Generation of Functional Lentoid Bodies From Human Induced Pluripotent Stem Cells Derived From Urinary Cells
Generation of Functional Lentoid Bodies From Human Induced Pluripotent Stem Cells Derived From Urinary Cells
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从源自泌尿细胞的人诱导多能干细胞生成功能性透镜体
DOI：
10.1167/iovs.16-20504
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发表时间：
2017-01-01
影响因子：
4.4
通讯作者：
Yao, Ke
中科院分区：
文献类型：
--
作者：
Fu, Qiuli;Qin, Zhenwei;Yao, Ke
目的。由于缺乏合适的体外细胞模型，白内障形成的病理机制在很大程度上仍不清楚。本研究的目的是建立一种稳定的人晶状体多能干细胞体外再生系统。方法：分离的人尿液细胞经4种山中因子感染后产生尿液中的人诱导多能干细胞(UIPSCs)，经诱导分化为晶状体祖细胞和类晶状体细胞(Lbs)。用实时荧光定量聚合酶链式反应、免疫染色和Western blotting检测晶状体特异性标志物的表达。结果建立了从UIPSCs分化为功能性LBS的“煎蛋”分化方法。UIPSC来源的LBS具有晶状体样形态和透明结构，并表达晶状体特异性标志物αA、αB、β和伽马晶体蛋白和MIP。在胚泡分化过程中，观察到胎盘标志物SIX1、EYA1、DLX3、PAX6和特异的早期晶状体标志物SOX1、PROX1、FOXE3、αA和αB晶状体蛋白。镜下可见晶状体囊膜附近的晶状体上皮细胞，以及未成熟和成熟的纤维样细胞。光学分析进一步证实了UIPSCs产生的LBS的放大能力(1.73)。结论我们的研究为UIPSCs产生功能性LBS提供了第一个证据，从而建立了一个可用于研究人类晶状体发育和白内障发生的体外系统，甚至可能用于药物筛选。
PURPOSE. The pathological mechanisms underlying cataract formation remain largely unknown on account of the lack of appropriate in vitro cellular models. The aim of this study is to develop a stable in vitro system for human lens regeneration using pluripotent stem cells.METHODS. Isolated human urinary cells were infected with four Yamanaka factors to generate urinary human induced pluripotent stem cells (UiPSCs), which were induced to differentiate into lens progenitor cells and lentoid bodies (LBs). The expression of lens-specific markers was examined by real-time PCR, immunostaining, and Western blotting. The structure and magnifying ability of LBs were investigated using transmission electron microscopy and observing the magnification of the letter "X,'' respectively.RESULTS. We developed a "fried egg'' differentiation method to generate functional LBs from UiPSCs. The UiPSC-derived LBs exhibited crystalline lens-like morphology and a transparent structure and expressed lens-specific markers alpha A-, alpha B-, beta-, and gamma-crystallin and MIP. During LB differentiation, the placodal markers SIX1, EYA1, DLX3, PAX6, and the specific early lens markers SOX1, PROX1, FOXE3, alpha A-, and alpha B-crystallin were observed at certain time points. Microscopic examination revealed the presence of lens epithelial cells adjacent to the lens capsule as well as both immature and mature fiber-like cells. Optical analysis further demonstrated the magnifying ability (1.73) of the LBs generated from UiPSCs.CONCLUSIONS. Our study provides the first evidence toward generating functional LBs from UiPSCs, thereby establishing an in vitro system that can be used to study human lens development and cataractogenesis and perhaps even be useful for drug screening.