Studies On The Characteristics Of Fetal Tissue-derived Human Mesenchymal Stem Cells And Its Immuno-Double-effect On Immune Hepatic Injury

Obiectives: Have emerged as a promising therapeutic modality for tissue regeneration and repair, MSCs have low immunogenicity and the ability to migrate to sites of tissue injury and rapidly expand and differentiate along its own, allogeneil or xenogeneic lineages to repair the damaged tissue without rejection. Compare to the MSCs from adult, the human MSCs from fetus are present earlier during development, less likely to be infected with pathogen and can be more easily harvested. Therefore, MSCs from fetus are the main source for transplant.It is known that transplant between individuals with different genetic background can induce the rejection, which has not been solved perfectly until now. It hinders the clinical application of transplant. MSC transplant have generated expectations since MSCs are capable of differentiating along multiple lineages and have low immunogenicity.Numerous studies have demonstrated that MSCs have powerful immunosuppressive function, and may be involved in the mechanism of the vanishing of graft rejection. Therefore, it is very important to show that the engraft of ectogenic MSCs do not induce tumorigenesis.Up to now, MSCs are largely studied for their uses in tissue repair, immunotolerance reestablishment and other fields. The clinical trials of MSCs therapy in cardiac muscle repair, bone disease and metabolic disease have been carried out all over the world. Several studies indicated that adoption of autogeneic or allogenetic MSCs by different administration routes (region/i.v./i.p.) has therapeutic action on experimental autoimmune encephalomyelitis (EAE)and collagenâ…¡-induced arthritis(CIA). Moreover, Le Blanc and his colleague have successfully harnessed MSCs to treat severe acute graft-versus-host disease(GVHD), and greatly reduced the incidence of GVHD. These results all suggest that MSCs could provide an ideal cell source in the treatment of immune-mediated diseases.In this study we examined the effect of hMSCs on ConA-induced hepatitis, and verified the therapeutic value and mechanism of hMSC in autoimmune hepatitis (AIH). Additionally, we validated the potential oncogenicity and promotion to hepatoma growth of hMSCs.Methods: hMSCs were isolated from aborted fetus's bone marrow or amniotic fluid. In order to certifficate the cells cultured in vitro are hMSC, we observed the cell morphology under a phase microscope, detected the phenotype by flow cytometry (FCM) and identified the potential for differentiation by culture cells under conditions that favored adipogenic and osteogenic differentiation. To confirm the immunomodulatory effect of hMSCs isolated from aborted fetus's bone marrow or amniotic fluid in vitro using MTS. Splenocytes stimulated by ConA were incubated in medium, the medium supernatant was harvested. hMSCs were incubated in the supernatant, and its features including proliferation, phenotype and chromosome caryotype were observed. ConA mice model were performed according to the protocol of Tiegs. The indexes of hepatic injury included the serum levels of alanine aminotransferase (ALT) , aspartate aminotransferase (AST) and C3, and the pathological lesion of liver. We used the indexes above to verify the effect of hMSCs with or without pretreated to the ConA mice model. To test if mice splenocytes cultivate supernatant pretreatment could induce the malignant growth of hMSCs, hMSCs with pretreated were subcutaneously inoculated in athymic mice. To test the influence of hMSCs to tumor, we used MHCC/97H (human hepatoma carcinoma cell) and H22(mouse hepatoma carcinoma cell) as the target cells of hMSC with or withou pretreated and detected the cell cycle, proliferation and the capability of invasion in vivo or in vitro.Results:1 The preparation and identification of hMSCs and its Immunosuppression. hMSCs were isolated from aborted fetus's bone marrow or amniotic fluid, and then was cultured as adherent cells. The cell of passage 3-5 displayed a morphology and phenotype typical of MSC, showing the potential for differentiation into adipogenic, chondrogenic and osteogenic cells.Compared to the control group, the proliferation of splenocytes induced by ConA in the hMSC-treated group was significantly suppressed in a dose-dependent fashion(P<0.05).2 hMSCs without pretreated can exacerbate the immunologic liver injury in ConA micehMSCs without pretreated could cut down the survival rate (P<0.05), heighten the serum