Generation of Human Lens Epithelial-Like Cells From Patient-Specific Induced Pluripotent Stem Cells
Generation of Human Lens Epithelial-Like Cells From Patient-Specific Induced Pluripotent Stem Cells
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DOI：
10.1002/jcp.25374
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发表时间：
2016-12-01
影响因子：
5.6
通讯作者：
Lu, Yi
中科院分区：
文献类型：
--
作者：
Li, Dan;Qiu, Xiaodi;Lu, Yi
白内障的发生始于动态的透镜上皮细胞（LEC）和邻近的纤维细胞。来源于细胞系的晶状体上皮细胞不能保持原代晶状体上皮细胞的晶体表达。目前的研究旨在从患者特异性诱导多能干细胞（iPSC）中有效地产生大量的人类LEC。从白内障手术中收集前透镜囊并用于培养原代hLEC。通过Oct 4、Sox 2、Klf 4和c-Myc的慢病毒转导从这些原代hLEC诱导iPSC。然后，通过从已建立的方法修改的定义的因子组合（Noggin、BMP 4/7、bFGF和EGF）的3步添加，将产生的iPSC再分化成hLEC。在再分化过程中，基于殖民地形态，使用玻璃挑取工具和克隆圆柱体分离感兴趣的菌落。在两个分离步骤后，产生LEC样细胞（LLC）群体，并通过qPCR、蛋白质印迹和免疫荧光染色通过透镜标记基因的表达来鉴定。该研究引入了一种改进的方案，通过在短时间内确定的因素从iPSC中分离LLC。该技术可用于晶状体相关疾病的机制研究。(C)2016 Wiley Periodicals，Inc.
Cataractogenesis begins from the dynamic lens epithelial cells (LECs) and adjacent fiber cells. LECs derived from cell lines cannot maintain the crystalline expression as the primary LECs. The current study aimed to efficiently generate large numbers of human LECs from patient-specific induced pluripotent stem cells (iPSCs). Anterior lens capsules were collected from cataract surgery and were used to culture primary hLECs. iPSCs were induced from these primary hLECs by lentiviral transduction of Oct4, Sox2, Klf4, and c-Myc. Then, the generated iPSCs were re-differentiated into hLECs by the 3-step addition of defined factor combinations (Noggin, BMP4/7, bFGF, and EGF) modified from an established method. During the re-differentiation process, colonies of interest were isolated using a glass picking tool and cloning cylinders based on the colony morphology. After two steps of isolation, populations of LEC-like cells (LLCs) were generated and identified by the expression of lens marker genes by qPCR, western blot and immunofluorescence staining. The study introduced a modified protocol to isolate LLCs from iPSCs by defined factors in a short time frame. This technique could be useful for mechanistic studies of lens-related diseases. (C) 2016 Wiley Periodicals, Inc.