Experimental Study Of Immunogenicity And Immunoregulation On Human Bone Marrow Derived Mesenchymal Stem Cells

Background:Bone defect caused by severe trauma,infection,tumors,osteonosus and so on is very common clinically,treated mainly with autogenous bone transplantation, allogeneic or xenogenic bone grafts,biomaterial replacement and other approaches. Autogenous bone grafting is the gold standard and the most effective methods for treating osseous defects,but autologous bone grafts often don't satisfy the amount of bone tissue needed for reconstruction and would bring patients new wound,the other ways have also no satisfied effect.In recent years tissue engineering bone brings new hope for bone defect repairing.MSCs(bone marrow derived mesenchymal stem cells) are considered as the optimal seed cells for bone tissue engineering.MSCs can be derived conveniently because they can be obtained through puncture only with small wounds and fewer complications, and be culturabel with rapid proliferation.Osteogenic induction in vivo and in vitro environment not only can differentiate into bone tissue,but also has more potential to differentiate.In addition,MSCs are also easily separated and cultured.In recent years,autologous osteoblast or MSCs for the individual animal and clinical treatment has produced good results.But MSCs content in the bone marrow is low,and decreasing with age and difficult in rapid amplification in vitro.Moreover,some autoimmune diseases significantly weaken its proliferation ability in MSCs.With increased culturing algebra "anti-differentiation" phenomenon and tumor growth may appear, accompanied with the loss of cell function and the safety.Therefore,autologous MSCs is limited owing to the use of sources and quantitative restrictions and can not meet "off the shelf" requirement.Establishing allogenic seed cell bank of MSCs is the shortcuts to solve these problems.Previous studies in lower-class animals showed that grafting in engineered tendon,cartilage and bone constructed using allogeneic seeding cells elicit no visible immunologic reaction and may influence on repairing bone defect,but people know few about the immunogenicity and immunoregulation of hMSCs after Osteogenic differentiated and treated by IFN-γ.The aim of the study is to investigate the the immunogenicity and immunoregulation of hMSCs after Osteogenic differentiated and treated by IFN-γ,it will prepare for the further application of allogeneic hMSCs.Objective:(1)To observed hMSCs isolated and cultured in vitro and induced osteoblast,and its characteristics in cell biology were;(2)To investigate the immunogenicity of hMSCs,Osteogenic cells differentiated from mesenchymal stem cells(DOC) and treated after IFN-γby FACS,RT-PCR and WB;(3)To explore the immunomodulatory mechanisms of hMSCs,DOC and treated after IFN-γby MLR under different conditions on peripheral blood mononuclear and cytokine secretion by ELISA.Material and methods:1.The biological characteristics of hMSCs.(1) Under aseptic conditions,about 20ml bone marrow was aspirated from ailiac crest of volunteer. hMSCs obtained by density gradient centrifugation and purification from bone marrow, were cultured with 15%fetal bovine serum in the culture medium F12-DMEM.(2) The growth curve and the growth cycle of hMSCs and DOC cells are detected and compared.(3) (The expression of CD14,CD29,CD34,CD44,CD45,CD105,CD166 in the third generation cell are dectcted by FACS.4) hMSCs differentiated into osteoblast were identified using tetracycline,alizarin red;collagenâ… and osteocalcin immunohistochemical staining.2.Experiment on immunogenicity of hMSCs,DOC,hMSCs+IFN-γ,DOC+IFN-γ.(1) Undifferentiated hMSCs was used as control group,investigated the expression of CD40,CD40L,CD80,CD86,HLA-â… ,HLA-â…¡of hMSCs,hMSCs+IFN-γ;DOC,DOC+IFN-γin day6,day12,day18.(2)Gene expression of B7-H1,B7-H4,HLA-B,HLA-DRA,HLA-DRB of hMSCs,hMSCs+IFN-γ;DOC,DOC+IFN-γin day6,day12,day18 were detected by RT-PCR.(3) Gene expression of B7-H1,B7-H4 of hMSCs,hMSCs+IFN-γ; DOC,DOC+IFN-γin day6,day12,day 18 were detected by WB.3.Experiment on immunoregulation of hMSCs,DOC,hMSCs+IFN-γ,DOC+IFN-γ. (1) By mixed lymphocyte reaction(MLR)the following were observed:â‘ Effect on PBMCs proliferation stimulated by allograft PBMCs of different magnitude of hMSCs,hMSCs+IFN-γ;DOC,DOC+IFN-γin dayl8;â‘¡Effect on PBMCs proliferation of hMSCs, hMSCs+IFN-γ;DOC,DOC+IFN-γin day18 by mitogen-stimulated;â‘¢Effect on PBMCs proliferation stimulated by allograft PBMCs and mitogen of different days of 10⁴hMSCs,hMSCs+IFN-γ;DOC,DOC+IFN-γin day18;(2) To detected the expression of IL-2,IL-4,IL-10,TNF-α,TGF-β1 and PGE2 in their culture supernatant by ELISA.Results:1.The biological characteristics of human MSCs.