**4. Chemoattractants involved in migration of hematopoietic stem cells**

Chemoattractants play an important role in directing migration of hematopoietic stem and progenitor cells to the bone marrow. Several studies have demonstrated that Stromal cell Derived Factor 1 (SDF-1), also known as CXC chemokine ligand 12 (CXCL12) (Tashiro et al., 1993) acts as a chemoattractant for hematopoietic stem and progenitor cells and is important for their transendothelial migration (Aiuti et al., 1997; Naiyer et al., 1999; Mohle et al., 1998; Kim & Broxmeyer, 1998; Glass et al., 2011). Further investigation, utilizing a large panel of CC and CXC chemokines, suggested that the only chemokine capable of inducing migration of murine hematopoietic stem and progenitor cells appears to be SDF-1 (Liesveld et al., 2001; Wright et al., 2002). Although the chemokine receptors CCR3 and CCR9 were also expressed at mRNA level, their ligands could not induce migration (Wright et al., 2002). Similarly, examination of a panel of chemokines and cytokines in transendothelial migration assays revealed that SDF-1 is also important for migration of human hematopoietic progenitors through a confluent layer of endothelial cells (Liesveld et al., 2001). However, to a lesser extent, also other chemokines and cytokines, including CCL2 (MCP-1), CCL5 (RANTES), CXCL10 (IP-10), IL-8 and SCF could also induce transendothelial migration (Liesveld et al., 2001). In addition, LTD4, a ligand for CysLT(1), a G protein-coupled receptor recognizing inflammatory mediator of the cysteinyl leukotriene family, which is highly expressed in hematopoietic progenitors, has been demonstrated to up-regulate CD49d/CD29 and CD49e/CD29 dependent adhesion of hematopoietic progenitors (Boehmler et al., 2009) and to induce chemotaxis and in vitro transendothelial migration (Bautz et al., 2001). Recently, a role for the proteolysis-resistant bioactive lipids sphingosine-1-phosphate and ceramide-1-phosphate in regulation of bone marrow homing has been suggested. Conditioning of mice for transplantation resulted in enhanced levels of these lipids in the bone marrow. In addition, both lipids appear to be chemoattractants for hematopoietic stem and progenitor cells (Kim et al., 2011).

Molecular Mechanisms Underlying Bone Marrow Homing of Hematopoietic Stem Cells 191

Several proteolytic enzymes have been implicated in negatively regulating migration of hematopoietic stem cells by cleaving and inactivating SDF-1, including matrix metalloproteinases (MMP) 2/9 (Heissig et al., 2002; Sweeney et al., 2002; McQuibban et al., 2001), CD26 (Christopherson et al., 2002), carboxypeptidase M (Marquez-Curtis et al., 2008), carboxypeptidase N (Davis et al., 2005), neutrophil elastase (Petit et al., 2002; Levesque et al., 2002), cathepsin G (Petit et al., 2002; Levesque et al., 2002) and cathepsin K (Kollet et al., 2006). Cleavage of SDF-1 by several individual MMPs at Ser4-Leu5 bond of SDF-1 Nterminal domain has, for example, been demonstrated to result in reduced binding capacity of SDF-1 for CXCR-4 and reduced chemoattractant activity for hematopoietic stem and progenitor cells (McQuibban et al., 2001; Cho et al., 2010). Another protein involved in regulation of the activity of SDF-1 is the membrane-bound extracellular peptidase CD26 (DPPIV). It has been shown that a small number of umbilical cord blood derived CD34+CXCR4+ cells express CD26 and can therefore cleave the N-terminal part of SDF-1 at 2-proline (Christopherson et al., 2002). Functional studies showed that truncated SDF-1 lacks the ability to induce migration of CD34+ cells. In addition, inhibition of endogenous CD26 activity appears to be sufficient to enhance the migratory capacity of CD34+ cells towards SDF-1, indicating that CD26 abrogates SDF-1 induced migration of hematopoietic progenitors (Christopherson et al., 2002; Christopherson et al., 2003; Christopherson et al.,

A third class of SDF-1 inhibitors includes the carboxypeptidases M and N (Marquez-Curtis et al., 2008; Davis et al., 2005). Carboxypeptidase N, which is present in human serum and plasma (Davis et al., 2005), can efficiently and specifically cleave SDF-1 at the carboxyterminal lysine (K68) resulting in reduced SDF-1 activity and inhibition of SDF-1 mediated induction of migration of hematopoietic progenitors (Davis et al., 2005). In contrast, carboxypeptidase M is a membrane bound zinc-dependent peptidase that cleaves carboxyterminal basic residues. This particular carboxypeptidase is expressed by stromal cells and CD34+ cells from both bone marrow and mobilized peripheral blood (Skidgel & Erdos, 1998; Marquez-Curtis et al., 2008). Carboxypeptidase M mediated cleavage of SDF-1 results in reduced chemotactic activity of hematopoietic stem and progenitor cells, which can be rescued by addition of the carboxypeptidase inhibitor DL-2-mercaptomethyl-3-guanidino-

Whereas high SDF-1 expression in the bone marrow is essential for normal bone marrow homing of hematopoietic stem and progenitor cells and lodging of those cells in the hematopoietic stem cell niche, during mobilization SDF-1 levels should conversely be decreased. Upon administration of G-CSF, which is used to mobilize HSPCs, an accumulation of various proteolytic enzymes including MMP-9, neutrophil elastase and cathepsin G or K (Petit et al., 2002; Levesque et al., 2002) has been observed in mouse bone marrow which correlated with a gradual decrease in SDF-1 in the bone marrow, but not circulation (Petit et al., 2002). In addition, also an enhanced SDF-1 plasma level was shown to result in up-regulation of MPP-9 in bone marrow cells and mobilization of hematopoietic stem and progenitor cells (Heissig et al., 2002). The importance of MPP-9 for mobilization of hematopoietic stem cells was demonstrated utilizing MMP-9 deficient mice. A high SDF-1 level in plasma was not sufficient to induce mobilization of hematopoietic progenitors in these mice (Heissig et al., 2002). In addition, in primary myelofibrosis, which is a chronic

ethylthiopropanoic acid (Marquez-Curtis et al., 2008).

**4.2 Regulation of SDF-1 activity** 

2006).

The role of SDF-1 in migration of hematopoietic stem and progenitor cells will be discussed below in more detail.

#### **4.1 SDF-1 and bone marrow homing**

SDF-1 is produced by several types of bone marrow cells (Maekawa & Ishii, 2000). In the adult human bone marrow, SDF-1 was found to be expressed by endothelial cells and along the endosteum region (Peled et al., 2000; Ponomaryov et al., 2000). SDF-1 plays an important role in many processes, including immune surveillance, proliferation, differentiation and survival of many cell types (Aiuti et al., 1997; Bleul et al., 1996; Bleul et al., 1998; Cashman & Eaves, 2000; Lataillade et al., 2000). In addition, SDF-1 is considered to be essential for migration of hematopoietic stem cells to the bone marrow (Imai et al., 1999; Peled et al., 1999a; Wright et al., 2002). To date, two receptors for SDF-1 have been identified, of which CXCR4 (LESTR/fusin), a seven-transmembrane domain G-protein coupled receptor, appears to be the most prominent (Heesen et al., 1997; Loetscher et al., 1994). CXCR4 is expressed by a variety of cell types, including hematopoietic stem and progenitor cells, T lymphocytes, endothelial, stromal and neuronal cells (Nagasawa et al., 1996; Ma et al., 1998; Mohle et al., 1998; Loetscher et al., 1994). Recently, CXCR7, another SDF-1 receptor, has been identified (Tarnowski et al., 2010). However, CXCR7 is expressed at low levels in normal human CD34+ hematopoietic stem and progenitor cells and does not appear to be important for migration of those cells. In contrast, CXCR7 is highly expressed in several human myeloid leukemic cell lines and is thought to play a role in adhesion and, to a lesser extent, also in migration of those cells (Tarnowski et al., 2010).

