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Transretinal migration of astrocytes and brain/spinal cord-like cells arising from subretinally transplanted human retinal organoids

ABSTRACT: Subretinal transplantation of human retinal organoid-derived cells can potentially restore vision lost in photoreceptor dystrophies. Prior studies analyzed the maturation, integration, and function of transplanted organoid cells in the subretinal region, but detailed molecular analysis of transplanted photoreceptor and of cells that migrate to other retinal layers has not been conducted. Here, we characterized migratory and non-migratory organoid-derived cells following subretinal transplantation into a dystrophic host. Retinal organoids derived from Crx-tdTomato+ H9 human embryonic stem cells (hESC) (aged D134) were transplanted into immunodeficient Rd1/NS mice. Graft-derived cells using single-cell RNA sequencing and histological analysis were analyzed 4.5 months later. Age-matched human retinal organoids maintained in vitro served as controls. Graft-derived cells migrated to all retinal layers, and underwent long-distance tangential migration. Transcriptomic analysis of transplanted cells identified two cell types not found in cultured controls – PAX2-positive retinal astrocytes, and ARX/NKX2-2/HOXC8-positive brain/spinal cord-like neural precursors. Some migrating cells were proliferative, but overall less proliferation was observed in the transplants than the cultured organoids. Transplanted rod and cone photoreceptors remained in the subretinal space, and were more mature than age-matched photoreceptors in cultured organoids. This study shows that extrinsic signals present in the degenerative subretinal space promote maturation of photoreceptors in transplanted retinal organoids, while also inducing formation of migratory cell populations that are not normally derived from retinal progenitors, either in cultured organoids or in vivo. This has important implications for the design and safety of cell-based therapies for treating photoreceptor dystrophies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE197847 | GEO | 2023/05/10

REPOSITORIES: GEO

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Project description:Photoreceptor disorders are collectively known as retinal degeneration (RD), and include retinitis pigmentosa (RP), cone-rod dystrophy and age related macular degeneration (AMD). These disorders are largely genetic in origin; individual mutations in any one of >200 genes cause RD, making mutation specific therapies prohibitively expensive. A better treatment plan, particularly for late stage disease, may involve stem cell transplants into the photoreceptor or ganglion cell layers of the retina. Stem cells from young mouse retinas can be transplanted, and can form photoreceptors in adult retinas. These cells can be grown in tissue culture, but can no longer form photoreceptors. We have used microarrays to investigate differences in gene expression between cultured retinal progenitor cells (RPCs) that have lost photoreceptor potential, postnatal day 1 (pn1) retinas and the postnatal day 5 (pn5) retinas that contain transplantable photoreceptors. We have also compared FACS sorted Rho-eGFP expressing rod photoreceptors from pn5 retinas with Rho-eGFP negative cells from the same retinas. We have identified over 300 genes upregulated in rod photoreceptor development in multiple comparisons, 37 of which have been previously identified as causative of retinal disease when mutated. It is anticipated that this research should bring us closer to growing photoreceptors in culture and therefore better treatments for RD. This dataset is also a resource for those seeking to identify novel retinopathy genes in RD patients. We extracted whole retinas from postnatal day 1 (Pn1) and postnatal day 5 (Pn5) mice, and compared them with cultured RPCs derived from pn5 retinas, using Affymetrix mouse 430A_2 arrays. We also extracted cells from Rho-eGFP Pn5 retinas and FACS sorted them. GFP+ve cells represent immature rod photoreceptors, as they express the Rho-eGFP fusion protein, which is only expressed in rods. GFP-ve cells represent all other retinal neurons. These samples were amplified and compared using Affymetrix mouse 430A_2arrays, by Source Biosciences GMBH, Berlin, Germany. Results from immature rods were then compared with those from other retinal neurons, while results from whole Pn5 retinas were compared with Pn1 retinas (which don't yet express rod specific genes), and RPCs, which are glial precursors. RPCs were also compared with Pn1 retinas. Genes which showed changed expression profiles in at least 3/4 of comparisons were prioritised for further investigation.

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Transretinal migration of astrocytes and brain/spinal cord-like cells arising from subretinally transplanted human retinal organoids

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