OBJECTIVETo investigate the effects of oxygen concentration and reactive oxygen species (ROS) on the biological characteristics of hematopoietic stem cells (HSC) and to analyzed the relationship among the oxygen concentration, ROS and the biological characteristics of mouse HSC through simulation of oxygen environment experienced by PB HSC during transplantation.

METHODSThe detection of reactive oxygen species (ROS), in vitro amplification, directional differentiation (BFU-E, CFU-GM, CFU-Mix), homing of adhesion molecules (CXCR4, CD44, VLA4, VLA5, P-selectin), migration rate, CFU-S of NOD/SCID mice irradiated with sublethal dose were performed to study the effect of oxgen concentration and reactive oxygen species on the biological characteristics of mouse BM-HSC and the relationship among them.

RESULTSThe oxygen concentrations lower than normal oxygen concentration (especially hypoxic oxygen environment) could reduce ROS level and amplify more Lin(-) c-kit(+) Sca-1(+) BM HSC, which was more helpful to the growth of various colonies (BFU-E, CFU-GM, CFU-Mix) and to maintain the migratory ability of HSC, thus promoting CFU-S growth significantly after the transplantation of HSC in NOD/SCID mice irradiated by a sublethal dose. BM HSC exposed to oxygen environments of normal, inconstant oxygen level and strenuously thanging of oxygen concentration could result in higher level of ROS, at the same time, the above-mentioned features and functional indicators were relatively lower.

CONCLUSIONThe ROS levels of BM HSC in PB HSCT are closely related to the concentrations and stability of oxygen surrounding the cells. High oxygen concentration results in an high level of ROS, which is not helpful to maintain the biological characteristics of BM HSC. Before transplantation and in vitro amplification, the application of antioxidancs and constant oxygen level environments may be beneficial for transplantation of BMMSC.

Animals ; Cell Differentiation ; Culture Media ; chemistry ; Erythroid Precursor Cells ; cytology ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Hematopoietic Stem Cells ; cytology ; metabolism ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Oxygen ; chemistry ; Reactive Oxygen Species ; metabolism

BACKGROUND: For autologous hematopoietic stem cell transplantation (HSCT), the volume of infused and DMSO contained in graft are the major causes of complications related to infusion. In this study, we evaluated feasibility of cryopreserving peripheral blood stem cell collects (PBSCC) at high cell concentration. METHODS: PBSCC from 40 patients with multiple myeloma or lymphoma were split and cryopreserved at two different concentrations of TNCs; one for standard concentration (SC) (2x108 cells/mL) and the other for high concentration (HC) (3x108 cells/mL). The viability of total nucleated cells and CD34+ cell count were examined before cryopreservation and after thawing. CFU-GM was examined with thawed products. Data were analyzed as two groups between good mobilizer (GM) and poor mobilizer (PM). RESULTS: There were no differences in TNC viability between SC and HC of all patients (P=0.0656) and PM (P=0.9658), however HC of GM showed significantly lower viability than SC (P=0.0314). CD34+ cell viability did not differ between SC and HC. CD34+ cell recovery was decreased in HC of all patients (P=0.459) and GM (P=0.0164), but no differences between SC and HC in PM (P=0.9658). CFU-GM clonogenic efficiency between SC and HC was not different in all patients (P=0.0635) and PM (P=0.8984), but was decreased in HC of GM (P=0.0427). CONCLUSION: Cryopreservation of PBSCC at 3x108 cells/mL seems to have minimal adverse effect on the quality of PBSC after thawing, particularly in PM. This approach may help to reduce infusion related complications while decreasing the cost of processing and storage of PBSCC.
Antigens, CD34 ; Cell Count ; Cell Survival ; Cryopreservation ; Dimethyl Sulfoxide ; Feasibility Studies* ; Granulocyte-Macrophage Progenitor Cells ; Hematopoietic Stem Cell Transplantation ; Humans ; Lymphoma ; Multiple Myeloma ; Peripheral Blood Stem Cell Transplantation ; Stem Cells* ; Transplants

OBJECTIVETo investigate the impact of cryopreservation duration of umbilical cord blood (UCB) on quality of hematopoietic stem cell and outcome of clinical transplantation.

