The Therapeutic Effect And Mechanism Of Human Umbilical Cord Mesenchymal Stem Cell-derived Exosomes In Acute Kidney Injury

ObjectiveAcute kidney injury(AKI)is a common critical clinical disorder associated with high morbidity and mortality.There is no definitive therapy to treat established AKI.Exosomes derived from mesenchymal stem cells(MSC-exos),as a cell-free therapy,have been demonstrated with great potential in the treatment of AKI by virtue of their intrinsic cargoes.Nevertheless,the underlying mechanisms remain largely unknown.This study mainly explored:(1)The biodistribution and efficacy of MSC-exos in the ischemia-reperfusion(I/R)-induced AKI(I/R-AKI)animal model;(2)The specific mechanism of MSC-exos on renal tubular repair;(3)The effects of hollow-fiber 3D culture system on the yield and therapeutic efficacy of MSC-exos on AKI.This study provided experimental and theoretical basis for understanding the novel mechanism of MSC-exos on the treatment of AKI and promoting its clinical transformation.This study includes three parts:Part one:The therapeutic effects of MSC-exos on ischemic AKIMethods:Human umbilical cord mesenchymal stem cells(huc MSCs)were identified by surface markers,osteogenesis,adipogenesis,and cartilage formation.Huc MSCs within the P6 generation were cultured for an additional 48 h in FBS-free media.The FBS-free culture supernatants were collected for isolating exosomes(MSC-exos).MSC-exos were characterized by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA),and western blot analysis of exosome markers.The biodistribution of MSC-exos in murine I/R-AKI was imaged by the IVIS spectrum imaging system.The mechanism of MSC-exos homing to injuried kidney was proved through techniques such as antibody blocking and si RNA.Finally,the therapeutic efficacy of MSC-exos was investigated in I/R-AKI mice.Results:Huc MSCs presented a homogeneous population of spindle fibroblast-like cells.Flow cytometry analysis revealed that huc MSCs were highly positive(>97%)for MSC surface markers including CD29,CD44,CD73 and CD90,but negative(<1%)for hematopoietic stem cell(HSC)surface markers CD34 and CD45.When cultured in osteogenic,adipogenic or chondragenic media,huc MSCs were able to differentiate into osteoblasts,adipocytes or chondroblasts.TEM showed typical bilayer membrane vesicles of MSC-exos.NTA analysis indicated the median diameter of MSC-exos was 134 nm.Ex vivo imaging showed that MSC-exos was efficiently homing to the ischemic kidney and predominantly accumulated in proximal tubules by virtue of the VLA-4 and LFA-1 on MSC-exos surface.MSC-exos alleviated murine ischemic AKI and decreased renal tubular injury in a dose-dependent manner.Conclusion:MSC-exos exhibit preferential tropism to injured kidney and localize to proximal tubules in ischemic AKI.The mechanism is mainly mediated by the binding of integrins VLA-4 and LFA-1 on MSC-exos to VCAM-1 and ICAM-1 on damaged renal tubules.Furthermore,MSC-exos could ameliorate ischemic AKI and promote tubular repair in a dose-dependent manner.Part two:The mechanism of MSC-exos on repairing ischemic AKIMethods:We labeled proliferating cells with Ki-67 and cells in G2/M phase withp-H3 staining.Hypoxia/reoxygenation(H/R)was used to construct cell injury models.The cell cycle arrest,proliferation and apoptosis of tubular epithelial cells(TECs)were evaluated in vivo and in HK-2 cells.The exosomal miRNAs of MSC-exos were profiled by high-throughput miRNA sequencing.One of the most enriched miRNAs in MSC-exos was knockdown by transfecting miRNA inhibitor to huc MSCs.Then we investigated whether this candidate miRNA was involved in MSC-exos-mediated tubular repair.Results:MSC-exos significantly attenuated the cell cycle arrest and apoptosis of TECs both in vivo and in vitro.Mechanistically,miR-125b-5p,which was highly enriched in MSC-exos,repressed the protein expression of p53 in TECs,leading to not only up-regulation of CDK1 and Cyclin B1 to rescue G2/M arrest,but also modulation of Bcl-2 and Bax to inhibit TEC apoptosis.Finally,inhibiting miR-125b-5p could mitigate the protective effects of MSC-exos in I/R mice.Conclusion:We demonstrated that MSC-exos ameliorate ischemic AKI and promote tubular repair by targeting the cell cycle arrest and apoptosis of TECs through miR-125b-5p/p53 pathway.This study provides a novel insight into the role of MSC-exos in renal tubule repair and highlights the potential of MSC-exos as a promising therapeutic strategy for AKI.Part three:The effect of 3D culture on the yield of MSC-exos and its therapeutic efficacy for AKIMethods:MSCs were isolated from fresh human umbilical cord and cultured in two-dimensional(2D)flasks.2×10~8 MSCs were inoculated into the hollow fiber bioreactor for 3D culture.The culture supernatants were collected every one or two days for isolating exosomes.Exosomes from 2D(2D-exos)and 3D cultures(3D-exos)were characterized by transmission electron microscopy,nanoparticle tracking analysis,and western blot analysis of exosome markers.The yield of exosomes from2×10~8 MSCs seeded in 2D and 3D culture system was compared,based on protein quantification.The therapeutic efficacy of 2D-exos and 3D-exos was investigated in a murine model of cisplatin-induced AKI in vivo and in vitro.Results:3D culture did not significantly change the surface markers of MSCs,as well as the morphology,size,and exosomal markers of 3D-exos when compared to those of 2D-exos.Compared with conventional 2D culture,the 3D culture system increased total exosome production up to 19.4 fold.3D-exos were more concentrated in the harvested supernatants(15.5 fold)than 2D-exos,which led to a higher exosome collection efficiency of 3D culture system.In vivo,both 2D-exos and 3D-exos significantly alleviated cisplatin-induced murine AKI evidenced by improved renal function,attenuated pathological changes of renal tubules,reduced inflammatory factors,and repressed T cells and macrophages infiltration.Impressively,3D-exos were more effective than 2D-exos.Moreover,3D-exos were taken up by TECs with improved efficiency,thereby exhibiting superior anti-inflammatory effects and improved viability of TECs in vitro.Conclusions:Our findings demonstrated that the hollow-fiber 3D culture system provides an efficient strategy for the continuous production of MSC-exos which has enhanced therapeutic potential for cisplatin-induced AKI.Summary and main innovations:In this study,we explored the biodistribution and therapeutic effect of MSC-exos on I/R-AKI,and investigated its specific mechanism on renal tubular repair.Furthermore,we established a novel strategy to produce 3D-exos with a hollow fiber bioreactor-based 3D culture system.The therapeutic efficacy of 2D-exos and 3D-exos was investigated in a murine model of cisplatin-induced AKI.The main innovations are as following:1.We found MSC-exos exhibited preferential tropism to injured kidney and localized to proximal tubules in I/R-AKI.This was mainly mediated by the binding of integrins VLA-4 and LFA-1 on MSC-exos to VCAM-1 and ICAM-1 on damaged renal tubules.2.MSC-exos could significantly attenuate the cell cycle arrest and apoptosis of TECs.Mechanistically,MSC-exos could deliver miR-125b-5p to TECs and promote tubular repair by targeting the cell cycle arrest and apoptosis of TECs through down-regulating p53.3.We found that the hollow-fiber 3D culture system could more efficiently produce MSC-exos in a continuous way compared with conventional 2D culture system.Furthermore,MSC-exos derived from the 3D culture system experted superior therapeutic potential in treatment of the cisplatin-induced AKI mice.