UCLA

Human pluripotent stem cells (hPSCs) can be derived from the inner cell mass of a developing blastocyst or through transcription factor mediated reprogramming. hPSCs can be maintained indefinitely in culture and have the capacity to differentiate into any cell type found in the body, making them powerful tools for studying development, modeling diseases, and potential regenerative medicine therapies. Before we can utilize the full potential of these cells, there are many unanswered questions remaining regarding their basic growth and survival. While hPSCs are regularly grown as compact colonies and passaged as small clumps, when dissociated to single cells they survive poorly and undergo apoptosis rapidly after dissociation. This poor survival as single cells impairs the use of hPSCs in common techniques that require the isolation of specific clonal populations, including genetic manipulation. Towards this end, we have taken several approaches, including candidate targeting and large scale screening, to identify modifiers of survival in hPSCs. One such small molecule that significantly improved the survival of dissociated hPSCs was the Rho kinase (ROCK) inhibitor HA-1077. Using shRNA we confirmed that inhibiting ROCK activity in hPSCs improves their survival by inhibiting dissociation-induced apoptosis. We also explored the role of cell-cell junctions between hPSCs in regulating survival. We found that blocking the activity of E-cadherin in dissociated cells reduces their survival and plating efficiency, even when treated with a ROCK inhibitor. Furthermore, we identified multiple junction proteins that are disrupted after dissociation of hPSC colonies, representing novel targets with potential to regulate survival of hPSCs. Finally, we developed improved high throughput assays for screening large libraries of small molecules to search for novel regulators of hPSC survival. We identified several novel small molecules, and validated that most function through inhibition of ROCK, suggesting that ROCK activity is a key modulator of survival in hPSCs. The data reported here represent a comprehensive evaluation of how survival is regulated in hPSCs. Further this work is one of the first to generate and compare large scale screening platforms for hPSC survival. With minor modifications, these screening platforms can be adapted to examine other aspects of hPSC fate such as self-renewal and differentiation. Improving our ability to grow, maintain, and manipulate hPSCs will enhance their use as a model for human disease and development and a source of future cell-based regenerative therapies.