**4. Therapeutic mobilization of BM-derived stem cells in myocardial regeneration**

Hematologists have used the concept of BM-derived stem cell mobilization using pharmacological agents such as G-CSF for a long time. Based on the available clinical experience and safety profile of these therapies, pharmacological stem cell mobilization in the setting of AMI has gained increasing enthusiasm. Mobilized BM-SPCs are either harvested for further transplantation or allowed for spontaneous homing to the infarcted myocardium and has demonstrated various degrees of success (Engelmann *et al.,* 2006; Ince *et al.,* 2005; Zohlnhöfer *et al.,* 2006). Similar to BMCs transplantation studies, the heterogeneous methodologies of the included studies diluted the effect. The overall lack of efficacy with G-CSF BMCs mobilization in the setting of acute myocardial infarction is somewhat incongruent with the salutary effects of BMCs transplantation in humans and G-CSF therapy in animal models for myocardial regeneration.

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:

**4. Therapeutic mobilization of BM-derived stem cells in myocardial** 

CSF therapy in animal models for myocardial regeneration.

Hematologists have used the concept of BM-derived stem cell mobilization using pharmacological agents such as G-CSF for a long time. Based on the available clinical experience and safety profile of these therapies, pharmacological stem cell mobilization in the setting of AMI has gained increasing enthusiasm. Mobilized BM-SPCs are either harvested for further transplantation or allowed for spontaneous homing to the infarcted myocardium and has demonstrated various degrees of success (Engelmann *et al.,* 2006; Ince *et al.,* 2005; Zohlnhöfer *et al.,* 2006). Similar to BMCs transplantation studies, the heterogeneous methodologies of the included studies diluted the effect. The overall lack of efficacy with G-CSF BMCs mobilization in the setting of acute myocardial infarction is somewhat incongruent with the salutary effects of BMCs transplantation in humans and G-

Bright field.

**regeneration** 

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

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 insights in this regard.

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 following the acute event.
