Dr. Laflamme began by noting that hESCs are derived from pre-implantation stage blastocysts. The cells have several properties that render them useful for regenerative medicine, including reproducible protocols for isolation and expansion, an indefinite proliferative capacity, and pluripotency. These cells have undisputed potential for regenerating damaged cardiac tissue; 0.1-1.0% of the cells in hESC embryoid bodies are cardiomyocytes (Gepstein L. Circ Res 2002;91:866-876). Protocols for the directed differentiation of hESCs employ growth factors such as BMP-4 and serial activin A (Laflamme MA, et.al. Nat Biotechnol 2007;25:1015-1024) combined with enrichment steps. The cardiac phenotype is then characterized using expected cardiac markers such as cTroponin I and sarcMHC. Moreover, cardiac-type action potentials in hESC-derived cardiomyocytes are unambiguous.
However, controlling differentiation is a challenge, and the specific application(s) of the cells must be considered. Human myocardium can be grafted in uninjured rat hearts, and these implants are robust and will proliferate (Laflamme MA, et.al. Am J Pathol 2005;167:663-671). These results have led researchers to ask whether hESC-derived cardiomyocytes could be used to establish viable grafts in the infarcted rodent heart and preserve cardiac function. Delivery of an enriched population of directly differentiated, hESC-derived cardiomyocytes via a pro-engraftment “cocktail” reliably remuscularized the infarct zone in engrafted rats (Laflamme MA, et.al. Nat Biotechnol 2007;25:1015-1024). Evidence of a surviving human graft was seen in 100% of recipient rats at four weeks with no teratomas or human grafts in distant organs. Furthermore, these hESC-derived cardiomyocytes preserved fractional shortening and enhanced regional wall thickening at four weeks post-transplant. The efficacy of hESC-derived cardiomyocyte grafting in rodent infarct models has subsequently been confirmed by four independent labs.
Remaining challenges to use this approach for regenerative medicine include increasing the purity of the cardiac cell population, preventing immune rejection, attenuating cell death post-implantation, and enhancing graft maturation, organization, and integration. To increase purity, potential solutions include guided differentiation, sorting for cardiomyocytes or more restricted progenitor populations, and genetic selection. Suppression of the immune response can be achieved through immunosuppression, hematopoietic chimerisms, somatic cell nuclear transfer, and the use of iPS cells. To attenuate cell death post-transplantation, improvement in pro-survival factors and tissue engineering may be helpful. Tissue engineering may also be useful to enhance the graft maturation, organization, and integration processes.
Tissue engineering may resolve some of these issues; scaffolds that mimic the microstructure of cardiac tissue could be pre-seeded to help pull cardiomyocytes into the scaffold. These scaffolds can recapitulate native tissue architecture. Also, scaffold-free cardiac patches that can be vascularized using endothelial cells and fibroblasts are being explored.
In conclusion, Dr. Laflamme noted that hESC-derived cardiomyocytes have an unambiguous cardiac phenotype. Directed differentiation of hESCs with growth factors results in efficient and reasonably pure cardiogenesis. hESC-derived cardiomyocytes can also form implants of proliferating human myocardium in the rodent heart. Graft survival in infarcted hearts is enhanced greatly by the use of a combinatorial pro-survival cocktail. Delivery of differentiated hESC-derived cardiomyocytes in such a cocktail results in remuscularization and preserved cardiac function. Finally, tissue engineering approaches hold significant promise for improving the state of maturation and tissue organization of hESC-derived cardiac implants.
One attendee asked if directed differentiation of SCs is an accepted idea throughout the field. Dr. Laflamme noted that the concept has been validated in vitro in several situations. He noted, however, that much work remains to be done with respect to clinical application.
Another participant inquired whether the contractility seen in these implants is real. Dr. Laflamme noted that it is; the transplanted cells can electromechanically integrate with the host tissue. However, this coupling can also drive arrhythmias, so additional work needs to be carried out to control the process.
One participant asked if the hESC-derived cardiomyocytes produce proteins or factors not produced by normal cardiomyocytes. Dr. Laflamme noted that skeletal muscle and ectodermal, endothelial, and neural cells have not been observed in these experiments. The cells gradually stop proliferating in vivo; generation of new cells usually tapers off over the course of ten weeks. One commenter noted that a similar pattern has been observed in the brain.
Dr. Laflamme stated that it is critical to note that this field is immature and that additional work is necessary to translate these results to the clinic. In addition to ensuring the safety and efficacy of transplanted cells, additional effort must be directed toward reducing the immune rejection of transplanted cells. He noted that the National Heart, Lung, and Blood Institute has recently recommended to establish a Cardiovascular and Pulmonary Progenitor Cell Biology Consortium that will address these issues.