Mouse transplantation studies have been performed to investigate the importance of SDF-1 in migration of hematopoietic stem cells to the bone marrow. Pre-treatment of human CD34+CD38-/low cells with a blocking antibody against CXCR4 has, for example, been demonstrated to be sufficient to impair their capacity to home to the bone marrow of immune deficient NOD/SCID mice or 2m deficient NOD/SCID mice (Peled et al., 1999b; Kollet et al., 2001; Kollet et al., 2002; Oberlin et al., 1996). In addition, up-regulation of CXCR4 expression by incubation with hematopoietic cytokines (SCF and IL-6) (Peled et al., 1999b) or over-expression of CXCR4 by viral transduction (Brenner et al., 2004; Kahn et al., 2004) resulted in enhanced bone marrow homing of human CD34+ and CD34+CD38- cells in NOD/SCID mice, which correlated with enhanced engraftment levels 6 weeks after transplantation (Peled et al., 1999b; Kollet et al., 2001; Kollet et al., 2002). Similarly, fetal liver hematopoietic stem and progenitor cells deficient for CXCR4 displayed a reduced bone marrow homing capacity compared to wild type cells (Ma et al., 1998). In addition to bone marrow homing, SDF-1 also appears to play a critical role in retention of hematopoietic stem cells in the hematopoietic stem cell niche. Enhancing the level of SDF-1 in plasma, but not bone marrow, utilizing adenoviral vectors (Hattori et al., 2001) or sulfated glycans (Sweeney et al., 2000; Frenette & Weiss, 2000; Sweeney et al., 2002) resulted in mobilization of CXCR4 expressing hematopoietic stem and progenitor cells (Hattori et al., 2001; Sweeney et al., 2002). Similarly, treatment of C3H/HeJ mice or healthy human volunteers with AMD3100, a selective CXCR4 antagonist, enhanced the number of HSCs and neutrophils in peripheral blood, again suggesting a role for CXCR4 and SDF-1 in HSC retention in BM (Broxmeyer et al., 2005).

#### **4.2 Regulation of SDF-1 activity**

190 Advances in Hematopoietic Stem Cell Research

The role of SDF-1 in migration of hematopoietic stem and progenitor cells will be discussed

SDF-1 is produced by several types of bone marrow cells (Maekawa & Ishii, 2000). In the adult human bone marrow, SDF-1 was found to be expressed by endothelial cells and along the endosteum region (Peled et al., 2000; Ponomaryov et al., 2000). SDF-1 plays an important role in many processes, including immune surveillance, proliferation, differentiation and survival of many cell types (Aiuti et al., 1997; Bleul et al., 1996; Bleul et al., 1998; Cashman & Eaves, 2000; Lataillade et al., 2000). In addition, SDF-1 is considered to be essential for migration of hematopoietic stem cells to the bone marrow (Imai et al., 1999; Peled et al., 1999a; Wright et al., 2002). To date, two receptors for SDF-1 have been identified, of which CXCR4 (LESTR/fusin), a seven-transmembrane domain G-protein coupled receptor, appears to be the most prominent (Heesen et al., 1997; Loetscher et al., 1994). CXCR4 is expressed by a variety of cell types, including hematopoietic stem and progenitor cells, T lymphocytes, endothelial, stromal and neuronal cells (Nagasawa et al., 1996; Ma et al., 1998; Mohle et al., 1998; Loetscher et al., 1994). Recently, CXCR7, another SDF-1 receptor, has been identified (Tarnowski et al., 2010). However, CXCR7 is expressed at low levels in normal human CD34+ hematopoietic stem and progenitor cells and does not appear to be important for migration of those cells. In contrast, CXCR7 is highly expressed in several human myeloid leukemic cell lines and is thought to play a role in adhesion and, to a lesser extent, also in migration of those cells (Tarnowski et al.,

Mouse transplantation studies have been performed to investigate the importance of SDF-1 in migration of hematopoietic stem cells to the bone marrow. Pre-treatment of human CD34+CD38-/low cells with a blocking antibody against CXCR4 has, for example, been demonstrated to be sufficient to impair their capacity to home to the bone marrow of immune deficient NOD/SCID mice or 2m deficient NOD/SCID mice (Peled et al., 1999b; Kollet et al., 2001; Kollet et al., 2002; Oberlin et al., 1996). In addition, up-regulation of CXCR4 expression by incubation with hematopoietic cytokines (SCF and IL-6) (Peled et al., 1999b) or over-expression of CXCR4 by viral transduction (Brenner et al., 2004; Kahn et al., 2004) resulted in enhanced bone marrow homing of human CD34+ and CD34+CD38- cells in NOD/SCID mice, which correlated with enhanced engraftment levels 6 weeks after transplantation (Peled et al., 1999b; Kollet et al., 2001; Kollet et al., 2002). Similarly, fetal liver hematopoietic stem and progenitor cells deficient for CXCR4 displayed a reduced bone marrow homing capacity compared to wild type cells (Ma et al., 1998). In addition to bone marrow homing, SDF-1 also appears to play a critical role in retention of hematopoietic stem cells in the hematopoietic stem cell niche. Enhancing the level of SDF-1 in plasma, but not bone marrow, utilizing adenoviral vectors (Hattori et al., 2001) or sulfated glycans (Sweeney et al., 2000; Frenette & Weiss, 2000; Sweeney et al., 2002) resulted in mobilization of CXCR4 expressing hematopoietic stem and progenitor cells (Hattori et al., 2001; Sweeney et al., 2002). Similarly, treatment of C3H/HeJ mice or healthy human volunteers with AMD3100, a selective CXCR4 antagonist, enhanced the number of HSCs and neutrophils in peripheral blood, again suggesting a role for CXCR4 and SDF-1 in HSC retention in BM (Broxmeyer et

below in more detail.

2010).

al., 2005).

**4.1 SDF-1 and bone marrow homing** 

Several proteolytic enzymes have been implicated in negatively regulating migration of hematopoietic stem cells by cleaving and inactivating SDF-1, including matrix metalloproteinases (MMP) 2/9 (Heissig et al., 2002; Sweeney et al., 2002; McQuibban et al., 2001), CD26 (Christopherson et al., 2002), carboxypeptidase M (Marquez-Curtis et al., 2008), carboxypeptidase N (Davis et al., 2005), neutrophil elastase (Petit et al., 2002; Levesque et al., 2002), cathepsin G (Petit et al., 2002; Levesque et al., 2002) and cathepsin K (Kollet et al., 2006). Cleavage of SDF-1 by several individual MMPs at Ser4-Leu5 bond of SDF-1 Nterminal domain has, for example, been demonstrated to result in reduced binding capacity of SDF-1 for CXCR-4 and reduced chemoattractant activity for hematopoietic stem and progenitor cells (McQuibban et al., 2001; Cho et al., 2010). Another protein involved in regulation of the activity of SDF-1 is the membrane-bound extracellular peptidase CD26 (DPPIV). It has been shown that a small number of umbilical cord blood derived CD34+CXCR4+ cells express CD26 and can therefore cleave the N-terminal part of SDF-1 at 2-proline (Christopherson et al., 2002). Functional studies showed that truncated SDF-1 lacks the ability to induce migration of CD34+ cells. In addition, inhibition of endogenous CD26 activity appears to be sufficient to enhance the migratory capacity of CD34+ cells towards SDF-1, indicating that CD26 abrogates SDF-1 induced migration of hematopoietic progenitors (Christopherson et al., 2002; Christopherson et al., 2003; Christopherson et al., 2006).

