Mesenchymal Stem Cell Exosomes-derived MicroRNA-223-3p Inhibit The Early Stage Development Of Murine Acute Graft-versus-host Disease

Objective:To explore the effect of micro RNA from different tissue mesenchymal stem cell-derived exosomes on the development of murine acute graft-versus-host disease(a Gv HD).Methods:(1)MSCs derived from murine compact bone and human umbilical cord were isolated and c?lture-expanded,the immuophenotypic of MSCs was analyzed by flow cytometry.Moreover,the adipogenic and osteogenic induced differentiation ability were validated by Oil-red-O staining and alkaline phosphatase staining.The key adipocyte transcription factor of C/EBP? and PPAR? and osteogenic differentiation transcription factor of Osteocalin and Runx2 for MSCs were measured by quantitative reverse transcriptase polymerase chain reaction(q-PCR).Next,MSCs were c?ltured with serum-free medium and collected the supernatant.The MSCs-derived exosomes were isolated by exosome isolation kit and analyzed the exosome size,morphological features,surface markers using flow cytometry,western blot,Nano-tracking technology and transmission electron microscope.(2)To assess the biological function of MSCs secrete exosome in vivo.C57BL/6 mice were injected MSCs by lateral tail vein.After 48 hours transplantation,the peripheral blood serum were collected and isolated exosomes by exosome isolation kit.The surface marker of exosome was determined by western blot,and mi R-223-3p in blood serum was detected by q-PCR.(3)Co-C?lture of MSC-derived exosome with human umbilical vein endothelial cell(HUVEC)for 24 h,and then assessed the expression of mi R-223-3p and ICAM-1 in co-culture HUVEC by q-PCR and western blot.Furthermore,HUVEC were transfected with mi R-223-3p mimic and NC,and then measured the expression of transcription level of mi R-223-3p and protein level of ICAM-1 by q-PCR and western blot.(3)The spleenocytes and bone marrow nuclear cells from C57BL/6 mice(H-2Kb)were infused into BALB/c recipient mice(H-2Kd)to establish an allogeneic bone marrow transplantation model.MSCs or mi R-223-3p mimic and NC were injected into lethally irradiated(8 Gy)C57BL/6 mice thro?gh lateral tail vein.The condition of the animals was monitored,including growth appearance,survival,clinical score of Gv HD,and white cell number.The migration ability of donor lymphocyte after bone marrow transplantation and the expression of inflammatory factor IFN-?were assessed by flow cytometry.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression of inflammatory factor in blood serum.The homing of donor lymphocyte in the spleen,lung,intestines and liver was observed by freezing microtome assay.Res?lts:(1)MSCs were isolated from murine compact bone and human umbilical cord,these cells exhibited the typical fibroblast-like cells morphology;both adherent cells were positive for Sca-1,CD29,CD105,CD73,CD166 and negative for CD31,CD34,CD45,and MHCII.Adipogenic differentiation of MSCs was apparent after 14 days of induction,indicated by accum?lation of oil-red-O staining lipid-rich vesicles.Under osteogenic conditions for 2 weeks,MSCs also co?ld form aggregates or nod?les displaying alkaline phosphatase activity.In agreement with the res?lts of differentiation assays,q-PCR analysis also demonstrated that MSCs displayed corresponding transcriptional expression of C/EBP? and paroxysm proliferation activated receptor gamma 2(PPAR?2),Osteocalin and Runx2 under specific adipogenic and osteogenic inductive c?ltures,respectively.These res?lts strongly indicated that the adherent cell isolated from murine compact bone and human umbilical cord were MSCs.Moreover,MSCs were c?ltured in free fetal calf serum for 24 hours and then collected the c?lture supernatant for purified exosomes by an exosome isolation kit.Flow cytometry analysis demonstrated that the exosome was positive for CD63 and CD81 in human umbilical cord MSCs-derived exosome,and the expression of CD63 and TSG101 in exosome from human and mouse MSCs was discovered by western blot.Nano-tracking technology revealed that the size of exosome was more than 100 nm in diameter,and was confirmed to be present on the vesicles with exosomal and larger shed membrane features by transmission electron microscope analysis.These res?lts s?ggest that the exosome was purified from human and mouse MSCs.Next,mi RNA CHIP was performed on these two kinds of MSC-derived exosomes.Using bioinformatics screening and comparison method,the high expression of mi RNA-223-3p in human and mouse MSC-derived exosomes was identified.(2)To further confirm the biological function of exosome,the mouse MSCs were injected into C57BL/6 mice thro?gh lateral tail vein.After transplantation for 48 hours,the exosome in the peripheral blood serum were collected and isolated exosomes.western blot analysis determined that exosome from blood serum was positive for CD63 and TSG101.The expression of mi R-223-3p in exosome was positive by q-PCR.There is a significant difference between MSC-treated group and non MSC-treated group(P(27)0.05).These res?lts support that MSC promotes the high expression of mi RNA-223-3p in vivo.(3)To address the mechanism of MSC-derived exosomes,the MSC-derived exosomes were co-c?ltured with HUVEC.Q-PCR analysis revealed that mi RNA-223-3p was overexpression in co-c?ltured HUVEC.Comparing with the control group,there is a significant difference(P(27)0.05).western blot analysis showed that the target gene of mi RNA-223-3p was ICAM-1,and down-expressed of ICAM-1 in HUVEC coc?ltue system.These data s?ggest that MSC-derived exosome reg?lated the expression of ICAM-1 in HUVEC thro?gh transfering mi RNA-223-3p into the HUVEC.Furthermore,mi RNA-223-3p mimic and NC were tranfected into HUVEC,the res?lts showed that mi RNA-223-3p mimic co?ld promote mi RNA-223-3p expression and inhibit the target gene of ICAM-1expression in transcription level and protein level.(4)To analyze whether mi R-223 contributes to murine Gv HD development,the mi RNA-223-3p mimic and NC were infused Gv HD mice thro?gh lateral tail vein.The res?lts showed that mi RNA-223-3p mimic infusion dramatically delayed the development of a Gv HD.Injection of mi RNA-223-3p mimic significantly enhanced the survival of Gv HD mice,and recovered the clinical symptosm,including diets,weight,activity and hematopoietic system.Flow cytometry analysis demonstrated that mi RNA-223-3p co?ld inhibit the migration of donor lymphocytes to recipiet spleen and down reg?late the expression of IFN-?.The homing of donor lymphocyte in the spleen,lung,intestines and liver was undetected by freezing microtome assay.ELISA assay revealed that mi RNA-223-3p co?ld suppress the secretion of IFN-?in blood serum.Conclusion: MSC-derived exosomes dramatically inhibited the expression of ICAM-1 in HUVEC by overexpression of mi RNA-223-3p,and further suppressed the homing ability of donor lymphocytes and secreting IFN-? in a Gv HD mice.These res?lts s?ggest that MSC-derived exosome co?ld inhibit the early stage development of murine a GvHD by miRNA-223-3p.