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Reliability of human retinal organoid generation from hiPSC-derived neuroepithelial cysts

ABSTRACT: Reliability of human retinal organoid generation from hiPSC-derived neuroepithelial cysts

PROVIDER: PRJNA993408 | ENA |

REPOSITORIES: ENA

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Reliability of human retinal organoid generation from hiPSC-derived neuroepithelial cysts

Project description:The application potential of human induced pluripotent stem cells (hiPSC) derived human retinal organoids (HRO) relies on the robustness and transferability of the methodology for their generation. Standardized strategies and parameters to effectively assess, compare and optimize organoid protocols have been started to be established, but are not completed yet. To advance this, we explored the efficiency and reliability of a differentiation protocol that facilitates retina generation by formation of neuroepithelial cysts from hiPSC clusters. Here, we tested seven different hiPSC lines, which reproducibly generated HROs. Histologic and ultrastructural analyses support regulated HRO differentiation and maturation. The different hiPSC lines appeared to be a larger source of variance than experimental rounds. Whereas previous reports showed that HRO in several other protocols contain a rather low numbers of cones compared to rods, HROs derived by the cyst protocol consistently are cone-richer and with an comparable ratio of cones, rods, and Müller glia. Additionally, we devised a potential strategy to systematically evaluate different protocols side-by-side through parallel differentiation from the same hiPSC batches: The cyst-protocol was compared to a conceptually different protocol based on cell aggregate formation from single hiPSCs. Comparison of four hIPSC lines showed that both protocols reproduced key characteristics of retinal epithelial structure and cell composition, but the cyst-protocol provided a higher HRO yield. Further, while cyst-derived HROs maintained stable at least up to date 250, whereas single hiPSC-derived HROs showed spontaneous pathologic changes already by day 200. Overall, our data provide insight into the efficiency, reproducibility, and stability of the cyst-protocol for HRO generation, which will be useful for further organoid system optimization, as well as basic and translational research applications.

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