INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells

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 INS (GFP/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. INS (GFP/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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00125-011-2379-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users.