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【Objective】 To investigate the feasibility of differentiation of human AB plasma hematopoietic stem/progenitor cells (HSCs/HPCs) from peripheral blood into mature erythrocytes. 【Methods】 Hematopoietic stem/progenitor cells were induced to be differentiated into mature erythrocytes in the medium supplemented with 5% FBS, 3% FBS + 2% human AB plasma and 8% human AB plasma, respectively, and inoculated in 24-well culture plate at the density of 1×106/mL. Cell proliferation and morphological changes were observed in three different groups. Flow cytometry was used to detect erythroid terminal differentiation markers, i. e. GPA, Band3 and α4(α4-integrin), and late erythroid cell enucleation in different group. The effects of different culture conditions on HSCs/HPCs differentiation into mature erythrocytes were compared. 【Results】 The cell growth and proliferation multiples of the three groups (8% human AB plasma, 5% FBS and 3% FBS+ 2% human AB plasma) were 2 573±116 vs 2 514±246 vs 2 539±119(P>0.05), respectively. The morphological changes of the three groups were similar. With the extension of culture time, the cells differentiated from proerythroblasts to basophils, polychromatic erythroblasts and positive erythroblasts, and almost all of them differentiated into erythrocytes enucleation on day 21. GPA expression and enucleation rate(%) of the three groups were 97.17±1.91 vs 94.95±1.61 vs 96.15±1.38, and 85.1±3.26 vs 86.93±5.96 vs 86.5±3.36(P>0.05), respectively. 【Conclusion】 The differentiation of HSCs/HPCs from peripheral blood plasma into mature erythrocytes from human AB was similar to that of fetal bovine serum.
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Objective To explore the effect of ginsenoside Rg1 on leukemia stem cells through comparing the biological senescence characteristics of HSCs in the patients with leukemia and healthy people,and provide new ideas and methods for leukemia prevention and treatment.Methods Fifteen cases of normal bone marrow in normal group and sixteen cases of chronic myeloid leukemia in leukemia group were divided into control group and Rg1 group,respectively.The control group used the conventional culture.The Rg1 group used the culture system with 10 μg/mL ginsenoside Rg1,other conditions were the same as control group.The bone marrow mononuclear cell of all groups were extracted after 2 days,and the CD34 +/CD38-cells population was isolated and purified by immunomagnetic adsorption cell sorting(MACS).The purity of the cells and cell cycles phase were detected by flow cytometry.Cell viability was detected by trypan blue staining.The percentage of positive cells was detected by SA-β-gal staining.CCK-8 detected the CD34 +/CD38-proliferation ability of each group.Results The ratio of CD34 +/CD38-cell population was (1.76 ± 0.34) ％ in every 1 × 106 BMNCs before sorting;the proportion of CD34 +/CD38-cell population per 1 × 106 cells after immunomagnetic sorting was (91.15 ± 2.41)％.The positive rate of SA-β-gal staining in human bone marrow CD34 +/CD38-cells of leukemia Rg1 group was significantly higher than that in leukemia control group,the difference was not significant (P ＞ 0.05);meanwhile there was no significant difference between normal control group and normal Rg1 group,but higher than that in leukemia control group,the difference was significant(P ＜ 0.05).CCK-8 results showed that the proliferation of CD34 +/CD38-cells was significantly increased in leukemia control group than those in the other groups.The survival rate of CD34 +/CD38-cells in human bone marrow was 99.1％ in all groups.Cell cycle phase results showed that the G1 arrest of CD34 +/CD38-cells in leukemia control group was significantly lower than those in the other three groups.Conclusion CD34 +/CD38-cells in chronic myeloid leukemia patients may be caused by some chronic myeloid leukemia.Ginsenoside Rg1 can effectively delay the process of aging.
