BACKGROUND: Maintaining the quality of cryopreserved cord blood is crucial. In this pilot study, we describe the results of the internal quality control program for a cord blood bank thus far. METHODS: Donated cord blood units unsuitable for transplantation were selected for internal quality control once a month. One unit of cord blood, aliquoted into 21 capillaries, was cryopreserved and thawed annually to analyze the total nucleated cell count, CD34⁺ cell count, cell viability test, and colony-forming units assay. RESULTS: No significant differences in the variables (total nucleated cell count, cell viability, CD34⁺ cell count) were observed between samples cryopreserved for one and two years. Upon comparing the variables before cryopreservation and post thawing with the capillaries of one year of storage, cell viability and CD34⁺ cell counts decreased significantly. The use of cord blood samples in capillaries, which can be easily stored for a long period, was similar to the methods used for testing segments attached to the cord blood unit. CONCLUSIONS: The results of this study may be useful for determining the period during which the quality of cryopreserved cord blood units used for transplantation is maintained.
Antigens, CD34/metabolism ; Blood Banks ; Cell Count ; Cell Survival ; Cryopreservation/*standards ; Fetal Blood/*cytology ; Humans ; Pilot Projects ; Quality Control ; Republic of Korea ; Time Factors

Axl encodes the tyrosine-protein kinase receptor, participating in the proliferation and migration of many cells. This study examined the role of Axl in functions of endothelial progenitor cells (EPCs). Axl was detected by RT-PCR and Western blotting in both placentas and EPCs from normal pregnancy and preeclampsia patients. The Axl inhibitor, BMS777-607, was used to inhibit the Axl signalling pathway in EPCs. Cell proliferation, differentiation, migration and adhesion were measured by CCK-8 assay, cell differentiation assay, Transwell assay, and cell adhesion assay, respectively. Results showed the expression levels of Axl mRNA and protein were significantly higher in both placentas and EPCs from preeclampsia patients than from normal pregnancy (P<0.05). After treatment with BMS777-607, proliferation, differentiation, migration and adhesion capability of EPCs were all significantly decreased. Our study suggests Axl may play a role in the function of EPCs, thereby involving in the pathogenesis of preeclampsia.
Adult ; Aminopyridines ; pharmacology ; Blood Pressure ; Case-Control Studies ; Cell Adhesion ; drug effects ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Female ; Fetal Blood ; cytology ; enzymology ; Gene Expression Regulation ; Gestational Age ; Human Umbilical Vein Endothelial Cells ; drug effects ; enzymology ; pathology ; Humans ; Placenta ; metabolism ; physiopathology ; Pre-Eclampsia ; blood ; genetics ; physiopathology ; Pregnancy ; Primary Cell Culture ; Protein Kinase Inhibitors ; pharmacology ; Proto-Oncogene Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Pyridones ; pharmacology ; RNA, Messenger ; antagonists & inhibitors ; genetics ; metabolism ; Receptor Protein-Tyrosine Kinases ; antagonists & inhibitors ; genetics ; metabolism ; Stem Cells ; drug effects ; enzymology ; pathology

PURPOSE: To develop a model based on non-invasive clinical and ultrasonographic parameters for predicting the likelihood of subsequent histologic chorioamnionitis in women with preterm premature rupture of membranes (PPROM) and to determine whether the inclusion of invasive test results improves the predictive value of the model. MATERIALS AND METHODS: This retrospective cohort study included 146 consecutive women presenting with PPROM (20-33 weeks). Transvaginal ultrasonographic assessment of cervical length was performed. Maternal serum C-reactive protein (CRP) levels and white blood cell (WBC) counts were measured after amniocentesis. Amniotic fluid (AF) obtained by amniocentesis was cultured, and interleukin-6 (IL-6) levels and WBC counts were determined. The primary outcome measure was histologic chorioamnionitis. RESULTS: Risk scores based on serum CRP concentrations and gestational age (model 1) were calculated for each patient. The model was shown to have adequate goodness of fit and an area under the receiver operating characteristic curve (AUC) of 0.742. When including AF test results (e.g., AF IL-6 levels) in model 1, serum CRP concentrations were found to be insignificant, and thus, were excluded from model 2, comprising AF IL-6 levels and gestational age. No significant difference in AUC was found between models 1 and 2. CONCLUSION: For women with PPROM, the newly developed model incorporating non-invasive parameters (serum CRP and gestational age) was moderately predictive of histologic chorioamnionitis. The inclusion of invasive test results added no predictive information to the model in this setting.
Adult ; *Amniocentesis ; Amniotic Fluid/*cytology/microbiology ; C-Reactive Protein/*metabolism ; Chorioamnionitis/blood/*diagnosis/metabolism ; Cohort Studies ; Female ; Fetal Membranes, Premature Rupture/*blood ; *Gestational Age ; Humans ; Infant, Newborn ; Interleukin-6/blood ; Leukocyte Count ; Predictive Value of Tests ; Pregnancy ; ROC Curve ; Retrospective Studies ; Sensitivity and Specificity