(1)The characteristics of hMSCs:cells appearance presented cambiform or triangular-shaped,fibroblasts-like and vortex-like.Four days of hMSCs of original generation,the fibroblast-like cell colony was observed,and hMSCs have fulfilled the bottom of the monolayer cultural bottle in 9-12 days,while the passage cells having done in 4-7 days;There was no obvious difference in adherence of DOC and hMSCs.Doubling time in Growth curve of hMSCs was 38.1h,and DOC was 39.7h.Compared with the cell cycle of DOC,hMSCs accounted for larger proportion in S+G2 phase of cell cycle,relatively small proportion in G1 phase.These suggested that hMSCs proliferated and growed faster than DOC.(2) The third generation was showed by FACS that expression of CD29,CD44,CD105 and CD166 was strong positive in the cultured cells,and expression of CD14,CD34 and CD45 is negative.(3) On 16^thd after osteogenic induction,there were a lot of calcium deposition in the massive cells with Alizarin red and tetracycline staining;there were lots of brown-positive cells by osteocalcin and collagenâ… immunocytochemical staining.2.Experiment on immunogenicity of hMSCs,hMSCs+IFN-γ,DOC,DOC+IFN-γ. (1) Undifferentiated hMSCs was used as control group,results by FACS showed that expression of HLA-â… increased in hMSCs+IFN-γgroup and DOC+IFN-γgroup(P＜0.05); morever,expression of HLA-â…¡increased significantly(P＜0.01).(2) Undifferentiated hMSCs was used as control group,results by RT-PCR showed that expression of B7-H1,B7-H4,HLA-B,HLA-DRA,HLA-DRB significantly increased expression in hMSCs+ IFN-γgroup and DOC+IFN-group(P＜0.01).(3) Undifferentiated hMSCs was used as control group,results by WB showed that expression of B7-H1,B7-H4 significantly increased expression in hMSCs+ IFN-γgroup and DOC+IFN-group(P＜0.01).3.Experiment on immunoregulation of hMSCs,hMSCs+IFN-γ,DOC,DOC+IFN-γ. (1) By mixed lymphocyte reaction(MLR)the following were founded:â‘ Different magnitude of hMSCs,hMSCs+IFN-γ;DOC,DOC+IFN-γin day18 can inhibit the proliferation of PBMCs stimulated by allograft PBMCs.The inhibition effects correlated with cell quantity positively,there is statistics evaluation between 10⁵ magnitude and 10³ magnitude(P＜0.01).â‘¡Different magnitude of hMSCs,DOC can inhibited the proliferation of PBMCs stimulated by mitogen.Besides the 10³ magnitude,Different magnitude of hMSCs+IFN-γ,DOC+IFN-γin day18 can inhibited the proliferation of PBMCs stimulated by mitogen,The inhibition effects correlated with cell quantity positively,PBMC proliferation.Inhibition effects correlated with cell quantity positively.â‘¢In different days,10⁴ magnitude hMSCs and DOC can inhibited the proliferation of PBMCs stimulated by allograft PBMCs and mitogen.The lowest peak appear in sixth day;Besides the second day,10⁴ magnitude hMSCs+IFN-γand DOC+IFN-γcan inhibited the proliferation of PBMCs.(2) hMSCs,hMSCs+IFN-γ;DOC,DOC+IFN-γin day18 could secrete IL-2,IL-4,IL-10,TNF-αin low level;TGF-β1,and PGE2 in high level; compared to the group without the stimulation of IFN-γ,all of cytokine increase of group stimulated after IFN-γand some cytokine have statistics evaluation(P＜0.01).Conclusions:(1) The cells obtained from dissociating red marrow by density gradient centrifugation have matched the hMSCs features in aspects of appearance,cell surface marker and multi-directional differentiation ability,it can be differentiated into osteoblast, and express collagenâ… and osteocalcin under the inducible conditions.(2) In vitro experiments show the progeny of hMSCs and DOC remains low immuogenicity,but the proinflammatory cytokine stimulation enhance the immunogenicity.(3) Besides PHA group,Different magnitude of hMSCs,hMSCs+IFN-γ;DOC,DOC+IFN-γin day18 can inhibited the proliferation of PBMCs stimulated by allograft PBMCs.The inhibition effects correlated with cell quantity positively,which can regulate immune responses by probably secreting cytokines level for immune regulatory role.In short,allogeneic hMSCs may be good and effective choice for tissue engineering.