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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE237007 | GEO | 2023/09/26

REPOSITORIES: GEO

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Project description:The loss of cone photoreceptor cells, which are critical for optimal daylight vision, have the great impact on vision during retinal degenerations. Retinal differentiation of human induced pluripotent stem cell (hiPSC) sources could provide a renewable source of cone photoreceptors towards developing a cone cell replacement therapy to treat blindness. Demonstration of comparable gene expression profiles between human foetal and stem cell-derived cones at equivalent stages is required to progress the cell transplantation approach into the patient, as it is hypothesised stem cell-derived cones are required to show high levels of developmental recapitulation of the in vivo generated cones. In this study, the AAV2/9.pR2.1.GFP reporter was used to specifically label L/M-opsin cone photoreceptors in human foetal retinal samples, obtained from the MRC-Wellcome Trust Human Developmental Biology Resource, at a range of developmental stages. The L/M-opsin cone population represent the majority cone cell types in the adult human retina and are the only photoreceptors present within the fovea. Using fluorescence activated cell sorting, L/M-opsin GFP+ cones and GFP- retinal populations, alongside total foetal retinal samples containing all retinal cell tytpes, were isolated and processed for bulk RNA sequencing and downstream comparative analysis. Using DESeq2 differential gene expression analyses, statistically significant genes enriched within the GFP+ human foetal LM-opsin cone populations were determined which led to the identification of a cone enriched gene signature of human L/M-opsin cone photoreceptors. The AAV2/9.pR2.1.GFP reporter was applied to hiPSC-derived retinal cultures to isolate and process cone-like cell populations for RNA sequencing using the same strategy developed within the human foetal retina. Applying the cone enriched gene signature to the transcriptome of hiPSC-derived GFP+ samples at equivalent developmental stages revealed some expression similarities in genes found to be enriched within the late foetal L/M-opsin cone photoreceptors. This analysis overall revealed an intermediate stage of cone differentiation was achieved within the hiPSC-derived samples and the comparison to human foetal L/M-opsin gene express profiles suggesting further differentiation of hiPSC-derived sample is required.

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

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