Endocrine cells develop within pancreatic bud-like structures derived from mouse ES cells differentiated in response to BMP4 and retinoic acid

We have examined factors affecting the in vitro differentiation of Pdx1GFP/w ESCs to pancreatic endocrine cells. Inclusion of Bone Morphogenetic Protein 4 (BMP4) during the first four days of differentiation followed by a 24-hour pulse of retinoic acid (RA) induced the formation of GFP+ embryoid bodies (EBs). GFP expression was restricted to E-cadherin+ tubes and GFP bright (GFPbr) buds, reminiscent of GFP+ early foregut endoderm and GFPbr pancreatic buds observed in Pdx1GFP/w embryos. These organoid structures developed without further addition of exogenous factors between days 5 and 12, suggesting that day 5 EBs contained a template for the subsequent phase of development. EBs treated with nicotinamide after day 12 of differentiation expressed markers of endocrine and exocrine differentiation, but only in cells within the GFPbr buds. Analysis of Pdx1GFP/w ESCs modified by targeting a dsRed1 gene to the Ins1 locus (Pdx1GFP/wIns1RFP/w ESCs) provided corroborating evidence that insulin positive cells arose from GFPbr buds, mirroring the temporal relationship between pancreatic bud development and the formation of endocrine cells in the developing embryo. The readily detectable co-expression of GFP and RFP in grafts derived from transplanted EBs demonstrated the utility of Pdx1GFP/wIns1RFP/w ESCs for investigating pancreatic differentiation in vitro and in vivo.