Potential role of exosome associated proteins from iPSC derived MSCs for early complications of diabetic retinopathy

Abstract

Purpose : We have developed a consistent and scalable cell source of induced MSCs (iMSCs) generated by differentiating the induced pluripotent stem cells (iPSC) derived from urinary epithelial cells through a non-insertional reprogramming approach. We tested the hypothesis that intravitreal delivery of iMSC exosomes will ameliorate retinal vascular and neuronal damage induced in diabetes.

Methods : Conditioned medium from iMSC that were exposed to TNFα/IFNγ in serum-free media was used to purify exosomes by ultracentrifugation method. Differential expression of proteins was analyzed by proteomic analysis. About 8-month-old Ins2^Akita/+ mice were intravitreally injected with exosomes (5mg protein/2μL) or saline. Littermate Ins2^+/+ receiving saline served as control. After 3 weeks post IVT, mice were tested for vascular permeability by FITC-BSA leakage method, visual function by ERG and OKN, and assessed for changes in dendritic complexity and postsynaptic targets by immunohistology.

Results : About 463 proteins were identified in the exosomes. Differential expression analysis revealed ~33 proteins enriched (>1.5 fold) in primed exosomes compared to unprimed exosomes. While Ins2^+/+ mice had minimal vascular leakage (30.7±6.0 RFU), Ins2^+/Akita mice that received saline showed 70.7±10.7 RFU with a significant reduction in Ins2^+/Akita mice that received exosomes (38.2±10.8 RFU, p<0.03). Similarly, a significant reduction in visual acuity (0.25±0.2 vs. 0.3±0.01 c/d, p<0.04), increased in contrast sensitivity threshold (57±8.6 vs. 39.1±6.5 %, p<0.05) and decreased b-wave amplitudes (220.2±7.8 vs. 306.2±16.3 mV, p<0.02) in Ins2^+/Akita mice were improved with exosomes. Finally, immunohistology and confocal microscopy revealed loss of dendrites in cone bipolar (Secretagogin), rod bipolar (PKCα), and horizontal cells (Calbindin D28k) in Ins2^+/Akita mice regenerated in mice that received exosomes.

Conclusions : Our data showed that a single intravitreal injection of iMSC-exosomes in Ins2^Akita/+ mice, reversed the ongoing vascular leakiness, regeneration of dendrite retraction, and re-establishment of synaptic disruption to rescue visual function. Future studies aiming at in-depth analysis of differentially expressed proteins within the exosomes will help establish the molecular processes leading to changes in the diabetic retina post iMSC-exosome transplantation.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.