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S. Bhattacharya, C. Del Debbio, I. Ahmad; Prospective Isolation and Regulation of Müller Stem Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1276.
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We have proposed a regenerative approach to retinal degeneration by activating a subset of Müller cells that possesses stem cell properties and potential to differentiate into retinal neurons (Das et al., 2006, Dev. Biol. 299:283). The success of such an approach requires a predictable activation and subsequent directed differentiation of Müller cells to replace degenerated neurons. This may entail recapitulation of the developmental program inherent in retinal stem cells/progenitors and its sequential alteration for photoreceptor cell fate determination. Towards this goal we have begun to prospectively isolate activated Müller cells from different neurotoxin models of retinal degeneration and examine their cellular and molecular phenotypes in response to perturbation of different signaling pathways.
Adult rats were treated with N-methyl-N-nitrosourea (MNU) or N-methyl-D-aspartic acid (NMDA) to perturb outer and inner retina, respectively, with or without intraocular injection of activators of Wnt/Notch/Shh signaling. Retina was examined for morphological and immunohistochemical changes besides subjected to Hoechst dye efflux assay for SP cell profiling, a measure of stem cell phenotype of activated Müller cells (Das et al., 2006, Dev. Biol. 299:283). Müller SP cells in MNU and NMDA-treated retina were sorted by FACS for comparative transcription profiling.
MNU treatment led to a significant reduction in thickness of the outer nuclear layer while those treated with NMDA resulted in the thinning of the inner retina, compared to controls. Both treatments led to an increase in the incorporation of BrdU in cells that expressed Müller cell markers and ABCG2, the molecular determinant of SP cell phenotype. SP cells were undetectable in controls as well as in MNU and NMDA-treated retina. However, in neurotoxin-treated retina, exposed to activator of Wnt /Notch/Shh pathways, SP cells were readily detected and consisted of 0.2-0.3% of total cells. These cells were BrdU-positive and expressed Müller cell-specific markers. Sorted Müller SP cells and SP cells from different stages of retinal development are subjected to microarray analyses to compare cell-specific transcription profiles.
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