The Fuction And Safety Research Of SCRG1 In Chondrogenic Differentiation Of Mesenchymal Stem Cells

Objectives:Cartilage defect is a common disease which was caused by many situations,such as trauma,tumors,autoimmune diseases and aging etc.The ability of cartilage regeneration is very weak,once the cartilage defect appears,it is difficult to repair,and cartilage degeneration will eventually occur,which seriously affects the health of the patient.Tissue engineering for cartilage regeneration is a promising treatment option.we can replace and repair damaged cartilage through tissue engineering cartilage.The source of seed cells was the key facor of cartilage tissue engineering,which is based on the rapid preparation of mesenchymal stem cells(MSCs)differentiation.The MSCs have a strong ability to differentiate into cartilage.At present,MSC-based cartilage regeneration has achieved promising results.A series of clinical trials have been carried out,and it is a promising cartilage defect repair method.However,there still remaining many problems.The prominent manifestation is that it takes a long time for cartilage differentiation,which takes about 3-4 weeks.At the same time,the underlying mechanism of chondrogenic differentiation is still unclear and requires deeply research.Since the chondrogenic differentiation of MSCs is an extremely complex process,there are many factors involved in each stage,especially a series of biologically active factors,which may be the target of chondrogenic differentiation.Therefore,it is important to identify a single transcription factor to promote cartilage differentiation.According to previous studies,we found that Scrapie-Responsive Gene 1(SCRG1)may promoting the differentiation of MSCs into cartilage,and is expected to clarify the mechanism of UCMSCs into cartilage differentiation.According to this purpose,we explored the function of SCRG1 in the chondrogenic differentiation of UCMSCs in this experiment,and tried to clarify the possible mechanism,to provide reliable and fast-prepared seed cells for cartilage tissue regeneration.In recent years,using UCMSCs to treat a variety of diseases has been widely carried out,but at the same time,due to its own stemness and allogeneic sources,there may be exist safety risks during the treatment,especially the administratiion of viruses to interfere with gene expression,which increases potential risks in stem cell therapy.Therefore,this study also explored the safety of UCMSCs administration.It provides the latest scientific evidence for the safety of UCMSCs in cartilage tissue regenerative medicine,and our research may benefit for the clinical application of UCMSCs.Methods:Part 1:The functional research of SCRG1 on chondrogenic differentiation of UCMSCs in vitro1.Identification of UCMSCs:UCMSCs were isolated by tissue-adherent culture methods.The surface markers(including CD90,CD73,CD105;CD45,CD34,CD 19,CD11b,HLA-DR)were detected by flow cytometry,and the chondrogenic differentiation ability was used to detect differentiation pluripotency of UCMSCs.Our identification results should meet the standards of The International Society for Cellular Therapy(ISCT).The chondrogenic differentiation of UCMSCs was induced by microsphere culture method,and the chondrogenic differentiation was detected by alician blue staining.After that,the quantitative real-time polymerase chain reaction(qRT-PCR)and immunohistochemistry(IHC)were used to detect the gene and protein expression of SCRG1 during cartilage differentiation.2.Overexpressing of SCRG1 in UCMSCs:RNA interference(RNAi)and gene overexpression techniques were used to intervene the gene expression level of SCRG1 in UCMSCs,and the expression level of SCRG1 protein was detected by immunocytochemistry(ICC).Then chondrogenic differentiation was induced in vitro.The chondrogenic differentiation ability of UCMSCs was identified by alician blue staining,and the expression levels of genes and proteins such as SOX9,ACAN,COL2A1 related to chondrogenic differentiation were detected by qRT-PCR and IHC.Cytometric Bead Array(CBA)method was used to measure the expression of inflammatory factors in UCMSCs after overexpression of SCRG1.Part 2:The mechanism of SCRG1 in the chondrogenic differentiation of UCMSCsUCMSCs and UCMSCs overexpressing SCRG1(UCMSCOE-SCRG1)were collected,and the different expressed genes between the two