ScRNA-seq analysis of in vivo rat choroid and rat choroid in vitro co-cultured with human pluripotent stem cell (hPSC) derived endothelial cells
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ABSTRACT:
ORGANISM(S): Rattus norvegicus Homo sapiens
PROVIDER: GSE235990 | GEO | 2024/03/13
REPOSITORIES: GEO
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Project description:The activity and survival of retinal photoreceptors depend on support functions performed by the retinal pigment epithelium (RPE) and on oxygen and nutrients delivered by blood vessels in the underlying choroid. By combining single cell and bulk RNA sequencing, we categorized mouse RPE/choroid cell types and characterized the tissue-specific transcriptomic features of choroidal endothelial cells. We found that choroidal endothelium adjacent to the RPE expresses high levels of Indian Hedgehog, and identified its downstream target as stromal GLI1+ mesenchymal stem cell-like cells. Genetic impairment of Hedgehog signaling in vivo induced significant loss of choroidal mast cells, as well as an altered inflammatory response and exacerbated visual function defects after retinal damage. Our studies reveal the cellular and molecular landscape of adult RPE/choroid and uncover a Hedgehog-regulated choroidal immunomodulatory signaling circuit. These results open new avenues for the study and treatment of retinal vascular diseases and choroid-related inflammatory blinding disorders.
2020-02-28 | GSE135167 | GEO
Project description:To compare the gene expression profiles of unpassaged, proliferating HUVEC and human iris, retinal and choroidal microvascular endothelial cells. Gene expression profiling revealed significant differences between HUVEC and ocular microvascular endothelial cells suggesting that HUVE cells may not be a suitable surrogate when studying pathophysiological mechanisms of ocular disorders. There were significant differences in the gene expression of important cell signalling pathways in human retinal and choroidal ECs. These differences may be important in the mechanisms and treatment of choroidal and retinal neovascularisation. 12 arrays are included. Endothelial cells were derived from 4 tissues: iris, retina, choroid and human umbilical vein. RNA extracts from cells were hybridised to Affymetrix HGU133plus2 arrays in triplicate.
2010-12-31 | E-GEOD-20986 | biostudies-arrayexpress
Project description:Purpose: The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch’s membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Methods: ECs from P5 and P30 mice were labeled in vivo by retro-orbital injection of fluorescently-labeled anti-VE-Cadherin. After 10 minutes, mice were euthanized, eyeballs were enucleated and the anterior segment was discarded. After removal of the neural retina, RPE/choroid was mechanically dissected from the sclera and digested. ECs were isolated by flow cytometry and processed immediately for RNA extraction. Results: Transcriptome analyses show that whereas P5 choroid EC transcriptome is preferentially enriched in cell cycle- and chromosome-related transcripts, reflecting an immature phenotype, the transcriptome of adult (P30) choroid ECs is enriched in genes encoding proteins involved in ‘biological adhesion’, including a variety of extracellular matrix (ECM)-related genes. Conclusion: these results strongly suggest that mature choroid ECs actively participate in extracellular matrix assembly and regulation.
2017-05-01 | GSE95835 | GEO
Project description:Purpose: The goals of this study were to identify cell specific expression patterns from infant and aged choroid, with a particular emphasis on how endothelial gene expression changes with age. Methods: Independent libraries were prepared for infant and adult choroidal cells from seventeen human donors. Trephine punch biopsies were used to acquire central RPE and choroidal tissue (infants: 4 mm punch biopsy; adults: 8 mm hemisected punch biopsy). Poly-A selected RNA transcripts underwent 150 base-pair paired-end sequencing with an Illumina HiSeq 2000. Reads were mapped to the human genome hg19 with STAR (version = 020201) and quantified with the featureCounts function from the Rsubread package (version = 1.28.1). Differential expression analysis was performed with edgeR (version = 3.26.8). Results: Differential expression analysis identified genes enriched in infant versus adult libraries. Differentially expressed genes (p-value < 0.00001, FDR < 0.001, cpm >2, logFC > 1) were input into WebGestalt for over-representation analysis. Aged choroidal samples were enriched in proinflammatory genes. Conclusions: This study provides a transcriptomic comparison of infant and aged human donor choroid at the bulk and single-cell levels. The aged choroid, in particular the aged vasculature, is enriched in pro-inflammatory genes.