A third class of SDF-1 inhibitors includes the carboxypeptidases M and N (Marquez-Curtis et al., 2008; Davis et al., 2005). Carboxypeptidase N, which is present in human serum and plasma (Davis et al., 2005), can efficiently and specifically cleave SDF-1 at the carboxyterminal lysine (K68) resulting in reduced SDF-1 activity and inhibition of SDF-1 mediated induction of migration of hematopoietic progenitors (Davis et al., 2005). In contrast, carboxypeptidase M is a membrane bound zinc-dependent peptidase that cleaves carboxyterminal basic residues. This particular carboxypeptidase is expressed by stromal cells and CD34+ cells from both bone marrow and mobilized peripheral blood (Skidgel & Erdos, 1998; Marquez-Curtis et al., 2008). Carboxypeptidase M mediated cleavage of SDF-1 results in reduced chemotactic activity of hematopoietic stem and progenitor cells, which can be rescued by addition of the carboxypeptidase inhibitor DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (Marquez-Curtis et al., 2008).

Whereas high SDF-1 expression in the bone marrow is essential for normal bone marrow homing of hematopoietic stem and progenitor cells and lodging of those cells in the hematopoietic stem cell niche, during mobilization SDF-1 levels should conversely be decreased. Upon administration of G-CSF, which is used to mobilize HSPCs, an accumulation of various proteolytic enzymes including MMP-9, neutrophil elastase and cathepsin G or K (Petit et al., 2002; Levesque et al., 2002) has been observed in mouse bone marrow which correlated with a gradual decrease in SDF-1 in the bone marrow, but not circulation (Petit et al., 2002). In addition, also an enhanced SDF-1 plasma level was shown to result in up-regulation of MPP-9 in bone marrow cells and mobilization of hematopoietic stem and progenitor cells (Heissig et al., 2002). The importance of MPP-9 for mobilization of hematopoietic stem cells was demonstrated utilizing MMP-9 deficient mice. A high SDF-1 level in plasma was not sufficient to induce mobilization of hematopoietic progenitors in these mice (Heissig et al., 2002). In addition, in primary myelofibrosis, which is a chronic

Molecular Mechanisms Underlying Bone Marrow Homing of Hematopoietic Stem Cells 193

investigated. Deletion of Vav1 in hematopoietic stem cells has been demonstrated to result in impaired responses to SDF1, dysregulated Rac/Cdc42 activation and a reduction of in vitro migration. In addition, intravital microscopy assays revealed that transplantation of Vav1 deficient hematopoietic stem and progenitor cells results in impaired early localization near nestin(+) perivascular mesenchymal stem cells after transplantation (Sanchez-Aguilera et al., 2011). Recently, another upstream regulator of Rac activity has been identified. In contrast to Rac, the activity of R-Ras, a member of the Ras family, is inhibited upon SDF-1 stimulation. Deletion of R-Ras resulted in enhanced levels of Rac1/2 activity, while expression of a constitutively active R-Ras mutant resulted in down-regulation of Rac1 activity. Deletion of R-Ras in hematopoietic stem and progenitor cells resulted in increased directional migration. This phenotype could be reversed by inhibition of Rac. Furthermore, R-Ras deficient mice showed enhanced responsiveness to G-CSF for progenitor cell mobilization and exhibited decreased bone marrow homing (Sanchez-Aguilera et al., 2011). Another important mediator of hematopoietic progenitor cell migration is the GTPase Rho (Bug et al., 2002; Ghiaur et al., 2006; Gottig et al., 2006). It has been demonstrated that SDF-1 mediated release of intracellular Ca2+ stores requires activation of Rho GTPases, but not Rac or Cdc42 (Henschler et al., 2003). Depletion of intracellular Ca2+ resulted in reduced SDF-1 induced migration and bone marrow homing of hematopoietic progenitors (Henschler et al., 2003). In addition, over-expresssion of dominant negative RhoA by retroviral transduction in mouse cells (C57BL/6J mice) resulted in decreased migration of hematopoietic progenitor cells towards SDF-1 and reduced integrin-mediated adhesion (Henschler et al., 2003). Furthermore, over-expression of RhoH, a GTPase deficient type of Rho (Sahai & Marshall, 2002), in hematopoietic stem and progenitor cells resulted in impaired activation of Rac GTPases, defective actin polymerization and impaired chemotaxis. In contrast, inhibition of RhoH expression in these cells conversely stimulated SDF-1–induced migration in vitro (Gu et al., 2005). In addition, it has been demonstrated that Epac1, a nucleotide exchange protein for the GTPase Rap1, which is directly activated by cAMP, can also improve the adhesive and migratory capacity CD34+ hematopoietic progenitor cells (Carmona et al., 2008),

suggesting that Rap1 may also play a role in bone marrow homing.

bone marrow homing will be discussed in the next section.

Endolyn (CD164), a type I integral transmembrane silomucin (Chan et al., 2001; Zannettino et al., 1998), which is recruited to CXCR4 upon SDF-1 stimulation (Forde et al., 2007) was shown to play an important role in SDF-1 mediated migration of human CD133+ hematopoietic stem and progenitor cells (Forde et al., 2007). Inhibition of CD164 in CD133+ cells with 103B2, a specific mAb, resulted in a reduction of migration towards SDF-1, but not CCL1, CCL5, CCL17, CCL19, CCL20, CCL21, CCL22 and CXCL3. A similar inhibition in SDF-1 mediated migration of CD133+ cells was observed after siRNA mediated knock-down of CD164 (Forde et al., 2007). Knock-down of CD164 resulted in a significant reduction in SDF-1 mediated activation of PI3K and PKCζ (Forde et al., 2007). Both PI3K and PKC have been implicated in playing an important role in SDF-1 mediated migration of CD34+ cells. Inhibition of PKC, for example, reduced SDF-1 induced migration of CD34+ cells and reduced engraftment levels after transplantation (Petit et al., 2005). Furthermore, injection of inhibitory PKC pseudosubstrate peptides resulted in mobilization of murine progenitors to the circulation, suggesting an important role for PKCin SDF-1-dependent regulation of hematopoietic stem and progenitor cell motility and localization (Petit et al., 2005) The role of PI3K in regulation of

myeloproliferative neoplasm characterized by constitutive mobilization of hematopoietic stem and progenitor cells into the peripheral blood (Migliaccio et al., 2008), both a high level of truncated SDF-1 and enhanced levels of proteases, including dipeptidyl peptidase-IV (CD26), neutrophil elastase, matrix metalloproteinase-2 (MMP-2), MMP-9, and cathepsin G have been observed (Cho et al., 2010). Taken together, these studies demonstrated that SDF-1 plays an important role in integrin-mediated firm arrest of human HSPCs, facilitate their transendothelial migration, and regulate bone marrow homing and retention of HSPCs in the hematopoietic stem cell niche.

