The study was purposed to explore the effect of panel reactive antibody (PRA) serum from patients with β-thalassemia on proliferation and apoptosis of the CD34(+)cells from cord blood and its mechanism. CD34(+) cells of umbilical cord blood were incubated with different sera and complement respectively. After incubation, the samples were centrifuged and the supernatants were collected for lactate dehydrogenase (LDH) detection, and the CD34(+) cells were harvested and measured for the apoptosis by flow cytometry with Annexin V/PI. The intracellular DNA synthesis were also quantified by [(3)H]TdR incorporation using liquid scintillation counter. The results showed that concentration of LDH in PRA positive groups was higher as compared with control group, and the DNA synthesis of CD34(+) cells in PRA positive groups were inhibited. There were no differences in the percentage of cell apoptosis and necrosis among different groups. It is concluded that thalassemic serum PRA impairs the cell membrane, inhibits the DNA synthesis, which can be increased by addition of the complement, but PRA had no significant effect on apoptosis of CD34(+) cells.
Antibodies ; blood ; immunology ; Antigens, CD34 ; Apoptosis ; immunology ; Cell Proliferation ; Child ; Fetal Blood ; cytology ; Humans ; beta-Thalassemia ; blood ; immunology ; pathology

This study was aimed to investigate the specific anti-leukemia cell effect of cytotoxic T lymphocytes (CTLs) induced by HL-60 or K562 cell-sensitized dendritic cells (DCs) from umbilical cord blood. 12 units of human umbilical cord blood (UCB) were collected and the mononuclear cells (MNCs) were isolated from UCB, then cultured with granulocyte monocyte colony- stimulating factor (GM-CSF), interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and EPO for 3 - 4 weeks. Flow cytometry was used to determine the number of DCs and cell surface antigens before and after culture with monoclonal antibodies including CD83, CD1a, CD11c and CDw123. HL-60 and K562 were frozen-thawed, and released their tumor antigen peptides (TAP). The CTLs were produced by sensitizing T lymphocytes with DC-loaded HL-60 and K562 cell antigens. The test of (3)H-TdR incorporation was used to detect the immunostimulation activity of DCs. MTT assay was applied to evaluate specific cytotoxicity of CTL on leukaemia cells. The results indicated that the MNCs of UCBs cultured with GM-CSF, IL-3, EPO and SCF were shown to differentiate into CD1a(+) CD11c(+) CD83(+) CDw123(+) DCs. Numbers of DCs from UCBs remarkably increased in 2 - 4 weeks and then decreased. After culture with cytokines DCs increased (10.6 - 28.2) x 10(5)/ml in actual numbers. The CTL induced by DC pulsed with HL-60, K562 frozen-thawed lysates were effective to kill HL-60 and K562. Cytotoxicity of CTL to HL60 and K562 were (42.04 +/- 8.46)% and (31.25 +/- 11.07)% respectively. It is concluded that the MNCs of UCBs cultured with cytokines of GM-CSF, SCF, EPO and IL-3 can differentiate into CD1a(+), CD83(+), CD11c(+) and CDw123(+) DCs. The CTL induced by DCs pulsed with HL-60, K562 frozen-thawed lysates can effectively kill HL-60 and K562. These DCs as antigen presenting cells play an important role in cancer immunotherapy.
Dendritic Cells ; cytology ; immunology ; Fetal Blood ; cytology ; immunology ; HL-60 Cells ; Humans ; K562 Cells ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVEMesenchymal stem cells (MSCs) were adult stem cells which contribute to the regeneration of mesenchymal tissues such as bone cartilage, muscle, ligament, tendon, adipose and stroma. Due to the multipotential ability and self-renewal capacity, the mesenchymal stem cells can be applied in many fields, such as the seed cells in tissues engineering, cell therapy and gene therapy. To enhance the clinical use of MSCs, the investigators studied the isolation and expansion of MSCs from adult bone marrow, fetal bone marrow and human umbilical cord blood, and investigated their biological identities.

METHODSTwo kinds of incubation systems containing L-DMEM or MSC special culture medium were used to purify and expand MSCs. The growth, purification and proliferative abilities of 3 kinds of MSCs were observed and their immunophenotypes were determined by flow-cytometry.