2020-04-23 | GSE149099 | GEO
Project description:The choroid is the vascular layer situated between the outer sclera and the inner retina. Together with the ciliary body and the iris it forms the uvea. Because of its rich blood supply it is also called the nutritive layer and it supplies oxygen and nutrition to the retinal pigment epithelium and the outer retina. Due to its vascular nature it acts as a heat sink and helps in thermoregulation within the eye. It also contains a pigment “melanin”, which absorbs excess light within the eye and prevents scattering. . The human choroid is thickest posteriorly where it measures 0.2 mm, whereas anteriorly it measures 0.1 mm.. The aim of this study was to characterize the choroid proteome to generate a resource for future studies on the choroid . The method used in this study combined bRPLC and LC-MS/MS analysis of the proteins isolated from the three cadaver samples of healthy donors. Subsequently, we classified the proteins based on gene ontology and pathway analysis. A total of 5,249 non redundant proteins were identified in the human choroid. Gene ontology classification pinpointed proteins involved in protein metabolism, regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism, transport, cell growth and/or maintenance and immune response. Several proteins identified in normal choroid have previously been identified in human choroid where these proteins are related to glaucoma, ocular inflammation, toxoplasmic retinochoroiditis, diabetic retinopathy, open-angle glaucoma and Grave’s disease. The top canonical pathway in which the choroid proteins participated are EIF2 signaling, integrin signalling, mitochondrial dysfunction, regulation of eIF4 and p70S6K signaling and clathrin-mediated endocytosis signalling. Around 800 choroid proteins were related to infectious diseases. The results of this study illustrate the largest number of proteins identified in human choroid and may serve as a valuable resource for future investigations of choroid biology and disease. Proteomic analysis of choroid proteins could provide versatile information to understand choroidal functions and the underlying pathogenesis of choroidal pathologies.
2018-04-25 | PXD002273 | Pride
Project description:Purpose: The goals of this study were to identify cell specific expression patterns of the retinal pigment epithelium (RPE) and a variety of choroidal cell types originating from the macula and the periphery of human donor eyes, with a particular emphasis on identifying expression signatures of choroidal endothelial cells. Methods: Independent libraries were prepared for macular and peripheral samples of combined RPE/choroid from seven donors in two single cell sequencing experiments. In the first experiment, 8-mm macular and peripheral punches of RPE/choroid from Donors 1-3 were digested in papain prior to cryopreservation and subsequent GEM barcoding/library construction. Donors 1-2 had no noted ophthalmologic disease documented, while Donor 3 was diagnosed with age-related macular degeneration. In the second experiment, 12-mm macular and peripheral punches of RPE/choroid from Donors 4-7 were digested in collagenase II prior to cryopreservation, CD31-magentic bead enrichment and subsequent GEM barcoding/library construction. Donors 5-7 had no noted ophthalmologic disease documented, while Donor 4 was diagnosed with age-related macular degeneration. In both experiments, libraries were sequenced on a HiSeq4000. Sequenced reads were mapped to the human genome build hg19 will CellRanger(v3.0.1) and filters removed cells likely to be doublets or cells with a high proportion of mitochondrial reads. Clustering of cells with similar expression profiles was performed with Seurat (v3.0.2). Results: In the first experiment with unenriched RPE/choroid from donors 1-3, we recovered 4,355 cells post filtering with 40,177,477 corresponding reads. A total of 2,167 cells originated from the macula and 2,168 cells originated from the periphery. A total of 11 clusters were identified, and highly enriched genes in each cluster were used to classify clusters into their presumed dominant cell type. Differential expression analysis was performed between cells of macular and peripheral origin in each cluster and between donors with and without a history of age-related macular degeneration. Results: In the second experiment with CD31-enriched RPE/choroid from donors 4-7, we recovered 14,234 cells post filtering with 135,742,297 corresponding reads. A total of 7,647 cells originated from the macula and 6,587 cells originated from the periphery. A total of 8,521 of these cells were presumed endothelial cells. A total of 13 clusters were identified, and highly enriched genes in each cluster were used to classify clusters into their presumed dominant cell type(s). Differential expression analysis was performed between cells of macular and peripheral origin in each cluster and between donors with and without a history of age-related macular degeneration. Within each of the endothelial clusters (Clusters 5-8), differential expression analysis was performed between each cluster and the remaining endothelial cells. Conclusions: This study provides a large atlas of single-cell level gene expression patterns of the human retinal pigment epithelium and choroidal cell types. We identify expression patterns of most expected choroidal cell populations and describe macular versus peripheral regional differences. We additionally identify enriched transcripts in specific cell populations in age-related macular degeneration. We characterize gene expression patterns along the choroidal vascular tree, and identify populations of arterial, capillary, and venous endothelial cells. Our results show that single-cell sequencing can be performed on human RPE/choroid after cryopreservation, and that CD31 magnetic bead enrichment can be employed to enrich for endothelial cells for single-cell sequencing.
