Adult ; Antigens, Differentiation, T-Lymphocyte ; blood ; Female ; Humans ; Liver Cirrhosis, Biliary ; blood ; pathology ; Male ; Middle Aged

The aims of this study were to analyze the composition of umbilical cord blood cells (UCBC), to examine the characteristics of dendritic cells (DC) before and after culture, to search the method of differentiation and increase of DC in vitro and to appraise surface antigen from UCBC. Twelve units of umbilical cord blood were collected from May 2002 to September 2002. Peripheral blood mononuclear cells of 9 cases were collected from healthy adult donors. The nature of UCBC was freshly determined and then UCBC were cultured for 1, 2, 3 and 4 weeks with granulocyte-monocyte colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and EPO. Method of flow cytometry was used to determine the number of DC and cell surface antigens before and after culture by using monoclonal antibodies. The monoclonal antibodies included CD4, CD8, CD19, CD34, CD38, CD83, CD1a, CD11c and CDw123. The results showed that amounts of CD34+ progenitors in peripheral blood cells were 0.02 x 10(5)/ml, and amounts of CD34+ progenitors in human UCBC were 0.22 x 10(5)/ml. UCBC cultured for 1, 2, 3 and 4 weeks with GM-CSF, IL-3, EPO and SCF were shown to differentiate into CD1a+ CD11c+ CD83+ CDw123+ DC. Numbers of DC from UCBC remarkably generated in 2-4 weeks and then decreased in number. By culture with cytokines DC increased up to (10.6 - 28.2) x 10(5)/ml in actual numbers. It is concluded that the mononuclear cells of UCB are able to differentiate into CD1a+, CD83+, CD11c+ and CDw123+ DC when UCBC are cultured with proper cytokines of GM-CSF, SCF, EPO and IL-3 for 2-4 weeks. These DCs as antigen presenting cells are possibly effective in cancer immunotherapy.
Antigens, CD1 ; blood ; Antigens, CD34 ; blood ; Blood Cell Count ; Cell Differentiation ; Cytokines ; pharmacology ; Dendritic Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Infant, Newborn

This study was aimed to investigate a beneficial approach for resolving the deficiency of blood source, preventing the infection resulting from blood transfusion and overcoming the knotty match of patients with rare blood group by using massive expansion of erythroid cells from cord blood CD34(+) cells in vitro. The CD34(+) cells from human cord blood were cultured in serum-free medium supplemented with stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO) for 1 week, then expansion and differentiation of CD34(+) cells into erythroid cells were supported by co-culture with human mesenchymal stem cells (MSCs) derived from bone marrow for 2 weeks. The results indicated that after culture for 23 days, the expansion multiple of total cell number reached 2.52 x 10(5), and over 95% of these cells were erythroid cells as compared with less than 1% of myelomonocytic (CD14(+) or CD15(+)) cells and megakaryocytic (CD41(+)) cells. However, the culture system without MSC support was significantly disadvantaged both in expansion ability and ratio of erythroid cells when compared with MSC supporting system. It is concluded that the erythroid cells can be produced from CD34(+) cells in large scale by culturing in the system comprised of cytokine sets and MSC feeders, in which MSCs can support the proliferation and differentiation of erythroid cells.
Antigens, CD34 ; Cell Culture Techniques ; methods ; Cell Differentiation ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans

OBJECTIVE: To compare the efficacy of directional erythroid differentiation in different serum free culture systems and to screen the optimal culture systems for inducing the differentiation of umbilical cord blood hematopoietic stem and progenior cells (HSPC) to erythroid cells. METHODS: The CD34 cells from umbilical blood munonuclear cells were sorted by using the magnetic beads, and were inoculated into 3 different of culture systems (system 1, 2 and 3 respectively), to induce erythrold differentiation by 3 stage culture. The living cells were counted in different differentiation stages and were observed by Wright-Giemsa staining; the expression of CD71 and CD235a on cell surface was detected by flow cytometry, the erythroid differentiation pteency was detected via colony-forming test. RESULTS: The ability of system 2 to promote the HSPC proliferation was the strongest, the efficacy of system 3 to promote the erythroid differentiation of HSPC was the most optimal; the proliferation ability of cells cultured in system 2 for 2-15 days all was higher than that of cells cutured in system 1 and 3 (P＜0.05). The flow cytometry detection showed that the expression of CD71 and CD235a on surface of cells cultured in system 3 was the highest, the CD235a percentage on day 15 of differentiation in system 3 was (92.33±3.89)%, that in system 2 was (84.67±3.12)%, while that in system 1 was (72.17±6.83)% (P＜0.05). Cell morplologic detection showed that throid differentiation was accelerated on day 12, the percentage of orthochromatic erythrocytes in system 3 was (67.67±2.08)% which was 10.69 and 25.34 times higher than that in system 2 and 1 respectively (P＜0.05). The colony-forming test showed the ratio of BFU-E in system 3 increased gradually on day 3-9 (r=0.99, P＜0.05), which was significanlly higher than that in system 2 and 1 on day 9 (90.35±5.52% vs 77.06±2.26% and 74.50±3.95%). CONCLUSION Culture system 3 is the most effective serum-free erythroid differentiation system, and the culture system 2 is the most powerful HSPC proliferation system. This study results provide a technical basis for further efficiently increasing and inducing the erythroid proliferation and differentiation of HSPC, and also provide culture system in vitro for the clinical application and basic research.
Antigens, CD34 ; Cell Differentiation ; Cells, Cultured ; Culture Media, Serum-Free ; Erythroid Precursor Cells ; Fetal Blood ; Humans

The expression of inducible co-stimulator (ICOS) in peripheral blood T lymphocytes from the patients with systemic lupus erythematosus (SLE) and the role in the pathogenesis of SLE was investigated. By using two-color immunofluorescent staining and flow cytometric assay, the expression levels of ICOS in peripheal blood T lymphocytes from 33 patients with SLE and 16 healthy volunteers were detected. SLE diseases activity index (SLEDAI) of the patients with SLE was used to evaluate the disease activity. The correlation between the ICOS expression and SLEDAI was analyzed among the groups. The results showed that the expression levels of ICOS in T lymphocytes in active SLE group was markedly higher than those in the control and inactive SLE groups (both P< 0.01). There was no significant difference in the expression levels of ICOS between the inactive SLE and the control groups (P>0.05). In active SLE and inactive SLE groups, positive linear correlation was found between the levels of the ICOS expression in T lymphocytes and SLEDAI (r=0. 711, P=0.001; r=0.561, P=0.03). It was suggested that the expression of ICOS in peripheral blood T lymphocytes from the patients with active SLE was up-regulated and and ICOS might be related to the pathogenesis of SLE.
Antigens, Differentiation, T-Lymphocyte/*biosynthesis ; Antigens, Differentiation, T-Lymphocyte/genetics ; Lupus Erythematosus, Systemic/blood ; Lupus Erythematosus, Systemic/etiology ; Lupus Erythematosus, Systemic/*immunology ; T-Lymphocytes/*immunology

OBJECTIVETo investigate the relationship between atrial fibrillation (AF) and serum soluble CD163.

METHODSA total of 336 patients with heart valve disease were included in this study, including 167 with AF and 169 with sinus rhythm. The clinical data were compared between the two grops, and Logistic regression analysis was used to identify the risk factors associated with AF.

RESULTSThe levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), tumor necrosis factor (TNF), interleukin-6 (IL - 6), high-sensitivity C-reactive protein (hs-CRP) and left atrial diameter (LAD) all differed significantly between the two groups (P<0.05). Serum soluble CD163 levels in AF patients were significantly higher than those in patients with sinus rhythm (P<0.05). Serum soluble CD163 was positively correlated with TNF (r=0.244, P=0.244), IL-6 (r=0.186, P=0.186), hs-CRP (r=0.183, P=0.183) and LAD (r=0.194, P=0.194) in patients with AF. Logistic regression analysis showed that LAD, IL-6, TNF, hs-CRP and CD163 were all associated with AF. ROC curve analysis showed that the area under curve of serum soluble CD163 was 0.861 in patients with AF (CI 95%: 0.820-0.901, P<0.01) with a sensitivity and a specificity of 80.8 and 76.9%, respectively.

CONCLUSIONSerum soluble CD163 level may be a risk factor for AF, and an increased soluble CD163 level may indicate active inflammation in AF patients.

Antigens, CD ; blood ; Antigens, Differentiation, Myelomonocytic ; blood ; Atrial Fibrillation ; blood ; C-Reactive Protein ; analysis ; Heart Atria ; pathology ; Humans ; Inflammation ; blood ; Interleukin-6 ; blood ; Lipoproteins, HDL ; blood ; Lipoproteins, LDL ; blood ; Receptors, Cell Surface ; blood ; Risk Factors ; Tumor Necrosis Factor-alpha ; blood

To study the possibility of separation and culture of human umbilical cord blood adherent cell (HUCBAC), the umbilical cord blood CD34(+) cells were cultured in Dexter system in order to evaluate and observe the biological behavior of adherent cells in vitro. The results showed that all cells were cultured with Dexter system. By day 9-14 (at a median of 11.2 days), adherent cell colonies formed and reached their maximum at 15-22 days (mean 19.6 days), by day 28, all adherent cells spread over the bottom of Petri dish. By means of light microscopy, these cells were found to differentiate into three kinds of cells in culture of 28 days: fibroblast-liked cell, macrophage liked cell and small-round cells. The ratio of these three kinds of cells was 56.8%, 38%, 5.5% respectively. Cytochemistry assay revealed that the positive rate reached 100% in NSE stain and PAS stain; the adherent cell by ALP stain were shown 35% positive, but in POX stain the result was negative. Immunohistochemistry stain revealed that the positive rate of cord adherent cells for CD106, CD29, CD44, CD45, CD50, Fn, Ln, collagen IV etc reached 96%, 93%, 98%, 68%, 72%, 92%, 74%, 83% respectively. It is concluded there are hematopoietic adherent precursors in cord blood CD34(+) cells and the HUCBAC shows some biological behavior of hematopoietic stromal cells.
Antigens, CD34 ; blood ; Cell Adhesion ; immunology ; Cell Differentiation ; immunology ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans ; Hyaluronan Receptors ; blood ; Immunohistochemistry ; Integrin beta1 ; blood ; Leukocyte Common Antigens ; blood ; Vascular Cell Adhesion Molecule-1 ; blood

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

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 induce differentiation of CD(34)+ cells from varying sources into epithelial cells for potential application of blood vessel prostheses.

METHODSCD(34)+ cells were isolated from canine peripheral blood and bone marrow or human umbilical cord blood by an immune magnetic cell sorting system. The isolated CD(34)+ cells were induced to differentiate into endothelial cells in a liquid culture with VEGF. Endothelial cells were evaluated by immunocytochemistry and transmission electron microscopy. CD(34)+ cells were seeded on PTFE prostheses, which were harvested and observed by electron microscopy.

RESULTSThe average isolated CD(34)+ cells from peripheral blood, bone marrow and umbilical cord blood were (26.30+/-2.42)%,(41.84 +/-3.65)%and (74.62+/-4.46)%, respectively. The number of CD(34)+ cells increased with the culture duration and reached to the highest level at the 14th d. Immunostaining showed positive signal for CD31 in endothelial cells and positive for factor VIII in cell culture. Transmission electron microscopy found Weibel-Palade bodies in the cytoplasm. Scanning electron microscopy demonstrated a single- layer of flat cells in the innermost layer of PTFE prostheses, and the cells were not of tropism.

CONCLUSIONCD(34)+ cells isolated from peripheral blood, bone marrow and umbilical cord blood can be induced into endothelial cells; bone marrow and umbilical cord blood can be used as the sources of seeding cells for endothelialization of prostheses.

Antigens, CD34 ; analysis ; Blood Vessel Prosthesis Implantation ; methods ; Cell Differentiation ; Endothelial Cells ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Immunohistochemistry