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Angshumonik Angbohang, Karen Eastlake, Silke Becker, Na Wu, G Astrid Limb; Upregulation of the Notch and Wnt signalling pathways by HB-EGF in adult human Müller stem cells in vitro. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1371.
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Müller glial stem cells (MCSs) spontaneously regenerate the retina of adult zebrafish after injury. MSCs can be isolated from adult human eye and induced to proliferate in vitro by HB-EGF stimulation. HB-EGF has been shown to initiate MSC derived retinal regeneration by activating the Notch and Wnt signalling pathways in the zebrafish. Since Notch inhibition in human MSC promotes their differentiation into retinal ganglion cells (RGCs) in vitro, and activation of the Wnt signalling pathway can promote MSC growth, it would be important to investigate whether HB-EGF activation of these cells may trigger interactive signals between the Notch and Wnt signalling pathways that can control adult stem cell growth and differentiation in vitro
Müller stem cells were cultured on matrigel coated flasks in DMEM media containing 2% foetal calf serum in the presence or absence of HB-EGF and γ-secretase inhibitor RO4929097. mRNA and protein isolated from these cells were examined for expression of molecules of the Notch and Wnt signalling pathways and RGC marker Brn3b using RT-PCR and Western-blot analysis. Morphological changes were examined by phase contrast microscopy.
Culture of Müller stem cells with exogenous HB-EGF, caused an increase in gene expression of the Notch downstream target Hes1 and the canonical Wnt signalling components Wnt2b and β-catenin. We found that the γ-secretase inhibitor RO4929097 induced down-regulation in HB-EGF gene expression, as well as in gene and protein expression of the Notch downstream target Hes1. MSCs cultured with RO4929097 acquired a neuronal-like morphology, displaying a very thin elongated cytoplasm with bright phase cell body and the formation of multiple neurites. It also induced up-regulation of Brn3b gene expression, a marker of RGC. The Wnt signalling ligand Wnt2b gene and protein expression were also down-regulated in these cells by this inhibitor.
The results showed that HB-EGF causes activation of both the Notch and Wnt signalling pathways in adult human Müller stem cells. Since this appears to be an evolutionary conserved interaction observed during the spontaneous retinal regeneration observed in zebrafish, it would be important to investigate whether activation of these pathways is suppressed in the adult human eye and whether they can be modulated to induce endogenous retina regeneration in the adult human eye.
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