2000), vasculature (Brighton et al., 1992), skin (Mizuno & Glowacki, 1996), adipose tissue (Zuk et al., 2001) and umbilical cord (O. K. Lee et al., 2004). Secondly, MSCs can differentiate into multiple cell types of mesodermal, endodermal, and epidermal origin such as bone (Pereira et al., 1995), cartilage (Pereira et al., 1998), fat (Umezawa et al., 1991), muscle (Ferrari et al., 1998), cardiomyocytes (Makino et al., 1999), and neurons (Kohyama et al., 2001). Such a surprising high plasticity of MSCs might be explained by the expression of a variety of gene families in undifferentiated MSCs. Several recent studies have shown that MSCs express several embryonic stem cell markers (pluripotent markers) such as Oct4, Nanog, alkaline phosphatase and SSEA-4, and SOX2 (Park & Patel, 2010; Pierantozzi et al., 2011; Riekstina et al., 2009). It also has been demonstrated that the translational and transcriptional machinery in MSCs responsible for the expression of multiple genes typical of several derivatives of three germ layers are not silenced, rather operating at the low level (Blondheim et al., 2006; Tondreau et al., 2008). Most importantly, at appropriate environmental conditions in vitro and in vivo MSCs can upregulate the expression of these genes and exhibit several characteristics of mature cells of different tissues such as heart (Choi, Kurtz, & Stamm, 2011; Hattan et al., 2005; Makino et al., 1999), lever (Stock et al., 2010) and central nervous system (Alexanian, 2010). While it still many controversy concerning transdifferentiation of MSCs these recent data suggest that MSCs could be ideal autologous source of easily reprogrammable cells. Harboring such a high plasticity these cells, in contrast to adult and other tissue specific stem cells and progenitors, could be manipulated more easily (Niibe et al., 2011).

Mesenchymal Stem Cells in CNS Regeneration 239

generation of neural cells from feline and human BM-derived MSCs (hMSC) (Alexanian, 2010; Z. Zhang, Maiman, Kurpad, Crowe, & Alexanian, 2011). In these studies, neural induction was achieved by exposing cells simultaneously to inhibitors of DNA methylation and histone deacetylation and pharmacological agents that increased cAMP levels. The main idea of this methodological approach was the reactivation of pluripotency-associated genes in MSCs simultaneously exposing them to neural-inducing factors. Neurally modified MSCs by this methodology, in contrast to naïve MSCs, express several neural progenitor and mature neural markers demonstrated by real time RT-PCR, western blot, ELISA and

> **B3T GFAP DAPI**

> **B3T GFAP DAPI**

**B3T GFAP DAPI** 

**<sup>e</sup> <sup>f</sup> g h**

**a b c d**

**<sup>i</sup> j k <sup>l</sup>**

**<sup>m</sup> <sup>n</sup> o p** 

**B3T GFAP DAPI** 

**B3T GFAP DAPI** 

**q r s**

Fig. 1. Expression of neural markers nestin, Sox2, A2B5, NCAM, B3T, GFAP, MAP-2, and NeuN in hMSCs (a-d) and NI-hMSC grown 24h, 1, 2, 3 weeks in neural induction medium (e-t). NI-hMSCs grown an additional week in neuronal induction medium were generated

**MAP2 NeuN DAPI** 

**MAP2 NeuN DAPI** 

**MAP2 NeuN DAPI**

**MAP2 NeuN DAPI** 

**MAP2 NeuN DAPI** 

**t** 

**B3T MAP2 DAPI** 

**v u w** 

immunocytochemistry Fig.1. and Fig.2.

**DAPI A2B5** 

**A2B5 NCAM DAPI** 

**NCAM DAPI** 

**A2B5 NCAM DAPI** 

**A2B5 NCAM DAPI** 

**A2B5 NCAM DAPI** 

> **B3T MAP2 DAPI**

cells with long axon- and dendrite-like extensions (v-w). Bars 40um.

**Nestin Sox2 DAPI** 

**Nestin Sox2 DAPI Nestin Sox2** 

**Nestin Sox2 DAPI** 

**Nestin Sox2 DAPI** 

**Nestin Sox2 DAPI** 

**B3T MAP2 DAPI** 

With the promise that MSCs present for the development of new cell therapies, researchers have pursued a broad range of investigations for their therapeutic utilization (Parekkadan & Milwid; Picinich, Mishra, Glod, & Banerjee, 2007; Stappenbeck & Miyoshi, 2009; Wang, Liao, & Tan, 2011). During the last two decades an overwhelming amount of basic and preclinical research has been accumulated that demonstrates the therapeutic usefulness of MSCs in the treatment of several diseases and injuries such as neurodegenerative diseases (Joyce et al.), spinal cord and brain injuries (Y. Jiang et al.), cardiovascular diseases (Trivedi, Tray, Nguyen, Nigam, & Gallicano), diabetes mellitus (Y. H. Zhang et al., 2009) and diseases of the skeleton (Chanda, Kumar, & Ponnazhagan). In most of these studies, treatment with MSCs results in substantial functional benefit and these pre-clinical studies have led to the initiation of a number of clinical trials worldwide.

MSCs have been used in clinical trials since 1995 and, currently, more than 180 trials are registered with ClinicalTrials.gov for the treatment of several diseases including numerous neurological disorders and injuries such amyotrophic lateral sclerosis, stroke, parkinson's disease, Alzheimer's disease, brain and spinal cord injuries.

#### **2. In vitro neural differentiation potential of MSCs**

Demonstration of neural differentiation potential of MSCs in several in vitro and in vivo studies suggests the potential usefulness of MSCs in the treatment of various CNS disorders. This potential has led to extensive studies to further explore the neural plasticity of these cells (Azizi, Stokes, Augelli, DiGirolamo, & Prockop, 1998; Kopen, Prockop, & Phinney, 1999; Munoz-Elias, Marcus, Coyne, Woodbury, & Black, 2004).

During the last several years, numerous in vitro neural induction protocols to produce neural cells from MSCs have been reported. In most induction experiments, MSCs were simply exposed to growth factors, neurotrophic factors or factors favoring neural cell differentiation (Bi et al., 2010; M. Chen et al., 2000; Q. Chen et al., 2005; Joannides et al., 2003; B. J. Kim, Seo, Bubien, & Oh, 2002; S. S. Kim et al., 2005; Kondo, Johnson, Yoder, Romand, & Hashino, 2005; Lim et al., 2008; Long, Olszewski, Huang, & Kletzel, 2005; Padovan et al., 2003; Sanchez-Ramos et al., 2000; Zeng et al., 2011). Other studies have used different culture media, supplemented with individual or various combinations of chemical and pharmacological agents, such as DMSO, b-mercaptoethanol, 5-bromo-2-deoxyuridine (BrdU), butylated hydroxyanisole, forskolin, and dibutyryl cyclic AMP (Ankeny, McTigue, & Jakeman, 2004; W. Deng, Obrocka, Fischer, & Prockop, 2001; Episkopou, 2005; Hermann et al., 2006; Jori et al., 2005; S. S. Kim et al., 2005; Lu, Blesch, & Tuszynski, 2004; Munoz-Elias, Woodbury, & Black, 2003; Tio, Tan, Lee, Wang, & Udolph, 2010; Yang, Wu, & Xiao, 2005; L. Zhang, Seitz, Abramczyk, Liu, & Chan, 2011). Other methods to induce MSCs into cells with neural characteristics include: transfection of MSCs with Noggin and Notch transcription factors (Dezawa et al., 2004; Kohyama et al., 2001); manipulation with surface proteins of culture substrate (Qian & Saltzman, 2004); co-culturing MSCs with NSCs or neural cells (Alexanian, 2005; Chu, Yu, Zhang, & Yu, 2008; Krampera et al., 2007; Wislet-Gendebien et al., 2005; Y. Q. Zhang et al., 2010); and growing MSCs as spheres in cultures (Shiota et al., 2007), transfection of MSCs with microRNA-9 (Jing et al., 2011). In several other studies, MSCs were turned into multipotent stage and then induced into neural cell lineages, by exposing them to appropriate neural differentiation conditions (Alexanian, 2007; Kohyama et al., 2001; Qu et al., 2004). Recently, we proposed an original method for efficient

With the promise that MSCs present for the development of new cell therapies, researchers have pursued a broad range of investigations for their therapeutic utilization (Parekkadan & Milwid; Picinich, Mishra, Glod, & Banerjee, 2007; Stappenbeck & Miyoshi, 2009; Wang, Liao, & Tan, 2011). During the last two decades an overwhelming amount of basic and preclinical research has been accumulated that demonstrates the therapeutic usefulness of MSCs in the treatment of several diseases and injuries such as neurodegenerative diseases (Joyce et al.), spinal cord and brain injuries (Y. Jiang et al.), cardiovascular diseases (Trivedi, Tray, Nguyen, Nigam, & Gallicano), diabetes mellitus (Y. H. Zhang et al., 2009) and diseases of the skeleton (Chanda, Kumar, & Ponnazhagan). In most of these studies, treatment with MSCs results in substantial functional benefit and these pre-clinical studies have led to the

MSCs have been used in clinical trials since 1995 and, currently, more than 180 trials are registered with ClinicalTrials.gov for the treatment of several diseases including numerous neurological disorders and injuries such amyotrophic lateral sclerosis, stroke, parkinson's

Demonstration of neural differentiation potential of MSCs in several in vitro and in vivo studies suggests the potential usefulness of MSCs in the treatment of various CNS disorders. This potential has led to extensive studies to further explore the neural plasticity of these cells (Azizi, Stokes, Augelli, DiGirolamo, & Prockop, 1998; Kopen, Prockop, & Phinney,

During the last several years, numerous in vitro neural induction protocols to produce neural cells from MSCs have been reported. In most induction experiments, MSCs were simply exposed to growth factors, neurotrophic factors or factors favoring neural cell differentiation (Bi et al., 2010; M. Chen et al., 2000; Q. Chen et al., 2005; Joannides et al., 2003; B. J. Kim, Seo, Bubien, & Oh, 2002; S. S. Kim et al., 2005; Kondo, Johnson, Yoder, Romand, & Hashino, 2005; Lim et al., 2008; Long, Olszewski, Huang, & Kletzel, 2005; Padovan et al., 2003; Sanchez-Ramos et al., 2000; Zeng et al., 2011). Other studies have used different culture media, supplemented with individual or various combinations of chemical and pharmacological agents, such as DMSO, b-mercaptoethanol, 5-bromo-2-deoxyuridine (BrdU), butylated hydroxyanisole, forskolin, and dibutyryl cyclic AMP (Ankeny, McTigue, & Jakeman, 2004; W. Deng, Obrocka, Fischer, & Prockop, 2001; Episkopou, 2005; Hermann et al., 2006; Jori et al., 2005; S. S. Kim et al., 2005; Lu, Blesch, & Tuszynski, 2004; Munoz-Elias, Woodbury, & Black, 2003; Tio, Tan, Lee, Wang, & Udolph, 2010; Yang, Wu, & Xiao, 2005; L. Zhang, Seitz, Abramczyk, Liu, & Chan, 2011). Other methods to induce MSCs into cells with neural characteristics include: transfection of MSCs with Noggin and Notch transcription factors (Dezawa et al., 2004; Kohyama et al., 2001); manipulation with surface proteins of culture substrate (Qian & Saltzman, 2004); co-culturing MSCs with NSCs or neural cells (Alexanian, 2005; Chu, Yu, Zhang, & Yu, 2008; Krampera et al., 2007; Wislet-Gendebien et al., 2005; Y. Q. Zhang et al., 2010); and growing MSCs as spheres in cultures (Shiota et al., 2007), transfection of MSCs with microRNA-9 (Jing et al., 2011). In several other studies, MSCs were turned into multipotent stage and then induced into neural cell lineages, by exposing them to appropriate neural differentiation conditions (Alexanian, 2007; Kohyama et al., 2001; Qu et al., 2004). Recently, we proposed an original method for efficient

initiation of a number of clinical trials worldwide.

disease, Alzheimer's disease, brain and spinal cord injuries.

**2. In vitro neural differentiation potential of MSCs** 

1999; Munoz-Elias, Marcus, Coyne, Woodbury, & Black, 2004).

generation of neural cells from feline and human BM-derived MSCs (hMSC) (Alexanian, 2010; Z. Zhang, Maiman, Kurpad, Crowe, & Alexanian, 2011). In these studies, neural induction was achieved by exposing cells simultaneously to inhibitors of DNA methylation and histone deacetylation and pharmacological agents that increased cAMP levels. The main idea of this methodological approach was the reactivation of pluripotency-associated genes in MSCs simultaneously exposing them to neural-inducing factors. Neurally modified MSCs by this methodology, in contrast to naïve MSCs, express several neural progenitor and mature neural markers demonstrated by real time RT-PCR, western blot, ELISA and immunocytochemistry Fig.1. and Fig.2.

Fig. 1. Expression of neural markers nestin, Sox2, A2B5, NCAM, B3T, GFAP, MAP-2, and NeuN in hMSCs (a-d) and NI-hMSC grown 24h, 1, 2, 3 weeks in neural induction medium (e-t). NI-hMSCs grown an additional week in neuronal induction medium were generated cells with long axon- and dendrite-like extensions (v-w). Bars 40um.

Mesenchymal Stem Cells in CNS Regeneration 241

very rare event and, therefore, can not be account for massive transdifferentiation demonstrated in numerous recent studies. In addition, MSCs can be induced into neural-like cells with several neural inducing factors, without being grown in co-cultures with NSCs. A few other reports suggested that some of these investigations suffered from artifacts created by in vitro chemical stress (Lu et al., 2004; Neuhuber et al., 2004). Nevertheless, Tondreau and colleagues have recently found significant upregulation of neural genes and downregulation of chondrogenic, osteogenic, adipogenic and myogenic genes in neurally differentiated MSCs as demonstrated by microarray analysis (Tondreau et al., 2008). In addition, a numerous studies suggest that with appropriate neural induction protocols, MSCs could produce mature neuron-like cells that exhibit multiple neuronal properties and traits, such as action potential, synaptic transmission, secretion of neurotrophic factors and dopamine, and demonstration of spontaneous post-synaptic current (Alexanian, Maiman, Kurpad, & Gennarelli, 2008; Bonilla et al., 2005; Greco, Zhou, Ye, & Rameshwar, 2008; Hermann et al., 2004; Y. Jiang et al., 2003; S. S. Kim et al., 2008; Mareschi et al., 2009; Trzaska et al., 2009; Wislet-Gendebien et al., 2005). Whether these neurally modified MSCs can produce fully functional neural cells in vitro and vivo is still under intensive investigations.

One of the first discoveries that demonstrate the pluripotent nature of adult MSCs in vivo, came from Ferrari et al. who clearly showed that adult murine BM contained cells capable of differentiation into skeletal muscle (Ferrari et al., 1998). In the past decade or more, several other studies have documented the ability of adult BM-derived cells to differentiate into liver and epithelium (Petersen et al., 1999; Theise, Badve, et al., 2000; Theise, Nimmakayalu, et al., 2000), endothelium (Kawamoto et al., 2001; Kawamoto et al., 2003; Takahashi et al., 1999), heart (Kucia et al., 2004; Orlic et al., 2001; Tomita et al., 1999), and brain (Brazelton, Rossi, Keshet, & Blau, 2000; Eglitis & Mezey, 1997; Mezey, Chandross, Harta, Maki, & McKercher, 2000). These striking observations indicate that there are BM cells that can migrate to distant sites and participate in repair of tissues across germ layer boundaries. In the most striking examples, BM cells injected in to the blastocyst contributed to most somatic cell lineages, including neural (Y. Jiang et al., 2002). These discoveries have led to extensive studies to further explore the neural differentiation potential of MSCs in intact,

However, multiple studies conducted during the last decade showed that MSCs transplanted into the intact, injured or diseased CNS environments do not differentiate or only a small portion of cells produce neural phenotypes (Alexanian, Kwok, Pravdic, Maiman, & Fehlings, 2010; Castro et al., 2002; J. Deng, Petersen, Steindler, Jorgensen, & Laywell, 2006). In contrast, MSCs transplanted in developing embryonic brain or in neurogenic areas of the adult brain expressed heterogeneous traits characteristic of radial glia, subventricular zone progenitors, migratory cells, parenchymal neurons, and glia (Azizi et al., 1998; Kopen et al., 1999; J. M. Li et al., 2011; Munoz-Elias et al., 2004). The fate of MSCs consequently appeared to be regulated by multiple influences, presumably including different microenvironments. These are in close analogy with studies in which pluripotent or highly immature NSCs were used. In a similar way, transplanted cells generated different neural phenotypes when transplanted into one of the few neurogenic areas of the brain [35,36] but remained undifferentiated or differentiated predominantly into the glial cells

**3. In vivo neural differentiation potential of MSCs** 

injured and diseased CNS.