content of ALT and AST (P<0.05) and intensify the ConA-induced liver injury (P<0.05).These findings indicated hMSCs without pretreated could exacerbate the immunologic liver injury in ConA mice.3 The promotion to immunologic injury induced by ConA of hMSCs without pretreated were independent on the expression of HLA-A,B,C or the serum content of C3HLA-A,B,C(-)/HLA-DR(-) and HLA-A,B,C(+)/HLA-DR(-) hMSCs without pretreated could both significantly increase the serum ALT levels(P<0.05). These results indicated the promotion to immunologic injury of hMSCs without pretreated were independent on the expression of HLA-A,B,C.Multiple linear regression was used to analyze the relationship between C3 and ALT, excluding the influence of time and group, there was no relationship between ALT and C3 (P>0.05). The above result showed that the promotion to immunologic injury of hMSCs without pretreated was independent on the serum contents of C3.4 hMSCs without pretreated can induce light damage of liver cells in normal miceCompared with the NS/NS group,the focus lytic necrosis, apoptotic body and inflammation in the district of portal in the ambitus of acini hepatis and the district of portal in the NS/na?ve hMSC group were different(P<0.05), indicated that hMSCs without pretreated could induce light damage of liver cells in normal mice.5 hMSCs with pretreated can extenuate the immunologic liver injury in ConA micehMSCs with pretreated could increase the survival rate (P<0.05), cut down the serum content of ALT and AST (P<0.05) and extenuate the ConA-induced liver injury (P<0.05). These findings indicated hMSCs with pretreated could protect the liver injury ConA mice .6 The decrease of FasL expression on hepatic tissue and the increase in the Treg cell ratio in splenocytes may involve in the protective effect of hMSCs with pretreated.hMSCs with pretreated can suppress the expression of FasL (P<0.05) and increase the ratio of Treg cell in splenocytes in the earlier period(3h) (P<0.05).7 The expression of IL-6, IL-8 and RANTES were increased in hMSCs with pretreated.By detecting the expression of inflammatory factor in the cultivate supernatant of hMSCs with or without pretreated by antibody array, we could find that the hMSC with pretreated expressed more IL-6, IL-8 and RANTES than hMSC without pretreated, and it may involve the protective effect of hMSCs with pretreated.8 Pretreatment could not induce the malignant growth of hMSCsThe hMSCs with pretreated by mice splenocytes cultivate supernatant has the same phenotype(CD29+, CD44+, CD90+, CD105+;HLA-DR-, CD14-, CD19-, CD34-, CD45-, CD31-) and caryotype of chromosome (2n=46)with hMSCs with or without pretreated proliferate arrest by blocking cell cycle at G0/G1 stage(P<0.05). Subcutaneous inoculation of hMSCs with pretreated in athymic mice, and no tumor emerge. So pretreated hMSCs by mice splenocytes cultivate supernatant could not induce the malignant growth of hMSC. 9 The effect of hMSCs with or without pretreated on the growth of hepatica cells in vivo or vitrohMSCs with or without pretreated can induce the MHCC/97H or H22 cells by blocking cell cycle at G0/G1 stage (P<0.05), and the inhibitory intensity between two type hMSC was not statistically significant (P>0.05). And hMSCs with or without pretreated had nothing to do with the capability of migration in vitro of MHCC/97H cell (P>0.05).Subcutaneous inoculation of MHCC/97H and H22 in athymic and BABL/c mice respectively, then hMSCs with or without pretreated. hMSCs with or without pretreated had nothing to do with the life span, tumor growth and capability of migration of tumor cell in vivo(P>0.05).Conclusions:1 Use of adherence-digest-serial subcultivation can harvest functional hMSCs successfully.2 hMSCs inhibit the proliferation of T cells stimulated by ConA.3 As we know, this is the first report that hMSCs with or without pretreated has opposite function on the ConA mice.4 hMSCs without pretreated can significantly exacerbate the immunologic liver injury in ConA mice.5 hMSCs with pretreated can powerfully protect the ConA mice.6 The promotion to immunologic injury of ConA mice by hMSCs without pretreated is independent on the expression of HLA-A,B,C and the serum content of C3.7 The decrease of FasL expression on hepatic tissue and the increase in the Treg cell ratio in splenocytes are related to the protective effect of hMSCs with pretreated.8 The expression of IL-6, IL-8 and RANTES is increased in hMSC with pretreated.9 hMSCs isolated from aborted fetus's bone marrow does not have the risk of tumorigenesis.