RESULTS(1) The shapes of 3 kinds of cells were same. There was no difference in number and size. The colonies formed early in adult bone marrow MSCs. (2) There was no difference in the expansion speed of the 3 kinds of MSCs, but after the colonies confluenced there had no touching constrain in MSCs from umbilical cord blood and fetal bone marrow. When the colonies confluenced, the cells also had proliferation ability. But in adult bone marrow, the touching constrain was significant. (3) MSCs had strong self-renewal capacity. After primary culture approximately 5 - 6 x 10(5) MSCs were obtained from 8 x 10(6) MNC of bone marrow and 25 x 10(6) MNC of umbilical cord blood. After passage 3, passage 5 and passage 10, the investigators could get 10(7), 10(8) and 10(10) MSCs, respectively. (4) Along with the increase in the passage and prolonging of culture time, the ability of expansion decreased, but they maintained good puripotentiality. After passage 2, passage 3 and passage 5, the purity of MSCs was 90%, 95% and 99%, respectively. (5) Three kinds of MSCs were all positive for CD(29), CD(44), CD(59), CD(90), CD(105), CD(166) and all negative for the markers of hematopoietic cells such as CD(11a), CD(14), CD(33), CD(34), CD(28), CD(45). All the important GVHD correlation markers were negative, such as HLA-DR, B7-1 (CD(80)), B7-2 (CD(86)), CD(40) and CD(40L). There were no differences in the phenotype among the 3 kinds of MSCs cells. (6) The 2 kinds of culture mediums used did not markedly affect isolation and expansion of MSCs, and the biological properties of MSCs.

CONCLUSIONS(1) Human MSCs could be isolated from many kinds of human tissues, and they had no difference in their origin; (2) Human MSCs maintained good puripotentiality and self-renewal capacity. Therefore, they could meet with the need of clinical tissue engineering. (3) The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier but had broad clinical use.

Bone Marrow Cells ; cytology ; immunology ; Cell Culture Techniques ; Cell Differentiation ; immunology ; Cell Division ; immunology ; Fetal Blood ; cytology ; immunology ; Humans ; Immunophenotyping ; Mesenchymal Stromal Cells ; cytology ; immunology ; Time Factors

OBJECTIVETo explore the specific anti-leukemia immune response of CD8+ cytotoxic T lymphocyte (CTL) derived from cord blood (CB) ex vivo and evaluate the feasibilities and values of the CTL for specific immunotherapy.

METHODSDendritic cells (DC) were induced from mononuclear cells (MNC) by combination cytokines in 10 CB samples. Loading U937 cell lysate antigen on the mature DC, they could stimulate the lymphocytes of the same origin to generate CTL. MidiMACS was used to isolate CD8+ CTL. Analysis of DC was performed by inverted microscopy, scanning electron microscopy and flow cytometry. Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the cytotoxicity of the CTL.

RESULTSCocultured with GM-CSF, IL-4, TNF-alpha and PGE2, CB-MNC could be induced into functional DC with typical morphology. The mean cytotoxicity of CD8+ CTL to U937 cells was significant stronger than that of CD8- CTL and TL at the same E: T ratios. The mean cytotoxicity rate of CD8+ CTL to U937 cells was higher than that to K562 cells [(66.36 +/- 12.43)% vs (41.97 +/- 14.24)%] at E: T ratio of 40: 1 (P < 0.05). The cytotoxicity of CD8- CTL to K562 cells showed no difference from that to U937 cells (P > 0.05).

CONCLUSIONMature CB-DC loading U937 cell antigens could induce CB-T lymphocytes to generate leukemia-specific CD8+ CTL. The cytotoxicity of the CD8+ CTL is specific against U937 cells and is more potent than that of CD8- CTL.

CD8-Positive T-Lymphocytes ; cytology ; immunology ; Dendritic Cells ; cytology ; immunology ; Fetal Blood ; cytology ; immunology ; Humans ; In Vitro Techniques ; K562 Cells ; T-Lymphocytes, Cytotoxic ; immunology ; U937 Cells

Colony-Forming Units Assay ; Cryopreservation ; methods ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Immunotoxins ; immunology ; Leukocytes, Mononuclear ; cytology ; Ricin ; immunology ; T-Lymphocytes ; cytology ; immunology ; Time Factors