Fig. 2. Morphological and immunocytochemical characterization of unmodified and NIfMSCs. Expression of neural markers B3T, NCAM, A2B5, MAP2, NeuN, NF, Nurr1, TH and ChAT in unmodified fMSCs (a-f) and in NI-fMSCs grown for 72h in neural induction medium (g-r).

Despite these studies, there is an intense ongoing debate about the nature of these differentiation responses. For example, some recent reports suggested that cell fusion could account for transdifferentiation (Terada et al., 2002). However, spontaneous cell fusion is a

Fig. 2. Morphological and immunocytochemical characterization of unmodified and NIfMSCs. Expression of neural markers B3T, NCAM, A2B5, MAP2, NeuN, NF, Nurr1, TH and ChAT in unmodified fMSCs (a-f) and in NI-fMSCs grown for 72h in neural induction

Despite these studies, there is an intense ongoing debate about the nature of these differentiation responses. For example, some recent reports suggested that cell fusion could account for transdifferentiation (Terada et al., 2002). However, spontaneous cell fusion is a

medium (g-r).

very rare event and, therefore, can not be account for massive transdifferentiation demonstrated in numerous recent studies. In addition, MSCs can be induced into neural-like cells with several neural inducing factors, without being grown in co-cultures with NSCs.

A few other reports suggested that some of these investigations suffered from artifacts created by in vitro chemical stress (Lu et al., 2004; Neuhuber et al., 2004). Nevertheless, Tondreau and colleagues have recently found significant upregulation of neural genes and downregulation of chondrogenic, osteogenic, adipogenic and myogenic genes in neurally differentiated MSCs as demonstrated by microarray analysis (Tondreau et al., 2008). In addition, a numerous studies suggest that with appropriate neural induction protocols, MSCs could produce mature neuron-like cells that exhibit multiple neuronal properties and traits, such as action potential, synaptic transmission, secretion of neurotrophic factors and dopamine, and demonstration of spontaneous post-synaptic current (Alexanian, Maiman, Kurpad, & Gennarelli, 2008; Bonilla et al., 2005; Greco, Zhou, Ye, & Rameshwar, 2008; Hermann et al., 2004; Y. Jiang et al., 2003; S. S. Kim et al., 2008; Mareschi et al., 2009; Trzaska et al., 2009; Wislet-Gendebien et al., 2005). Whether these neurally modified MSCs can produce fully functional neural cells in vitro and vivo is still under intensive investigations.

#### **3. In vivo neural differentiation potential of MSCs**

One of the first discoveries that demonstrate the pluripotent nature of adult MSCs in vivo, came from Ferrari et al. who clearly showed that adult murine BM contained cells capable of differentiation into skeletal muscle (Ferrari et al., 1998). In the past decade or more, several other studies have documented the ability of adult BM-derived cells to differentiate into liver and epithelium (Petersen et al., 1999; Theise, Badve, et al., 2000; Theise, Nimmakayalu, et al., 2000), endothelium (Kawamoto et al., 2001; Kawamoto et al., 2003; Takahashi et al., 1999), heart (Kucia et al., 2004; Orlic et al., 2001; Tomita et al., 1999), and brain (Brazelton, Rossi, Keshet, & Blau, 2000; Eglitis & Mezey, 1997; Mezey, Chandross, Harta, Maki, & McKercher, 2000). These striking observations indicate that there are BM cells that can migrate to distant sites and participate in repair of tissues across germ layer boundaries. In the most striking examples, BM cells injected in to the blastocyst contributed to most somatic cell lineages, including neural (Y. Jiang et al., 2002). These discoveries have led to extensive studies to further explore the neural differentiation potential of MSCs in intact, injured and diseased CNS.

However, multiple studies conducted during the last decade showed that MSCs transplanted into the intact, injured or diseased CNS environments do not differentiate or only a small portion of cells produce neural phenotypes (Alexanian, Kwok, Pravdic, Maiman, & Fehlings, 2010; Castro et al., 2002; J. Deng, Petersen, Steindler, Jorgensen, & Laywell, 2006). In contrast, MSCs transplanted in developing embryonic brain or in neurogenic areas of the adult brain expressed heterogeneous traits characteristic of radial glia, subventricular zone progenitors, migratory cells, parenchymal neurons, and glia (Azizi et al., 1998; Kopen et al., 1999; J. M. Li et al., 2011; Munoz-Elias et al., 2004). The fate of MSCs consequently appeared to be regulated by multiple influences, presumably including different microenvironments. These are in close analogy with studies in which pluripotent or highly immature NSCs were used. In a similar way, transplanted cells generated different neural phenotypes when transplanted into one of the few neurogenic areas of the brain [35,36] but remained undifferentiated or differentiated predominantly into the glial cells

Mesenchymal Stem Cells in CNS Regeneration 243

In most reported studies, transplanted MSCs either do not differentiate, or only very small percentage of cells survive and produce neural cells in vivo. This led to studies to elucidate whether neural modification of MSCs will promote cell survival and neural differentiation

Several recent studies suggest that neural modification of MSC prior to their transplantation can exhibit even higher beneficial therapeutic effect then naïve MSCs. In one of these studies Sung-Rae Cho et al. showed that transplantation of neurally differentiated MSCs derived from bone marrow promoted functional recovery in spinal cord injured rats and the latency of somatosensory evoked potentials were significantly improved compared with those of naïve MSCs and PBS controls (Cho et al., 2009). Furthermore, transplanted cells prelabeled with BrdU also differentiated into neural lineage cells that expressed specific markers for astrocytes and oligodendrocytes 4 weeks after transplantation, even though the number of integrated cells was not abundant. However, these differentiated cells did not survive longer than 8 weeks post transplantation, which was similar to what was reported in a previous studies (4). Because injured rats showed significant motor recovery at a relatively early stage after transplantation, and only a small number of transplanted cells survived in the injured spinal cord for a limited period, authors concluded that trophic or paracrine

Recently, we also demonstrated that transplanted neurally induced hMSCs (NI-hMSCs) promoted tissue preservation and improved locomotor recovery of injured animals (Alexanian et al., 2011). Motor recovery that consisted of hindlimb weight support and consistent hindlimb stepping was significantly different at 2-12 weeks post-recovery in the group that was transplanted with NI-hMSCs when compared with the control groups that

Histological studies of spinal cord sections at specified distances rostral and caudal to the epicenter demonstrated that at the epicentre and 1mm caudal and rostral from it the percentage of the eriochrome cyanine-positive spared white matter was significantly larger in NI-hMSCs treated group than that in the PBS group (Fig.4.A,B). While there was no significant difference between naïve hMSCs and PBS groups, there was a modest trend for increased white matter sparing in hMSCs-treated versus PBS-treated spinal cords

Stereological assessments of injured spinal cord tissues demonstrated a modest reduction in the percentage of cystic cavities in the NI-hMSCs and hMSCs treated groups versus PBS group (Fig.4.C) (Fig.5). Although no statistically significant difference had been noticed between groups (Fig.4.C), the difference found between NI-HMSCs and PBS was very close

Immunohistochemistry data showed that NI-hMSCs were survived at post transplantation weeks 1-12. Analysis of the spinal cord slices of two weeks treated animals revealed that 85% percent of survived cells were positive to B3T (Fig.6.a,b,c,d). A small percentage of cells (2%) was positive to GFAP (Fig.4.e) and 5% to Sox2 (Fig.6.f). By 12 weeks the number of surviving cells declined to 15-20% of that at week 2 and only 10% of survived cells were

of transplanted cells in intact and injured CNS.

support could be the main factors for functional improvement.

to the significance level adopted in the study (p<0.05).

received hMSCs and PBS (Fig.3).

positive to B3T (Fig.6.g,h,i).

(Fig.4.B).

when transplanted into injured or non-neurogenic areas [37]. However, when late-stage precursors and immature neurons were transplanted into non-neurogenic or injured brain and spinal cord, more neural differentiation was observed [38,39]. This indicates that, while microenvironment can play a decisive role in determining the fate of engrafted MSCs or NSCs, the intrinsic state of these transplanted cells is another important factor for the commitment of cells to a particular phenotype. MSCs which presumably committed to mesodermal lineages most probably will not produce neural cells in intact, injured or diseased CNS and therefore, manipulation of cells into neural fate maybe required before transplantation. In fact, several recent studies showed that neurally modified MSCs transplanted into intact or damaged CNS exhibited higher ability to generate cells positive to various neural markers (Alexanian et al., 2008; Alexanian, Michael, Zhang, & Maiman, 2011; Cho et al., 2009).

#### **4. Therapeutic effects of naïve and neurally modified MSCs in CNS disorders and their underlying mechanisms**

Experimental treatments of CNS disorders can be broadly grouped into the two distinct but interrelated strategies of neuroprotection and neurorepair/neuroregeneration. Neuroprotection refers to inhibition of the death of CNS parenchymal cells in traumantic and neurodegenerative CNS, neurorepair/neuroregeneration refers to the replacement of lost neural cells, stimulation of endogenous neural progenitors and/or regeneration of severed axons or sprouting of intact axons to innervate denervated targets in injured or diseased CNS. MSCs have been used for all of these strategies and exhibited beneficial therapeutic effect in several animal models of CNS injury and neurodegenerative diseases.

#### **4.1 MSC in CNS injury (traumatic spinal cord and brain injury, ischemia/stroke)**

Recent multiple studies demonstrated that naïve or neurally modified MSCs derived from different tissue sources exerted therapeutic effect in several animal models of spinal cord injury (SCI). However, the precise mechanisms by which transplantation of MSCs promote functional recovery after SCI is still unclear. A number of mechanisms have been suggested, including the promotion of axon regeneration, neuroprotection, modulation of the immune responses, and trans-differentiation into neural cell types (Chamberlain, Fox, Ashton, & Middleton, 2007; Dezawa, 2002; Enzmann, Benton, Talbott, Cao, & Whittemore, 2006; Keilhoff, Goihl, Stang, Wolf, & Fansa, 2006). The immunosuppressive properties of MSCs (Bartholomew et al., 2002a; Corcione et al., 2006; Di Nicola et al., 2002; X. X. Jiang et al., 2005) may combine to reduce the acute inflammatory response to SCI and hence reduce cavity formation as well as decrease astrocyte and microglia/macrophage reactivity (Abrams et al., 2009; Himes et al., 2006; Neuhuber, Timothy Himes, Shumsky, Gallo, & Fischer, 2005) in injured spinal cords. The therapeutic effect of MSCs on axonal growth could be exerted by creation of a favorable environments such as cellular bridges, guiding strands and scaffolds, secretion of trophic factors, cytokines and production of extracellular matrix (Fuhrmann et al., 2010; Gu et al., 2010; Hofstetter et al., 2002; Neuhuber et al., 2004). The neuroprotective mechanism of MSCs could be multifactorial, such as modulation of immune response and provision of trophic factors (Uccelli, Benvenuto, Laroni, & Giunti, 2011). Whether MSCs therapeutic effect can be exerted via cell replacement is still one of the most debated issues.

when transplanted into injured or non-neurogenic areas [37]. However, when late-stage precursors and immature neurons were transplanted into non-neurogenic or injured brain and spinal cord, more neural differentiation was observed [38,39]. This indicates that, while microenvironment can play a decisive role in determining the fate of engrafted MSCs or NSCs, the intrinsic state of these transplanted cells is another important factor for the commitment of cells to a particular phenotype. MSCs which presumably committed to mesodermal lineages most probably will not produce neural cells in intact, injured or diseased CNS and therefore, manipulation of cells into neural fate maybe required before transplantation. In fact, several recent studies showed that neurally modified MSCs transplanted into intact or damaged CNS exhibited higher ability to generate cells positive to various neural markers (Alexanian et al., 2008; Alexanian, Michael, Zhang, & Maiman,

**4. Therapeutic effects of naïve and neurally modified MSCs in CNS disorders** 

Experimental treatments of CNS disorders can be broadly grouped into the two distinct but interrelated strategies of neuroprotection and neurorepair/neuroregeneration. Neuroprotection refers to inhibition of the death of CNS parenchymal cells in traumantic and neurodegenerative CNS, neurorepair/neuroregeneration refers to the replacement of lost neural cells, stimulation of endogenous neural progenitors and/or regeneration of severed axons or sprouting of intact axons to innervate denervated targets in injured or diseased CNS. MSCs have been used for all of these strategies and exhibited beneficial therapeutic effect in several animal models of CNS injury and neurodegenerative

**4.1 MSC in CNS injury (traumatic spinal cord and brain injury, ischemia/stroke)** 

Recent multiple studies demonstrated that naïve or neurally modified MSCs derived from different tissue sources exerted therapeutic effect in several animal models of spinal cord injury (SCI). However, the precise mechanisms by which transplantation of MSCs promote functional recovery after SCI is still unclear. A number of mechanisms have been suggested, including the promotion of axon regeneration, neuroprotection, modulation of the immune responses, and trans-differentiation into neural cell types (Chamberlain, Fox, Ashton, & Middleton, 2007; Dezawa, 2002; Enzmann, Benton, Talbott, Cao, & Whittemore, 2006; Keilhoff, Goihl, Stang, Wolf, & Fansa, 2006). The immunosuppressive properties of MSCs (Bartholomew et al., 2002a; Corcione et al., 2006; Di Nicola et al., 2002; X. X. Jiang et al., 2005) may combine to reduce the acute inflammatory response to SCI and hence reduce cavity formation as well as decrease astrocyte and microglia/macrophage reactivity (Abrams et al., 2009; Himes et al., 2006; Neuhuber, Timothy Himes, Shumsky, Gallo, & Fischer, 2005) in injured spinal cords. The therapeutic effect of MSCs on axonal growth could be exerted by creation of a favorable environments such as cellular bridges, guiding strands and scaffolds, secretion of trophic factors, cytokines and production of extracellular matrix (Fuhrmann et al., 2010; Gu et al., 2010; Hofstetter et al., 2002; Neuhuber et al., 2004). The neuroprotective mechanism of MSCs could be multifactorial, such as modulation of immune response and provision of trophic factors (Uccelli, Benvenuto, Laroni, & Giunti, 2011). Whether MSCs therapeutic effect can be exerted via cell replacement is still one of the most debated issues.

2011; Cho et al., 2009).

diseases.

**and their underlying mechanisms** 

In most reported studies, transplanted MSCs either do not differentiate, or only very small percentage of cells survive and produce neural cells in vivo. This led to studies to elucidate whether neural modification of MSCs will promote cell survival and neural differentiation of transplanted cells in intact and injured CNS.

Several recent studies suggest that neural modification of MSC prior to their transplantation can exhibit even higher beneficial therapeutic effect then naïve MSCs. In one of these studies Sung-Rae Cho et al. showed that transplantation of neurally differentiated MSCs derived from bone marrow promoted functional recovery in spinal cord injured rats and the latency of somatosensory evoked potentials were significantly improved compared with those of naïve MSCs and PBS controls (Cho et al., 2009). Furthermore, transplanted cells prelabeled with BrdU also differentiated into neural lineage cells that expressed specific markers for astrocytes and oligodendrocytes 4 weeks after transplantation, even though the number of integrated cells was not abundant. However, these differentiated cells did not survive longer than 8 weeks post transplantation, which was similar to what was reported in a previous studies (4). Because injured rats showed significant motor recovery at a relatively early stage after transplantation, and only a small number of transplanted cells survived in the injured spinal cord for a limited period, authors concluded that trophic or paracrine support could be the main factors for functional improvement.

Recently, we also demonstrated that transplanted neurally induced hMSCs (NI-hMSCs) promoted tissue preservation and improved locomotor recovery of injured animals (Alexanian et al., 2011). Motor recovery that consisted of hindlimb weight support and consistent hindlimb stepping was significantly different at 2-12 weeks post-recovery in the group that was transplanted with NI-hMSCs when compared with the control groups that received hMSCs and PBS (Fig.3).

