In-depth characterisation of Retinal Pigment Epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSC).
In-depth characterisation of Retinal Pigment Epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSC).
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DOI：
10.1007/s12017-014-8308-8
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发表时间：
2014-09
影响因子：
3.5
通讯作者：
Weber, Bernhard H. F.
中科院分区：
文献类型：
--
作者：
Brandl, Caroline;Zimmermann, Stephanie J.;Milenkovic, Vladimir M.;Rosendahl, Sibylle M. G.;Grassmann, Felix;Milenkovic, Andrea;Hehr, Ute;Federlin, Marianne;Wetzel, Christian H.;Helbig, Horst;Weber, Bernhard H. F.
诱导多能干细胞（iPSC）衍生的视网膜色素上皮（RPE）已被广泛认为是对源自原发性RPE病理的人类眼部疾病建模的有前景的工具。在这里，我们描述了成人皮肤成纤维细胞成功重编程为iPSC，并将其分化为具有天然RPE结构和功能特征的纯可膨胀RPE细胞。从皮肤活检材料建立成纤维细胞培养物，随后在用OCT 4、SOX 2、KLF 4和L-Myc进行多顺反子慢病毒转导后重编程。成纤维细胞来源的iPSC显示出典型的形态、染色体完整性和独特的干细胞标志物谱。随后的分化导致可膨胀的色素六角形RPE细胞。细胞显示成熟RPE标志物RPE 65、RLBP和BEST 1的稳定RNA表达。免疫标记证实BEST 1定位于基底外侧质膜，扫描电子显微镜显示iPSC衍生的RPE细胞顶侧有典型的微绒毛。在细胞培养期间，跨上皮电阻维持在高水平，表明紧密连接的功能形成。证明了VEGF-A的分泌能力。猪感光细胞外节的喂养揭示了这些细胞的吞噬功能。IPSC衍生的RPE细胞在冷冻保存后很大程度上保持了这些特性。总之，我们的研究强调，成人真皮成纤维细胞可以作为iPSC衍生的RPE的宝贵资源，其特征高度令人联想到真正的RPE细胞。这将允许其广泛应用于建立RPE相关人类疾病的细胞模型。本文的在线版本（doi：10.1007/s12017 - 014 - 8308 - 8）包含补充材料，可供授权用户使用。
Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here, we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4, SOX2, KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology, chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65, RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane, and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together, our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases. The online version of this article (doi:10.1007/s12017-014-8308-8) contains supplementary material, which is available to authorized users.
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影响因子：
2.9
作者：
Mekala, Subba Rao;Vauhini, Vasundhara;Mariappan, Indumathi
通讯作者：
Mariappan, Indumathi
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2
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Yamanaka, Shinya
影响因子：
64.5
作者：
Takahashi, Kazutoshi;Tanabe, Koji;Yamanaka, Shinya
通讯作者：
Yamanaka, Shinya
影响因子：
3.3
作者：
Rowland, Teisha J.;Blaschke, Alison J.;Clegg, Dennis O.
通讯作者：
Clegg, Dennis O.