This study was purposed to investigate the biological characteristics and immunogenicity changes of ex vivo expanded megakaryocyte progenitors from human umbilical cord blood CD34(+) cells in order to provide experimental basis for clinical application of ex vivo expanded umbilical cord blood megakaryocyte progenitor cells. Mononuclear cells (MNCs) were obtained from umbilical cord blood by Ficoll-Hyapaque density gradient separation. CD34(+) cells were enriched by magnetic cell sorting (MACS). The selected CD34(+) cells were seeded in serum-free medium stimulated with thrombopoietin (TPO, 50 ng/ml), interleukin 11 (IL-11, 50 ng/ml), and heparin (25 U/ml) for 14 days. The immunophenotyping (CD34(+), CD41a(+), CD61(+), CD34(+) CD41a(+) and CD34(+) CD61(+) cells) of amplificated products, matured megakaryocyte apoptosis, and expression of human leukocyte antigen (HLA) class I and class II molecules were measured by fluorescence-activated cell sorter (FACS). The number of colony-forming units-megakaryocyte (CFU-Mk) was also evaluated by CFU-Mk assay. The results showed that the umbilical cord blood CD34(+) mononuclear cells could be effectively differentiated into megakaryocytes. The peak expression ratios of CD41a(+) and CD61(+) cells were all at 14th days, while that of CD34(+) CD41(+) and CD34(+) CD61(+) cells were at 7th day [(3.41 +/- 2.80)% and (1.89 +/- 1.43)%, respectively]. The expansion times of large and small CFU-Mk reached peak at 7th day (20.66 +/- 32.79) and 10th day (435.62 +/- 482.65), respectively. The apoptotic rates of megakaryocytes at 7th, 10th, 14th day were (19.48 +/- 9.64)%, (26.87 +/- 9.03)%, and (52.46 +/- 11.74)%, respectively. The apoptotic rate of megakaryocytes had no significant difference in 7 and 10 days culture (p > 0.05), while that significantly increased in culture for 14 day culture, compared with culture for 7 and 10 days (p < 0.05, respectively). The expression of HLA class I and class II molecules on megakaryocytes decreased along with the prolongation of expansion time and sharply decreased in 0 to 10 days. It is concluded that the cytokines of TPO, IL-11, and heparin can promote the expansion of megakaryocyte progenitors from umbilical cord blood CD34(+) mononuclear cells effectively in vitro. The peaked expansion times of large CFU-Mk, the peaked expression ratios of CD34(+) CD41(+) and CD34(+) CD61(+) cells were all at 7th day. So the culture for 7 days appeared to be the optimal duration of expanding megakaryocyte progenitors.
Antigens, CD34 ; immunology ; Cell Differentiation ; Cell Division ; Cell Separation ; Cells, Cultured ; Fetal Blood ; cytology ; immunology ; Humans ; Megakaryocyte Progenitor Cells ; cytology

OBJECTIVEThe low rate of engraftment in children with beta-thalassemia has seriously restricted the popularity of the hematopoietic stem cell transplantation (HSCT). Panel reactive antibody (PRA) has been regarded as one of the important factors for the success of kidney transplantation. Poly-transfusion before transplantation is associated with the production of PRA. Also PRA is produced in the children with beta-thalassemia major who need poly-transfusion for life. PRA might be one of factors inducing the low rate of engraftment in children with beta-thalassemia. This study focused on observing the effect of PRA on the proliferation, differentiation, apoptosis and necrosis of cord blood CD34(+) cells in vitro by incubating the cord blood CD34(+) cells with serum containing PRA.

METHODSeven samples of cord blood were collected and the HLA typing for every sample was done. Seven sera positive for PRA and seven negative sera were selected respectively. Mononuclear cells (MNCs) were obtained by Ficoll-Hypaque density gradient centrifugation. CD34(+) cells were isolated from MNCs by positive selection using an immunomagnetic separation (CD34(+) progenitor cell isolation kit and auto-MACS). The CD34(+) cells of umbilical cord blood were incubated with the serum and complement in the following groups: A (absence of serum), B (presence of PRA positive serum), C (presence of PRA positive serum and complement), D (presence of complement), and E (presence of PRA negative serum). After incubation the samples were centrifuged and the supernatant was collected for LDH detection. At the same time the CD34(+) cells were harvested for assessing the expression of Annexin V and CD95 of the CD34(+) cells by flow cytometry and also for the detection of the DNA synthesis by (3)H-TaR incorporation. Meanwhile the cells were inoculated into the methylcellulose cultural system. The proliferation and hematopoietic potential of the CD34(+) cell of cord blood by the colony formation assay were detected on the day 10.