Histological studies of spinal cord sections at specified distances rostral and caudal to the epicenter demonstrated that at the epicentre and 1mm caudal and rostral from it the percentage of the eriochrome cyanine-positive spared white matter was significantly larger in NI-hMSCs treated group than that in the PBS group (Fig.4.A,B). While there was no significant difference between naïve hMSCs and PBS groups, there was a modest trend for increased white matter sparing in hMSCs-treated versus PBS-treated spinal cords (Fig.4.B).

Stereological assessments of injured spinal cord tissues demonstrated a modest reduction in the percentage of cystic cavities in the NI-hMSCs and hMSCs treated groups versus PBS group (Fig.4.C) (Fig.5). Although no statistically significant difference had been noticed between groups (Fig.4.C), the difference found between NI-HMSCs and PBS was very close to the significance level adopted in the study (p<0.05).

Immunohistochemistry data showed that NI-hMSCs were survived at post transplantation weeks 1-12. Analysis of the spinal cord slices of two weeks treated animals revealed that 85% percent of survived cells were positive to B3T (Fig.6.a,b,c,d). A small percentage of cells (2%) was positive to GFAP (Fig.4.e) and 5% to Sox2 (Fig.6.f). By 12 weeks the number of surviving cells declined to 15-20% of that at week 2 and only 10% of survived cells were positive to B3T (Fig.6.g,h,i).

Mesenchymal Stem Cells in CNS Regeneration 245

spared white matter through the entire T8 spinal cord segment. (C) Graph representing

Fig. 5. Representative three-dimensionally reconstructed images of the lesion cavities through T8 injured spinal cord segments of NI-hMSCs and PBS treated animals.

Fig. 6. Transplanted NI-hMSCs survived 2 weeks after transplantation and expressed neural markers such as B3T (a-c, b and c are the higher magnifications of the marked area in the image a), and GFAP (d-f). By 12 weeks the number of surviving cells declined to 15-20% of

comparison of the volumes of lesion cavities.

Fig. 3. Locomotor recovery (BBB) scores for the post spinal cord injury (DPI-days post injury) behavioral analysis. The asterisks (\*) and (\*\*) indicates a significant differences between the NI-hMSCs transplanted group compaired to the PBS and PBS+HMSCs groups respectively. Asterisk (\*\*\*) indicates a significant differences between the hMSCs transplanted group compaired to the PBS.

Fig. 4. Analysis of white matter sparing and lesion cavity volumes in NI-hMSCs, hMSCs, and PBS treated groups. (A) Representative spinal cord cross-sections extending 500um rostral and caudal from the lesion epicenter. (B) Graph representing the percentages of

Fig. 3. Locomotor recovery (BBB) scores for the post spinal cord injury (DPI-days post injury) behavioral analysis. The asterisks (\*) and (\*\*) indicates a significant differences between the NI-hMSCs transplanted group compaired to the PBS and PBS+HMSCs groups

Fig. 4. Analysis of white matter sparing and lesion cavity volumes in NI-hMSCs, hMSCs, and PBS treated groups. (A) Representative spinal cord cross-sections extending 500um rostral and caudal from the lesion epicenter. (B) Graph representing the percentages of

respectively. Asterisk (\*\*\*) indicates a significant differences between the hMSCs

transplanted group compaired to the PBS.

spared white matter through the entire T8 spinal cord segment. (C) Graph representing comparison of the volumes of lesion cavities.

Fig. 5. Representative three-dimensionally reconstructed images of the lesion cavities through T8 injured spinal cord segments of NI-hMSCs and PBS treated animals.

Fig. 6. Transplanted NI-hMSCs survived 2 weeks after transplantation and expressed neural markers such as B3T (a-c, b and c are the higher magnifications of the marked area in the image a), and GFAP (d-f). By 12 weeks the number of surviving cells declined to 15-20% of

Mesenchymal Stem Cells in CNS Regeneration 247

The therapeutic effect of MSCs was also demonstrated in animal models of stroke. Several recent studies showed that transplantation of MSCs, derived from bone marrow, into rodent cerebral ischemia models can reduce infarct size and improve functional outcome (18,27,50,52,83,85,106,110,111,139,144). MSCs derived from adipose tissue (ADSCs) also showed therapeutic effect in rat model of cerebral ischemia (150). Importantly, treatment of ischemic animals with neurally induced ADSCs resulted in better functional recovery and

To test the clinical relevance of these observations, recently, a phase I clinical trial was conducted. A feasibility and safety of transplantation of autologous human MSCs in stroke patients was the main objective of this trail (51). In this study the autologous MSCs were delivered intravenously 36–133 days post-stroke. All patients had magnetic resonance angiography to identify vascular lesions, and magnetic resonance imaging prior to cell infusion and at intervals up to 1 year after. Neurological status was scored using the National Institutes of Health Stroke Scale and modified Rankin scores. The results of this study showed that the median daily rate of National Institutes of Health Stroke Scale change was 0.36 during the first week post-infusion, compared with a median daily rate of change of 0.04 from the first day of testing to immediately before infusion. No central nervous system tumors, abnormal cell growths or neurological deterioration was observed, and there was no evidence for venous thromboembolism, systemic malignancy or systemic infection in any of the patients following stem cell infusion. Thus the stroke is another potential target

There is currently a great deal of interest in the use of MSCs to treat several neurological diseases such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and

Recently, a number of studies have examined the ability of MSCs to differentiate into dopamine-producing cells, re-innervate the striatum, and ameliorate behavioral deficits in Parkinsonian models. Varying degrees of success have been achieved in vitro, including dopaminergic marker expression, and dopamine secretion in response to depolarization (Dezawa et al., 2004; Fu et al., 2006; Guo et al., 2005; Suon, Yang, & Iacovitti, 2006; Trzaska, Kuzhikandathil, & Rameshwar, 2007; Trzaska & Rameshwar, 2011). In addition, engraftment and functional improvement were demonstrated following transplantation of undifferentiated (Hellmann, Panet, Barhum, Melamed, & Offen, 2006; Y. Li et al., 2001) and neurally differentiated MSCs (Dezawa et al., 2004; Fu et al., 2006) in hemiparkinsonian rodents. However, only relatively low efficiencies of dopaminergic differentiation were achieved, and comparisons between the varying methods have not been performed, resulting in difficulties with identifying the optimal methodology. These studies suggest that complex mechanisms might underline the therapeutic effect of MSCs in these animal parkinsonian models and neuroprotection could be the most important ones (P. H. Lee & Park, 2009). Despite all these promising data several issues remain to be resolved including the optimal method for inducing a dopaminergic phenotype from MSCs, engraftment and survival capabilities of MSCs, optimal sites for transplantation, potential immunological responses to MSC grafts, and whether neural differentiation prior to transplantation

more reduction in hemispheric atrophy in comparison to unmodified ADSCs (150).

for MSCs therapy.

multiple sclerosis.

**4.2 MSCs in neurodegenerative diseases** 

provides engraftment advantages.

that at week 2 and only 10% of survived cells were positive to B3T (g,h,i). The images h and i are higher magnifications of g. HM stands for human anti**-**mitochondrial antibody.

Thus, MSCs, either neurally modified or not, may provide an alternative source of autologous adult stem cells that could be useful for replacing damaged neural cells in injured spinal cord and/or providing support to spinal cord tissue cells.

Over the last decade or so, MSCs have been also used in experimental repair of the injured brain. Chopp and Li initially demonstrated transplanted MSCs promote functional recovery in rats with traumatic brain injury and attributed the beneficial effects of MSCs to the enhancement of endogenous restorative and regenerative processes (Chopp & Li, 2002). Later, Chopp and his group showed that MSCs treated with neurotrophins NGF and BDNF in vitro led to a higher number of engrafted cells after transplantation into the adult rat brain and improved motor function. A small number of cells stained for either astrocytic or neuronal markers (Mahmood, Lu, Wang, & Chopp, 2002), but were far too few to provide cellular replacement. This group also reported that i.v. administration of MSCs 1 day after brain injury in the rat brain resulted in an increase in BDNF and NGF (Mahmood et al., 2002). Both intracerebral and i.v. MSC administration promoted endogenous progenitor cell proliferation after traumatic brain injury (Mahmood, Lu, & Chopp, 2004), and functional recovery was dose dependent and persisted for at least 3 months (Mahmood, Lu, Qu, Goussev, & Chopp, 2006). Recently another group confirmed the therapeutic effect of human MSCs (hMSCs) in a rat model of TBI and demonstrated that expression of neurotrophic growth factors was induced by MSC treatment (H. J. Kim, Lee, & Kim, 2010). Furthermore, they observed an increase in phosphorylation of the cell survival signaling molecule, Akt, followed by decreased caspase-3 activation. These results suggest that the therapeutic effects of hMSCs transplantation may involve promotion of antiapoptotic activity as a result of secreted growth factors (H. J. Kim et al., 2010).

A single Phase I study using bone marrow-derived MSCs in children after isolated TBI has recently been completed (Cox et al., 2011). In this study, 10 children age 5–14 years with a Glasgow coma scale score of 5–8 were treated with 6 × 106 bone marrow-derived mononuclear cells per kg body weight delivered intravenously within 48 hours of an isolated TBI. To determine the safety of administration, systemic and cerebral hemodynamics, laboratory parameters, chest radiographs, and serial clinical assessments were monitored. Additionally, serial cerebral magnetic resonance imaging neuropsychologic evaluation, and functional outcome measures were obtained as preliminary measures of efficacy. There were no identifiable adverse events with close monitoring of the neurologic, pulmonary, renal, hepatic, and hematologic systems. Functional and neuropsychological testing, including the Glasgow Outcome Scale, the Pediatric Injury Functional Outcome Scale, and the Wechsler Abbreviated Scale of Intelligence, revealed recovery consistent with (or improved from) expected baselines. Magnetic resonance imaging volumetric data revealed no significant change in grey matter, white matter, intracranial volume, or CSF space at 1 and 6 months as measured relative to expected norms. Authors concluded that bone marrow harvest and intravenous mononuclear cell infusion as treatment for severe TBI in children is logistically feasible and safe.

that at week 2 and only 10% of survived cells were positive to B3T (g,h,i). The images h and i

Thus, MSCs, either neurally modified or not, may provide an alternative source of autologous adult stem cells that could be useful for replacing damaged neural cells in

Over the last decade or so, MSCs have been also used in experimental repair of the injured brain. Chopp and Li initially demonstrated transplanted MSCs promote functional recovery in rats with traumatic brain injury and attributed the beneficial effects of MSCs to the enhancement of endogenous restorative and regenerative processes (Chopp & Li, 2002). Later, Chopp and his group showed that MSCs treated with neurotrophins NGF and BDNF in vitro led to a higher number of engrafted cells after transplantation into the adult rat brain and improved motor function. A small number of cells stained for either astrocytic or neuronal markers (Mahmood, Lu, Wang, & Chopp, 2002), but were far too few to provide cellular replacement. This group also reported that i.v. administration of MSCs 1 day after brain injury in the rat brain resulted in an increase in BDNF and NGF (Mahmood et al., 2002). Both intracerebral and i.v. MSC administration promoted endogenous progenitor cell proliferation after traumatic brain injury (Mahmood, Lu, & Chopp, 2004), and functional recovery was dose dependent and persisted for at least 3 months (Mahmood, Lu, Qu, Goussev, & Chopp, 2006). Recently another group confirmed the therapeutic effect of human MSCs (hMSCs) in a rat model of TBI and demonstrated that expression of neurotrophic growth factors was induced by MSC treatment (H. J. Kim, Lee, & Kim, 2010). Furthermore, they observed an increase in phosphorylation of the cell survival signaling molecule, Akt, followed by decreased caspase-3 activation. These results suggest that the therapeutic effects of hMSCs transplantation may involve promotion of antiapoptotic

A single Phase I study using bone marrow-derived MSCs in children after isolated TBI has recently been completed (Cox et al., 2011). In this study, 10 children age 5–14 years with a Glasgow coma scale score of 5–8 were treated with 6 × 106 bone marrow-derived mononuclear cells per kg body weight delivered intravenously within 48 hours of an isolated TBI. To determine the safety of administration, systemic and cerebral hemodynamics, laboratory parameters, chest radiographs, and serial clinical assessments were monitored. Additionally, serial cerebral magnetic resonance imaging neuropsychologic evaluation, and functional outcome measures were obtained as preliminary measures of efficacy. There were no identifiable adverse events with close monitoring of the neurologic, pulmonary, renal, hepatic, and hematologic systems. Functional and neuropsychological testing, including the Glasgow Outcome Scale, the Pediatric Injury Functional Outcome Scale, and the Wechsler Abbreviated Scale of Intelligence, revealed recovery consistent with (or improved from) expected baselines. Magnetic resonance imaging volumetric data revealed no significant change in grey matter, white matter, intracranial volume, or CSF space at 1 and 6 months as measured relative to expected norms. Authors concluded that bone marrow harvest and intravenous mononuclear cell infusion as treatment for severe TBI in children is logistically feasible and

are higher magnifications of g. HM stands for human anti**-**mitochondrial antibody.

injured spinal cord and/or providing support to spinal cord tissue cells.

activity as a result of secreted growth factors (H. J. Kim et al., 2010).

safe.

The therapeutic effect of MSCs was also demonstrated in animal models of stroke. Several recent studies showed that transplantation of MSCs, derived from bone marrow, into rodent cerebral ischemia models can reduce infarct size and improve functional outcome (18,27,50,52,83,85,106,110,111,139,144). MSCs derived from adipose tissue (ADSCs) also showed therapeutic effect in rat model of cerebral ischemia (150). Importantly, treatment of ischemic animals with neurally induced ADSCs resulted in better functional recovery and more reduction in hemispheric atrophy in comparison to unmodified ADSCs (150).

To test the clinical relevance of these observations, recently, a phase I clinical trial was conducted. A feasibility and safety of transplantation of autologous human MSCs in stroke patients was the main objective of this trail (51). In this study the autologous MSCs were delivered intravenously 36–133 days post-stroke. All patients had magnetic resonance angiography to identify vascular lesions, and magnetic resonance imaging prior to cell infusion and at intervals up to 1 year after. Neurological status was scored using the National Institutes of Health Stroke Scale and modified Rankin scores. The results of this study showed that the median daily rate of National Institutes of Health Stroke Scale change was 0.36 during the first week post-infusion, compared with a median daily rate of change of 0.04 from the first day of testing to immediately before infusion. No central nervous system tumors, abnormal cell growths or neurological deterioration was observed, and there was no evidence for venous thromboembolism, systemic malignancy or systemic infection in any of the patients following stem cell infusion. Thus the stroke is another potential target for MSCs therapy.

#### **4.2 MSCs in neurodegenerative diseases**

There is currently a great deal of interest in the use of MSCs to treat several neurological diseases such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and multiple sclerosis.

Recently, a number of studies have examined the ability of MSCs to differentiate into dopamine-producing cells, re-innervate the striatum, and ameliorate behavioral deficits in Parkinsonian models. Varying degrees of success have been achieved in vitro, including dopaminergic marker expression, and dopamine secretion in response to depolarization (Dezawa et al., 2004; Fu et al., 2006; Guo et al., 2005; Suon, Yang, & Iacovitti, 2006; Trzaska, Kuzhikandathil, & Rameshwar, 2007; Trzaska & Rameshwar, 2011). In addition, engraftment and functional improvement were demonstrated following transplantation of undifferentiated (Hellmann, Panet, Barhum, Melamed, & Offen, 2006; Y. Li et al., 2001) and neurally differentiated MSCs (Dezawa et al., 2004; Fu et al., 2006) in hemiparkinsonian rodents. However, only relatively low efficiencies of dopaminergic differentiation were achieved, and comparisons between the varying methods have not been performed, resulting in difficulties with identifying the optimal methodology. These studies suggest that complex mechanisms might underline the therapeutic effect of MSCs in these animal parkinsonian models and neuroprotection could be the most important ones (P. H. Lee & Park, 2009). Despite all these promising data several issues remain to be resolved including the optimal method for inducing a dopaminergic phenotype from MSCs, engraftment and survival capabilities of MSCs, optimal sites for transplantation, potential immunological responses to MSC grafts, and whether neural differentiation prior to transplantation provides engraftment advantages.