Objective To observe the effect of the expanded human umbilical cord blood CD34+cells in ischemic limb of mice and analyse the relationship between the CD34+cells and angiogenesis. Methods Human umbilical cord blood was collected and CD34+cells were separated for expanding. Mice limbs ischemia models were established (n=15) and randomly divided into three groups:expanded CD34+cells group (n=5),fresh CD34+cells group (n=5),and control group(n=5). CD34+cells were detected by DiI dye tracing and antihuman nuclear antigen antibody(HNA) immunohistochemical staining. The improvement of blood reperfusion was evaluated by indicators including limb temperature,CD31 staining,and transforming growth factor-β1 (TGF-β1) mRNA expression. Results On days 14 (t=5.421,P=0.001;t=0.616,P=0.000) and 28(t=10.780,P=0.000; t=12.123,P=0.000),both expanded CD34+cells group and fresh CD34+cells group enjoyed better temperature improvement. Days 28 later,the vascular densities in the expanded CD34+cells group and the fresh CD34+cells group were 592.3±24.6 (t=26.386,P=0.000) and 530.7±25.5 (t=21.502,P=0.000),which were significantly higher than that in control group 219.7±19.9. The TGF-β1 mRNA expression in the expanded CD34+cells group and the fresh CD34+cells group were (0.578±0.050) copies (t=12.376,P=0.000) and (0.504±0.080) copies (t=7.098,P=0.000),both over control group [(0.224±0.040)copies]. Conclusions In vitro culture of cord blood CD34+cells can emigrate to ischemic zone and induce angiogenesis to alleviate ischemia. Thus,it may provide a treatment option for lower limb ischemia.
Animals ; Antigens, CD34 ; metabolism ; Cell Transplantation ; Cells, Cultured ; Extremities ; physiopathology ; Fetal Blood ; cytology ; Humans ; Ischemia ; therapy ; Mice ; Neovascularization, Physiologic ; Random Allocation ; Transforming Growth Factor beta1 ; metabolism

To demonstrate the effect of AB serum on terminal erythroid differentiation ex vivo.
 Methods: After separation of CD34+ cells from cord blood, the cells were cultured and divided into a control group and an experimental group. The effects of AB serum were examined by the expressions of different markers (GPA, Band3 and α4-integrin) for erythroblast differentiation and enucleation by flow cytometry. 
 Results: The CD34+ cells were successfully differentiated to enucleated red blood cells. There were evident differences among the expressions of GPA, Band3 and α4-integrin between the 2 groups. The percentage of GPA positive cells in the experimental group was bigger than that in the control group in every time point. The expression of Band3 in the experimental group was higher than that in the control group. The expression of α4-integrin in the experimental group was lower than that in the control group. In addition, the enucleation rate in the experimental group was higher than that in the control group.
 Conclusion: AB serum can promote the cell differentiation and enucleation during terminal erythroid differentiation in vitro.
ABO Blood-Group System ; blood ; physiology ; Anion Exchange Protein 1, Erythrocyte ; metabolism ; Antigens, CD34 ; blood ; Cell Differentiation ; genetics ; physiology ; Cell Nucleus ; Cells, Cultured ; Erythrocytes ; physiology ; ultrastructure ; Erythropoiesis ; genetics ; physiology ; Fetal Blood ; cytology ; physiology ; Flow Cytometry ; Glycophorins ; metabolism ; Humans ; Integrin alpha4beta1 ; metabolism

Pulmonary arterial hypertension (PAH) causes right ventricular failure due to a gradual increase in pulmonary vascular resistance. The purposes of this study were to confirm the engraftment of human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) placed in the correct place in the lung and research on changes of hemodynamics, pulmonary pathology, immunomodulation and several gene expressions in monocrotaline (MCT)-induced PAH rat models after hUCB-MSCs transfusion. The rats were grouped as follows: the control (C) group; the M group (MCT 60 mg/kg); the U group (hUCB-MSCs transfusion). They received transfusions via the external jugular vein a week after MCT injection. The mean right ventricular pressure (RVP) was significantly reduced in the U group after the 2 week. The indicators of RV hypertrophy were significantly reduced in the U group at week 4. Reduced medial wall thickness in the pulmonary arteriole was noted in the U group at week 4. Reduced number of intra-acinar muscular pulmonary arteries was observed in the U group after 2 week. Protein expressions such as endothelin (ET)-1, endothelin receptor A (ERA), endothelial nitric oxide synthase (eNOS) and matrix metalloproteinase (MMP)-2 significantly decreased at week 4. The decreased levels of ERA, eNOS and MMP-2 immunoreactivity were noted by immnohistochemical staining. After hUCB-MSCs were administered, there were the improvement of RVH and mean RVP. Reductions in several protein expressions and immunomodulation were also detected. It is suggested that hUCB-MSCs may be a promising therapeutic option for PAH.
Animals ; Cytokines/metabolism ; Disease Models, Animal ; Endothelin-1/metabolism ; Fetal Blood/*cytology ; Gene Expression Regulation/drug effects ; Hemodynamics ; Humans ; Hypertension, Pulmonary/chemically induced/*therapy ; Hypertrophy, Right Ventricular/physiopathology ; Immunohistochemistry ; Lung/metabolism/pathology ; Male ; Matrix Metalloproteinase 2/metabolism ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/*cytology/metabolism ; Monocrotaline/toxicity ; Nitric Oxide Synthase Type III/metabolism ; Pulmonary Artery/pathology ; Rats ; Rats, Sprague-Dawley ; Receptor, Endothelin A/metabolism

