Benazra Marion, Lecomte Marie-José, Colace Claire, Müller Andreas, Machado Cécile, Pechberty Severine, Bricout-Neveu Emilie, Grenier-Godard Maud, Solimena Michele, Scharfmann Raphaël, Czernichow Paul, Ravassard Philippe
Institut du cerveau et de la moelle (ICM), Biotechnology & Biotherapy Team, 75013 Paris, France; CNRS UMR7225, 75013 Paris, France; INSERM U1127, 75013 Paris, France; Université Pierre et Marie Curie, 75013 Paris, France.
Endocells, Pépinière d'entreprises Institut du Cerveau et de la Moelle, 75007 Paris, France.
Mol Metab. 2015 Oct 20;4(12):916-25. doi: 10.1016/j.molmet.2015.09.008. eCollection 2015 Dec.
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.
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.
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.
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.
获得表型上接近真正成年β细胞的永生化人胰岛β细胞系，是更好地理解人β细胞生理学并开发糖尿病新疗法的主要工具。在此，我们获得了一种新的条件永生化人β细胞系EndoC-βH3，其中通过简单添加他莫昔芬即可有效去除永生化转基因。
我们使用慢病毒介导的基因转移，将他莫昔芬诱导型CRE（CRE-ERT2）稳定整合到最近开发的条件永生化EndoC βH2细胞系中。对所得的EndoC-βH3细胞系在他莫昔芬处理前后进行细胞增殖、胰岛素含量和胰岛素分泌方面的表征。
我们表明，表达CRE-ERT2的EndoC-βH3细胞在培养中可大量扩增。我们建立了优化的他莫昔芬处理方法，以有效切除永生化转基因，导致细胞增殖停滞。此外，胰岛素表达提高了12倍，胰岛素含量增加了23倍，达到每百万细胞2μg胰岛素。如此大量的增加伴随着葡萄糖刺激后胰岛素分泌的增强。我们进一步观察到，经他莫昔芬处理的细胞在培养中维持稳定功能达5周。
EndoC βH3细胞系是一种强大的工具，它允许通过简单有效的程序大量生产功能性非增殖性人β细胞。此类细胞接近真正的人β细胞，并且在培养中维持稳定表型达5周。
