Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Transl Vis Sci Technol
2022 Apr 01;114:4. doi: 10.1167/tvst.11.4.4.
Show Gene links
Show Anatomy links
Derivation and Characterization of Murine and Amphibian Müller Glia Cell Lines.
Gallo RA
,
Qureshi F
,
Strong TA
,
Lang SH
,
Pino KA
,
Dvoriantchikova G
,
Pelaez D
.
???displayArticle.abstract???
Purpose: Müller glia (MG) in the retina of Xenopus laevis (African clawed frog) reprogram to a transiently amplifying retinal progenitor state after an injury. These progenitors then give rise to new retinal neurons. In contrast, mammalian MG have a restricted neurogenic capacity and undergo reactive gliosis after injury. This study sought to establish MG cell lines from the regeneration-competent frog and the regeneration-deficient mouse.
Methods: MG were isolated from postnatal day 5 GLAST-CreERT; Rbfl/fl mice and from adult (3-5 years post-metamorphic) X laevis. Serial adherent subculture resulted in spontaneously immortalized cells and the establishment of two MG cell lines: murine retinal glia 17 (RG17) and Xenopus glia 69 (XG69). They were characterized for MG gene and protein expression by qPCR, immunostaining, and Western blot. Purinergic signaling was assessed with calcium imaging. Pharmacological perturbations with 2'-3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) and KN-62 were performed on RG17 cells.
Results: RG17 and XG69 cells express several MG markers and retain purinergic signaling. Pharmacological perturbations of intracellular calcium responses with BzATP and KN-62 suggest that the ionotropic purinergic receptor P2X7 is present and functional in RG17 cells. Stimulation of XG69 cells with adenosine triphosphate-induced calcium responses in a dose-dependent manner.
Conclusions: We report the characterization of RG17 and XG69, two novel MG cell lines from species with significantly disparate retinal regenerative capabilities.
Translational Relevance: RG17 and XG69 cell line models will aid comparative studies between species endowed with varied regenerative capacity and will facilitate the development of new cell-based strategies for treating retinal degenerative diseases.
Figure 1. Immunophenotypic characterization of mouse MG cell line RG17. (A) Phase contrast images and immunofluorescence staining of primary MG cells and passage 20 RG17 cells. RG17 demonstrate typical MG morphology in culture and express many well-characterized MG markers. Scale bar, 50 µm and applies to all immunofluorescence images. (B) Expression of characteristic MG genes in primary MG and RG17 cells (passages 20–25). The geometric means and geometric standard deviations (N = 3 independent cultures) are graphed. An unpaired t-test was applied with a P value of ≤0.05 considered statistically significant. (C) Western blot detection of MG markers from primary MG and passage 20 RG17 cells. (D) The doubling rate of RG17 cells does not vary significantly between passages 5 and 20. Data points are mean ± standard deviation.
Figure 2. Immunophenotypic characterization of X laevis MG cell line XG69. (A) Phase contrast images and immunofluorescence staining of primary MG and XG69 cell line at passage 20. (B) Expression of MG genes in primary MG and XG69 cells (passages 20–25). The geometric means and geometric standard deviations (N = 3 independent cultures) are graphed. An unpaired t-test was applied with a P value of ≤0.05 considered statistically significant. (C) Western blot detection of MG markers from primary MG and passage 20 XG69 cells. Frog MG do not express RPE markers ZO-1 and RPE65 unlike primary Xenopus RPE cells. (D) The doubling rate of XG69 cells does not vary significantly through 20 passages. Data points are mean ± standard deviation.
Figure 3. Calcium responses elicited by P2X7 activation in RG17 cells. (A) Heat map shows calcium responses of RG17 cells to 50 µM of ATP and the combination of 50 µM of ATP and 1 µM of P2X7 noncompetitive antagonist P2X7 (n = 41 cells). Each row is a single cell, the x axis is time, and the change of the fluorescent intensity over baseline (ΔF/F) is indicated by the color scale, where fluorescence intensity increases from purple to red. (B) Confocal images of the RG17 intracellular calcium responses. Scale bar, 100 µm. (C) Representative traces of calcium responses of individual RG17 cells as well as the average trace to ATP and KN-62. (D) KN-62 mitigated the ATP-induced calcium response in RG17 cells (unpaired t-test of the area under the curve; **** P < 0.0001). (E) Heat map shows calcium responses of RG17 cells to 50 µM of BzATP (n = 50 cells). Each row is a single cell, the x axis is time, and ΔF/F is indicated by the color scale, where fluorescence intensity increases from purple to red. (F) Representative traces of calcium responses of individual RG17 cells to BzATP. (G) Average trace of the RG17 calcium response to BzATP (n = 50 cells).
Figure 4. ATP elicits calcium responses in XG69 cells. (A) Heat map of calcium responses from all recorded XG69 cells to 50, 100, and 300 µM of ATP (n = 40 cells). Each row is a single cell, the x axis is time, and ΔF/F is indicated by the color scale, where fluorescence intensity increases from purple to red. (B) Representative calcium response traces from individual XG69 cells. (C) Average trace of calcium responses (n = 40 cells). (D) XG69 cells exhibit intracellular calcium increases with a dose–response to ATP stimulation (ordinary one-way analysis of variance of the area under the curve, *P < 0.05, **P < 0.01).
Augustine,
Characterization of a Spontaneously Immortalized Murine Müller Glial Cell Line QMMuC-1.
2018, Pubmed
Augustine,
Characterization of a Spontaneously Immortalized Murine Müller Glial Cell Line QMMuC-1.
