A human beta cell line with drug inducible excision of immortalizing transgenes
A human beta cell line with drug inducible excision of immortalizing transgenes
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DOI：
10.1016/j.molmet.2015.09.008
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发表时间：
2015-12-01
影响因子：
8.1
通讯作者：
Ravassard, Philippe
中科院分区：
文献类型：
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作者：
Benazra, Marion;Lecomte, Marie-Jose;Ravassard, Philippe
目的：获得表型上接近真正成人β细胞的永生化人胰腺β细胞系代表了更好地理解人β细胞生理学和开发糖尿病新疗法的主要工具。在这里，我们推导出一个新的条件永生化的人β细胞系，EndoC-β H3，其中永生化的转基因可以有效地删除通过简单地添加tamoxifen.Methods：我们使用慢病毒介导的基因转移稳定整合到最近开发的条件永生化的EndoC β H2线的他莫昔芬诱导形式的CRE（CRE-ERT 2）。在他莫昔芬处理细胞增殖、胰岛素含量和胰岛素分泌之前和之后，对所得的EndoC-β H3系进行表征。结果：我们表明，表达CRE-ERT 2的EndoC-β H3可以在培养物中大量扩增。我们建立了一种优化的他莫昔芬治疗，以有效地切除永生化转基因，导致增殖停滞。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2 μ g胰岛素。这种大量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，他莫昔芬处理的细胞保持稳定的功能为5周culture.Conclusions：EndoC β H3细胞系是一个强大的工具，允许，使用一个简单而有效的程序，大量生产的功能性非增殖的人β细胞。这样的细胞接近于真正的人β细胞，并且在培养物中保持稳定的表型5周。(C)2015年，作者。由爱思唯尔有限公司出版。这是CC BY许可下的开放获取文章（http：//creativecommons. org/许可证/by/4.0/）。
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. (C) 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).