Development of an optimized 5-stage protocol for the in vitro preparation of insulin-secreting cells from mouse ES cells
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In order to produce insulin-secreting cells with a high value of glucose-stimulated insulin secretion (GSIS) from mouse embryonic stem cells, we have developed an optimized 5-stage protocol by referring to culture conditions so far reported elsewhere. This protocol is characterized by 4 points: (1) use of an activin-free medium in the first stage, (2) use of gelatin/fibronectin coated culture dishes in 1–4 stages throughout, (3) removal of undifferentiated cells by cell sorter at the end of 4th stage, and (4) sedimental culture in the 5th stage. GSIS value of the produced cells reached 2.4, that was at a higher rank of those so far reported. The produced cells were transplanted in diabetes model mice but no remedy effect was observed. Then transplantation was conducted in pre-diabetes model mice, in which GSIS was impaired without affecting insulin producing function. The transplantation of 5 × 106 cells resulted in a marked improvement of glucose tolerance within 20 days. This effect decreased but was still observed at 120 days post-transplantation. This demonstrates the feasibility of the novel optimized protocol.
KeywordsGlucose-stimulated insulin secretion Insulin-secreting cells Mouse ES cells Pre-diabetes model mice
We thank Dr. Niwa for the donation of feeder free EB3 cells, and Dr. Okitsu for excellent technical support on transplantation. The work was supported in part by the Strategic Research Promotion Program, the Ministry of Education, Culture, Sports, Science, and Technology, on the research subject “Development of Next Generation Bioresources”.
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