Optimization of a Method to Isolate and Culture Adult Porcine, Rats and Mice Müller Glia in Order to Study Retinal Diseases

Pereiro, Xandra; Ruzafa, Noelia; Acera, Arantxa; Urcola, Javier Aritz; Vecino, Elena

doi:10.3389/fncel.2020.00007

Cited by 20 publications

(17 citation statements)

References 43 publications

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“…We have recently established a method to purify and culture adult Müller glia cells from mouse, rat and pig retinas, and we have previously shown that cultured murine Müller glia cells express GFAP, Glutamine Synthetase (GS), p75 NTR , and Cralbp. Flow cytometry indicated that >94% of all the cells in these cultures express p75 NTR [ 43 ]. In order to further characterize our Müller glia culture system, we used a combination of markers normally expressed in Müller glia cells, including the cytoskeletal protein Vimentin ( Figure 2 A,A’, green) and the transcription factors Lhx2, Pax6, and Sox2 ( Figure 2 B–D, red).…”

Section: Resultsmentioning

confidence: 99%

Effects of Adult Müller Cells and Their Conditioned Media on the Survival of Stem Cell-Derived Retinal Ganglion Cells

Pereiro

Miltner

Torre

et al. 2020

Cells

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Retinal neurons, particularly retinal ganglion cells (RGCs), are susceptible to the degenerative damage caused by different inherited conditions and environmental insults, leading to irreversible vision loss and, ultimately, blindness. Numerous strategies are being tested in different models of degeneration to restore vision and, in recent years, stem cell technologies have offered novel avenues to obtain donor cells for replacement therapies. To date, stem cell–based transplantation in the retina has been attempted as treatment for photoreceptor degeneration, but the same tools could potentially be applied to other retinal cell types, including RGCs. However, RGC-like cells are not an abundant cell type in stem cell–derived cultures and, often, these cells degenerate over time in vitro. To overcome this limitation, we have taken advantage of the neuroprotective properties of Müller glia (one of the main glial cell types in the retina) and we have examined whether Müller glia and the factors they secrete could promote RGC-like cell survival in organoid cultures. Accordingly, stem cell-derived RGC-like cells were co-cultured with adult Müller cells or Müller cell-conditioned media was added to the cultures. Remarkably, RGC-like cell survival was substantially enhanced in both culture conditions, and we also observed a significant increase in their neurite length. Interestingly, Atoh7, a transcription factor required for RGC development, was up-regulated in stem cell-derived organoids exposed to conditioned media, suggesting that Müller cells may also enhance the survival of retinal progenitors and/or postmitotic precursor cells. In conclusion, Müller cells and the factors they release promote organoid-derived RGC-like cell survival, neuritogenesis, and possibly neuronal maturation.

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Section: Resultsmentioning

confidence: 99%

Effects of Adult Müller Cells and Their Conditioned Media on the Survival of Stem Cell-Derived Retinal Ganglion Cells

Pereiro

Miltner

Torre

et al. 2020

Cells

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“…In the case of MIO-M1, it has been observed that these cells express marker genes of neural progenitors as well as postmitotic neuronal cells and various opsins [ 42 , 43 ]. Thus, primary cells are often considered as more physiologically similar to in vivo cells [ 44 ]. Therefore, we also analyzed primary equine RMG.…”

Section: Discussionmentioning

confidence: 99%

Cell Surface Profiling of Retinal Müller Glial Cells Reveals Association to Immune Pathways after LPS Stimulation

