Comparison of expression profile between human Müller cells, HMCL-I and HMCL-II
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE3118 | GEO | 2006/08/12
SECONDARY ACCESSION(S): PRJNA92951
REPOSITORIES: GEO
Similar Datasets
Project description:Rat Retinal Müller cells from diabetic rats (diabetes duration 6 months) compared to Rat Retinal Müller cells from healthy rats. Diabetes was induced by streptozotozine. Diabetic rats were treated with small doses of insulin to prevent catabolism. Keywords: ordered
2004-11-17 | E-GEOD-1979 | biostudies-arrayexpress
Project description:MM-CM-<ller cells are the principal glial cells in the retina. Alterations in MM-CM-<ller cell behaviour are observed in retinal tissue from patients with proliferative diabetic retinopathy. The purpose of this study was to compare gene and protein expression profile of normal human MM-CM-<ller cells (NHMC) with two spontaneously human MM-CM-<ller cell lines generated from type 1 (HMCL-I) and type 2 (HMCL-II) diabetic donors using Serial Analysis Gene Expression (SAGE). Approximatively 50 000 tags were sequenced for each of the three SAGE libraries. Identification of the transcripts was obtained by matching the 15bp (CATG + 11bp) with the UniGene and GenBank databases. Classification of the genes was based upon the updated information of the genome directory found at the NCBI website. SAGE allowed us to characterize the entire transcriptome of human MM-CM-<ller cells and to compare these data with those from MM-CM-<ller cells of type 1 and 2 diabetic donors. Polyadenylated RNA was extracted with the Oligotex mRNA Purification System (Qiagen), annealed with the biotin-5 -T18-3 primer and converted to cDNA with the cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). The resulting cDNA library was digested with NlaIII (anchoring enzyme), and the 3M-bM-^@M-^Y restriction fragments were isolated with streptavidin-coated magnetic beads (Dynal Biotech, Oslo, Norway) and separated into two populations. Each population was ligated to one of the two annealed linker pairs and extensively washed to remove unligated linkers. The tag beside the most 3M-bM-^@M-^Y NlaIII restriction site (CATG) of each transcript was released by digestion with BsmFI (tagging enzyme). The blunting kit from Takara Co. (Otsu, Japan) was used for the blunting and ligation of the two tag populations. The resulting ligation products containing the ditags were amplified by PCR with an initial denaturation step of 1 min at 950C, followed by 22 cycles of 20 s at 940C, 20 s at 600C and 2 s at 720C with 27 bp primers. The PCR product was digested with NlaIII, and the band containing the ditags was extracted from the acrylamide gel. The purified ditags were self-ligated to form concatemers. The concatemers of 500 to 1800bp were isolated by agarose gel. The resulting DNA fragments were ligated into the SphI site of pUC19 and cloned into UltraMAX DH5M-oM-^AM-!FT (Invitrogen). White colonies were screened by PCR to select long inserts for automated sequencing. Sequence files were analyzed by the SAGEana program, a modification of SAGEparser (ftp://ftp.pbrc.edu/public/eesnyder/SAGE/). Tags corresponding to linker sequences were discarded, and duplicate concatemers were counted only once. We used the comparative count display (CCD) test to identify the transcripts that were differentially expressed significantly (P<0.05) between the groups with more than a twofold change. The CCD test makes a key-by-key comparison of two key-count distributions by generating a probability that the frequency of any key in the distribution differs by more than a given fold factor from the other distribution.
