Abstract 3228: Development of a Novel Two-dimensional Directed Differentiation System for Generation of Cardiomyocytes From Human ES Cells and iPS Cells
Background: Human iPS (hiPS) cells hold great promise for treating ischemic heart disease. We have successfully generated hiPS cells from peripheral blood of coronary artery disease (CAD) patients (hiPS-ECP1). Despite studies on the generation of cardiomyocytes (CMs) from human ES (hES) cells or hiPS cells with three dimensional (3D) or 2D methods, it was either low or unclear with regard to the rate or yield of generation of contracting-CM phenotypes. Accordingly in this study, we sought to develop a novel 2D directed differentiation system that enables to highly enrich contracting CMs from hES cells and hiPS cells and to determine their characteristics.
Methods and Results: To induce differentiation into CMs from undifferentiated hES cells (H1) or hiPS cells (hiPS-ECP1) without EB formation, these cells cultured on feeder cells were directly transferred onto Matrigel-coated plates and cultured under conditioned media for one day, followed by incubation for several days under bFGF-removed hES cell media and subsequently cultured in DMEM supplemented with 20% FBS. Around 13–15 days of culture, contracting clusters began to appear which increased in numbers reaching ~90% of initially seeded hES or hiPS colonies over 7 days. We next manually isolated contracting clusters using micro-dissection and characterized them. These cells express β-MHC (>95 %), cardiac troponin T (>90%), Nkx2.5 (>95%), GATA-4 (>95%) and MEF2c (>95%), suggesting differentiated or organized CMs. There were no significant differences in the yield of CMs between hES cells and patient-derived hiPS cells. Electrophysiologic studies demonstrated that the isolated CMs showed each of the 3 major action potentials types (nodal, atrial and ventricular). Of note both hES and hiPS cells show a highly enriched ventricular-CM phenotype (61% and 78%).
Conclusions: Here, we developed a 2D system to differentiate hES- and hiPS cells into mature CMs at high yield. Taken together, our system will provide a novel and efficient option to generate CMs from hES- or hiPS cells, and will enable the use of patient-derived hiPS cells for autologous cardiac regeneration therapy.