METHODS605 units of UCB which had been used in clinical transplantation were previously cryopreserved for 820 (88-2651) days in average. UCB was detected for total nucleated cell count, CD34+ cells count, cell recovery rate, cell viability and CFU-GM after thawing.

RESULTSNo statistical correlation was found between cryopreservation duration and cell recovery rate, cell viability. CFU-GM decreased along with the extension of cryopreservation duration (P=0.011), ranging between 109.6 and 105.7/1 × 10⁵. There was no significant difference on hematopoietic reconstitution time, graft failure, acute GVHD and overall survival among groups with different cryopreservation duration.

CONCLUSIONCryopreservation duration has no significant effect on cell recovery rate, cell viability and clinical transplantation outcome. Extension of cryopreservation duration may reduce CFU-GM of stem cells with fluctaion still in normal range. UCB could maintain cell viability and function to achieve satisfactory clinical transplantation outcome even when thawed after 3 to 7 years' cryopreservation.

Cell Count ; Cell Survival ; Cryopreservation ; Fetal Blood ; Graft vs Host Disease ; Granulocyte-Macrophage Progenitor Cells ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Humans ; Treatment Outcome

BACKGROUNDRadiation-induced injury after accidental or therapeutic total body exposure to ionizing radiation has serious pathophysiological consequences, and currently no effective therapy exists. This study was designed to investigate whether transplantation of allogeneic murine compact bone derived-mesenchymal stem cells (CB-MSCs) could improve the survival of mice exposed to lethal dosage total body irradiation (TBI), and to explore the potential immunoprotective role of MSCs.

METHODSBALB/c mice were treated with 8 Gy TBI, and then some were administered CB-MSCs isolated from C57BL/6 mice. Survival rates and body weight were analyzed for 14 days post-irradiation. At three days post-irradiation, we evaluated IFN-γ and IL-4 concentrations; CD4(+)CD25(+)Foxp3(+) regulatory T cell (Treg) percentage; CXCR3, CCR5, and CCR7 expressions on CD3(+) T cells; and splenocyte T-bet and GATA-3 mRNA levels. CB-MSC effects on bone marrow hemopoiesis were assessed via colony-forming unit granulocyte/macrophage (CFU-GM) assay.

RESULTSAfter lethal TBI, compared to non-transplanted mice, CB-MSC-transplanted mice exhibited significantly increased survival, body weight, and CFU-GM counts of bone marrow cells (P < 0.05), as well as higher Treg percentages, reduced IFN-γ, CXCR3 and CCR5 down-regulation, and CCR7 up-regulation. CB-MSC transplantation suppressed Th1 immunity. Irradiated splenocytes directly suppressed CFU-GM formation from bone marrow cells, and CB-MSC co-culture reversed this inhibition.

CONCLUSIONAllogeneic CB-MSC transplantation attenuated radiation-induced hematopoietic toxicity, and provided immunoprotection by alleviating lymphocyte-mediated CFU-GM inhibition, expanding Tregs, regulating T cell chemokine receptor expressions, and skewing the Th1/Th2 balance toward anti-inflammatory Th2 polarization.

Animals ; Bone and Bones ; cytology ; Cytokines ; metabolism ; Female ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Male ; Mesenchymal Stem Cell Transplantation ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Whole-Body Irradiation ; adverse effects

