Comparison Research Of Immunomodulatory Function Of MSCs Derived From Bone Marrow And Fetal Liver

Research Background:Since mesenchymal stem cells(MSCs)firstly found out,through all these decades,we've got to know that MSCs have more advantages of capacities in suppressing and modulating harmful immune responses through diverse molecular mechanisms.Those derived from adult bone marrow have become the"Golden Standard" for clinical therapy,as comparing with other resources.These cells are greatly used in cell therapies of immune-mediated diseases and treatments of disease transplantation.A wide range of pre-clinical studies and limited number of clinical trials related to MSCs have not only shown the promising safety and efficacy profiles but have also revealed changes in regulating T cells(Tregs)frequency and function.However,the mechanisms underlying this important observation are still missing.Cell to cell contact,production of soluble factors,reprogramming of antigen presenting cells into tolerogenic phenotypes have emerged as possible mechanisms by which MSCs produce an immunomodulatory environment for Tregs expansion.We and others demonstrated that adult bone-marrow MSCs(BM-MSCs)suppress adaptive immune responses not only directly through inhibiting the proliferation of CD 4+("helper")and CD 8+("cytotoxic")T cells but also indirectly through their capability of Tregs induction.In parallel,we demonstrated that fetal liver MSCs(FL-MSCs)display much longer lasting immunomodulatory properties compared to BM-MSCs,by inhibiting directly the proliferation and suppressing the activation of CD 4+and CD 8+T cells.Therefore,we want to further investigated if FL-MSCs would exert their strong immunosuppressive effect also indirectly through induction of Tregs.Research objective:BM-MSCs regarded as the best well-known golden standard among all the other resources,but still,it has its own shortages,like invasive acquiring methods,limited obtaining resources and severe senescence problems.So,at the same time,we are trying hard to find other substitutes.Later on,perinatal and newborn associated tissues have came to be an ideal solution.Here,in our work,we select FL-MSCs as a promising cell resource,we want to compare with BM-MSCs,by checking the functional differences to find out whether it is suitable or not for cell therapies,or maybe they're even better to replace BM-MSCs.Research methods:1)MSCs are isolated,cultured,passaged and cryopreserved in vitro both from FL and adult BM,then they are characterized according to three aspects:surface antigen expression,multilineage differentiation capability and proliferation potential.2)Mononuclear cells are freshly isolated from the spleen of C57BL/6 mice(age range from 6 to 12 weeks)and human peripheral blood,by passing through magnet,we can purely sort CD3+CD25-T cells out,then these cells would be in vitro cultured,passaged and cryopreserved.3)Through different in-vitro mixed combination ratios of MSCs and T lymphocytes,we perform co-cultures of FL-MSCs or BM-MSCs with murine or human CD 3+CD 25-T cells to investigate immunosuppressive effects of MSCs on T cells,both on CD 4+T conventional cells and CD 8+T conventional cells.4)Then we choose one certain ratio to continue co-culture of MSCs and T lymphocytes,check the expression of typical activation markers related to T cells,like GITR,TNFR 2,ICOS,both on CD 4+T conventional cells and CD 8+T conventional cells separately.5)Upon performing co-culture experiments,we observed an increasing trend of Tregs in FL-MSCs group than BM-MSCs ones,so we quantified this difference of capability in converting T conventional cells into Tregs,also checked the phenotypical activation markers of these induced Tregs(iTregs)and further performed in-vitro MLR(Mix Lymphocytes Reaction)experiments to see if them would be more effective to exert their functions.Subject results:1)Both type MSCs are successfully isolated and well adherent to the culture plate,showing a homogeneous spindle-shaped morphology,and a similar expression of typical characterized markers,also similar capacity to multi-differentiation.However,FL-MSCs showed a much better proliferation capacity over BM-MSCs in our 8-day assay,as well as displaying a longer proliferate period time.2)Freshly isolated T lymphocytes are successfully isolated and then purely sorted and co-cultured with MSCs.3)After stimulating the CFSE labeled T cells with anti CD 3/CD 28 beads,co-cultures with MSCs would inhibit proliferation capacity of CD 4+and CD 8+conventional T cells at all the ratios,and FL-MSCs are better than BM-MSCs in that.4)With the results of expression of activation markers,FL-MSCs can show a stronger capability in modulating them towards a less active phenotype when compared to BM-MSCs,both in suppressing CD 4+and CD 8+conventional T cells.5)FL-MSCs are more capable in converting CD 3+CD 25-Foxp 3-T cells into CD 3+CD 25+Foxp 3+T cells,no matter CD 3+CD 4+CD 25+Foxp 3+or CD 3+CD 8+CD 25+Foxp 3+,and these FL-MSCs iTregs are turned out to be more efficient in hindering conventional T cells to exert functions later.Research conclusions:These results highlighted the immunosuppressive activity of FL-MSCs on T cells and showed for the first time that one of the possible main immunoregulatory mechanisms of FL-MSCs is through the induction of activated and functional Tregs.