OBJECTIVE: To explore the effect of hypoxia-supported umbilical cord mesenchymal stem cell (UC-MSC) on the expansion of cord blood mononuclear cell （MNC） in vitro. METHODS: The isolated cord blood mononuclear cells were inoculated on the preestablished umbilical cord mesenchymal stem cell layer and cultured under hypoxic conditions (3％ O₂) and the experimental groups were normoxia (MNCs were cultured under normoxic conditions), hypoxia (MNCs were cultured under hypoxic conditions), UC-MSC (MNCs were cultured with UC-MSC under normoxic conditions), and UC-MSC+hypoxia (MNCs were cultured with UC-MSC under hypoxic conditions). To further investigate the combinational effect of 3 factors of SCF+FL+TPO (SFT) on expansion of cord blood MNCs in vitro in hypoxia-supported UC-MSC culture system, the experiments were further divided into group A (MNCs were cultured with UC-MSC and SFT under normoxic conditions), group B (MNCs were cultured with UC-MSC under hypoxic conditions), group C (MNCs were cultured with UC-MSC and SFT under hypoxic conditions). The number of nucleated cells (TNC), CD34⁺ cell, CFU and CD34⁺CXCR4⁺, CD34⁺CD49d⁺, CD34⁺CD62L⁺ cells of each groups were detected at 0, 7, 10 and 14 days, respectively. RESULTS: Compared with group hypoxia and UC-MSC, group UC-MSC+hypoxia effectively promoted the expansion of TNC, CD34⁺ cell and CFU, and upregulated the expression level of adhesion molecule and CxCR4 of the cord blood CD34⁺ cell（P<0.05）. After culturing for 14 days, compared with group A and group B, group C effectively promoted the expansion of cord blood MNC at different time points（P<0.05）, and the effect of group A was better than that of group B at 7 and 10 days（P<0.05）. CONCLUSION Hypoxia-supported UC-MSC efficiently promoted the expansion and expression of adhesion molecule and CXCR4 of cord blood CD34⁺ cell, and the effect of expansion could be enhanced when SFT 3 factors were added.
Humans ; Cells, Cultured ; Fetal Blood ; Cell Proliferation ; Umbilical Cord/metabolism* ; Mesenchymal Stem Cells ; Antigens, CD34/metabolism* ; Hypoxia/metabolism*

This study was aimed to investigate the function defect of partial homing receptor on cord blood hematopoietic stem cells (CBHSC) and explore efficacy and feasibility of intervention in vitro. The expression and activity of active groups in P, E-selectin ligands on CD34+ cells from cord blood, bone marrow and peripheral blood were detected by flow cytometry; meanwhile the expression of active groups in selectin ligands on CD34+ cells treated by fucosyl transferase in vitro was determined by flow cytometry. The results indicated that the expression levels of CD26 on the surface of stem/progenitor cells (CD34+) from cord blood, bone marrow and peripheral blood were (7.62+/-0.63)%, (6.35+/-0.89)% and (6.18+/-0.91)% (p>0.05) respectively. And the activities of CD26 of the three sources of stem cells were 67.15 U/1000 cells (1 U=1 pmol/min), 26.85 U/1000 cells and 20.95 U/1000 cells respectively, in which the activity of CD26 on surface of CD34+ from cord blood was significantly higher than that from other both sources (p<0.01). The expression levels of P-selectin ligand on the stem/progenitor cells three kinds were (83.46+/-6.33)%, (15.65+/-0.89)% and (80.17+/-6.85)%, and the expression levels of E-selectin ligand on stem/progenitor cells of three kinds were (25.31+/-1.03)%, (26.34+/-0.89)% and (29.79+/-1.78)% respectively. The expression of E-selectin ligand on the surface of cord blood stem/progenitor cell CD34+ increased from (25.31+/-1.03)% to (63.23+/-1.08)% after glycosylation engineering. It is concluded that there is no significant difference of the expression of CD26 between the three sources of stem/progenitor cells, but the activity of CD26 in cord blood was obviously higher than that in bone marrow and peripheral blood. The expression of P-selectin ligand on bone marrow stem/progenitor cell was lower than that on stem cells of cord blood and peripheral blood. Glycosylation engineering can promote and elevate the expression of E-selectin ligand on the surface of CD34+ cells from cord blood.
Antigens, CD34 ; metabolism ; Bone Marrow Cells ; cytology ; metabolism ; Cells, Cultured ; Dipeptidyl Peptidase 4 ; metabolism ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Receptors, Fibroblast Growth Factor ; metabolism ; Sialoglycoproteins ; metabolism ; Stem Cells ; cytology ; metabolism

