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Understanding and manipulating pancreatic ␤-cell proliferation is a major challenge for pancreas biology and diabetes therapy. Recent studies have raised the possibility that human ␤-cells can undergo dedifferentiation and give rise to highly proliferative mesenchymal cells, which retain the potential to redifferentiate into ␤-cells. To directly test whether cultured ␤-cells dedifferentiate, we applied genetic lineage tracing in mice. Differentiated ␤-cells were heritably labeled using the Cre-lox system, and their fate in culture was followed. We provide evidence that mouse ␤-cells can undergo dedifferentiation in vitro into an insulin-, pdx1-, and glut2-negative state. However, dedifferentiated ␤-cells only rarely proliferate under standard culture conditions and are eventually eliminated from cultures. Thus, the predominant mesenchymal cells seen in cultures of mouse islets are not of a ␤-cell origin. Diabetes

OBJECTIVE-In vitro expansion of ␤-cells from adult human islets could solve the tissue shortage for cell replacement therapy of diabetes. Culture of human islet cells typically results in Ͻ16 cell doublings and loss of insulin expression. Using cell lineage tracing, we demonstrated that the expanded cell population included cells derived from ␤-cells. Understanding the molecular mechanisms involved in ␤-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 ␤-cell dedifferentiation and proliferation in vitro. RESEARCH DESIGN AND METHODS-Isolated human isletswere dissociated into single cells. ␤-Cells were genetically labeled using a Cre-lox system delivered by lentiviruses. Cells were analyzed for changes in expression of components of the NOTCH pathway during the initial weeks in culture. HES-1 expression was inhibited by a small hairpin RNA (shRNA), and the effects on ␤-cell phenotype were analyzed.RESULTS-Human ␤-cell dedifferentiation and entrance into the cell cycle in vitro correlated with activation of the NOTCH pathway and downregulation of the cell cycle inhibitor p57. Inhibition of HES-1 expression using shRNA resulted in significantly reduced ␤-cell replication and dedifferentiation.CONCLUSIONS-These findings demonstrate that the NOTCH pathway is involved in determining ␤-cell fate in vitro and suggest possible molecular targets for induction of ␤-cell redifferentiation following in vitro expansion. Diabetes