Immunosuppressive Agents Modulate Function, Growth, and Survival of Cardiomyocytes and Endothelial Cells Derived from Human Embryonic Stem Cells

Abstract

Cardiac cell replacement therapy by using human embryonic stem cell (hESC) derivatives remains a potential approach to regenerate myocardium. The major hurdles to clinical application of this technology are immunogenicity and post-transplantation cell death. Here we examined the effects of calcineurin-targeting immunosuppressants cyclosporine A (CsA) and FK506, as well as rapamycin and a selective inhibitor of calcineurin-binding downstream nuclear factor of activated T-cell (NFAT) transcription factor VIVIT on the proliferative activity, function, and survival of hESC-derived cardiomyocytes (hESC-CM) and endothelial cells (hESC-EC) in culture. As shown by automated microscopy, treatments with CsA, FK506, and rapamycin all decreased proliferation, reducing the percentage of hESC-CM and hESC-EC with the mitotic marker Ki67(+) by as much as 60% and 74%, respectively. Administration of the cell permeable analogue 11R-VIVIT protein did not modulate their proliferative activity. All immunosuppressants reversed the proapoptotic effect of chelerythrine in hESC-CM demonstrating an inhibitory role of calcineurin/NFAT and mammalian target of rapamycin (mTOR) pathways in hESC-CM survival (using apoptotic marker caspase-3), whereas the protection was less obvious in hESC-EC exposed to H2O2. Immunosuppressants did not affect cell viability in hESC-EC. Our results show that immunosuppressants reduce proliferation, while offsetting cell loss to a smaller extent by reduction in apoptosis of hESC-CM. Immunosuppressant therapy would be compatible with stem cell transplantation, but the resulting reduction in graft expansion capabilities would potentially necessitate implantation of increased cell numbers when immunosuppressants are given. The effects of NFAT-binding immunosuppressant molecules, which do not affect hESC-CM proliferation, may point the way forward for new classes of compounds better suited to cell implantation.