Gladstone Institutes, 1650 Owens Street, San Francisco, CA 94158, USA.
Gladstone Institutes, 1650 Owens Street, San Francisco, CA 94158, USA; Developmental and Stem Cell Biology Program, University of California, 1675 Owens St, San Francisco, CA 94158, USA.
Stem Cell Res. 2021 May;53:102368. doi: 10.1016/j.scr.2021.102368. Epub 2021 Apr 27.
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, labor-intensive, 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.
年龄相关性黄斑变性和 Best 病和色素性视网膜炎等遗传性失明形式可由视网膜色素上皮 (RPE) 的退化引起。源自患者来源的诱导多能干细胞 (iPSC) 的 RPE 对于研究疾病机制和开发治疗策略都非常有价值。然而，体外产生 iPSC 衍生的 RPE 的方案通常效率低下、劳动强度大、通量低，并且在细胞系之间和批次内高度可变。在这里，我们报告了一种使用强力霉素诱导表达眼区转录因子 OTX2、PAX6 和 MITF 并与 RPE 允许的培养基相结合生成 iPSC-RPE 的稳健、可扩展的方法。强力霉素的添加诱导这些转录因子在外源性表达Best 病患者和野生型 iPSC 中，以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 单层表现出功能特征，包括通过色素沉着吸收光、极性驱动的流体运输和吞噬作用。通过这种方法，我们实现了高效且易于扩展的分化，而无需机械分离或富集方法，生成适用于体外研究的 RPE 培养物。
