The overall goal of this proposal is to define the mechanisms that underlie the formation of endocrine cells in the pancreas and to apply this knowledge to instruct human embryonic stem cells (hESCs) to produce functional insulin-secreting beta cells. During the past funding period, work under this grant has determined that ductal progenitors in the pancreas are the major source of endocrine cells during embryonic development. Mechanistic studies in the PI's laboratory have further shown that the transcription factor Sox9 is necessary to bestow competence upon ductal progenitors to initiate endocrine gene expression programs. Moreover, we have demonstrated that Sox9 expression is under control of the Fgf and Notch signaling pathways, suggesting that progenitors need to be exposed to Fgf and Notch signals for endocrine cell differentiation to be initiated. In preliminary studies presented to support a continuation of these studies, we show that Fgf and Notch signaling are aberrantly regulated in current differentiation protocols of hESCs towards pancreatic endocrine beta cells. We hypothesize that the aberrant Fgf and Notch signaling environment accounts for the malfunction of beta- like cells produced in vitro. In this continuing renewal application, the PI proposes a combination of mouse genetic and hESC-based approaches to (a.) further define the mechanisms by which Fgf and Notch signaling orchestrate endocrine cell development and (b.) apply this knowledge to generate functional endocrine cells from hESCs in vitro. To better understand the specific signaling environment necessary for endocrine cell differentiation, Aim 1 will investigate how the Fgf and Notch signaling pathways coordinately control the specification and differentiation of endocrine cells. To aid these experiments, the PI's laboratory has developed unique genetic mouse models.
In Aim 2, we will employ a novel live imaging technology established in the PI's laboratory to monitor the initiation of endocrine cell differentiation at single cell resolution in real time. Based on evidence in othe tissues and organs, experiments under this aim will explore a possible connection between cell division, Notch activity, and the initiation of cell differentiation.
In Aim 3, we will apply paradgms learned from our mouse genetic experiments to direct hESCs towards the beta cell lineage. Experiments under this Aim will directly test how changes in Fgf and Notch signaling affect the maturity of endocrine cells produced from hESCs in vitro. Preliminary evidence from the PI's laboratory suggests that endocrine cell maturity can be improved by providing a Fgf and Notch signaling environment that more closely resembles the environment during normal development. Together, these experiments will aid the identification of culture conditions that support the differentiation of functional beta cells from hESCs in vitro.

Public Health Relevance

Loss of pancreatic insulin-producing beta cells is the hallmark of type 1 diabetes and advanced cases of type 2 diabetes. Studies supported by this grant have led to the discovery of fundamental mechanisms by which beta cells are generated during development and adulthood. The proposed work leverages key findings made during the previous funding period to appropriately instruct human embryonic stem cells (hESCs) to produce functional beta cells in culture.

National Institute of Health (NIH)
Research Project (R01)
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Cellular Aspects of Diabetes and Obesity Study Section (CADO)
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Sato, Sheryl M
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University of California San Diego
Schools of Medicine
La Jolla
United States
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Shih, Hung Ping; Sander, Maike (2014) Pancreas development ex vivo: culturing embryonic pancreas explants on permeable culture inserts, with fibronectin-coated glass microwells, or embedded in three-dimensional Matrigelâ„¢. Methods Mol Biol 1210:229-37
Xie, Ruiyu; Everett, Logan J; Lim, Hee-Woong et al. (2013) Dynamic chromatin remodeling mediated by polycomb proteins orchestrates pancreatic differentiation of human embryonic stem cells. Cell Stem Cell 12:224-37
Jin, Liang; Feng, Tao; Shih, Hung Ping et al. (2013) Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel. Proc Natl Acad Sci U S A 110:3907-12
Kopp, Janel L; Dubois, Claire L; Schaffer, Ashleigh E et al. (2011) Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas. Development 138:653-65
Dubois, Claire L; Shih, Hung Ping; Seymour, Philip A et al. (2011) Sox9-haploinsufficiency causes glucose intolerance in mice. PLoS One 6:e23131
Seymour, Philip A; Sander, Maike (2011) Historical perspective: beginnings of the beta-cell: current perspectives in beta-cell development. Diabetes 60:364-76
Dorrell, Craig; Erker, Laura; Schug, Jonathan et al. (2011) Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice. Genes Dev 25:1193-203
Carpentier, Rodolphe; Suner, Regina Espanol; van Hul, Noemi et al. (2011) Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells. Gastroenterology 141:1432-8, 1438.e1-4
Kopp, Janel L; Dubois, Claire L; Hao, Ergeng et al. (2011) Progenitor cell domains in the developing and adult pancreas. Cell Cycle 10:1921-7
Schaffer, Ashleigh E; Freude, Kristine K; Nelson, Shelley B et al. (2010) Nkx6 transcription factors and Ptf1a function as antagonistic lineage determinants in multipotent pancreatic progenitors. Dev Cell 18:1022-9

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