Abstract
Purpose: :
Adult human Müller stem cells represent a potential source of cells for use in cell-based therapies to treat retinal degenerative diseases. This project aimed to develop in vitro methods to differentiate these cells towards a photoreceptor fate.
Methods: :
Cells were cultured for various periods of time in the presence of extracellular matrix (basement membrane proteins (bMP) or laminin) and combinations of various growth and differentiation factors including retinoic acid, taurine, fibroblast growth factor, insulin like growth factor and DAPT. Gene expression of markers of photoreceptor precursors (CRX, NRL, Nr2e3) was studied by RT-PCR, whilst analysis of phenotypic differentiation was performed by immunohistochemistry and confocal microscopy.
Results: :
A significant increase in the expression of Crx, Nrl and Nr2e3 was observed in Müller stem cells cultured on laminin or bMP in the presence of retinoic acid, taurine, insulin like growth factor, fibroblast growth factor or DAPT. All individual factors induced an increased expression of these genes but combinations of some of these factors had a more significant effect not only on gene expression (CRX, p=0.001, NRL, p=0.0285) but also on the phenotypic changes observed on these cells after 5-7 days in culture.
Conclusions: :
It has been shown that post mitotic retinal progenitors are ontogenetically ideal to replace photoreceptors in experimental models of photoreceptor cell death. This study shows that Müller stem cells can be induced to differentiate towards photoreceptor precursors in vitro by using a short time protocol based on extracellular matrix substrates and exogenous factors. It suggests that this strategy may potentially be used to generate a population of ontogenetically suitable cells for transplantation into models of photoreceptor degeneration that could in principle be scaled up under GMP conditions and readily translated into human therapies.
Keywords: differentiation • Muller cells • photoreceptors