Mesenchymal Stem Cells in CNS Regeneration 249

useful vehicles for delivering anti-Abeta activity and thus exhibiting the maxium

The potentials of MSCs as a therapy for autoimmune neurological diseases arose from some unexpected observations. Therapies with MSCs were originally based on their similarities to most adult stem cells and the possibility that they might regenerate tissues through their ability to differentiate into mesodermal tissues and perhaps other embryonic lineages. The unexpected observation that MSCs inhibited T cell proliferation both in vitro (Di Nicola et al., 2002) and in vivo (Bartholomew et al., 2002b) introduced the possibility that MSCs might be effective in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS) halting the (auto)immune attack to myelin antigens and promoting nervous tissue repair through their integration in the central nervous system (CNS). During the last few years, animal experiments in the EAE mouse model of MS showed that intraventricular, intraperitoneal or intravenous injection of human or murine BM-MSCs significantly improved clinical outcomes (Bai et al., 2009; Gordon et al., 2008; Kassis et al., 2008; Zappia et al., 2005; J. Zhang et al., 2005) (Zappia et al., 2005, Bai et al., 2009, Gordon et al., 2008, Zhang et al., 2005, Kassis et al., 2008). Chopp's group, in addition to observing functional recovery in EAE mice, demonstrated that small percentage of transplanted MSCs was integrated and expressed neural markers (J. Zhang et al., 2006). Their observations therefore suggested that some transdifferentiation had occurred. Overall, these pioneer studies demonstrated the therapeutic efficacy of MSCs in a model of CNS autoimmunity, but they left open the question whether their integration in the nervous system was essential for their therapeutic

Recently, a phase I trial was initiated to evaluate the safety and feasibility of intrathecal injection of autologous BM-MSCs in MS patients (Karussis et al., 2010). The initial findings of this trial support the possibility of migration of MSCs from their site of injection (lumbar area of the cerebrospinal fluid) to the brain ventricles and spinal cord parenchyma. Preliminary data of this trail also demonstrated the immunomodulatory effect of MSC in human neurological diseases. The authors concluded that the early clinical stabilization and improvement in some of the patients could be related to these immunomodulating effects. The possibility of neuroprotection and neuroregeneration through transdifferentiation of MSCs into cells of the neuronal or glial lineage, although theoretically viable, has yet to be

Promising results from this study will support further clinical trials to evaluate the long term safety and the potential clinical efficacy of MSC transplantation in the treatment of MS.

Although the curative effect of MSCs has been demonstrated in several animal models of CNS injury and neurodegeneration as well as in early human clinical trials of neurological disorders, the mechanisms that are responsible for these beneficial therapeutic effects are still poorly understood. Analysis of accumulated literature in this area suggest the following main mechanisms that may underlie the therapeutic effect of naive or neurally modified MSCs: 1) neurorepair (replacement of damaged or diseased neural cells by neurally transdifferentiated MSCs), 2) neuroprotection (modulation of immune response and inflammation, provision of trophic factors that could prevent neural cell apoptosis and

therapeutic effect on AD (Habisch et al., 2010).

benefits.

proved by neuroimaging studies.

**5. Conclusions** 

Unlike Parkinson disease, which is a slower degenerative disease and affects a specific area of the brain, amyotrophic lateral sclerosis (ALS) presents quite a challenge for cellular therapy because of the distributed cell loss throughout the body and the requirement to properly reinnervate muscle tissue. Transplantation of wild-type BM cells into irradiated SOD1 transgenic mouse models of ALS demonstrated a delay in disease onset and an increase in life span (Corti et al., 2004). Minimal neural differentiation was detected, thus the authors concluded that functional improvement was likely due to trophic effects. Another study showed that transplantation of human MSCs into SOD1 ALS mice significantly delayed disease onset and progression, in addition to increasing lifespan (56). The human cells survived more than 20 weeks in the xenogenic model, and were able to migrate into the brain and spinal cord and differentiate into neuroglial cells (Zhao et al., 2007). Initial clinical studies began in 2003, when Mazzini et al took autologous MSCs from seven ALS patients and expanded them in culture (Mazzini et al., 2003). The cells were directly transplanted into the spinal cord, and did not result in toxicity or uncontrolled proliferation. Three months after transplantation, four patients experienced a mild reduction in muscle strength decline in the lower limbs. In a long term follow-up of the patients, the same group reported, after 36 months, that four of the seven patients showed a significant reduction in the linear decline of lung function and ALS functional rating scale (Mazzini et al., 2006). Though these preliminary clinical studies are encouraging, further studies are warranted.

Research on the role of MSCs in Alzheimer's disease (AD) is in its infancy. However, a recent study showed positive results in an AD rat model (Wu, Li, Feng, & Wang, 2007). Transplantation of BM-derived MSCs into the hippocampus of rats injected with ß amyloid protein to mimic AD demonstrated significant improvement based on the Morris Water Maze test (Wu et al., 2007). The authors suggested that the MSCs transdifferentiated into cholinergic cells and improved the cognitive ability of the AD rat models. Another group recently showed that transplanted MSCs exerted anti-apoptotic effect in an acutely-induced AD mice model produced by injecting Abeta intrahippocampally (J. K. Lee, Jin, & Bae, 2010). The same group also showed that intracerebral transplantation of BM-MSCs into APP/PS1 mice significantly reduced amyloid beta-peptide (Abeta) deposition (J. K. Lee, Jin, Endo, et al., 2010). Interestingly, these effects were associated with restoration of defective microglial function, as evidenced by increased Abeta-degrading factors, decreased inflammatory responses, and elevation of alternatively activated microglial markers. Furthermore, APP/PS1 mice treated with BM-MSCs had decreased tau hyperphosphorylation and improved cognitive function. Thus, BM-MSCs can modulate immune/inflammatory responses in AD mice, ameliorate their pathophysiology, and improve the cognitive decline associated with Abeta deposits. These results demonstrate that BM-MSCs are a potential new therapeutic agent for AD. Interestingly, Stroch A. et al and his group recently detected the functional induction of two genes upon neuroectodermal conversion of human adult MSCs, namely F-spondin and neprilysin (CD10), with a 4,992 + or - 697-fold and 692 + or - 226-fold increase of mRNA levels in converted cells compared to MSCs, respectively (Habisch et al., 2010). These genes are known to be involved in the formation and degradation of Abeta peptides, respectively. Consistently, co-incubation of the neuroectodermally converted MSCs with HEK-293 cells stably expressing amyloid precursor protein (APP) lead to a significant cell dose-dependent decrease of Abeta peptides. These in vitro results indicate that neurally modified MSCs might be even more

Unlike Parkinson disease, which is a slower degenerative disease and affects a specific area of the brain, amyotrophic lateral sclerosis (ALS) presents quite a challenge for cellular therapy because of the distributed cell loss throughout the body and the requirement to properly reinnervate muscle tissue. Transplantation of wild-type BM cells into irradiated SOD1 transgenic mouse models of ALS demonstrated a delay in disease onset and an increase in life span (Corti et al., 2004). Minimal neural differentiation was detected, thus the authors concluded that functional improvement was likely due to trophic effects. Another study showed that transplantation of human MSCs into SOD1 ALS mice significantly delayed disease onset and progression, in addition to increasing lifespan (56). The human cells survived more than 20 weeks in the xenogenic model, and were able to migrate into the brain and spinal cord and differentiate into neuroglial cells (Zhao et al., 2007). Initial clinical studies began in 2003, when Mazzini et al took autologous MSCs from seven ALS patients and expanded them in culture (Mazzini et al., 2003). The cells were directly transplanted into the spinal cord, and did not result in toxicity or uncontrolled proliferation. Three months after transplantation, four patients experienced a mild reduction in muscle strength decline in the lower limbs. In a long term follow-up of the patients, the same group reported, after 36 months, that four of the seven patients showed a significant reduction in the linear decline of lung function and ALS functional rating scale (Mazzini et al., 2006). Though these preliminary clinical studies are encouraging, further studies are warranted.

Research on the role of MSCs in Alzheimer's disease (AD) is in its infancy. However, a recent study showed positive results in an AD rat model (Wu, Li, Feng, & Wang, 2007). Transplantation of BM-derived MSCs into the hippocampus of rats injected with ß amyloid protein to mimic AD demonstrated significant improvement based on the Morris Water Maze test (Wu et al., 2007). The authors suggested that the MSCs transdifferentiated into cholinergic cells and improved the cognitive ability of the AD rat models. Another group recently showed that transplanted MSCs exerted anti-apoptotic effect in an acutely-induced AD mice model produced by injecting Abeta intrahippocampally (J. K. Lee, Jin, & Bae, 2010). The same group also showed that intracerebral transplantation of BM-MSCs into APP/PS1 mice significantly reduced amyloid beta-peptide (Abeta) deposition (J. K. Lee, Jin, Endo, et al., 2010). Interestingly, these effects were associated with restoration of defective microglial function, as evidenced by increased Abeta-degrading factors, decreased inflammatory responses, and elevation of alternatively activated microglial markers. Furthermore, APP/PS1 mice treated with BM-MSCs had decreased tau hyperphosphorylation and improved cognitive function. Thus, BM-MSCs can modulate immune/inflammatory responses in AD mice, ameliorate their pathophysiology, and improve the cognitive decline associated with Abeta deposits. These results demonstrate that BM-MSCs are a potential new therapeutic agent for AD. Interestingly, Stroch A. et al and his group recently detected the functional induction of two genes upon neuroectodermal conversion of human adult MSCs, namely F-spondin and neprilysin (CD10), with a 4,992 + or - 697-fold and 692 + or - 226-fold increase of mRNA levels in converted cells compared to MSCs, respectively (Habisch et al., 2010). These genes are known to be involved in the formation and degradation of Abeta peptides, respectively. Consistently, co-incubation of the neuroectodermally converted MSCs with HEK-293 cells stably expressing amyloid precursor protein (APP) lead to a significant cell dose-dependent decrease of Abeta peptides. These in vitro results indicate that neurally modified MSCs might be even more useful vehicles for delivering anti-Abeta activity and thus exhibiting the maxium therapeutic effect on AD (Habisch et al., 2010).

The potentials of MSCs as a therapy for autoimmune neurological diseases arose from some unexpected observations. Therapies with MSCs were originally based on their similarities to most adult stem cells and the possibility that they might regenerate tissues through their ability to differentiate into mesodermal tissues and perhaps other embryonic lineages. The unexpected observation that MSCs inhibited T cell proliferation both in vitro (Di Nicola et al., 2002) and in vivo (Bartholomew et al., 2002b) introduced the possibility that MSCs might be effective in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS) halting the (auto)immune attack to myelin antigens and promoting nervous tissue repair through their integration in the central nervous system (CNS). During the last few years, animal experiments in the EAE mouse model of MS showed that intraventricular, intraperitoneal or intravenous injection of human or murine BM-MSCs significantly improved clinical outcomes (Bai et al., 2009; Gordon et al., 2008; Kassis et al., 2008; Zappia et al., 2005; J. Zhang et al., 2005) (Zappia et al., 2005, Bai et al., 2009, Gordon et al., 2008, Zhang et al., 2005, Kassis et al., 2008). Chopp's group, in addition to observing functional recovery in EAE mice, demonstrated that small percentage of transplanted MSCs was integrated and expressed neural markers (J. Zhang et al., 2006). Their observations therefore suggested that some transdifferentiation had occurred. Overall, these pioneer studies demonstrated the therapeutic efficacy of MSCs in a model of CNS autoimmunity, but they left open the question whether their integration in the nervous system was essential for their therapeutic benefits.

Recently, a phase I trial was initiated to evaluate the safety and feasibility of intrathecal injection of autologous BM-MSCs in MS patients (Karussis et al., 2010). The initial findings of this trial support the possibility of migration of MSCs from their site of injection (lumbar area of the cerebrospinal fluid) to the brain ventricles and spinal cord parenchyma. Preliminary data of this trail also demonstrated the immunomodulatory effect of MSC in human neurological diseases. The authors concluded that the early clinical stabilization and improvement in some of the patients could be related to these immunomodulating effects. The possibility of neuroprotection and neuroregeneration through transdifferentiation of MSCs into cells of the neuronal or glial lineage, although theoretically viable, has yet to be proved by neuroimaging studies.

Promising results from this study will support further clinical trials to evaluate the long term safety and the potential clinical efficacy of MSC transplantation in the treatment of MS.

#### **5. Conclusions**

Although the curative effect of MSCs has been demonstrated in several animal models of CNS injury and neurodegeneration as well as in early human clinical trials of neurological disorders, the mechanisms that are responsible for these beneficial therapeutic effects are still poorly understood. Analysis of accumulated literature in this area suggest the following main mechanisms that may underlie the therapeutic effect of naive or neurally modified MSCs: 1) neurorepair (replacement of damaged or diseased neural cells by neurally transdifferentiated MSCs), 2) neuroprotection (modulation of immune response and inflammation, provision of trophic factors that could prevent neural cell apoptosis and

Mesenchymal Stem Cells in CNS Regeneration 251

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Human mesenchymal stem cells express neural genes, suggesting a neural

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#### **6. References**


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Plasticity of cultured mesenchymal stem cells: switch from nestin-positive to

mice can improve the cognitive ability of an Alzheimer's disease rat model.

neuron-like cells and show SMN protein expression]. Zhonghua Yi Xue Za Zhi,

Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis

of human bone marrow mesenchymal stem cells into neuron-like cells in vitro.

stromal cell treatment improves neurological functional recovery in EAE mice. Exp

cells reduce axonal loss in experimental autoimmune encephalomyelitis mice. J

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producing cells derived from rat bone marrow and their autologous


Riekstina, U., Cakstina, I., Parfejevs, V., Hoogduijn, M., Jankovskis, G., Muiznieks, I., et al.

Sanchez-Ramos, J., Song, S., Cardozo-Pelaez, F., Hazzi, C., Stedeford, T., Willing, A., et al.

Shiota, M., Heike, T., Haruyama, M., Baba, S., Tsuchiya, A., Fujino, H., et al. (2007). Isolation

Stappenbeck, T. S., & Miyoshi, H. (2009). The role of stromal stem cells in tissue regeneration

Stock, P., Bruckner, S., Ebensing, S., Hempel, M., Dollinger, M. M., & Christ, B. (2010). The

Suon, S., Yang, M., & Iacovitti, L. (2006). Adult human bone marrow stromal spheres

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Theise, N. D., Nimmakayalu, M., Gardner, R., Illei, P. B., Morgan, G., Teperman, L., et al. (2000). Liver from bone marrow in humans. Hepatology, 32(1), 11-16. Tio, M., Tan, K. H., Lee, W., Wang, T. T., & Udolph, G. (2010). Roles of db-cAMP, IBMX and

Tomita, S., Li, R. K., Weisel, R. D., Mickle, D. A., Kim, E. J., Sakai, T., et al. (1999).

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Trzaska, K. A., King, C. C., Li, K. Y., Kuzhikandathil, E. V., Nowycky, M. C., Ye, J. H., et al.

bone marrow mesenchymal stromal cells. BMC Genomics, 9, 166.

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**12** 

*Spain* 

**Therapeutic Potential of MSCs** 

José Ramón Lamas, Pilar Tornero-Esteban

and Benjamín Fernández-Gutiérrez *Hospital Clínico San Carlos, Madrid* 

**in Musculoskeletal Diseases (Osteoarthritis)** 

The Musculoskeletal or locomotor system is defined by a complex interconnection of different body parts functionally arranged in two main sub-systems: the skeletal system, composed of the bones, and secondly the muscle system. In addition other structures such as tendons, ligaments and other connective tissue joins both sub-systems providing

Musculoskeletal disorder or more generically Rheumatic disease (RD) is a term used to describe over 200 different disorders involving different connective tissue. Depending on the main target affected, RDs can be grouped into different pathologies. Joints are structures commonly affected in RDs, as it is the location where bone, tendon, ligament and muscle meet. Joint disorders are generically termed as arthropathies, and it is only when inflammation occurs, in one or more joints, that the disorder is called arthritis. However, RDs also include systemic disorders (autoimmune diseases affecting multiple organs), dorsopathies (back disorders), soft tissue disorders (involving muscles, tendons, etc.), and osteopathies/chondropathies (e.g., disorders

The prevalence of RDs in the elderly has erroneously been associated only with ageing; however RDs may develop at any time even in childhood (Manners, 2003, Mariller, 2005). Musculoskeletal disorders constitute the most common cause of severe chronic pain and physical disability, thus they are considered a public health problem that affect millions of individuals and constitute a major burden on health care, a situation which is aggravated by

Osteoarthritis is the most prevalent type of inflammatory arthritis (Spahn, 2011). Although it has long been considered to be primarily a cartilage disorder, induced by accumulated mechanical stress, as occurs in many other arthropathies the contribution of an inflammatory component is well established; sometimes produced by an autoimmune response, leading to chronic joint inflammation, destruction and cartilage loss. Little is

**1. Introduction** 

**1.1 Musculoskeletal disorders (MSDs)** 

related to bone density and structure-like osteoporosis).

an increasingly aging population (Bansback, 2005, Loza, 2008).

functional and structural support.

