The major findings of this study are: (a) hESC-derived hCM obtained through p38MAP kinase inhibitor-directed differentiation, and purified by Percoll-gradient centrifugation, confer functional benefit when transplanted into injured mouse mycardium; (b) ultrasound-guided injections of hCM into mouse hearts 3 days post-MI results in improvement of cardiac function, smaller infarct size, less deleterious LV remodeling, and an increase in the peri-infarct and scar wall thickness at day 60 post-therapy compared with injection of hFF or medium; (c) treatment with hCM results in an increase in capillary bed area and an increase in arteriole number compared with treatment with hFF or medium; (d) hCM therapy results in a decrease in native cardiomyocyte apoptosis and an increase in CM proliferation; (e) these benefits are achieved despite a low retention rate of hCM in the myocardium and without cell differentiation after therapy at day 60, suggesting a predominantly paracrine mechanism; (f) hCM therapy is safe and does not result in teratoma formation at 60 days of follow-up in the immunodeficient mouse model; (g) human fetal fibroblast therapy does not result in cardiac functional improvement post-MI in this model. While our group and others had previously demonstrated enrichment of hESC-derived hCM with p38MAPK inhibition (13
), the effectiveness of these cells in vivo
has not been studied until now. In addition, other groups have examined the potential therapeutic benefits of hESC-derived hCM in rodent models of MI (23
); however, they did not evaluate hCM specifically derived by p38MAPK inhibition. As the proposed mechanism by which p38MAPK inhibition preferentially directs mesodermal differentiation at the expense of ectoderm (13
) differs from other methods for directed differentiation of hCM (23
), it was not obvious that these cells would behave similarly to other hESC-derived cardiac cells in vivo
Our results demonstrate that injection of hCM attenuates adverse left ventricular remodeling and improves cardiac function compared with hFF and control medium. The benefits are achieved despite a low myocardial retention rate of the cells. These findings are reminiscent of studies with adult bone marrow cells, where significant functional improvement was achieved post-MI despite a low retention rate of the transplanted cells (15
). In contrast with adult stem cells, we hypothesized that hCM would result in higher cell retention and differentiation, but this was not seen. Despite low cell retention, however, treatment with hCM resulted in an increase in capillary bed area and an increase in arteriole number compared with treatment with hFF or stem cell differentiation medium. In addition to the beneficial effects on blood vessel numbers, hCM treatment resulted in a decrease in native CM apoptosis and an appreciable increase in the proliferation of these cells. In contrast with some studies that have shown an initial functional benefit followed by a fall-off in this benefit over time (25
), the improvement in cardiac function seen in this study at the early time-point (day 28) persisted through the entire study (60 days post-MI).
Other groups citing evidence of low engraftment with other sources of cardiac stem cells have proposed paracrine mechanisms that include recruitment of resident cardiac stem cells (30
), inhibition of fibrosis (32
), cardioprotection (probably through secretion of hepatocyte growth factor (HGF), transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1, stanniocalcin 1, and granulocyte-macrophage colony-stimulating factor (GM-CSF)) and promotion of neoangiogenesis (33
). Another hypothesis is that hCM may modulate the inflammatory microenvironment of ischemic tissue. Our data showing smaller infarct size and an increase in peri-infarct wall thickness ( and ), and an increase in capillary bed area and arteriole number (), suggest that paracrine effects leading to inhibition of fibrosis and neoangiogenesis, respectively, are probably responsible for the functional effects observed in our studies.
Over the past few years, several studies have reported improvement in cardiac function with transplantation of cardiomyocytes or cardiac progenitor cells derived from hESC (23
). The majority of injected cells in these reports disappeared within days after transplantation. The goal of this study was to test whether a new method for differentiation (i.e. p38MAPK inhibition) and the mode of delivery (i.e. ultrasound-guided closed-chest several days after MI) might lead to better engraftment and therefore a greater functional benefit. While a higher rate of engraftment and larger measureable improvement in function were not observed, our experiments show that effects similar to those seen with spontaneously differentiated hCM can be achieved with hCM obtained using a directed differentiation method shown previously by us and others to improve the yield of hCM in vitro
). These data also suggest that open-heart delivery immediately following infarct may not be necessary to achieve previously seen desired effects with hESC-derived hCM.
Undifferentiated hESC produce germ-line tumors, or teratomas, when introduced into a physiologic environment (21
). As such, methods that either produce highly purified cardiac cells derived from hESC, or eliminate undifferentiated cells (20
), are critical to avoiding teratoma formation. With the isolation method described in this report, it is encouraging that we did not detect teratomas in the heart. It should be noted that, in this rodent study, we transplanted human cells into an immunodeficient mouse model. Further investigation is required to evaluate whether hESC would result in teratoma formation when transplanted into immunosuppressed recipients.
In summary, we report that highly purified hCM can be obtained for therapeutic purposes by directed differentiation of hESC into hCM with p38MAPK inhibition, and subsequent purification by Percoll-gradient centrifugation, and that these cells improve cardiac function post-MI and result in significantly smaller scar size, higher capillary density and increased numbers of arterioles, even when introduced by closed-chest injection. These benefits were achieved despite a very low cell retention rate at 60 days post-therapy, as assessed by quantitative PCR and immunohistochemistry. These studies also confirm that therapy with hCM produced and delivered in this manner does not result in teratoma formation.