Abstract
Purpose: :
Increasing evidence suggests that Müller glia can function as stem cells in the adult mammalian retina. However, their ability to proliferate, dedifferentiate and generate retinal neurons is limited. The mechanisms imposing these limitations are poorly understood. This study tested the roles of DNA methylation and brain derived neurotrophic factor (BDNF) in regulating gene expression and differentiation potential of Müller-derived retinal stem cells.
Methods: :
Conditionally immortalized mouse Müller glia (ImM10) cultured as spheres in serum-free medium with 20 ng/ml EGF and FGF2 for 5 days, with 0 or 10µM 5-Aza-deoxycytidine (AzadC) for the last 48 hours. Following priming with 20ng/ml EGF, cells were cultured for 5 or 25 days in differentiating medium containing 0 or 20ng/ml BDNF, without AzadC (n=3 for each condition). Following differentiation, mRNA and protein expression were analyzed by quantitative RT-PCR and immunohistochemistry.
Results: :
In differentiation cultures without AzadC pretreatment, BDNF (5 days) significantly increased expression of 14 of 16 genes tested vs. no BDNF controls (p=0.001). Increased genes included pluripotency [Pou5f1 (3.8 fold), Nanog2 (only detected in BDNF treated cells), Sox2 (2.6-fold)], retinal ganglion cell-specific [Atoh7 (93-fold), Pou4f2 (>10,000 fold), Pou4f3 (152-fold), Isl1 (only detected in BDNF treated cells)] and retinal pigment epithelium genes [Rpe65 (only detected in BDNF-treated cells), Bestrophin1 (143-fold)]. Cralbp increased (21-fold), but expression of Vimentin was unchanged. Prior AzadC treatment increased expression of the same gene-set, but yielded larger increases for Pou4f2 (1.2-fold), EphB2 (1.4-fold) and Mitf (2-fold) (p=0.001 vs. BDNF without AzadC). In long-term cultures (25 days) in BDNF, with or without prior demethylation, expression of all genes up-regulated at 5 days declined significantly (p≤0.009), except for Mitf (4.15-fold) and EphB1 (6.55-fold) in demethylated samples (p=0.001 vs. 5 day BDNF controls without AzadC). For both AzdC treated and untreated groups, immunostaining revealed increased expression of PAX6 (day 5 and 25) and ZO1 (day 25) with BDNF differentiation.
Conclusions: :
Our data suggests a role of BDNF in regulation of pluripotent, RGC developmental and retinal pigment epithelium specific genes. The additive effect of BDNF treatment and DNA demethylation are consistent with epigenetic limitations on the proliferative and neurogenic potential of Müller glia in vitro. Reversing epigenetic silencing of neuronal genes together with enhancing neuronal survival offers a potential strategy for increasing neurogenesis from Müller-derived stem cells.
Keywords: Muller cells • gene/expression • retinal culture