ScRNA-seq experiments on human foreskin fibroblasts, foreskin fibroblasts induced directly to retina pigment epithelium, and induced pluripotent stem cell derived retina pigment epithelium.
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE166935 | GEO | 2021/12/01
REPOSITORIES: GEO
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Project description:Stargardt retinopathy is an inherited form of macular degeneration caused by mutations in gene ABCA4 and characterized by the accumulation of lipid-rich deposits in the retinal pigment epithelium (RPE), RPE atrophy, and photoreceptor cell death. Inadequate mechanistic insights into pathophysiological changes occurring in Stargardt RPE have contributed to its lacking treatments. Here we show that ABCA4 knockout or Stargardt patient’s induced pluripotent stem cells-derived RPE (STGD1-iRPE) differentiate normally but display intracellular lipid and ceramide deposits reminiscent of the disease phenotype. STGD1-iRPE also shows defective photoreceptor outer segment (POS) processing and reduced cathepsin B activity, indicating higher lysosomal pH. Lipid deposits in STGD1-iRPE are reduced by increasing the activity of ABCA1, a lipid transporter, and ABCA4 ortholog. Overall, our work suggests that ABCA4 is involved in POS and lipid handling in RPE cells and provides guidance for ongoing gene therapy approaches to target both RPE and photoreceptor cells for an effective treatment.
2022-11-16 | GSE198362 | GEO
Project description:Retinal pigment epithelial cells differentiated from human induced pluripotent stem cells (hiPSCs), called iRPE, are being explored as a cell-based therapy for the treatment of retinal degenerative diseases, especially age-related macular degeneration (AMD). The success of RPE implantation is believed to be linked to the use of biomimetic scaffolds that simulate Bruch’s membrane and aid in its maturation and integration. Current practices widely utilize fibrous scaffolds from synthetic polymers, such as polycaprolactone (PCL), rather than from natural biomaterials. Here, we tested the hypothesis that naturally derived fibrous scaffolds, with their unique translational potential, can provide favorable conditions permissive for the maturation of iRPE in vitro. We show that the culture of iRPE on natural, soy protein-derived nanofibrous scaffolds match the results of similar cells cultured on synthetic PCL nanofibrous scaffolds. The mechanical properties of soy scaffolds better emulate the native Bruch’s membrane, which would likely limit the foreign body reaction triggered by mismatch of host and biomaterial strength. Additionally, soy scaffolds exhibit unique biochemistry that improves biocompatibility and have tunable crosslinking to control degradation rates. Comparative transcriptome analysis demonstrates that iRPE maturation on nanofibrous scaffolds, either natural or synthetic, yielded more consistent outcomes than on other non-fibrous substrates. Taken together, our studies suggest that the maturation of cultured RPE for subsequent clinical applications will benefit from the use of nanofibrous scaffolds derived from natural proteins.
2020-07-10 | GSE153645 | GEO
Project description:The retinal pigment epithelium (RPE) provides vital support to photoreceptor cells and its dysfunction is associated with the onset and progression of age-related macular degeneration (AMD). Surgical provision of RPE cells may ameliorate AMD and thus it would be valuable to develop sources of patient-matched RPE cells for this application of regenerative medicine. We describe here the generation of functional RPE-like cells from fibroblasts that represent an important step toward that goal. We identified candidate master transcriptional regulators of RPEs using a novel computational method and then used these regulators to guide exploration of the transcriptional regulatory circuitry of RPE cells and to reprogram human fibroblasts into RPE-like cells. The RPE-like cells share key features with RPEs derived from healthy individuals, including morphology, gene expression and function, and thus represent a step toward the goal of generating patient-matched RPE cells for treatment of macular degeneration. Expression analysis was performed on induced retinal pigment epithelium-like cells.
