**2. Isolation and functional characteristics of BM-derived pluripotent stem cells**

The bone marrow acts as a reservoir for a heterogeneous pool of tissue-committed and non-committed stem cells. These populations contain progenitors that aid in the chimerism and cellular turnover of different organs as well as very rare populations of pluripotent and non-committed stem cells. The old dogma that adult tissues lack pluripotent stem cells (PSCs) has been continuously challenged during the last decade through multiple studies that isolated PSCs from adult humans' and animals' tissues. These populations were distinguished based on their morphology with small cell size, large nucleus demonstrating euchromatin and large nucleus to cytoplasm ratio. Furthermore, cell surface markers as well as nuclear transcription factors, such as SSEA1/4, Oct4 and Nanog, have been deployed.

Very small embryonic like stem cells (VSELs) represent a rare yet pluripotent population of adult stem cells. They have been initially described by Dr. Ratajczak's group in the murine BM based on their expression of Sca1 (murine stem cell marker) and lack of expression of CD45 (pan–leukocytic marker) and differentiated lineage (Lin) markers (Kucia *et al.,* 2006; Zuba-Surma *et al.,* 2008). Following their isolation from murine tissues, VSELs were subsequently isolated from human BM, umbilical cord blood (CB) and peripheral blood based on the lack of expression of Lin/CD45 and the expression of the stem cell markers CD133, CXCR4 and CD34. **Figure 1** illustrates the flow cytometry protocol for identifying and isolating VSELs from murine and human samples. VSELs were further characterized using a multi-dimensional approach comprising molecular, protein and cell imaging techniques to confirm their pluripotent features (Zuba-Surma *et al.* 2008). VSELs are morphologically similar to embryonic stem cells demonstrating small diameter compared to more committed progenitors/stem cells with large nucleus containing open-type chromatin surrounded with thin rim of cytoplasm and multiple mitochondria (Zuba-Surma *et al.* 2008).

VSELs exhibit multiple embryonic and pluripotent surface and nuclear embryonic markers such as Oct4, SSEA1/4, Nanog, and Rex1. In vivo and in vitro studies have demonstrated the capability of VSELs to differentiate into multiple cell lines across germ lines including cardiomyocytes (Kucia *et al.* 2006).

The bone marrow harbors other multi- and pluri-potent stem cell populations such as the mesenchymal stem cells (MSC) (Hattan *et al.,* 2005; Kawada *et al.,* 2004), multipotent adult progenitor cells (MAPC) (Jiang *et al.,* 2002), and marrow-isolated multilineage inducible cells (MIAMI) (D'Ippolito *et al.,* 2004). Similar populations with cardiac differentiation potential

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

**2. Isolation and functional characteristics of BM-derived pluripotent stem** 

The bone marrow acts as a reservoir for a heterogeneous pool of tissue-committed and non-committed stem cells. These populations contain progenitors that aid in the chimerism and cellular turnover of different organs as well as very rare populations of pluripotent and non-committed stem cells. The old dogma that adult tissues lack pluripotent stem cells (PSCs) has been continuously challenged during the last decade through multiple studies that isolated PSCs from adult humans' and animals' tissues. These populations were distinguished based on their morphology with small cell size, large nucleus demonstrating euchromatin and large nucleus to cytoplasm ratio. Furthermore, cell surface markers as well as nuclear transcription factors, such as

Very small embryonic like stem cells (VSELs) represent a rare yet pluripotent population of adult stem cells. They have been initially described by Dr. Ratajczak's group in the murine BM based on their expression of Sca1 (murine stem cell marker) and lack of expression of CD45 (pan–leukocytic marker) and differentiated lineage (Lin) markers (Kucia *et al.,* 2006; Zuba-Surma *et al.,* 2008). Following their isolation from murine tissues, VSELs were subsequently isolated from human BM, umbilical cord blood (CB) and peripheral blood based on the lack of expression of Lin/CD45 and the expression of the stem cell markers CD133, CXCR4 and CD34. **Figure 1** illustrates the flow cytometry protocol for identifying and isolating VSELs from murine and human samples. VSELs were further characterized using a multi-dimensional approach comprising molecular, protein and cell imaging techniques to confirm their pluripotent features (Zuba-Surma *et al.* 2008). VSELs are morphologically similar to embryonic stem cells demonstrating small diameter compared to more committed progenitors/stem cells with large nucleus containing open-type chromatin surrounded with thin rim of cytoplasm and multiple

VSELs exhibit multiple embryonic and pluripotent surface and nuclear embryonic markers such as Oct4, SSEA1/4, Nanog, and Rex1. In vivo and in vitro studies have demonstrated the capability of VSELs to differentiate into multiple cell lines across germ lines including

The bone marrow harbors other multi- and pluri-potent stem cell populations such as the mesenchymal stem cells (MSC) (Hattan *et al.,* 2005; Kawada *et al.,* 2004), multipotent adult progenitor cells (MAPC) (Jiang *et al.,* 2002), and marrow-isolated multilineage inducible cells (MIAMI) (D'Ippolito *et al.,* 2004). Similar populations with cardiac differentiation potential

their differentiation into cardiomyocytes (Fukuhara *et al.,* 2005).

SSEA1/4, Oct4 and Nanog, have been deployed.

mitochondria (Zuba-Surma *et al.* 2008).

cardiomyocytes (Kucia *et al.* 2006).

**cells** 

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 pluripotent potential.

**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 R3), while hematopoietic stem cells (HSCs) as CD45+/Lin-/CD34+ cells (region R4). **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 CD45+/Lin-/Sca-1+ cells (region R4).

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.

Bone Marrow Derived Pluripotent Stem Cells in

Wojakowski *et al.* 2009).

cardiac MRI in humans (Wojakowski*, et al.* 2006).

regeneration.

Ischemic Heart Disease: Bridging the Gap Between Basic Research and Clinical Applications 429

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

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

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;

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

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

committed stem cells such as HSPCs and endothelial progenitor cells (EPCs).

well as functional capacity of EPCs in diabetic patients (Fadini *et al.,* 2006).
