Objective: Pancreatic ductal cells are a potential source for progenitor cells. The differentiation of ductal cells into islet-like cells has been investigated, however, the efficiency of this process is low. Islet neogenesis and regeneration has been observed during pregnancy and Prolactin (PRL) is one of the involved hormones. As activation of PRL-PRLR signaling pathway is known to contribute to islet regeneration, we herein investigated whether PRL could induce the differentiation of pancreatic progenitor cells to islet-like cells in vitro.
Methods: Primary mouse and human pancreatic ductal cells were obtained by CK-19 labelling and MACS sorting of digested pancreatic tissue, and selected cells were cultured with recombinant mouse prolactin (50 ng/mL) and recombinant human prolactin (100 ng/mL), respectively. Subsequently, the cell clusters formed by the pancreatic ductal cells were stained by anti-insulin, anti-GCK, anti-GLUT2, anti-PC1/3 Abs. In vitro, glucose stimulated insulin secretion (GSIS) was quantified. Prolactin related signaling pathway, glucose transportation and insulin secretion related proteins were assessed by immunoblotting. Lastly, the in vitro formed islet-like cells were transplanted under the kidney capsule of diabetic mice. To assess the role for blood glucose regulation of transplanted islet-like cells, blood glucose, c-peptide and intravenous glucose tolerance tests were performed and immunofluorescence staining for insulin and glucagon was performed on the grafts.
Results: At day 7, the pancreatic ductal cells, also called progenitor cells, expressed CK-19; at day 14, the ductal cells were aggregated and formed sphere-shaped cell clusters. At day 21, the cell clusters stained positive for insulin, GCK, GLUT2 and PC1/3. By in vitro GSIS test, we measured a 2.58±0.32-fold increase of insulin secretion in response to a 16.7 mM glucose stimulation. Immunoblotting demonstrated that PDX-1, MafA, Ptf1a, Sox9, HNC6, Nkx6.1, Ngn3, NeuroD, Pax6, CK-19 were expressed. At day 21, the cell clusters expressed PDX-1, Nkx6.1, Nkx2.2, NeuroD, Pax6, insulin and glucagon. In vivo studies showed that diabetic mice transplanted with islet-like cells, maintained blood glucose levels below 300 mg/dL with c-peptide detected in the serum. Further tissue staining indicated that the transplanted islet-like cells contained both β-cells and α-cells.
Conclusion: Out study shows that in vitro stimulation by prolactin might differentiate pancreatic ductal cells into islet-like cells and might present a new source for insulin-producing cells.