Dataset Information

Restoration of mesenchymal RPE by transcription factor-mediated reprogramming

ABSTRACT: TGFbeta-mediated epithelial-to-mesenchymal transition (EMT) is a major component of the wound healing response and a negative determinant of retinal pigment epithelial (RPE) differentiation after periods of sustained sub-confluent culture or repetitive passage. Inhibition of TGFbeta signaling using receptor kinase inhibitors forestalls the onset of passage-dependent EMT and can restore the capacity to differentiate to cells that previously underwent the mesenchymal switch [Radeke et al., 2015, Genome Med. 7:58]. However, even with the sustained inhibition of mesenchymal gene expression using TGFbeta signaling inhibitors the cells eventually lose the capacity to attain a characteristic phenotype. This suggests that there are additional mechanisms at play that contributeto the prevention of RPE differentiation after protracted periods of wound stimulus and mitosis. In this study we investigate the non-TGFbeta-mediated processes that contribute to the demise of the RPE phenotype after extended periods of proliferative wound response. Using comparative transcriptomics, we show that with increasing passage there is a downregulation of RPE genes, misregulation of cell cycle genes, a decline in proliferative potential that cannot be prevented or reversed by inhibition of TGFbeta signaling using the TGFbeta receptor kinase inhibitor A-83-01. Importantly, among the RPE genes with decreased expression are several transcription factors known to be critical for RPE development. Exogenous expression of MYCN and OTX2 in conjunction with A-83-01 treatment restored the ability of passage 7 RPE to differentiate. Taken together, these results demonstrate that the loss of capacity to differentiate as a result of chronic wound stimulus is a product of both TGFbeta pathway-dependent increases in mesenchymal gene expression and a TGFbeta pathway-independent loss of RPE programming.

ORGANISM(S): Homo sapiens

PROVIDER: GSE78740 | GEO | 2016/12/01

SECONDARY ACCESSION(S): PRJNA313410

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:Age-related macular degeneration (AMD) is a leading cause of blindness. Most vision loss occurs following the transition from a disease of deposit formation and inflammation to a disease of neovascular fibrosis and/or cell death. Here, we investigate how protracted wound stimulus leads to seminal changes in gene expression and the onset of a self-sustained state of wound response in retinal pigmented epithelial (RPE) cells. Using a human fetal RPE cell culture model and a systems level transcriptome analysis, we show that prolonged subconfluent culture resulting from repeated passage, leads to terminal acquisition of a mesenchymal-like phenotype post-confluence accompanied by altered expression of >40% of the transcriptome. In contrast, at subconfluence <5% of transcripts have >2-fold expression changes after repeated passage. Protein-protein interaction analysis reveals a core set of genes comprising two interconnected modules with functions pertaining to wound response and cell division. Among the wound response genes are the TGF-beta pathway activators: TGFB1, TGFG2, INHBA, INHBB, GDF6, CTGF, and THBS1. Small molecule inhibition of TGFBR1/ACVR1B mediated signaling both forestalls and reverses the passage-dependent loss of epithelial potential. Moreover, a disproportionate number of RPE wound response genes have altered expression in neovascular and geographic AMD; including key members of the TGF-beta pathway. In conclusiton, in RPE cells the switch to a terminal mesenchymal-like state following protracted or repeated wound stimulus is driven by activation of a self-perpetuating TGF-beta feedback loop. Targeted inhibition of TGF-beta signaling may be an effective approach towards retarding AMD progression and producing RPE cells in quantity for research and cell based therapies. Transcriptome profiles were determined from 44 human fetal RPE cultures of varying passage number, seeding density, culture maturity, and/or growth factor or small molecule treatment. Probes were labeled with Cy3 or Cy5 and Agilent whole genome microarrays were hybridized with a pair of Cy3 and Cy5 probes. After background subtraction and LOWESS correction to adjust for dye-dependent effects the net intensity values were determined as described in the Data Processing section and the entire data set was quantile normalized. The values reported in the Series Matrix are the quantile normalized net intensity values of the gene specific probes.

Project description:Ocular pigment epithelium (PE) cells promote the generation of T regulators (PE-induced Treg cells). Moreover, T cells exposed to PE acquire the capacity to suppress the activation of bystander T cells via TGFbeta. Membrane-bound TGFbeta on iris PE cells interacts with TGFbeta receptors on T cells, leading to the conversion of T cells to CD8(+) Treg cells via a cell contact-dependent mechanism. Conversely, soluble forms of TGFbeta produced by retinal PE cells can convert CD4(+) T cells into Treg cells in a manner that is independent of cell contact. In this study, we looked at the expression of immunoregulatory factors (TGFbeta, thrombospondins, CD59, IL-1 receptor antagonist, etc.) in PE cells as identified via an oligonucleotide microarray. Several thrombospondin-binding molecules were detected, and thus we focused subsequent analyses on thrombospondins. Via the conversion of latent TGFbeta to an active form that appears to be mediated by thrombospondin 1 (TSP-1), cultured iris PE and retinal PE cells induce a PE-induced Treg cell fate. After conversion, both ocular PE and PE-induced Treg cells express TSP-1. Regulatory T cell generation was amplified when the T cells also expressed TSP-1. In addition, PE-induced Treg cells significantly suppressed activation of bystander T cells via TSP-1. These results strongly suggest that the ability of ocular PE and PE-induced Treg cells to suppress bystander T cells depends on their capacity to produce TSP-1. Thus, intraocular TSP-1 produced by both ocular parenchymal cells and regulatory T cells is essential for immune regulation in the eye. Experiment Overall Design: Comparison of gene expression pattern in Mouse Primary Cultured Cell Lines (RPE, IPE, CBPE). Experiment Overall Design: Adult (6- to 8-week-old) C57BL/6 purchased from CLEA Japan Inc. (Tokyo, Japan) were used as donors ocular pigment epithelium (PE). To cultivate iris PE (IPE) and ciliary body PE (CBPE), iris and ciliary body tissues in the eyes were dissected out, and then incubated in 1 mg/ml Dispase and 0.05 mg/ml DNase I (both from Boehringer-Mannheim, Mannheim, Germany) for 1 hr. To cultivate retinal PE (RPE) Eyes were cut into two parts along a circumferential line posterior to the ciliary process, creating a ciliary body-deficient posterior eyecup. The eyecup was then incubated in 0.2% trypsin (Biowhitaker) for 1 hr. These tissues were triturated to make a single cell suspension, and then re-suspended in the medium.

Dataset Information

Restoration of mesenchymal RPE by transcription factor-mediated reprogramming

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure