| Background:Hematopoietic stem cells(HSCs)are a special kind of cells,possessing the ability of both multi-potency and self-renewal.And it can be developed into a variety of blood cells.HSCs are widely used in the treatment of malignant blood diseases,hereditary diseases,severe immunodeficiency disease and so on.At present,the main source of HSCs are bone marrow(BM),peripheral blood(PB),and cord blood(CB).It has been demonstrated that bone marrow(BM)reconstituting HSCs reside within a subpopulation of bone marrow or PB-derived mononuclear cells of a few percent which express the surface antigen CD34.Among the many surface markers of HSCs,CD34 is the most commonly used surface antigen,so this molecular marker is often used to enrich HSC.CB has become an important source of HSCs because of its low HLA matching requirements and low graft-versus-host disease(GVHD)incidence.However,the number of HSCs in a single CB unit is insufficient,and the low doses of HSC infusion can lead to a detention in neutrophils and platelet recovery time.Morover,it can increase risk of secondary infection and bleeding.Solving these problems depends largely on the progress of HSC basic research.In order to solve the problem of insufficient number of HSCs in a single CB unit,the researchers have been trying to use a variety of methods for expansion of CB-derived HSC in vitro.So that to expand the scope of its clinical application and to accelerate the recovery of hematopoietic and immune systems after transplantation.However,there is a huge difference between the culture environment in vitro and the BM microenvironment in vivo.When HSCs were expandeding in vitro,their physiological function may change,and which may affect the proliferation,differentiation,homing efficiency,etc.These problems directly affect the effect of HSCs transplantation.Therefore,it is necessary to study the effect of culture environment on the physiological function of HSCs in vitro,and optimize the culture conditions in vitro.To ensure that cultured HSCs can maintain normal physiological functions.In order to mimic the microenvironment in the BM,oxygen concentration and small molecule are important factors to be considered in the process of HSC culture in vitro.Oxygen concentration is an important physical parameter in HSCs culture condition and BM microenvironment in vivo.The oxygen concentration in the common culture system can reach 21%,while in the hematopoietic microenvironment of BM is only about 1-5%.This significant difference between the two conditions may have an important effect on the proliferation,differentiation and physiological function of HSCs.Previous studies have demonstrated that HSCs expansion and self-renewal can be effectively promoted by certain factors/signals in vitro.It has been found that the function of HSCs can be modulated by addition small biological and chemical molecules to the culture medium.We selected three small molecules,testosterone,epinephrine and norepinephrine,to be added to the CD34+ cell culture medium,and cultured in normoxia and hypoxia(1%),respectively.To observe the effect of combination of hormone and different oxygen concentration on HSCs proliferation,antigenic phenotype and gene expression,and to explore a better HSCs culture condition in vitro.Many studies have shown that there is a difference in the HSC concentration of CB between male and female newborns,does this mean that neonatal gender is also a factor affecting CB transplantation?Until now there is no report on this issue.We focused on the effects of neonatal gender as an independent factor on expansion,cell phenotype,and gene expression profiles of CD34+ cells.And whether this difference has a clinical value for the selection of CB remains to be studied.Reasonable selection of the best CB unit for transplantation can improve the HSC transplantation efficacy and reduce the waste of this process.So as to achieve the greatest benefits,which is the focus of our research.ObjectivesTo study the effect of testosterone on the expansion,antigen phenotype and gene expression of CB-derivea CD34+ cells at different oxygen concentrations.To study the effect of neonatal gender on the amplification,antigen phenotype and gene expression profile of CB-derived CD34+ cells.Methods1.To study the effect of testosterone on the expansion,antigen phenotype and gene expression of CB-derived CD34+ cells at different oxygen concentrations.Twelve cord blood samples(all male newborns)were collected.Mononuclear cells(MNCs)were isolated by density gradient centrifugation,and CD34+ cells were enriched by MACS,and the purity of the cells was identified by FACS.Then the CD34+ cells were performed CFU assay,the testosterone,epinephrine and norepinephrine were added to the methylcellulose medium under normoxia(20%)or hypoxia(1%),respectively.The effects of these three hormones on CFU were observed under different oxygen concentration.The testosterone,which had a positive effect on CFU,was screened among the three hormones.The umbilical cord mesenchymal stem cells(MSCs)were passaged to 5-7 generations and irradiated with y-rays as a feeder layer.Then CD34+ cells and feeder layers were co-cultured.The effects of testosterone on CD34+ cells at different oxygen concentrations were