2019-10-30 | GSE135922 | GEO
Project description:Purpose: The goals of this study were to identify cell specific expression patterns from infant and aged choroid, with a particular emphasis on how endothelial gene expression changes with age. Methods: Independent libraries were prepared for infant and adult choroidal cells from four human donors. Macular and peripheral samples from single-cell donors 18-21 were acquired, analyzed, and uploaded in a previous experiment (GSE135922). In single-cell donors 22-25, new reads were acquired from a 12-mm macula-centered punch. Choroidal punches gently dissociated in collagenase II for 1 hours before cryopreservation. Thawed cells were enriched endothelial cells by incubating with anti-CD31-microbeads. Both CD31-positive and CD31-negative libraries from single-cell donors 22-25 were sequenced on an Illumina NovaSeq system. Sequenced reads were mapped to the human genome build hg19 with CellRanger(v3.0.1) and filtered to remove cells likely to be doublets or cells with a high proportion of mitochondrial reads. Libraries from single-cell donors 22-25 were aggregated with donors 18-21 using canonical correlation analysis. Feature selection was performed with the variance stabilizing transformation before integration was performed with Seurat (v3.0.1). Results: A total of 37,070 cells were recovered after filtering with 415,740,738 corresponding reads. A total of 17,229 cells were recovered from infant libraries while 19,841 cells were recovered from the adult libraries. Clusters were assigned to known choroidal cell types based on expression of distinguishing marker genes. Differential expression analysis identified genes enriched in each population of cells originating from infant versus adult libraries. Particular emphasis was given to genes enriched in infant choriocapillaris endothelial cells. Conclusions: This study provides a transcriptomic comparison of infant and aged human donor choroid at the bulk and single-cell levels. The aged choroid, in particular the aged vasculature, is enriched in pro-inflammatory genes.
2020-04-23 | GSE149100 | GEO
Project description:ScRNA-seq analysis of in vivo rat choroid and rat choroid in vitro co-cultured with human pluripotent stem cell (hPSC) derived endothelial cells
| PRJNA988130 | ENA
Project description:To investigate the tRF/tiRNA expression pattern in the development of choroidal neovascularization, we built a laser-induced choroidal neovascularization mouse model and performed the tRF/tiRNA sequencing on RPE-choroid-sclera complexes at different time points after model establishment, while the tissues from mice without treatment were used as control group.
2022-08-05 | GSE210099 | GEO
Project description:To investigate the differences in gene expression between matched human macular and peripheral choroidal endothelial cells (CEC) and matched human macular inner and outer CEC. The gene expression profiles of the unpassaged cell types were compared using Affymetrix GeneChip arrays. 9 arrays are included. 6mm diameter samples were dissected from 3 regions of the eye: peripheral inner choroid, macular outer choroid and macular inner choroid. RNA extracts from cells were hybridised to Affymetrix HGU133plus2 arrays in triplicate.
2013-12-31 | E-GEOD-51246 | biostudies-arrayexpress