Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids
Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids
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DOI：
10.1073/pnas.1901572116
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发表时间：
2019-05-28
影响因子：
11.1
通讯作者：
Liu, Wei
中科院分区：
文献类型：
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作者：
Kim, Sangbae;Lowe, Albert;Liu, Wei
视杆和视锥光感受器是人类视网膜中的感光细胞。视杆细胞在周边视网膜中占主导地位，而视锥细胞在黄斑中富集，黄斑负责中心视力和视敏度。黄斑变性对视力的影响最大，目前无法治愈。在这里，我们报告了使用改进的视网膜分化系统从hESC分化的富含视锥细胞的人类视网膜类器官的产生、转录组分析和功能验证。在细胞外基质的诱导下，hESC的聚集体形成由具有前神经外胚层/外胚层命运（包括视网膜细胞命运）的上皮细胞组成的单腔囊肿。然后，将囊肿整体传代，贴附于培养表面，并生长，形成集落，在其中发现视网膜祖细胞斑块。在温和的细胞脱落后，视网膜祖细胞自组装成视网膜上皮细胞-视网膜类器官-在搅拌培养中分化成分层的富含锥体的视网膜组织。电子显微镜显示感光细胞外节分化。时间进程视网膜类器官的大量RNA测序分析表明，体外视网膜分化在细胞分化标志物和视网膜疾病基因的时间表达以及mRNA选择性剪接中重演了体内视网膜发生。8个月视网膜类器官的单细胞RNA测序分析鉴定了视锥细胞和视杆细胞簇，并证实了定量显微镜最初揭示的视锥富集。值得注意的是，来自视网膜类器官和人类黄斑的视锥细胞具有相似的单细胞转录组，视杆细胞也是如此。视网膜类器官中的视锥细胞具有电生理功能。总的来说，我们已经建立了富含视锥细胞的视网膜类器官和转录组的参考，这些转录组是视网膜研究的宝贵资源。
Rod and cone photoreceptors are light-sensing cells in the human retina. Rods are dominant in the peripheral retina, whereas cones are enriched in the macula, which is responsible for central vision and visual acuity. Macular degenerations affect vision the most and are currently incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids differentiated from hESCs using an improved retinal differentiation system. Induced by extracellular matrix, aggregates of hESCs formed single-lumen cysts composed of epithelial cells with anterior neuroectodermal/ectodermal fates, including retinal cell fate. Then, the cysts were en bloc-passaged, attached to culture surface, and grew, forming colonies in which retinal progenitor cell patches were found. Following gentle cell detachment, retinal progenitor cells self-assembled into retinal epithelium-retinal organoid-that differentiated into stratified cone-rich retinal tissue in agitated cultures. Electron microscopy revealed differentiating outer segments of photoreceptor cells. Bulk RNA-sequencing profiling of time-course retinal organoids demonstrated that retinal differentiation in vitro recapitulated in vivo retinogenesis in temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-sequencing profiling of 8-mo retinal organoids identified cone and rod cell clusters and confirmed the cone enrichment initially revealed by quantitative microscopy. Notably, cones from retinal organoids and human macula had similar single-cell transcriptomes, and so did rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-rich retinal organoids and a reference of transcriptomes that are valuable resources for retinal studies.