#### **4.3 Molecular mechanisms underlying SDF-1 mediated regulation of migration**

To understand the molecular mechanism underlying migration of hematopoietic stem and progenitor cells, research has focused on identifying the downstream effectors of SDF-1 and CXCR4. SDF-1 has been demonstrated to induce the activity of the integrins CD11a/CD18 (Peled et al., 2000) and CD49/CD29 (Hidalgo et al., 2001; Peled et al., 2000) on CD34+ cells which allows interaction with their substrates ICAM-1 and VCAM-1, respectively.

Small guanosine triphosphatases (GTPases) that belong to the Ras superfamily of GTPases , including Rho, Rac and Cdc42, have been demonstrated to be involved in SDF-1 mediated homing and migration of hematopoietic stem and progenitor cells (Fuhler et al., 2008; del Pozo et al., 1999). The activity of Rho GTPases can be induced by tyrosine kinase receptors (Taylor & Metcalfe, 2000; Timokhina et al., 1998), integrin receptors (del Pozo et al., 2004) and chemokine receptors including SDF-1 (Cancelas et al., 2005; del Pozo et al., 1999; Fuhler et al., 2008; Shirvaikar et al., 2011). It has been demonstrated in *in vitro* assays that SDF-1 induced chemo-attraction is mediated, at least in part, by Rac (del Pozo et al., 1999; Shirvaikar et al., 2011; Wysoczynski et al., 2005). In addition, analysis of Rac2 deficient mice revealed that Rac2 is essential for lodging of HSPCs in the bone marrow. Deletion of Rac2 resulted, for example, in reduced adhesion and enhanced mobilization of hematopoietic stem cells to the circulation. Furthermore, Rac2 deficiency resulted in enhanced SDF-1 induced migration of hematopoietic stem and progenitor cells (Yang et al., 2001). An enhanced activation of Cdc42 and Rac1 was observed in these cells, suggesting a compensatory role of Cdc42 and Rac1 with regard to migration, but not adhesion (Yang et al., 2001). In addition, it was shown that SDF-1 mediated Rac activation is impaired in CD34+ cells from MDS patients. CD34+ cell from patients with myelodysplastic syndrome exhibit reduced F-actin polymerization and migration towards SDF-1 compared to normal CD34+ cells (Fuhler et al., 2008). While pharmacological inhibition of Rac1 activity in a human myeloblastic cell line (HL-60) with NSC23766 was sufficient to abrogate SDF-1 induced actin assembly and migration, over-expression of active Rac in HL-60 cells conversely restored both F-actin polymerization and migration, suggesting that Rac is essential for SDF-1–induced migration in these cells (Fuhler et al., 2008). Although overexpression of active Rac in CD34+ cells from patients with myelodysplastic syndrome resulted in increased F-actin polymerization and enhanced motility, directional migration toward SDF-1 was not improved (Fuhler et al., 2008). These studies suggest that SDF-1 mediated induction of Rac activity is important for migration of both normal and malignant hematopoietic progenitors (Fuhler et al., 2008). The role of the hematopoietic-specific guanine nucleotide exchange factor Vav1, which is an upstream regulator of Rac activity, in localization and engraftment of hematopoietic stem and progenitor cells has also been

myeloproliferative neoplasm characterized by constitutive mobilization of hematopoietic stem and progenitor cells into the peripheral blood (Migliaccio et al., 2008), both a high level of truncated SDF-1 and enhanced levels of proteases, including dipeptidyl peptidase-IV (CD26), neutrophil elastase, matrix metalloproteinase-2 (MMP-2), MMP-9, and cathepsin G have been observed (Cho et al., 2010). Taken together, these studies demonstrated that SDF-1 plays an important role in integrin-mediated firm arrest of human HSPCs, facilitate their transendothelial migration, and regulate bone marrow homing and retention of HSPCs in

**4.3 Molecular mechanisms underlying SDF-1 mediated regulation of migration** 

which allows interaction with their substrates ICAM-1 and VCAM-1, respectively.

To understand the molecular mechanism underlying migration of hematopoietic stem and progenitor cells, research has focused on identifying the downstream effectors of SDF-1 and CXCR4. SDF-1 has been demonstrated to induce the activity of the integrins CD11a/CD18 (Peled et al., 2000) and CD49/CD29 (Hidalgo et al., 2001; Peled et al., 2000) on CD34+ cells

Small guanosine triphosphatases (GTPases) that belong to the Ras superfamily of GTPases , including Rho, Rac and Cdc42, have been demonstrated to be involved in SDF-1 mediated homing and migration of hematopoietic stem and progenitor cells (Fuhler et al., 2008; del Pozo et al., 1999). The activity of Rho GTPases can be induced by tyrosine kinase receptors (Taylor & Metcalfe, 2000; Timokhina et al., 1998), integrin receptors (del Pozo et al., 2004) and chemokine receptors including SDF-1 (Cancelas et al., 2005; del Pozo et al., 1999; Fuhler et al., 2008; Shirvaikar et al., 2011). It has been demonstrated in *in vitro* assays that SDF-1 induced chemo-attraction is mediated, at least in part, by Rac (del Pozo et al., 1999; Shirvaikar et al., 2011; Wysoczynski et al., 2005). In addition, analysis of Rac2 deficient mice revealed that Rac2 is essential for lodging of HSPCs in the bone marrow. Deletion of Rac2 resulted, for example, in reduced adhesion and enhanced mobilization of hematopoietic stem cells to the circulation. Furthermore, Rac2 deficiency resulted in enhanced SDF-1 induced migration of hematopoietic stem and progenitor cells (Yang et al., 2001). An enhanced activation of Cdc42 and Rac1 was observed in these cells, suggesting a compensatory role of Cdc42 and Rac1 with regard to migration, but not adhesion (Yang et al., 2001). In addition, it was shown that SDF-1 mediated Rac activation is impaired in CD34+ cells from MDS patients. CD34+ cell from patients with myelodysplastic syndrome exhibit reduced F-actin polymerization and migration towards SDF-1 compared to normal CD34+ cells (Fuhler et al., 2008). While pharmacological inhibition of Rac1 activity in a human myeloblastic cell line (HL-60) with NSC23766 was sufficient to abrogate SDF-1 induced actin assembly and migration, over-expression of active Rac in HL-60 cells conversely restored both F-actin polymerization and migration, suggesting that Rac is essential for SDF-1–induced migration in these cells (Fuhler et al., 2008). Although overexpression of active Rac in CD34+ cells from patients with myelodysplastic syndrome resulted in increased F-actin polymerization and enhanced motility, directional migration toward SDF-1 was not improved (Fuhler et al., 2008). These studies suggest that SDF-1 mediated induction of Rac activity is important for migration of both normal and malignant hematopoietic progenitors (Fuhler et al., 2008). The role of the hematopoietic-specific guanine nucleotide exchange factor Vav1, which is an upstream regulator of Rac activity, in localization and engraftment of hematopoietic stem and progenitor cells has also been

the hematopoietic stem cell niche.

investigated. Deletion of Vav1 in hematopoietic stem cells has been demonstrated to result in impaired responses to SDF1, dysregulated Rac/Cdc42 activation and a reduction of in vitro migration. In addition, intravital microscopy assays revealed that transplantation of Vav1 deficient hematopoietic stem and progenitor cells results in impaired early localization near nestin(+) perivascular mesenchymal stem cells after transplantation (Sanchez-Aguilera et al., 2011). Recently, another upstream regulator of Rac activity has been identified. In contrast to Rac, the activity of R-Ras, a member of the Ras family, is inhibited upon SDF-1 stimulation. Deletion of R-Ras resulted in enhanced levels of Rac1/2 activity, while expression of a constitutively active R-Ras mutant resulted in down-regulation of Rac1 activity. Deletion of R-Ras in hematopoietic stem and progenitor cells resulted in increased directional migration. This phenotype could be reversed by inhibition of Rac. Furthermore, R-Ras deficient mice showed enhanced responsiveness to G-CSF for progenitor cell mobilization and exhibited decreased bone marrow homing (Sanchez-Aguilera et al., 2011).

