BC Children's Hospital Research Institute, Vancouver, BC, Canada; Department of Surgery, University of British Columbia, Vancouver, BC, Canada.
Department of Pharmacology, University of Alberta, Edmonton, AB, Canada; Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada.
Mol Metab. 2024 Nov;89:102017. doi: 10.1016/j.molmet.2024.102017. Epub 2024 Aug 23.
Human embryonic stem cell (hESC; SC)-derived pancreatic β cells can be used to study diabetes pathologies and develop cell replacement therapies. Although current differentiation protocols yield SCβ cells with varying degrees of maturation, these cells still differ from deceased donor human β cells in several respects. We sought to develop a reporter cell line that could be used to dynamically track SCβ cell functional maturation.
To monitor SCβ cell maturation in vitro, we created an IAPP-2A-mScar and INSULIN-2A-EGFP dual fluorescent reporter (INS;IAPP) hESC line using CRISPR/Cas9. Pluripotent SC were then differentiated using a 7-stage protocol to islet-like cells. Immunohistochemistry, flow cytometry, qPCR, GSIS and electrophysiology were used to characterise resulting cell populations.
We observed robust expression of EGFP and mScarlet fluorescent proteins in insulin- and IAPP-expressing cells without any compromise to their differentiation. We show that the proportion of insulin-producing cells expressing IAPP increases over a 4-week maturation period, and that a subset of insulin-expressing cells remain IAPP-free. Compared to this IAPP-free population, we show these insulin- and IAPP-expressing cells are less polyhormonal, more glucose-sensitive, and exhibit decreased action potential firing in low (2.8 mM) glucose.
The INS;IAPP hESC line provides a useful tool for tracking populations of maturing hESC-derived β cells in vitro. This tool has already been shared with 3 groups and is freely available to all.
人胚胎干细胞（hESC；SC）衍生的胰腺β细胞可用于研究糖尿病病理和开发细胞替代疗法。尽管目前的分化方案产生了具有不同成熟程度的 SCβ细胞，但这些细胞在几个方面仍与已故供体人类β细胞不同。我们试图开发一种报告细胞系，可用于动态跟踪 SCβ细胞功能成熟。
为了在体外监测 SCβ细胞的成熟，我们使用 CRISPR/Cas9 技术创建了一个 IAPP-2A-mScar 和 INSULIN-2A-EGFP 双荧光报告（INS；IAPP）hESC 系。然后使用 7 阶段方案将多能 SC 分化为胰岛样细胞。免疫组织化学、流式细胞术、qPCR、GSIS 和电生理学用于表征产生的细胞群体。
我们观察到胰岛素和 IAPP 表达细胞中 EGFP 和 mScarlet 荧光蛋白的强烈表达，而不会影响其分化。我们表明，在 4 周的成熟过程中，表达 IAPP 的胰岛素产生细胞的比例增加，并且一部分胰岛素表达细胞仍然没有 IAPP。与这种无 IAPP 群体相比，我们表明这些胰岛素和 IAPP 表达细胞的多激素性更低、对葡萄糖更敏感，并且在低（2.8mM）葡萄糖下表现出减少的动作电位发射。
INS；IAPP hESC 系为在体外跟踪成熟的 hESC 衍生β细胞群体提供了有用的工具。该工具已经与 3 个小组共享，并且可供所有人免费使用。
