INS-GFP hESCs facilitate isolation of in vitro derived INSULIN expressing cells

Differentiation of INSGFP/w hESCs using published protocols demonstrated that all GFP+ cells co-expressed insulin, confirming the fidelity of the reporter gene. INS-GFP+ cells also co-expressed glucagon and somatostatin, confirming prior studies regarding the polyhormonal nature of early hESC derived insulin-expressing cells. INSGFP/w hESCs were employed to develop a 96 well format spin Embryoid Body (EB) differentiation protocol that utilized the recombinant protein based fully defined medium, APEL. Like INS-GFP+ cells generated with other methods, those derived using the spin EB protocol expressed a collection of pancreatic related transcription factors including ISL1, PAX6 and NKX2.2. However, in contrast to previous methods, the spin EB protocol yielded INS-GFP+ cells that also co-expressed the beta-cell transcription factor, NKX6.1 and comprised a substantial proportion of monohormonal insulin+ cells.