The aim of this study was to investigate the effect of GW003 on the ability of granulocyte colony forming in vitro of bone marrow cells. The bone marrow samples was collected from normal rhesus, the patients with leukemia in stages of remission and chemotherapy respectively, and the nucleated cells were separated and cultured for 12 days after addition of different concentrations of GW003 or rhG-CSF, or G-CSF mutant. Then the amount of colony-forming unit-granulocyte-macrophage was counted. The results indicated that GW003 could enhance the ability of bone marrow nucleated cells of rhesus to forming CFU-GM in vitro, and its effect was much better than that of rhG-CSF or G-CSF mutant at the same concentration(®). The GW003 showed dose-response relationship to CFU-GM level (r = R(2) = 0.965, P = 0.003, in a certain concentration), the GW003 also could enhance CFU-GM formation of marrow nucleated cells in leukemic patients, especially for patients receiving chemotherapy. The GW003 could relieve the marrow suppression caused by chemotherapy significantly. It is concluded that the GW003 can significantly improve the ability of bone marrow cells to form granulocyte colony in vitro as well as effectively alleviate bone marrow suppression.
Adult ; Animals ; Bone Marrow Cells ; drug effects ; Cell Line, Tumor ; Colony-Forming Units Assay ; Female ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Granulocyte-Macrophage Progenitor Cells ; cytology ; drug effects ; Granulocytes ; drug effects ; Humans ; Macaca mulatta

OBJECTIVETo explore the influence of 1,4-benzoquinone (1,4-BQ) on proliferation of human bone marrow haematopoietic stem cells (hBM-HSCs) and human bone marrow mesenchymal stem cells (hBM-MSCs).

METHODSThe bone marrow samples were collected from a healthy donor. Methylcellulose semi-solid culture medium was used to culture the mononuclear cells of bone marrow in different culture systems. Colony-forming unit (CFU) assay was utilized to evaluate the proliferation of hBM-HSCs exposed to 1,4-BQ at the doses of 10, 25, 50 and 100 µmol/L and to observe the influence of 1,4-BQ on the Colony-forming unit-erythroid (CFU-E)/Burst-forming unit-erythroid (BFU-E), Colony-forming unit-granulocyte, macrophage (CFU-GM), Colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) in hBM-MSCs. MTT assay was used to detect the proliferation of hBM-MSCs exposed to 1,4-BQ at the doses of 1, 5, 10, 25, 50, 100, 200, 500 and 1000 µmol/L for 24 h, respectively, after hBM-MSCs were isolated, cultured and expanded.

RESULTSThe results of CFU assay indicated that numbers of CFU-E/BFU-E, CFU-GM and CFU-GEMM in 25, 50 and 100 µmol/L groups significantly decreased, as compared with control group (P < 0.05). However, no significant difference was found between the 10 µmol/L group and the control group. The results of MTT assay showed that the cellular viability of hBM-MSCs exposed to 1,4-BQ at the doses of 50 ∼ 200 µmol/L for 24 h significantly decreased in a dose-depended manner. When the exposure dose was higher than 200 µmol/L, the cellular viability of hBM-MSCs was lower than 5% which was significantly lower than that of control group (P < 0.05). When the exposure dose was lower than 25 µmol/L, there was no significant difference of cellular viability between exposure group and control group (P > 0.05).

CONCLUSIONThe results of the present study demonstrated that 1,4-BQ could inhibit the colony forming of hBM-HSCs and the relative viability of hBM-MSCs in vitro. The hematotoxicity induced by 1,4-BQ may be related to inhibiting the proliferation capacity of hBM-HSCs.

Benzoquinones ; toxicity ; Bone Marrow Cells ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Erythroid Precursor Cells ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo investigate in vitro characteristics of colony-forming cells (CFC) in patients with myelodysplastic syndrome (MDS) and to compare that in patients with non-severe aplastic anemia (NSAA).

METHODSData of in vitro CFC and correlation with other related laboratory tests in 155 newly diagnosed MDS patients were analyzed retrospectively, and to compare with data of in vitro CFC in 122 newly diagnosed NSAA patients.

