在哺乳动物眼睛发育的早期,VSX2,BRN3b,RCVRN表达标记神经视网膜祖细胞(NRP),视网膜神经节细胞,和光感受器(PR),分别。从人类诱导的多能干细胞(hiPSC)产生视网膜类器官的能力具有模拟人类视网膜发育和视网膜疾病的巨大潜力。然而,没有方法允许同时,目前存在对发育过程中多种特定视网膜细胞类型的实时监测。
在hiPSC中CRISPR/Cas9介导的同源定向修复(HDR)促进了VSX2(祖细胞)的替换,BRN3b(神经节),和RCVRN(光感受器)终止密码子,其序列编码与Cerulean融合的病毒P2A肽,绿色荧光蛋白,和mCherry报告基因,分别,以产生称为PGP1的三重转基因报道HiPSC系。这是通过将HDR模板和sgRNA/Cas9载体共电穿孔到hiPSC中,然后进行抗生素选择来实现的。PGP1hiPSC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,显示与靶基因表达的起始一致的三个荧光报告基因的表达。还通过荧光激活的细胞分选分析了解聚的类器官,并测试了荧光群体的靶向基因的表达。
从PGP1系形成的视网膜类器官表达与NRP分化一致的适当荧光蛋白,RGC,和PR。从PGP1系产生的类器官表达的转录本与所有主要视网膜细胞类型的发展一致。
PGP1系列提供了一个强大的新工具来研究视网膜发育,视网膜重新编程,和治疗药物筛选。
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, real-time monitoring of multiple specific retinal cell types during development currently exist.
CRISPR/Cas9-mediated homology-directed repair (HDR) in hiPSCs facilitated the replacement of the
VSX2 (Progenitor), BRN3b (Ganglion), and RCVRN (Photoreceptor) 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.
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.
The PGP1 line offers a powerful new tool to study retinal development, retinal reprogramming, and therapeutic drug screening.