Project description:To study the function of AMD-associated gene POLDIP2 in retinal pigment epithelial (RPE) cells, we used CRISPR/Cas to knockout POLDIP2 in the human RPE cell line ARPE19. We then performed RNA-seq to profile the transcriptome of wildtype ARPE19 and POLDIP2 knockout.
Project description:Low-level infection is believed to play a role in the degradation of the outer blood retinal barrier, which is composed of retinal pigment epithelial (RPE) cells. By investigating immunopathogenic West nile virus (WNV) infected RPE via microarray, we sought to find key genes involved in a low-level viral infection, which are vital for normal immune responses. We infected primary human RPE cells and an human RPE cell line with WNV and extracted RNA to hybridize onto Affymetrix arrays. We were interested in the global expression of differentially regulated genes in WNV infected RPE cells, at 24h post infection. We were also interested in seeing the differences in response between infected primaries and the infected ARPE19 cell line.
Project description:We generated hiPSCs from patients fibloblast with retinitis pigmentosa (RP) using retrovirus and Sendai virus vectors, which we differentiated into hiPSC derived retinal pigment epithelium using two different methods (SDIA and SFEB methods). We investigated whether these hiPSC-RPE colonies, which were differentiated from various cell lines and methods, showed similar gene expression patterns to those of native RPE. We classified hiPSC-RPE, hiPSCs, and fibroblasts from RP patients, hRPE (commercially available human fetal RPE, Lonza) , ARPE19 (a human RPE cell line), and other human tissues from 54,675 probe sets using microarray data.
Project description:The purpose of this study is to determine the changes in gene expression by a human retinal pigment epithelium (RPE) cell line (ARPE-19) in response to combination treatment of TGF and TNF, which induces phenotypic changes in vitro that mimic the EMT (Epithelial-to-Mesenchymal Transition). For this purpose, total RNA was extracted from TGF and TNF-treated ARPE-19 cells and differential gene expression between each time point (0, 1, 6, 16, 24, 42, and 60 hours) was determined using genechip arrays (Affymetrix, Human Genome U133). Experiment Overall Design: ARPE19 cell lines treated with TGF and TNF for 0, 1, 6, 16, 24, 42, and 60 hour. Each experiment were repeated three times. But 1 hour experiment was repeated two times.
Project description:Aberrant epithelial-mesenchymal transition (EMT) is involved in pathological processes including fibrotic disorders and cancer invasion and metastasis. Alterations of the cell-extracellular matrix (ECM) interaction also contribute to those pathological settings. However, the functional interplay between EMT and cell-ECM interaction is poorly understood. Here, we show that tumor necrosis factor (TNF)-alpha, a potent mediator of inflammation, induces EMT-associated fibrosis in retinal pigment epithelial cells, and that this is regulated by hyaluronan (HA)-CD44-Moesin interaction. TNF-alpha elicits both HA synthesis and Moesin phosphorylation through protein kinase C activation, promoting binding of CD44 to the newly synthesized HA. The HA-CD44-Moesin interaction leads to cell-cell dissociation through actin remodeling and increased cellular motility associated with mesenchymal phenotype. Furthermore, we established an in vivo model of TNF-alpha-induced fibrosis in the mouse eye, and the ocular fibrosis was completely suppressed in CD44-null mice. Therefore, HA production and its interaction with CD44 plays essential role in TNF-alpha-induced-EMT, and the interference of the complex formation can be a new strategy for the fibrotic disorders. ARPE19 cell lines were treated with TGF and TNF for 6 and 42 hour. Each experiment were repeated three times. But 1hour experiment was repeated two times. For this submission, total RNA was extracted from TGF- or TNF-treated ARPE-19 cells and differential gene expression between each time point (6 and 42 hours) was determined using genechip arrays (Affymetrix, Human Genome U133).
Project description:ARPE19 cells, derived from human retinal pigment epithelium (RPE), were cultured in serum supplemented media, then the serum was removed, and culture continued in serum free media. We use this culture system as a model for the early stages of Age-related macular degeneration. RNA-Seq was performed on RNAs from seven time points: day0 (serum supplemented), day1, 3, 4, 5, 6, and 9 serum deprived samples.
