Investigating the Bmp4 pathway regulation effect on Photoreceptors' neurite outgrowth in in vitro and ex vivo models

Abstract

Purpose : Pluripotent-derived photoreceptor transplants are a promising treatment for outer retinal degenerative diseases, which are manifested by the loss of photoreceptor cells but with relatively preserved other retinal layers. A significant challenge, however, is the ability of the transplanted cells to functionally integrate with the host retina through the extension of neurites and synaptogenesis. The goal of this study is to investigate the effect of various molecules and on the ability of rat photoreceptor precursors (rPRP) to extend neurites and to understand the mechanisms which regulate these processes. We focused on RhoA, its related kinase (ROCK), Taurine (Tau) and glial cells media on neurite extension.

Methods : rPRP cells derived from neonatal SD rats were cultured on PLL/laminin coated plates. The cultures were treated with ara-C to inhibit glial cells proliferation, followed by treatment with various molecules. After three days, the cells were stained for the PRP marker CRX and actin and the length of their extensions was measured. To characterize signal pathways involved in neurite extension, RNA-seq analysis was performed on cell cultures treated with ROCK inhibitor (Y27632) and compared to a control group. The results were validated by qPCR of selected genes and were tested on conditioned media (CM) and Tau with the same methods.
Furthermore, we established an ex-vivo model in which dissociated rPRP cells were seeded on degenerated retinas, to investigate neurite extension toward the inner retinal cells.

Results : Following ROCKi treatment, CRX positive cells exhibited dose dependent increase in cell percentage with processes and increase in neurites length Glial condition media and Tau had a weaker effect on the cells. RNA-seq analysis identified several upregulated pathways. qPCR analysis for these 3 treatments showed an upregulation of Bmp4 of more than 2-fold. Preliminary results of Bmp4 treatment exhibited neurite extension and retraction by inhibition of this pathway. Finally, rPRP cells seeded on the explant model with the mentioned treatments extended neurites towards the retina bipolar cells after 24h.

Conclusions : The findings presented here provide a foundation for improving neurite extension and synapse formation between the host retina and transplanted cells, addressing one of the major challenges in cell replacement-based vision restoration.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.