Differential expression of neuronal genes in Müller glia in two- and three-dimensional cultures.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Basic Sciences, College of Optometry, University of Houston, Houston, Texas 77204-2020, USA.
M Joseph
MJ
Phillips
Deborah C
DC
Otteson
D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology*; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D009419:Nerve Tissue Proteins / Q000235:genetics*; D058953:Neural Stem Cells / Q000378:metabolism; D055495:Neurogenesis / Q000502:physiology*; D009457:Neuroglia / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D055351:Retinal Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018874:Spheroids, Cellular / Q000648:ultrastructure; D054457:Tissue Scaffolds
D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D016222:Fibroblast Growth Factor 2; D004815:Epidermal Growth Factor