BACKGROUND Adult human pancreatic beta cells are the "gold standard" for studies on diabetes pathogenesis, but their use is limited by insufficient availability and variable quality. An important effort has recently taken place to differentiate beta cells from human induced pluripotent stem cells (iPSCs) and validate their use for diabetes research. We presently used a 7-stage protocol to generate beta cells from human iPSC and evaluated whether these cells are responsive to the pro-inflammatory cytokines (IFNγ, IL-1β, or IFNα) that play a role in type 1 diabetes. METHODS The iPSC-derived islet-like cell clusters contained 40-50% beta and 10-15% alpha cells and expressed the receptors for IFNγ, IL-1β, or IFNα. Cells were exposed to either IFNγ (1000 U/mL) + IL-1β (50 U/mL) or IFNα alone (2000 U/mL) for 24/48 h. Apoptosis was quantified using Hoechst/propidium iodide staining or the RealTime Glo Apoptosis Kit (Promega). After treatment, CXCL10 secretion was quantified by ELISA. The expression of multiples genes (Ins, Gcg, Nkx2.2, Nkx6.1, Pdx1, Mafa, BiP, Chop, Atf3, CXCL10, CXCL9, CCL5, and HLA-ABC) was quantified by RT-qPCR. Phosphorylation state and total expression of STAT1/STAT2, as well as expression of PDL1 and of the ER chaperone BiP, were quantified by Western blotting. The co-localization of HLA-ABC or cleaved caspase-3 and Ins/Gcg expression was assessed by immunohistochemistry. The presence of HLA-ABC at the plasma membrane was measured by flow cytometry. RESULTS IFNγ + IL-1β and IFNα induced apoptosis of the cells after 48 h of exposure. Cleaved caspase-3 co-localized mostly but not exclusively with Ins+ cells. Exposure to IFNγ + IL-1β induced a pro-inflammatory phenotype, including increased CXCL10, CXCL9, and CCL5 expression; CXCL10 secretion; and HLA-ABC expression. HLA overexpression was confirmed at the protein level by Western blotting and flow cytometry. Exposure to IFNγ + IL-1β (but not IFNα) also induced beta cell dedifferentiation and endoplasmic reticulum stress (increase in BiP, Chop, and Atf3 mRNA expression). Phosphorylation of STAT1 was stimulated already after 1 h by IFNγ + IL-1β and IFNα, while phosphorylation of STAT2 was only activated by IFNα at 1-4 h. PDL1 expression was increased by both IFNγ + IL-1β and IFNα. CONCLUSIONS Our data show that human iPSC-derived beta cells respond to pro-inflammatory cytokines IL-1β + IFNγ and IFNα, by activating the same pathogenic processes as adult human primary beta cells. These cells thus represent a valuable tool for future research on the pathogenesis of type 1 diabetes.

中文翻译：

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促炎性细胞因子在人iPSC衍生的β细胞中诱导细胞死亡，炎症反应和内质网应激。

背景技术成年人类胰腺β细胞是糖尿病发病机理研究的“金标准”，但是其使用受到可用性和质量变化的限制。最近，人们进行了重要的努力，以将β细胞与人诱导的多能干细胞（iPSC）区别开来，并验证其在糖尿病研究中的用途。我们目前使用7阶段协议从人iPSC生成β细胞，并评估这些细胞是否对在1型糖尿病中起作用的促炎细胞因子（IFNγ，IL-1β或IFNα）有反应。方法源自iPSC的胰岛样细胞簇包含40-50％的β细胞和10-15％的α细胞，并表达IFNγ，IL-1β或IFNα的受体。将细胞暴露于IFNγ（1000 U / mL）+IL-1β（50 U / mL）或单独的IFNα（2000 U / mL）24/48小时。使用Hoechst /碘化丙啶染色或RealTime Glo细胞凋亡试剂盒（Promega）对细胞凋亡进行定量。治疗后，通过ELISA定量CXCL10分泌。通过RT-qPCR定量多个基因（Ins，Gcg，Nkx2.2，Nkx6.1，Pdx1，Mafa，BiP，Chop，Atf3，CXCL10，CXCL9，CCL5和HLA-ABC）的表达。蛋白质印迹状态定量STAT1 / STAT2的磷酸化状态和总表达，以及PDL1和ER伴侣BiP的表达。通过免疫组织化学评估HLA-ABC或裂解的caspase-3和Ins / Gcg表达的共定位。通过流式细胞术测量在质膜处HLA-ABC的存在。结果IFNγ+IL-1β和IFNα暴露48 h后可诱导细胞凋亡。切开的半胱天冬酶3主要但并非唯一与Ins +细胞共定位。暴露于IFNγ+IL-1β会诱发促炎表型，包括增加的CXCL10，CXCL9和CCL5表达。CXCL10分泌物；和HLA-ABC表达。通过蛋白质印迹和流式细胞术在蛋白质水平上证实了HLA过表达。暴露于IFNγ+IL-1β（而不是IFNα）也会诱导β细胞去分化和内质网应激（BiP，Chop和Atf3 mRNA表达增加）。STAT1的磷酸化已在1 h后被IFNγ+IL-1β和IFNα刺激，而STAT2的磷酸化仅在1-4 h被IFNα激活。IFNγ+IL-1β和IFNα均可增加PDL1表达。结论我们的数据表明，人类iPSC衍生的β细胞通过激活与成年人类原代β细胞相同的致病过程来响应促炎性细胞因子IL-1β+IFNγ和IFNα。