| Summary: | Müller glia mediates retina regeneration in zebrafish. Despite exhibiting Müller glial with stem cell (hMSC) characteristics in vitro by the human retina, retinal regeneration mediated by Müller glia following disease or injury has not been demonstrated. Notch, Wnt, TGFβ and HBEGF signalling is well known to regulate retinal neurogenesis and inflammation, but the roles of these molecules in the neural differentiation of hMSC are not known. This thesis aimed to establish whether there is an interaction between these signalling pathways and the role that these factors play during retinal ganglion cell (RGC) and photoreceptor differentiation of hMSC. The research showed that inhibition of Notch signalling caused downregulation of components of the canonical Wnt signalling pathway in these cells, as demonstrated by a decrease in mRNA expression of the Wnt ligand WNT2B and its target genes WISP-1 and AXIN2. Addition of TGFβ1 did not significantly change the expression of the Notch signalling target HES1 or the RGC marker BRN3A/B. Culture of hMSC with a combination of factors that induce their photoreceptor differentiation (FGF2, taurine, retinoic acid and Insulin growth factor; collectively called FTRI), markedly upregulated the expression of components of the canonical Wnt signalling pathway, including WNT2B, DKK1 and active β-catenin. Although FTRI did not modify mRNA expression of WNT5B, a component of the non-canonical/planar cell polarity Wnt pathway, it upregulated its secretion. Furthermore, TGFβ1 not only decreased WNT2B expression, but inhibited FTRI-induced photoreceptor differentiation of hMSC, as determined by expression of the photoreceptor markers NR2E3, rhodopsin and recoverin. Inhibition of TGFβ1 signalling by an ALK5 inhibitor prevented TGFβ1 induced changes in the expression of the two Wnt ligands examined. More importantly, inhibition of the canonical Wnt signalling by XAV-939 prevented FTRI-induced photoreceptor differentiation. Similarly, HBEGF, a factor shown to be upregulated by FTRI also decreased Wnt signalling components such as WNT2B, WISP-1, DKK1 and AXIN2. Inhibition of HBEGF by its specific inhibitor CRM197 prevented photoreceptor differentiation. These observations suggest that both Notch and Wnt signalling pathways can regulate the neurogenicity of hMSC in vitro and that TGFβ as well as HBEGF play important roles in mediating key pathways leading to either RGC or photoreceptor differentiation of hMSC. Targeting components of both Notch and Wnt pathways may constitute targets for potential induction of endogenous regeneration of the human retina and this merits further studies.
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