Abstract
Purpose :
Mesenchymal stem/stromal cells (MSC) are well known for immunomodulation; however, the mechanisms involved in their benefits in the ischemic retina are unknown. In this study, we tested the hypothesis that MSC via upregulation of transcription factor forkhead box P3 (Foxp3) in T cells elicit immune modulation and thus protect against ischemic retinal damage
Methods :
MSCs were generated from urine epithelial cells derived induced pluripotent stem cells (iPSC) through non-insertional reprogramming (iMSCs). Mitochondria transfer from MSC to immune cells was assessed by confocal microscopy, and oxygen consumption rates (OCR) were measured using Seahorse Flux Bioanalyzer. Activated splenocytes co-cultured with iMSC in differentiation medium were assessed with anti-Foxp3 antibody and analyzed by flow cytometry. Unilateral retinal ischemia reperfusion (I/R) were done in adult C57BL/6 mice by transiently elevating the intraocular pressure for 1 h. Uninjured eyes served as I/R controls. After 1 day of reperfusion, the animals were randomized to receive intravitreal iMSC (1000 cells/1mL) or saline (1mL). After 7 days, the retinal function was assessed by Electroretinogram (ERG). The retinal extracts were processed by qRT-PCR, and the retinal flat mounts were processed by confocal microscopy for Foxp3+ cells.
Results :
In in-vitro cultures, iMSC transferred mitochondria to immune cells via F-actin nanotubes, significantly increased OCR for basal respiration and ATP production, suppressed effector T cells, and promoted differentiation of CD4+CD25+ Tregs in co-culture with mouse splenocytes. In in-vivo studies, iMSCs in I/R eye significantly increased Tregs in the retina compared to saline injected I/R eyes (63.4 ± 14.29 v/s 29.99 ± 6.69 cells/mm2, p<0.05, anova). Furthermore, iMSC injected I/R eyes had decrease in retinal inflammation (IL1b: 6.09±1.54 v/s 27.08±8.69 fold p<0.01; Ccl2: 3.52±0.96 v/s 13.6±2.7 fold p<0.001,anova) and improved b-wave amplitudes compared to saline injected I/R eyes (at 1cd.s.m2 139±48 v/s 48±10 µvolt, p=0.05, t-test).
Conclusions :
Our study demonstrates that iPSC derived MSCs can transfer mitochondria to T cells to enhance differentiation into Foxp3 Tregs. Additionally, our current data demonstrates that MSC can improve the retina's immune function through upregulation of Tregs to decrease inflammation to reduce I/R injury-induced retinal degeneration
This is a 2021 ARVO Annual Meeting abstract.