groups were tested by RNA sequencing(RNAseq),and then these molecules with significant differences were selected by bioinformatics analysis.Next,based on gene enrichment analysis(Gene Set Enrichment Analysis,GSEA),we found out the gene sets which related to stem cell differentiation,screen out the differentially expressed molecules according to this gene set,and PCR and WB was used to verify the expression of these molecules.Then,the role of this signaling molecules in SCRG1 which promoting the chondrogenic differentiation of UCMSCs was verified by the recovery experiments.Including recombinant protein,RNAi and gene overexpression were used to detect the expression of cartilage-related genes and upstream and downstream pathways.Part 3:The safety and function study of SCRG1 enhancing chondrogenic differentiation of UCMSCs in vivo1.The safety assessment of UCMSCs after overexpression of SCRG1:The expression level of oncogenes,including C-MYC,K-RAS,H-RAS and tumor suppressor genes RA and P53 were detected after overexpression of SCRG1 in UCMSCs.Cell proliferation ability was detected by CCK-8,and then the clone formation assay(Clony-Forming Unit Assays,CFU)was used to detect the changes of clonogenic ability of UCMSC,UCMSCOE-NC and UCMSCOE-SCRGI group.Finally,the tumorigenicity of UCMSC,UCMSCOE-NC and UCMSCOE-SCRG1 group was detected by subcutaneous allogeneic cell transplantation in nude mice.2.The functional assessment of UCMSCsOE-SCRG1 in vivo:(1)The animal model of acute cartilage defect:The skin and joint capsule were surgically incised,and the knee joint was exposed.An acute cartilage defect model was established in the trochlear groove of the distal femur of the rabbit knee joint.Then,UCMSCs were immediately transplanted to the defect site,and the joint capsule and skin were closed.The repair period is 12 weeks,and magnetic resonance imaging(Magnatic Resonance Imaging,MRI)was used to detect the repairment of UCMSCs in cartilage defects.After the observation period was ended,the femur head were collected and stained for pathology,including hematoxylin-eosin(HE),alician blue,and safranin "O"/fast green staining,and finally evaluate by the International Cartilage Repair Society(ICRS)gross morphology assessment scale and the histology scoring system.(2)The animal model of chronic osteoarthritis:The modified Hulth method was used to established chronic osteoarthritis model,and the animal model was treated with UCMSCs.MRI was used to detect the repair of cartilage defects.After the treatment,the samples were collected and stained for pathology,including HE and safranin "O"fast green staining.The modified mankin score was used to evalueate the repairment of chronic osteoarthritis in rabbit knee.The expression of inflammatory factors in synovial fluid was detected by enzyme linked immunosorbent assay(ELISA).Results:Part 1:SCRG1 promotes chondrogenic differentiation of UCMSCs in vitro1.The UCMSCs were successfully isolated through tissue-adherent culture methods,and can be proliferated and stably passaged in vitro.The morphology,surface molecular markers and multi-lineage differentiation ability of the isolated cells was met the international identification standards of mesenchymal stem cells.The isolated cells were spindle-shaped,similar to fibroblasts.Flow analysis showed that the cells positively expressed CD90,CD73,and CD105;negatively expressed CD45,CD34,CD 19,CD11b,and HLA-DR.The isolated UCMSCs have the ability of osteogenic,adipogenic and chondrogenic differentiation.2.The gene and protein levels of SCRG1 increased during the chondrogenic differentiation of UCMSCs.After RNAi interfered with the expression of SCRG1,the results of qRT-PCR showed that the gene levels of SOX9,ACAN,and COL2A1 were decreased,and the results of IHC showed that the protein levels of ACAN and COL2A1 were decreased.These results indicating that the chondrogenic differentiation ability of UCMSCs was reduced.3.After overexpression of SCRG1,qRT-PCR results showed that the gene levels of SOX9,ACAN and COL2A1 were increased,and the IHC results showed that the expressions level of ACAN and COL2A1 protein were increased,and the chondrogenic differentiation of UCMSCs was enhanced.The results of CBA assay showed that the expression of inflammatory factors in UCMSCs remained unchanged after overexpression of SCRG1 in vitro.Part 2:SCRG1 promotes chondrogenic