Effect Of Hypoxia-Supported Umbilical Cord Mesenchymal Stem Cells On The Expansion Of Cord Blood Mononuclear Cells In Vitro

Umbilical cord blood has become an important source of hematopoietic stem cell transplantation due to its easy collection,convenient freezing,low immunogenicity,low incidence of GVHD,and wide range of sources.At present,it is widely used in the treatment of a variety of blood system diseases,congenital defects and solid tumors.However,due to the insufficient number of hematopoietic stem/progenitor cells contained in a single piece of cord blood,it can lead to implantation failure,delayed hematopoietic and immune reconstruction,which limits the clinical application of cord blood,especially the high failure rate of cord blood transplantation in adult patients and large-weight children.Therefore,in vitro expansion of cord blood can solve the problem of insufficient cord blood.However,the amplification effect of cord blood is closely related to the culture system containing mesenchymal stem cells,cytokine combination,oxygen conditions,implanting adhesion molecules and the expression of CXCR4.Experimental purposeUsing umbilical cord mesenchymal stem cells as trophoblast cells,the effect of umbilical cord mesenchymal stem cells on in vitro expansion of human umbilical cord blood mononuclear cells under hypoxia support was observed,and the effect of SCF+FL+TPO（SFT）3 factor combination on in vitro expansion of umbilical cord blood mononuclear cells in hypoxia supported umbilical cord mesenchymal stem cell culture system was further investigated.Experimental method1.Umbilical cord mesenchymal stem cells and umbilical cord blood mononuclear cells were used as research objects,and both umbilical cord and umbilical cord blood were provided by the obstetrics and gynecology department of our hospital.2.Establishment of umbilical cord mesenchymal stem cell trophoblast:The umbilical cord of newborn babies of healthy mothers was inoculated in a disposable sterile petri dish with the size of 100mm by tissue adhesion method.DMEM/F12culture medium containing 1%penicillomycin and 10%fetal bovine serum was added into the dish and incubated in a 5%O₂incubator at 37℃.After 3d,the culture medium was fully changed,the non-adherent cells were fully removed,and the medium was changed in half every 3-4d,and cultured until the cells were grown and fused,and the immunophenotype of the cells was identified by flow cytometry.3.In vitro expansion,culture and experimental grouping of umbilical blood mononuclear cells:The isolated umbilical cord blood MNC was inoculated on the pre-established UC-MSC layer and cultured in groups under hypoxia（3%O₂）and normal oxygen conditions.The experimental group was divided into four groups:normal oxygen（normal oxygen+cord blood MNC）,hypoxia（hypoxia+cord blood MNC）,UC-MSC（normal oxygen+UC-MSC+cord blood MNC）,UC-MSC+hypoxia（hypoxia+UC-MSC+cord blood MNC）.The number of TNC,CD34⁺cells,CFU and CD34⁺CXCR4⁺,CD34⁺CD49d⁺and CD34⁺CD62L⁺cells in each group were detected and compared on day 0 and 7 of culture.4.In order to further investigate the effect of SCF+FL+TPO（SFT）on the expansion of umbilical cord blood MNC in vitro in hypoxic-supported UC-MSC culture system,The experiment was further divided into three groups:group A（normal oxygen+UC-MSC+SFT+cord blood MNC）,Group B（hypoxia+UC-MSC+cord blood MNC）and group C（hypoxia+UC-MSC+SFT+cord blood MNC）.The number of TNC,CD34⁺cells,CFU and the number of CD34⁺CXCR4⁺,CD34⁺CD49d⁺and CD34⁺CD62L⁺cells in each group were detected on 0,7,10 and 14 days.5.Statistical analysis:SPAA statistical software was used for data analysis.All measurement data were expressed as,and ANOVA was used for comparison between groups.P<0.05 was considered statistically significant.Experimental result1.After 5 days of UC-MSC culture,small fusiform adherent cells could be seen.After a week,the cells began to prolifate rapidly,forming cell colonies of varying sizes.14d cells were spindle-shaped slender cells arranged closely,with a fusion degree of about 85%-90%,and were parallel or swirly dense growth.2.Compared with the hypoxia group and the UC-MSC+hypoxia group,the number of TNC,CD34⁺cells and CFU in cord blood can be effectively expanded（P<0.05）,and the expression of adhesion molecules and CXCR4 on cord blood CD34⁺cells can be significantly increased（P<0.05）.3.After 14 days of culture,the level of detection indicators at each time point in group C was significantly higher than that in groups A and B（P<0.05）,and the amplification effect of group A at 7 and 10 days was significantly better than that in group B（P<0.05）.Conclusion1.Hypoxia and umbilical cord mesenchymal stem cell layer can promote in vitro expansion of cord blood MNC.2.Hypoxia supported umbilical cord mesenchymal stem cell layer can effectively expand cord blood MNC and increase the expression of adhesion molecules and CXCR4 on cord blood CD34⁺cells.3.Adding SCF+FL+TPO3 factor combination into hypoxic cord mesenchymal stem cell culture system can further improve the amplification effect of cord blood MNC.