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C. W. Guo, S. Singhal, A. J. Wong, B. Bhatia, P. T. Khaw, G. Limb; Evidence for Wnt Signalling in Müller Cell Line MIO-M1. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1268.
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© ARVO (1962-2015); The Authors (2016-present)
To fully understand the factors regulating Müller stem cell differentiation and proliferation we examined the expression of various genes and proteins of the Wnt signaling pathway in MIO-M1 cells. We investigated the effects of growth factors and extracellular matrix (ECM) proteins on the expression of various components of the Wnt signaling pathway, as well as the effects of modulation of Wnt signaling on the expression of known downstream factors implicated in this signaling.
The Müller stem cell line MIO-M1 was cultured in the presence of growth factors such as FGF2 and insulin, and the basement membrane protein matrigel. RT-PCR, western blots and immunostaining were performed to examine the gene and protein expression of various Wnt pathway components. Down regulation of this pathway was achieved by addition of the Wnt antagonist Dickkopf-1 (DKK1). The effects of this treatment on cell proliferation was examined by immunostaining for the proliferative marker Ki-67.
Genes of the Wnt pathways such as Wnt2b, Wnt4a, Frizzled (FZD) 1, 4, 7, β-Catenin and LEF1 are constitutively expressed in Müller stem cells under baseline conditions. However, gene expression of WNT3A and FZD5 were not detected in Müller stem cells. Growth factors and ECM proteins promoted upregulation of FZD1 and WNT5B, and downregulation of FZD7. Addition of recombinant DKK1 induced downregulation of β-Catenin expression. This was confirmed by gene and protein expression, as well as immunostaining.
Since Wnt proteins play an important role in stem cell regulation, the present findings that adult human Müller stem cells constitutively express various components of this pathway, further confirm the progenic ability of these cells. In vitro modulation of this pathway may potentially be used to generate specific neural cell populations required for cell based therapies to repair diseased retina.
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