Characterization of Stem-cell derived Retinal Pigmented Epithelium (RPE)
ABSTRACT: To assess the geome-wide similarities between primary fetal retinal pigmented epithelium (RPE) and stem-cell derived RPE, we performed whole genome microarray expression on primary RPE and both embryonic stem cell (ESC) derived RPE and induced pluripotent stem cell (iPSC) derived RPE. We found ES-derived RPE better resembles fetal RPE than iPS-derived RPE. Gene expression was measured in primary fetal RPE, ES-derived RPE, iPS-derived RPE. ES cells and BJ fibroblasts were used as controls.
Project description:We show that Retinal pigment epithelium (RPE) secreted-factor, pigment epithelium derived factor (PEDF) secreted/derived from primary or iPSC-derived retinal pigment epithelium (RPE)RPE, dramatically inhibitsed the cell growth of iPSCs. PEDF was detected abundantly in culture supernatant media of primary and iPSC-derived RPE. We examined the gene expression in primary RPE and iPS-derived RPE. Two samples: RPE derived from 253G1 iPSC, Primary RPE.
Project description:Retinal pigment epithelium (RPE) cells can be obtained through in vitro differentiation of both embryonic stem cell (ESC) and induced pluripotent stem cells (iPSC) for cell replacement therapy. We have previously identified 87 signature genes relevant to RPE cell differentiation and function through transcriptome analysis of both human ESC- and iPSC-derived RPE as well as normal fetal RPE. Here, we profiled miRNA expression through small RNA-seq in human ESCs and their RPE derivatives. Much like conclusions drawn from our previous transcriptome analysis, we found that the overall miRNA landscape in RPE is distinct from ESCs and other differentiated somatic tissues. We also profiled miRNA expression during intermediate stages of RPE differentiation and identified unique subsets of miRNAs that are gradually up- or downregulated, suggesting dynamic regulation of these miRNAs is associated with the RPE differentiation process. Indeed, the down-regulation of a subset of miRNAs during RPE differentiation is associated with up-regulation of RPE-specific genes, such as RPE65, which is exclusively expressed in RPE. We conclude that miRNA signatures can be used to classify different degrees of in vitro differentiation of RPE from human pluripotent stem cells. We suggest that RPE-specific miRNAs likely contribute to the functional maturation of RPE in vitro, similar to the regulation of RPE-specific mRNA expression. Study miRNA in ESC-derived RPE
Project description:To assess the geome-wide similarities between primary fetal retinal pigmented epithelium (RPE) and stem-cell derived RPE, we performed whole genome microarray expression on primary RPE and both embryonic stem cell (ESC) derived RPE and induced pluripotent stem cell (iPSC) derived RPE. We found ES-derived RPE better resembles fetal RPE than iPS-derived RPE. Overall design: Gene expression was measured in primary fetal RPE, ES-derived RPE, iPS-derived RPE. ES cells and BJ fibroblasts were used as controls.
Project description:Retinal Pigment Epithelium (RPE) derived from two human embryonic stem cell lines, H1 and H9, were compared with human fetal RPE (hfRPE) using RNA-seq. Nominally, the transcriptome of H1-derived RPE showed greater overlap with hfRPE. For cells maintained in the medium used to differentiate RPE, 6.2% (H1-RPE) and 4.2% (H9-RPE) of the transcripts were expressed in amounts that were statistically different from hfRPE (false discovery rate: 5%). After adaptation to the serum-free medium, SFM-1, only 1.0 % (H1-RPE) and 1.9% (H9-RPE) were expressed in amounts that were statistically different. For RPE signature genes, statistical differences were observed for 1.0 % (H1-RPE) and 1.9% (H9-RPE) of the transcripts. For some barrier-function related mRNAs the statistical differences were greater than these small differences would predict. For adhesion proteins and plasma membrane transporters, the differences were as great as 6.9% and 4.3%, respectively. After adaptation to SFM-1, the statistical differences between H1- and H9-RPE were only 0.4% for all transcripts, 1.4% for signature genes, and 0.7% for membrane transporters. No statistical differences were observed for the transcripts related to tight junctions, adhesion junctions or ion channels. In summary, SFM-1 promoted the maturation of stem cell-derived RPE, and the statistical difference between two stem cell lines was minimal. RNA sequencing of hfRPE from three fetuses (reference sample) and three independent isolates of RPE derived from the H1, and three from the H9, human embryonic stem cell (hESC) lines. The cultures were maintained in a serum-free medium, SFM-1. Comparisons were also made to cultures maintained in the medium used to differentiate the cells, KSR.
