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

Content Provider	Europe PMC
Author	Micallef, S. J. Li, X. Schiesser, J. V. Hirst, C. E. Yu, Q. C. Lim, S. M. Nostro, M. C. Elliott, D. A. Sarangi, F. Harrison, L. C. Keller, G. Elefanty, A. G. Stanley, E. G.
Abstract	Aims/hypothesisWe aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS+) cells derived in vitro.MethodsHomologous 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.ResultsDifferentiation 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/interpretationINS 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 materialThe 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.
ISSN	0012186X
Journal	Diabetologia
Volume Number	55
PubMed Central reference number	PMC3268987
Issue Number	3
PubMed reference number	22120512
e-ISSN	14320428
DOI	10.1007/s00125-011-2379-y
Language	English
Publisher	Springer-Verlag
Publisher Date	2011-11-26
Publisher Place	Berlin/Heidelberg
Access Restriction	Open
Rights License	This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. © The Author(s) 2011
Subject Keyword	Diabetes Gene targeting GFP Human embryonic stem cells Insulin
Content Type	Text
Resource Type	Article
Subject	Endocrinology, Diabetes and Metabolism Internal Medicine