Significant progress has been made in cell replacement therapy for neural retinal diseases using retinal cells differentiated from human pluripotent stem cells. Low tumorigenicity and the ability to mature to form synaptic junctions make precursor cells a promising donor source. Here, we attempted to improve the yield of photoreceptor precursor cells in three-dimensional retinal organoids from human embryonic stem cells (hESCs). A CRX-tdTomato-tagged hESC line was generated to track retinal precursors in 3D retinal organoids. COCO, a multifunctional antagonist of the Wnt, TGF-β, and BMP pathways, was employed to 3D organoid differentiation schemes for enhanced photoreceptor precursor cells. Organoid fluorescence intensity measurement was used to monitor retinalization tendency with the number of precursors further checked by flow cytometry. Signature gene expression during organoid differentiation were assessed by qPCR and immunocytochemistry after COCO supplementation. CRX-positive cells can be spatiotemporally tracked by tdTomato without affecting retinalization during retinal organoid differentiation. Fluorescence intensity of organoids, which turned out highly consistent with flow cytometry measurement, allowed us to determine the differentiation efficiency of precursors during organoid culturing directly. Using COCO as an auxiliary supplement, rather than alone, can yield an increased number of photoreceptor precursors in the early stage of organoid differentiation. Over a longer time-frame, photoreceptor precursors enhanced their fate of cones and decreased fate of rods after treatment with COCO. Tracing with the CRX-reporter system showed that in retinal organoids derived from human pluripotent stem cells, COCO increased the differentiation efficiency of photoreceptor precursors and cones.

中文翻译：

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COCO可提高早期人类胚胎干细胞来源的视网膜类器官中感光细胞前体分化的效率。

使用从人多能干细胞分化而来的视网膜细胞，在神经视网膜疾病的细胞替代治疗中取得了重大进展。低致瘤性和成熟形成突触连接的能力使前体细胞成为有希望的供体来源。在这里，我们试图提高人类胚胎干细胞（hESCs）的三维视网膜类器官中感光细胞前体细胞的产量。生成了CRX-tdTomato标签的hESC系，以追踪3D视网膜类器官中的视网膜前体。WCO，TGF-β和BMP途径的多功能拮抗剂COCO被用于3D类器官分化方案，以增强感光细胞前体细胞。使用类器官荧光强度测量来监测视网膜化趋势，并通过流式细胞术进一步检查前体的数量。补充COCO后，通过qPCR和免疫细胞化学评估类器官分化过程中的签名基因表达。tdTomato可以时空追踪CRX阳性细胞，而不会在视网膜类器官分化过程中影响视网膜形成。类器官的荧光强度与流式细胞仪测量高度一致，这使我们能够直接确定类器官培养过程中前体的分化效率。在类器官分化的早期，使用COCO作为辅助补充剂（而不是单独使用）可以产生数量增加的感光受体前体。在更长的时间内，用COCO处理后，光感受器前体增强了视锥细胞的命运，并降低了视杆细胞的命运。