Project description:Using the paradigm of in vitro differentiation of hESCs/iPSCs into retinal pigment epithelial (RPE) cells, we have recently profiled mRNA and miRNA transcriptomes to define a set of RPE mRNA and miRNA signature genes implicated in directed RPE differentiation. In this study, in order to understand the role of DNA methylation in RPE differentiation, we profiled genome-scale DNA methylation patterns using the method of reduced representation bisulfite sequencing (RRBS). We found dynamic waves of de novo methylation and demethylation in four stages of RPE differentiation. Integrated analysis of DNA methylation and RPE transcriptomes revealed a reverse-correlation between levels of DNA methylation and expression of a subset of miRNA and mRNA genes that are important for RPE differentiation and function. Gene Ontology (GO) analysis suggested that genes undergoing dynamic methylation changes were related to RPE differentiation and maturation. We further compared methylation patterns among human ESC- and iPSC-derived RPE as well as primary fetal RPE (fRPE) cells, and discovered that specific DNA methylation pattern is useful to classify each of the three types of RPE cells. Our results demonstrate that DNA methylation may serve as biomarkers to characterize the cell differentiation process during the conversion of human pluripotent stem cells into functional RPE cells. Study of DNA methylation patterns during RPE differentiation of pluripotent stem cells.
Project description: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.
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: To investigate the effects of T cell-derived cytokines on gene and protein expression of chemokines in a human RPE cell line (ARPE-19). Methods: We used an in vitro co-culture system in which the RPE and CD3/28-activated T cells were separated by a membrane. Differential gene expression in the RPE cells of chemokine genes was quantified using three different microarrays. Protein expression was determined by single- and multiplex ELISA and immunoblotting. Results: Co-culture with activated T cells increased RPE mRNA and protein expression of chemokines CCL2 (MCP-1), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CXCL1 (GRO-α), IL8 (CXCL8), CXCL9 (MIG), CXCL10 (IP10), CXCL11 (ITAC), and CX3CL1 (fractalkine). The secretion of CCL7 and CXCL9, 10, and 11 was polarized in the apical direction. Using recombinant human cytokines and neutralizing antibodies we identified IFNgamma and TNFalpha as the two major T cell-derived cytokines responsible for the RPE response. For CCL5, CXCL9, 10, 11, 16, and CX3CL1 we observed a synergistic effect of IFNgamma and TNFalpha in combination. CCL20, CXCL1 and 6, and IL8 were negatively regulated by IFNgamma. Conclusions: RPE cells responded to exposure to T cell-derived cytokines by upregulating expression of several chemokines related to microglial, T cell, and monocyte chemotaxis and activation. This inflammatory stress response may have implications for immune homeostasis in the retina, and for the further understanding of inflammatory ocular diseases such as uveitis and age-related macular degeneration. Differentiated ARPE19 grown on inserts in serum-free media was basolaterally stimulated for 48h with activated T cells or recombinant cytokines (IFNg and/or TNFa) or neutralizing antibodies (aIFNg and/or aTNFa)
Project description:The retinal pigment epithelium (RPE) is a polarized cell layer that is critical for photoreceptor function and survival. It’s unique relationship to the photoreceptors and its specific physiology makes the RPE a critical determinant of human vision. Therefore we performed global expression profiling of native and cultured human fetal and adult RPE and determined a unique set of highly-expressed genes (called the “signature” set) by comparing the observed RPE gene profiles to the Novartis expression database (SymAtlas: http://wombat.gnf.org/index.html) of 78 tissues. A comparative analysis of transcriptomes from human fetal and adult RPE, primary cultures and commonly-used cell lines was performed. Using selection criteria of at least 10-fold higher expression in each of three RPE preparations, we identified 154 RPE signature genes, which were validated by qRT-PCR analysis in RPE and in an independent set of 11 tissues.
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 repeated wound stimulus leads to seminal changes in gene expression and the onset of a perpetual state of stimulus-independent wound response in retinal pigmented epithelial (RPE) cells, a cell-type central to the etiology of AMD. Using a human fetal RPE cell culture model that considers monolayer disruption and subconfluent culture as a proxy for wound stimulus, we have shown that prolonged wound stimulus leads to terminal acquisition of a mesenchymal phenotype post-confluence and altered expression of more than 40% of the transcriptome (see GEO:GSE62224). In contrast, at subconfluence fewer than 5% of expressed transcripts have 2-fold or greater expression differences after repeated passage. Protein-protein and pathway interaction analysis of the genes with passage-dependent expression levels in subconfluent cultures reveals a 158-node interactome comprised of two interconnected modules with functions pertaining to wound response and cell division. Among the wound response genes are the TGFb pathway activators: TGFB1, TGFB2, INHBA, INHBB, GDF6, CTGF, and THBS1. Significantly, inhibition of TGFBR1/ACVR1B mediated signaling using receptor kinase inhibitors both forestalls and reverses the passage-dependent loss of epithelial potential. In this RNA-Seq based transcriptome analysis we show that the TGFb receptor kinase inhibitor, A-83-01, largely reverses the effects of passage and restores the transcriptome profile of Passage 4 RPE highly similar to that seen in differentiated Passage 0 RPE. Examination of mRNA expression in three different primary fetal RPE donor lines in 32 day old passage 0, passage 3, and passage 3 treated with 500 nM A-83-01 cultures