A human beta cell line with drug inducible excision of immortalizing transgenes

作者：Benazra Marion; Lecomte Marie Jose; Colace Claire; Mueller Andreas; Machado Cecile; Pechberty Severine; Bricout Neveu Emilie; Grenier Godard Maud; Solimena Michele; Scharfmann Raphael; Czernichow Paul; Ravassard Philippe^*

来源：Molecular Metabolism, 2015, 4(12): 916-925.

DOI：10.1016/j.molmet.2015.09.008

• Cell engineering
• Human pancreatic beta cell line
• Conditional immortalization
• Tamoxifen inducible CRE
• Human beta cell function

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-beta H3 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 beta H2 line. The resulting EndoC-beta H3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion.
Results: We showed that EndoC-beta H3 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 mu g 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 beta H3 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.

• 出版日期2015-12