Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Effect of cAMP and seeding density on RPE redifferentiation

ABSTRACT: Retinal pigment epithelium (RPE) cell integrity is critical to the maintenance of retinal function. Many retinopathies such as age-related macular degeneration (AMD) are caused by the degeneration or malfunction of the RPE cell layer. Replacement of diseased RPE with healthy, stem cell derived RPE is a potential therapeutic strategy for treating AMD. Human embryonic stem cells (hESC) differentiated into RPE progeny have potential to provide an unlimited supply of cells for transplantation but challenges around scalability and efficiency of the differentiation process still remain. Using hESC-derived RPE as a cellular model, we sought to understand mechanisms that could be modulated to increase RPE yield following differentiation. Our data show that activation of the cAMP pathway increases proliferation of dissociated RPE in culture, in part through inhibition of TGFβ signalling. This in turn results in enhanced uptake of epithelial identity. In line with these findings, targeted manipulation of the TGFβ pathway with small molecules produces an increase in efficiency of RPE re-epithelialization. Taken together, these data highlight mechanisms that promote epithelial fate acquisition in stem cell derived RPE. Modulation of these pathways has potential to favorably impact upon scalability and clinical translation of hESC-derived RPE as a cell therapy. A sample of Gene Pool™ cDNA, from human fetal normal brain tissue (Invitrogen D8830-01) is included for reference.

ORGANISM(S): Homo sapiens

SUBMITTER: Alex Gutteridge

PROVIDER: E-MTAB-3878 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Age-related macular degeneration (AMD) is caused by the degeneration of the macular photoreceptors. This is preceded by development of subretinal protein aggregates (drusen) and degeneration of the macular retinal pigment epithelium (RPE). The underlying pathogenesis remains unknown, but the immune system is suspected to play a key role in it. Aging of the immune system, immunosenescence, includes changes in T cell sub-populations and results in low-level chronic inflammation. Because AMD exclusively occurs in patients over 55 years of age, we hypothesize that aging of the T cell compartment may be implicated in AMD pathogenesis. Methodology and principal findings: Using an in vitro co-culture system, we investigated the effects of activated T cells on a human RPE cell line (ARPE-19). Differential gene expression in the RPE cells of complement factor genes was identified using microarrays, and selected gene transcripts from the alternative complement pathway were validated by q-RT-PCR. Protein expression was determined by ELISA and immunoblotting. Co-culture with activated T cells increased RPE mRNA and protein expression of complement component C3, factors B, H, H-like 1, CD46, CD59, and clusterin, in a dose-dependent manner. Conclusions: RPE cells responded to inflammatory assault caused by exposure to T cell-derived cytokines by upregulating expression of many complement factors from the alternative pathway. This may have implications for RPE ability to deal with complement attack, and opsonization and removal of debris from the RPE-choroidal interface. We speculate that regulation of the complement system in response to inflammatory assault may play a vital role in RPE pathology and drusen biogenesis. Experiment Overall Design: 10 samples investigating variations of co-cultures of RPE cells and T-cells. Variable parameters include the cell type, the co-culturing of the counter-part cell type and the orientation of the system (apical vs basolateral).

Project description:Purpose: Age-related degeneration (AMD) is a major cause of blindness in developed countries. The molecular pathogenesis of early events in AMD is poorly understood. We investigated differential gene expression in samples of human retinal pigment epithelium (RPE)/choroid from early AMD and control maculas using exon-based arrays. Methods: Gene expression levels in nine early AMD and nine control human donor eyes were assessed using Affymetrix Human Exon ST 1.0 arrays. Two controls did not pass quality control and were removed. Differentially expressed genes were annotated using DAVID, and gene set enrichment analysis (GSEA) was performed on RPE-specific and endothelium-associated gene sets. CFH genotype was also assessed and differential expression was analyzed with respect to high AMD risk (YH/HH) and low AMD risk (YY) genotypes. Results: Seventy-five genes were identified as differentially expressed (raw p-value < 0.01; >50% fold change, mean log2 expression level in AMD or control M-bM-^IM-% median of all average gene expression values); however, no genes were significant (adj. p-value < 0.01) after correction for multiple hypothesis testing. Of 52 genes with decreased expression in AMD (fold change < 0.5; raw p-value < 0.01), 18 genes were identified by DAVID analysis as associated with vision or neurological processes. GSEA of RPE-associated and endothelium-associated genes revealed a significant decrease in genes typically expressed by endothelial cells in the early AMD group compared to controls, consistent with previous histologic and proteomic studies. Analysis with respect to CFH genotype indicated decreased expression of ADAMTS9 in eyes with high-risk genotypes (fold change = -2.61; raw p-value = 0.0008). Conclusions: GSEA results suggest that RPE transcripts are preserved or elevated in early AMD, concomitant with loss of endothelial cell marker expression. These results are consistent with the notion that choroidal endothelial cell dropout occurs early in the pathogenesis of AMD. Using AltAnalyze (ver. 2.0.7 beta), we analyzed nine early AMD and nine control eyes using Affymetrix Human Exon ST 1.0 arrays. Following initial processing in AltAnalyze, two control arrays were identified as potential outliers by tests implement in arrayQualityMetrics, a package for R.

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Effect of cAMP and seeding density on RPE redifferentiation

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