The study was aimed to explore the influence of co-culture ex vivo of CD34+ cells from two units of cord blood (CB) on the homing-related adherent molecule expression of each other. Mesenchymal stem cells (MSCs) were obtained from human bone marrow. Two units of CB CD34+ cells were co-cultured on 12 Gy gamma-ray irradiated MSC layer. Their adherent molecule expressions were assessed by flow cytometry. The results showed that the purity of the isolated CD34+ cells was (98.25+/-0.93)%. After co-culture on MSC layer for 6 days, the proportion of CD34+ cells of each unit was dropped to (60.4+/-6.32)% and (60.2+/-5.12)% respectively, but there was no significant difference from the control groups. The expressions of CD44, CD62L, CD184 and CD26 on CD34+ cells of each unit remained unaffected. The expression of CD162 was downregulated and CD54 was first increased but then dropped to the level before co-culture. But there was no significant difference between the experimental and control groups. In conclusion, co-culture of CD34+ cells from two units of CB may have no effects on the adherent molecule expressions of each other.
Antigens, CD34 ; metabolism ; Bone Marrow Cells ; cytology ; Cell Adhesion Molecules ; metabolism ; Coculture Techniques ; Fetal Blood ; cytology ; metabolism ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo study both the release of HMGB1 from irradiation-treated mesenchymal stem cells (MSCs) and the effects of HMGB1 on human cord blood CD34(+) hematopoietic progenitor cell proliferation and differentiation.

METHODSMSCs were obtained from human bone marrow. HMGB1 released by the MSCs after treatment with 12 Gy gamma-ray irradiation was determined by enzyme linked immunosorbent assay (ELISA). CD34(+) cells were positively selected with a MACS CD34 isolation kit. The freshly isolated CD34(+) cells were cultured in the presence of HMGB1 for 6 days. Phenotype of cultured cells surface molecules (CD13, CD14, CD11c, CD41 and CD71) were analyzed by flow cytometry. The proliferation and differentiation capacities of cord blood HSCs were assayed by colony forming cell assay. The receptors of HMGB1 (RAGE, TLR2 and TLR4) on cord blood CD34(+) cells were detected by flow cytometry.

RESULTSHMGB1 level in the supernatant \[(4.3 +/- 0.9) ng/ml\] of the irradiated MSC was significantly higher than that in control \[(0.4 +/- 0.2) ng/ml\] (P < 0.01). Human cord blood CD34(+) cells expressed the HMGB1 receptors RAGE, TLR2 and TLR4. The HMGB1-treated CD34(+) cells contained higher proportions of CD13(+) \[(32.6 +/- 5.9)% vs (18.4 +/- 3.8)%\], CD14(+)\[(25.4 +/- 4.4)% vs (12.6 +/- 2.7)%\], CD11c(+) \[(20.3 +/- 3.9)% vs (9.8 +/- 2.1)%\], CD71(+) \[(47.1 +/- 7.4)% vs (26.6 +/- 4.6)%\] cells compared with control group did. But HMGB1 did not induce the generation of CD41(+) cells \[(1.3 +/- 0.5)% vs (1.1 +/- 0.4)%\]. Furthermore, HMGB1 profoundly induced the growth of BFU-E, CFU-GM and total CFU in a dose-dependent manner, and this effect was partially inhibited by TLR2 and TLR4 antibodies.

CONCLUSIONHuman MSC treated with gamma-ray irradiation can release HMGB1, which can induce the proliferation and differentiation of human cord CD34(+) cells.

Antigens, CD34 ; metabolism ; Cell Differentiation ; Cells, Cultured ; Fetal Blood ; cytology ; HMGB1 Protein ; Hematopoietic Stem Cells ; cytology ; Humans

OBJECTIVETo explore the effects of bone marrow mesenchymal stem cells (MSCs) on in vitro expansion potential, the adherent molecules expression of cord blood (CB) CD34(+) cells.

