Figure 1.

Pancreatic differentiation and PAX4 overexpression in hESCs. (A) Pancreatic endocrine differentiation according to Gage et al.⁴ modified from Rezania et al.³ Stage-specific sequential development of hESCs from undifferentiated cells through foregut-derived endodermal progenitors to hormone-positive endocrine cells. (B) Adenoviral infection of 11 d differentiated hESCs yields eGFP expression (green) by 24 h post-viral delivery which persists through the culture period. (C) 21-d differentiated pancreatic endocrine cultures were immunostained for eGFP (green) to mark expression of the adenoviral vector and synaptophysin (red) to mark the endocrine cell population as a portion of the total cells (DAPI, white). (D) Quantification of the total number of eGFP-positive cells as a percentage of the total number of nuclei. (E) Infection of day 11 cells with an adenoviral human PAX4 expression construct resulted in a dose-dependent increase in PAX4 transcript levels as measured by RT-qPCR of day 21 samples relative to expression in adult human islets. (F) PAX4 delivery was also associated with nuclear immunoreactivity (PAX4, green), in contrast to rare cytoplasmic PAX4 immunoreactivity seen in uninfected and control infected day 21 cultures (nuclei, blue). * Indicates significant overexpression of human PAX4 compared with control virus (Ad eGFP) at the same dose (P < 0.05 by 1-way ANOVA with Bonferroni post-hoc test). Scale bar is 200 μm in (B), 100 μm in (C) and 25 μm in (F).