Application And Precision Exploration Of Human Wharton’s Jelly Mesenchymal Stem Cells And Their Small Extracellular Vesicles In Tissue Injury Repair

BackgroundHuman umbilical cord Wharton’s Jelly mesenchymal stem cells(WJ-MSCs)have become one of the ideal seed cells in tissue engineering regenerative medicine with their advantages of convenient collection,non-invasive invasion and abundant sources.WJ-MSCs show attractive application prosepcts in promoting wound healing,inhibiting scar hyperplasia and promoting cartilage regeneration.Small extracellular vesicles(s EVs)are important paracrine components that mediate the positive results of MSCs therapy.They carry a large number of bioactive substances and can simulate the biological functions of MSCs and were also considered natural nanotherapeutic agents.Therefore,MSCs and their derivatives s EVs are hot new regenerative medicine tools in terms of therapeutic characteristics.It is of great significance to explore the efficacy and potential molecular mechanisms of MSCs-based cell therapy and MSCs derivatives-based cell-free therapy in tissue injury diseases for the selection of clinical strategies.In addition,on the basis of effective treatment,we need to consider the precision of treatment.At present,there are great challenges in promoting the clinical transformation of MSCs,such as the uneven effect of cell therapy and poor repeatability.The fundamental reason is the inherent heterogeneity of MSCs.Obviously,comprehensive characterization and separation of homologous and effective dominant functional subsets are important measures for the development of precise MSCs treatment programs.Fortunately,single cell multimodal omics provides strong technical support for the realization of this goal.ObjectiveIn part 1.WJ-MSCs derivatives based cell-free treatment:To explore the therapeutic effect and mechanism of s EVs derived from WJ-MSCs in treating osteoarthritis(OA),and provide a theoretical basis for cell-free treatment.In part 2.WJ-MSCs based precision therapy:To analyze the heterogeneity and differential functional sources of cultured WJ-MSCs,and to analyze the spatial gene expression patterns of WJ-MSCs in congenetic umbilical cord tissues,and to establish a screening scheme for dominant functional subpopulation with enhanced wound repair properities.MethodsIn part 1.(1)WJ-MSCs were isolated by tissue block culture method and cultured in vitro.The surface markers were detected by flow cytometry.(2)The s EVs were extracted from WJMSC culture supernatant(WJMSC-s EVs)by size exclusion chromatography,and morphological analysis,surface marker detection and particle size analysis were performed.(3)In vitro experiments:Chondrocyte inflammation model was established and treated with different concentrations of WJMSC-s EVs(1,5,10,15μg/ml).The proliferation and migration of chondrocytes were detected,and the polarization effect of WJMSC-s EVs on macrophages was observed by co-culture with RAW264.7 cells.(4)In vivo experiments:A rat OA model was induced by anterior cruciate ligament rupture.Intra-articular injections were performed 4 weeks following surgery.Sham group,Control group(OA animals were treated with 200μL PBS once a week for 4weeks),WJ-MSCs group(OA animals were treated with 5×10~5cells/200μL one time),and WJMSC-s EVs group(OA animals were treated with 200μL WJMSC-s EVs(30μg)once a week for 4 weeks).At 9 weeks after operation,the knee specimens were collected for gross observesion,micro CT scanning,histopathological examination and pathological grading.Pathological changes of cartilage tissue in each group were observed by pathological section of knee joint.(5)Proteomics and micro RNA sequencing were used to elucidate the molecular mechanisms.In part 2.(1)Single-cell RNA sequencing:fresh umbilical cords from 3healthy maternal puerpera were collected.We isolated WJ-MSCs from Wharton’s jelly of umbilical cord using tissue block culture methods,and obtained passage 3(P3)cells by in vitro expansion.After the preparation of single-cell suspensions,sc RNA-seq and bioinformatics analysis were performed to draw the heterogeneity maps of WJ-MSCs in different donors,different generations and different subpopulations from transcriptome level.Global transcriptional patterns are obtained in combination with bulk RNA sequencing to comprehensively characterize gene expression changes.