Ning Ke, Luo Ziming, Kowal Tia J, Tran Matthew, Majumder Rishab, Jarin Trent M, Wu Albert Y, Goldberg Jeffrey L, Sun Yang
Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, CA, USA.
Palo Alto Veterans Administration, Palo Alto, CA, USA.
Stem Cells Int. 2023 Jan 13;2023:6494486. doi: 10.1155/2023/6494486. eCollection 2023.
Primary cilia are conserved organelles found in polarized mammalian cells that regulate neuronal growth, migration, and differentiation. Proper cilia formation is essential during eye development. Our previous reports found that both amacrine and retinal ganglion cells (RGCs) contain primary cilia in primate and rodent retinas. However, whether primary cilia are present in the inner retina of human retinal organoids remains unknown. The purpose of this study is to characterize the primary cilia distribution in human embryonic stem cell (hESC-derived retinal organoid development.
Retinal organoids were differentiated from a hESC line, harvested at various developmental timepoints (day 44-day 266), and immunostained with antibodies for primary cilia, including Arl13b (for the axoneme), AC3, and Centrin3 (for the basal body). AP2, Prox1, GAD67, Calretinin, GFAP, PKC, and Chx10 antibodies as well as Brn3b-promoted tdTomato expression were used to visualize retinal cell types.
A group of ciliated cells were present in the inner aspects of retinal organoids from day 44 to day 266 in culture. Ciliated Chx10-positive retinal progenitor cells, GFAP-positive astrocytes, and PKC-positive rod-bipolar cells were detected later during development (day 176 to day 266). Ciliation persisted during all stages of retinal developmental in AP2-positive amacrine cells, but it was decreased in Brn3b-positive retinal ganglion cells (RGCs) at later time points. Additionally, AC3-positive astrocytes significantly decreased during the later stages of organoid formation.
Amacrine cells in retinal organoids retain cilia throughout development, whereas RGC ciliation gradually and progressively decreases with organoid maturation.
初级纤毛是存在于极化哺乳动物细胞中的保守细胞器，可调节神经元的生长、迁移和分化。在眼睛发育过程中，正确的纤毛形成至关重要。我们之前的报告发现，在灵长类和啮齿类动物的视网膜中，无长突细胞和视网膜神经节细胞（RGCs）都含有初级纤毛。然而，人类视网膜类器官的内视网膜中是否存在初级纤毛仍不清楚。本研究的目的是表征人类胚胎干细胞（hESC）来源的视网膜类器官发育过程中初级纤毛的分布。
从一个hESC系分化出视网膜类器官，在不同发育时间点（第44天至第266天）收获，并使用针对初级纤毛的抗体进行免疫染色，包括用于轴丝的Arl13b、AC3以及用于基体的Centrin3。使用AP2、Prox1、GAD67、钙视网膜蛋白、GFAP、PKC和Chx10抗体以及Brn3b促进的tdTomato表达来可视化视网膜细胞类型。
在培养的第44天至第266天，视网膜类器官内部存在一群纤毛细胞。在发育后期（第176天至第266天）检测到有纤毛的Chx10阳性视网膜祖细胞、GFAP阳性星形胶质细胞和PKC阳性视杆双极细胞。在AP2阳性无长突细胞的视网膜发育的所有阶段，纤毛化持续存在，但在后期时间点，Brn3b阳性视网膜神经节细胞（RGCs）中的纤毛化减少。此外，在类器官形成的后期阶段，AC3阳性星形胶质细胞显著减少。
视网膜类器官中的无长突细胞在整个发育过程中保留纤毛，而随着类器官成熟，RGC的纤毛化逐渐减少。
