P510Modulating cell survival of human embryonic stem cell derivatives by immunosuppressive drugs

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Abstract

Cardiac cell replacement therapy by using human embryonic stem cell 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 (CsA; 0.2 µM), and FK506 (10 ng/mL), as well as rapamycin (RAP; 10 ng/mL) on the proliferative activity, function and survival of hESC-derived cardiomyocytes (hESC-CM) and endothelial cells (hESC-EC) in culture. Undifferentiated H7 hESC were treated with a set of growth factors to generate hESC-CM and hESC-EC. As shown by automated microscopy, treatments with CsA, FK506 and RAP all decreased proliferation (percentage of mitotic marker Ki67 + cells, p < 0.001) of single hESC-CM and hESC-EC. Administration of cell permeable 11R-VIVIT protein, a selective inhibitor of calcineurin-binding downstream NFAT transcription factor did not modulate the proliferative activity of hESC-CM or hESC-EC. Thus, NFAT-independent calmodulin/calcineurin pathways may play a role in the proliferative activity of both cell types. All immunosuppressants reversed pro-apoptotic effect of Chelerythrine (10µM) in hESC-CM demonstrating an inhibitory role of calcineurin/NFAT as well as mTOR pathways in survival of hESC-CM (for apoptotic marker caspase-3, p < 0.01), with no effect on cell necrosis. In hESC-EC and in endothelial control HUVEC, CsA, FK506, RAP and VIVIT did not affect cell viability. Our results show that immunosuppressants reduce proliferation with offsetting cell loss to a smaller extent by reduction in apoptosis of hESC-CM. Immunosuppressants would be safe for transplantation but the resulting reduction in graft expansion capabilities would potentially necessitate implantation of increased cell numbers when immunosuppressants are given.

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