**1.2 Aetiology of osteoarthritis** 

transplantation in the duodenal wall for treating diabetes. Anat Rec (Hoboken), 292(5), 728-735.


### **Therapeutic Potential of MSCs in Musculoskeletal Diseases (Osteoarthritis)**

José Ramón Lamas, Pilar Tornero-Esteban and Benjamín Fernández-Gutiérrez *Hospital Clínico San Carlos, Madrid Spain* 

#### **1. Introduction**

260 Tissue Regeneration – From Basic Biology to Clinical Application

Zhang, Y. Q., Zeng, X., He, L. M., Ding, Y., Li, Y., & Zeng, Y. S. (2010). NT-3 gene modified

differentiate into neuron-like cells in vitro. Anat Sci Int, 85(2), 61-67. Zhang, Z., Maiman, D. J., Kurpad, S. N., Crowe, M. J., & Alexanian, A. R. (2011). Feline Bone

292(5), 728-735.

Cytotherapy, 9(5), 414-426.

Tissue Eng, 7(2), 211-228.

transplantation in the duodenal wall for treating diabetes. Anat Rec (Hoboken),

Schwann cells promote TrkC gene modified mesenchymal stem cells to

Marrow-Derived Mesenchymal Stem Cells Express Several Pluripotent and Neural Markers and Easily Turn into Neural-Like Cells by Manipulation with Chromatin Modifying Agents and Neural Inducing Factors. Cellular reprogramming, 13(5). Zhao, C. P., Zhang, C., Zhou, S. N., Xie, Y. M., Wang, Y. H., Huang, H., et al. (2007). Human

mesenchymal stromal cells ameliorate the phenotype of SOD1-G93A ALS mice.

Multilineage cells from human adipose tissue: implications for cell-based therapies.

Zuk, P. A., Zhu, M., Mizuno, H., Huang, J., Futrell, J. W., Katz, A. J., et al. (2001).

#### **1.1 Musculoskeletal disorders (MSDs)**

The Musculoskeletal or locomotor system is defined by a complex interconnection of different body parts functionally arranged in two main sub-systems: the skeletal system, composed of the bones, and secondly the muscle system. In addition other structures such as tendons, ligaments and other connective tissue joins both sub-systems providing functional and structural support.

Musculoskeletal disorder or more generically Rheumatic disease (RD) is a term used to describe over 200 different disorders involving different connective tissue. Depending on the main target affected, RDs can be grouped into different pathologies. Joints are structures commonly affected in RDs, as it is the location where bone, tendon, ligament and muscle meet. Joint disorders are generically termed as arthropathies, and it is only when inflammation occurs, in one or more joints, that the disorder is called arthritis. However, RDs also include systemic disorders (autoimmune diseases affecting multiple organs), dorsopathies (back disorders), soft tissue disorders (involving muscles, tendons, etc.), and osteopathies/chondropathies (e.g., disorders related to bone density and structure-like osteoporosis).

The prevalence of RDs in the elderly has erroneously been associated only with ageing; however RDs may develop at any time even in childhood (Manners, 2003, Mariller, 2005). Musculoskeletal disorders constitute the most common cause of severe chronic pain and physical disability, thus they are considered a public health problem that affect millions of individuals and constitute a major burden on health care, a situation which is aggravated by an increasingly aging population (Bansback, 2005, Loza, 2008).

#### **1.2 Aetiology of osteoarthritis**

Osteoarthritis is the most prevalent type of inflammatory arthritis (Spahn, 2011). Although it has long been considered to be primarily a cartilage disorder, induced by accumulated mechanical stress, as occurs in many other arthropathies the contribution of an inflammatory component is well established; sometimes produced by an autoimmune response, leading to chronic joint inflammation, destruction and cartilage loss. Little is

Therapeutic Potential of MSCs in Musculoskeletal Diseases (Osteoarthritis) 263

vehicle during new tissue formation would be the optimal scenario for the repairing process. However, this seemingly straightforward schema can be complicated depending on the tissue to repair, which in turn determines not only the type of cells to use but also the number and the mode of application. Thus, current challenges in musculoskeletal regenerative medicine cover several topics under study including: (1) the better understanding of cell biology, (2) the synthesis of new biomaterials for extracellular matrices (*scaffolds*), and (3) the definition of the best combination of cells, biologically active

Given that cells are the main building blocks of regenerative therapies, their availability and their commitment to a specific lineage are major limitations. The cells can be of autologous (host-derived) or allogeneic origin (non-host derived). Other sources, are cells of xenogeneic (from individuals of another species), syngeneic or isogeneic (isolated from genetically identical organisms or highly inbred individuals, respectively) origin are only constrained to

Obviously, the most logical approach for the regeneration of joint degraded cartilage, consists in the direct re-establishment of its main functional component, the chondrocytes. Joint cartilage is a connective tissue with special characteristics. It consists of chondrocytes that secrete a cartilage-specific extracellular matrix (ECM) made of collagens, mainly type II collagen, and different proteoglycans. The chondrocytes do not have direct cell-to-cell contact, thus each cell acts as a functional unit responsible for the production and maintenance of the ECM in its surrounding. These characteristics, in addition to the cartilage avascularity, explain the difficulties involved in repairing this tissue, because

The first approach, and the gold standard for years in joint orthopaedic surgery, has been the autologous chondrocyte implantation (ACI), after harvesting, from healthy cartilage biopsies and expanded in culture. So far, thousands of ACIs have been clinically applied with encouraging results in the short- and mid-term, but their long-term efficiency needs further confirmation (Alvarez-Dolado, 2007). The effectiveness of the technique is limited by some major drawbacks, including the absence of appropriate sources of suitable hyaline cartilage and the additional damage caused at the site of biopsy. Other important issues arise during chondrocyte expansion *in vitro* and further transplantation. In culture, chondrocytes easily dedifferentiate losing their chondrogenic phenotype and their redifferentiation potential and once it occurs, about half of the ACIs show evidence of chondrocyte hypertrophy, indicating the formation of a bone-like tissue. Finally, the occurrence of poor adhesion between the new and the original tissue is common and in those cases where scaffolds are used, the biomechanical properties obtained do not achieve the expected results. These problems have raised the need for alternative cell sources with chondrogenic potential for cartilage tissue engineering, a requisite accomplished by the stem

Under normal conditions body tissues are subjected to a continuous process of repair and regeneration of damaged and dead cells by means of a pool of progenitor or stem cells,

molecules and vehicles to promote growth and differentiation.

chondroprogenitor cell access to the damaged site is very limited.

cells, and in particular by mesenchymal stem cells (MSCs).

**2.2 Regenerative medicine using MSCs** 

**2.1 Regenerative medicine using mature chondrocytes** 

experimental models.

known about underlying molecular mechanisms. Its initiation and progression appear to be independent processes associated with different risk factors (Worthington, 2005). In addition to biomechanical stress on articular cartilage, the involvement of other tissues of the affected joint, such as the synovium, ligaments, periarticular muscles, and nerves, have also been proposed in OA aetiology and progression (Brandt, 2006). Several studies suggest that the subchondral bone is likely to be the most important structural element in both pain generation and disease progression. At least in its generalised form, OA shows features of a systemic musculoskeletal disease with a metabolic component and a genetic predisposition leading to the formation of a defective cartilage matrix (Aspden, 2008, Zhu, 2009). Unfortunately, despite advances in research, little is known about OA's exact etiology and pathogenetic mechanisms.

Currently there is no known cure for OA, and modern treatments only manage to reduce pain and maintain joint movement as much as possible. For many years the only known options for OA treatment were disease-modifying drugs, in mild cases, and several types of surgery depending on the affectation of articular structures. Among the surgery choices, arthroscopy and joint arthroplasty are the most common. The first is used in people with moderate lesions of articular cartilage or bone, in order to alleviate pain for a short time and to allow the joints to move more easily. Although it does not seem to treat the arthritis itself, sometimes the relief can delay the use of other more aggressive surgeries (Laupattarakasem, 2008). Total or partial joint arthroplasty is the ultimate surgical treatment when joint damage can be seen on radiographs. It involves surgery to replace the ends of bones, mostly in the hip, knee and shoulder thereby creating new surfaces. However, surgery is not recommended in those cases where the patient's health is precarious, due to serious risk of infections, and because after surgery, long periods of physical rehabilitation are needed. Moreover, the prostheses have a lifespan of 10 to 20 years, after which they require substitution.

In this therapeutic context, it is easy to understand that there is a real and urgent need for new and alternative treatments to circumvent the relatively low efficiency of existing therapies. This is where the emerging potential of regenerative medicine becomes increasingly important as the most promising method to restore, maintain or improve tissue structure and joint function (Bruder, 1997, Mackay, 1998, Pittenger, 1999, Zavan, 2007).

#### **2. Regenerative medicine in rheumatic diseases**

Broadly speaking, the term "regenerative medicine" refers to a new field in biomedical research focused on the development of therapeutic approaches allowing the body to replace and regenerate damaged or diseased cells, and ultimately the function of tissues and organs. This goal is achieved by means of a combination of approaches that include the use of soluble molecules, biomaterials, tissue engineering, gene therapy, stem cell transplantation and the reprogramming of cell and tissue types.

In the context of musculoskeletal disease, and in particular the reconstruction of articular defects caused by trauma or disease, the goal is to deliver cells that become competent in the defect site, initially optimizing biomechanics, and ultimately initiating new tissue production. Sometimes, as occurs in the case of soft tissue repair, an additional implant vehicle(s), is required to transport and constrain the implanted cells in the defect site and to provide mechanical stability to the surgical site. The progressive biodegradation of the

known about underlying molecular mechanisms. Its initiation and progression appear to be independent processes associated with different risk factors (Worthington, 2005). In addition to biomechanical stress on articular cartilage, the involvement of other tissues of the affected joint, such as the synovium, ligaments, periarticular muscles, and nerves, have also been proposed in OA aetiology and progression (Brandt, 2006). Several studies suggest that the subchondral bone is likely to be the most important structural element in both pain generation and disease progression. At least in its generalised form, OA shows features of a systemic musculoskeletal disease with a metabolic component and a genetic predisposition leading to the formation of a defective cartilage matrix (Aspden, 2008, Zhu, 2009). Unfortunately, despite advances in research, little is known about OA's exact etiology and

Currently there is no known cure for OA, and modern treatments only manage to reduce pain and maintain joint movement as much as possible. For many years the only known options for OA treatment were disease-modifying drugs, in mild cases, and several types of surgery depending on the affectation of articular structures. Among the surgery choices, arthroscopy and joint arthroplasty are the most common. The first is used in people with moderate lesions of articular cartilage or bone, in order to alleviate pain for a short time and to allow the joints to move more easily. Although it does not seem to treat the arthritis itself, sometimes the relief can delay the use of other more aggressive surgeries (Laupattarakasem, 2008). Total or partial joint arthroplasty is the ultimate surgical treatment when joint damage can be seen on radiographs. It involves surgery to replace the ends of bones, mostly in the hip, knee and shoulder thereby creating new surfaces. However, surgery is not recommended in those cases where the patient's health is precarious, due to serious risk of infections, and because after surgery, long periods of physical rehabilitation are needed. Moreover, the prostheses have a

In this therapeutic context, it is easy to understand that there is a real and urgent need for new and alternative treatments to circumvent the relatively low efficiency of existing therapies. This is where the emerging potential of regenerative medicine becomes increasingly important as the most promising method to restore, maintain or improve tissue structure and joint function (Bruder, 1997, Mackay, 1998, Pittenger, 1999, Zavan, 2007).

Broadly speaking, the term "regenerative medicine" refers to a new field in biomedical research focused on the development of therapeutic approaches allowing the body to replace and regenerate damaged or diseased cells, and ultimately the function of tissues and organs. This goal is achieved by means of a combination of approaches that include the use of soluble molecules, biomaterials, tissue engineering, gene therapy, stem cell

In the context of musculoskeletal disease, and in particular the reconstruction of articular defects caused by trauma or disease, the goal is to deliver cells that become competent in the defect site, initially optimizing biomechanics, and ultimately initiating new tissue production. Sometimes, as occurs in the case of soft tissue repair, an additional implant vehicle(s), is required to transport and constrain the implanted cells in the defect site and to provide mechanical stability to the surgical site. The progressive biodegradation of the

lifespan of 10 to 20 years, after which they require substitution.

**2. Regenerative medicine in rheumatic diseases** 

transplantation and the reprogramming of cell and tissue types.

pathogenetic mechanisms.

vehicle during new tissue formation would be the optimal scenario for the repairing process. However, this seemingly straightforward schema can be complicated depending on the tissue to repair, which in turn determines not only the type of cells to use but also the number and the mode of application. Thus, current challenges in musculoskeletal regenerative medicine cover several topics under study including: (1) the better understanding of cell biology, (2) the synthesis of new biomaterials for extracellular matrices (*scaffolds*), and (3) the definition of the best combination of cells, biologically active molecules and vehicles to promote growth and differentiation.

#### **2.1 Regenerative medicine using mature chondrocytes**

Given that cells are the main building blocks of regenerative therapies, their availability and their commitment to a specific lineage are major limitations. The cells can be of autologous (host-derived) or allogeneic origin (non-host derived). Other sources, are cells of xenogeneic (from individuals of another species), syngeneic or isogeneic (isolated from genetically identical organisms or highly inbred individuals, respectively) origin are only constrained to experimental models.

Obviously, the most logical approach for the regeneration of joint degraded cartilage, consists in the direct re-establishment of its main functional component, the chondrocytes. Joint cartilage is a connective tissue with special characteristics. It consists of chondrocytes that secrete a cartilage-specific extracellular matrix (ECM) made of collagens, mainly type II collagen, and different proteoglycans. The chondrocytes do not have direct cell-to-cell contact, thus each cell acts as a functional unit responsible for the production and maintenance of the ECM in its surrounding. These characteristics, in addition to the cartilage avascularity, explain the difficulties involved in repairing this tissue, because chondroprogenitor cell access to the damaged site is very limited.

The first approach, and the gold standard for years in joint orthopaedic surgery, has been the autologous chondrocyte implantation (ACI), after harvesting, from healthy cartilage biopsies and expanded in culture. So far, thousands of ACIs have been clinically applied with encouraging results in the short- and mid-term, but their long-term efficiency needs further confirmation (Alvarez-Dolado, 2007). The effectiveness of the technique is limited by some major drawbacks, including the absence of appropriate sources of suitable hyaline cartilage and the additional damage caused at the site of biopsy. Other important issues arise during chondrocyte expansion *in vitro* and further transplantation. In culture, chondrocytes easily dedifferentiate losing their chondrogenic phenotype and their redifferentiation potential and once it occurs, about half of the ACIs show evidence of chondrocyte hypertrophy, indicating the formation of a bone-like tissue. Finally, the occurrence of poor adhesion between the new and the original tissue is common and in those cases where scaffolds are used, the biomechanical properties obtained do not achieve the expected results. These problems have raised the need for alternative cell sources with chondrogenic potential for cartilage tissue engineering, a requisite accomplished by the stem cells, and in particular by mesenchymal stem cells (MSCs).