Another important mediator of hematopoietic progenitor cell migration is the GTPase Rho (Bug et al., 2002; Ghiaur et al., 2006; Gottig et al., 2006). It has been demonstrated that SDF-1 mediated release of intracellular Ca2+ stores requires activation of Rho GTPases, but not Rac or Cdc42 (Henschler et al., 2003). Depletion of intracellular Ca2+ resulted in reduced SDF-1 induced migration and bone marrow homing of hematopoietic progenitors (Henschler et al., 2003). In addition, over-expresssion of dominant negative RhoA by retroviral transduction in mouse cells (C57BL/6J mice) resulted in decreased migration of hematopoietic progenitor cells towards SDF-1 and reduced integrin-mediated adhesion (Henschler et al., 2003). Furthermore, over-expression of RhoH, a GTPase deficient type of Rho (Sahai & Marshall, 2002), in hematopoietic stem and progenitor cells resulted in impaired activation of Rac GTPases, defective actin polymerization and impaired chemotaxis. In contrast, inhibition of RhoH expression in these cells conversely stimulated SDF-1–induced migration in vitro (Gu et al., 2005). In addition, it has been demonstrated that Epac1, a nucleotide exchange protein for the GTPase Rap1, which is directly activated by cAMP, can also improve the adhesive and migratory capacity CD34+ hematopoietic progenitor cells (Carmona et al., 2008), suggesting that Rap1 may also play a role in bone marrow homing.

Endolyn (CD164), a type I integral transmembrane silomucin (Chan et al., 2001; Zannettino et al., 1998), which is recruited to CXCR4 upon SDF-1 stimulation (Forde et al., 2007) was shown to play an important role in SDF-1 mediated migration of human CD133+ hematopoietic stem and progenitor cells (Forde et al., 2007). Inhibition of CD164 in CD133+ cells with 103B2, a specific mAb, resulted in a reduction of migration towards SDF-1, but not CCL1, CCL5, CCL17, CCL19, CCL20, CCL21, CCL22 and CXCL3. A similar inhibition in SDF-1 mediated migration of CD133+ cells was observed after siRNA mediated knock-down of CD164 (Forde et al., 2007). Knock-down of CD164 resulted in a significant reduction in SDF-1 mediated activation of PI3K and PKCζ (Forde et al., 2007). Both PI3K and PKC have been implicated in playing an important role in SDF-1 mediated migration of CD34+ cells. Inhibition of PKC, for example, reduced SDF-1 induced migration of CD34+ cells and reduced engraftment levels after transplantation (Petit et al., 2005). Furthermore, injection of inhibitory PKC pseudosubstrate peptides resulted in mobilization of murine progenitors to the circulation, suggesting an important role for PKCin SDF-1-dependent regulation of hematopoietic stem and progenitor cell motility and localization (Petit et al., 2005) The role of PI3K in regulation of bone marrow homing will be discussed in the next section.

Molecular Mechanisms Underlying Bone Marrow Homing of Hematopoietic Stem Cells 195

migration experiments through a confluent layer of human umbilical vein endothelial cells revealed that the observed reduction in firm adhesion does not ameliorate the induced migratory capacity of CD34+ cells pre-treated with a PKB inhibitor (Buitenhuis et al., 2010). In addition, ectopic expression of constitutively active PKB in CD34+ cells conversely induced firm adhesion and reduced the basal level of migration. Although it cannot be excluded that transient activation of PI3K/PKB activity by SDF-1 is important for induction of migration, these studies suggest that prolonged activation of PKB activity is detrimental for migration of CD34+ cells. The role of PI3K in regulation of bone marrow homing was initially examined utilizing mice deficient for SHIP (SH2-containing inositol-5'-phosphatase), a negative regulator of PI3K (Damen et al., 1996). Transplantation of lethally irradiated recipients with HSCs from SHIP deficient mice resulted in diminished repopulation, suggesting that constitutive activation of PI3K impairs the ability of HSCs to home to and to be retained in the hematopoietic stem cell niche in the bone marrow. Assessment of bone marrow homing revealed that SHIP–/– hematopoietic stem and progenitor cells indeed traffic to the bone marrow and spleen with significantly reduced efficiency compared to wild type cells. Although it is evident that constitutive activation of PI3K plays a critical role in regulation of hematopoiesis per se (Buitenhuis et al., 2008), these results indicate that the inability of SHIP deficient hematopoietic stem cells to engraft and sustain long-term hematopoiesis can be, at least partially, explained by their impaired ability to home to the bone marrow (Desponts et al., 2006). Deletion of Phosphate and tensin homologue (PTEN), another critical negative regulator of PI3K signaling that dephosphorylates PI(3,4,5)P3 resulting in the formation of PI(4,5)P2 (Maehama & Dixon, 1998) only decreased bone marrow homing when PTEN deficient HSCs were transplanted into non-irradiated recipients. These results suggest that, although PTEN deficient hematopoietic stem cells are capable of migrating to the bone marrow, their performence is reduced compared to competeting wild-type hematopoietic stem cells when vacant niches are limited (Zhang et al., 2006). Although both PTEN and SHIP act on the main product of PI3K activity, PI(3,4,5)P3, the products generated are distinct, which could explain the differences between SHIP and PTEN deficient hematopoietic stem cells in terms of bone marrow homing (Dowler et al., 2000; Golub & Caroni, 2005). Recent findings demonstrated that, similar to deletion of SHIP, constitutive activation of PKB in human hematopoietic progenitors cells is sufficient to significantly inhibit homing of these cells to the bone marrow and spleen of 2 microglobulin -/- NOD/SCID mice (Buitenhuis et al., 2010). In contrast, although transplantation of C57 BL/6 mice with bone marrow cells from *5-fluorouracil* treated mice that ectopically expressed constitutively active PKB resulted in reduced engraftment levels, bone marrow homing was only modestly impaired 18 hours after transplantation (Kharas et al., 2010). To investigate whether inhibition of PKB activity would be sufficient to conversely improve bone marrow homing, human hematopoietic progenitor cells, pre-treated with a PKB inhibitor for 24 or 48 hours, were injected into recipient mice. Flow cytometric analysis, 22 hours after transplantation, revealed that transient inhibition of PKB activity prior to transplantation is sufficient to improve bone marrow homing (Buitenhuis et al., 2010). In addition, while constitutive activation of PKB appears to be detrimental for bone marrow homing, engraftment levels and hematopoietic recovery, inhibition of PKB activity prior to transplantation, resulting in an induction of bone marrow homing, conversely enhanced engraftment levels in recipient mice. Together, these studies demonstrated that correct regulation of PI3K/PKB is essential for migration of hematopoietic stem and progenitor cells to the bone marrow after transplantation, which is essential for optimal engraftment and hematopoietic recovery (Buitenhuis et al., 2010; Desponts et al., 2006; Kharas et al., 2010).

