Downregulation of the canonical Wnt signalling pathway by TGF-β1 inhibits photoreceptor differentiation of adult human Müller stem cells

Abstract

Purpose: Müller glia regenerate the adult zebrafish retina after injury. Müller glia with stem cell characteristics (hMSC) can be isolated from the adult human retina and induced to differentiate into photoreceptors in vitro. Activation of Wnt signalling promotes proliferation and differentiation of neural progenitors, whilst transforming growth factor-β (TGF-β), highly upregulated during retinal gliosis, is known to promote regeneration of the zebrafish retina. We investigated whether TGF-β may control the Wnt signalling pathway and whether it may influence the ability of human Müller glia to differentiate into photoreceptors in vitro

Methods: hMSC were differentiated into photoreceptors by culture with FGF-2, Taurine, Retinoic acid and IGF (FTRI). Cells were also cultured with or without TGF-β1, TGF-β inhibitors and Wnt inhibitor XAV939. mRNA and protein isolated from these cells were examined for expression of molecules of Wnt signalling pathways and photoreceptor markers NR2E3 and Rhodopsin using RT-PCR, western-blot and confocal microscopy

Results: Culture of hMSC with FTRI increased gene and protein expression of WNT2B and β-catenin, components of Wnt canonical signalling pathway. Whilst TGFβ1 decreased gene and protein expression of WNT2B and β-catenin, it also inhibited differentiation of hMSC into photoreceptors as judged by decreased expression of NR2E3 and Rhodopsin. TGF-β type I receptor inhibitor SB431542 and Smad3 inhibitor SIS3 blocked the TGF-β inhibition of WNT2B. In addition, cells undergoing photoreceptor differentiation showed a decrease in DKK1 expression, an antagonist of Wnt signalling pathway. Addition of XAV939 to hMSC undergoing differentiation prevented upregulation in gene and protein expression of NR2E3.

Conclusions: The results suggest that downregulation of canonical Wnt signalling pathway by TGF-β1 inhibits photoreceptor differentiation of hMSC. This could be due to inhibition of the expression of Wnt ligands in these cells. There is no evidence that adult human retina regenerates despite harbouring Müller glia with stem cell characteristics. Since TGF-β is highly upregulated during gliosis, it may be possible that this cytokine may inhibit potential regenerative ability of these cells. It would be therefore important to investigate how these pathways can be modulated in order to induce endogenous regeneration of adult human retina.