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Using an iron overload mouse model, we explored the protective effect of deferasirox (DFX) and N-acetyl-L-cysteine (NAC) on injured bone marrow hematopoietic stem/progenitor cells (HSPC) induced by iron overload. Mice were intraperitoneally injected with 25 mg iron dextran every 3 days for 4 weeks to establish an iron overload (Fe) model. DFX or NAC were co-administered with iron dextran in two groups of mice (Fe+DFX and Fe+NAC), and the function of HSPCs was then examined. Iron overload markedly decreased the number of murine HSPCs in bone marrow. Subsequent colony-forming cell assays showed that iron overload also decreased the colony forming capacity of HSPCs, the effect of which could be reversed by DFX and NAC. The bone marrow hematopoiesis damage caused by iron overload could be alleviated by DFX and NAC.
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Animals , Male , Acetylcysteine/pharmacology , Triazoles/pharmacology , Benzoates/pharmacology , Hematopoietic Stem Cells/drug effects , Iron Chelating Agents/pharmacology , Free Radical Scavengers/pharmacology , Iron Overload/prevention & control , Protective Agents/pharmacology , Reference Values , Time Factors , Reproducibility of Results , Treatment Outcome , Reactive Oxygen Species/analysis , Colony-Forming Units Assay , Disease Models, Animal , Flow Cytometry , Hematopoiesis/drug effects , Mice, Inbred C57BLABSTRACT
AIM:To construct AFT024-SCF cell line and HPC-Lhx2 cell line for confirming the biological function of AFT024-SCF.METHODS:The HPC-Lhx2 cell line, AFT024-SCF cell line and AFT024-GFP cell line were constructed by retro-viral infection.The expression of stem cell factor(SCF) in AFT024-SCF cells was detected by real-time PCR and Western blot.SCF in the supernatant of AFT024-SCF was detected with ELISA.The supernatant of AFT024-SCF and AFT024-GFP were collected and then diluted (1:10) with basic IMDM medium.So we made 4 culture medium:AFT024-SCF medium was used for experiment group, AFT024-GFP medium was used for endogenous negative control, IM-DM basic medium was used for exogenous negative control, and IMDM basic medium with SCF was used for positive con-trol.SCF-dependent HPC-Lhx2 cell line was cultured in these 4 different medium for 72 h.According to MTT method and colony forming experiment, the biological function of AFT024-SCF was confirmed by the proliferation ability of SCF-depend-ent HPC-Lhx2 cell line.RESULTS:SCF was highly expressed in AFT024-SCF cells.After cultured for 72 h, neither IM-DM basic medium nor GFP-AFT024 medium support HPC-Lhx2 cell line proliferation.However, AFT024-SCF medium supported HPC-Lhx2 cell line expansion as well as the positive control medium.CONCLUSION:AFT024-SCF cells ex-press SCF successfully and recombinant SCF can be replaced by the supernatant of AFT024-SCF culture medium for expan-ding HPC-Lhx2 cell line in vitro.
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Objective: To investigate the mechanism of ginsenoside Rg1 against the hematopoietic stem/progenitor cell (HSC/HPC) senescence during serial transplantation. Methods: Male mice Sca-1+HSC/HPC isolated and purified by magnetic activated cell sorting (MACS) were transplanted for three generations to establish aging in vivo model of mice. The female recipient mice radiated lethal dose from 60Co γ ray were divided into four groups: the control, model, Rg1-treated (20 mg/kg), and Rg1 prevention (20 mg/kg) groups. The mice were administered once daily for 30 d. The expression of p16INK4a, p19Arf, p53, and p21Cip1/Waf1 mRNA was detected by quantitative PCR. The expression of p16INK4a, p21Cip1/Waf1, CDK4, CDK2, CyclinD1, and CyclinE proteins was examined by Western blotting. Results: Compared with the model group, the expression of p16INK4a, p19Arf, p53, p21Cip1/Waf1mRNA and p16INK4a, p21Cip1/Waf1, CyclinD1 proteins was down-regulated, and the expression of CDK4, CDK2, and CyclinE proteins was up-regulated in both Rg1-treated and Rg1 prevention groups. The all index changes of mice in Rg1 prevention group were significantly higher than those of mice in Rg1-treated group. Conclusion: Rg1 could prevent and treat the Sca-1+HSC/HPC senescence during serial transplantation in which the signaling pathways of p16INK4a-Rb and p19Arf-p53-p21Cip/Waf1 may play an important role.
