Taken together these findings suggest a working model

Taken together, these findings suggest a working model to define CS-derived cell lineages with cardiovascular differentiation potential from the adult human heart (Figure 7E). In this model, CD90 expression segregates adult human CS-derived rac1 inhibitor with differing cardiomyogenic differentiation. Our study suggests the presence of a “true” adult CPC versus a mesenchymal/myofibroblast cell that can express cardiac proteins but not differentiate into a true CM.
Experimental Procedures
Author Contributions
Acknowledgments
Introduction To avoid these hazards, the availability of functional SA node (SAN) cells for transplantation or the de novo generation of such cells in vivo is required. Correspondingly, to obtain biological pacemaker cells for future therapy, two possible approaches are being addressed. One approach is to convert beating myocardium into pacemaker cells in situ via genetic manipulations (i.e., direct reprogramming). In this regard, the early key transcription factor TBX3 led to cells with incomplete pacemaker characteristics (Bakker et al., 2012). More recently, viral overexpression of TBX18, a member of the same transcription factor family, was reported to enable reprogramming of chamber myocardium toward nodal cells (Kapoor et al., 2013). Thus far, however, this approach requires integration of viruses, prohibiting a spatially and timely controllable expression of TBX18, which would mimic the in vivo situation in the developing embryo. TBX18 is only transiently expressed in the head part of the developing sinus node, whereas TBX3 is permanently expressed in the entire node in vivo (Wiese et al., 2009). In addition, the efficiency of Tbx18-based direct reprogramming is low (Kapoor et al., 2013). Another approach relies on the transplantation of in-vitro-generated “biological pacemakers” derived from pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) (Wobus et al., 1991; Kehat et al., 2002). In this regard, Kleger et al. (2010) postulated that the small-molecule compound EBIO enhances the formation of nodal cells from murine ESCs to some extent. However, they did not address the actual ability of the cells to pace ventricular myocardium, and again the cells’ beating rates were low. Moreover, on the electrophysiological level, they did not discriminate between relatively mature pacemaker cells and similar spontaneously contracting early/intermediate cell types (Kleger et al., 2010; Maltsev et al., 1994; David and Franz, 2012). More recently, a cell population purified based on Alcam (CD166) expression was described to give rise to nodal cells (Scavone et al., 2013). It will be interesting to see whether this approach can be transferred to the human system, as surface marker expression specificity often differs among species. As an alternative, in this work we further developed our previous method for cardiomyocyte-subtype forward programming of ESCs via single transcription factors (David et al., 2008, 2009; David and Franz, 2012). We applied this approach to TBX3 and found that it led to a doubling of functional pacemaker cells, but alone was still insufficient to obtain pure populations of these cells. This may be related to the insufficiency of TBX3 as a single factor for direct reprogramming (Bakker et al., 2012). However, when we combined our approach with Myh6 promoter-based antibiotic selection (Klug et al., 1996), we achieved cell aggregates that consisted exclusively of spontaneously beating cardiomyocytes, with beats per minute (bpm) rates close to those of a mouse heart. Of these cells, >80% corresponded to the desired pacemaker phenotypes bearing full functionality at the levels of protein expression, electrophysiological parameters, and Ca2+ signaling characteristics, as well as potency to robustly pace murine myocardium ex vivo. Therefore, we provide and example of highly enriched, stem cell-derived pacemaker cell populations that possess all of the properties characteristic of that cell type, which may be an important prerequisite for future cell therapies and in vitro drug evaluation.