Abstract 5594: Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences Undergo Cardiogenesis in Defined Conditions

Abstract

Human induced pluripotent stem (iPS) cells hold great promise for cardiovascular research and therapies, but the lentiviral generated iPS cells contain transgene and vector sequences inserted into the genome that could result in insertional mutagenesis. The recent generation of iPS cells using oriP/EBNA1(Epstein-Barr nuclear antigen-1)-based episomal vectors avoids genomic integrations and brings iPS cells closer to clinical applications. However, the ability of vector-free iPS cells to generate cardiomyocytes (CMs) is unknown. The aim of this study was to test the ability of vector-free iPS cells to undergo cardiac differentiation in embryoid bodies (EBs) as well using directed differentiation in defined medium. Four iPS clones derived from foreskin fibroblasts (iPS-DF6 –9 –9T, iPS-DF6 –9 –12T, iPS-DF19 –9 –7T, iPS-DF19 –9 –11T) were maintained either on mouse embryonic fibroblasts for EB formation, or in feeder-free TeSR medium for directed differentiation. EBs formed contracting outgrowths with variable efficiency among 4 clones (6.1±3.0%, 10.4±3.0%, 0.4±0.2% and 1.6±0.6%, respectively). Likewise, directed differentiation using sequential treatment with Activin A (100 ng/ml) and BMP4 (10 ng/ml) resulted in the formation of contracting CMs, but flow cytometry for cTnT-positive cells demonstrated 10 –15 fold more CMs were obtained using directed differentiation than with the EB method. RT-PCR comparing iPS cell-derived CMs with undifferentiated iPS cells demonstrated strong upregulation of cardiac genes (NKX2–5, TNNT2, MYH6, ACTN2, HPPA, PLN, MYL2 and MYL7) and concomitant downregulation of pluripotency genes (OCT4 and NANOG). Immunolabeling of cTnT, alpha-actinin, MLC2a and MLC2v showed organized sarcomeres in single iPS cell-derived CMs. Sharp microelectrode recordings revealed spontaneous action potentials comparable to those observed in embryonic stem cell-derived CMs (n = 34 cells of 5 microdissected EB outgrowths). Our findings demonstrate that vector-free iPS cells readily undergo cardiogenesis in EBs and that directed differentiation under defined conditions greatly increases cardiogenesis efficiency. Thus, vector-free iPS cells have growing appeal for cardiovascular research and cardiac repair applications.