RESULTThe concentration of LDH in group A was (20.71 +/- 2.81) U/L, which was significantly lower than that in group B (64.28 +/- 5.12) U/L and group C (84.29 +/- 4.99) U/L. The concentration of LDH in group B was significantly lower than that in group C, while there were no significant differences in the concentration of LDH among groups A, D and E (P > 0.05). The cpm in group A was (22 629 +/- 3288), which was significantly higher than that in group B (4598 +/- 2178) and group C (1626 +/- 1192). And the cpm in group B was significantly higher than that in group C. There were no significant differences in the cpm among groups A, D and E (P > 0.05). On day 10 of culture, the total colonies, granulocyte-macrophage colony forming unit (CFU-GM), mixed colony forming unit (CFU-GEMM) and erythroid burst colony forming unit (BFU-E) in group A were significantly higher than that in group B and C. The total colonies, CFU-GM and CFU-GEMM in group B were significantly higher than those in group C. No significant differences were found in the total colonies, CFU-GM, CFU-GEMM and BFU-E among groups A, D and E (P > 0.05). There were no statistically significant differences in the CD95 and Annexin V expression among all the groups (P > 0.05).

CONCLUSIONPRA could impair the membrane, decrease the DNA synthesis, and inhibit the colony formation of CD34(+) cord blood cells, which could be strengthened by the presence of the complement at the given concentration in our study. PRA had no significant influence on the apoptosis of CD34(+) cells in vitro.

Antibodies ; immunology ; Antigens, CD34 ; Apoptosis ; immunology ; Cell Differentiation ; immunology ; Cell Proliferation ; Cells, Cultured ; Child ; Fetal Blood ; cytology ; immunology ; metabolism ; Flow Cytometry ; Humans ; Quorum Sensing ; immunology ; beta-Thalassemia ; immunology

This study was purposed to investigate the difference of nucleated cell (NC) count, CD34(+) cell ratio and expansion multiple, cell cycle and colony formation capability in in vitro expanded human umbilical cord blood CD34(+) cells from HOXB4-transfecting directly and HOXB4-transfected human umbilical cord mesenchymal stem cells (HUCMSC) by means of prepared feeder layers of HUCMSC. The HUCMSC were divided into 2 groups:first group, in which HOXB4 gene was transfected into HUCMSC by using lentiviral vecfor, and feeder layers were set up; and second group in which feeder layers for HUCMSC of non-transfected HOXB4 gene were set up. The CD34(+) cells were separated from HUCB by magmatic activated cell sorting(MACS). After culture in medium with cytokines for 2 days, CD34(+) cells were divided into 5 groups, including control group and experimental group. The control groups included CD34(+) cells as group A (blank control group) and GFP-CD34(+) cells as group B (negative control group) and experimental groups included HOXB4-CD34(+) cells as group C, HUCMSC+CD34(+) cells as group D, HOXB4-HUCMSC+ CD34(+) cells as group E and cells in all groups were cultured in vitro. The number of nucleated cells were counted at day 6, 10, 14 of culture and CD34 immunophenotypes, cell cycle and colony forming capability were measured at day 10 of culture in different conditions. The results indicated that HOXB4 gene could be transfected into HUCMSC by lentiviral vector and feeder layers were set up successfully. After culture for 14 days, the nucleated cells in 5 groups could be amplified effectively, and the expansion levels in 5 groups were in order HOXB4-HUCMSC+CD34(+) cell group> HOXB4-CD34(+) cell group>HUCMSC+CD34(+) cell group> control groups (P < 0.05). At day 10 of in vitro expansion the CD34(+) cell percentage decreased significantly in all groups, while the number of CD34(+) cell increased in experiment groups, which were in order HOXB4-CD34(+) cells group> HOXB4-HUCMSC+CD34(+) cell group>HUCMSC+CD34(+) cell group>control groups (P < 0.05). The cell cycle detection showed that the percentage of cells in S+G2/M phase in experiment groups were higher than that in control groups (P < 0.05), and percentage of cells in HOXB4-HUCMSC+CD34(+) cells group was higher (41.57%) than that in HOXB4-CD34(+) cells group(37.87%) and HUCMSC+CD34(+) cell group (28.65%) (P < 0.05). There was no statistical difference in the CFU number between HOXB4-HUCMSC+CD34(+) cell group and HOXB4-CD34(+) cell group, which were both higher than that in HUCMSC+CD34(+) cell group and control groups (P < 0.05).It is concluded that the CD34(+) cells cultured on HOXB4-HUCMSC feeder layers can be amplified significantly and kept the characteristics of stem cells, The feeder lager of HOXB4-HUCMSC is relative safe for amplification of CD34(+) cells in vitro, it possesses the potential useful value.
Antigens, CD34 ; immunology ; Cell Separation ; Cells, Cultured ; Fetal Blood ; cytology ; Homeodomain Proteins ; genetics ; Humans ; Mesenchymal Stromal Cells ; cytology ; immunology ; Transcription Factors ; genetics ; Transfection ; Umbilical Cord ; cytology ; immunology

