Pancreatic Differentiation of ARX ko hESCs.

(A) Schematic representation of the in vitro pancreatic endocrine differentiation method as previously described by Bruin et al. (2014). (B) Flow cytometry of wild type (WT), ARX knockout (ARX ko) clone 1 (C1), and ARX ko clone 2 (C2) at the end of definitive endoderm (CXCR4), foregut (PDX1), and pancreatic progenitor (NKX6.1) stages. (C-H) Media composition and cellular content analysis of differentiating wild type and ARX ko hESCs. Glucagon, C-peptide and somatostatin were assayed from un-stimulated media samples collected from days 11 to 26 (C, E, G; left column); a static sequential glucose stimulated hormone release assay was performed on day 24 of culture which included 1 hour treatments of low glucose (2 mM), high glucose (25 mM), and potassium chloride (KCl, 30 mM) (C, E, G; right column). (D, F, H) total hormone content measurements of day 26 WT and ARX ko cell lysate samples normalized for total protein content. (N = 4 independent differentiation trials for all data sets). * indicates p < 0.05 wild type vs ARX ko C1 and C2 at indicated time point or treatment based on a one-way ANOVA and Bonferroni post hoc test.