Transcription factor overexpression drives reliable differentiation of retinal pigment epithelium from human induced pluripotent stem cells
Transcription factor overexpression drives reliable differentiation of retinal pigment epithelium from human induced pluripotent stem cells
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DOI：
10.1016/j.scr.2021.102368
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发表时间：
2021-05-05
影响因子：
1.2
通讯作者：
Conklin, Bruce R.
中科院分区：
文献类型：
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作者：
Dewell, Tessa E.;Gjoni, Ketrin;Conklin, Bruce R.
老年性黄斑变性和遗传性失明如Best病和视网膜色素变性可由视网膜色素上皮(RPE)退化引起。从患者来源的诱导多能干细胞(IPSCs)产生的RPE对于疾病机制的研究和治疗策略的发展都是有价值的。然而，体外生产IPSC来源的RPE的方法通常效率低、劳动强度大、产量低，并且在细胞系之间和批次内高度可变。在这里，我们报告了一种健壮的，可扩展的方法来产生IPSC-RPE，使用多西环素诱导的眼场转录因子OTX2，PAX6和MITF的表达与RPE允许的培养液配对。强力霉素的加入可以诱导这些转录因子在Best疾病患者和野生型IPSCs中外源表达，从而有效地产生具有特征形态和基因表达的RPE单层。此外，这些RPE单层还具有多种功能，包括通过色素沉着吸收光、极性驱动的流体传输和吞噬作用。通过这种方法，我们实现了高效和易于扩展的分化，而不需要机械分离或浓缩方法，生成了适用于体外研究的RPE培养物。
Age-related macular degeneration and genetic forms of blindness such as Best Disease and Retinitis Pigmentosa can be caused by degeneration of the Retinal Pigment Epithelium (RPE). RPE generated from patient-derived induced pluripotent stem cells (iPSCs) is valuable for both the study of disease mechanisms and development of therapeutic strategies. However, protocols to produce iPSC-derived RPE in vitro are often inefficient, laborintensive, low-throughput, and highly variable between cell lines and within batches. Here, we report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in Best Disease patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies.