title: Insulin-expressing colonies developed from murine embryonic stem cell-derived progenitors.
abstract: 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 beta-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 beta-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.
authors: Department of Gene and Cell Medicine and Surgery, Mount Sinai School of Medicine, New York, NY 10029-6574, USA. hsun.ku@mssm.edu
authors: Hsun Teresa
authors: HT
authors: Ku
authors: Jing
authors: J
authors: Chai
authors: Yoon-Jung
authors: YJ
authors: Kim
authors: Peter
authors: P
authors: White
authors: Sheetal
authors: S
authors: Purohit-Ghelani
authors: Klaus H
authors: KH
authors: Kaestner
authors: Jonathan S
authors: JS
authors: Bromberg
mesh_terms: D000818:Animals; D002449:Cell Aggregation; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D020869:Gene Expression Profiling; D007328:Insulin / Q000235:genetics*; D007515:Islets of Langerhans / Q000166:cytology; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D007328:Insulin