Stepwise Differentiation of CA1S Cells

Ninety percent confluent cultures of CA1S cells were differentiated as previously described in detail [26 (link)] and summarized in Fig 2A. At day 5, 11, and 17 of the culture, developing cells were assessed for expression of CXCR4 (definitive endoderm), PDX1 (foregut endoderm), and NKX6.1 (pancreatic endoderm) by flow cytometry (n = 4 independent differentiation trials) as previously described [26 (link)] using antibodies described in S2 Table. Hormone content of 24-hour static media samples (end of each stage), static sequential glucose/KCl release (day 24; 1 hour each in 2 mM glucose, 25 mM glucose, 30 mM KCl) and total content (day 26) were assayed by radioimmunoassay as previously described [3 (link)] (C-peptide; HCP-20K, glucagon; GL-32K, both Millipore) or enzyme-linked immunoassay (somatostatin; EK-060-03, Phoenix Pharmaceuticals Inc., Burlingame, CA, USA) (n = 3–4 samples for each treatment/timepoint of 4 trials). Total protein samples were prepared by vortexing snap frozen cell pellets in RIPA lysis buffer and total protein content was assessed by BCA assay (Pierce Biotechnology, Rockford, IL, USA).