#### **2.2 Regenerative medicine using MSCs**

Under normal conditions body tissues are subjected to a continuous process of repair and regeneration of damaged and dead cells by means of a pool of progenitor or stem cells,

Therapeutic Potential of MSCs in Musculoskeletal Diseases (Osteoarthritis) 265

cells giving rise to a variety of cells which can form connective tissues such as chondrocytes, osteocytes, adipocytes and tenocytes (Pittenger, 1999), (Figure 1) and second: in contrast to most other adult stem cells, they can be isolated from a diversity of accessible tissues, such as bone marrow, fat tissues and umbilical cord blood (Chanda, 2010, Moretti, 2010, Romanov, 2005). Moreover, they can be isolated and identified through their adhesion potential in culture and by the expression of several "positive markers", represented by the transmembrane proteins CD90, CD73, CD105 and CD166. Additionally, MSCs are easily expanded *in vitro* without losing their "stemness" and/or self-renewal capacity (Bianco,

MSCs have been shown to differentiate *in vitro* into bone, cartilage, muscle, tendon, and fat, and possibly also into cardiomyocytes and hepatocytes (Conget, 1999, Chivu, 2009, Dennis, 1999, Pereira, 1995, Pittenger, 1999, Remy-Martin, 1999). Finally, from an immunological point of view, an important property of MSCs, especially for their use in rheumatic diseases, resides in their potent immunosuppressive and anti-inflammatory functions, the lack of induction of graft rejection and their chemotactic properties, similar to immune cells in response to injury on sites of inflammation (Le Blanc, 2004, Spaeth, 2008). As such, these cells are currently being considered for their potential use in cell and gene therapy, in a large number of human diseases, and particularly in a variety of clinical musculoskeletal conditions, including the repair of cartilage defects, tendon/ligament and

As occurs during embryogenesis, the generation of new cartilage (chondrogenesis) involves the MSCs progression through different stages in a tightly regulated process coordinated by multiple signalling pathways which include the Wnt, Notch or TGF (Quintana, 2009, Roelen, 2003). In particular, the Wnt/β-catenin signalling pathway plays a crucial role in cartilage reparation, since it participates in the differentiation of MSCs into osteoblasts or chondrocytes during osteogenesis and/or chondrogenesis (Day, 2005, Gaur, 2005, Hill, 2005). Markedly, alteration in any of the aforementioned pathways, as occurs in some

*In vitro* chondrogenesis is routinely performed by culturing MSCs in three dimensional scaffolds made of different biomaterials such as collagen, fibrin, agarose, alginate, chitosan or hyaluronic acid of natural or synthetic origin, or a combination of both types (Li, 2005, Lisignoli, 2005, Necas, 2010, Zhou, 2008). In addition, these scaffolds can be supplemented with soluble factors such as TGF-β, growth factors, bone morphogenetic proteins (BMPs),

Application of engineered MSCs for cartilage regeneration has been addressed by the slight modifications of two main approaches widely tested in different OA animal models with

In the first, MSCs are seeded on 3D scaffolds with the presence or absence of soluble factors (growth factors and/or cytokines) and the resulting structure is used to repair the cartilage defect (Zscharnack, 2010). The second approach, consists of the direct administration of MSCs, (loaded or not in 3D scaffolds) without previous differentiation (Thorpe, 2010).

diseases, can lead to detrimental effects during the regeneration process.

etc. to facilitate the chondrogenic differentiation of MSCs.

2001, Caplan, 2000, Reiser, 2005).

**2.3 MSCs and cartilage repair in OA** 

encouraging results (Figure 2).

bone.

which have the capacity to differentiate into the specialised cell type being replaced. 'Stem cells' is a generic term to describe a variety of cells which share two common characteristics: (1) their self-renewal potential and (2) their capacity to give rise to different tissues. However their "potency", or differentiation potential is variable and therefore there exists a hierarchy according to stem cell types. The most versatile, the totipotent embryonic stem cells (ESCs) give rise to other embryonic or extra embryonic adult stem cells (ASCs) with pluri- multi- or uni- potentiality. Pluripotent stem cells are descendants of totipotent cells and can differentiate into cells derived from the endoderm, mesoderm and ectoderm germ layers. Multipotent stem cells can produce only cells of a closely related family of cells, e.g., hematopoietic stem cells and MSCs. Finally, unipotent cells only produce one cell type, but retain their self-renewal properties, a feature that distinguishes them from other non-stem cells.

Fig. 1. Diagram of the mesenchymal stem cell lineage and its differentiation potential

MSCs have the potential to differentiate into several cell types of mesodermal origin including bone, cartilage, muscle, bone marrow stroma, tendon/ligament, fat, dermis. Thus these cells are optimal candidates in regenerative medicine strategies intended to restore these connective tissues. Adapted from (Caplan, 2007).

Although for the purposes of regenerative medicine, ESCs could be considered the optimal candidates; their clinical use in human therapies is still controversial due to ethical issues. In the context of musculoskeletal diseases, the least compromised are mesenchymal stem cells (MSCs) and are of particular interest for several reasons. First, they are the progenitors of cells giving rise to a variety of cells which can form connective tissues such as chondrocytes, osteocytes, adipocytes and tenocytes (Pittenger, 1999), (Figure 1) and second: in contrast to most other adult stem cells, they can be isolated from a diversity of accessible tissues, such as bone marrow, fat tissues and umbilical cord blood (Chanda, 2010, Moretti, 2010, Romanov, 2005). Moreover, they can be isolated and identified through their adhesion potential in culture and by the expression of several "positive markers", represented by the transmembrane proteins CD90, CD73, CD105 and CD166. Additionally, MSCs are easily expanded *in vitro* without losing their "stemness" and/or self-renewal capacity (Bianco, 2001, Caplan, 2000, Reiser, 2005).

MSCs have been shown to differentiate *in vitro* into bone, cartilage, muscle, tendon, and fat, and possibly also into cardiomyocytes and hepatocytes (Conget, 1999, Chivu, 2009, Dennis, 1999, Pereira, 1995, Pittenger, 1999, Remy-Martin, 1999). Finally, from an immunological point of view, an important property of MSCs, especially for their use in rheumatic diseases, resides in their potent immunosuppressive and anti-inflammatory functions, the lack of induction of graft rejection and their chemotactic properties, similar to immune cells in response to injury on sites of inflammation (Le Blanc, 2004, Spaeth, 2008). As such, these cells are currently being considered for their potential use in cell and gene therapy, in a large number of human diseases, and particularly in a variety of clinical musculoskeletal conditions, including the repair of cartilage defects, tendon/ligament and bone.

#### **2.3 MSCs and cartilage repair in OA**

264 Tissue Regeneration – From Basic Biology to Clinical Application

which have the capacity to differentiate into the specialised cell type being replaced. 'Stem cells' is a generic term to describe a variety of cells which share two common characteristics: (1) their self-renewal potential and (2) their capacity to give rise to different tissues. However their "potency", or differentiation potential is variable and therefore there exists a hierarchy according to stem cell types. The most versatile, the totipotent embryonic stem cells (ESCs) give rise to other embryonic or extra embryonic adult stem cells (ASCs) with pluri- multi- or uni- potentiality. Pluripotent stem cells are descendants of totipotent cells and can differentiate into cells derived from the endoderm, mesoderm and ectoderm germ layers. Multipotent stem cells can produce only cells of a closely related family of cells, e.g., hematopoietic stem cells and MSCs. Finally, unipotent cells only produce one cell type, but retain their self-renewal properties, a feature that distinguishes them from other non-stem

Fig. 1. Diagram of the mesenchymal stem cell lineage and its differentiation potential

these connective tissues. Adapted from (Caplan, 2007).

MSCs have the potential to differentiate into several cell types of mesodermal origin including bone, cartilage, muscle, bone marrow stroma, tendon/ligament, fat, dermis. Thus these cells are optimal candidates in regenerative medicine strategies intended to restore

Although for the purposes of regenerative medicine, ESCs could be considered the optimal candidates; their clinical use in human therapies is still controversial due to ethical issues. In the context of musculoskeletal diseases, the least compromised are mesenchymal stem cells (MSCs) and are of particular interest for several reasons. First, they are the progenitors of

cells.

As occurs during embryogenesis, the generation of new cartilage (chondrogenesis) involves the MSCs progression through different stages in a tightly regulated process coordinated by multiple signalling pathways which include the Wnt, Notch or TGF (Quintana, 2009, Roelen, 2003). In particular, the Wnt/β-catenin signalling pathway plays a crucial role in cartilage reparation, since it participates in the differentiation of MSCs into osteoblasts or chondrocytes during osteogenesis and/or chondrogenesis (Day, 2005, Gaur, 2005, Hill, 2005). Markedly, alteration in any of the aforementioned pathways, as occurs in some diseases, can lead to detrimental effects during the regeneration process.

*In vitro* chondrogenesis is routinely performed by culturing MSCs in three dimensional scaffolds made of different biomaterials such as collagen, fibrin, agarose, alginate, chitosan or hyaluronic acid of natural or synthetic origin, or a combination of both types (Li, 2005, Lisignoli, 2005, Necas, 2010, Zhou, 2008). In addition, these scaffolds can be supplemented with soluble factors such as TGF-β, growth factors, bone morphogenetic proteins (BMPs), etc. to facilitate the chondrogenic differentiation of MSCs.

Application of engineered MSCs for cartilage regeneration has been addressed by the slight modifications of two main approaches widely tested in different OA animal models with encouraging results (Figure 2).

In the first, MSCs are seeded on 3D scaffolds with the presence or absence of soluble factors (growth factors and/or cytokines) and the resulting structure is used to repair the cartilage defect (Zscharnack, 2010). The second approach, consists of the direct administration of MSCs, (loaded or not in 3D scaffolds) without previous differentiation (Thorpe, 2010).

Therapeutic Potential of MSCs in Musculoskeletal Diseases (Osteoarthritis) 267

However, there are still some unanswered questions about the mechanism by which MSCs perform the repair; but several possibilities have been outlined, such as the following: (1) the secretion of cytokines to enhance repair (Chen, 2008); (2) the modulation of immune (Aggarwal, 2005, Gerdoni, 2007, Karussis, 2008, Le Blanc, 2007, Ren, 2008) and inflammatory responses (Gupta, 2007, Ortiz, 2007); (3) stimulation of the proliferation of tissue endogenous stem cells (Lee, 2006, Munoz, 2005); and (4) the rescue of damaged cells (Spees, 2006, Spees, 2003). Finally, MSCs are the subject in a controversy where their contradictory effects *in vitro* and *in vivo* on tumour cell growth have been called into question. Recent studies have shown that MSCs can increase the proliferation of tumor cells *in vitro* and promote tumor growth *in vivo* by increasing the neovascularization (Suzuki, 2011, Tian, 2011). This is a major concern that should be carefully considered, particularly in conditions

Our group has been focused for several years on the study of the biology of articular cartilage in the OA pathogenesis and the potential of MSCs in regeneration of damaged cartilage due to this disease. Some of the issues addressed include the basic research and the clinical trials to validate the translational efficacy in the clinic of MSC implantation. Much of our work in this field has been based on the use of modern techniques, that include proteomics and genomics approaches in combination with bioinformatics and genetic

Proteomics is considered and emerging field with widespread potential applications to shape how rheumatic diseases are diagnosed, prognosticated, and clinically managed (Camafeita, 2009, Vanarsa, 2010). A key methodological advance in the classical twodimensional gel electrophoresis (2-DE) has been the emergence of multiplexing twodimensional fluorescence difference gel electrophoresis (2D-DIGE) (Unlu, 1997). The 2D-DIGE technique circumvents many of the issues associated with traditional 2-DE, providing more sensitivity, high reproducibility and a wide dynamic range of detection (Alban, 2003, Viswanathan, 2006). It consists in the labelling of lysine groups on protein extracts with fluorescent dyes with different emission spectra before isoelectric focusing (IEF). Protein samples are further labelled with Cy3 and Cy5 fluorescent dyes, while Cy2 dye is used to label the internal standard, which consists of a pooled sample comprising equal amounts of all samples to be compared. Then the three samples are electrophoresed on a single 2D gel, which allows both direct quantitative comparisons within each gel and the normalization of quantitative abundance values for each protein between gels. The combination of 2D-DIGE with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) provides a powerful tool for identifying disease-related proteins (Stults, 2005). (Figure 3). Genomic approaches have been developed through DNA microarray analysis, a proven "state of the art" technology for the simultaneous screening of expression levels in

Another strategy, different from the previous two, is the systemic application of MSCs, that have been shown to promote tissue repair by formation of fibrocartilage-like tissues in response to damaged subchondral bone (Chang, 2011), which is likely due to the intrinsic

where tumoral malignancies are present.

**3. Studies carried out in our group** 

large numbers of genes (Licatalosi, 2010). (Figure 4)

ability of MSCs to migrate into injured or inflamed tissues.

validation.

Fig. 2. MSC-based tissue engineering and cell therapy for cartilage repair and regeneration. Once MSCs are isolated and expanded, both tissue engineering and cell therapy approaches are suited for regeneration. In the *ex vivo* approach cells are loaded *in vitro* onto the scaffolds under appropriate stimuli and after a short incubation to insure attachment, the cell-scaffold composites are implanted. Another strategy is based on local injection into the affected joint. Adapted from (Caplan, 2007).

However, there are still some unanswered questions about the mechanism by which MSCs perform the repair; but several possibilities have been outlined, such as the following: (1) the secretion of cytokines to enhance repair (Chen, 2008); (2) the modulation of immune (Aggarwal, 2005, Gerdoni, 2007, Karussis, 2008, Le Blanc, 2007, Ren, 2008) and inflammatory responses (Gupta, 2007, Ortiz, 2007); (3) stimulation of the proliferation of tissue endogenous stem cells (Lee, 2006, Munoz, 2005); and (4) the rescue of damaged cells (Spees, 2006, Spees, 2003). Finally, MSCs are the subject in a controversy where their contradictory effects *in vitro* and *in vivo* on tumour cell growth have been called into question. Recent studies have shown that MSCs can increase the proliferation of tumor cells *in vitro* and promote tumor growth *in vivo* by increasing the neovascularization (Suzuki, 2011, Tian, 2011). This is a major concern that should be carefully considered, particularly in conditions where tumoral malignancies are present.

#### **3. Studies carried out in our group**

266 Tissue Regeneration – From Basic Biology to Clinical Application

Fig. 2. MSC-based tissue engineering and cell therapy for cartilage repair and regeneration. Once MSCs are isolated and expanded, both tissue engineering and cell therapy approaches are suited for regeneration. In the *ex vivo* approach cells are loaded *in vitro* onto the scaffolds under appropriate stimuli and after a short incubation to insure attachment, the cell-scaffold composites are implanted. Another strategy is based on local injection into the affected joint.

Adapted from (Caplan, 2007).

Our group has been focused for several years on the study of the biology of articular cartilage in the OA pathogenesis and the potential of MSCs in regeneration of damaged cartilage due to this disease. Some of the issues addressed include the basic research and the clinical trials to validate the translational efficacy in the clinic of MSC implantation. Much of our work in this field has been based on the use of modern techniques, that include proteomics and genomics approaches in combination with bioinformatics and genetic validation.

Proteomics is considered and emerging field with widespread potential applications to shape how rheumatic diseases are diagnosed, prognosticated, and clinically managed (Camafeita, 2009, Vanarsa, 2010). A key methodological advance in the classical twodimensional gel electrophoresis (2-DE) has been the emergence of multiplexing twodimensional fluorescence difference gel electrophoresis (2D-DIGE) (Unlu, 1997). The 2D-DIGE technique circumvents many of the issues associated with traditional 2-DE, providing more sensitivity, high reproducibility and a wide dynamic range of detection (Alban, 2003, Viswanathan, 2006). It consists in the labelling of lysine groups on protein extracts with fluorescent dyes with different emission spectra before isoelectric focusing (IEF). Protein samples are further labelled with Cy3 and Cy5 fluorescent dyes, while Cy2 dye is used to label the internal standard, which consists of a pooled sample comprising equal amounts of all samples to be compared. Then the three samples are electrophoresed on a single 2D gel, which allows both direct quantitative comparisons within each gel and the normalization of quantitative abundance values for each protein between gels. The combination of 2D-DIGE with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) provides a powerful tool for identifying disease-related proteins (Stults, 2005). (Figure 3). Genomic approaches have been developed through DNA microarray analysis, a proven "state of the art" technology for the simultaneous screening of expression levels in large numbers of genes (Licatalosi, 2010). (Figure 4)

Another strategy, different from the previous two, is the systemic application of MSCs, that have been shown to promote tissue repair by formation of fibrocartilage-like tissues in response to damaged subchondral bone (Chang, 2011), which is likely due to the intrinsic ability of MSCs to migrate into injured or inflamed tissues.