Lorenz

Hirmer

Schmalen

et al. 2021

Cells

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Retinal Müller glial cells (RMG) are involved in virtually every retinal disease; however, the role of these glial cells in neuroinflammation is still poorly understood. Since cell surface proteins play a decisive role in immune system signaling pathways, this study aimed at characterizing the changes of the cell surface proteome of RMG after incubation with prototype immune system stimulant lipopolysaccharide (LPS). While mass spectrometric analysis of the human Müller glia cell line MIO-M1 revealed 507 cell surface proteins in total, with 18 proteins significantly more abundant after stimulation (ratio ≥ 2), the surfaceome of primary RMG comprised 1425 proteins, among them 79 proteins with significantly higher abundance in the stimulated state. Pathway analysis revealed notable association with immune system pathways such as “antigen presentation”, “immunoregulatory interactions between a lymphoid and a non-lymphoid cell” and “cell migration”. We could demonstrate a higher abundance of proteins that are usually ascribed to antigen-presenting cells (APCs) and function to interact with T-cells, suggesting that activated RMG might act as atypical APCs in the course of ocular neuroinflammation. Our data provide a detailed description of the unstimulated and stimulated RMG surfaceome and offer fundamental insights regarding the capacity of RMG to actively participate in neuroinflammation in the retina.

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“…The culture medium was changed every 4-5 days and cells were passaged biweekly at a ratio of 1:3. Müller cells were identified by their characteristic bipolar morphology and immunostained with glutamine synthetase and glial fibrillary acidic protein (data not shown) (25). The purity of Müller cells was >90% after 2-3 passages.…”

Section: Methodsmentioning

confidence: 95%

High glucose induces HSP47 expression and promotes the secretion of inflammatory factors through the IRE1α/XBP1/HIF‑1α pathway in retinal Müller cells

et al. 2021

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Diabetic retinopathy, a common complication of diabetes, is the leading cause of blindness globally. Müller cells are key players in diabetes-associated retinal inflammation and dysfunction. However, the pathological changes of Müller cells in response to high glucose (HG) and the underlying mechanism remain unclear. The aim of the present study was to investigate the key role of heat shock protein 47 (HSP47) in HG-induced unfolded protein and inflammatory responses. Primary mouse Müller cells were starved in serum-free DMEM overnight and then treated with HG (30 mM) for 0, 6, 12 or 24 h. It was observed that HG (30 mM) significantly induced the protein expression of HSP47, inositol-requiring transmembrane kinase and endonuclease-1α (IRE1α) and spliced X-box-binding protein 1 (XBP1s) in primary mouse Müller cells compared with the untreated group. In addition, the immunoprecipitation results revealed that HSP47 directly interacted with IRE1α, and this interaction was significantly enhanced by HG exposure for 12 or 24 h compared with the untreated group. Furthermore, small interfering RNA-mediated silencing of HSP47 significantly suppressed HG-induced activation of the IRE1α/XBP1s/hypoxia inducible factor-1 subunit α (HIF-1α) pathway and upregulation of the mRNA expression levels of the inflammatory cytokines vascular endothelial growth factor, platelet-derived growth factor subunit B, inducible nitric oxide synthase and angiopoietin-2 in Müller cells. Furthermore, overexpression of IRE1α or HIF-1α partially attenuated HSP47-siRNA-mediated inhibition of inflammatory cytokine expression in Müller cells. Collectively, these results indicated that HG may induce HSP47 expression and promote the inflammatory response through enhancing the interaction between HSP47 and IRE1α, and activating the IRE1α/XBP1s/HIF-1α pathway in retinal Müller cells.

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Optimization of a Method to Isolate and Culture Adult Porcine, Rats and Mice Müller Glia in Order to Study Retinal Diseases

Cited by 20 publications

References 43 publications

Effects of Adult Müller Cells and Their Conditioned Media on the Survival of Stem Cell-Derived Retinal Ganglion Cells

Effects of Adult Müller Cells and Their Conditioned Media on the Survival of Stem Cell-Derived Retinal Ganglion Cells

Cell Surface Profiling of Retinal Müller Glial Cells Reveals Association to Immune Pathways after LPS Stimulation

High glucose induces HSP47 expression and promotes the secretion of inflammatory factors through the IRE1α/XBP1/HIF‑1α pathway in retinal Müller cells

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