METHODSMSCs were obtained from human bone marrow and their differentiation function and phenotype were identified. CB CD34(+) cells were expanded in culture systems with or without MSC layer. Hematopoietic progenitor cells and adhesion molecules expression were assessed by semisolid culture assay and flow cytometry.

RESULTSThy-1, SH2, SB10, CD44, CD13, CD49e and CD29 were highly expressed on MSCs with no expressions of CD34, CD45, HLA-DR, CD14 and CD31. The MSCs could differentiate into adipocytes and osteoblasts under specific induction conditions. After culturing on MSCs layer with supplement of cytokines for 8 days, the absolute numbers of nuclear cells, CD34(+), CD34(+)CD38(-), CD34(+)CD62L(+) cells and CFU-Cs were increased by 145.57 +/- 17.89, 37.47 +/- 13.78, 69.78 +/- 50.07, 10.74 +/- 5.89 and 20.73 +/- 5.54-folds, respectively, being significantly higher than that cultured with cytokines alone. The expression of ALCAM, VLA-alpha4, VLA-alpha5, VLA-beta1, HCAM, PECAM and LFA-1 on CD34(+) cells remained unaffected. The expressions of ICAM-1 and L-selectin were downregulated during expansion, while the absolute numbers of CD34(+)CD62L(+) and CD34(+)CD54(+) cells were increased.

CONCLUSIONSMSCs layer improves expansion of CB CD34(+) cells while inhibiting their differentiation and retaining their homing ability.

Antigens, CD34 ; Cell Adhesion Molecules ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Mesenchymal Stromal Cells

OBJECTIVETo study the effect of ex vivo expansion on the migration ability and the CXCR4 expression of umbilical cord blood (CB) hematopoietic stem/progenitor cells (HSPC).

METHODSCD(34)(+) cells isolated from fresh CB samples were cultured in a serum-free and stroma-free culture system. On day 7, 10 and 14, CD(34)(+) cells were re-selected from the expanded cells, and the expression of CXCR4 and the transmigration ability of these CD(34)(+) cells were evaluated respectively and compared with those of the precultured fresh CD(34)(+) cells.

RESULTS(1) SDF-1 induced a higher migration percentage of fresh or expanded CB CD(34)(+) cells than that of uninduced ones. (2) Both of the uninduced and SDF-1-induced migrations were slightly reduced in the first week and then much more reduced in the second week expansion (P < 0.05). (3) The number of the CD(34)(+)CXCR4(+) cells were significantly increased during the culture period, but there was a downtrend of CXCR4 expression on CD(34)(+) subset; the expression levels on day 10 and 14 were lower than that on day 0.

CONCLUSIONSThe expanded HSPC would sustain the chemotactic activity during one-week-culture, but with further extended culture time their intrinsic homing potential would be partly impaired.

Antigens, CD34 ; genetics ; metabolism ; Cell Culture Techniques ; Cell Movement ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; metabolism ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Male ; Pregnancy

OBJECTIVE: To induce hematopoietic progenitor/stem cells of umbilical cord blood to differentiate into mature megakaryocytes and platelets in vitro and to investigate the mechanism of production of platelets. METHODS: The CD34+ cells were sorted from umbilical cord blood by magnetic activated cell sorting (MACS) and then cultured in vitro with optimized medium to be differentiated into mature megakaryocytes and platelets. The cultured cells and the platelet-like particles were isolated from the culture and were checked by the fluorescence-activated cell sorter (FACS), immunohistochemistry assays, light microscope,electron microscope and platelet aggregation tests. RESULTS: The cultured megakaryocytes were detected with proplatelets and both the cultured cells and the platelet-sized particles were found to have the same structure with the normal megakaryocytes and platelets by light and electron microscope. The immunohistochemistry assays revealed the cultured cells expressed GP II b III a with a positivity of 95% which was a special antigen for platelets and megakaryocytes. Culture-derived platelet-sized particles aggregated in response to thrombin as the plasma derived-platelets did. The cultured platelets had the same positivity of CD41 as the platelets from platelet rich plasma. CONCLUSION The hematopoietic progenitor/stem cells can be induced to differentiate into purified and mature megakaryocytes and platelets. It provides a practical way to study the mechanism of platelets production.
Antigens, CD34 ; metabolism ; Blood Platelets ; cytology ; Cell Differentiation ; physiology ; Cells, Cultured ; Fetal Blood ; cytology ; metabolism ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Megakaryocytes ; cytology