OBJECTIVETo observe the anti-virus effects of andrographolide (AD) on the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) signaling pathway when immunological cells were infected with H1N1.

METHODSLeukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line (16HBE). The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium (MTT) assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunosorbent assay (ELISA) detection while immunological cells for real-time polymerase chain reaction (RT-PCR).

RESULTSThe optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-β promoter stimulator-1 (IPS-1), interferon regulatory factor (IRF)-7, IRF-3 and nuclear transcription factor κB (NF-κB) mRNA levels increased significantly (P<0.05). When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF-κB decreased significantly in each group with significant statistic differences (P<0.05).

CONCLUSIONSThe RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Antiviral Agents ; pharmacology ; Cells, Cultured ; Coculture Techniques ; DEAD Box Protein 58 ; DEAD-box RNA Helicases ; genetics ; metabolism ; Dendritic Cells ; drug effects ; immunology ; virology ; Diterpenes ; pharmacology ; Fetal Blood ; cytology ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; immunology ; Influenza, Human ; drug therapy ; immunology ; virology ; Interferon-beta ; genetics ; metabolism ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Leukocytes, Mononuclear ; drug effects ; immunology ; virology ; Macrophages ; drug effects ; virology ; NF-kappa B ; genetics ; metabolism ; Promoter Regions, Genetic ; drug effects ; immunology ; RNA, Messenger ; metabolism ; Signal Transduction ; drug effects ; genetics ; immunology

This study was aimed to investigate the effect of high mobility group box1(HMGB1) and/or stromal cell derived factor-1(SDF-1) on the migration of cord blood CD34⁺ cells, and to explore whether HMGB1 promotes cord blood CD34⁺ cell migration via SDF-1/CXCR4 axis. Cord blood mononuclear cells were isolated by Ficoll-Paque density centrifugation, CD34⁺ cells were collected by a positive immunoselection procedure (CD34 MicroBeads) according to the manufacturer's instructions, the purity of the isolated CD34⁺ cells was detected by flow cytometry. In vitro chemotaxis assays were performed using Transwell cell chambers to detect cells migration. 1 × 10⁵ cells/well cord blood CD34⁺ cells were added into the upper chambers. Different concentrations of HMGB1 and/or SDF-1 (0, 10, 25, 50, 100, 200 ng/ml) were used to detect the optimal concentrations of HMGB1 and/or SDF-1 for inducing migration of cord blood CD34⁺ cells. Freshly isolated cord blood CD34⁺ cells express CXCR4 (SDF-1 receptor), and HMGB1 receptor TLR-2,TLR-4 and RAGE. To explore which receptors were required for the synergy of HGMB1 and/or SDF-1 on cells migration, the anti-SDF-1, anti-CXCR4 and anti-RAGE antibodies were used to detect the effect of HGMB1 alone or with SDF-1 on cord blood CD34⁺ cells migration. The results showed that the purity of CD34⁺ cells isolated from cord blood mononuclear cells by magnetic cell sorting was 97.40 ± 1.26%, the 25 ng/ml SDF-1 did not induce migration of cord blood CD34⁺ cells, whereas the optimal migration was observed at 100 ng/ml. HMGB1 alone did not induce migration up to 100 ng/ml. The dose test found that the the best synergistic concentrations for cells migration were 100 ng/ml HMGB1 combined with 50 ng/ml SDF-1. The blocking test showed that both the anti-SDF-1 (4 µg/ml) and anti-CXCR4 (5 µg/ml) antibodies could block cell migration induced by HMGB1 or combined with SDF-1, but the cord blood CD34⁺ cells in the presence of anti-RAGE, anti-TLR-2 and anti-TLR-4 antibodies did not modify the response to SDF-1 in the presence of HMGB1. It is concluded that both HMGB1 and SDF-1 can induce cord blood CD34⁺ cells migration, HMGB1 enhances SDF-1-induced migration exclusively via CXCR4 and in a RAGE and TLR receptors-independent manner, the exact mechanism needs to be further explored.
Antigens, CD34 ; metabolism ; Cell Movement ; Chemokine CXCL12 ; metabolism ; Fetal Blood ; cytology ; metabolism ; Flow Cytometry ; HMGB1 Protein ; metabolism ; Humans ; Receptors, CXCR4 ; metabolism

OBJECTIVETo investigate the enhanced cytotoxicity against leukemia cells of natural Killer (NK) cells from cord blood (CB) after expansion in vitro.

