**Bone Marrow Derived Pluripotent Stem Cells in Ischemic Heart Disease: Bridging the Gap Between Basic Research and Clinical Applications**

Ahmed Abdel-Latif1, Ewa Zuba-Surma2 and Mariusz Z. Ratajczak3 *1Gill Heart Institute and Division of Cardiovascular Medicine, University of Kentucky, and Lexington VA Medical Center, Lexington, KY 2Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, 3Stem Cell Biology Institute, James Graham Brown Cancer Center, University of Louisville, Louisville, KY,* 

*1,3USA 2Poland* 

### **1. Introduction**

424 Advances in Hematopoietic Stem Cell Research

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real-time TaqMan PCR for routine quantitation of cytomegalovirus DNA in crude leukocyte lysates from stem cell transplant patients. *J Virol Methods* Vol.110, No.1, The prevalence of ischemic heart disease and acute myocardial infarction (AMI) has increased to alarming rates in the United States and the western world (Roger *et al.,* 2011). Patients who survive the initial AMI suffer ischemic cardiomyopathy (ICM) which is often complicated by high mortality and poor overall prognosis (Braunwald *et al.,* 2000; McMurray *et al.,* 2005). Despite significant advances in medical therapy and revascularization strategies, the prognosis of patients with AMI and ischemic cardiomyopathy remains dismal (Levy D *et al.,* 2002; Roger VL *et al.,* 2004). The last decade has demonstrated significant progress and rapid translation of myocardial regenerative therapies particularly those utilizing stem cells isolated from adult tissues (Abdel-Latif *et al.,* 2007).

Studies examining the potential therapeutic use of bone marrow (BM)-derived cells in myocardial regeneration have overshadowed the growing evidence of innate cardiac reparatory mechanisms. Several studies have demonstrated the capability of cardiomyocytes to replenish through poorly understood innate mechanisms. Follow up of cardiac transplantation patients have demonstrated continuous replenishment of cardiomyocytes by recipient's derived cells through poorly understood mechanisms (Quaini *et al.,* 2002). There is growing evidence that BM-derived cells are responsible, at least in part, for organ chimerism including cardiomyocyte chimerism (de Weger *et al.,* 2008; Deb *et al.,* 2003). Animal studies have confirmed this to be a dynamic process responding to significant injury such as myocardial infarction and peaks in the peri-infarct zone (Hsieh *et al.,* 2007). Although this process appears to be robust enough to achieve the renewal of approximately 50% of all cardiomyocytes in the normal life span, very little is known about its underpinnings (Bergmann *et al.,* 2009).

Bone Marrow Derived Pluripotent Stem Cells in

pluripotent potential.

Ischemic Heart Disease: Bridging the Gap Between Basic Research and Clinical Applications 427

have been also isolated from skeletal muscle and other tissues (Abdel-Latif *et al.,* 2008a). However, it is conceivable that different investigators have isolated, using different methods, the same or very similar populations and named them differently. It is also possible that these populations at least in part contain VSELs which might explain their

**Panel A:** Gating strategy for isolating human cord blood (CB)-derived VSELs. Morphology of total CBderived nucleated cells is shown on dot-plot representing FSC and SSC parameters related to cell size and granularity/ complexity, respectively. All objects larger than 2m are enclosed in region R1 and further visualized on histogram showing the expression of markers of mature hematopoietic cells (lineage markers; Lin). Cells not expressing differentiated hematopoietic markers (Lin- in region R2) are then analyzed for CD34 and CD45 expression. VSELs are identified as CD45-/Lin-/CD34+ cells (region

**Panel B**: Sorting of murine bone marrow (BM)-derived VSELs. Morphology of total murine BM-derived nucleated cells is shown on dot-plot presenting FSC and SSC parameters and all objects in range of 2- 10m in diameter are included in region R1. Lymphocytic cells including stem cell fraction is further analyzed for Sca-1 and differentiated hematopoietic lineages markers (Lin) expression and only Sca-1+/Lin- cells are included in region R2. Cells from this region are further seperated based on CD45 expression. Murine VSELs are distinguished as CD45-/Lin-/Sca-1+ cells (region R3), while HSCs as

Fig. 1. Strategy for flow cytometric analysis of human and murine Very Small Embryonic-Like and hematopoietic stem cells. Briefly, BM is flushed from the femurs and tibias. Nucleated cells are isolated by lysis of red blood cells and cells are then gated on based on the cell size (>2 m). Of note, lysis is preferred for isolating VSELs rather than Ficoll gradient that we have shown to lose some of the VSELs due to their small size.

R3), while hematopoietic stem cells (HSCs) as CD45+/Lin-/CD34+ cells (region R4).

CD45+/Lin-/Sca-1+ cells (region R4).

Complex innate reparatory mechanisms are initiated by myocardial ischemia interacting with different elements of the immune system, the infarcted myocardium and bone marrow stem cells, culminating in BM-stem and progenitor cells' (SPCs) mobilization as we and others have demonstrated (Abdel-Latif *et al.,* 2010; Walter *et al.,* 2007; Wojakowski *et al.,* 2009). However, very little is known about the mechanisms and clinical significance of this mobilization. Animal studies show that mobilized BM-derived cells (BMCs) repopulate the infarct border, however the significance of this mobilization is unclear given the low rate of their differentiation into cardiomyocytes (Fukuhara *et al.,* 2005).