OBJECTIVETo cultivate hematopoietic stem/progenitor cells (CD34(+)CD38(-)) isolated from umbilical cord blood (UCB) long for the observation of cell growth and expansion in vitro, surface marker expression, and chromosomal complements.

METHODSBy flow cytometry CD34-FITC and CD38-PE labeled CD34(+) and CD38(-) stem/progenitor cells were isolated from UCB. The cells were cultivated in vitro for 6 months in a stem cell culture system with addition of six kinds of cell growth factors (IL-3, IL-6, GM-CSF, Epo, SCF, IGF-1). One month after cultivation, cultured cells were investigated for surface marker expression by flow cytometry and karyotype by G banding method.

RESULTSAfter 7-12 days cultivation, the CD34(+)CD38(-) stem/progenitor cells began proliferation. The proliferation rate and the peak proliferation duration were greater in 1 cell/well cultivation conditions than in 10 cells/well. The cells remained CD34(+)CD38(-) and their karyotypic characteristics remained unchanged.

CONCLUSIONCD34(+)CD38(-) stem/progenitor cells from UCB may provide a larger than original amount of stem/progenitor cells for transplantation after long-term cultivation in vitro.

ADP-ribosyl Cyclase 1 ; immunology ; Adult ; Antigens, CD34 ; immunology ; Cell Proliferation ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; immunology ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans ; Immunophenotyping ; Karyotyping

In order to investigate the influence of cytokine combinations on proliferation and differentiation of human umbilical cord blood CD34(+) cells into megakaryocytes/platelets in vitro, the CD34(+) cells from human umbilical cord blood were amplified in serum-free medium StemSpan(SFEM) supplemented with several cytokine combinations by three-phase culture system. The effects of the cytokine combinations were compared. The results showed that at day 14 of the first culture phase, the CD34(+) cells cultured with cytokine combinations SCF + TPO + FL + IL-3 were amplified (11 000 ± 1 000) times, which were significantly higher than that of cells cultured with SCF + TPO + FL, but were not significantly different from that of cells cultured with SCF + TPO + IL-3 or SCF + TPO + FL + IL-3+ hydroxyl-corticosteroids. At day 7 of the second culture phase, the CD34(+) cells cultured with cytokine combination SCF + TPO + FL + IL-11 were amplified by (204666.7 ± 11718.9) times, which were significantly higher than that of cells cultured with SCF + TPO + FL + IL-3, but were not significantly different from that of cells cultured with SCF + TPO + FL + IL-11 + BMP4 + VEGF. At day 3 and day 6, the CD34(+) platelet-like cells accounted for about (39.8 ± 1.9)%, (39.7 ± 2.6)% and (25.5 ± 1.4)%, (23.1 ± 3.5)% cultured with SCF + TPO + FL + IL-11 and SCF + TPO + FL + IL-11 + BMP4 + VEGF, and significantly higher than that of the cells cultured with SCF + TPO + FL + IL-3. It is concluded that the cytokine combination of SCF + TPO + FL + IL-3 is most suitable cytokines combination for the amplification of CD34(+) hematopoietic progenitor cells. The cytokine combination of SCF + TPO + FL + IL-11 is preferred for the proliferation and differentiation of megakaryocytes, this study lays an experimental basis for investigating the proliferation and differentiation of CD34(+) into megakaryocytes/platelets in vitro.
Antigens, CD34 ; immunology ; Blood Platelets ; cytology ; Cell Differentiation ; Fetal Blood ; cytology ; immunology ; Humans ; Interleukin-11 ; pharmacology ; Interleukin-3 ; pharmacology ; Megakaryocytes ; cytology ; Stem Cell Factor ; pharmacology ; Thrombopoietin ; pharmacology