Dabrafenib Another major limitation of the adult cardiac
Another major limitation of the adult cardiac stem cell field is the inability to get full differentiation from putative stem/progenitor Dabrafenib in vitro or in vivo. In fact, although expression of cardiac genes and proteins is demonstrated frequently, the levels, relative to authentic CMs, and their ability to form sarcomeres and display calcium transients are rarely quantified (Barile et al., 2007; Bearzi et al., 2007; He et al., 2011). In vivo engraftment studies have often demonstrated low-level retention of cells that, whereas expressing sarcomeric proteins, do not morphologically appear to resemble adult CMs (Li et al., 2010). This is also true of the CMs differentiated from human CS (Davis et al., 2009). The basis for this paradox is unknown but is a critical issue to resolve if these cells are to be used therapeutically. Despite these limitations, the therapeutic efficacy of autologous CS-derived cells after myocardial infarction was tested in a recent clinical trial by Makkar et al. (2012). The authors reported that the therapy appeared safe, reduced myocardial scar, and increased viable myocardium, but there was no global improvement in patients’ cardiac function.
In the present study, we sought to determine the identity of the cell(s) with cardiogenic potential in CS generated from human hearts. We describe two distinct populations of CS-derived cells based on their expression of CD90 and their cardiomyogenic potential. The first cell population is enriched for the cardiac transcription factor NKX2.5 and ISL1 but negative for mesenchymal stem cell/fibroblast surface marker CD90 and negative for cardiac stem cell marker C-KIT. These CD90− cells can differentiate into SMCs, ECs, and CMs, which form complete sarcomeres and beat spontaneously but at a very low frequency. The second cell population appears to be a cardiac mesenchymal/fibroblast cell that is positive for CD90 and Periostin but is NKX2.5low and ISL1low. This cell demonstrated endothelial and SMC differentiation but only incomplete cardiac differentiation into CM-like cells without sarcomere formation or calcium cycling. These results may account for previous reports of multiple stem cells in the heart (Barile et al., 2007; Beltrami et al., 2003; Davis et al., 2010; Deb et al., 2003; Laflamme et al., 2002; Murry et al., 2004; Toma et al., 2002) and suggest that a cell with a phenotype consistent with a mesenchymal stem cell or myofibroblast exists in the adult heart that is capable of differentiating into CM-like cells that are not authentic mature CMs.
Discussion Tissue-specific stem or progenitor cells are notoriously difficult to expand and propagate in vitro in an undifferentiated state. Various culture systems have been developed in an attempt to maintain stemness in these cells, including a 3D culture system based on sphere formation. We utilized CS generated from fetal and adult hearts to attempt to identify an aCPC that has the capacity to differentiate into CMs, SMCs, and ECs. Fetal and adult CS-derived cells differentiated into vascular lineages like SMCs and ECs with similar efficiencies. However, whereas functional CMs were easily derived from fetal CS, the majority of adult CS-derived CMs did not form sarcomeres or exhibit calcium cycling despite expression of relevant cardiac-specific genes. We cloned cells from adult CS and demonstrated two distinct populations of cells with differing cardiovascular differentiation potential. The first cell type has properties consistent with a CPC (CD90−/ISL1hi/Periostinlo), including potential to differentiate into more mature CMs, although the percentage of mature CMs obtained was very low (<1%). This was not simply a technical issue related to our differentiation conditions because multiple published protocols were tested (Figures S7A–S7C). Likewise, the fact that cells isolated from CS, which were derived directly from CD90− cells, demonstrated cardiogenic differentiation suggests that CM dedifferentiation cannot explain these results. Although these results support the conclusion that CSs contain an aCPC with cardiogenic potential, it is unlikely that this differentiation could explain any therapeutic effect given its rarity. Furthermore, the explanation for why formation of mature CMs occurs at such a low efficiency even from clones with cardiogenic potential is unclear. Whether it is related to limitations of the artificial in vitro differentiation conditions or represents a block to differentiation in these cells will require further studies. Regardless, generation of sufficient aCPCs in vitro to regenerate injured hearts will require the discovery of novel techniques to expand and differentiate these cells.