**3. BM-derived pluripotent stem cells are mobilized in the peripheral circulation following myocardial ischemia in animal models and humans**

Myocardial ischemia, particularly large myocardial infarction, produce multiple stimuli include various chemokines, cytokines, kinins, bioactive lipids and members of the complement cascade, that lead to the mobilization and subsequent homing of BMSPCs. Indeed, several reports have confirmed that mobilization of stem cells originating from the BM occurs in response to myocardial ischemic injury (Grundmann *et al.,* 2007; Kucia *et al.,* 2004b; Leone *et al.,* 2005; Massa *et al.,* 2005; Shintani *et al.,* 2001; Wojakowski *et al.,* 2006) and heart failure (Valgimigli *et al.,* 2004). Similar observations were noted in patients with acute neurological ischemia (Paczkowska *et al.,* 2005) and patients with extensive skin burn (Drukala *et al.,* 2011).

Stimuli responsible for the mobilization and homing of BMSCs in the setting of myocardial ischemia show similarities and differences with those involved in hematopoietic stem cells (HSCs) homing to the BM. The role of stromal cell derived factor (SDF-1) and its receptor (CXCR4) axis in the retention of hematopoietic stem/progenitor cells (HSPCs) in bone marrow is undisputed (Kucia *et al.,* 2005; Lapidot *et al.,* 2002), however, its role in the mobilization and homing of BM-SPCs to a highly proteolytic microenvironment, such as the ischemic/infarcted myocardium, is somewhat less certain. Studies have demonstrated that multiple members of the metalloproteinases (MMP) family, such as MMP2, MMP9 and MMP13, are upreagulated in the myocardium following infarction (Peterson *et al.,* 2000). The elevated levels of the MMPs contribute to the degradation of chemokines such as SDF-1 and the byproduct of this degradation acts as an inhibitor the sole SDF-1 receptor, CXCR4 (McQuibban *et al.,* 2001; McQuibban *et al.,* 2002). In support of this hypothesis, Agrawal *et al* demonstrated that the conditional deletion of CXCR4 in cardiomyocytes did not influence the recovery of left ventricular (LV) function, reduce the scar size or alter the homing of MSCs to the myocardium following myocardial infarction (Agarwal *et al.,* 2010). Thus, there is growing evidence that other mechanisms beside the SDF-1/CXCR4 axis are contributing to the mobilization and homing of BM-SPCs in AMI and other conditions (Jalili *et al.,* 2010; Ratajczak *et al.,* 2010). These data suggest an important interplay between the complement cascade, the immune system, cathelicidins, low levels of SDF-1, and sphingosine-1 phosphate (S1P) and other bioactive lipids in the mobilization and homing of HSPCs. Our preliminary data suggest that these complex interactions might be involved in the mobilization of BM-SPCs in acute myocardial ischemia as well (unpublished data). Clinically, pharmacological modulators of S1P receptors are already approved by the FDA and can be utilized to enhance BM-SPC mobilization in the setting of ischemic heart disease. Similarly, modulation of the complement cascade can be also utilized in this process similar to their role in the mobilization of HSPCs.

The first evidence for the mobilization of CD34+ mononuclear cells in AMI was demonstrated by Shintani *et al* (Shintani *et al.* 2001). The authors demonstrated successful in vitro differentiation of circulating BM-SPCs into endothelial cells that expressed CD31, VEcadherin and the kinase insert domain receptor (KDR) (Shintani*, et al.* 2001). Leone *et al* demonstrated that the levels of circulating CD34+ cells in the setting of AMI were higher when compared to patients with mild chronic stable angina and healthy controls. The magnitude of CD34+ cell mobilization correlated with the recovery of regional and global LV function recovery as well as other functional LV parameters (Leone *et al.* 2005). Similarly,

Myocardial ischemia, particularly large myocardial infarction, produce multiple stimuli include various chemokines, cytokines, kinins, bioactive lipids and members of the complement cascade, that lead to the mobilization and subsequent homing of BMSPCs. Indeed, several reports have confirmed that mobilization of stem cells originating from the BM occurs in response to myocardial ischemic injury (Grundmann *et al.,* 2007; Kucia *et al.,* 2004b; Leone *et al.,* 2005; Massa *et al.,* 2005; Shintani *et al.,* 2001; Wojakowski *et al.,* 2006) and heart failure (Valgimigli *et al.,* 2004). Similar observations were noted in patients with acute neurological ischemia (Paczkowska *et al.,* 2005) and patients with extensive skin burn

