Prospective purification and characterization of Müller glia in the mouse retina regeneration assay

Abstract

Reactive gliosis is an umbrella term for various glia functions in neurodegenerative diseases and upon injury. Specifically, Müller glia (MG) in some species readily regenerate retinal neurons to restore vision loss after insult, whereas mammalian MG respond by reactive gliosis-a heterogeneous response which frequently includes cell hypertrophy and proliferation. Limited regeneration has been stimulated in mammals, with a higher propensity in young MG, and in vitro compared to in vivo, but the underlying processes are unknown. To facilitate studies on the mechanisms regulating and limiting glia functions, we developed a strategy to purify glia and their progeny by fluorescence-activated cell sorting. Dual-transgenic nuclear reporter mice, which label neurons and glia with red and green fluorescent proteins, respectively, have enabled MG enrichment up to 93% purity. We applied this approach to MG in a mouse retina regeneration ex vivo assay. Combined cell size and cell cycle analysis indicates that most MG hypertrophy and a subpopulation proliferates which, over time, become even larger in cell size than the ones that do not proliferate. MG undergo timed differential genomic changes in genes controlling stemness and neurogenic competence; and glial markers are downregulated. Genes that are potentially required for, or associated with, regeneration and reactive gliosis are differentially regulated by retina explant culture time, epidermal growth factor stimulation, and animal age. Thus, MG enrichment facilitates cellular and molecular studies which, in combination with the mouse retina regeneration assay, provide an experimental approach for deciphering mechanisms that possibly regulate reactive gliosis and limit regeneration in mammals.

Retinal cell death dependent reactive proliferative gliosis in the mouse retina

“…We have previously shown and confirmed here (Fig. S2d ) that the vast majority of proliferating cells are MG by immunostaining for KI67 15 , 24 , an antigen expressed during the active stages of the cell cycle. MG proliferation and nuclear displacement into the ONL started on DEV 2 and further increased until DEV 4 (3 ± 0.4 cells compared to 0 ± 0.000 cells in control, N ≥ 3, Fig.…”

“…EGF treatment stimulates highly limited MG-derived neuronal regeneration in vivo 13 . Recent work has shown that regulated molecular programs control regeneration, and possibly limit it 7 , 15 , 24 . Retinal-damage-induced mechanisms might differentially trigger, regulate, and restrict glial responses, like regeneration or scarring, which possibly depend on the species and type of disease, and are incompletely understood.…”

“…However, MG reactive gliosis in damaged mouse retina is frequently associated with an activation of cell cycle related regulators 19 , 45 . Here, we utilize our recently developed mouse retinal regeneration assay that facilitates studies in an easily controlled environment and recapitulates some major steps of reactive gliosis in mammals in vivo and in regenerating zebrafish retina 15 , 21 , 24 : Organotypic explant culture of juvenile mouse retina induces extensive neuronal cell loss and MG become reactive, involving cell hypertrophy, cell displacement and gliotic hallmark gene expression changes. More than 50% of MG re-enter the cell cycle upon EGF stimulation, some reprogram into a stem cell or neurogenic state, and very few differentiate into neuronal-like cells.…”

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

“…Much of the current information about Müller cells, their activity and dysfunction has been derived from animal models, and mainly from cultured Müller cells. However, while there are several protocols for the isolation of Müller cells from different mammalian species, these generally rely on the use of tissue from neonatal animals to get primary cell cultures (Table 1), probably due to the loss of regeneration potential as mammals age (Fischer et al, 2002;Schafer and Karl, 2017). However, degenerative retinal diseases are more frequent in adults and consequently, in vitro model systems developed with adult cells would be more useful to investigate the physiological and pathological events that occur in the mature retina.…”

Photobiomodulation Mediates Neuroprotection against Blue Light Induced Retinal Photoreceptor Degeneration

“…With regard to our study, an ex vivo application, especially whole eyeballs cultivation, is favorable as it combines the efficiency and control common to in vitro techniques with close imitation of the in vivo environment. Ex vivo models are common experimental designs to examine retinal tissue regeneration with a highly controlled setting, e.g., by the delivery of equal amounts of light to the retina by a constant distance [29,31,32]. We chose blue light (BL) irradiation as a damaging model of irradiated photoreceptors and analyzed cellular and molecular action of RL or NIRL after post-treatment [29,30].…”