RESULTSMedian number of burst-forming units-erythroid (BFU-E) was 9 (0 - 157)/10(5) bone marrow mononuclear cells (BMMNC), colony forming unit-erythroid (CFU-E) 30 (0 - 425)/10(5)BMMNC and colony forming unit-granulocytes/macrophages (CFU-GM) 14 (0 - 125)/10(5)BMMNC in patients with MDS, being significantly lower than those in healthy control; number of BFU-E and/or CFU-E was lower than the lower limit of normal control in 66 cases (42.6%), CFU-GM lower in 3 cases (1.9%) and BFU-E and/or CFU-E with CFU-GM lower in 70 cases (45.2%). Cluster/CFU-GM ratio was significantly lower in low blast group (MDS < 5% blast in bone marrow smear) than that in high blast group (MDS ≥ 5% blast) (0.65 vs 1.0, P = 0.049). In all MDS patients, cluster had positive correlation with each type of CFC (r = 0.415, 0.338, 0.642 for BFU-E, CFU-E, CFU-GM, respectively, P = 0.000), but had negative correlation with neutrophil alkaline phosphatase (N-ALP) positive rate and scores (r(rate) = -0.315, P = 0.001 and r(scores) = -0.257, P = 0.006). The median number of each type of CFC was significantly higher in MDS group than that in NSAA group (BFU-E 9 vs 5/10(5)BMMNC, P = 0.017; CFU-E 30 vs 19.5/10(5)BMMNC, P = 0.023; CFU-GM 14 vs 10/10(5)BMMNC, P = 0.003, respectively). Positive correlation between BFU-E and CFU-E were revealed in both MDS and NSAA group (r(MDS) = 0.712, P = 0.000 and r(NSAA) = 0.757, P = 0.000), with a lower correlation coefficient in MDS (P < 0.05).

CONCLUSIONSEarly onset MDS present markedly decreased hematopoietic progenitor cells (HPC), and particularly in erythroid progenitors extensively and severely. The number of BFU-E, CFU-E and CFU-GM can reflect HPC number in vivo but not stand for normal hematopoietic clones, the number of clusters represent pathologic HPC clones but not exactly leukemic blasts.

Adolescent ; Adult ; Aged ; Anemia, Aplastic ; pathology ; Bone Marrow Cells ; pathology ; Cells, Cultured ; Child ; Child, Preschool ; Female ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; pathology ; Retrospective Studies ; Stem Cells ; Young Adult

This study was purposed to investigate the role of post-thaw infused donor cells for predicting engraftment and hematopoietic reconstitution after unrelated cord blood transplantation (UCBT). The retrospective analysis was performed on clinical data of 97 children with malignant or non-malignant diseases received single unit UCBT from August 1999 to April 2010. The impact of pre-freezing and post-thaw cell dose of total nucleated cells (TNC), CD34(+) cells and colony-forming units-granulocyte/macrophage (CFU-GM) on engraftment and hematological recovery after UCBT was analyzed. Unrelated donors were from Guangzhou cord blood bank (GZCBB) entirely. The results indicated that the pre-freezing TNC (/kg) (mean ± SD: 7.65 × 10⁷ ± 4.26 × 10⁷; median: 6.34 × 10⁷), CD34(+)cells (/kg) (mean ± SD: 4.64 × 10(5) ± 4.47 × 10⁵; median: 3.03 × 10⁵) and CFU-GM (/kg) (mean ± SD: 0.79 × 10⁵ ± 1.09 × 10⁵; median: 0.57 × 10⁵) showed a good correlation with their post-thaw counterparts including TNC(/kg) (mean ± SD: 6.98 × 10⁷ ± 4.12 × 10⁷; median: 6.00 × 10⁷), CD34(+)cells (/kg)(Mean ± SD: 6.86 × 10⁵ ± 8.56 × 10⁵; Median: 4.17 × 10⁵), and CFU-GM (/kg) (mean ± SD: 0.52 × 10⁵ ± 0.52 × 10⁵; median: 0.39 × 10⁵) (r = 0.952, p < 0.001; r = 0.794, p < 0.001; r = 0.478, p < 0.001). Either the pre-freezing or post-thaw number of infused CFU-GM was significant higher in patients who achieved engraftment (n = 70) than those who suffered graft failure (n = 22) (p = 0.023 and 0.011, respectively), but no significant difference of TNC and CD34(+) cells dose (pre-freezing or post-thaw) were found between these two groups. Pre-freezing CFU-GM, TNC, CD34(+) cell dose negatively correlated with the time of neutrophil engraftment (r = -0.285, p = 0.018; r = -0.396, p = 0.002; r = -0.373, p = 0.002), as well as the post-thaw number of TNC and CD34(+) cells (r = -0.260, p = 0.031; r = -0.483, p < 0.001), whereas only pre-freezing CD34(+) cells showed a significant correlation with platelet engraftment time (r = -0.352, p = 0.013). It is concluded that the CFU-GM amount is useful for predicting engraftment of UCBT, while pre-freezing hematopoietic cell doses show superior correlation with the speed of engraftment and hematopoietic reconstitution than their post-thaw counterparts in pediatric recipients, suggesting that it is essential to perform hematopoietic potency assay on each cord blood unit prior to listing or release for administration.
Adolescent ; Antigens, CD34 ; blood ; Blood Banks ; Child ; Child, Preschool ; Cord Blood Stem Cell Transplantation ; methods ; Female ; Fetal Blood ; cytology ; Graft Survival ; Granulocyte-Macrophage Progenitor Cells ; Humans ; Infant ; Male ; Retrospective Studies ; Tissue Donors