In addition to regulating the activity of downstream effectors, SDF-1 has also been demonstrated to regulate the expression of specific target genes. Stimulation of peripheral blood mononuclear, Jurkat or HeLa cells has, for example, been demonstrated to result in a rapid increase in expression of the ubiquitin-specific protease 17 (USP17) (de la Vega et al., 2011). A role for this protease in regulation of migration of hematopoietic progenitor cells has been examined in vitro. Inhibition of USP17 in these cells showed decreased chemotaxis towards SDF-1, whereas over-expression of USP17 conversely resulted in increased chemotaxis. Interestingly, CXCR4 levels were not affected by inhibition or over-expression of USP17, suggesting that USP17 modulates the down-stream signaling of the CXCR4 receptor. shRNA mediated inhibition of USP17 expression resulted in decreased polymerization of actin and tubulin and reduced membrane ruffling. In addition, upon SDF-1 stimulation, the GTPases, RAC1, Cdc42 and RhoA were not transported to the plasma membrane, thereby prohibiting their activiation (de la Vega et al., 2011). In addition, CD9, a member of the tetraspanin superfamily (Boucheix et al., 1991) that is widely expressed in hematopoietic and non-hematopoietic cells, has been shown to be a SDF-1 responsive gene. Microarray analysis with human umbilical cord blood derived CD34+ cells revealed that short-term exposure to SDF-1 resulted in up-regulation of CD9 mRNA expression both in CD34+ CD38+ and CD34+ CD38-/low cells (Leung et al., 2011). A role for CD9 in migration and adhesion of human cord blood derived hematopoietic stem and progenitor cells was investigated utilizing a neutralizing CD9 antibody (Leung et al., 2011). Although actin polymerization was not affected, the calcium influx and transendothelial migration towards a SDF-1 gradient was reduced by this antibody (Leung et al., 2011). In contrast, adhesion of progenitor cells to fibronectin and human umbilical vein endothelial cells was enhanced (Leung et al., 2011). Transplantation experiments revealed that in NOD/SCID mice, pretreatment of human CD34+ cells with a neutralizing CD9 antibody resulted in inhibition of homing to bone marrow and spleen. However, enhanced CD9 expression in CD34+ cells with ingenol 3,20-dibenzoate (IDB), a protein kinase C agonist which was shown to induce CD9 expression in CD34+ cells, did not result in enhanced bone marrow homing (Desmond et al., 2011).

#### **5. The PI3K/PKB signalling module and bone marrow homing**

Correct regulation of the Phosphatidylinositol-3-Kinase (PI3K) / Protein Kinase B (PKB/c-Akt) signaling module is essential for multiple processes during hematopoiesis. Phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2, the most important substrate for PI3K, can be phosphorylated upon extracellular stimulation, resulting in the formation of phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3) (Hawkins et al., 2006). PI(3,4,5)P3 subsequently serves as an anchor for pleckstrin homology (PH) domain-containing proteins, such as Protein Kinase B (PKB/ c-akt) (Burgering & Coffer, 1995). Activation of PI3K and its downstream effector Protein Kinase B (PKB/c-Akt) has been observed in leukemic cell lines stimulated with SDF-1 (Ganju et al., 1998). A positive role for PI3K/PKB in regulation of SDF-1 induced migration of hematopoietic stem cells was therefore suggested. However, it has been shown that Protein Phosphatase 2A plays an important role in positively regulating SDF-1 mediated migration of human hematopoietic progenitors by inhibition of PKB activity (Basu et al., 2007). Similarly, inhibition of PKB activity in CD34+ cells for over 24 hours appears to be sufficient to reduce their adhesion to bone marrow derived stromal cells and to induce their basal migratory capacity (Buitenhuis et al., 2010). Transwell

In addition to regulating the activity of downstream effectors, SDF-1 has also been demonstrated to regulate the expression of specific target genes. Stimulation of peripheral blood mononuclear, Jurkat or HeLa cells has, for example, been demonstrated to result in a rapid increase in expression of the ubiquitin-specific protease 17 (USP17) (de la Vega et al., 2011). A role for this protease in regulation of migration of hematopoietic progenitor cells has been examined in vitro. Inhibition of USP17 in these cells showed decreased chemotaxis towards SDF-1, whereas over-expression of USP17 conversely resulted in increased chemotaxis. Interestingly, CXCR4 levels were not affected by inhibition or over-expression of USP17, suggesting that USP17 modulates the down-stream signaling of the CXCR4 receptor. shRNA mediated inhibition of USP17 expression resulted in decreased polymerization of actin and tubulin and reduced membrane ruffling. In addition, upon SDF-1 stimulation, the GTPases, RAC1, Cdc42 and RhoA were not transported to the plasma membrane, thereby prohibiting their activiation (de la Vega et al., 2011). In addition, CD9, a member of the tetraspanin superfamily (Boucheix et al., 1991) that is widely expressed in hematopoietic and non-hematopoietic cells, has been shown to be a SDF-1 responsive gene. Microarray analysis with human umbilical cord blood derived CD34+ cells revealed that short-term exposure to SDF-1 resulted in up-regulation of CD9 mRNA expression both in CD34+ CD38+ and CD34+ CD38-/low cells (Leung et al., 2011). A role for CD9 in migration and adhesion of human cord blood derived hematopoietic stem and progenitor cells was investigated utilizing a neutralizing CD9 antibody (Leung et al., 2011). Although actin polymerization was not affected, the calcium influx and transendothelial migration towards a SDF-1 gradient was reduced by this antibody (Leung et al., 2011). In contrast, adhesion of progenitor cells to fibronectin and human umbilical vein endothelial cells was enhanced (Leung et al., 2011). Transplantation experiments revealed that in NOD/SCID mice, pretreatment of human CD34+ cells with a neutralizing CD9 antibody resulted in inhibition of homing to bone marrow and spleen. However, enhanced CD9 expression in CD34+ cells with ingenol 3,20-dibenzoate (IDB), a protein kinase C agonist which was shown to induce CD9 expression in CD34+ cells, did not result in enhanced bone marrow homing (Desmond

**5. The PI3K/PKB signalling module and bone marrow homing** 

Correct regulation of the Phosphatidylinositol-3-Kinase (PI3K) / Protein Kinase B (PKB/c-Akt) signaling module is essential for multiple processes during hematopoiesis. Phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2, the most important substrate for PI3K, can be phosphorylated upon extracellular stimulation, resulting in the formation of phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3) (Hawkins et al., 2006). PI(3,4,5)P3 subsequently serves as an anchor for pleckstrin homology (PH) domain-containing proteins, such as Protein Kinase B (PKB/ c-akt) (Burgering & Coffer, 1995). Activation of PI3K and its downstream effector Protein Kinase B (PKB/c-Akt) has been observed in leukemic cell lines stimulated with SDF-1 (Ganju et al., 1998). A positive role for PI3K/PKB in regulation of SDF-1 induced migration of hematopoietic stem cells was therefore suggested. However, it has been shown that Protein Phosphatase 2A plays an important role in positively regulating SDF-1 mediated migration of human hematopoietic progenitors by inhibition of PKB activity (Basu et al., 2007). Similarly, inhibition of PKB activity in CD34+ cells for over 24 hours appears to be sufficient to reduce their adhesion to bone marrow derived stromal cells and to induce their basal migratory capacity (Buitenhuis et al., 2010). Transwell

et al., 2011).