(2)Functional verification of dominant functional subpopulations in wound repair:the dominant functional subpopulation with high wound repair ability was preliminarily determined based on the sc RNA-seq data.We identified the subpopulation marker genes and then used the flow cytometry sorting technology to enrich the subpopulation.Transwell co-culture system was used to compare the effects of dominant functional subpopulation and unsorted WJ-MSCs on the proliferation and migration of wound healing-related cells.Then we established wild-type AB strain zebrafish skin wound model,each zebrafish per group received a 10μL subcutaneous injection of respective cells(1×10~4cells)or PBS at the wound edge.The wound area was measured at 0,5,15and 30 days after operation,and wound healing rate was calculated.(3)Spatial transcriptome:two fresh umbilical cords homologous to sc RNA-seq were selected to prepare the tissue blocks with a length of 3 cm were prepared at the maternal and fetal ends of umbilical cord,respectively.ST sequencing technology was used to draw the spatial expression maps between and within wharton’s jelly tissue regions.SPOTlight analysis was performed to integrate single cell and ST data.ResultsIn part 1.(1)P3 WJ-MSCs highly expressed CD44,CD73,CD105,CD90,but low expressed CD34,CD45,CD19,HLA-DR.WJMSC-s EVs had typical disc morphology with an average diameter of about 69.66 nm.They highly expressed ALIX,TSG101 and CD63,but did not express Calnexin.(2)In vitro,WJMSC-s EVs promote chondrocyte proliferation and migration in a concentration-dependent manner(15μg/ml group had the best effect).In addition,WJMSC-s EVs can promote the polarization of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages.(3)In vivo experiments,WJ-MSCs and WJMSC-s EVs can promote the regeneration of articular cartilage in rats,improve pathological scores,and significantly up-regulate the expression of Collagen II and down-regulate the expression of MMP13 and ADAMTS5.(4)Proteomics and micro RNA sequencing results showed that the biological processes involved in the molecules carried by WJMSC-s EVs contributed significantly to the treatment of OA.In particular,let-7e-5p contained in WJMSC-s EVs was found to be a potential core molecule for promoting cartilage regeneration via regulating the level of STAT3 and IGF1R.In part 2.(1)We mapped transcriptome profiles of WJ-MSCs at the single-cell level.38125 single cells were screened for downstream analysis.Four subpopulations of WJ-MSCs were identified,and then gene expression profiles,cell communication networks and transcriptional regulation networks of different WJ-MSCs subpopulations were presented at the single-cell level.(2)Through single-cell marker gene expression and functional annotation,we determined that biofunctional type＿MSC had high wound healing ability and could be sorted depending on the selectable markers(S100A9,CD29 and CD142).In vitro experiments showed that S100A9~+CD29~+CD142~+cells could significantly promote the proliferation and migration of wound healing-related cells compared with the control group.In vivo zebrafish skin wound model found that this subpopulation can accelerate wound repair and enhanced re-epithelization.(3)The spatial gene expression profiles of wharton’s jelly tissues in different regions were mapping.We identified subpopulations including wharton’s jelly-derived myofibroblast cells,human umbilical cord perivascular cells,epithelial cells,and epithelial cells,and confirmed the existence and distribution of subpopulations by immunofluorescence.In addition,through the analysis of differential expressed genes and functional enrichment,it was found that the gene set of fetal segment was related to skin development,neutrophil activation involved in immune response and regulation of angiogenesis.Most importantly,the result of SPOTlight demonstrated that fetal segment contains more biofunctional type＿MSC with wound repair function.Conclusion1.Our data showed that WJMSC-s EVs had a positive protective effect on cartilage and subchondral bone injury caused by OA.In addition,WJMSC-s EVs can carry specific micro RNAs and proteins to maintain cartilage homeostasis by reversing inflammatory-mediated chondrocyte changes.Therefore,compared with their parent cells and complex synthetic nanoparticles,natural WJMSC-s EVs would be the preferred cell-free nanotherapeutic agent.2.This is the first study to comprehensively analyze the heterogeneity of human umbilical cord tissue and homologous cultured WJ-MSCs by combination of single-cell RNA sequencing and spatial transcriptome data.Our results reveal the heterogeneity of WJ-MSCs in donor,generation,subpopulation and space,and finding the presence of WJ-MSCs subpopulation with efficient wound repair capabilities in fetal region of the umbilical cord.The results not only provide data supplement for traditional bulk RNA sequencing and single cell sequencing,but also inspire us to treat specific diseases in a more targeted manner,providing a new direction for precise treatment of stem cells.