Embryonic Stem Cell Microenvironment Reverses Hydrogen Dioxide-induced Retinal Pigment Epithelium Cellular Senescence by Activating the TGFβ Signaling Pathway

Abstract

Purpose : Retinal pigment epithelium (RPE) cellular senescence is an important pathogenic factor of age-related macular degeneration (AMD). We previously demonstrated that embryonic stem cells (ESCs) microenvironment can reverse somatic cellular senescence. Accordingly, we hypothesize that ESCs microenvironment (ESCMe) can reverse hydrogen dioxide (H₂O₂)-induced RPE cellular senescence.

Methods : Human primary RPE cells were treated with 0-600 μM H₂O₂ for 4h to identify the final concentration. RPE cells were divided into three groups: (1) R: cells cultured for 48h; (2) RH: cells treated with H₂O₂ for 4h and then cultured for 44h; and (3) RHE: cells treated with H₂O₂ for 4h and then co-cultured with ESCs at a 1:2 ratio for 44h and finally isolated. The cell counting kit 8 (CCK-8) assay, senescence-related β-galactosidase (SA-β-GAL) staining, cell cycle distribution, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were measured. P21^WAF1/CIP1, cyclin A2 and cyclin B1 were detected by RT-PCR and western blotting. RNA-seq was used to reveal specific mechanisms. The two-tailed t-test was used for statistical analysis (RH vs R; RHE vs RH).

Results : Cells showed decreased proliferation and increased SA-β-GAL⁺ rates with increasing doses of H₂O₂. 400 μM H₂O₂ was finally used. RPE cells in RH group showed decreased proliferation, G₀/G₁ fraction (p＜0.0001) and levels of cyclin A2 (0.27-fold) and cyclin B1 (0.68-fold), and increased SA-β-GAL⁺ rate (p＜0.0001), ROS (p＜0.0001), MMP (p＜0.0001), G₂/M fraction (p＜0.0001) and p21^WAF1/CIP1 expression (3.93-fold) compared to cells in R group. However, RPE cells in RHE group showed increased proliferation and levels of cyclin A2 (1.70-fold) and cyclin B1 (1.77-fold), and decreased SA-β-GAL⁺ rate (p=0.008 ), ROS (p=0.003), MMP (p=0.0021) and p21^WAF1/CIP1 (0.77-fold) expression compared to cells in RH group. There was no statistically significant difference in cell cycle distribution. RNA-seq revealed that TGF-β1 (2.14-fold), ID1 (2.17-fold) and ID3 (3.55-fold) were up-regulated and ID4 (0.35-fold) was down-regulated in RHE group, which were further verified by RT-PCR and western blotting.

Conclusions : ESCMe can effectively reverse H₂O₂-induced RPE cellular senescence, possibly by activating the TGFβ pathway. The cell cycle changed by ESCMe in senescent RPE cells will be further studied.

This is a 2020 ARVO Annual Meeting abstract.