2015-09-23 | E-GEOD-64264 | biostudies-arrayexpress
Project description:Purpose: The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier. Primary cultures of RPE can model the barrier, but are very sensitive to culture conditions. We examined how the neural retina regulates the RPE transcriptome in a culture model of embryonic development. Attention focused on the tight junctional genes essential for barrier function. Methods: Chick RPE from embryonic day 7 (E7) or embryonic day 14 (E14) was cultured on filters in a serum free medium. Media conditioned by organ culture of neural retinas was applied to the apical surface of the cultured RPE. Fetal bovine serum (2%) was included in some experiments. When the transepithelial electrical resistance (TER) reached a plateau, total RNA was isolated to probe the chick genome on Affymetrix microarrays. The expression tight junctional mRNAs was confirmed by real-time PCR and immunoblotting the protein. Results: The TER was slightly increased by serum, but increased 2-3X by retinal conditioned medium. Serum diminished the effect of retinal conditioned medium. In basal conditions, 86% of the transcriptome expressed during development in vivo was also expressed in culture. Approximately 5% of the transcriptome expressed in culture was absent in vivo. E14 retinal conditioned medium affected 15% of the transcriptome in E7 cultures (24% if serum was included), but only 1.9% in E14 cultures (12% with serum). Examination of 610 genes important for RPE function revealed that mRNAs for 17% were regulated by retinal conditioned medium alone in E7 cultures, compared to 6.2% for E14. For tight junctions, retinal conditioned medium had the most affect on members of the claudin family. These results were confirmed by quantitative real-time PCR. Besides regulating mRNA levels, immunoblotting and immunocytochemistry suggested additional mechanisms whereby retinal secretions regulated protein expression and localization. Conclusions: Gene expression in primary cultures of embryonic RPE resembled the native tissue, but expression, and differentiation, is improved when elements of the normal extracellular environment are replicated in culture. For a small group of proteins, retinal secretions were required to maintain in vivo-like expression in culture. Albeit insufficient, retinal secretions promoted differentiation of immature RPE and helped maintain the properties of more mature RPE. Keywords: Tissue interactions, retina, retinal pigment epithelium, blood-retinal barrier, tight junctions
2008-09-30 | GSE10538 | GEO
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.
2013-03-20 | E-GEOD-36695 | biostudies-arrayexpress
Project description:Age-related macular degeneration (AMD) is a leading cause of blindness. Vision loss is caused by the loss of the retinal pigment epithelium (RPE) and photoreceptors and/or retinal and choroidal angiogenesis. Here we use AMD patient specific RPE cells with the Y402H high-risk polymorphism in the complement factor H to perform a comprehensive analysis of EVs, their cargo and role in disease pathology. We show that AMD RPE is characterised by enhanced and polarised EV secretion. Transcriptomic, proteomic and lipidomic analyses demonstrate that AMD RPE EVs carry RNA, proteins and lipids that reflect changes in the parental RPE and mediate key AMD pathological processes including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. We demonstrate that exposure of control RPE to AMD RPE apical EVs leads to the formation of stress vacuoles, cytoskeletal destabilization, and abnormalities in the morphology of the nucleus in the recipient cells. Treatment of retinal organoids with apical AMD RPE EVs leads to disrupted neuroepithelium and appearance of cytoprotective alpha B crystallin immunopositive cells, with some co-expressing retinal progenitor cell markers Pax6 or Vsx2, suggesting activation of regenerative pathways upon injury. These findings indicate that AMD RPE EVs mediated signalling have an important role in disease progression.
2022-01-21 | MSV000088713 | MassIVE
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.
