Bioprinted 3D Outer Retina Barrier Uncovers RPE-dependent Choroidal Phenotype in Advanced Macular Degeneration
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA887751 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21830500.fastq.gz | Fastqsanger.gz | |||
| SRR21830501.fastq.gz | Fastqsanger.gz | |||
| SRR21830502.fastq.gz | Fastqsanger.gz | |||
| SRR21830503.fastq.gz | Fastqsanger.gz | |||
| SRR21830504.fastq.gz | Fastqsanger.gz |
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2013-12-31 | E-GEOD-51246 | biostudies-arrayexpress
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Project description:Background: To identify and functionally annotate cell type-specific gene expression in the human retinal pigment epithelium (RPE), a key tissue involved in age-related macular degeneration and retinitis pigmentosa. Methodology: RPE, photoreceptor and choroidal cells were isolated from selected freshly frozen healthy human donor eyes using laser microdissection. RNA isolation, amplification and hybridization to 44k microarrays was carried out according to Agilent specifications. Bioinformatics was carried out using Rosetta Resolver, David and Ingenuity software. Principal Findings: Our previous 22k analysis of the RPE transcriptome showed that the RPE has high levels of protein synthesis, strong energy demands, is exposed to high levels of oxidative stress and a variable degree of inflammation. We currently use a complementary new strategy aimed at the identification and functional annotation of RPE-specific expressed transcripts. This strategy takes advantage of the multilayered cellular structure of the retina and overcomes a number of limitations of previous studies. In triplicate, we compared the transcriptomes of RPE, photoreceptor and choroidal cells and we deduced RPE specific expression. We identified at least 114 entries with RPE-specific gene expression. Thirty-nine of these 114 genes also show high expression in the RPE, comparison with the literature showed that 85% of these 39 were previously identified to be expressed in the RPE. In the group of 114 RPE specific genes there was an overrepresentation of genes involved in (membrane) transport, vision and ophthalmic disease. More fundamentally, we found RPE-specific involvement in the RAR-activation, retinol metabolism and GABA receptor signaling pathways. Conclusions: In this study we provide a further specification and understanding of the RPE transcriptome by identifying and analyzing genes that are specifically expressed in the RPE. Six RPE samples, three phot samples, three choroid samples
2010-06-08 | E-GEOD-20191 | biostudies-arrayexpress
Project description:To investigate the roles of microRNAs in the aqueous humor of patients with typical age-related macular degeneration and polypoidal choroidal vasculopathy using next-generation sequencing.
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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.
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Project description:Age related macular degeneration (AMD) is a leading cause of legal blindness. Vision loss is caused by the retinal pigment epithelium (RPE) and photoreceptors loss and/or retinal and choroidal angiogenesis. Here we report enhanced polarised secretion of extracellular vesicles (EVs) in complement factor H (CFH) Y402H AMD patient specific RPE. As indicated by multi ‘omics analyses, AMD RPE EVs carry a repertoire of RNA, proteins and lipids that reflect disease changes in the RPE cells of origin and mediate pathological processes such as oxidative stress, cytoskeletal dysfunction and angiogenesis. We demonstrate that exposure of control RPE to AMD RPE EVs leads to the formation of numerous cytoplasmic stress vacuoles, cytoskeletal destabilization, and abnormalities in the morphology of the nucleus in the recipient cells. Treatment of laminated retinal organoids containing photoreceptor cells with apical AMD RPE EVs leads to disrupted neuroepithelium and appearance of enlarged cells immunopositive for cytoprotective alpha B crystallin. The presence of cells expressing alpha B crystallin and progenitor cells markers Pax6 or Vsx2 are consistent with the activation of regenerative pathways upon injury. These findings suggest that AMD RPE EVs act as signalling messengers in the outer retina and may be involved in disease progression.
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