Laboratory of Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, 325027, China.
Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Science Key Laboratory, Beijing, 100730, China.
Stem Cell Res Ther. 2020 Aug 24;11(1):366. doi: 10.1186/s13287-020-01883-5.
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.
使用人多能干细胞分化的视网膜细胞进行神经视网膜疾病的细胞替代治疗已取得显著进展。前体细胞具有低致瘤性和成熟形成突触连接的能力，是一种很有前途的供体来源。在此，我们尝试从人胚胎干细胞（hESC）中提高三维视网膜类器官中光感受器前体细胞的产量。
生成了一个带有 CRX-tdTomato 标签的 hESC 系，以在 3D 视网膜类器官中追踪视网膜前体细胞。COCO 是 Wnt、TGF-β 和 BMP 通路的多功能拮抗剂，用于 3D 类器官分化方案以增强光感受器前体细胞。使用类器官荧光强度测量来监测视网膜化趋势，并用流式细胞术进一步检查前体细胞的数量。通过 qPCR 和补充 COCO 后的免疫细胞化学评估类器官分化过程中的特征基因表达。
CRX 阳性细胞可以通过 tdTomato 进行时空追踪，而不会影响视网膜类器官分化过程。类器官的荧光强度与流式细胞术测量结果高度一致，使我们能够直接确定类器官培养过程中前体细胞的分化效率。将 COCO 用作辅助补充剂（而不是单独使用）可以在类器官分化的早期增加光感受器前体细胞的数量。在更长的时间范围内，COCO 处理后光感受器前体细胞增强了其向视锥细胞的命运，减少了向视杆细胞的命运。
使用 CRX 报告系统进行追踪表明，在人多能干细胞来源的视网膜类器官中，COCO 提高了光感受器前体细胞和视锥细胞的分化效率。