differentiation of UCMSCs via Wnt5aThe different expressed genes were screened out by RNAseq,and GSEA analysis was used to find the enriched signal pathway.The top enriched gene set was "Stem Cell Pluripotency Regulation Signaling Pathway(KO04550)",which was related to stem cell totipotency.In this gene set,we found that after overexpression of SCRG1,UCMSCs’ Stem cell totipotency decreased,and our results showed that the decrease of stem cell totipotency was mainly related to the activation of WNT signaling pathway.The PCR and WB results showed that the expression of Wnt5a protein was increased after SCRG1 overexpression,and further WB detection results showed that the canonical WNT signaling pathway was inhibited,suggesting that Wnt5a exert functions through activating non-canonical Wnt/β-catenin signaling pathway.In the recovery experiment,the addition of recombinant proteins Wnt5a(rhWnt5a)and SCRG1(rhSCRG1)could reverse the changes in wnt/β-catenin signaling pathway caused by SCRG1 knockdown.Administration of rhWnt5a can promote the chondrogenic differentiation of UCMSCs in vitro.Part 3:UCMSCs overexpressing SCRG1 are a safe source of seed cells for cartilage regeneration and promote the repair of cartilage defect in vivo1.There was no tumorigenic in UCMSCOE-SCRG1:The expressions of oncogenes(C-MYC,K-RAS)in UCMSC,UCMSCOE-NC and UCMSCOE-SCRG1 group were down-regulated,and H-RAS was slightly increased.The expression of tumor suppressor genes RA and P53 did not decrease significantly.The cell growth curve results showed that the proliferation of UCMSCs was inhibited after overexpression of SCRG1.Colony-Forming Unit Assays(CFU)assay results showed that UCMSC,UCMSCOE-NC and UCMSCOE-SCRG1 group was failed to form clones in vitro,and there was no change in the ability of colony formation between these groups.The results of subcutaneous allogeneic cell transplantation in nude mice showed that no nodules were formed under the skin in the UCMSC,the UCMSCOE-NC and the UCMSCOE-SCRG1 group during the 12 weeks observation,and no tumor nodules were formed in the brain,liver,lung and other tissues.2.UCMSCOE-SCRG1 promoted the repairment of acute cartilage defects:In vivo experiments,we eastablished an acute cartilage defects model in the distal trochlear groove of the femur head of the rabbit knee joint,and then used Matrigel as a cell scaffold and transplanted it into the defect.The results showed that UCMSCs overexpressing SCRG1 can promote the repair of acute cartilage defects.The groos appearance is similar to the surrounding normal tissue.HE staining result shows a large number of cell proliferation at the defect.Alicia blue and safranin "O"/fast green staining shows that there are a large number of chondrocytes and extrachondral matrix in the repaired region,suggesting that there is cartilage tissue regeneration in the defect.The cartilage repair gross score and tissue repair score were all significantly different from the control group.We successfully established a chronic osteoarthritis animal model.After administration of UCMSCs,HE and safranin "O"/fast green staining results and Mankin score showed that UCMSCOE-NC group and UCMSCOE-SCRG1 group had a protective effect on chronic osteoarthritis.The results of ELISA showed that the pro-inflammatory factors in the synovial fluid of the UCMSCOE-NC group and the UCMSCOE-SCRG1 group were lower than those of the Blank group.Conclusions:1.In this study,human UCMSCs meeting international standards were successfully isolated.The expression of SCRG1 increased during the chondrogenic differentiation of UCMSCs,and the expression level was positively correlated with the chondrogenic differentiation ability of UCMSCs.2.After overexpression of SCRG1 in UCMSCs,the totipotency of the cells was reduced,and the cells differentiated into chondrocytes.The expression of Wnt5a,a signaling ligand of the WNTA signaling family,was increased.The canonical Wnt/β-catenin signaling pathway was inhibited by Wnt5a,and promotes the differentiation of UCMSCs into chondrocytes finally.3.UCMSCs and UCMSCOE-SCRG1 did not appear cancerous in vitro and did not have tumorigenicity in vivo.Transplantation of UCMSCOE-SCRG1 could promote the repair of acute cartilage defects.UCMSCOE-SCRG1 can be used as a source of seed cells for safe and rapid preparation of cartilage tissue engineering regenerative medicine.