INSGFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells
SJ Micallef, X Li, JV Schiesser, CE Hirst, QC Yu, SM Lim, MC Nostro, DA Elliott, F Sarangi, LC Harrison, G Keller, AG Elefanty, EG Stanley
DIABETOLOGIA | SPRINGER | Published : 2012
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-..View full abstract
We thank R. Mayberry, K. Koutsis and A. Bruce for the provision of hESCs, S. Hawes for the provision of human fetal pancreas RNA, FlowCore for flow cytometric sorting, and Monash Micro Imaging for confocal microscopy and time-lapse video services. All work involving experimentation with human ES cell lines was performed under approval of the Monash University Human Ethics Committee (2002-225MC). This work was supported by the Australian Stem Cell Centre (ASCC), The National Health and Medical Research Council (NHMRC) of Australia and the Juvenile Diabetes Research Foundation. M. C. Nostro was supported by a postdoctoral fellowship from the McEwen Centre for Regenerative Medicine. L. C. Harrison is a Senior Principal Research Fellow, and A. G. Elefanty and E. G. Stanley are Senior Research Fellows of the NHMRC.