Abstract 20724: Matrix-Promoted Efficient Cardiac Differentiation of Human iPS and ES Cells
Efficient and reproducible approaches for differentiating human iPS and ES cells to cardiomyocytes (CMs) are needed for research and potential clinical applications. The purpose of this study was to test the ability of matrix in combination with growth factors to promote the initial epithelial-mesenchymal transition (EMT) of the epiblast-like iPS and ES cells leading to the mesoderm formation and cardiogenesis. Vector-free and lenti-viral generated iPSCs (DF19-9-11T, DF6-9-9T and IMR90 C4), and ESCs (H1 and H9) were seeded on matrigel coated plates in mTeSR1 media and grown as a monolayer, followed by matrigel overlay and sequential treatment with Activin A, BMP4 and bFGF. We tested the application of matrigel overlays at various times during the differentiation and found that the combination of matrigel overlays after 3 days of monolayer culture and with Activin A addition (5–6 days) dramatically increased the EMT compared to no overlays as evidenced by electron micrographs showing the transformation of monolayer polarized epithelial-like cells to multilayer mesonchymal-like cells. Furthermore, the matrigel overlays significantly changed the concentration-dependent response to Activin A induction of T and SOX17 expression. RT-PCR analysis demonstrated the sequential upregulation of mesendoderm markers (T, MIXL1 and MESP1), followed by cardiac transcription factors (GATA4, ISL1 and NKX2-5) and cardiac myofilament proteins (TNNT2, MYL2, MYL7). By day 8 confluent contracting cells were observed and large CMs networks formed demonstrated by cTnT and MLC2a immunolabeling. Flow cytometry revealed that a high percentage of the cells present after 15 days were cTnT+ CMs (91±3%, 77±3%, 50±6%, 45±4% and 71±7% for DF19-9-11T, DF6-9-9T, IMR90 C4, H1 and H9, respectively). The yield from one starting ES/iPS cell ranged from 4–7 CMs. Sharp microelectrode recordings demonstrated spontaneous action potentials of similar properties for the iPS and ES cell-derived CMs as previously observed. Our results demonstrate that matrix facilitated EMT and growth factor delivery promote mesoderm formation and enable efficient and reproducible cardiac differentiation among different iPS and ES cell lines.
- © 2010 by American Heart Association, Inc.