2013-03-20 | GSE36695 | GEO
Project description:Purpose: The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier. Primary cultures of RPE can model the barrier, but are very sensitive to culture conditions. We examined how the neural retina regulates the RPE transcriptome in a culture model of embryonic development. Attention focused on the tight junctional genes essential for barrier function. Methods: Chick RPE from embryonic day 7 (E7) or embryonic day 14 (E14) was cultured on filters in a serum free medium. Media conditioned by organ culture of neural retinas was applied to the apical surface of the cultured RPE. Fetal bovine serum (2%) was included in some experiments. When the transepithelial electrical resistance (TER) reached a plateau, total RNA was isolated to probe the chick genome on Affymetrix microarrays. The expression tight junctional mRNAs was confirmed by real-time PCR and immunoblotting the protein. Results: The TER was slightly increased by serum, but increased 2-3X by retinal conditioned medium. Serum diminished the effect of retinal conditioned medium. In basal conditions, 86% of the transcriptome expressed during development in vivo was also expressed in culture. Approximately 5% of the transcriptome expressed in culture was absent in vivo. E14 retinal conditioned medium affected 15% of the transcriptome in E7 cultures (24% if serum was included), but only 1.9% in E14 cultures (12% with serum). Examination of 610 genes important for RPE function revealed that mRNAs for 17% were regulated by retinal conditioned medium alone in E7 cultures, compared to 6.2% for E14. For tight junctions, retinal conditioned medium had the most affect on members of the claudin family. These results were confirmed by quantitative real-time PCR. Besides regulating mRNA levels, immunoblotting and immunocytochemistry suggested additional mechanisms whereby retinal secretions regulated protein expression and localization. Conclusions: Gene expression in primary cultures of embryonic RPE resembled the native tissue, but expression, and differentiation, is improved when elements of the normal extracellular environment are replicated in culture. For a small group of proteins, retinal secretions were required to maintain in vivo-like expression in culture. Albeit insufficient, retinal secretions promoted differentiation of immature RPE and helped maintain the properties of more mature RPE. Experiment Overall Design: RPE were isolated from chicken embryos on embryonic day 7 or 14 and cultured, as described <Rahner C, Fukuhara M, Peng S, Kojima S, Rizzolo LJ. The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development. J Cell Sci. 2004;117:3307-18>. E7 is when tight junctions of the RPE begin to form; E14 is when tight junctions achieve their mature morphological appearance in vivo. The cells were cultured in medium that contains or lacks E14 retinal conditioned medium. Because serum inhibits the effect of retinal conditioned medium, serum was added to some cultures, resulting in 4 culture conditions for each age. To isolate total RNA, the RNeasy Protect kit (Qiagen) was used according to the manufacturer's protocols. For each culture, 3 independent preparations were used for analysis on Affymetrix microarrays of the chicken genome (Santa Clara, CA). The quality of the total RNA was assessed by the Keck Center, Yale University using formamide gels and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA)
2008-11-01 | E-GEOD-10538 | biostudies-arrayexpress
Project description:The retinal pigment epithelium (RPE) provides vital support to photoreceptor cells and its dysfunction is associated with the onset and progression of age-related macular degeneration (AMD). Surgical provision of RPE cells may ameliorate AMD and thus it would be valuable to develop sources of patient-matched RPE cells for this application of regenerative medicine. We describe here the generation of functional RPE-like cells from fibroblasts that represent an important step toward that goal. We identified candidate master transcriptional regulators of RPEs using a novel computational method and then used these regulators to guide exploration of the transcriptional regulatory circuitry of RPE cells and to reprogram human fibroblasts into RPE-like cells. The RPE-like cells share key features with RPEs derived from healthy individuals, including morphology, gene expression and function, and thus represent a step toward the goal of generating patient-matched RPE cells for treatment of macular degeneration.
2015-09-23 | GSE64264 | GEO
Project description:The developmental pathway of the neural retina (NR) and retinal pigment epithelium (RPE) has been revealed by extensive research in mice. However, the molecular mechanisms underlying the development of the human NR and RPE, as well as the interactions between these two tissues, have not been well defined. Here, we analyzed 2,421 individual cells from human fetal NR and RPE using single-cell RNA sequencing (RNA-seq) technique and revealed the tightly regulated spatiotemporal gene expression network of human retinal cells. We identified major cell classes of human fetal retina and potential crucial transcription factors for each cell class. We dissected the dynamic expression patterns of visual cycle and ligand-receptor interaction related genes in the RPE and NR. Moreover, we provided a map of disease-related genes for human fetal retinal cells and highlighted the importance of retinal progenitor cells as potential targets of inherited retinal diseases. Our findings captured the key in vivo features of the development of the human NR and RPE and offered insightful clues for further functional studies.
2019-07-04 | GSE107618 | GEO