2018,
Pubmed
Bernardos,
Late-stage neuronal progenitors in the retina are radial Müller glia that function as retinal stem cells.
2007,
Pubmed
Blackshaw,
Genomic analysis of mouse retinal development.
2004,
Pubmed
Bringmann,
Müller glial cells in retinal disease.
2012,
Pubmed
Bringmann,
Müller cells in the healthy and diseased retina.
2006,
Pubmed
Bringmann,
Upregulation of P2X(7) receptor currents in Müller glial cells during proliferative vitreoretinopathy.
2001,
Pubmed
Das,
Muller cells of chicken retina synthesize 11-cis-retinol.
1992,
Pubmed
Dvoriantchikova,
Development and epigenetic plasticity of murine Müller glia.
2019,
Pubmed
Dyer,
Control of Müller glial cell proliferation and activation following retinal injury.
2000,
Pubmed
Fini,
Thyroid hormone signaling in the Xenopus laevis embryo is functional and susceptible to endocrine disruption.
2012,
Pubmed
,
Xenbase
Fischer,
Müller glia are a potential source of neural regeneration in the postnatal chicken retina.
2001,
Pubmed
Fries,
Identification of P2Y receptor subtypes in human muller glial cells by physiology, single cell RT-PCR, and immunohistochemistry.
2005,
Pubmed
Gallo,
Effects of Mitomycin-C and 5-Fluorouracil on Ocular Adnexal Sebaceous Carcinoma Cells.
2022,
Pubmed
Germer,
Modification of glutamine synthetase expression by mammalian Müller (glial) cells in retinal organ cultures.
1997,
Pubmed
Hamon,
Linking YAP to Müller Glia Quiescence Exit in the Degenerative Retina.
2019,
Pubmed
,
Xenbase
Hamon,
Müller glial cell-dependent regeneration of the neural retina: An overview across vertebrate model systems.
2016,
Pubmed
Hengst,
Inhibitors of the Cip/Kip family.
1998,
Pubmed
,
Xenbase
Hoang,
Gene regulatory networks controlling vertebrate retinal regeneration.
2020,
Pubmed
Jablonski,
Targeted disruption of Müller cell metabolism induces photoreceptor dysmorphogenesis.
2000,
Pubmed
,
Xenbase
Jorstad,
Stimulation of functional neuronal regeneration from Müller glia in adult mice.
2017,
Pubmed
Kittipassorn,
Characterization of the novel spontaneously immortalized rat Müller cell line SIRMu-1.
2019,
Pubmed
Langhe,
Müller glial cell reactivation in Xenopus models of retinal degeneration.
2017,
Pubmed
,
Xenbase
Lawrence,
MIO-M1 cells and similar muller glial cell lines derived from adult human retina exhibit neural stem cell characteristics.
2007,
Pubmed
Lenkowski,
Müller glia: Stem cells for generation and regeneration of retinal neurons in teleost fish.
2014,
Pubmed
Limb,
In vitro characterization of a spontaneously immortalized human Müller cell line (MIO-M1).
2002,
Pubmed
Makhmutova,
Pancreatic β-Cells Communicate With Vagal Sensory Neurons.
2021,
Pubmed
Marino,
Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum.
2000,
Pubmed
Martinez-De Luna,
Müller glia reactivity follows retinal injury despite the absence of the glial fibrillary acidic protein gene in Xenopus.
2017,
Pubmed
,
Xenbase
Otteson,
A conditional immortalized mouse muller glial cell line expressing glial and retinal stem cell genes.
2010,
Pubmed
Pannicke,
P2X7 receptors in Müller glial cells from the human retina.
2000,
Pubmed
Phillips,
Differential expression of neuronal genes in Müller glia in two- and three-dimensional cultures.
2011,
Pubmed
Powell,
Zebrafish Müller glia-derived progenitors are multipotent, exhibit proliferative biases and regenerate excess neurons.
2016,
Pubmed
Powell,
Analysis of DNA methylation reveals a partial reprogramming of the Müller glia genome during retina regeneration.
2013,
Pubmed
Raymond,
Molecular characterization of retinal stem cells and their niches in adult zebrafish.
2006,
Pubmed
Roesch,
The transcriptome of retinal Müller glial cells.
2008,
Pubmed
Sarthy,
Establishment and characterization of a retinal Müller cell line.
1998,
Pubmed
Todd,
Comparative Biology of Vertebrate Retinal Regeneration: Restoration of Vision through Cellular Reprogramming.
2022,
Pubmed
Todd,
Efficient stimulation of retinal regeneration from Müller glia in adult mice using combinations of proneural bHLH transcription factors.
2021,
Pubmed
Tomi,
Expression and regulation of L-cystine transporter, system xc-, in the newly developed rat retinal Müller cell line (TR-MUL).
2003,
Pubmed
Turner,
A common progenitor for neurons and glia persists in rat retina late in development.
,
Pubmed
Uckermann,
Glutamate-evoked alterations of glial and neuronal cell morphology in the guinea pig retina.
2004,
Pubmed
Ueki,
Transgenic expression of the proneural transcription factor Ascl1 in Müller glia stimulates retinal regeneration in young mice.
2015,
Pubmed
Wurm,
Purinergic signaling involved in Müller cell function in the mammalian retina.
2011,
Pubmed
Xue,
CRALBP supports the mammalian retinal visual cycle and cone vision.
2015,
Pubmed
Yao,
Restoration of vision after de novo genesis of rod photoreceptors in mammalian retinas.
2018,
Pubmed
Young,
Cell differentiation in the retina of the mouse.
1985,
Pubmed