migration experiments through a confluent layer of human umbilical vein endothelial cells revealed that the observed reduction in firm adhesion does not ameliorate the induced migratory capacity of CD34+ cells pre-treated with a PKB inhibitor (Buitenhuis et al., 2010). In addition, ectopic expression of constitutively active PKB in CD34+ cells conversely induced firm adhesion and reduced the basal level of migration. Although it cannot be excluded that transient activation of PI3K/PKB activity by SDF-1 is important for induction of migration, these studies suggest that prolonged activation of PKB activity is detrimental for migration of CD34+ cells. The role of PI3K in regulation of bone marrow homing was initially examined utilizing mice deficient for SHIP (SH2-containing inositol-5'-phosphatase), a negative regulator of PI3K (Damen et al., 1996). Transplantation of lethally irradiated recipients with HSCs from SHIP deficient mice resulted in diminished repopulation, suggesting that constitutive activation of PI3K impairs the ability of HSCs to home to and to be retained in the hematopoietic stem cell niche in the bone marrow. Assessment of bone marrow homing revealed that SHIP–/– hematopoietic stem and progenitor cells indeed traffic to the bone marrow and spleen with significantly reduced efficiency compared to wild type cells. Although it is evident that constitutive activation of PI3K plays a critical role in regulation of hematopoiesis per se (Buitenhuis et al., 2008), these results indicate that the inability of SHIP deficient hematopoietic stem cells to engraft and sustain long-term hematopoiesis can be, at least partially, explained by their impaired ability to home to the bone marrow (Desponts et al., 2006). Deletion of Phosphate and tensin homologue (PTEN), another critical negative regulator of PI3K signaling that dephosphorylates PI(3,4,5)P3 resulting in the formation of PI(4,5)P2 (Maehama & Dixon, 1998) only decreased bone marrow homing when PTEN deficient HSCs were transplanted into non-irradiated recipients. These results suggest that, although PTEN deficient hematopoietic stem cells are capable of migrating to the bone marrow, their performence is reduced compared to competeting wild-type hematopoietic stem cells when vacant niches are limited (Zhang et al., 2006). Although both PTEN and SHIP act on the main product of PI3K activity, PI(3,4,5)P3, the products generated are distinct, which could explain the differences between SHIP and PTEN deficient hematopoietic stem cells in terms of bone marrow homing (Dowler et al., 2000; Golub & Caroni, 2005). Recent findings demonstrated that, similar to deletion of SHIP, constitutive activation of PKB in human hematopoietic progenitors cells is sufficient to significantly inhibit homing of these cells to the bone marrow and spleen of 2 microglobulin -/- NOD/SCID mice (Buitenhuis et al., 2010). In contrast, although transplantation of C57 BL/6 mice with bone marrow cells from *5-fluorouracil* treated mice that ectopically expressed constitutively active PKB resulted in reduced engraftment levels, bone marrow homing was only modestly impaired 18 hours after transplantation (Kharas et al., 2010). To investigate whether inhibition of PKB activity would be sufficient to conversely improve bone marrow homing, human hematopoietic progenitor cells, pre-treated with a PKB inhibitor for 24 or 48 hours, were injected into recipient mice. Flow cytometric analysis, 22 hours after transplantation, revealed that transient inhibition of PKB activity prior to transplantation is sufficient to improve bone marrow homing (Buitenhuis et al., 2010). In addition, while constitutive activation of PKB appears to be detrimental for bone marrow homing, engraftment levels and hematopoietic recovery, inhibition of PKB activity prior to transplantation, resulting in an induction of bone marrow homing, conversely enhanced engraftment levels in recipient mice. Together, these studies demonstrated that correct regulation of PI3K/PKB is essential for migration of hematopoietic stem and progenitor cells to the bone marrow after transplantation, which is essential for optimal engraftment and hematopoietic recovery (Buitenhuis et al., 2010; Desponts et al., 2006; Kharas et al., 2010).

Molecular Mechanisms Underlying Bone Marrow Homing of Hematopoietic Stem Cells 197

mediated regulation of HSC migration has been investigated extensively. Thus far, multiple downstream effectors have been identified, including CD164, the GTPases Rac, Rho, and Cdc42, and the signalling molecules PI3K and PKC. In addition, the SDF-1 responsive genes CD9, USP17, both implicated in regulation of hematopoietic stem cell migration, have been indentified. Finally, SDF-1 has been demonstrated to induce the activity of integrins which allows interaction with their substrates. Although activation of PI3K and its downstream effector Protein Kinase B (PKB/c-Akt) has been observed in leukemic cell lines stimulated with SDF-1, suggesting a positive role for PI3K/PKB in regulation of SDF-1 induced migration of hematopoietic stem cells, the above described studies clearly implicate the PI3K/PKB signalling module in playing a critical role in negatively regulating migration

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of HSCs and bone marrow homing.

**7. References** 

The molecular mechanisms underlying PKB mediated regulation of migration and bone marrow homing are, thus far, incompletely understood. Although PKB mediated inhibition of migration has been demonstrated to involve RAC1 (Farooqui et al., 2006), NFAT (Yiu & Toker, 2006; Yoeli-Lerner et al., 2005) and p27Kip1 (Baldassarre et al., 2005; Viglietto et al., 2002; Wu et al., 2006) in non-hematopoietic cell lines, their importance for migration of hematopoietic stem and progenitor cells remains to be investigated. As described above, adhesion and migration of HSCs depend on correct integrin and selectin expression and regulation of integrin activity. PKB and its downstream effector GSK-3 have initially been shown to play an important role in recycling of the CD49e/CD29 and CD51/CD61 (v) integrins to the membrane in NIH 3T3 fibroblasts, resulting in enhanced cell spreading and adhesion (Roberts et al., 2004). Ectopic expression of PKB in human hematopoietic stem and progenitor cells has been demonstrated to enhance the level of CD49d, while inhibition of PKB activity conversely reduces expression of both CD49d and CD18 (Buitenhuis et al., 2010), providing a potential mechanism by which PKB induces adhesion and inhibits migration. Although it is evident that integrins play an important role in adhesion and migration of cells, the importance of these molecules in PKB mediated inhibition of migration remains to be investigated. In addition, CXCR4 expression has been demonstrated to be reduced in SHIP deficient hematopoietic stem cells, suggesting that activation of PI3K also impairs their response to SDF-1(Zhang et al., 2006).