Therapeutic Potential of MSCs in Musculoskeletal Diseases (Osteoarthritis) 269

Fig. 4. Schematic representation of the DNA microarray methodology.

DNA microarrays are commonly used to detect messenger RNAs (mRNA), referred to as expression profiling. The method consists in the fluorescently labelling of RNA while the RNA

sometimes incorporated into this step. Two-color labeling allows two samples or conditions, to be hybridized to the same array and their gene expression profiles compared via the difference in the fluorescence of the two samples. Statistical post-processing of the fluorescence data is

The breakdown of cartilage in OA involves the degradation of the extracellular matrix macromolecules and the altered expression of chondrocyte proteins necessary for normal joint function (Lane Smith, 2000). Thus the screening of proteins with altered expression in chondrocytes from patients with end stage OA compared to control subjects could expand the knowledge of the pathological processes implicated in the damage of articular cartilage in OA. Elucidation of the phenotypical alterations occurring in OA is important for the ascertainment of disease aetiology and for the development of effective treatments for OA.

is converted into complementary DNA (cDNA). Amplification of sequences by PCR is

usually necessary to eliminate artifacts and false results from the data obtained.

**3.1 Proteomic studies in Chondrocytes and MSCs in osteoarthritis** 

Fig. 3. Schematic representation of the 2D-DIGE methodology

2D-DIGE used for the analysis of protein differential expression in MSCs and chondrocytes of patients with osteoarthritis (sample A) compared to control subjects (sample B). CHAPS, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate; Em, Emission; Ex, Excitation; IEF, isoelectrofocusing. MM, Molecular mass; pI, Isoelectric point; SDS PAGE, polyacrylamide gel electrophoresis in the presence of Sodium Dodecyl Sulphate.

Fig. 3. Schematic representation of the 2D-DIGE methodology

2D-DIGE used for the analysis of protein differential expression in MSCs and chondrocytes of patients with osteoarthritis (sample A) compared to control subjects (sample B). CHAPS,

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate; Em, Emission; Ex, Excitation; IEF, isoelectrofocusing. MM, Molecular mass; pI, Isoelectric point; SDS PAGE,

polyacrylamide gel electrophoresis in the presence of Sodium Dodecyl Sulphate.

Fig. 4. Schematic representation of the DNA microarray methodology. DNA microarrays are commonly used to detect messenger RNAs (mRNA), referred to as expression profiling. The method consists in the fluorescently labelling of RNA while the RNA is converted into complementary DNA (cDNA). Amplification of sequences by PCR is sometimes incorporated into this step. Two-color labeling allows two samples or conditions, to be hybridized to the same array and their gene expression profiles compared via the difference in the fluorescence of the two samples. Statistical post-processing of the fluorescence data is usually necessary to eliminate artifacts and false results from the data obtained.

#### **3.1 Proteomic studies in Chondrocytes and MSCs in osteoarthritis**

The breakdown of cartilage in OA involves the degradation of the extracellular matrix macromolecules and the altered expression of chondrocyte proteins necessary for normal joint function (Lane Smith, 2000). Thus the screening of proteins with altered expression in chondrocytes from patients with end stage OA compared to control subjects could expand the knowledge of the pathological processes implicated in the damage of articular cartilage in OA. Elucidation of the phenotypical alterations occurring in OA is important for the ascertainment of disease aetiology and for the development of effective treatments for OA.

Therapeutic Potential of MSCs in Musculoskeletal Diseases (Osteoarthritis) 271

extraction. After 2D-DIGE, differentially expressed proteins were excised from the gel, digested with trypsin and analysed by a MALDI-TOF MS mass spectrometer. Protein identification after peptide mass fingerprinting (PMF) enabled the identification of 27 proteins (14 decreased and 13 increased) in OA chondrocytes. The cellular localization, biological process and molecular functions and of the identified proteins obtained from the

In this study, a significant differential expression pattern was observed for 27 different chondrocyte proteins. These included an elevated number of cytoskeletal binding proteins cytoskeleton binding, protein disruption, apoptosis and glycolysis proteins displayed a significantly changed expression in OA chondrocytes. Overall, the results suggested the deregulated production in OA cartilage of proteins pertaining to key cellular processes essential for the proper functioning of the chondrocytes, which may have direct effects on

A similar approach was also carried out to study the differential proteome of bone marrow MSCs (BM-MSCs) (Rollin, 2008b) from patients with OA *vs.* MSCs control, obtained from patients with hip fracture without OA signs. In this study we demonstrated the existence of specific alterations in the proteome content of bone marrow MSCs from patients with OA. Once classified into different groups, according to their biological function, the majority of proteins that changed at least 1.5-fold, belonged to the metabolic enzymes, cytoskeleton/motility and transport categories. Markedly, most proteins related to cytoskeleton/motility were down-regulated in MSCs from OA patients. Considering previous evidences supporting that MSCs can home to some tissues, particularly when injured or inflamed, the mechanisms underlying migratory capacity, as a key event for tissue repair by MSCs, were also studied *in vitro* using PDGF as chemoattractant. Our results demonstrated a significant increase in the motility of MSC of OA patients. Together with the differential expression of metabolic and cytoskeleton proteins we concluded that an activation of OA BM-MSCs occurs in response to chemotactic signals sent by the altered

online FatiGO ontology database are summarized in (Figure 5).

subchondral bone in an attempt to heal damaged tissues.

**3.2 Gene expression alterations in bone marrow MSCs in osteoarthritis** 

Our previous experimental data obtained in MSCs proteomic studies indicate an increased migratory capacity of BM-MSCs to the damaged tissues, likely to initiate and/or enhance the wound repair process. In this context, it is known that transforming growth factor-β (TGF-β) plays an important role in directing the cell fate choices in mesenchymal cells (Roelen, 2003). TGF-β induces the chondrogenic differentiation of MSC in the presence of dexamethasone or 3-dimensional cell aggregates (Mackay, 1998) and may act in conjunction with other microenvironmental factors on MSC differentiation. To assess the importance of TGF-β expression in MSCs from OA we comparatively studied by quantitative real-time PCR the expression of genes encoding the total TGF-β and those of the 3 isoforms of TGF-β (1, 2, 3) and TGF-β receptors (TBR-I, TBR-II, TBR-III) in primary cultures of BM-MSCs from patients with end stage OA and healthy control subjects (Rollin, 2008a). Our results showed that only TGF-β1 isoform was significantly increased in MSCs from OA. In addition, we also described an increased expression of TBR-II and TBR-III genes, but not of TBR-I in MSCs from OA. A possible explanation for this upregulated TGF-β upregulation in MSCs could be related to an stimulatory effect on

OA cartilage biology.

Fig. 5. Gene ontology annotation of the 27 changed proteins identified in OA chondrocytes.

Our first proteomic study dates from 2008 (Rollin, 2008c). Here human knee cartilage was obtained during total joint replacement surgery in six patients with clinical and radiological features of OA and six control samples from adult donors of similar age. Chondrocytes were isolated and cultured during 2-3 weeks at confluence in primary culture, before protein

Fig. 5. Gene ontology annotation of the 27 changed proteins identified in OA chondrocytes.

Our first proteomic study dates from 2008 (Rollin, 2008c). Here human knee cartilage was obtained during total joint replacement surgery in six patients with clinical and radiological features of OA and six control samples from adult donors of similar age. Chondrocytes were isolated and cultured during 2-3 weeks at confluence in primary culture, before protein extraction. After 2D-DIGE, differentially expressed proteins were excised from the gel, digested with trypsin and analysed by a MALDI-TOF MS mass spectrometer. Protein identification after peptide mass fingerprinting (PMF) enabled the identification of 27 proteins (14 decreased and 13 increased) in OA chondrocytes. The cellular localization, biological process and molecular functions and of the identified proteins obtained from the online FatiGO ontology database are summarized in (Figure 5).

In this study, a significant differential expression pattern was observed for 27 different chondrocyte proteins. These included an elevated number of cytoskeletal binding proteins cytoskeleton binding, protein disruption, apoptosis and glycolysis proteins displayed a significantly changed expression in OA chondrocytes. Overall, the results suggested the deregulated production in OA cartilage of proteins pertaining to key cellular processes essential for the proper functioning of the chondrocytes, which may have direct effects on OA cartilage biology.

A similar approach was also carried out to study the differential proteome of bone marrow MSCs (BM-MSCs) (Rollin, 2008b) from patients with OA *vs.* MSCs control, obtained from patients with hip fracture without OA signs. In this study we demonstrated the existence of specific alterations in the proteome content of bone marrow MSCs from patients with OA. Once classified into different groups, according to their biological function, the majority of proteins that changed at least 1.5-fold, belonged to the metabolic enzymes, cytoskeleton/motility and transport categories. Markedly, most proteins related to cytoskeleton/motility were down-regulated in MSCs from OA patients. Considering previous evidences supporting that MSCs can home to some tissues, particularly when injured or inflamed, the mechanisms underlying migratory capacity, as a key event for tissue repair by MSCs, were also studied *in vitro* using PDGF as chemoattractant. Our results demonstrated a significant increase in the motility of MSC of OA patients. Together with the differential expression of metabolic and cytoskeleton proteins we concluded that an activation of OA BM-MSCs occurs in response to chemotactic signals sent by the altered subchondral bone in an attempt to heal damaged tissues.

#### **3.2 Gene expression alterations in bone marrow MSCs in osteoarthritis**

Our previous experimental data obtained in MSCs proteomic studies indicate an increased migratory capacity of BM-MSCs to the damaged tissues, likely to initiate and/or enhance the wound repair process. In this context, it is known that transforming growth factor-β (TGF-β) plays an important role in directing the cell fate choices in mesenchymal cells (Roelen, 2003). TGF-β induces the chondrogenic differentiation of MSC in the presence of dexamethasone or 3-dimensional cell aggregates (Mackay, 1998) and may act in conjunction with other microenvironmental factors on MSC differentiation. To assess the importance of TGF-β expression in MSCs from OA we comparatively studied by quantitative real-time PCR the expression of genes encoding the total TGF-β and those of the 3 isoforms of TGF-β (1, 2, 3) and TGF-β receptors (TBR-I, TBR-II, TBR-III) in primary cultures of BM-MSCs from patients with end stage OA and healthy control subjects (Rollin, 2008a). Our results showed that only TGF-β1 isoform was significantly increased in MSCs from OA. In addition, we also described an increased expression of TBR-II and TBR-III genes, but not of TBR-I in MSCs from OA. A possible explanation for this upregulated TGF-β upregulation in MSCs could be related to an stimulatory effect on

Therapeutic Potential of MSCs in Musculoskeletal Diseases (Osteoarthritis) 273

were signal transduction, development and cell differentiation, which in turn are key functions in pluripotential cells. Based on the function of the proteins encoded by these genes, our results suggested that MSCs from patients with OA have a diminished differentiation and regenerative potentials that limit ther ability to generate a functional

Overall, in this study we provided a reference dataset of genes related to essential functions for the normal biology of MSCs that become altered in OA (Figure 4). We also described for the first time an association between the COL10A1 gene and OA susceptibility suggesting that underlying biological changes which occur during OA disease might be related, at least in part, to defects in the ECM and the formation of subchondral bone, an essential structure for providing joint stability. Moreover, in this work we also demonstrated that the expression of

Chronic degeneration is the most frequent cause for lesions of the rotator cuff, a set of four tendons connecting the scapula with the humeral head in shoulder. The most affected tendon of the cuff, frequently affected by tears, is the supraspinatus tendon. These lesions are increased in aged patients who frequently need reconstructive surgery. Surgical methods are often unsatisfactory due to inefficient recovery. In this context, MSC-based regenerative medicine offers a hopeful alternative for this type of treatment. In this sense, our group is performing several studies focused on the evaluation of the effectiveness of recovery after treatments consisting in surgical implantations of MSCs alone or in combination with a commercial membrane of type I collagen (OrthoadaptTM). This strategy has been previously tested in in a model of acute and chronic injury in rats with promising results allowing us to conduct a clinical trial in humans that is currently under

Treatment of osteoarthritis includes a combination of pharmacological and nonpharmacological measures aimed at relieving pain and the improvement of joint function. Initial treatment is dependent on the extent of the disease, age of patients and the joints affected, in descending order of frequency: Hip, Knee, Foot and Ankle. In mild cases, treatment begins by the use of simple analgesics (eg. paracetamol), (Nonsteroidal antiinflammatory drugs) NSAIDs (eg. ibuprofen and naproxen) or intermittent intra-articular administration (infiltration) of corticosteroids. However, although the symptoms and pain can be partially alleviated, adverse effects associated with conventional drug therapy is not recommended for long time periods. Moreover, treatment is often accompanied by nonpharmacological treatments, these include patient education and physical exercises to restore joint movement and to increase muscle strength, reduction of weight on painful joints. When joints are severely damaged treatment may require surgery. The most common surgical treatments are arthroscopic surgery, to trim damaged cartilage. Osteotomy, to change the alignment of a bone to relieve stress on the bone or joint. Arthrodesis or surgical fusion of bones, usually in the spine and the total or partial arthroplasty to replace the

multiple genes related to the wnt pathway were downregulated in OA patients.

**4. MSCs, conventional and other experimental treatments in OA** 

lineage of cells involved in musculoskeletal tissue homeostasis.

**3.3 Animal models in regenerative medicine (tendon repair)** 

development.

damaged joint with an artificial one.

mesenchymal cell proliferation in bone marrow allowing their expansion in response to the bone and cartilage damage characteristic of this disease.

More recently, another experimental approach was carried out by our group based on the comprehensive study of gene expression of MSCs using a DNA microarray expression analysis (Lamas, 2010). Gene expression profiles of MSCs from OA patients were compared to those of MSCs from healthy individuals. After integration of expression profiles into functional categories, by means of a gene ontology (GO)-based statistical analysis using GeneCodis 2.0 (Carmona-Saez, 2007), seventy-five genes from a list of 532 provided for comparison did not show annotations. The remaining 457 genes were grouped into different GO categories based on the subcellular location and functionality (Figure 6). Functional categories showing a major number of genes with downregulated expression in OA-MSCs

Fig. 6. Differential expression of genes in osteoarthritis mesenchymal stem cells (OA-MSCs) according to gene ontology (GO) categories.

The X-axis represents individual genes classified according to the gene ontology (GO) slim categories provided by the GeneCodis2 (Carmona-Saez, 2007) analysis. The Y-axis represents the fold variation in expression of OA-MSCs compared to control subjects (p<0.05). Only genes expressing at least twofold differences in expression were considered. A: Multicellular organismal development; B: Signal transduction; C: Cell differentiation; D: Cell-cell signaling; E: Cell proliferation; F: Metabolic Process; G: Carbohydrate Metabolic Process; H: Anatomical structure morphogenesis; I: Cytoskeleton organization; J: Response to stress; K: Cellular component organization; L: Response to external stimulus; M: Growth

mesenchymal cell proliferation in bone marrow allowing their expansion in response to

More recently, another experimental approach was carried out by our group based on the comprehensive study of gene expression of MSCs using a DNA microarray expression analysis (Lamas, 2010). Gene expression profiles of MSCs from OA patients were compared to those of MSCs from healthy individuals. After integration of expression profiles into functional categories, by means of a gene ontology (GO)-based statistical analysis using GeneCodis 2.0 (Carmona-Saez, 2007), seventy-five genes from a list of 532 provided for comparison did not show annotations. The remaining 457 genes were grouped into different GO categories based on the subcellular location and functionality (Figure 6). Functional categories showing a major number of genes with downregulated expression in OA-MSCs

Fig. 6. Differential expression of genes in osteoarthritis mesenchymal stem cells (OA-MSCs)

The X-axis represents individual genes classified according to the gene ontology (GO) slim

categories provided by the GeneCodis2 (Carmona-Saez, 2007) analysis. The Y-axis represents the fold variation in expression of OA-MSCs compared to control subjects (p<0.05). Only genes expressing at least twofold differences in expression were considered. A: Multicellular organismal development; B: Signal transduction; C: Cell differentiation; D: Cell-cell signaling; E: Cell proliferation; F: Metabolic Process; G: Carbohydrate Metabolic Process; H: Anatomical structure morphogenesis; I: Cytoskeleton organization; J: Response to stress; K: Cellular component organization; L: Response to external stimulus; M: Growth

according to gene ontology (GO) categories.

the bone and cartilage damage characteristic of this disease.

were signal transduction, development and cell differentiation, which in turn are key functions in pluripotential cells. Based on the function of the proteins encoded by these genes, our results suggested that MSCs from patients with OA have a diminished differentiation and regenerative potentials that limit ther ability to generate a functional lineage of cells involved in musculoskeletal tissue homeostasis.

