Immunobiology Of Umbilical Cord Mesenchymal Stem Cells And A Pilot Study Of Clinical Application

Objective To establish standard methods of isolation and expansion of umbilical cord mesenchymal stem cell(UC-MSCs). By studying the basic biological characteristics of UC-MSCs, its safety, its immunobiology and clinical treatment of graft- versus-host disease, with a view to establishing the UC-MSCs banks and to promoting clinical application of UC-MSCs.Methods The study was divided into five parts: 1. MSCs were separated from umbilical cord with two enzyme method and expanding cultured in vitro. Growth curves of cells were drawn and surface antigens were detected with flow cytometry. differentiation potentials were observed by inducing to osteoblasts and fat. tumorigeicities of cells were tested by soft agar cloning method and its acute toxicity by observing acute toxicity test. 2. Immunophenotype of UC-MSCs was detected by flow cytometry. The proliferation of allogeneic T lymphocytes was measured by Brdu method; The impact on the T cells subset and cytokine secretion were measured. 3. To establish mice acute GVHD model , bone marrow and spleen cells of male C57BL/ 6 mice were intravenously injected into female BALB/c mice. Thirty mice were randomized to three groups: negative control group, BMT group, aGVHD group. which received lethally irradiation by Cs137source. And then clinical manifestations, survival and pathology examinations were evaluated to judge GVHD severity,which could establish stable mice aGVHD model. 4. lethally irradiated female BALB/c mice were devided into three groups: negative control group, aGVHD group, therapy group. The degree of GVHD, survival , histopathological changes were observed in all the group after BMT. 5 Three patients with GVHD were treated by from September 2006 to February 2005. Among them , one patient was suffering from aGVHD and two were cGVHD. MSCs were seperated from human umbilical cord . After isolation and culture in vitro, UC-MSCs were transplanted into patients by intravenous infusion.Results The methods of culture and expansion of UC-MSCs in vitro were established. Adhesive cells were all positive for MSC-related antigens , such as CD29,CD44,CD166,CD105,CD106, but negative for CD34 , CD45 , CD31 , Multipication of cells was about 30 hours. Cell cycle showed that there was percentage of stem cells as G0 / G78.84 % and S+ G2+ M11.16 % cells respectively. UC-MSCs didn't form clone in soft agar and it had no acute toxicity through acute toxicity animal experiment. UC-MSCs had no HLA-DR, CD80, CD86 and could suppress proliferation of umbilical cord blood lymphocytes stimulated by mitogen. CD8 ~+ suppopulation was significantly decreased ( P < 0.05) , and CD4 ~+ suppopulation was changed mildly(P>0.05).ELISA showed that the secretion of IFN-γwas downregulated, and IL-4 was up regulated ( P < 0.01). Typical aGVHD could not be induced in BMT group. Clinical scores of recipients in aGVHD group was about 8,and all mice survived 14-30 days post transplantation. In animal experiments, clinical signs,survival and pathological changes were observed during the relative concentrated period. Clinical scores,survival, pathological change of small intestine, were significantly different between aGVHD group and therapy group ( P < 0.05). There was not side effect in three cases , GVHD improved in all of them.Conclusion:1 Two enzyme method is a good method to isolate and culture UC-MSCs. The cultured cells , with the biological characteristics of MSCs other than tumorigenic ability, can be amplified easily and passaged in vitro , providing a basis for UC-MSCs banks and clinical application.2 UC-MSCs can supress T cells proliferation, affect T cell subsets and cytokine secretion. The results indicate that UC-MSCs might negatively modulate T lymphocyte-mediated immunity, prevent and treat GVHD in allogeneic stem cells transplantation.3 Stable mice aGVHD model was successfully established, which could be used in evaluating the effect of anti-GVHD therapy. 4 UC-MSCs might efficiently control GVHD associated with allogeneic hematopoietic transplantation, opening new perspectives for clinical application5 UC-MSCs could be a simple , safe , and effective method to treat patient s with GVHD. This is worthy of further clinical trials.