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 a relatively preserved other retinal layers. A significant obstacle to this approach, however, is the ability of the transplanted cells to integrate and form new synapses with the host retina through the extension of neurites. The goal of this study is to investigate the effect of various molecules and growth factors on the ability of rat photoreceptor precursors (rPRP) to extend neurites. We focused on the role of RhoA, which is involved in cytoskeleton organization, and its related kinase (ROCK), 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, followed by treatment with various molecules. After three days, the cells were stained for both CRX (a PRP marker) and actin and the length of their extensions was measured. To characterize signal pathways involved in neurite extension, RNAseq analysis was performed on cell cultures treated with ROCKi (ROCK inhibitor). Furthermore, we established an ex vivo model in which rPRP cells were seeded on a degenerated retina, to investigate neurite extension toward the inner retinal cells.
Results :
Following ROCKi treatment, CRX positive cells exhibited a dose dependent increase in the percentage of cells with processes with a measured average neurite length of 3.97 µm ±2.42, 17.17 µm ±0.45,19.12 µm ±2.42 and 21.45 µm ±7.65 for control, 50µM, 100 µM and 200µM respectively. Glial condition media had a weaker effect on the cells, while BDNF and NGF had no effect. RNAseq analysis identified several pathways that were upregulated, including those related to phototransduction, CREB signaling, and YAP-controlled genes. The explant model showed that the retina was able to maintain its structural integrity for seven days without experiencing significant cell death. During which, rPRP cells seeded on the explant extended neurites towards the retina bipolar cells.
Conclusions :
The findings presented here provide a foundation for improving neurite extension and the formation of synapses 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 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.