Figure 1

Pdx1 promotes ES cell-differentiation into IPCs. (a) Schematic representation of the ES cell differentiation protocols. (b) ES cells were subjected to differentiation using a multi-step differentiation protocol. The Pdx1 expressing R1Pdx1AcGFP/RIP-Luc ES cells (20X magnification) were subjected to EB formation followed by differentiation using four different protocols. Immunostaining of ES cell-derived IPCs for pancreatic markers: The Pdx1 expressing ES cell-derived differentiating cells and IPCs using protocol D were characterized by confocal microscope (60X magnification) following immunostaining using Foxa2, Sox17, Ngn3, NeuroD, insulin, somatostatin, glucagon and C-peptide. The results indicate that the differentiating cells express Foxa2, Sox17, Ngn3, and NeuroD which are critical for the development of IPCs. The differentiation leads to the generation of IPCs and glucagon expressing isolated cell clusters. As compared to the IPCs there is an abundance of glucagon expressing cells. (c) The in vitro differentiation of R1Pdx1AcGFP/RIP Luc ES cells into IPCs was monitored by real-time quantitative RT-PCR at various stages of differentiation. As a positive control, 18S rRNA was used to normalize and validate the results. The data indicate progressive up-regulation of Foxa2, Isl1, Pax4, glucagon, insulin 1, insulin 2 and PC2/3 genes following the differentiation of ES cells into IPCs.