Opacification of lentoid bodies derived from human induced pluripotent stem cells is accelerated by hydrogen peroxide and involves protein aggregation
Opacification of lentoid bodies derived from human induced pluripotent stem cells is accelerated by hydrogen peroxide and involves protein aggregation
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过氧化氢加速源自人诱导多能干细胞的透镜状体的混浊，并涉及蛋白质聚集
DOI：
10.1002/jcp.28943
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发表时间：
2019-12-01
影响因子：
5.6
通讯作者：
Fu, Qiuli
中科院分区：
文献类型：
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作者：
Qin, Zhenwei;Zhang, Lifang;Fu, Qiuli
尽管最近在白内障药物开发方面取得了突破，但由于缺乏人类体外白内障疾病模型，进一步的改进受到限制。因此，本研究旨在产生合格的白内障疾病模型。使用“煎蛋”方法从人诱导多能干细胞（iPSC）分化的第25天（D25）的成熟晶状体体（LB）被连续培养（对照）或用紫外线（UV）辐射或过氧化氢（H2 O2）额外处理。光镜观察和平均灰度值评价LB的形态变化和混浊度。用免疫荧光和透射电子显微镜（TEM）检查它们的结构和晶体蛋白表达。采用真实的实时聚合酶链反应和蛋白质印迹法研究自噬在混浊LB中的潜在作用。成熟LB随时间的推移而变得浑浊，H2 O2加速了这种浑浊。免疫荧光检查和TEM显示，H2 O2处理和对照LB具有相似的形状、透镜囊和单层透镜上皮细胞（LEC）结构。然而，我们无法对经紫外线处理的LB进行进一步评估，因为LB的结构在经紫外线辐射处理时容易受损。与对照LB相比，H2 O2处理LB中含有聚集蛋白（αA-晶状体蛋白和αB-晶状体蛋白）斑点的细胞更丰富。此外，与原发性LEC中的LC 3 B水平相比，从年龄相关性白内障（ARC）患者获得的前透镜囊中的LC 3 B表达随年龄而降低，这与LB中的LC 3 B表达在D45时低于D25时一致。我们的研究发现，人iPSCs来源的LB随着时间的推移变得浑浊，伴随着蛋白质聚集，并且H2 O2加速了这种现象，这表明延长培养的LB可以作为体外ARC的人类模型。
Despite the recent breakthrough in cataract drug development, further improvements have been limited by the lack of human in vitro cataract disease models. This study, therefore, aims to generate a qualified cataract disease model. Mature lentoid bodies (LBs) on Day 25 (D25), which were differentiated from human induced pluripotent stem cells (iPSCs) using the “fried egg” method, were continually culturing (control) or extra treated with either ultraviolet (UV) radiation or hydrogen peroxide (H2O2). The LBs’ shape alteration and opacity were examined using light microscopy and mean gray value evaluation. Their structure and crystallin expression were examined using immunofluorescence and transmission electron microscopy (TEM). Real‐time polymerase chain reaction and western blot were used to investigate the potential role of autophagy in cloudy LBs. Mature LBs became cloudy with time which was accelerated by H2O2. Immunofluorescence examinations and TEM showed that the H2O2‐treated and control LBs had similar shapes, lens capsule, and monolayer lens epithelial cell (LEC) structures. However, we were unable to do further assessment of the UV‐treated LBs as the structures of LBs were easily damaged when treated with UV radiation. Cells containing aggregated protein (αA‐crystallin and αB‐crystallin) puncta were more abundant in the H2O2‐treated LBs as compared with control LBs. Moreover, LC3B expression decreased with age in anterior lens capsules obtained from age‐related cataracts (ARCs) patients as compared with LC3B levels in primary LECs, which is consistent with that LC3B expression in LBs was lower on D45 than on D25. Our study found that human iPSCs‐derived LBs became cloudy with time which was accompanied by protein aggregation, and this phenomenon was accelerated by H2O2, suggesting that LBs with extending culture may serve as a human model for in vitro ARCs.