2006-08-12 | E-GEOD-3118 | biostudies-arrayexpress
Project description:Rat Retinal Müller cells from diabetic rats (diabetes duration 6 months) compared to Rat Retinal Müller cells from healthy rats. Diabetes was induced by streptozotozine. Diabetic rats were treated with small doses of insulin to prevent catabolism. Keywords: ordered
2004-11-17 | GSE1979 | GEO
Project description:Diabetic retinopathy (DR), the leading cause of blindness in working-age adults, is thought to be primarily a microvascular complication of diabetes. As a central element of the retinal neurovascular unit, Müller cells, the major macroglia of the retina, are important for maintaining a healthy and functional retina and play a critical role in several pathological events during DR disease progression. Here, we aim to improve our understanding of Müller cell-specific signalling pathways that are altered during DR disease progression in order to develop novel gene therapy strategies that target Müller cells. We used a multi-omics approach on purified Müller cells from 6-month-old control and diabetic db/db mice, including (i) RNA-seq followed by oPOSSUM-3 transcription factor (TF) binding site cluster analysis, (ii) glial chromatin landscape analysis by ATAC-seq, and (iii) Müller cell proteomics by MS/MS mass spectrometry. This led to the identification of the glucocorticoid receptor (GR, gene ID: Nr3c1) most highly expressed in Müller cells. In cells from diabetic mice, GR mRNA and protein expression is significantly reduced and its target gene cluster downregulated in Müller cells. Importantly, GR was identified as a potential master regulator not only by oPOSSUM analysis based on differentially expressed mRNA in Müller cells, but also validated by the ATAC-seq approach. In an in vitro cortisol-stimulated retinal explant model cortisol not only increased GR phosphorylation, but also induced changes in the expression of known downstream GR target genes. Finally, we evaluated whether AAV-mediated GR overexpression in Müller cells improves retinal functionality and we found moderate improvements indicated by electroretinography. While synthetic and topical glucocorticoids are widely used in ophthalmology with undeniable beneficial effects, our study provides valuable new insight into the role of GR signalling and glial alterations in the diabetic retina. This supports the therapeutic concept of locally enhancing the GR signaling axis. Our findings of reduced GR levels in Müller cells of the diabetic retina suggests that therapeutic approaches should aim at increasing the expression of the receptor rather than adding more ligand.
2024-05-23 | PXD045085 | Pride
Project description:Müller cells are the main macroglial cells of the retina exerting a wealth of functions to maintain retinal homoeostasis. Upon pathological changes in the retina, they become gliotic with both protective and detrimental consequences. Accumulating data also provide evidence for a pivotal role of Müller cells in the pathogenesis of diabetic retinopathy (DR). While microglial cells, the resident immune cells of the retina are considered as main players in inflammatory processes associated with DR, the implication of activated Müller cells in chronic retinal inflammation remains to be elucidated. In order to assess the signaling capacity of Müller cells and their role in retinal inflammation, we performed in-depth proteomic analysis of Müller cell proteomes and secretomes after stimulation with INFγ, TNFα, IL-4, IL-6, IL-10, TGFβ1, TGFβ2 and TGFβ3. We used both, primary porcine Müller cells and the human Müller cell line MIO-M1 for our hypothesis generating approach. Our results point towards an intense signaling capacity of Müller cells, which reacted in a highly discriminating manner upon treatment with different cytokines. Stimulation of Müller cells results in a primarily pro-inflammatory phenotype with secretion of cytokines and components of the complement system. Furthermore, we observed evidence for mitochondrial dysfunction, implying oxidative stress after treatment with the various cytokines. Finally, both MIO-M1 cells and primary porcine Müller cells showed several characteristics of atypical antigen-presenting cells, as they are capable of inducing MHC class I and MHC class II with co-stimulatory molecules. In line with this, they express proteins associated with formation and maturation of phagosomes. Thus, our findings underline the importance of Müller cell signaling in the inflamed retina, indicating an active role in chronic retinal inflammation underlying the pathogenesis of diabetic retinopathy.
2022-02-16 | PXD028390 | Pride
Project description:Diabetic retinopathy (DR), the leading cause of blindness in working-age adults, is thought to be primarily a microvascular complication of diabetes. Müller cells, the major macroglia of the retina, are important for maintaining a healthy and functional retina and play a critical role in several pathological events during DR disease progression as they are a central element of the retinal neurovascular unit. Here, we aim to improve our understanding of Müller cell-specific signaling pathways that are altered during disease progression in order to develop novel gene therapy strategies targeting Müller cells in DR. To achieve this, we used a multi-omics approach on purified Müller cells from 6-month-old control and diabetic db/db mice, including (i) RNA-seq followed by oPOSSUM-3 transcription factor (TF) binding site cluster analysis, (ii) glial chromatin landscape analysis by ATAC-seq, and (iii) Müller cell proteomics by MS/MS mass spectrometry. This led to the identification of the glucocorticoid receptor (GR, gene ID: Nr3c1) as the most promising candidate. Its transcripts and protein were most highly expressed in Müller cells and significantly reduced in cells from diabetic mice, its target gene cluster was downregulated in Müller cells from diabetic animals, and importantly, it was identified as a potential master regulator not only by oPOSSUM analysis based on differentially expressed mRNA in Müller cells, but also validated by the ATAC-seq approach. Next, we investigated the effect of GR modulation in an in vitro cortisol-stimulated retinal explant model. Cortisol not only increased GR phosphorylation levels, but also induced changes in the expression of known downstream GR target genes. Finally, we evaluated whether AAV-mediated GR overexpression in Müller cells improves retinal functionality and found moderate functional improvements in electroretinogram recordings. Although synthetic and topical glucocorticoids are widely used in ophthalmology with undeniable beneficial effects, our study provides valuable new insights into the role of GR signaling and glial alterations in the diabetic retina and thus supports the therapeutic concept of locally enhancing the GR signaling axis. However, our finding of reduced GR levels in Müller cells of the diabetic retina suggests that therapeutic approaches should aim at increasing the expression of the receptor rather than adding more ligands. This may result in optimized local efficacy with fewer systemic unwarranted side effects.