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Embryonic stem cells(ESCs) are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos, possessing permanent self-renewal and indefinite proliferative capacity in vitro. And ESCs could differentiate into the hematopoietic cell fate. Therefore ESCs may provide an alternative for hematopoietic stem cell transplantation and blood cells transfusion. Furthermore, ESCs differentiation could also provide a powerful model system to better understand the hematopoietic development and the mechanism involved. The current status for efforts to differentiate ESCs into hematopoietic lineages were reviewed.
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Objective To study the effect of stromal cell-derived factor-1(SDF-1)/CXCR4 on the proliferation of CD34+ hematopoietic stem/progenitor cells(HSPCs) supported by bone marrow mesenchymal stem cells(MSCs).Methods Rat bone marrow MSCs were plated as feeder layer in long-term cultures(LTC) with bone marrow CD34+ cells in vitro.Cultures were weekly supplemented with SDF-1,anti-SDF-1 antibody,or anti-CXCR4 antibody separately for 5 weeks.The hematopoietic-supporting activity was evaluated by the number of CD34+ cells and colony forming cell(CFC).To assess the effect of SDF-1/CXCR4 on CD34+ cell proliferation cycle,killing assays were carried out and proliferation indexes were calculated.Expression of SDF-1 and CXCR4 in MSCs and CD34+ cells were determined by flow cytometry.SDF-1 in MSC-and CD34+ cell-conditioned medium was also measured by ELISA.Results The number of CD34+ cells,CFC and proliferation indexes were significantly increased in treat-ment with SDF-1(P
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Objective:To investigate whether VLA-5 and VLA-6 are involved in facilitating en-dothelium-oriented transmigration of hematopoietic stem/progenitor cells. Methods:Purified hu-man CD34+ cells were subject to ex vivo transmigration assay and blocking experiments throughtranswell filter inserts coated with human umbilical vein endothelial cells (HUVECs). Four-colorfluorescence-activated cell sorting (FACS) analysis was applied to detect the expression profilesof adhesion molecules and chemokine receptor CXCR-4 on CD34bright cells. Results:Stromal cell-derived factor (SDF)-1-induced transmigrations of both mobilized peripheral blood (mPB)-(56.6±20. 1)% and bone marrow (BM)- (15. 6±1. 8)% derived CD34+ cells were significantlyfacilitated through HUVECs-coated transwell filter insters compared with noncoated ones, whichwere efficiently blocked by preincubation of CD34+ cells with neutralizing antibodies to VLA-5,or VLA-6, or both of them; meanwhile the proportions of migrating CD34+ cells through bothHUVECs-coated and noncoated transwell filter inserts in BM were significantly lower than thosein mPB; the different percentages of migrating CD34+ cells between in mPB and BM were corre-lated with their variable expressions of VLA-5 and VLA-6, but not for VLA-4 or chemokine re-ceptor CXCR-4. Conclusion:Facilitating HS/PCs transmigrations through HUVECs are involvedin both VLA-5 and VLA-6.
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Objective To clone CD34 extracellular region encoding cDNA and to construct its eukaryon expression vector to explore the feasibility of its monoclonal antibody preparation by gene immunization. Methods Total RNA was extracted from KG-1a cell lines. CD34 extracellular region encoding genes were amplified by RT-PCR method and then confirmed by enzymatic lysis and DNA sequencing, and its eukaryon expression vector was constructed as pcDNA3.1-CD34. Twelve BABL/c mice aged 4-6 weeks were selected and randomly divided into 3 groups. Before immunization, 50?l 25% saccharin solution was injected into mouse musculus quadriceps femoris. Fifteen minutes later, blank control PBS, PBS diluted empty vector pcDNA3.1(50?g), or PBS diluted pcDNA3.1-CD34 was injected into the same site of the above three groups, respectively. Immunization was taken every two weeks for a total of three times. The antibody was detected regularly by FACS using tail blood from immunized mice. Results The results demonstrated that the length of CD34 cDNA was 886bp which was identical to the theoretical value and its sequence was confirmed by DNA sequencing which was identical to the registered sequence. The accuracy of CD34 expression vector recombination was confirmed by restriction enzymatic lysis. Hind III and EcoRI restriction enzymatic lysis sites were introduced into 5 and 3 terminals of amplified cDNA sequence, respectively. Terminate code TGA was also introduced into CD34 extracellular encoding cDNA. The expression vector possessing target genes was named as pcDNA3.1-CD34. FACS detection indicated that only 1/4(25%) immunized mice had a lower titer CD34 antibody in their tail vein serum 2-6 weeks after immunization, and the others did not show no antibody production. Conclusion The eukaryon expression vector pcDNA3.1-CD34, which express CD34 extracellular region, has been constructed. The feasibility of CD34 McAb preparation can primarily be confirmed by gene immunization.
