Human embryonic stem cell (hESC)-derived pancreatic alpha and beta cells can be used to develop cell replacement therapies to treat diabetes. However, recent published differentiation protocols yield varying amounts of alpha and beta cells amidst heterogeneous cell populations. To visualize and isolate hESC-derived alpha and beta cells, we generated a GLUCAGON-2AmScarlet and INSULIN-2A-EGFP dual fluorescent reporter (INSEGFPGCGmScarlet) hESC line using CRISPR/Cas9. We established robust expression of EGFP and mScarlet fluorescent proteins in insulin- and glucagon-expressing cells respectively without compromising the differentiation or function of these cells. We also showed the insulin- and glucagon-expressing bihormonal population at the maturing endocrine cell stage (Stage 6) of our pancreatic islet differentiation lose insulin expression over time, while maintaining an alpha-like expression profile, suggesting these bihormonal cells are cell autonomously fated to become alpha-like cells. We also demonstrated this cell line can be used to monitor hESC-derived insulin- and glucagonexpressing cells, and hESC-derived islet morphology in vivo by transplanting them into the anterior chamber of the eye in mice. Together, the INSEGFPGCGmScarlet hESC line provides an efficient strategy for tracking populations of hESC-derived beta- and alpha-like cells.

中文翻译：

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使用双报告基因人胚胎干细胞系在体外和体内追踪表达胰岛素和胰高血糖素的细胞


人胚胎干细胞 （hESC） 衍生的胰腺 α 和 β 细胞可用于开发治疗糖尿病的细胞替代疗法。然而，最近发表的分化方案在异质性细胞群中产生不同数量的 α 和 β 细胞。为了可视化和分离 hESC 衍生的 α 和 β 细胞，我们使用 CRISPR/Cas9 生成了 GLUCAGON-2AmScarlet 和 INSULIN-2A-EGFP 双荧光报告基因 （INSEGFPGCGmScarlet） hESC 系。我们分别在表达胰岛素和胰高血糖素的细胞中建立了 EGFP 和 mScarlet 荧光蛋白的稳健表达，而不会影响这些细胞的分化或功能。我们还显示，在我们胰岛分化的成熟内分泌细胞阶段（第 6 阶段）表达胰岛素和胰高血糖素的双激素群体随着时间的推移失去胰岛素表达，同时保持 α 样表达谱，表明这些双激素细胞是细胞自主命中注定成为 α 样细胞的。我们还证明，该细胞系可用于通过将 hESC 衍生的胰岛素和胰高血糖素压迫细胞移植到小鼠的眼睛前房中来监测体内 hESC 衍生的胰岛形态。总之，INSEGFPGCGmScarlet hESC 细胞系为追踪 hESC 来源的 β 和 α 样细胞群提供了一种有效的策略。