Optimization of a Method to Isolate and Culture Adult Porcine, Rats and Mice Muller Glia in Order to Study Retinal Diseases.
ABSTRACT: Müller cells are the predominant glial elements in the retina, extending vertically across this structure, and they fulfill a wealth support roles that are critical for neurons. Alterations to the behavior and phenotype of Müller glia are often seen in animal models of retinal degeneration and in retinal tissue from patients with a variety of retinal disorders. Thus, elucidating the mechanisms underlying the development of retinal diseases would help better understand the cellular processes involved in such pathological changes. Studies into Müller cell activity in vitro have been hindered by the difficulty in obtaining pure cell populations and the tendency of these cells to rapidly differentiate in culture. Most protocols currently used to isolate Müller glia use neonatal or embryonic tissue but here, we report an optimized protocol that facilitates the reliable and straightforward isolation and culture of Müller cells from adult pigs, rats and mice. The protocol described here provides an efficient method for the rapid isolation of adult mammalian Müller cells, which represents a reliable platform to study therapeutic targets and to test the effects of drugs that might combat retinal diseases.
Project description:BACKGROUND:Retinal organoids serve as excellent human-specific disease models for conditions affecting otherwise inaccessible retinal tissue from patients. They permit the isolation of key cell types affected in various eye diseases including retinal ganglion cells (RGCs) and Müller glia. AIM:To refine human-induced pluripotent stem cells (hiPSCs) differentiated into three-dimensional (3D) retinal organoids to generate sufficient numbers of RGCs and Müller glia progenitors for downstream analyses. METHODS:In this study we described, evaluated, and refined methods with which to generate Müller glia and RGC progenitors, isolated them via magnetic-activated cell sorting, and assessed their lineage stability after prolonged 2D culture. Putative progenitor populations were characterized via quantitative PCR and immunocytochemistry, and the ultrastructural composition of retinal organoid cells was investigated. RESULTS:Our study confirms the feasibility of generating marker-characterized Müller glia and RGC progenitors within retinal organoids. Such retinal organoids can be dissociated and the Müller glia and RGC progenitor-like cells isolated via magnetic-activated cell sorting and propagated as monolayers. CONCLUSION:Enrichment of Müller glia and RGC progenitors from retinal organoids is a feasible method with which to study cell type-specific disease phenotypes and to potentially generate specific retinal populations for cell replacement therapies.
Project description:The ability to regenerate the entire retina and restore lost sight after injury is found in some species and relies mostly on the epigenetic plasticity of Müller glia. To understand the role of mammalian Müller glia as a source of progenitors for retinal regeneration, we investigated changes in gene expression during differentiation of retinal progenitor cells (RPCs) into Müller glia and analyzed the global epigenetic profile of adult Müller glia. We observed significant changes in gene expression during differentiation of RPCs into Müller glia in only a small group of genes and found a high similarity between RPCs and Müller glia on the transcriptomic and epigenomic levels. Our findings also indicate that Müller glia are epigenetically very close to late-born retinal neurons, but not early-born retinal neurons. Importantly, we found that key genes required for phototransduction were highly methylated. Thus, our data suggest that Müller glia are epigenetically very similar to late RPCs; however, obstacles for regeneration of the entire mammalian retina from Müller glia may consist of repressive chromatin and highly methylated DNA in the promoter regions of many genes required for the development of early-born retinal neurons. In addition, DNA demethylation may be required for proper reprogramming and differentiation of Müller glia into rod photoreceptors.
Project description:Müller glia in the mammalian retina have some stem cell-like characteristics, although their capacity for neurogenesis remains limited both in vivo and in vitro. In vitro studies to date have used traditional two-dimensional (2D) cell culture to assess neuronal differentiation of Müller glia. The purpose of this study was to compare the effects of 2D and three-dimensional (3D) environments on Müller glial gene expression after growth factor stimulation.Conditionally immortalized mouse Müller glia cells (ImM10) were cultured under nonimmortalizing conditions with EGF/FGF2 to generate spheres that were differentiated in vitro on uncoated culture dishes (2D) or encapsulated in self-assembling, RADA-16 peptide hydrogels (3D) under identical media and growth factor supplementation conditions. Gene expression was analyzed using quantitative RT-PCR and immunocytochemistry. Cellular morphology was analyzed with light and confocal microscopy; sphere ultrastructure was analyzed with transmission electron microscopy.ImM10 Müller cells express numerous genes associated with neural stem cells and retinal progenitors in both normal growth conditions and sphere-forming conditions. When encapsulated in the 3D hydrogel, cells can migrate and send processes into the hydrogel. Many genes associated with neurogenesis, as well as retinal neuron-specific genes, are differentially expressed in 2D and 3D differentiation conditions.ImM10 Müller glia upregulate genes characteristic of retinal neurons after growth factor stimulation in vitro, and gene expression patterns are altered in 3D hydrogel cultures.
