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J. Hertz, J. L. Goldberg; Extrinsic Control of Human Ciliary Margin Retinal Progenitor Proliferation and Differentiation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1281.
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© ARVO (1962-2015); The Authors (2016-present)
What are the molecular signals that regulate how neural progenitor cells differentiate into particular neural cell fates? Whether looking at the differentiation of retinal ganglion cells (RGCs) from retinal progenitors, or of specific neuronal populations from their progenitors in the brain, a combination of intrinsic and extrinsic signals regulate cell fate specification. Among these, cell fate-specifying signals have been shown to be strongly dependent on environmental signals. The protein sonic hedgehog (Shh) was shown to be secreted by early developing RGCs and influences overall ganglion cell fate number in the developing. GDF11 was shown to repress expression of the transcription factor Math5 in order to produce the appropriate number of RGCs and control the time window of RGC competence. It is not yet known whether these secreted signals influence human adult ciliary margin derived retinal progenitors. In order to address these questions I have developed the following experiment:
Using biopsy samples of human ciliary margin, we cultured the cells using a neurosphere assay. The progenitors are cultured in the absence and presences of different concentrations of Shh and GDF-11. The retinal cells suspension are cultured in a serum free neurosphere media containing the mitogens EGF and bFGF at low density. Proliferation is measured by counting the number and average diameter of the neurospheres in culture. After one cell passage (10 days), the cells are gently dissociated with enzymes and subsequently plated on poly-d-lysine (PDL) and laminin coated culture dishes in RGC promoting media, which will not contain mitogens, in order to promote neural differentiation. After four days, the cells are fixed and immunostained for retinal ganglion cell markers including Brn-3a, Brn-3b, Thy1, Map2 and Tau. Using fluorescence microscopy, I measured the number of RGCs and observe morphological changes such as dendrite and axon formation. Statistical tools will be used to analyze the data to determine significance.
Shh plays a role in progenitor proliferation and differentiation in vitro. Cells plated in the presence of Shh produce more neurospheres then control media demonstrating that Shh signals these progenitors directly. Further data, including BrdU labeling can further demonstrate the proliferation differences. The ciliary margin stem cells exposed to Shh also demonstrated an increased number in differentiated RGCs in vitro.
Exogenous factors, such as Shh, influence progenitor proliferation and differentiation in adult human ciliary margin derived progenitors.
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