Exosomes from Adipose Derived Stromal Cells Mitigate Acute Radiation Injury in Mice

Abstract

Radiation-induced Gastrointestinal Syndrome (RIGS) results from a combination of lethal injury to intestinal crypts and the stromal microenvironment. Our previous studies demonstrated that bone marrow derived stromal cells (BMSC) mitigate RIGS and regenerate intestinal crypts. Mitigation of RIGS by transplantation of adipose derived stromal cells (AdSC) has also been reported. We hypothesized that the stromal cells secrete exosomes loaded with paracrine factors for regeneration of irradiated crypts. In this study, we examined the radio-mitigation effects of Ad-SC-derived exosomes in mouse intestinal organoids, in vitro, and in survival experiments after whole body irradiation (WBI) of mice. AdSC, isolated from the subcutaneous and visceral fat from hyperlipidemic, ApoE-deficient female mice, were cultured, and characterized using Flow Cytometry, fluorescence microscopy, and mesenchymal stem cell differentiation assay. Exosomes were isolated from culture supernatants using the Total Exosome Isolation Kit (ThermoFisher) and centrifuged at 10,000 g for 1 hour. For survival experiments, C57/Bl6 mice were irradiated with 9.4 WBI and animals were treated with exosomes 24 hours after WBI. Treatment groups included Intraperitoneal (IP) exosomes (100 ug, n = 7), Intravenous (IV) Exosomes (100 ug, n = 6), or PBS control (n = 6). Intestinal organoids were irradiated at 7 Gy and treated with growth media and exosomes. Post-irradiation crypt budding and proliferation were monitored. Kaplan-Meier Survival curves were generated to determine overall-survival (OS). Flow cytometry demonstrated that AdSC expressed vimentin (53.1%) and were negative for Mesenchymal Stem Cell (MSC) markers (CD90, CD105, CD29, and CD133). Immunofluorescent staining demonstrated that AdSC expressed alpha-SMA and Nestin, which are markers of myofibroblasts. AdSC also failed to differentiate into adipocytes, chondrocytes, and osteoblasts, indicating that these stromal cells were not mesenchymal. AdSC-derived exosomes showed remarkable benefit against radiation injury in vivo and in vitro. Post-irradiation survival (Day 30) in animals treated with IV exosome, IP exosome, and Control-PBS was 86%, 71%, and 33%, respectively (log-rank P < 0.001 for both IV exosome and IP exosome compared to control), suggesting a significant survival benefit. Exosome-treated mouse intestinal organoids and human iPSC-derived intestinal organoids (iHIOs) showed significant proliferation and budding post-irradiation compared to irradiated controls. These results indicate that the exosomes derived from adipose stromal cells serve as an excellent cell-free alternative for treatment of lethal radiation injuries.