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Effect of Ramoplanin treatment on hESC-RPE transcriptome during POS phagocytosis

ABSTRACT: we report the effect of Ramoplanin on the transcription profile of RPE cells in comparison to DMSO (Vehicle) or MFGE8+GAS6 treated cells in the presence and absence of POS

ORGANISM(S): Homo sapiens

PROVIDER: GSE132828 | GEO | 2020/11/25

REPOSITORIES: GEO

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Effect of Ramoplanin treatment on hESC-RPE transcriptome during POS phagocytosis

Project description:Effect of Ramoplanin treatment on hESC-RPE transcriptome during POS phagocytosis

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RNA-seq of mouse cultured retinal pigment epithelium (RPE) challenged with photoreceptor outer segments (POS).

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Project description:Retinal photoreceptors undergo daily renewal of their distal outer segments, a process indispensable for maintaining retinal health. Photoreceptor Outer Segment (POS) phagocytosis occurs as a daily peak, roughly about one hour after light onset. However, the underlying cellular and molecular mechanisms which initiate this process are still unknown. Here we show that, under constant darkness, mice deficient for core circadian clock genes (Per1 and Per2), lack a daily peak in POS phagocytosis. By qPCR analysis we found that core clock genes were rhythmic over 24h in both WT and Per1, Per2 double mutant whole retinas. More precise transcriptomics analysis of laser capture microdissected WT photoreceptors revealed no differentially expressed genes between time-points preceding and during the peak of POS phagocytosis. By contrast, we found that microdissected WT retinal pigment epithelium (RPE) had a number of genes that were differentially expressed at the peak phagocytic time-point compared to adjacent ones. We also found a number of differentially expressed genes in Per1, Per2 double mutant RPE compared to WT ones at the peak phagocytic time-point. Finally, based on STRING analysis we found a group of interacting genes which potentially drive POS phagocytosis in the RPE. This potential pathway consists of genes such as: Pacsin1, Syp, Camk2b and Camk2d among others. Our findings indicate that Per1 and Per2 are necessary clock components for driving POS phagocytosis and suggest that this process is transcriptionally driven by the RPE.

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Project description:Purpose: Primary retinal pigment epithelium (RPE) cells have a limited capacity to re-establish epithelial morphology and to remain native RPE function in vitro, and all commercially available RPE cell lines have drawbacks of morphology or function; therefore, the establishment of new RPE cell lines with typical characteristics of RPE would be helpful in further understanding of their physiological and pathological mechanism. Here, we firstly report a new spontaneously generated RPE line, fhRPE-13A, from a 13-week aborted foetus. We aimed to investigate its availability as a RPE model. Methods:RNA-seq data were mapped to the human genome assembly hg19. Global transcriptional data were analyzed by Weighted Gene Co-expression Network Analysis (WGCNA) and differentially expressed genes(DEGs) to identify gene expression profiles. The morphology and molecular characteristics were examined by immunofluorescence, transmission electron micrographs, PCR and western blot. Photoreceptor outer segments (POS) phagocytosis assay and transepithelial resistance measurement (TER) were performed to assess phagocytic activity and barrier function, respectively. Results: The fhRPE-13A cells showed typical polygonal morphology and the normal biological processes of RPE, had ability of POS phagocytosis in vitro, and expressed TYR and TYRP1 significantly higher than that in ARPE-19 cells. Conclusions: The fetal human RPE line fhRPE-13A is a valuable system for researching phagocytosis function and morphogenesis of RPE in vitro.

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Project description:One of the major biological functions accomplished by the retinal pigmented epithelium (RPE) is the clearance of shed photoreceptor outer segments (POS) through a multistep process referred to as phagocytosis. Phagocytosis helps maintain the viability of photoreceptors which otherwise could succumb to the high metabolic flux and photo-oxidative stress associated with visual processing. Regulatory mechanisms underlying phagocytosis in the RPE are not fully understood, although dysfunction of this process contributes to the pathogenesis of multiple human retinal degenerative disorders, including age-related macular degeneration (AMD). Here we present an integrated analysis of phagocytosing cultured-RPE cells.

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