2024-01-25 | GSE236627 | GEO
Project description:Non-mammalian vertebrates have a robust ability to regenerate injured retinal neurons from Müller glia cells (MG) that activate the proneural factor Achaete-scute homolog 1 (Ascl1/Mash1) and de-differentiate into progenitors cells. In contrast, mammalian MG have a limited regenerative response and fail to upregulate Ascl1 after injury. To test whether Ascl1 could restore a neurogenic potential to mammalian MG, we over-expressed Ascl1 in dissociated mouse MG cultures and intact retinal explants. Ascl1-infected MG upregulate retinal progenitor-specific genes, while downregulating glial genes. Furthermore, Ascl1 remodeled the chromatin at its targets from a repressive to active configuration. MG-derived progenitors differentiated into cells that exhibited neuronal morphologies, expressed retinal subtype-specific neuronal markers, and displayed neuron-like physiological responses. These results indicate that a single transcription factor, Ascl1, can produce a neurogenic state in mature Muller glia. Expresssion profiling was used to determine the genes that were changed after Ascl1 infection of P12 cultured Müller glia compared with those present in P0 progenitors and P7-P21 Müller glia Retinas were dissociated and FAC-sorted from Hes5-GFP mice at P0, P7, P10, P14 or P21 and submitted for profiling. WT Retinas were dissociated at P12, grown for 1 week in culture, and infected with lentiviruses expressing Ascl1 or GFP for four days. Total RNA was extracted and submitted for profiling.
2013-04-06 | E-GEOD-45835 | biostudies-arrayexpress
Project description:Müller cells are the primary glia of the neural retina and play crucial roles in maintaining the structural and functional homeostasis of the retina. Müller cells also possess certain levels of retinal regeneration capacity, especially in lower vertebrates. In this study, we deep sequenced the transcriptomes and methylomes of mouse Müller cells and early retinal progenitor cells (RPCs), as well as Müller cells from injured retinas and aged retinas, which will help to reveal molecular mechanisms governing Müller cells development, physiological functions and regeneration activities.
2019-11-12 | GSE124532 | GEO
Project description:The underlying pathomechanisms in diabetic retinopathy (DR) remains incompletely understood. The aim of this study was to add to the current knowledge about the particular role of retina Müller glial cells (RMG) in the initial processes of DR. Applying a mass spectrometric workflow, we investigated proteome changes of primary porcine RMG under short term diabetic cell culture treatment. We revealed global changes in RMG proteome indicated by a fundamental remodeling of ECM and focal adhesion processes which potentially leads to destabilization of the retinal inner limiting membrane. This is in line with our previous findings of a disrupted ILM in the retinae of a diabetic pig model and suggests an involvement of RMG in this process. Additionally, employing porcine organotypic retinal explant cultures, we demonstrate a specific decrease of RMG-associated SPP1 expression as a result of osmotic challenge induced by high glucose levels. We assume a loss of neuroprotective potential administrated by RMG and subsequent acceleration of neurodegenerative processes in DR.
2021-04-06 | PXD015879 | Pride
Project description:In order to identify the miRNAs in adult light damaged Muller glia of the the neural retina, we isolated the Müller glia from Rlbp-CreER: Stopf/f-tdTomato by means of fluorescent activated cell sorting and analyzed their miRNAs using NanoStrings Technologies®. We compared the data to that from and Dicer1-CKO Müller glia and wild type glia Müller glia (GSE103098). We found three miRNAs potentially regulating genes involved in stress response in both data sets.
2020-12-24 | GSE162993 | GEO