| Liver disease is one of the major diseases which caused great harizonaards to human health and social economy.There are two urgent problems need to be solved both in basic research and clinical applications on this disease,one is that the lacking of the suitable animal models which could really match to the biological characteristics of human's liver disease,and another is the shortage of donor liver for transplantation and the immune rejection.Macaques belonging to primates monkeys macaca animals,and also called rhesus monkey.As a kind of higher non-human primates,macaques has high genetic similarity with human being,thus it is generally accepted as an important experimental animal model for human disease research.Mesenchymal stem cells(MSCs)is a kind of adult connective tissue cells which exist in mesoderm and ectoderm tissues,it can be isolated and expanded easily from bone marrow and many tissues,and it can differentiate to many cell types such as hepatocyte and promote tissue regeneration.In addition,there is no ethical issues in MSCs compare with embryonic stem cell in clinical application,all those make MSCs one of the best cell types for regenerative medicine.In this study,we used macaque bone marrow-derived MSCs to perform a systematical preclinical research focus on stem cell transplantation therapy for liver disease.MSCs isolation and identification from bone marrow,cryopreserved condition exploring of MSCs for long time store,the potential compare of bone marrow and adipose-derived MSCs differentiation to hepatocyte-like cells,the effect of MSCs labeled with green fluorescent protein(GFP)and a new type superparamagnetic iron oxide called MIRB,and transplant MSCs to treat liver fibrosis animal models including mouse and macaque rhesus,for this present study,we also expected that the differentiation,engraftment and tracking of MSCs transplantation to the liver disease were explored,as well as important theoretical,technical and applied reference were provided for clinical research.In addition,as a comparation of MSCs therapy,we also studied the therapeutic effect that MSCs treat spontaneous osteoarthritisof rhesus monkeys.1.MSCs were isolated from macaque rhesus bone marrow using the method of differential attachment.After identified through surface marker,differentiation potential to adipogenesis,osteogenesis and chondrogenesis,and compared with the standard of The International Society of Cellular Therapy(ISCT),we finished the confirmation of the MSCs.2.The comparison of differentiation potential to hepatocyte-like cells between bone marrow-derived and adipose-derived MSCs were assayed through morphological observation,hepatic glycogen staining,urea detection and hepatic related genes expression analysis,and it was showed that the differentiation potential of bone marrow-derived MSCs was stronger than that of adipose-derived.3.MSCs were double labbeled with GFP(by lentiviral transfection)and new-style superparamgnetic iron oxide(MIRB)in this study.The labeling rate of GFP and MIRB were above 98%,and the proliferative activity and differentiative potential were all not affected after labeling.This double-label method could satisfy with both detection in vitro and tracking in vivo after MSCs transplantation.It is particularly helpful for understanding the mechanism of migration,homing,planting and destiny of MSCs.4.DMNA induced macaque liver fibrosis model and CCl4 induced mouse liver fibrosis model were established successfully.5.GFP labeled MSCs were transplanted into DMNA induced liver fibrosis macaque and CCl4 induced liver fibrosis mouse The effectiveness of MSCs transplantation treatment was evaluated by serology,pathology and iconography,the results showed that the degree of liver fibrosis were both improved after MSCs transplantation 1 month later in macaque and mouse.6.As a control,the MSCs transplantation therapy to spontaneous osteoarthritis in macaque were also studied preliminarily MSCs were transplanted into articular cavity of macaque with spontaneous osteoarthritis.After analyzed the results of articular width,angle of femur and tibia,relative serum proteins,X-ray images,MRI and athletic ability with 3 times MSCs transplantation,we found that allogeneic MSCs showed a positive effectiveness to macaque spontaneous osteoarthritis.7.For the aim of screening out an optimal condition on long-time cryopreservation for macaque rhesus bone marrow-derived MSCs,we assay an comparison on the effect to biological characteristics of MSCs which were vitrified cryopreserved using two different permeable cryoprotectants DMSO and EG respectively.,The results showed that there were no differences in morphology,surface markers and differentiation potential in MSCs between DMSO and EG vitrified cryopreservation,however,EG seems has the advantages over DMSO both in viability and proliferation activity.Further analysis of genes expression by RNA-seq gave the results of that,compared with non-cryopreserved MSCs,there were 6987 differential expressed genes exist in EG vitrification MSCs,and just 2524 differential expressed genes in DMSO vitrification MSCs.Among them,there were 1886 differential expressed genes exist between EG and DMSO vitrification MSCs.From the point of function,the differential expressed genes could classify to 8 for pluripotency,28 for differentiation,44 MSCs specific genes,4 for immunosuppression and immune regulation,26 for methylation regulation,21 for oxidative stress and lipid peroxidation,6414 for metabolism and 88 for apoptosis,necrosis and autophagy.Furtherly,the result of GO functions enrichment showed that differential expression genes were enriched in immune response function,immune system processes,hydrolase and the activity of cytokines.These results indicated that different cryoprotectants could result in gene differential expression in macaque bone marrow MSCs.Above all,a preclinical study of macaque bone marrow-derived MSCs transplantation for liver fibrosis and osteoarthritis and the influence comparison between two vitrified cryoprotectants on biological characteristics of MSCs was performed this time.All the results of this study would be the important reference for MSCs transplantation therapy on human liver disease and osteoarthritis,and it would also provide important information to the studies of mechanism on MSCs migration,homing and tumorigenicity. |
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