S. M. Kunisaki1, G. Jiang1, J. Di Bernardo1, T. J. Herron1, K. S. O’Shea1 1University Of Michigan,Ann Arbor, MI, USA
Introduction: Hypoplastic left heart syndrome (HLHS) is one of the most devastating congenital heart anomalies in children. The local delivery of replacement cells has been investigated as a promising adjunctive therapy aimed at enhancing cardiac function in these infants. The purpose of this study was to generate transgene-free cardiomyocytes derived from HLHS neonates as a prelude to subsequent autologous applications.
Methods: With IRB approval, neonatal dermal fibroblasts were harvested from HLHS (n=1) and control (n=2) foreskin samples prior to cellular reprogramming into induced pluripotent stem cells (iPSCs) using non-integrating Sendai RNA viral vectors expressing OCT4, SOX2, cMYC, and KLF4. The resultant iPSCs were then differentiated into cardiomyocytes by a modified Matrigel sandwich technique and characterized in multiple assays, including confocal immunofluorescence, quantitative gene expression, and electrophysiological testing.
Results: iPSCs were successfully generated from both HLHS and control dermal fibroblasts based on morphological characteristics and alkaline phosphatase staining. The pluripotency of HLHS iPSCs was confirmed by the expression of NANOG, OCT4, SOX2, SSEA3, TRA-1-81, and TERT, and by three germ layer lineage differentiation of embryoid bodies. Two weeks after induced differentiation, beating cells were identified and expressed MYH7, MF20, actinin, and MLC2v after six weeks in culture. Optical mapping of calcium transients and transmembrane voltage studies were consistent with that of cardiomyocytes (Graphic). There were no major differences noted between cardiomyocytes derived from HLHS and controls.
Conclusion: Beating human cardiomyocytes can be generated in a minimally invasive fashion in HLHS. This study supports the feasibility of autologous transgene-free cardiomyocyte-based therapies in the management of severe congenital heart disease.
