ScRNAseq comparison of iPSC-derived retinal organoids from Control and LCA7 patients
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE184080 | GEO | 2021/09/17
REPOSITORIES: GEO
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Project description:Mutations in the cone-rod homeobox (CRX) transcription factor lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs mutation, we established an in vitro model of CRX-LCA in retinal organoids that exhibit defective photoreceptor maturation by histology and gene profiling including diminished expression of visual opsins. Gene therapy by delivery of an additional correct CRX allele using an AAV vector partially restored photoreceptor phenotype and expression of phototransduction-related genes as revealed by single cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying a K88N mutation revealed loss of opsin expression as a common phenotype, which could also be alleviated by AAV-mediated overexpression of CRX. Our studies provide the proof-of-concept for development of gene therapy for dominant CRX-LCA and other CRX-retinopathies.
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Project description:Mutations in RP2 lead to a severe form of X-linked retinitis pigmentosa (XLRP). RP2 functions as a GTPase activating protein (GAP) for the small GTPase ARL3, which is essential for cilia function and for photoreceptor development and maintenance. The mechanisms of RP2 associated retinal degeneration in humans are poorly understood, and genetically engineered animal models of RP2 XLRP present with differing retinal phenotypes and slow degeneration suggesting potential species differences. Here, we developed CRISPR gene edited isogenic RP2 knock-out (RP2 KO) induced pluripotent stem cells (iPSC) and RP2 patient derived iPSC to produce 3D retinal organoids as a human retinal disease model. The isogenic RP2 KO retinal organoids and two unrelated RP2 patient iPSC lines produced retinal organoids with a defined photoreceptor cell layer (ONL) containing rod and cone photoreceptors. Strikingly the RP2 KO and RP2 patient derived organoids showed a thinning of the ONL by 180 days (D180) of culture, which was associated with a spike in cell death in the ONL at D150 of differentiation. RNA sequencing confirmed induction of cell death pathways in the RP2 null organoids at this stage. Photoreceptor cell death and ONL thinning was attributed to cells undergoing terminal rod cell differentiation characterized by reduced RHO expression and fewer rhodopsin immunoreactive photoreceptors. Gene augmentation with a human RP2 transgene in an AAV2/5 vector efficiently transduced RP2 KO organoids and led to high levels of RP2 expression in both rods and cones. Importantly, the viral transduction significantly increased ONL thickness and restored rhodopsin expression, suggesting rescue of the RP2 degeneration phenotype. These data show that 3D retinal organoids can be used to model molecular defects associated with inherited retinal disease, photoreceptor cell death and also to test potential therapies targeted to prevent photoreceptor degeneration.
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Project description: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.
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Project description:scRNAseq comparison of iPSC-derived retinal organoids from Control and LCA7 patients
| PRJNA763113 | ENA
Project description:Retinitis pigmentosa (RP) is an irreversible and inherited retinopathy. RPGR mutations are the most common causes of this disease. It remains challenging to decipher the mechanism of RPGR mutation because of the lack of appropriate study models. The substitution of patient-specific diseased retina without ethical restrictions is desired and iPSC-derived 3D retina is the best choice. In our experiment, we generated iPSCs from one RP patient with 2-bp frameshift mutation in the exon14 of RPGR gene, which were differentiated into retinal organoids. Also we generated iPSCs from a normal control and differentiated those control-iPSCs into healthy retinal organoids. Samples of patient- and control-retinal organoids at W0, W7, W13 (two replicates), W18 (two replicates) and W22 (two replicates for patient) were collected for RNA-seq. Corrected-iPSC were derived from CRISPR/Cas9-mediated gene correction. Then we collected the corrected-iPSC derived retinal organoids at W0, W7, W13 (two replicates), W18 (two replicates) and W22 (two replicates) for RNA-seq. Through the RNA-seq data, we demonstrate that patient-specific iPSC-dervied 3D retinae can recapitulate disease progress of Retinitis Pigmentosa through presenting defects in photoreceptors' gene profile. CRISPR/Cas9-mediated gene correction can rescue photoreceptor gene profile. Those transcriptome are consistent with the phenotype and function.
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