compared.The next step,the second CFU assay was used to identify the differentiation ability of the.hematopoietic progenitor cells(HPCs).The cell antigen phenotype CD34,CD38,and CD71 were identified after cocultured 7days.Meantime the expressions of HOXA9,HOXB2,HOXB4,BMI1,HOXC4,GATA-1,HIF-1?,C-MYB,HOXB6 and NFE2 were detected by Quantitative Real-time polymerase chain reaction(qRT-PCR)to study the effects of hypoxia and testosterone.2.To study the effect of neonatal gender on the expansion,cell phenotype and gene expression profile of CB-drived CD34+ cells.Human CB samples(n=43;20male,23 female)were collected,and the MNCs were separated by density gradient centrifugation,then the CD34+ cells were enriched by MACS.The purity of CD34+ cells was detected by FACS.The concentration of CD34+ cells were compared between male and female group.Purified CD34+ cells in two groups were carried out CFU assay.After 2 weeks,the number of CFUs and expanded cells were compared.Two groups of CD34+ cells were expanded in liquid medium for 7 days,then the total number of.cells and amplification curve were compared.The percentage of CD34+CD38-cells in both two groups were identified and compared by FACS.Microarray-based gene expression analysis:Whole Human Genome Oligo Microarray was performed to identify differentially expressed(DE)genes between the male and the female CD34+cell.Gene Ontology(GO)functional categories and Kyoto Encyclopedia of Genes and Genomes(KEGG)functional pathways were searched based on the Database for Annotation Visualization and Integrated Discovery(DAVID)database.Focus on understanding the difference between the two groups of hematopoietic cell signaling pathways.At last,to validate the expression of Gene chip.Two key molecules IL-7R(CD 127)and gp 130(CD 130)were verified in two groups by FACS.Results:1.Effects of testosterone on proliferation,antigen phenotype and gene expression of CB-derived CD34+ cells under two oxygen concentrations.The numbers of CFUs and total cells in testosterone group were significantly higher than those in other groups(P<0.01).In contrast,in the norepinephrine group,the numbers of CFUs and total cells in normoxia and hypoxia were significantly decreased(P<0.01).No CFU was observed in the epinephrine group.The CFU number of different types in the testosterone group were significantly higher than that in the other groups under normoxia and hypoxia,including CFU-GM,BFU-E,CFU-Mix,and CFU-E.And the number of CFU-E in the testosterone groups were significantly higher than that in other groups(P<0.01)Expansion of CD34+ cells and second CFU assay:During the first 3 days of CD34+ cells and MSC co-cultured,the cells rapidly entered the logarithmic growth period in two normoxia groups(Day 3:testosterone group 41.53 ± 4.31 ×105/mL vs control group 30.65 ± 2.74 × 105/mL,P>0.05),while in the two hypoxia group,CD34+ cells grew slowly(Day 3:hypoxia testosterone group 15.22 ± 3.41 × 105/mL vs hypoxia group 13.37 ± 2.10 × 105/mL;P>0.05).But after 3 days,the cells in two hypoxia groups grew rapidly,especially hypoxia testosterone group(Day 7:hypoxia testosterone group:118.44 ± 17.72 × 105/mL vs.hypoxia control group:79.04 ± 10.54× 105/mL;P<0.05).However,there was no significant difference between the two normoxia groups(day 7:normoxia testosterone group 162 ± 20.11 × 105/mL,vs.normoxia control group 150 ± 13.20 × 105/mL;P>0.05).Subsequently,the expanded CD34+ cells were perform a secondary CFU assay to observe the differential potential of HPCs.No significant differences were observed in the number and type of CFUs among the 4 groups(P>0.05).CD34+ cells were cultured for 7 days,cell phenotypes CD34,CD38,CD71 were identified by FACS.The results showed no difference in CD34+CD38+CD71+cell percentage between the two normoxia groups.(74.98 ± 8.79%vs 75.82 ± 9.50%,P>0.05).However,the difference was significant between hypoxia testosterone group and hypoxia control group(87.15 ± 10.13%vs 60.45 ± 6.58%,P<0.05).The percentage of hypoxia control group was the lowest(60.45 ± 6.58%),moreover,the proportion of CD34+CD38+CD71+ cells in hypoxia testosterone group was the highest among the four groups(87.15 ± 10.13%)10 hematopoietic genes were detected by qRT-PCR after cultured for 7 days,including stem cell specific genes HOXB4,BMI-1,C-MYB;erythrocyte-associated gene HOXB2,HOXB6;lymphocyte-associated gene HOXC4;granulocyte-associated gene HOXA9;megakaryocyte-associated gene GATA-1,NFE2;and hypoxia-associated gene HIF-?.The expression of HOXA9,HOXB2,HOXB4,BMI1,HOXC4,GATA-1,HIF-1? and C-MYB genes in the hypoxia testosterone group were all higher than the other three groups(P<0.01&P<0.05).However,there was no significant difference in gene expression levels among the other three groups(P>0.05).The expression of HOXB6 and NFE2 did not significantly differ among the four groups.2.The effect of newborn gender on the expansion,cell phenotype and gene expression profile of CB-derived CD34+ cells.The number of isolated MNCs was higher in the female group than in the male group(median 2.04 ± 0.20 × 106/mL vs.2.52 ± 0.38 × 106/mL;P<0.05).However,the percentage of CD34 + cells enriched by MACS was significantly higher in the male group(median 2.72 ± 0.17%)than in the female group(median 1.26 ± 0.09%,P<0.05).The male