In this study we have described methods

In this study we have described methods for the selective isolation of primitive hMSCs that may prove advantageous both experimentally and therapeutically. The identification of the combination marker LTV makes it possible to prospectively isolate functional hMSCs directly from BM. Using these cells, we may be able to tease out the complex molecular mechanisms that govern the undifferentiated state of hMSCs. Additionally, these markers may allow visualization of hMSCs in vivo, thereby helping to uncover their physiological and pathophysiological roles. The use of VCAM-1 as an REC-selective marker facilitates the isolation of enriched MSCs from a heterogeneous population of cultured MSCs that represents mixed CFU-Fs. Since we demonstrated that the coexistence of SECs alters the self-renewal and differentiation potency of RECs in culture, the isolation of VCAM-1+ Sirtinol from cultured hMSCs may prove useful therapeutically. In conclusion, our data should help to unravel the fundamental cellular and molecular biology of MSCs. This information is critical if MSCs are to be used effectively and safely in regenerative therapy.
Experimental Procedures
Acknowledgments
Introduction Pluripotent stem cells provide an exciting opportunity in the field of basic as well as regenerative biology because of their unique capacity to differentiate in culture into all somatic cells that form an individual. Current efforts are aimed at generating a preferred somatic cell type by manipulating growth factors added to culture media. While these efforts have advanced the field, derivation of a homogenous specific cell population from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) still remains as a major challenge in the field. Successful derivation of a desired somatic cell lineage from ESCs or iPSCs would likely to be advanced by comprehensive understanding of how specification of that particular cell lineage is accomplished in the developing embryo. As for blood and vessel development, tracking a receptor tyrosine kinase fetal liver kinase 1 (FLK-1) expression has been instrumental. Specifically, cell lineage tracing studies have demonstrated that FLK-1+ mesoderm contributes to primitive and definitive blood, endothelial cells, and cardiac and skeletal muscles (Lugus et al., 2009; Motoike et al., 2003). FLK-1+ (or KDR+ in human) mesoderm isolated from ESCs or embryos can generate hematopoietic, endothelial, and smooth muscle cells as well as cardiac cells (Choi et al., 1998; Faloon et al., 2000; Yamashita et al., 2000; Ema et al., 2003; Huber et al., 2004; Kennedy et al., 2007; Kattman et al., 2006; Moretti et al., 2006; Yang et al., 2008). Importantly, hemangiogenic or cardiogenic potential of the FLK-1+ mesoderm can be segregated by the platelet-derived growth factor receptor α (PDGFRα) expression in both mouse and human, such that, while the FLK-1+PDGFRα− cell population is enriched for the hemangiogenic potential, FLK-1+PDGFRα+ cell population is enriched for the cardiogenic potential (Kattman et al., 2011; Liu et al., 2012). Molecularly, there is an antagonistic relationship between the hemangiogenic and cardiogenic mesodermal outcome. For example, enforced Er71 expression leads to an increase in hematopoietic and endothelial cell output but a decrease in cardiac output. Conversely, Er71 deficiency results in defective hematopoietic and endothelial cell output but enhanced cardiac outcome (Lee et al., 2008; Liu et al., 2012). Similarly, the hematopoietic program is inhibited by enforced Mesp1 or Nkx2-5 expression, which promotes cardiac differentiation (Caprioli et al., 2011; Lindsley et al., 2008). Herein, we reasoned that hemangioblast generation from ESCs could be enhanced by inhibiting cardiac output with defined hemangiogenic factors. We presumed that the candidate factors should be preferentially expressed within the hemangioblast population, that they could individually skew toward the hemangiogenic output, and that the skewing effect could be most dramatic when the candidate factors were coexpressed. We identify ER71, GATA2, and SCL as core factors in hemangioblast development. Transient coexpression of these three factors during mesoderm formation stage robustly enhanced FLK-1+ hemangioblast (FLK-1+PDGFRα−) production while concomitantly inhibiting cardiac outcome from differentiating ESCs. Such FLK-1+ hemangioblasts generated functional endothelial and smooth muscle cells in culture as well as in ischemic mouse hindlimbs, resulting in improved blood perfusion and limb salvage.