Changes in Growth Factor Responsiveness during In Vitro Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes

Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) offer a promising source of non-proliferative CMs that can be used to identify conditions that stimulate heart regeneration. Realizing this potential requires efficient methods to generate matured hPSC-CMs that do not readily respond to exogenous cell cycle stimulation. Therefore, the goal of this project was to mature hPSC-CMs and test their cell cycle response in growth factor screens. Alignment on nano-grooved substrates was the first method investigated to mature hPSC-CMs. While aligned tissues formed and established functional syncytia, strong contractions resulted in tissue detachment from the substrate. Next, 3D aggregate suspension was explored as an approach to mature hPSC-CMs. Following 4 weeks in suspension, hPSC-CMs displayed a more mature morphology, gene expression profile, and electrophysiology. Importantly, matured hPSC-CMs were unresponsive to growth factor stimulation for cell cycle induction. Following stimulation of aggregates with bFGF and HRG for 24h, immature hPSC-CMs initiated DNA synthesis, while matured hPSC-CMs did not. Interestingly, both immature and matured hPSC-CMs exhibited ERK activation in response to stimulation. A significant increase in expression of cyclin dependent kinase (CDK) inhibitors, with a corresponding significant decrease in expression of most cyclins and CDKs, is a likely explanation for this result. To test the effect of 40 additional growth factors on cell cycle response, a screening platform was developed. The screen revealed overlapping and distinct hits in immature and matured hPSC-CMs. Transferring screening results to 3D, however, resulted in no effect in matured CMs, which was found to likely be the result of dedifferentiation in 2D, highlighting the need to perform cell cycle stimulation assays in a 3D environment that supports a mature CM state. Overall, these results provide insights into the development of adequate model CMs and experimental assays for discovering treatments that induce CM cell cycle re-entry for regenerative purposes.Ph.D