Redifferentiation of expanded human pancreatic β-cell-derived cells by inhibition of the NOTCH pathway

In vitro expansion of β-cells from adult human pancreatic islets would overcome donor β-cell shortage for cell replacement therapy for diabetes. Using a β-cell-specific labeling system we have shown that β-cell expansion is accompanied by dedifferentiation resembling epithelial-mesenchymal transition and loss of insulin expression. Epigenetic analyses indicate that key β-cell genes maintain open chromatin structure in expanded β-cell-derived (BCD) cells, although they are not transcribed. In the developing pancreas important cell-fate decisions are regulated by NOTCH receptors, which signal through the Hairy and Enhancer of Split 1 (HES1) transcription regulator.Wehave reported that BCD cell dedifferentiation and proliferation in vitro correlate with reactivation of the NOTCH pathway. Inhibition of HES1 expression using shRNA during culture initiation results in reduced β-cell replication and dedifferentiation, suggesting that HES1 inhibition may also affect BCD cell redifferentiation following expansion. Here, we used HES1 shRNA to down-regulate HES1 expression in expanded human BCD cells, showing that HES1 inhibition is sufficient to induce BCD cell redifferentiation, as manifested by a significant increase in insulin expression. Combined treatment with HES1 shRNA, cell aggregation in serum-free medium, and a mixture of soluble factors further stimulated the redifferentiation of BCD cells. In vivo analyses demonstrated the ability of the redifferentiated cells to replace β-cell function in hyperglycemic immunodeficient mice. These findings demonstrate the redifferentiation potential of ex vivo expanded BCD cells and the reproducible differentiating effect of HES1 inhibition in these cells.