METHODSNK cells was expanded on a layer of trophoblast cells with irradiated K562-mb15-41BBL cell line for 21 days. The levels of receptors on NK cells were detected by flow cytometry. Cytotoxicity of expanded NK cells against leukemia cells and specific ligand of immunoglobulin like(Ig- liKe)receptors were assessed using 51Cr released assay.

RESULTSThere were no differences of inhibitory receptors expression between fresh NK cells and expanded NK cells [CD158a:(16.77±11.65)% vs(14.37±11.12)%, P>0.05; CD158b: (42.48±18.11)% vs (40.92±19.02)%, P>0.05; NKG2A: (70.20±18.43)% vs (78.90±13.69)%, P>0.05], but higher activated receptors expression on expanded NK cells [NKp30: (54.10±13.27)% vs (4.14±2.05)%, P<0.05; NKp44: (72.10±17.30)% vs (0.52±1.16)%, P<0.05; NKp46: (80.63±14.01)% vs (44.19±6.19)%, P<0.05; NKG2D: (97.50±2.55)% vs (72.25±14.35)%, P<0.05]. Expanded NK cells showed higher cytotoxicity against leuKemia cell lines than fresh NK cells [K562: (74.3±3.6)% vs (55.3±4.2)%, P<0.05; Raji: (60.6±5.0)% vs (12.0±3.6)%, P<0.05]. CD158a⁻ CD158b⁻ NK cells had higher cytotoxicity on four types of target cells, but CD158a⁺CD158b⁻ CB-NK cell had lower cytotoxicity on 221-Cw4 and 221-Cw3Cw4 cells. CD158a⁻ CD158b⁺ CB- NK cells had lower cytotoxicity on 221-Cw3 and 221-Cw3Cw4, but CD158a⁺CD158b⁺ CB-NK cells had higher cytotoxicity on 721- 221 cells.

CONCLUSIONExpression of activated receptors of expanded NK cells were up-regulated, but no changes of inhibitory receptors. Expanded NK cells showed high cytotoxicity against leukemia cells and kept the specificity of ligand of Ig-like receptors, which could be beneficial to cell-therapy for tumor.

Cells, Cultured ; Coculture Techniques ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; K562 Cells ; Killer Cells, Natural ; cytology ; metabolism

PURPOSE: We evaluated the effect of human parathyroid hormone (hPTH) on the engraftment and/or in vivo expansion of hematopoietic stem cells in an umbilical cord blood (UCB)-xenotransplantation model. In addition, we assessed its effect on the expression of cell adhesion molecules. MATERIALS AND METHODS: Female NOD/SCID mice received sublethal total body irradiation with a single dose of 250 cGy. Eighteen to 24 hours after irradiation, 1x107 human UCB-derived mononuclear cells (MNCs) and 5x106 human UCB-derived mesenchymal stem cells (MSCs) were infused via the tail vein. Mice were randomly divided into three groups: Group 1 mice received MNCs only, Group 2 received MNCs only and were then treated with hPTH, Group 3 mice received MNCs and MSCs, and were treated with hPTH. RESULTS: Engraftment was achieved in all the mice. Bone marrow cellularity was approximately 20% in Group 1, but 70-80% in the hPTH treated groups. Transplantation of MNCs together with MSCs had no additional effect on bone marrow cellularity. However, the proportion of human CD13 and CD33 myeloid progenitor cells was higher in Group 3, while the proportion of human CD34 did not differ significantly between the three groups. The proportion of CXCR4 cells in Group 3 was larger than in Groups 1 and 2 but without statistical significance. CONCLUSION: We have demonstrated a positive effect of hPTH on stem cell proliferation and a possible synergistic effect of MSCs and hPTH on the proportion of human hematopoietic progenitor cells, in a xenotransplantation model. Clinical trials of the use of hPTH after stem cell transplantation should be considered.
Animals ; Bone Marrow/metabolism ; Cell Proliferation ; Female ; Fetal Blood/*cytology ; Flow Cytometry ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/*drug effects ; Humans ; Leukocytes, Mononuclear/*cytology ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/*cytology ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Parathyroid Hormone/*therapeutic use ; Stem Cells/cytology ; Transplantation, Heterologous