The purpose of this study was to synthesize the complex of magnetic nanoparticles and antibody, and to apply them to isolate the CD34(+) cells from umbilical cord blood, then to evaluate its separation efficiency. The complex of magnetic nanoparticles and antibody was used to separate CD34(+) cells from umbilical cord blood in the outer magnetic field because of its superparamagnetism, specific identification and function of combination with CD34(+) cells. Scanning electron microscopy was used to observe the morphology of the separated CD34(+) cells. Flow Cytometer was applied to evaluate the sorting efficiency of magnetic nanoparticles, liquid culture and colony culture were taken to assay proliferation and differentiation capacity of the separated CD34(+) cells. The results showed that the CD34(+) cells from umbilical cord blood were isolated fast and effectively by the complex of magnetic nanoparticles and monoclonal antibody. Moreover, the isolated CD34(+) cells still maintained its normal morphology, highly proliferative and differentiative capacity. It is concluded that the complex of magnetic nanoparticles and monoclonal antibody has been successfully synthesized and developed as a technique which efficiently and quickly isolates CD34(+) cells from umbilical cord blood.
Antigens, CD34 ; metabolism ; Fetal Blood ; cytology ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans ; Immunomagnetic Separation ; methods ; Magnetics ; Nanoparticles

The cell-surface expression and functional status of the CD95/Fas antigen on primitive hematopoietic progenitors isolated from human cord blood (CB) were studied. The CD34+ cells freshly isolated from CB displayed low CD95 expression. The combinations of cytokines such as SCF + FL could up-regulate the expression of CD95 in vitro culture and tumor necrosis factor-alpha (TNF-alpha) and interon-gamma (IFN-gamma) further increased the CD95 expression induced by positive cytokines. The functional status of CD95-mediated apoptosis were analyzed by incubation of CD34+ CB cells in the presence of anti-CD95 monoclonal antibodies (McAbs). The effects of anti-CD95 McAbs were measured by viable cell counting, flow cytometry, LTIC and CFU-C assays. A decrease of viable cells, CFU-C and LTIC numbers were observed in the presence of anti-CD95 McAbs and TNF-alpha or IFN-gamma. However, growth factor deprivation or the early-acting cytokine such as SCF and FL cross-linking to CD95 caused low apoptosis of CD34+ cells. The correlation of increased intracytoplasmic levels of bcl-2 and the presence of CD95 on fresh CB CD34+ cells suggested that bcl-2 might be involved in protecting against CD95-mediated apoptosis of CB CD34+ cells.
Antigens, CD34 ; Apoptosis ; Fetal Blood ; cytology ; Hematopoiesis ; Hematopoietic Stem Cells ; metabolism ; Humans ; Leukocytes, Mononuclear ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; fas Receptor ; metabolism

The cell-surface expression and functional status of the CD95/Fas antigen on primitive hematopoietic progenitors isolated from human cord blood (CB) were studied. The CD34+ cells freshly isolated from CB displayed low CD95 expression. The combinations of cytokines such as SCF + FL could up-regulate the expression of CD95 in vitro culture and tumor necrosis factor-alpha (TNF-alpha) and interon-gamma (IFN-gamma) further increased the CD95 expression induced by positive cytokines. The functional status of CD95-mediated apoptosis were analyzed by incubation of CD34+ CB cells in the presence of anti-CD95 monoclonal antibodies (McAbs). The effects of anti-CD95 McAbs were measured by viable cell counting, flow cytometry, LTIC and CFU-C assays. A decrease of viable cells, CFU-C and LTIC numbers were observed in the presence of anti-CD95 McAbs and TNF-alpha or IFN-gamma. However, growth factor deprivation or the early-acting cytokine such as SCF and FL cross-linking to CD95 caused low apoptosis of CD34+ cells. The correlation of increased intracytoplasmic levels of bcl-2 and the presence of CD95 on fresh CB CD34+ cells suggested that bcl-2 might be involved in protecting against CD95-mediated apoptosis of CB CD34+ cells.
Antigens, CD34 ; Antigens, CD95/*metabolism ; Apoptosis ; Fetal Blood/*cytology ; Hematopoiesis ; Hematopoietic Stem Cells/*metabolism ; Leukocytes, Mononuclear/metabolism ; Proto-Oncogene Proteins c-bcl-2/*metabolism