Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins
Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins
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DOI：
10.1002/term.1458
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发表时间：
2013-08-01
影响因子：
3.3
通讯作者：
Clegg, Dennis O.
中科院分区：
文献类型：
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作者：
Rowland, Teisha J.;Blaschke, Alison J.;Clegg, Dennis O.
人胚胎干细胞（hESC）和诱导多能干细胞（iPSC）的潜在应用是产生视网膜色素上皮（RPE）以治疗年龄相关性黄斑变性（AMD），这是一种常见但无法治愈的视网膜疾病。衍生自hESC（hESC-RPE）和iPSC（iPSC-RPE）的RPE细胞表达必需的RPE标志物，并且可以在动物模型中挽救视觉功能。然而，标准分化方案以低频率产生RPE细胞，特别是来自iPSC系，并且Matrigel和异种饲养细胞的常用与临床应用不相容。细胞外基质（ECM）可以影响分化，因此ECM组成的变化可以提高干细胞-RPE分化的频率。我们基于体内RPE ECM环境选择了几种纯化的ECM蛋白和底物，并测试了它们支持iPSC-RPE分化和维持的能力。在几乎所有测试的基底上分化的iPSC形成色素沉着区域，其中Matrigel和小鼠层粘连蛋白-111支持最高的色素沉着频率。尽管在大多数测试基质上培养的iPSC-RPE表达关键RPE基因，但只有六种基质支持具有正常RPE形态的融合单层的发育，包括基质胶和小鼠层粘连蛋白-111。在小鼠层粘连蛋白-111上分化的iPSC产生表达RPE蛋白的iPSC-RPE，并且在小鼠层粘连蛋白-111上分化的hESC产生高产量的功能性hESC-RPE。关键ECM蛋白的这种鉴定可以有助于未来的支架设计，并提供用于从与临床翻译相关的人多能干细胞合成、无异种、符合GMP的RPE生成的肽序列。版权所有（c）2012约翰威利父子有限公司
A potential application of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is the generation of retinal pigmented epithelium (RPE) to treat age-related macular degeneration (AMD), a common but incurable retinal disease. RPE cells derived from hESCs (hESC-RPEs) and iPSCs (iPSC-RPEs) express essential RPE markers and can rescue visual function in animal models. However, standard differentiation protocols yield RPE cells at low frequency, especially from iPSC lines, and the common use of Matrigel and xenogeneic feeder cells is not compatible with clinical applications. The extracellular matrix (ECM) can affect differentiation, and therefore changes in ECM composition may improve the frequency of stem cell-RPE differentiation. We selected several purified ECM proteins and substrates, based on the in vivo RPE ECM environment, and tested their ability to support iPSC-RPE differentiation and maintenance. iPSCs differentiated on nearly all tested substrates developed pigmented regions, with Matrigel and mouse laminin-111 supporting the highest pigmentation frequencies. Although iPSC-RPEs cultured on the majority of the tested substrates expressed key RPE genes, only six substrates supported development of confluent monolayers with normal RPE morphology, including Matrigel and mouse laminin-111. iPSCs differentiated on mouse laminin-111 produced iPSC-RPEs expressing RPE proteins, and hESCs differentiated on mouse laminin-111 resulted in high yields of functional hESC-RPEs. This identification of key ECM proteins may assist with future scaffold designs and provide peptide sequences for use in synthetic, xeno-free, GMP-compliant generation of RPE from human pluripotent stem cells relevant to clinical translation. Copyright (c) 2012 John Wiley & Sons, Ltd.