New hPSC SOX9 and INS Reporter Cell Lines Facilitate the Observation and Optimization of Differentiation into Insulin-Producing Cells,Stem Cell Reviews and Reports

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New hPSC SOX9 and INS Reporter Cell Lines Facilitate the Observation and Optimization of Differentiation into Insulin-Producing Cells
Stem Cell Reviews and Reports ( IF 4.5 ) Pub Date : 2021-08-19 , DOI: 10.1007/s12015-021-10232-9
Rabea Dettmer ₁ , Isabell Niwolik ₁ , Ilir Mehmeti ₁ , Anne Jörns ₁ , Ortwin Naujok ₁

Affiliation

Differentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. SOX9 plays a crucial role during development of the pancreas and particularly in the development of insulin-producing cells as SOX9⁺ cells form the source for NEUROG3⁺ endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2K^k-F2A-GFP2 reporter gene into the SOX9-locus and a P2A-mCherry reporter gene into the INS-locus mediated by CRISPR/CAS9-technology. The knock-ins enabled co-expression of the endogenous and reporter genes and report on the endogenous gene expression. Furthermore, FACS and MACS enabled the purification of pancreatic progenitors and insulin-producing cells. Using these cell lines, we established a new differentiation protocol geared towards SOX9⁺ cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells. Our new protocol offers an alternative route towards stem cell-derived beta cells, pointing out the importance of Wnt/beta-catenin inhibition and the efficacy of EGF for the development of pancreatic progenitors, as well as the significance of 3D culture for the functionality of the generated beta cells.

Graphic Abstract

中文翻译：

新的 hPSC SOX9 和 INS 报告细胞系有利于观察和优化胰岛素生成细胞的分化

人类多能干细胞分化为产生胰岛素的干细胞衍生的β细胞对于糖尿病的研究和治疗具有巨大的潜力。 SOX9在胰腺发育过程中发挥着至关重要的作用，特别是在胰岛素生成细胞的发育过程中，因为 SOX9 ⁺细胞形成了 NEUROG3 ⁺内分泌祖细胞的来源。为了轻松监测胰腺祖细胞和胰岛素产生细胞的分化效率，我们通过将 P2A-H-2K ^k -F2A-GFP2 报告基因敲入SOX9基因座和 P2A-mCherry 报告基因来生成新的报告基因系通过 CRISPR/CAS9 技术介导将基因插入INS位点。敲入使内源基因和报告基因共表达并报告内源基因表达。此外，FACS 和 MACS 能够纯化胰腺祖细胞和胰岛素产生细胞。利用这些细胞系，我们建立了一种针对 SOX9 ⁺细胞的新分化方案，以有效地将人类多能干细胞转化为葡萄糖反应性 β 细胞。我们的新方案为干细胞衍生的 β 细胞提供了另一种途径，指出了 Wnt/β-连环蛋白抑制的重要性和 EGF 对于胰腺祖细胞发育的功效，以及 3D 培养对于胰腺祖细胞功能的重要性生成的β细胞。

图文摘要

更新日期：2021-08-20

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