#### **6. Conclusion**

Allogeneic HSC transplantation is the preferred treatment modality for a number of hematological malignancies. To allow normal long-term hematopoiesis to occur after transplantation, correct regulation of homing of hematopoietic stem and progenitor cells to the bone marrow and subsequent lodging of those cells into the hematopoietic stem cell niche is essential. As described above, this is a coordinated multistep process that is regulated by chemokines, integrins and selectins. Initial tethering and rolling of hematopoietic stem and progenitor cells along the endothelial wall of blood vessels are the first steps in this process. It has been demonstrated that both P and E-selectin play an important role in rolling of HSCs. In addition to selectins, integrins are also implicated in playing an important role in regulation of bone marrow homing. Both studies with blocking antibodies and knockout mice have revealed that CD49d/CD29, CD49e/CD29, CD49f, and CD49d/ITGB7 play an important role in adhesion of hematopoietic stem and progenitor cells to endothelial cells and subsequent transendothelial migration. In addition, both CD49d/CD29 and CD49e/CD29 integrins appear to be involved in mediation of SDF-1– induced directional migration of CD34+ cells through the basal lamina. In addition, although, under normal circumstances, CD18 appears not to be essential for bone marrow homing of hematopoietic stem cells, CD18 can contribute to bone marrow homing when the function of CD49d/CD29 is compromised. Although multiple chemokines are capable of inducing transendothelial migration of hematopoietic stem cells, the chemokine SDF-1 appears to be the most prominent chemokine involved in bone marrow homing. In addition, SDF-1 also appears to play a critical role in retention of hematopoietic stem cells in the hematopoietic stem cell niche. Regulation of SDF-1 activity by a variety of proteolytic enzymes has been demonstrated to play an important role in migration of hematopoietic stem cells to and from the bone marrow. The molecular mechanism underlying SDF-1 mediated regulation of HSC migration has been investigated extensively. Thus far, multiple downstream effectors have been identified, including CD164, the GTPases Rac, Rho, and Cdc42, and the signalling molecules PI3K and PKC. In addition, the SDF-1 responsive genes CD9, USP17, both implicated in regulation of hematopoietic stem cell migration, have been indentified. Finally, SDF-1 has been demonstrated to induce the activity of integrins which allows interaction with their substrates. Although activation of PI3K and its downstream effector Protein Kinase B (PKB/c-Akt) has been observed in leukemic cell lines stimulated with SDF-1, suggesting a positive role for PI3K/PKB in regulation of SDF-1 induced migration of hematopoietic stem cells, the above described studies clearly implicate the PI3K/PKB signalling module in playing a critical role in negatively regulating migration of HSCs and bone marrow homing.

#### **7. References**

196 Advances in Hematopoietic Stem Cell Research

The molecular mechanisms underlying PKB mediated regulation of migration and bone marrow homing are, thus far, incompletely understood. Although PKB mediated inhibition of migration has been demonstrated to involve RAC1 (Farooqui et al., 2006), NFAT (Yiu & Toker, 2006; Yoeli-Lerner et al., 2005) and p27Kip1 (Baldassarre et al., 2005; Viglietto et al., 2002; Wu et al., 2006) in non-hematopoietic cell lines, their importance for migration of hematopoietic stem and progenitor cells remains to be investigated. As described above, adhesion and migration of HSCs depend on correct integrin and selectin expression and regulation of integrin activity. PKB and its downstream effector GSK-3 have initially been shown to play an important role in recycling of the CD49e/CD29 and CD51/CD61 (v) integrins to the membrane in NIH 3T3 fibroblasts, resulting in enhanced cell spreading and adhesion (Roberts et al., 2004). Ectopic expression of PKB in human hematopoietic stem and progenitor cells has been demonstrated to enhance the level of CD49d, while inhibition of PKB activity conversely reduces expression of both CD49d and CD18 (Buitenhuis et al., 2010), providing a potential mechanism by which PKB induces adhesion and inhibits migration. Although it is evident that integrins play an important role in adhesion and migration of cells, the importance of these molecules in PKB mediated inhibition of migration remains to be investigated. In addition, CXCR4 expression has been demonstrated to be reduced in SHIP deficient hematopoietic stem cells, suggesting that activation of PI3K

Allogeneic HSC transplantation is the preferred treatment modality for a number of hematological malignancies. To allow normal long-term hematopoiesis to occur after transplantation, correct regulation of homing of hematopoietic stem and progenitor cells to the bone marrow and subsequent lodging of those cells into the hematopoietic stem cell niche is essential. As described above, this is a coordinated multistep process that is regulated by chemokines, integrins and selectins. Initial tethering and rolling of hematopoietic stem and progenitor cells along the endothelial wall of blood vessels are the first steps in this process. It has been demonstrated that both P and E-selectin play an important role in rolling of HSCs. In addition to selectins, integrins are also implicated in playing an important role in regulation of bone marrow homing. Both studies with blocking antibodies and knockout mice have revealed that CD49d/CD29, CD49e/CD29, CD49f, and CD49d/ITGB7 play an important role in adhesion of hematopoietic stem and progenitor cells to endothelial cells and subsequent transendothelial migration. In addition, both CD49d/CD29 and CD49e/CD29 integrins appear to be involved in mediation of SDF-1– induced directional migration of CD34+ cells through the basal lamina. In addition, although, under normal circumstances, CD18 appears not to be essential for bone marrow homing of hematopoietic stem cells, CD18 can contribute to bone marrow homing when the function of CD49d/CD29 is compromised. Although multiple chemokines are capable of inducing transendothelial migration of hematopoietic stem cells, the chemokine SDF-1 appears to be the most prominent chemokine involved in bone marrow homing. In addition, SDF-1 also appears to play a critical role in retention of hematopoietic stem cells in the hematopoietic stem cell niche. Regulation of SDF-1 activity by a variety of proteolytic enzymes has been demonstrated to play an important role in migration of hematopoietic stem cells to and from the bone marrow. The molecular mechanism underlying SDF-1

also impairs their response to SDF-1(Zhang et al., 2006).

**6. Conclusion** 


Molecular Mechanisms Underlying Bone Marrow Homing of Hematopoietic Stem Cells 199

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**10** 

*1,2France 3USA* 

**Searching for the Key to Expand** 

Philippe Leboulch1,2,3 and Diana Tronik-Le Roux1,2

*2Inserm U962 and University Paris 11, CEA-iMETI, Fontenay-aux-Roses,* 

*3Harvard Medical School and Genetics Division, Brigham & Women's Hospital, Boston,* 

Stem cells are characterized by their capacity to self renew and differentiate into progressively restricted cells that ultimately become limited to a specific cell fate. The two

Embryonic stem cells (ESC) are mostly derived from the undifferentiated inner mass cells of a blastocyst. These cells give rise during development of the embryo to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. They do not contribute to the extra-embryonic membranes or the placenta. Ex-vivo, they can be cultured for extended periods of time and under the appropriate conditions, they can be also directed to differentiate into many specialized types of cells. These particular features are being exploited to use ESC as starting material for treatment of degenerative diseases and replacement of damaged organs. Although their potential is great, the promise of ESC-derived therapies will be unfulfilled unless several challenges are overcome. For example, the quite small production of ESC-

Unlike embryonic stem cells, the adult stem cells are already partially specialized. They have been found in most self-renewing tissues, including the skin, the brain, the intestinal epithelium and the hematopoietic system and have the primary role of maintaining and repairing the tissue in which they are found. They are located deep within organs in specialized areas known as the "stem cell niche" (Scadden, 2006). This microenvironment allows for their survival, self renewal, regulated proliferation and maintenance of their quiescence for long periods of time until the moment in which they are activated. *Ex vivo*, however, the capacity of stem cells to self-renew is limited, they exhibit poor survival and

One of the more intriguing but highly debated areas of stem cell biology was the phenomenon described as plasticity or transdifferentiation. Numerous reports expressed opposing views

broad types of mammalian stem cells are: embryonic stem cells and adult stem cells.

derived cells obtained or the active immune rejection of the ESC-derived graft.

consequently their numbers sharply declines during experimental manipulation.

**1. Introduction** 

**Hematopoietic Stem Cells** 

Jeanne Grosselin1,2, Karine Sii-Felice1,2,

*Innovative Therapies (iMETI), Fontenay-aux-Roses,* 

*1CEA, Institute of Emerging Diseases and* 

