Generation of a Retina Reporter hiPSC Line to Label Progenitor, Ganglion, and Photoreceptor Cell Types
Generation of a Retina Reporter hiPSC Line to Label Progenitor, Ganglion, and Photoreceptor Cell Types
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DOI：
10.1167/tvst.9.3.21
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发表时间：
2020-02-01
影响因子：
3
通讯作者：
Robinson, Michael L.
中科院分区：
文献类型：
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作者：
Lam, Phuong T.;Gutierrez, Christian;Robinson, Michael L.
目的：在哺乳动物眼发育早期，VSX2、BRN3b和RCVRN的表达分别标记视网膜神经前体细胞(NRPs)、视网膜神经节细胞(RGCs)和光感受器(PR)。从人诱导多能干细胞(HiPSC)中产生视网膜类器官的能力在模拟人类视网膜发育和视网膜疾病方面具有巨大的潜力。方法：CRISPR/Cas9介导的同源定向修复(HDR)促进了VSX2(ROGIGITOR)、BRN3b(神经节)和RCVRN(光感受器)终止密码子的替换，使编码病毒P2A肽的序列分别与Cerulean、绿色荧光蛋白和mCherry报告基因融合，产生了一个名为PGP1的三重转基因报告细胞系。这是通过将HDR模板和sgRNA/Cas9载体共电穿孔到HiPSCs中，然后选择抗生素来实现的。PGP1 hiPSC系的功能验证包括产生视网膜类器官的能力，所有主要的视网膜细胞类型，显示三个荧光报告基因的表达与靶基因表达的开始一致。结果：从PGP1系形成的视网膜有机体表达了与NRPs、RGCs和PR分化一致的适当的荧光蛋白。结论和翻译相关性：PGP1系为研究视网膜发育、视网膜重编程和治疗药物筛选提供了一个强大的新工具。
Purpose: Early in mammalian eye development, VSX2, BRN3b, and RCVRN expression marks neural retinal progenitors (NRPs), retinal ganglion cells (RGCs), and photoreceptors (PRs), respectively. The ability to create retinal organoids from human induced pluripotent stem cells (hiPSC) holds great potential for modeling both human retinal development and retinal disease. However, no methods allowing the simultaneous, realtime monitoring of multiple specific retinal cell types during development currently exist.Methods: CRISPR/Cas9-mediated homology-directed repair (HDR) in hiPSCs facilitated the replacement of the VSX2 (rogenitor), BRN3b (Ganglion), and RCVRN (hotoreceptor) stop codons with sequences encoding a viral P2A peptide fused to Cerulean, green fluorescent protein, and mCherry reporter genes, respectively, to generate a triple transgenic reporter hiPSC line called PGP1. This was accomplished by co-electroporating HDR templates and sgRNA/Cas9 vectors into hiPSCs followed by antibiotic selection. Functional validation of the PGP1 hiPSC line included the ability to generate retinal organoids, with all major retinal cell types, displaying the expression of the three fluorescent reporters consistent with the onset of target gene expression. Disaggregated organoids were also analyzed by fluorescence-activated cell sorting and fluorescent populations were tested for the expression of the targeted gene.Results: Retinal organoids formed from the PGP1 line expressed appropriate fluorescent proteins consistent with the differentiation of NRPs, RGCs, and PRs. Organoids produced from the PGP1 line expressed transcripts consistent with the development of all major retinal cell types.Conclusions and Translational Relevance: The PGP1 line offers a powerful new tool to study retinal development, retinal reprogramming, and therapeutic drug screening.