INSGFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells

Abstract

Aims/hypothesis: We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS+) cells derived in vitro. Methods: Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS+ cells. Results: Differentiation of INSGFP/w hESCs using published protocols demonstrated that all GFP+ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP + cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INSGFP/w hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used 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 suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP+ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS+ cells. Conclusions/interpretation: INS GFP/w hESCs are a valuable tool for investigating the nature of early INS+ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS+ cells from differentiating hESCs. © 2011 The Author(s).