Paul Langerhans Institute Dresden (PLID) of Helmholtz Center Munich at the University Clinic Carl Gustav Carus of TU Dresden, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany.
German Centre for Diabetes Research (DZD), Munich-Neuherberg, Germany.
Sci Rep. 2024 Aug 27;14(1):19863. doi: 10.1038/s41598-024-69645-4.
The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional β-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of β-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS and GCG cells and their resolution into mono-hormonal cells (INS, INS/GCG) as well as β-cell maturation (INS/MAFA) and function (INS). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal β-cells without affecting the number of α-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets.
人类多能干细胞（hPS）向胰腺内分泌细胞（包括功能性β细胞）分化的显著进展是基于对潜在发育机制的深入了解。然而，从内分泌祖细胞到单激素和成熟胰腺内分泌细胞的最终分化步骤仍未完全理解，这反映在 hPS 细胞衍生胰岛细胞（SC-islet 细胞）的剩余缺陷中，包括β细胞成熟和不同细胞系之间的变异性。必须以组合方式评估最终分化步骤的额外信号和修饰，以解决剩余问题，适当的报告基因系在这方面将是有用的。在这里，我们报告了 hPS 细胞报告基因系的生成和功能验证，这些报告基因系可以监测 INS 和 GCG 细胞的生成及其向单激素细胞（INS、INS/GCG）以及β细胞成熟（INS/MAFA）和功能（INS）的分化。报告基因 hPS 细胞系保持了多能性标志物的强广泛表达，并有效地分化为确定的内胚层和胰腺祖细胞（PP）细胞。所有系的 PP 细胞都有效地分化为胰岛细胞簇，这些细胞簇强烈表达相应的报告基因，并含有葡萄糖反应性、胰岛素产生细胞。为了证明这些 hPS 细胞报告基因系在高内涵活细胞成像方法中识别最佳分化条件的适用性，我们调整了我们的分化程序，以在微井中生成 SC-islet 簇。这允许对单个条件下的多个 SC-islet 进行活共聚焦成像，并且使用这种方法，我们发现最后阶段在分化中使用 N21 补充剂可以增加单激素β细胞的数量，而不影响 SC-islet 中α细胞的数量。本文描述的 hPS 细胞报告基因系和高内涵活细胞成像方法将能够有效地评估多个条件，以实现 SC-islet 的最佳分化和成熟。
