Regulation of pancreatic islet cell fate

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Project Summary/Abstract Significant research efforts are currently underway to understand and prevent the pathogeneses of Type 1 and Type 2 diabetes, both of which are associated with the gradual loss of functional insulin-producing b cells. Progress has been made with using human pluripotent stem cell (hPSC) populations as alternative sources of islet cells; however, the efficient production of pure populations of fully functional b cells has not yet been achieved. Furthermore, the current protocols result in highly variable differentiations depending on the stem cell source, differentiation procedure and user. In addition, both endogenous and stem cell-derived islet cells display inherent phenotypic instability and often undergo spontaneous dedifferentiation and/or reprogramming in pathophysiological conditions, which results in the loss of cellular identity and phenotypic dysfunction. These challenges suggest that there is still a great need to identify the precise transcriptional and epigenetic mechanisms that promote the specification and maintenance of islet cell identities. We and others have demonstrated that development and maintenance of b cell identity requires the continuous activity of several non-redundant transcriptional programs. Although many of the transcription factors that are essential for maintaining a and b cell phenotypes have been characterized, we lack a complete understanding of how cell-specific gene programs are regulated to specify and maintain these closely related cell lineages in mice, and there are varying reports related to their respective roles in human pancreas development. This proposal will allow us to gain a more complete understanding of the basic molecular mechanisms underlying islet cell differentiation and the maintenance of islet cell identity/function; this knowledge will pave the way to improved human islet cell differentiation protocols and the identification of better treatments for islet cell dysfunctions in diabetes. These studies are facilitated by increased access to novel and improved molecular technologies. Furthermore, our studies in rodent models are providing important biological and physiological context that will be directly translated into the hPSC differentiation system. Our first aim will characterize the differential molecular mechanisms that regulate a vs b cell programs in mice. We will focus on the combined molecular activities of NKX2.2, NKX6.1, KLF4 and CHD4 in regulating endocrine cell fate decisions. We will also take an unbiased approach using combined single cell ATAC-Seq and RNA-Seq to characterize the transcriptional landscape associated with islet cell fate decisions in vivo. The second aim will characterize the role of the chromatin modifier protein CHD4 in regulating b cell development and function, and in modulating NKX2.2 and NKX6.1 regulatory activities. The third aim will begin to apply this knowledge to the directed differentiation of human islet cells from hPSCs. We propose to identify the necessary and sufficient functions of NKX2.2 and NKX6.1 in regulating a and b cell fate decisions. We will also use this platform to identify the chromatin and gene expression landscapes regulated by NKX2.2 and NKX6.1 during these lineage choices.