Stimuli responsible for the mobilization and homing of BMSCs in the setting of myocardial ischemia show similarities and differences with those involved in hematopoietic stem cells (HSCs) homing to the BM. The role of stromal cell derived factor (SDF-1) and its receptor (CXCR4) axis in the retention of hematopoietic stem/progenitor cells (HSPCs) in bone marrow is undisputed (Kucia *et al.,* 2005; Lapidot *et al.,* 2002), however, its role in the mobilization and homing of BM-SPCs to a highly proteolytic microenvironment, such as the ischemic/infarcted myocardium, is somewhat less certain. Studies have demonstrated that multiple members of the metalloproteinases (MMP) family, such as MMP2, MMP9 and MMP13, are upreagulated in the myocardium following infarction (Peterson *et al.,* 2000). The elevated levels of the MMPs contribute to the degradation of chemokines such as SDF-1 and the byproduct of this degradation acts as an inhibitor the sole SDF-1 receptor, CXCR4 (McQuibban *et al.,* 2001; McQuibban *et al.,* 2002). In support of this hypothesis, Agrawal *et al* demonstrated that the conditional deletion of CXCR4 in cardiomyocytes did not influence the recovery of left ventricular (LV) function, reduce the scar size or alter the homing of MSCs to the myocardium following myocardial infarction (Agarwal *et al.,* 2010). Thus, there is growing evidence that other mechanisms beside the SDF-1/CXCR4 axis are contributing to the mobilization and homing of BM-SPCs in AMI and other conditions (Jalili *et al.,* 2010; Ratajczak *et al.,* 2010). These data suggest an important interplay between the complement cascade, the immune system, cathelicidins, low levels of SDF-1, and sphingosine-1 phosphate (S1P) and other bioactive lipids in the mobilization and homing of HSPCs. Our preliminary data suggest that these complex interactions might be involved in the mobilization of BM-SPCs in acute myocardial ischemia as well (unpublished data). Clinically, pharmacological modulators of S1P receptors are already approved by the FDA and can be utilized to enhance BM-SPC mobilization in the setting of ischemic heart disease. Similarly, modulation of the complement cascade can be also utilized in this process similar

The first evidence for the mobilization of CD34+ mononuclear cells in AMI was demonstrated by Shintani *et al* (Shintani *et al.* 2001). The authors demonstrated successful in vitro differentiation of circulating BM-SPCs into endothelial cells that expressed CD31, VEcadherin and the kinase insert domain receptor (KDR) (Shintani*, et al.* 2001). Leone *et al* demonstrated that the levels of circulating CD34+ cells in the setting of AMI were higher when compared to patients with mild chronic stable angina and healthy controls. The magnitude of CD34+ cell mobilization correlated with the recovery of regional and global LV function recovery as well as other functional LV parameters (Leone *et al.* 2005). Similarly,

**3. BM-derived pluripotent stem cells are mobilized in the peripheral circulation following myocardial ischemia in animal models and humans** 

(Drukala *et al.,* 2011).

to their role in the mobilization of HSPCs.

Wojakowski *et al* demonstrated the mobilization of multiple BM-SPCs populations in patients with AMI and found significant correlation between the number of circulating CD34+ cells and plasma SDF-1 levels (Wojakowski *et al.,* 2004). In their following publication, the authors demonstrated the correlation between circulating BM-SPCs and ejection fraction at baseline and lower brain natriuretic peptide (BNP) levels (Wojakowski *et al.* 2006). Interestingly, the mobilization of BM-SPCs is reduced by the successful revascularization of the culprit vessel in acute STEMI (Müller-Ehmsen *et al.,* 2005). However, the majority of the above mentioned studies have focused on the mobilization of partially committed stem cells such as HSPCs and endothelial progenitor cells (EPCs).

We and others have demonstrated the mobilization of pluripotent stem cells (PSCs) including VSELs in the setting of myocardial ischemia (Abdel-Latif *et al.* 2010; Wojakowski *et al.* 2009). The number of circulating VSELs was highest in patients with ST-elevation myocardial infarction (STEMI), particularly in the early phases following the injury, when compared to patients with lesser degrees of ischemia such as non STEMI (NSTEMI) and those with chronic ischemic heart disease (Abdel-Latif *et al.* 2010). The mobilization of PSCs appears to be related to the extent of myocardial ischemia and the degree of myocardial damage. Moreover, the ability of patients to mobilize PSCs in the peripheral circulation in response to AMI decreases with age, reduced global LV ejection fraction (LVEF) and diabetes supporting the notion of an age/comorbidity related decline in the regenerative capacity (Abdel-Latif*, et al.* 2010; Wojakowski*, et al.* 2009). Indeed, animal models confirm the reduction of number as well as pluripotent features of BM-derived VSELs with age (Zuba-Surma *et al.* 2008). Similarly, studies have demonstrated the reduction of number as well as functional capacity of EPCs in diabetic patients (Fadini *et al.,* 2006).

The pluripotent features of mobilized VSELs, including the presence of octamer-binding transcription factor-4 (Oct4) and stage specific embryonic antigen-4 (SSEA4), were confirmed both on the RNA and protein levels. Utilizing the capabilities of the ImageStream system, we demonstrated that circulating VSELs during AMI have very similar embryonic features similar to their BM and CB counterparts including the small size (7-8 m), large nucleus and high nuclear-to-cytoplasm ratio (**Figure 2**). Furthermore, circulating VSELs during AMI express markers of early cardiac and endothelial progenitors that suggest that the mobilization is rather specific and that circulating VSELs are destined to aid in the myocardial regeneration following injury (Abdel-Latif *et al.* 2010; Kucia *et al.* 2004b; Wojakowski *et al.* 2009).

