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Invest Ophthalmol Vis Sci. 2011 Mar 14;52(3):1439-49. doi: 10.1167/iovs.10-6400. Print 2011 Mar.

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

Author information

  • 1Department of Basic Sciences, College of Optometry, University of Houston, Houston, Texas 77204-2020, USA.

Abstract

PURPOSE:

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.

PMID:
21051699
[PubMed - indexed for MEDLINE]
PMCID:
PMC3101681
Free PMC Article

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