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PURPOSE: The umbilical cord blood has been considered as an alternative source of hematopoietic stem cells for transplantation. The CD34+ is known as a common stem cell antigen and the CD34+ CD38- immunophenotype reportedly defines a primitive subpopulation of progenitor cells. In this study the frequency of CD34+ and CD34+ CD38- hematopoietic stem/progenitor cells in placental/cord blood (UCB), and adult peripheral blood (PB) were determined. METHODS: Between July and September 1998, 27 collections of UCB were performed at the obstetric units of Hanyang University Hospital. Fifteen adult PB samples were also obtained for control. RESULTS: The frequency of total CD34+ cells in UCB was higher (1.13+/-0.58% vs 0.46+/-0.30%, P=0.0002) than PB and the frequency of CD34+ CD38- cells in UCB was also greater (0.02+/-0.02 vs 0.01+/-0.01, P=0.035) than PB. The majority of UCB- and PB-derived CD34+ cells expressed CD38- antigen (98.22+/-2.11% in UCB and 97.85+/-2.56% in PB). The frequency of CD38- expression by UCB derived CD34+ cells was slightly higher (not statistically significant) than that by PB derived CD34+ cells. The frequency of CD34+ cells was increased linearly with birth weight (r=0.413). CONCLUSION: These results suggest that UCB could be a useful source of highly primitive hematopoietic stem cells for marrow reconstitution after bone marrow ablation.
Subject(s)
Adult , Humans , Birth Weight , Bone Marrow , Fetal Blood , Hematopoietic Stem Cells , Stem CellsABSTRACT
Homeobox(HOX) gene is a kind of regulatory gene,which can regulate the embryonic development and cell differentiation,and participate in the regulation and control of hematopoietic stem/progenitor cell proliferation,differentiation and maturation,etc.Cluster A in HOX gene family is the master gene for proliferation and differentiation of hematopoietic stem/progenitor cells,which has an influence on the number of hematopoietic stem cells and the differentiation of hematopoietic stem cell into erythroid,myeloid,megakaryocytic and lymphatic system,and has a relationship with the generation of leukemia.In this article,study progress of Cluster A in HOX gene family and its relationship with leukemia were briefly stated.
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Objective:To detect the engraftment capability and hematopoietic potential of the expanded cells,establish an optimized project of ex vivo expansion and the SCID mice model of UCB transplantation.Methods:Human cord blood CD34 + cells were clutured in strom free liquid culture system,the sublethally irradiated SCID mice were injected with human expanded cord blood cells by tail vein.Four weeks after inocuration,BM cells of the survived animals are obtained from both femurs and tibias and assessed for the presence of human cells by immunofluorescence staining and PCR.Results:The cells and CFUs of group FL+TPO+SCF+IL 6 were increased significantly,and maintained some proportional CD34 + cells.The CFU of group SCF+IL 6+IL 3+GM CSF+EPO had reduced in week 2,CFC and CD34 + cells and Alu gene were detected in SCID mice BM.Conclusion:The cytokine combination with FL+TPO+SCF+IL 6 can effectively expand UCB CD34 + cells,and the expanded cells can engraft the BM of sublethally irradiated SCID mice and reconstitute hematopoiesis.