Adipose tissue derived mesenchymal stem cells differentiate towards RPE and rescue apoptotic RPE under oxidative stress, in vitro and in vivo.

Abstract

Purpose : Oxidative stress leads to degeneration and apoptosis of Retinal pigment epithelial cells (RPE) in age related macular degeneration (AMD). Subcutaneous adipose derived mesenchymal stem cells (ASCs) may serve a therapeutic tool for regenerating RPE. Here we evaluated ASCs’ protective effect on apoptosis of RPE, the differentiation potential of ASCs towards RPE, and the efficacy of sub-retinal transplantations of ASCs in a mouse model of AMD.

Methods : Human ASCs were harvested from subcutaneous fat of patients undergoing abdominoplasty. A co-culture of ASCs and human RPE, or human RPE alone, was subjected to oxidative stress by exposure to 1.5mM hydrogen peroxide (H2O2). H2O2 induced RPE apoptosis was measured by Annexin V/ propidium iodide staining and flow cytometry analysis. The differentiation potential of ASCs towards RPE was evaluated using td-tomato marked RPE and GFP marked ASCs seeded in a co-culture. GFP marked ASCs were then isolated by FACS sorter and analyzed for RPE and eyefield markers (BF1, Rx, MITF, PAX6, RPE65) by qRT-PCR and immunostaining. Finally, GFP marked ASCs were transplanted in the subretinal space of sodium iodate (NAIO3) treated mice compared to controls of subretinal saline injection. Evaluation of transplanted ASCs and endogenous RPE was studied by immunostaining for GFP and RPE65 of frozen sections at day 0, 7, 14, and 21 days post injection.

Results : Treatment of RPE with ASCs prevented H2O2 induced apoptosis (70% decrease, p <0.05). After 7 days in co-culture, ASCs upregulated RPE and eyefeild markers (BF1 1.9+ 0.2 Rx 4.5+0.8 MITF 1.9+0.17 PAX6 5.5+0.2 RPE65 7.7+2.6, folds of control ). In vivo, transplanted ASCs were located in the subretinal space of NAIO3 mice at days 0, 7, 14, and 21 post injections. Level of RPE65 was higher in ASCs treated mice at day 14 (4 folds increase in mean florescence of RPE65 compared to controls).

Conclusions : Treatment of apoptotic RPE with ASCs reduced RPE apoptosis in vitro. ASCs demonstrate a differentiation potential into RPE, evident by upregulation of RPE and eye field markers. Transplantation of ASCs in the subretinal space of NAIO3 mice resulted in an increase in RPE cell count compared to controls. ASCs may have therapeutic potential in regenerating RPE.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.