Abstract
Purpose :
The Ciliary Epithelium (CE) of adult mammalian eyes contains a quiescent population of retinal progenitor/stem cells that are able to proliferate, generate neurospheres in vitro and differentiate into functional retinal neurons. Despite this regenerative potential, the neuronal differentiation efficiency is low, probably due to the presence of regulatory mechanisms and inhibitory factors. Platelet-Activating Factor (PAF) is a lipid mediator present during retinal development. PAF and PAF receptor (PAFR) demonstrated inhibitory effects on retinal progenitor cell cycle and neuronal differentiation in mammalian developing retinas. We investigated the role of PAF and PAFR in CE cells during neurospheres formation
Methods :
CE cells from Balb/c wild type mice were separated from retina, sclera and iris. The Pigmented Epithelium (PE) cells were cultured with growth factors (FGF and EGF, 10 and 20 ng/ml) to form neurospheres, and treated with PAFR agonist (cPAF, 100 nM) or antagonist (PCA4248, 10 μM). After 7 days in culture, we analyzed neurospheres size and number, and expression of stem cells markers by PCR and Immunohistochemistry
Results :
Retinal progenitor cells within the neurospheres expressed PAFR protein and transcripts levels similarly to the original pigmented epithelial cells from CE. Neurospheres treated with PAFR agonist (cPAF) increased the expression of markers related to differentiated epithelial cells (palmd), and decreased progenitor/stem cell markers (pax6, nanog, sox2, and nestin). On the other hand, neurospheres treated with PAFR antagonist (PCA4248) increased the neurospheres in number and in progenitor/stem cell markers.
Conclusions :
Our results suggest that inhibition of PAF signaling through its receptor increased progenitor/stem cells markers expression and the number of neurospheres, probably trough the regulation of the cell cycle. However, activation of PAF signaling was able to decrease the stem cell profiles expression and increased differentiated cells transcripts. The information gleaned from this study may provide valuable insight into the cellular and molecular events that underlie the reprogramming response of CE cells and the mechanism of retinal differentiation
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.