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J. Phillips, M. Guirguis, K. Beach, L. Pillai, D. C. Otteson; A Conditionally Immortalized Müller Cell Line Acquires Retinal Stem Cell Characteristics. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5784. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the retinal stem cell characteristics of cultured, conditionally immortalized mouse Müller cells following epidermal growth factor (EGF) and fibroblastic growth factor 2 (FGF2) stimulation.
Müller cells (ImM10), isolated from retinas of postnatal day 10 Immortomouse pups as previously described, contain a transgene encoding an interferon gamma (IFNg) inducible, temperature sensitive, SV40 large T-antigen (TAg), that immortalizes cells when cultured at 33°C. Cultures were maintained in Neurobasal with 2% fetal bovine serum, IFNg (50U/ml), 2mM L-glutamine and antibiotics. To abolish TAg expression and promote sphere formation, cells were incubated at 39oC in serum-free Neurobasal containing B27 and M5 with EGF (20ng/ml), FGF2 (20ng/ml) and lacking IFNg. Total RNA was isolated using RNAeasyMicro columns (Qiagen) and reverse transcribed using oligo-dT primers. Relative quantification of Müller and stem cell gene expression was determined with quantitative realtime PCR using SYBR green on a Stratagene MX3005P with intron spanning, optimized primers. Wholemount immunocytochemistry was performed on paraformladelyde fixed spheres with Cy3 or Alexa Fluor 488 conjugated secondary antibodies.
After 7 days of growth factor stimulation, Müller cells detached from the dish and formed proliferating spheres typical of retinal and neural stem cells in culture. Compared to Müller cells in growth conditions, sphere cells showed a distinct change in their mRNA expression profile. Expression of Notch1, Hes1, and Pax6, typically expressed in retinal stem/progenitor cells, was upregulated in sphere cells. Mushashi1 was undetectable in Müller cells, but was abundant in sphere cells. The Müller cell gene vimentin was downregulated in spheres, while GFAP was slightly upregulated. Unexpectedly, nucleostemin levels remained similar between groups, Nestin was moderately downregulated and GS expression was upregulated in spheres. Immunostaining confirmed expression of the stem cell markers nestin and Pax6, the Müller cell markers GS, vimentin, and the activated Müller cell marker GFAP in sphere cells.
Conditionally immortalized mouse Müller glia form stem-like cells in vitro in response to growth factor stimulation. However, under current culture conditions, de-differentiation remained incomplete. Future studies will focus on further quantifying the changes in gene expression and identification of factors and substrates that will enhance the acquisition of stem-cell characteristics and promote neuronal differentiation.
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