HES1 Is involved in adaptation of adult human beta cells to proliferation in vitro

In vitro expansion of insulin-producing beta cells from adult human islets could solve the tissue shortage for cell-replacement therapy of diabetes. Propagation in culture of adult human islets from cadaveric donors results in 16 cell doublings and loss of insulin expression. Using cell-lineage tracing we recently demonstrated that the expanded cell population included cells derived from beta cells, which underwent dedifferentiation and were induced to replicate. Understanding the molecular mechanisms involved 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. However, this pathway is normally inactive in adult islets. Here we report that beta-cell dedifferentiation and entrance into the cell cycle in vitro involve activation of the NOTCH pathway, including a 5-fold upregulation in HES1 transcripts and a 4-10-fold upregulation in NOTCH1-3 transcripts. These changes correlate with a 3-fold downregulation of transcripts for the cell cycle inhibitor p57 and loss of insulin expression. HES1 upregulation in the cells was prevented upon addition of a gamma-secretase inhibitor, which prevents NOTCH cleavage, thus showing a NOTCH-dependent mechanism. Using cell-linage tracing, we showed that HES1 and the active nuclear form of NOTCH1 are not present in differentiated beta cells, however they are expressed in dedifferentiated beta cells that lost insulin and p57 expression. Inhibition of HES1 expression using small hairpin RNA upon culture initiation, completely prevented p57 downregulation and significantly reduced beta-cell replication. In addition, HES1 inhibition significantly reduced beta-cell dedifferentiation, as manifested by a higher level of insulin and beta-cell-specific transcription factor transcripts, a higher fraction of cells immunostained for insulin, higher insulin content, and higher insulin secretion in response to glucose. These findings suggest possible molecular targets for prevention of beta-cell dedifferentiation in culture, and induction of cell redifferentiation following in vitro expansion. (Diabetes, 2008 Jul 3).