Overall, in this study we provided a reference dataset of genes related to essential functions for the normal biology of MSCs that become altered in OA (Figure 4). We also described for the first time an association between the COL10A1 gene and OA susceptibility suggesting that underlying biological changes which occur during OA disease might be related, at least in part, to defects in the ECM and the formation of subchondral bone, an essential structure for providing joint stability. Moreover, in this work we also demonstrated that the expression of multiple genes related to the wnt pathway were downregulated in OA patients.

#### **3.3 Animal models in regenerative medicine (tendon repair)**

Chronic degeneration is the most frequent cause for lesions of the rotator cuff, a set of four tendons connecting the scapula with the humeral head in shoulder. The most affected tendon of the cuff, frequently affected by tears, is the supraspinatus tendon. These lesions are increased in aged patients who frequently need reconstructive surgery. Surgical methods are often unsatisfactory due to inefficient recovery. In this context, MSC-based regenerative medicine offers a hopeful alternative for this type of treatment. In this sense, our group is performing several studies focused on the evaluation of the effectiveness of recovery after treatments consisting in surgical implantations of MSCs alone or in combination with a commercial membrane of type I collagen (OrthoadaptTM). This strategy has been previously tested in in a model of acute and chronic injury in rats with promising results allowing us to conduct a clinical trial in humans that is currently under development.

#### **4. MSCs, conventional and other experimental treatments in OA**

Treatment of osteoarthritis includes a combination of pharmacological and nonpharmacological measures aimed at relieving pain and the improvement of joint function. Initial treatment is dependent on the extent of the disease, age of patients and the joints affected, in descending order of frequency: Hip, Knee, Foot and Ankle. In mild cases, treatment begins by the use of simple analgesics (eg. paracetamol), (Nonsteroidal antiinflammatory drugs) NSAIDs (eg. ibuprofen and naproxen) or intermittent intra-articular administration (infiltration) of corticosteroids. However, although the symptoms and pain can be partially alleviated, adverse effects associated with conventional drug therapy is not recommended for long time periods. Moreover, treatment is often accompanied by nonpharmacological treatments, these include patient education and physical exercises to restore joint movement and to increase muscle strength, reduction of weight on painful joints. When joints are severely damaged treatment may require surgery. The most common surgical treatments are arthroscopic surgery, to trim damaged cartilage. Osteotomy, to change the alignment of a bone to relieve stress on the bone or joint. Arthrodesis or surgical fusion of bones, usually in the spine and the total or partial arthroplasty to replace the damaged joint with an artificial one.

Therapeutic Potential of MSCs in Musculoskeletal Diseases (Osteoarthritis) 275

**Symptom Current approach Mode of action Advantages Disadvantages** 

Analgesic


Anti-inflammatory

reinforcement

Bone marrow stimulation

Tissue

trasplantation from autologous or allogeneic origin

Cellular therapy using committed cell lineages


Surgery not needed

Relatively inexpensive surgery

Age of donor eligible

Relatively well suited for medium cartilage defects

Relatively well suited for medium cartilage defects

Defect filling with hyaline cartilage in a short time

Adverse effects

recommended in some cases

Age of patients Inferior quality of neoformed

Availability of implants.

Poor integration and maintenance of implant.

Two sugeries needed

Small number of chondrocyte availability

Poor integration Do not show improvement over other conventional techniques

tissue

Donor morbidity.

Not

**Initial stages (non surgical)** 


**Structural damage of joint cartílage (surgical methods)** 






trasplantation (ACI)

**Structural damage of joint cartílage (trasplantation methods)** 

treatment

Pain and

Inflammation

Small defects <2.5cm2

Medium defects 2.5 to 4 cm2

Halfway between drug therapy and joint replacement surgery, several arthroscopic strategies combined with cell therapies have been developed for the treatment of cartilage injuries. The techniques used and the results obtained greatly vary depending on the size of the lesion. For smaller and medium sized cartilage defects, autologous osteochondral cylinder transfer or mosaicplasty has been widely used but its efficacy is limited by donor site morbidity and the poor integration of implants.

Techniques of cell therapy in OA were initially based on the stimulation of bone marrow by drilling, microfracture and abrasive chondroplasty to promote better access of pluripotential stem cells from subchondral vascular area, to the site of injury (Steinwachs, 2008; Chen, 2011). Although the microfracture has achieved good results in terms of functionality and reduction of pain, several limitations such as chondral defect size and age of the patient are major constraints. These methods only provide a partial filling of the defect with fibrocartilage without the characteristics of hyaline cartilage. More recently improvements of these cell therapies have been made using the implantation of cultured autologous chondrocytes in the defect site (ACI) and a variation of this technique, using collagen Type III/I scaffolds, MACI (Matrix-induced autologous chondrocyte implantation) (Strauss, 2011; Ventura, 2011). MACI was developed to enable the treatment of larger defects when cell engaged procedures such as ACI cannot be used or it is not indicated. The results of the ongoing studies in chondrocyte implantation show better results in the formation of a hyaline-like cartilage with similar characteristics and durability than normal hyaline cartilage. In any case, the major drawbacks are that the chondrocytes harvesting require additional surgery and only a small number of chondrocytes can be isolated from the explants. In addition these cells lose their phenotypic characteristics in culture, limiting their application in extensive chondral defects, such as those produced in osteoarthritis. Otherwise, allograft transplantation is limited by donor availability.

Table 1 shows a summary of the most common techniques used clinically and experimentally. However the number of combinations of treatment options with each strategy is unlimited and growing every day. A great number of studies involve animal models evaluating different scaffolds, number of cells and ambiental factors used, etc. However, given the complex variety of combinations, there are no well-conducted clinical trials in humans evaluating the efficacy of a particular method.

In summary, regarding OA, advances in research for the development of new technologies in the management of cartilage defects is currently unresolved. Actually any treatment method provides consistent and acceptable long-term clinical results, and in particular for treatment of large chondral defects. With evolving techniques, versatility, availability and differentiation potential of stem cells have become the hope to improve current treatments based on other more committed cells. Alone or in combination with different scaffold materials and environmental factors, including growth factors, signalling molecules and mechanical influence, these cells are exceptional candidates for engineer cartilage constructs *in vitro*. Several studies have shown an improvement in the quality of the new tissue formed, but its long-term efficacy and the mechanism by which it occurs are unknown. In this regard it has been postulated that the low intrinsic immunogenicity of MSCs along with its ability to reduce inflammation, are characteristics that determine the establishment of a less inflammatory environment that facilitates the repair.

Halfway between drug therapy and joint replacement surgery, several arthroscopic strategies combined with cell therapies have been developed for the treatment of cartilage injuries. The techniques used and the results obtained greatly vary depending on the size of the lesion. For smaller and medium sized cartilage defects, autologous osteochondral cylinder transfer or mosaicplasty has been widely used but its efficacy is limited by donor

Techniques of cell therapy in OA were initially based on the stimulation of bone marrow by drilling, microfracture and abrasive chondroplasty to promote better access of pluripotential stem cells from subchondral vascular area, to the site of injury (Steinwachs, 2008; Chen, 2011). Although the microfracture has achieved good results in terms of functionality and reduction of pain, several limitations such as chondral defect size and age of the patient are major constraints. These methods only provide a partial filling of the defect with fibrocartilage without the characteristics of hyaline cartilage. More recently improvements of these cell therapies have been made using the implantation of cultured autologous chondrocytes in the defect site (ACI) and a variation of this technique, using collagen Type III/I scaffolds, MACI (Matrix-induced autologous chondrocyte implantation) (Strauss, 2011; Ventura, 2011). MACI was developed to enable the treatment of larger defects when cell engaged procedures such as ACI cannot be used or it is not indicated. The results of the ongoing studies in chondrocyte implantation show better results in the formation of a hyaline-like cartilage with similar characteristics and durability than normal hyaline cartilage. In any case, the major drawbacks are that the chondrocytes harvesting require additional surgery and only a small number of chondrocytes can be isolated from the explants. In addition these cells lose their phenotypic characteristics in culture, limiting their application in extensive chondral defects, such as those produced in osteoarthritis.

Table 1 shows a summary of the most common techniques used clinically and experimentally. However the number of combinations of treatment options with each strategy is unlimited and growing every day. A great number of studies involve animal models evaluating different scaffolds, number of cells and ambiental factors used, etc. However, given the complex variety of combinations, there are no well-conducted clinical

In summary, regarding OA, advances in research for the development of new technologies in the management of cartilage defects is currently unresolved. Actually any treatment method provides consistent and acceptable long-term clinical results, and in particular for treatment of large chondral defects. With evolving techniques, versatility, availability and differentiation potential of stem cells have become the hope to improve current treatments based on other more committed cells. Alone or in combination with different scaffold materials and environmental factors, including growth factors, signalling molecules and mechanical influence, these cells are exceptional candidates for engineer cartilage constructs *in vitro*. Several studies have shown an improvement in the quality of the new tissue formed, but its long-term efficacy and the mechanism by which it occurs are unknown. In this regard it has been postulated that the low intrinsic immunogenicity of MSCs along with its ability to reduce inflammation, are characteristics that determine the establishment of a

site morbidity and the poor integration of implants.

Otherwise, allograft transplantation is limited by donor availability.

trials in humans evaluating the efficacy of a particular method.

less inflammatory environment that facilitates the repair.


Therapeutic Potential of MSCs in Musculoskeletal Diseases (Osteoarthritis) 277

Special thanks to Peter Clarke and his disinterested revision and editing of the manuscript

This work was supported by a research grant from Roche Farma S.A. and was also partially supported by the RETICS Program, RD08/0075 (RIER) from Instituto de Salud Carlos III.

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Brandt KD; Radin EL; Dieppe PA et al. (2006). Yet more evidence that osteoarthritis is not a cartilage disease. *Ann Rheum Dis*, Vol. 65, No. 10, pp. (1261-1264) , ISSN 0003-4967 Bruder SP; Jaiswal N; Haynesworth SE. (1997). Growth kinetics, self-renewal, and the

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finding significant concurrent annotations in gene lists. *Genome Biol*, Vol. 8, No. 1,

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and to Dr. Emilio Camafeita for providing some of the representations included.

**6. Acknowledgments** 

1615-9853

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No. 2, pp. (249-257) ISSN 1097-4644

(379 Suppl), pp. (S67-70) , ISSN 0009-921X

**7. References** 


Table 1. Conventional and experimental treatments in OA.

### **5. Conclusions and future perspectives**

The goal in regenerative medicine is based in a conceptually simple scheme: the development of new strategies to replace human cells or induce the regeneration of diseased or injured human tissues. Although during the last twenty years a considerable scientific progress has been done in this field, there are still many unanswered questions about key concepts concerning both tissue engineering and cell therapy.

Stem cells, in its two "flavours": embryonic and adult stem cells are the basis of regenerative medicine; however, biological differences between adult and embryonic stem cells and among adult stem cells found in different tissues is an important aspect which implication for therapeutic uses is not resolved. From the point of view of their clinical application, the source of the cells is of extreme importance. In the case of autologous cells that are not rejected by the patient's immune system their application is potentially safer than in allogeneic cells and more suitable for permanent tissue replacement. However, and for example, in cases where the recipient suffers from a genetic disorder, their application would be inappropriate. Future efforts should be done to minimise rejection, and to favour the banking and use of allogeneic adult cells. Among adult stem cells, the MSCs are of paramount importance for the treatment of several rheumatic diseases. Besides their plasticity and regenerative potential they show immunosuppressive and antiinflammatory characteristics *in vitro* and proven in preclinical and clinical studies.

Future studies will need to focus on the particular cell biology of MSCs including the biochemical signal transduction pathways involved in maintaining and enhancing chondrogenic differentiation, but also in the mechanisms implicated in immunomodulation. Other important aspects that need further research include the evaluation of safety and efficacy of local or systemic modes of admistration of MSCs; the mechanisms of cell to cell communication, such as microvesicles transporting RNAs, cytokines, etc.; the behaviour of MSCs in different niches; the design of specialised engineered scaffolds, to enable the efficient repair of a variety of tissues; and finally, the implementation and use of genetic reprogramming strategies.

#### **6. Acknowledgments**

Special thanks to Peter Clarke and his disinterested revision and editing of the manuscript and to Dr. Emilio Camafeita for providing some of the representations included.

This work was supported by a research grant from Roche Farma S.A. and was also partially supported by the RETICS Program, RD08/0075 (RIER) from Instituto de Salud Carlos III.

#### **7. References**

276 Tissue Regeneration – From Basic Biology to Clinical Application

**Symptom Current approach Mode of action Advantages Disadvantages** 

Cellular therapy

The goal in regenerative medicine is based in a conceptually simple scheme: the development of new strategies to replace human cells or induce the regeneration of diseased or injured human tissues. Although during the last twenty years a considerable scientific progress has been done in this field, there are still many unanswered questions about key

Stem cells, in its two "flavours": embryonic and adult stem cells are the basis of regenerative medicine; however, biological differences between adult and embryonic stem cells and among adult stem cells found in different tissues is an important aspect which implication for therapeutic uses is not resolved. From the point of view of their clinical application, the source of the cells is of extreme importance. In the case of autologous cells that are not rejected by the patient's immune system their application is potentially safer than in allogeneic cells and more suitable for permanent tissue replacement. However, and for example, in cases where the recipient suffers from a genetic disorder, their application would be inappropriate. Future efforts should be done to minimise rejection, and to favour the banking and use of allogeneic adult cells. Among adult stem cells, the MSCs are of paramount importance for the treatment of several rheumatic diseases. Besides their plasticity and regenerative potential they show immunosuppressive and antiinflammatory

Future studies will need to focus on the particular cell biology of MSCs including the biochemical signal transduction pathways involved in maintaining and enhancing chondrogenic differentiation, but also in the mechanisms implicated in immunomodulation. Other important aspects that need further research include the evaluation of safety and efficacy of local or systemic modes of admistration of MSCs; the mechanisms of cell to cell communication, such as microvesicles transporting RNAs, cytokines, etc.; the behaviour of MSCs in different niches; the design of specialised engineered scaffolds, to enable the efficient repair of a variety of tissues; and finally, the implementation and use of genetic

Better early results

using stem cells Potential Expensive

restoration

Medium and long-term results not available

Expensive

Infections Expiration


implantation (MACI)

Mesenchymal Stem Cells (MSCs)

> 4 cm2 Total arthroplasty Joint replacementt Joint

Table 1. Conventional and experimental treatments in OA.

concepts concerning both tissue engineering and cell therapy.

characteristics *in vitro* and proven in preclinical and clinical studies.

**5. Conclusions and future perspectives** 

Large defects

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**13** 

*USA* 

**Stem Cell-Mediated Intervertebral** 

Namath S. Hussain, Vickram Tejwani and Mick Perez-Cruet

*Oakland University William Beaumont School of Medicine, Royal Oak, Michigan* 

Currently, degenerative disk disease (DDD) and the subsequent chronic lower back pain that results from it represent a significant source of morbidity and mortality worldwide. The available treatment modalities such as pain therapy and surgical interventions aim to provide symptomatic relief; however, they do not address the underlying pathophysiology of DDD. The disease also has high societal health care costs (Chan et al., 2006; Cassinelli et al, 2001). Many modalities exist for symptomatic treatment of this condition, including bed rest, massage, stretching, strengthening exercises, physical therapy, epidural injections and other pain management therapies, and spinal surgery. Most conservative therapies are

Fig. 1. Anatomy of the spine with the compartmentalization of the IVD.

**1. Introduction** 

**Disc Regeneration** 

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