Figure 3. Adherent culture-induced β cell de-differentiation is reversible.
(A) RCU reporter mice are made by crossing mice homozygous for the Insulin2-Cre transgene with mice doubly-homozygous for Rosa26-lox-stop-lox-H2BmCherry and Ucn3-GFP. Insulin expression in RCU progeny is permanently marked by red nuclear fluorescence, and Ucn3 expression is marked by green cytoplasmic fluorescence. (B) Pancreas sections of PBS-treated control and S961-treated diabetic RCU mice. Ucn3-GFP is reduced in diabetic mice, but not in controls, and Ucn3 expression returns after remission from diabetes. All images show live (unstained) reporter fluorescence. (C) De-differentiation and re-differentiation of RCU islets cultured in vitro. Islets from adult RCU mice were isolated and plated on 804G matrix for 1 week (left and middle panel). Note islet spreading and loss of Ucn3-GFP in the de-differentiated islets (middle panel). After 7 days, the de-differentiated islets were transplanted into euglycemic SCID mice for 3 weeks (right panel) after which time the transplants show the return of Ucn3 expression in β cells.
Figure 3—figure supplement 1. RCU mice show nuclear insulin expression-coupled mCherry and Ucn3-derived cytoplasmic GFP.
Figure 3—figure supplement 2. Ucn3 and insulin expression are down regulated in islets grown in adherent culture.
Figure 3—figure supplement 3. β cells lose glucose-stimulated insulin secretion upon de-differentiation in culture.
