Retinal ganglion cell-conditioned medium and surrounding pressure alters gene expression and differentiation of rat retinal progenitor cells

Abstract

Loss of retinal ganglion cells is implicated in glaucoma and high intraocular pressure. Factors that affect the differentiation of retinal progenitor cells into retinal ganglion cells remain unclear. The present study aimed to investigate the effects of retinal ganglion cell-conditioned medium on gene expression and differentiation in retinal progenitor cells, and the effects of surrounding pressure on the survival and differentiation of retinal progenitor cells. Retinal progenitor cells and retinal ganglion cells were isolated from rats. Immunofluorescence staining of Nestin and Thy1 was performed to identify rat retinal progenitor cells and retinal ganglion cells, respectively. Retinal progenitor cells and ganglion cells were cultured for 48 h under surrounding pressure of 0, 20, 40, 60 and 80 mmHg. Cellular apoptosis was detected using a caspase-3 assay kit. In addition, the culture supernatant of rat retinal ganglion cells was collected. Retinal progenitor cells were cultured in the presence or absence of retinal ganglion-conditioned medium for 72 h under normal pressure. Gene expression of Nestin, paired box protein 6 (PAX6), Thy1 and brain-specific homeobox/POU domain protein 3 (Brn-3) in retinal progenitor cells was detected by reverse transcription-quantitative polymerase chain reaction. Retinal progenitor cells were cultured in retinal ganglion-conditioned medium for 72 h under surrounding pressure of 0 and 40 mmHg, respectively, and flow cytometry was utilized to evaluate the effects of pressure on the differentiation of retinal progenitor cells into retinal ganglion cells. The results demonstrated that isolated retinal progenitor cells were Nestin-positive and retinal ganglion cells were Thy1-positive, suggesting successful isolation. The activity of caspase-3 increased in retinal progenitor cells and retinal ganglion cells in a pressure-dependent manner. When the surrounding pressure reached 40, 60 and 80 mmHg, the activity of caspase-3 in retinal progenitor cells and ganglion cells increased significantly compared with cells that were not under pressure. Compared with retinal progenitor cells cultured without ganglion-conditioned medium, those cultured with ganglion-conditioned medium had significantly decreased expression levels of Nestin and PAX6, and increased expression levels of Thy1 and Brn3. Compared with 0 mmHg pressure, retinal progenitor cells cultured in ganglion-conditioned medium under 40 mmHg pressure had increased percentages of Thy1-positive cells. In conclusion, the apoptosis of rat retinal progenitor cells and retinal ganglion cells was pressure-dependent. Retinal ganglion cell-conditioned medium increased the differentiation of retinal progenitor cells into retinal ganglion-like cells, and the differentiation increased as surrounding pressure increased. Current study provides insights that may contribute to the efforts of developing a treatment for glaucoma.