Retinoic acid promotes the generation of pancreatic endocrine progenitor cells and their further differentiation into beta-cells

Abstract

The identification of secreted factors that can selectively stimulate the generation of insulin producing beta-cells from stem and/or progenitor cells represent a significant step in the development of stem cell-based beta-cell replacement therapy. By elucidating the molecular mechanisms that regulate the generation of beta-cells during normal pancreatic development such putative factors may be identified. In the mouse, beta-cells increase markedly in numbers from embryonic day (e) 14.5 and onwards, but the extra-cellular signal(s) that promotes the selective generation of beta-cells at these stages remains to be identified. Here we show that the retinoic acid (RA) synthesizing enzyme Raldh1 is expressed in developing mouse and human pancreas at stages when beta-cells are generated. We also provide evidence that RA induces the generation of Ngn3(+) endocrine progenitor cells and stimulates their further differentiation into beta-cells by activating a program of cell differentiation that recapitulates the normal temporal program of beta-cell differentiation.

Figure 1. Raldh1 expression in mouse and human embryonic pancreas.

(A) In situ hybridization of an e13.5, e14.5 and e15.5 mouse pancreas using a Raldh1 probe. (B) qRT-PCR using cDNA prepared from human fetal pancreas (n = 3 for week 8, 9, 10, 11, 13, 14 and 18, n = 2 for week 12, 15, 17 and 21, n = 1 for week 19 and 20) with RALDH1 and INSULIN specific primers. (C) In situ hybridizations of week 12, week 14 and week 17 human fetal pancreas using a RALDH1 probe. (D) The stomach-duodenal region from a wild-type and an Ipf1/RAR403 transgenic neonatal mouse.

Figure 2. RA stimulates β-cell differentiation in vitro.

(A) Representative sections of control and RA-treated e10.5 dorsal pancreatic explants cultured for 6 days and analyzed for insulin (green) and glucagon (red) expression. (B) Quantification of insulin and glucagon positive cells in control pancreatic explants and pancreatic explants exposed to RA for 6 days (n = 6 for both) displayed as % positive cells/total number of cells. (C) Representative sections of control and retinol-treated e10.5 pancreatic explants cultured for 6 days and stained with antibodies against insulin (green). (D) Quantification of insulin expression in control and retinol-treated pancreatic buds (n = 2 for both). Scale bar = 40 µM in (A) and 30 µM in (C). Data show mean±S.E.M, *p<0.05.

Figure 3. RA induces Ngn3 expression in vitro.

(A) Representative sections of a control and RA-treated e10.5 dorsal pancreatic explants cultured for 2 days and stained with antibodies specific for Ngn3. (B) Quantification of Ngn3⁺ cells in control and RA-treated explants cultured for 2 or 4 days (n = 4 for each condition) displayed as % Ngn3 expressing cells/total number of cells. (C) qRT-PCR using Bhlhb2, Hes1 and NeuroD specific primers on cDNA from control and RA treated e10.5 pancreatic explants cultured for 2 (n = 13) or 4 days (n = 9). Data represent the mean value±S.E.M. *p<0.05, **p<0.01, for RA treated explants versus controls.

Figure 4. Temporal changes in the expression of Bhlh and homeobox genes in response to RA.

(A) Quantification of Ins⁺ cells in control pancreatic explants (n =  6) and pancreatic explants exposed to 25 nM RA for the first 2 (n = 3) or 4 days (n = 4) of the 6 day in vitro culture period displayed as % positive cells/total number of cells. Data show mean±S.E.M, *p<0.05 **p<0.01. (B) qRT-PCR using Pax4 specific primers on cDNA from control and RA treated e10.5 pancreatic explants cultured for two (n = 13) or four days (n = 9). Data represent the mean value±S.E.M. *p<0.05, **p<0.01. (C) 6d RA treated explants double-stained for insulin (green) and Ipf1/Pdx1, Nkx6.1, Hb9 or C-peptide (all red). (D) Schematic model summarizing the multiple proposed roles of RA during pancreatic development. Our data provides evidence that RA sequentially specifies: i) proendocrine cells; ii) committed pre-β-cells; and finally iii) differentiated β-cells. Scale bar = 30 µM in (C) (7,5 µM for the enlarged inserts).