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Benazra M.,Institute Du Cerveau Et Of La Moelle Icm | Benazra M.,French National Center for Scientific Research | Benazra M.,French Institute of Health and Medical Research | Benazra M.,University Pierre and Marie Curie | And 20 more authors.
Molecular Metabolism | Year: 2015

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-βH3 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 βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. Results: We showed that EndoC-βH3 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 βH3 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. © 2015 The Authors. Source

Scharfmann R.,University of Paris Descartes | Pechberty S.,University of Paris Descartes | Pechberty S.,Pepiniere Dentreprises Institute Du Cerveau Et Of La Moelle | Hazhouz Y.,Pepiniere Dentreprises Institute Du Cerveau Et Of La Moelle | And 11 more authors.
Journal of Clinical Investigation | Year: 2014

Diabetic patients exhibit a reduction in βcells, which secrete insulin to help regulate glucose homeostasis; however, little is known about the factors that regulate proliferation of these cells in human pancreas. Access to primary human βcells is limited and a challenge for both functional studies and drug discovery progress. We previously reported the generation of a human βcell line (EndoC-βH1) that was generated from human fetal pancreas by targeted oncogenesis followed by in vivo cell differentiation in mice. EndoC-βH1 cells display many functional properties of adult βcells, including expression of βcell markers and insulin secretion following glucose stimulation; however, unlike primary βcells, EndoC-βH1 cells continuously proliferate. Here, we devised a strategy to generate conditionally immortalized human βcell lines based on Cre-mediated excision of the immortalizing transgenes. The resulting cell line (EndoC-βH2) could be massively amplified in vitro. After expansion, transgenes were efficiently excised upon Cre expression, leading to an arrest of cell proliferation and pronounced enhancement of βcell-specific features such as insulin expression, content, and secretion. Our data indicate that excised EndoC-βH2 cells are highly representative of human βcells and should be a valuable tool for further analysis of human βcells. Source

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