The above evidence suggest an innate, yet poorly understood, reparatory mechanism that culminates in the mobilization of BMSCs including pluripotent and embryonic like stem cells in acute myocardial injury. This mobilization correlates with the recovery of LV function and other LV functional parameters. Therefore, mobilization of PSCs in myocardial ischemia is a relevant and clinically significant process. Future studies aiming at selective mobilization of PSCs rather than the non-selective actions of agents such as granulocyte colony stimulating factor (G-CSF) may prove beneficial in the field of myocardial regeneration.

Indeed, there is evidence that the mobilization of CXCR4+ cells in the setting of AMI is correlated with LVEF recovery as well as myocardial reperfusion when assessed with cardiac MRI in humans (Wojakowski*, et al.* 2006).

Bone Marrow Derived Pluripotent Stem Cells in

(Abdel-Latif *et al.* 2008b).

insights in this regard.

following the acute event.

Ischemic Heart Disease: Bridging the Gap Between Basic Research and Clinical Applications 431

The largest study utilizing G-CSF in the setting of acute myocardial infarction was the REVIVAL-2 trial that included 114 patients (Zohlnhöfer*, et al.* 2006). The study randomized AMI patients to 10 g/kg of G-CSF vs. placebo and left ventricular functional parameters were assessed using cardiac MRI (CMR). The study demonstrated no significant difference in the tested parameters between patients treated with G-CSF or placebo. However, baseline characteristics in the study population showed normal or near normal LV function and therefore the expected benefit is minimal. Patient selection was a methodological flaw that plagued some of the studies that utilized G-CSF. Indeed, with careful examination of the available literature, patients with reduced LV function at baseline as well as those treated within the first 36 hours following AMI benefited the most (Abdel-Latif *et al.,* 2008b; Achilli *et al.,* 2010). On the other hand, safety concerns regarding a potentially increasing evidence of instent restenosis (Kang *et al.,* 2004) and recurrent ischemia (Hill *et al.,* 2005) have halted subsequent clinical trials. However, it is important to note that these safety concerns were not confirmed in large studies (Zohlnhöfer*, et al.* 2006) or in the cumulative meta-analyses

Beyond the methodological flaws encountered in human trials, this lack of efficacy can be explained by multiple factors. While G-CSF and similar therapies mobilize a wide array of BMSPCs in the peripheral blood, homing factors may not be sufficient to guide them to the myocardial infarct zone. Indeed, the homing of c-Kit+ cells to the infarcted myocardium improved when G-CSF therapy was combined with local administration of SDF-1 (Askari *et al.,* 2003). The myocardial levels of chemoattractants peaks within 24-72 hours following injury (Kucia *et al.,* 2004a; Ma *et al.,* 2005; Wang *et al.,* 2006) and therefore delayed therapy in some human trials may have missed the homing window to the infarct zone. Similarly, the addition of Flt-3 to G-CSF therapy improved outcomes in animal models (Dawn *et al.,* 2008). Moreover, different cytokines are known to preferentially mobilize somewhat different subsets of BMCs (Hess *et al.,* 2002; Neipp *et al.,* 1998). Future studies investigating the characteristics of G-CSF-mobilized cells will be necessary to glean additional mechanistic

Recently, a combined approach with stem cell mobilization and enhanced homing using therapies known to increase local SDF-1 or CXCR4 antagonists have been proposed and is currently being tested (Jujo *et al.,* 2010; Zaruba *et al.,* 2009). Going forward, the beneficial effects of BM-derived stem cell mobilization may be augmented by selective mobilization of undifferentiated BMSCs rather than differentiated inflammatory cells. It is also important to remember that some of the G-CSF arbitrated effects can be mediated by its direct effect on cardiomyocytes which are known to express G-CSF receptor (Shimoji *et al.,* 2010). G-CSF therapy may be inducing the proliferation of cardiomyocytes or the differentiation of resident cardiac stem cells. On a similar note, G-CSF therapy upregulates Akt (Ohtsuka *et al.,* 2004) and may result in reducing apoptosis of ischemic cardiomyocytes if utilized early

The use of BM-derived cells in myocardial regeneration has moved rapidly from the basic research lab to the clinical arena. The results from these studies varied widely probably secondary to the heterogeneous methodologies used with an overall marginal benefit with

**5. BM-derived stem cell transplantation for myocardial repair** 

Fig. 2. Representative ImageStream images of VSEL and hematopoietic stem/ progenitor cell (HSPC) circulating in peripheral blood following acute ST-elevation myocardial infarction. Cells were stained against: 1) hematopoietic lineages markers (Lin) and CD45 to be detected in one channel (FITC, green), 2) marker of pluripotency Oct4 (PE, yellow) and 3) stem cell antigen CD34 (PE-Cy5, cyan). Nuclei are stained with 7-aminoactinomycin D (7- AAD, red). Scales represents 10 m. VSELs are identified based on the lack of expression of both Lin and CD45 markers and positive staining for CD34 antigen and nuclear appearence of Oct-4 transcription factor (**Upper Panel**). HSCs are identified as cell expressing Lin and/ or CD45 markers as well as CD34 antigen; however, negative for Oct-4 (**Lower Panel**). BF: Bright field.