CB CD34+ cells formed more CFUs and total cells than those in the female group(118 ± 20 vs.89 ± 8;12.8 ± 2.5 × 106/mL vs.9.2 ± 3.3 × 106/mL,P<0.05).It was no difference about CFU morphology between male and female groups were observed using microscope.But the number of CFU-Mix in male group was more than that in female group(2015 vs 9±2;P<0.05).There were no significant differences in the numbers of CFU-GM,BFU-E and CFU-E between the two groups(P>0.05).The male CB CD34+ cells exhibit better amplification efficiency and growth curve than female group(P<0.05).According to FACS analysis,there were no statistical differences in the fractions of CD34+CD38' cells between male and female group on day 0(2.0±0.14%vs.1.5±0.07%;P>0.05)and day 7(21.7 ± 2.0%vs.18.5 ± 1.1%;P>0.05).Microarray-based gene expression analysis:DE genes number analysising showed there were 1,205 genes(8.8%of total genes)that showed at least 2.0-fold upregulation in the male group.There were 1,313 genes(9.6%of total genes)that were upregulated at least 2.0-fold in the female group.DE genes that were upregulated by more than 6-fold in male group included the following functions:Y-chromosome specificity,anti-tumor immunity,chemokines,T cell development,arginine and histidine metabolism,and signal transduction.Among them,there were about 30%of the DE genes releated Y chromosome function.DE genes upregulated by more than of 6-fold are shown in female group included those involved in X-chromosome specificity,fatty acid metabolism,cholinergic receptor,inhibition of tumor,and chemokines.Among them,more chemokine DE genes expression increased,including CCL4,CCL3,CCL3L3,CXCL10.Biological function classification showed that the top five upregulated biological processes in the male group were sister chromatid segregation,chromosome segregation,neural precursor cell proliferation,mitotic sister chromatid segregation,and positive regulation of cell proliferation.In contrast,the top five upregulated biological processes in the female group were platelet activation,response to,wounding,wound healing,cell activation,and blood coagulation.DE genes categorized into six common biological functions:We compared six functional categories:cell adhesion,growth,immunity,stimulus response,proliferation,and others.The result showed that the genes involved in the response to stimulation were both significantly higher in the male and female groups(37%vs.45%)Signal pathway analysis showed that the Top 3 up-regulated signaling pathways in women were graft versus host disease,malaria,and African trypanosomiasis,and the Top 3 up-regulated signaling pathways in the male group were arginine biosynthesis,hematopoietic cell lines,cytokine receptor interactions.Hematopoietic cell lineage signaling pathway showed that the male group showed high expression of CD5,CD8,CD20,CD21,CD24,CD126,CD127,IL-7,which are mainly related to lymphocyte function.In contrast,the female group exhibited high expression of CD41,CD42,CD61,and TPO,which are mainly related to platelet function.Cytokine-cytokine receptor interaction signaling pathway showed the CXCL12-CXCR4 pathway was upregulated in the male group,and the expression levels of IL6ST(gp130)IL-7 and IL-7R were significantly higher in male group.In the female group,more cytokines were expressed in CD34+ cells,including CCL2,CCL3,CCL4,CCL5,CXCL3,CXCL6,CXCL7,CXCL10,1L1A,1L1B,IL6,TPO and TNF.In order to verify the results of gene chip hybridization,we used FACS to validate the expression of two key molecules in the male group,gp130(CD 130)and IL-7R(CD127).We found that proportion of CD34+CD127+ cells and CD34+CD130+cells were all higher in the male group than in the female group(3.73 ± 0.04%vs.1.72 ± 0.02%and 2.60 ± 0.01%vs.0.82 ± 0.02%,respectively;P<0.05).These results are consistent with those of the gene chip.Conclusions:1.The presence of testosterone in normoxia or hypoxia conditions allows CD34+ cells to form more CFU,especially CFU-E.2.Hypoxia was not conducive to CD34+ cell expansion,but the addition of testosterone could improve this phenomenon.3.The combination of hypoxia and testosterone could make more CD34+ cells differentiate into erythroid progenitor cells.And it was beneficial to the expression of hematopoietic genes.4.CD34+ cells derived from male neonatal CB formed more CFUs,especially CFU-Mix.And they had better amplify ability than female.5.KEGG analysis showed:The hematopoietic cell line signal pathway and cytokine-cytokine receptor signaling pathway of male CD34+ cells were up-regulated,which were favorable for partial expression of lymphocyte-associated genes and T/B lymphocyte development.6.The CXCL12-CXCR4 signaling pathway was upregulated in the male CD34+ cells,which was beneficial to maintain the characteristics and function of HSC.7.The expression of chemokines in the female CD34+ cells were significantly higher than in male.This may be beneficial to the migration/chemotaxis of HSC.And platelet maturation related gene expression increased,may exhibit increased platelet activation and coagulation functions.8.The expression of IL-7R(CD 127)and gp 130(CD 130)were detected by FACS,the male group was higher than female group.The result was consistent with gene chip. |
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