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HES1 IS INVOLVED IN ADAPTATION OF ADULT HUMAN BETA CELLS TO PROLIFERATION IN VITRO

Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel

Objective: UU In vitro expansion of beta cells from adult human islets could solve the tissue shortage for cell-replacement therapy of diabetes. Culture of human islet cells typically results in <16 cell doublings and loss of insulin expression. Using cell-lineage tracing we demonstrated that the expanded cell population included cells derived from beta cells. Understanding the molecular mechanisms involved in beta-cell fate in vitro is crucial for optimizing expansion and redifferentiation of these cells. In the developing pancreas important cell-fate decisions are regulated by NOTCH receptors, which signal through the Hairy and Enhancer of Split (HES) 1 transcriptional regulator. Here we investigated the role of the NOTCH signaling pathway in beta-cell dedifferentiation and proliferation in vitro.

Research Design and Methods: Isolated human islets were dissociated into single cells. Beta cells were genetically labeled using a Cre-loxP system delivered by lentiviruses. Cells were analyzed for changes in expression of components of the NOTCH pathway during the initial weeks in culture. HES1 expression was inhibited by a small hairpin RNA, and the effects on beta-cell phenotype were analyzed.

Results: Human beta-cell dedifferentiation and entrance into the cell cycle in vitro correlated with activation of the NOTCH pathway and dowregulation of the cell cycle inhibitor p57. Inhibition of HES1 expression using small hairpin RNA resulted in significantly reduced beta-cell replication and dedifferentiation.

Conclusions: These findings demonstrate that the NOTCH pathway is involved in determining beta-cell fate in vitro and suggest possible molecular targets for induction of beta-cell redifferentiation following in vitro expansion.

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