| IntroductionAllogeneic hematopoietic stem cell transplantation(Allo-HSCT)– using blood or marrow as the source– provides curative therapy for a substantial number of patients with hematological malignancies .HLA matched related or unrelated donors are the best selections. However, the odds are that only 25~30% of patients will have a matched sibling to act as donor, and the chance of finding a matched unrelated donor is even lower(one in 50~100 thousand). Another potential alternative donor is a genetically haploidentical family member. Such a donor is readily available in 90% of patients, which will increase the therapeutical opportunity for patients who have no HLA matched donors if the HLA barrier could be overcome suitably.80% of patients, who have received HLA haploidentical transplants, would suffer from graft verus host disease (GVHD).GVHD, the main complication after Allo-HSCT, accounts for morbidity and mortality post transplantation. To explore safe and efficient method to prevent incidence and severity of GVHD become the focus of scientists and clinicians. The critical function of multipotent mesenchymal stromal cells(MSC) include regulation of hematopoietic cell proliferation, differentiation and maturation. The interaction between MSC and immune cell has also been studied. It was demonstrated that MSC possess the ability to regulate immunity, evidenced by its effect on the GVHD prevention in large amount of animal model and clinical research. However, the application of MSC is limited in clinic due to relative difficult collection and multipotential differentiation. Our laboratory has previously isolated a novel population of adherent fibroblast-like cells from human umbilical cord blood (hUCB) CD34+cells, called hUCB-derived stromal cells (hUCBDSCs) and confirmed their supportive effect on hematopoisis in vitro. Previous studies have usually investigated human MSC-allogeneic immune cell reaction in vitro. To test the feasibility of replacing human immune cells with xenogeneic counterparts in vitro is of significance in establishing an animal model for further in vivo study of immunological properties of human MSC, such as their inhibitory effect on GVHD. In the present study, we focused on immunological properties of hUCBDSCs and their effect on xenogeneic immune cells in vitro and GVHD in mouse subjected to MHC haploidentical transplantation. MethodsPart A Experiment in vitro1. CD34+cells were isolated from human umbilical cord blood using 6% gelatin, percoll separating medium(density,1.077g/L) and magnetic bead selection and cultured in DMEM medium containing 12.5%HS, 12.5%FBS, 10-6mol/L hydrocortisone,10ng/mlSCF and 10ng/ml bFGF. Culture was replaced by fresh medium after 48 h, and then, it was demi-depopulated weekly and fresh medium was added to it. After the density of cells had risen above 80% confluence, hUCB-derived stromal cells were subcultured with 1:2ratio under the same culture medium and condition;2. The expression of HLA-I,HLA-II and costimulatory molecules such as CD80, CD86, CD40 and CD154 on freshly isolated and cryopreserved and resuscitated hUCBDSC was tested using flow cytometry;3. CD3+ T cells was isolated and enriched from spleen in mouse using 70% percoll combined with Mouse T cell Enrichment Columns;4. BM-DC was cultured and induced in medium containing rmGM-CSF and LPS after isolation of bone marrow mononuclear cells;5. hUCBDSC was cocultured with T cells stimulated by PHA or DC. The proliferation of the T cells was tested by CCK-8;6. hUCBDSC was cocultured with T cells stimulated by PHA. Cell cycle, apoptosis, the subset of CD4+Treg and Th1/Th2 in T cells were analyzed by flow cytometry;7. hUCBDSC was cocultured with BM-DC. Morphology was observed using electron microscope. The expression of MHC-Ⅱ,CD80,CD86 on BM-DC was tested by flow cytometry. T cells was stimulated by BM-DC after coculturation with hUCBDSC to analyze the function of BM-DC;Part B Experiment in vivo1. Recipient (B6×BALB/c)F1 mice were irradiated with a single dose of 750 cGy using a 60Coγ-ray source at a dose rate of approximately 31 cGy/min. BMC (1×107) and SP (1×10~7) were injected intravenously into recipient donor C57BL/6 mice within 4 hours of conditioning2. hUCBDSC(1×107) was infused simultaneously or on one week after transplantation of BMC and SP. Survival time, GVHD clinical appearance and pathohistological manifestation were observed;3. The subset of CD4+Treg and Th1/Th2 in splenic T cells and expression of MHC-Ⅱ,CD80,CD86 on splenic DC were analyzed in recipient mice using flow cytometry on 1w,2w,3w and 4w post transplantation.ResultsPart A Experiment in vitro1. 84.1±2.9% of hUCBDSC expressed HLA-I, but there was nearly no expression of HLA-II (1.6±0.3%). The levels of costimulator expression were as follows: CD80 (0.8±0.1%), CD86 (0.8±0.1%),CD40 (0.6±0.1%), and CD40L (0.5±0.1%), and cell cryopreservation did not influence the expression of aboved molecules;2. The addition of hUCBDSCs in in vitro culture did not evoke xenogeneic T-cell proliferation; on the contrary, it suppressed the proliferation induced by PHA or allogeneic dendritic cells;3. The proportion of T cell in G1 and S phase was 62.1±3.7% and 35.6±2.7% after T cells were stimulated by PHA for 72 hours. The percentage of T cells in G1 phase was increased to 84.3±3.6%, and in S phase was decreased to 12.3±1.5% when PHA stimulated T cells were cocultured with hUCBDSC;4. There are no significant difference statistically on the percentage of apopotic cell between T cells alone and T cells cocultured with hUCBDSC;5. There was a significant increase in the proportion of CD4+Treg (12.1±1.4% vs 1.2±0.3%, p < 0.01) when T cells were stimulated by PHA in the presence of hUCBDSCs compared with control;6. The ratio of Th1/Th2 subset in T cells alone was 1.4±0.1%, significantly higher than in T cells cocultured with hUCBDSC (0.7±0.1%, p<0.01);7. The surface projection and organelle in BM-DC was significantly decreased evidenced by observation using electron microscope. The expression of MHC-Ⅱ,CD80,CD86 on BM-DC was down-regulated. The stimulatory ability of BM-DC was significantly attenuated. Part B Experiment in vivo1. The MHC haploidentical HSCT GVHD model in mouse has been successfully constructed ecidenced by typical GVHD clinical appearance and pathohistological changes;2. The long-time survival ratio of recipient mice was increased from 20% to 60% and 40% after infusion of hUBDSC on day0 or on week. In addition, the GVHD clinical appearance has been attenuated significantly, and the injury degree of targeted tissue was significantly decreased.3. The subset of CD4+Treg in splenic T cells in recipient mice subjected to hUCBDSC infusion was increased significantly, and Th1/Th2 subset was decreased. Expression of MHC-Ⅱ,CD80,CD86 on splenic DC were down-regulated significantly.Conclusion1. HUCBDSC are immunoprivileged evidenced by lower or negligible expression of MHC-II, costimulatory molecules including CD80,CD86,CD40 and CD154 on cellular surface and non stimulatory effect on xenogeneic T cells. Interruption of cell cycle, induction of CD4+Treg, polarization of Th1/Th2 subset and interference with BM-DC maturation may be involved in its possible mechanisms rather than apoptosis.2. HUCBDSC could prolong survival time of mice subjected to MHC haploidentical HSCT, and attenuate GVHD clinical appearance and pathohistological change of target tissue. In accordance with results in vitro, possible mechanisms include induction of CD4+Treg, polarization of Th1/Th2 subset and interference with BM-DC maturation.
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