Hyaluronic Acid Hydrogels Induce mTOR Activation to Enhance Isolated Rod Photoreceptor Survival in Vitro

Research into photoreceptor development and degeneration has been greatly hindered by the lack of methods for in vitro culture of photoreceptors. Conventional 2-dimensional (2D) culture of photoreceptors leads to profound cell death and morphological changes. We found that culturing dissociated photoreceptors in hyaluronic acid (HA) – based hydrogels greatly enhances their survival in vitro. We then investigated the molecular mechanism that mediates the prosurvival effect of the HA hydrogels on the photoreceptors.

 

Rod photoreceptors were isolated by cell sorting from NrlGFP mice and cultured in 2D or in HA gels. Alive cells stained for Hoechst (nuclei), expressed GFP (Nrl-driven) and did not stain for EthD-1 (dead cell dye). To assess whether the differential stiffness of the HA gels compared to 2D tissue culture plastic mediated the prosurvival effect, we cultured photoreceptors in HA gels of varied stiffness. We found that altering substrate stiffness did not affect rod photoreceptor survival. To investigate the role of HA, we cultured photoreceptors in hydrogels without HA, or added soluble HA in 2D culture conditions. The presence of HA significantly enhanced rod survival, irrespective of whether the culture is performed in 2D or 3D. In order to decipher the molecular pathways induced by HA, immunostaining for phospho-mTOR as well as small inhibitor experiments were conducted. We used rapamycin, IWR1e and Y27632 to inhibit the mTOR, canonical Wnt and ROCK pathways, respectively. HA induced an increase in phospho-mTOR staining from 1.53% to 27.2%, and treatment with rapamycin abolished the prosurvival effect of the HA hydrogels. Treatment with IWR1e or Y26732 attenuated but did not eliminate the prosurvival effect of HA on rods. When either of IWR1e or Y26732 was combined with rapamycin, no additional decrease in rod survival was observed as compared to rapamycin alone. Lastly, when IWR1e and Y26732 were combined in the absence of rapamycin, the prosurvival effect of HA was abolished.

We conclude that the HA component of the HA-based hydrogels mediates photoreceptor survival through activating the mTOR pathway. The upstream mediators are canonical Wnt signaling and the RhoA/ROCK pathway. Our hydrogel represents a promising substrate for in vitro culture of stem cell-derived photoreceptors to study their development or perform screening assays. The pathways we identified may constitute compelling targets for investigation in animal models of retinal degeneration.