Project description:Müller glia, the most abundant glia of vertebrate retina, have an elaborate morphology characterized by a vertical stalk that spans the retina and branches in each retinal layer. Müller glia play diverse, critical roles in retinal homeostasis, which are presumably enabled by their complex anatomy. However, much remains unknown, particularly in mouse, about the anatomical arrangement of Müller cells and their arbors, and how these features arise in development. Here we use membrane-targeted fluorescent proteins to reveal the fine structure of mouse Müller arbors. We find sublayer-specific arbor specializations within the inner plexiform layer (IPL) that occur consistently at defined laminar locations. We then characterize Müller glia spatial patterning, revealing how individual cells collaborate to form a pan-retinal network. Müller cells, unlike neurons, are spread across the retina with homogenous density, and their arbor sizes change little with eccentricity. Using Brainbow methods to label neighboring cells in different colors, we find that Müller glia tile retinal space with minimal overlap. The shape of their arbors is irregular but nonrandom, suggesting that local interactions between neighboring cells determine their territories. Finally, we identify a developmental window at postnatal Days 6 to 9 when Müller arbors first colonize the synaptic layers beginning in stereotyped inner plexiform layer sublaminae. Together, our study defines the anatomical arrangement of mouse Müller glia and their network in the radial and tangential planes of the retina, in development and adulthood. The local precision of Müller glia organization suggests that their morphology is sculpted by specific cell to cell interactions with neurons and each other.
Project description:The profile of miRNAs in mature glia is not well characterized, and most studies have been done in cultured glia. In order to identify the miRNAs in adult and young (postnatal day 11/12) Müller glia of the neural retina, we isolated the Müller glia from Rlbp-CreER: Stop<sup>f/f</sup>-tdTomato mice by means of fluorescent activated cell sorting and analyzed their miRNAs using NanoStrings Technologies<sup>®</sup>. In freshly isolated adult Müller glia, we identified 7 miRNAs with high expression levels in the glia, but very low levels in the retinal neurons. These include miR-204, miR-9, and miR-125-5p. We also found 15 miRNAs with high levels of expression in both neurons and glia, and many miRNAs that were enriched in neurons and expressed at lower levels in Müller glia, such as miR-124. We next compared miRNA expression of acutely isolated Müller glia with those that were maintained in dissociated culture for 8 and 14 days. We found that most miRNAs declined in vitro. Interestingly, some miRNAs that were not highly expressed in adult Müller glia increased in cultured cells. Our results thus show the miRNA profile of adult Müller glia and the effects of cell culture on their levels.
Project description:Identification of the signaling pathways that influence the reprogramming of Müller glia into neurogenic retinal progenitors is key to harnessing the potential of these cells to regenerate the retina. Glucocorticoid receptor (GCR) signaling is commonly associated with anti-inflammatory responses and GCR agonists are widely used to treat inflammatory diseases of the eye, even though the cellular targets and mechanisms of action in the retina are not well understood. We find that signaling through GCR has a significant impact upon the ability of Müller glia to become proliferating Müller glia-derived progenitor cells (MGPCs). The primary amino acid sequence and pattern of GCR expression in the retina is highly conserved across vertebrate species, including chickens, mice, guinea pigs, dogs and humans. In all of these species we find GCR expressed by the Müller glia. In the chick retina, we find that GCR is expressed by progenitors in the circumferential marginal zone (CMZ) and is upregulated by Müller glia in acutely damaged retinas. Activation of GCR signaling inhibits the formation of MGPCs and antagonizes FGF2/MAPK signaling in the Müller glia. By contrast, we find that inhibition of GCR signaling stimulates the formation of proliferating MGPCs in damaged retinas, and enhances the neuronal differentiation while diminishing glial differentiation. Given the conserved expression pattern of GCR in different vertebrate retinas, we propose that the functions and mechanisms of GCR signaling are highly conserved and are mediated through the Müller glia. We conclude that GCR signaling directly inhibits the formation of MGPCs, at least in part, by interfering with FGF2/MAPK signaling.
