biostudies-literatureUnknown55(3)Micallef SJ<h4>Aims/hypothesis</h4>We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS⁺) cells derived in vitro.<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusions/interpretation</h4>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.Diabetologia694-706https://www.ebi.ac.uk/biostudies/studies/S-EPMC3268987biostudies-literatureINS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells.PMC3268987Micallef SJLim SMNostro MCHirst CEHarrison LCElliott DAYu QCElefanty AGLi XStanley EGKeller GSarangi FSchiesser JVfalseINS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells.<h4>Aims/hypothesis</h4>We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS⁺) cells derived in vitro.<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusions/interpretation</h4>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.2012-01-01T00:00:00Z2012 Mar2024-12-04T03:08:00.503Z2019-03-27T00:48:36ZS-EPMC32689872212051210.1007/s00125-011-2379-y