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Insulin-Expressing Colonies Developed From Murine Embryonic Stem Cell–Derived Progenitors

¹ Departments of Gene and Cell Medicine and Surgery, Mount Sinai School of Medicine, New York, New York
² Institute for Diabetes, Obesity, and Metabolism and Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
³ Recanati/Miller Transplantation Institute, Mount Sinai School of Medicine, New York, New York

Address correspondence and reprint requests to H. Teresa Ku, Box 1496, Mount Sinai School of Medicine, New York, NY 10029-6574. E-mail: hsun.ku{at}mssm.edu

Abbreviations: bHLH, basic helix-loop-helix; CM, conditioned media; EGFP, enhanced green fluorescent protein; E, embryonic day; ICFU, insulin-expressing colony-forming unit; NEA, nicotinamide, exendin-4, and activin ßB; Ngn3, neurogenin 3; Pdx-1, pancreatic duodenal homeobox–1; VEGF, vascular endothelial growth factor

Previous studies describe a unique culture method for the commitment of murine embryonic stem cells to early endocrine pancreata. In this report, early pancreatic-like ß-cell progenitors were enriched and a colony assay devised to allow these progenitors to differentiate into insulin-expressing colonies in vitro. An embryonic stem cell line with enhanced green fluorescent protein (EGFP) inserted into one allele of neurogenin 3 (Ngn3), a marker for pancreatic endocrine progenitors, was differentiated. During the late stage of culture, 20–30% of cells were Ngn3-EGFP⁺. Gene expression profiling using the PancChip microarray platform demonstrated that Ngn3-EGFP⁺ cells differentially express endocrine-related genes. A novel semisolid culture method was developed to support the formation of individual insulin/C-peptide–expressing colonies from dissociated single cells. Approximately 0.1–0.6% of Ngn3-EGFP⁺ cells gave rise to insulin-expressing colonies, a three- to fivefold enrichment of ß-cell–like progenitors, or insulin-expressing colony-forming units (ICFUs), compared with nonsorted cells. All of the single colonies expressed insulin II, while 69% coexpressed insulin I and 44% coexpressed glucagon. Some single colonies expressed insulin I, insulin II, and Pdx-1 (pancreatic duodenal homeobox–1), but not glucagon. In other colonies, glucagon expression overlapped with C-peptide II in double immunostaining analysis, suggesting heterogeneity among the ICFUs and their resulting colonies. Together, these results demonstrate that progenitors that have the potential to give rise to insulin-expressing cells can be derived from murine embryonic stem cells.

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