ABSTRACT
Innovative treatments to cure type 1 diabetes are being actively researched. Among the different strategies, the replacement of ß-cells has given promising results. Classically, islets from cadaveric donors are transplanted into diabetic patients, but recently phase I clinical trials that use stem cell-derived ß-cells have been started. Such protocols require either an immunosuppressive treatment or the macroencapsulation of the ß-cells. They involve cell aggregation and the exposure of the cells to hypoxia. Using an engineered human ß-cell, we have addressed these two problems: a novel human ß-cell line called EndoC-ßH3 was cultured as single cells or aggregated clusters. EndoC-ßH3 cells were also cultured at normal atmospheric oxygen tension (pO2 = 21%) or hypoxia (pO2 = 3%) in the presence or absence of modulators of the hypoxia-inducible factor 1α (HIF1α) pathway. Cell aggregation improved glucose-stimulated insulin secretion, demonstrating the benefit of cell-cell contacts. Low oxygen tension decreased ß-cell viability and their sensitivity to glucose, but did not alter insulin production nor the insulin secretion capacity of the remaining cells. To investigate the role of HIF1α, we first used a HIF stabilizer at pO2 = 21%. This led to a mild decrease in cell viability, impaired glucose sensitivity, and altered insulin secretion. Finally, we used a HIF inhibitor on EndoC-ßH3 pseudoislets exposed to hypoxia. Such treatment considerably decreased cell viability. In conclusion, aggregation of the EndoC-ßH3 cells seems to be important to improve their function. A fraction of the EndoC-ßH3 cells are resistant to hypoxia, depending on the level of activity of HIF1α. Thus, these cells represent a good human cell model for future investigations on islet cell transplantation analysis.
ABSTRACT
OBJECTIVES: Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-ßH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. METHODS: We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC ßH2 line. The resulting EndoC-ßH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. RESULTS: We showed that EndoC-ßH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 µg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. CONCLUSIONS: EndoC ßH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture.