Project description:Adult zebrafish generate new neurons in the brain and retina throughout life. Growth-related neurogenesis allows a vigorous regenerative response to damage, and fish can regenerate retinal neurons, including photoreceptors, and restore functional vision following photic, chemical, or mechanical destruction of the retina. Müller glial cells in fish function as radial-glial-like neural stem cells. During adult growth, Müller glial nuclei undergo sporadic, asymmetric, self-renewing mitotic divisions in the inner nuclear layer to generate a rod progenitor that migrates along the radial fiber of the Müller glia into the outer nuclear layer, proliferates, and differentiates exclusively into rod photoreceptors. When retinal neurons are destroyed, Müller glia in the immediate vicinity of the damage partially and transiently dedifferentiate, re-express retinal progenitor and stem cell markers, re-enter the cell cycle, undergo interkinetic nuclear migration (characteristic of neuroepithelial cells), and divide once in an asymmetric, self-renewing division to generate a retinal progenitor. This daughter cell proliferates rapidly to form a compact neurogenic cluster surrounding the Müller glia; these multipotent retinal progenitors then migrate along the radial fiber to the appropriate lamina to replace missing retinal neurons. Some aspects of the injury-response in fish Müller glia resemble gliosis as observed in mammals, and mammalian Müller glia exhibit some neurogenic properties, indicative of a latent ability to regenerate retinal neurons. Understanding the specific properties of fish Müller glia that facilitate their robust capacity to generate retinal neurons will inform and inspire new clinical approaches for treating blindness and visual loss with regenerative medicine.
Project description:Vision impairments and blindness caused by retinitis pigmentosa result from severe neurodegeneration that leads to a loss of photoreceptors, the specialized light-sensitive neurons that enable vision. Although the mammalian nervous system is unable to replace neurons lost due to degeneration, therapeutic approaches to reprogram resident glial cells to replace retinal neurons have been proposed. Here, we demonstrate that retinal Müller glia can be reprogrammed in vivo into retinal precursors that then differentiate into photoreceptors. We transplanted hematopoietic stem and progenitor cells (HSPCs) into retinas affected by photoreceptor degeneration and observed spontaneous cell fusion events between Müller glia and the transplanted cells. Activation of Wnt signaling in the transplanted HSPCs enhanced survival and proliferation of Müller-HSPC hybrids as well as their reprogramming into intermediate photoreceptor precursors. This suggests that Wnt signaling drives the reprogrammed cells toward a photoreceptor progenitor fate. Finally, Müller-HSPC hybrids differentiated into photoreceptors. Transplantation of HSPCs with activated Wnt functionally rescued the retinal degeneration phenotype in rd10 mice, a model for inherited retinitis pigmentosa. Together, these results suggest that photoreceptors can be generated by reprogramming Müller glia and that this approach may have potential as a strategy for reversing retinal degeneration.
Project description:Müller glial cells are the source of retinal regeneration in fish and birds; although this process is efficient in fish, it is less so in birds and very limited in mammals. It has been proposed that factors necessary for providing neurogenic competence to Müller glia in fish and birds after retinal injury are not expressed in mammals. One such factor, the proneural transcription factor Ascl1, is necessary for retinal regeneration in fish but is not expressed after retinal damage in mice. We previously reported that forced expression of Ascl1 in vitro reprograms Müller glia to a neurogenic state. We now test whether forced expression of Ascl1 in mouse Müller glia in vivo stimulates their capacity for retinal regeneration. We find that transgenic expression of Ascl1 in adult Müller glia in undamaged retina does not overtly affect their phenotype; however, when the retina is damaged, the Ascl1-expressing glia initiate a response that resembles the early stages of retinal regeneration in zebrafish. The reaction to injury is even more pronounced in Müller glia in young mice, where the Ascl1-expressing Müller glia give rise to amacrine and bipolar cells and photoreceptors. DNaseI-seq analysis of the retina and Müller glia shows progressive reduction in accessibility of progenitor gene cis-regulatory regions consistent with the reduction in their reprogramming. These results show that at least one of the differences between mammal and fish Müller glia that bears on their difference in regenerative potential is the proneural transcription factor Ascl1.
Project description:Retinal neurons and Müller glia are generated from a common population of multipotent retinal progenitor cells. We purposed to identify Müller glia-specific molecular signatures during retinal development. Using transgenic mice carrying the Hes1 promoter (pHes1) followed by EGFP, we purified EGFP-positive Müller glia and other EGFP-negative retinal cells from developing retinas and subjected them to RNA sequencing analysis. Gene expression pattern of EGFP-positive cell was similar to genes expressed in retinal progenitors, and they were downregulated in other cell lineages. Then, we examined the modification profiles of H3K27me3 and H3K4me3 by referring to chromatin immunoprecipitation-sequencing data of rods and other cells. Clustering of the H3K4me3 and H3K27me3 values followed by ontology analysis revealed a high incidence of transcription factors including Hes1 in clusters with high H3K27me3 levels. Hes1 expression level decreased dramatically, and the H3K27me3 level at the Hes1-locus was upregulated strongly during retinal development. Furthermore, the Hes1 expression level was upregulated in an Ezh2-knockout retina. These results suggest that downregulation of Müller glia-related genes in other lineage rather than upregulation of them in Müller glia contributed Müller-specific molecular features, and a role for modified H3K27me3 in suppressing Müller glia-related genes in other retinal cell lineages to avoid unfavorable expression.