Investigating regulators controlling differentiation potential of ES cells

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SUMMARY Pluripotent stem cells, such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can proliferate indefinitely in vitro without changes in their characteristics (self-renewal) while keeping their potential to give rise to almost all cell types in adult organisms (pluripotency). Due to such exceptional characteristics, ES and iPS cells have been extensively studied and used as tools for understanding the molecular basis of early embryo development and also serve as useful instruments in drug discovery and establishing various disease models. To fully utilize their potential in therapeutic applications, it is crucial to completely understand how these two unique characteristics are modulated. Prior studies have largely focused on understanding of self-renewal, allowing us to better illuminate the regulatory mechanisms mediated by key transcription factors (TFs), signaling pathways, and other associated genomic features. On the other hand, understanding of exit mechanisms from self-renewal towards cell fate specification, and factors involved in proper differentiation of pluripotent stem cells have not yet been systematically examined. The long-term objective of the proposed research is to investigate regulators controlling differentiation potential of pluripotent stem cells. In our previous research supported by NIGMS awards, we have revealed multiple TFs, epigenetic regulators, and genomic features that influence the differentiation potential of ES cells. Among those factors, we showed that Yap1, a transcriptional co- regulator, downstream of the Hippo pathway, is dispensable for self-renewal but required for differentiation of ES cells. We furthermore revealed the roles of Yap1 in safeguarding ES cells from excessive cell death during differentiation. We additionally observed that cell density, tightly linked to the Hippo signaling activity, significantly affects global gene expression programs of not only self-renewing ES cells, but also their differentiation potential. However, underlying mechanisms of ES cell differentiation in the context of cell density and survival vs. death decision have been elusive. To address this critical gap in knowledge, our objectives of the proposal will be 1) to determine, at the single cell level, how the survival vs. death decision is made when ES cells differentiate, 2) to define outcomes of density- dependent gene expression signatures and enhancer usage during ES cell self-renewal and differentiation, and 3) to identify effectors controlling density-dependent gene expression programs and elucidate their regulatory mechanisms. The information obtained from the proposal will provide novel insights into the reproducibility issues in biomedical studies caused by inconsistencies in cell density between different experimental techniques. Furthermore, outcomes of this proposal will provide a foundation for manipulation of stem cells to control cell fates towards desired lineages and contribute to the advances in stem cell-based cell therapies.