BACKGROUND AND OBJECTIVELeukemic microenvironment has a major role in the progression of leukemia. Leukemic cells can induce reversible changes in microenvironmental components, especially the stromal function which results in improved growth conditions for maintaining the malignant leukemic cells. This study aimed to investigate the survival advantage of leukemic cells over normal hematopoietic cells in stromal microenvironment in long term.

METHODSThe mice were injected intraperitoneally with N-N' ethylnitrosourea (ENU) to induce leukemia; the mice received injection of normal saline were used as control. At 180 days after ENU induction, the mice were killed and the bone marrows were cultured for 19 days. Colony-forming assays were used to analyze the formation of various cell colonies. The expression of Sca-1, CD146, VEGFR2, CD95, pStat3, pStat5, and Bcl-xL in marrow cells were detected by flow cytometry.

RESULTSLong-term leukemic bone marrow culture showed abnormal elongated stromal fibroblasts with almost absence of normal hematopoietic cells. Adherent cell colonies were increased, but CFU-F and other hematopoietic cell colonies were significantly decreased in leukemia group (P<0.001). Primitive progenitor-specific Sca-1 receptor expression was decreased with subsequent increased expression of CD146 and VEGFR-2 in leukemic bone marrow cells. Decreased Fas antigen expression with increased intracellular pStat3, pStat5 and Bcl-xL proteins were observed in leukemic bone marrow cells.

CONCLUSIONSStromal microenvironment shows altered morphology and decreased maturation in leukemia. Effective progenitor cells are decreased in leukemia with increased leukemia-specific cell population. Leukemic microenvironment plays a role in promoting and maintaining the leukemic cell proliferation and survivability in long term.

Animals ; Antigens, Ly ; metabolism ; Bone Marrow Cells ; metabolism ; pathology ; CD146 Antigen ; metabolism ; Cell Count ; Cells, Cultured ; Colony-Forming Units Assay ; Erythroid Precursor Cells ; metabolism ; pathology ; Ethylnitrosourea ; Female ; Fibroblasts ; metabolism ; pathology ; Granulocyte-Macrophage Progenitor Cells ; metabolism ; pathology ; Granulocytes ; metabolism ; pathology ; Hematopoiesis ; Hematopoietic Stem Cells ; metabolism ; pathology ; Leukemia ; chemically induced ; metabolism ; pathology ; Male ; Membrane Proteins ; metabolism ; Mice ; Myeloid Progenitor Cells ; metabolism ; pathology ; Phenotype ; STAT3 Transcription Factor ; metabolism ; STAT5 Transcription Factor ; metabolism ; Tumor Microenvironment ; physiology ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism ; bcl-X Protein ; metabolism ; fas Receptor ; metabolism

This study is to analyze microcosmic significance of Chinese medicine composing principle "principal, assistant, complement and mediating guide" and it's fuzzy mathematic quantitative law. According to molecular biology and maximal membership principle, fuzzy subset and membership functions were proposed. Using in vivo experiment on the effects of SiWu Decoction and its ingredients on mice with radiation-induced blood deficiency, it is concluded that DiHuang and DangGui belonged to the principal and assistant subset, BaiShao belonged to the contrary complement subset, ChuanXiong belonged to the mediating guide subset by maximal membership principle. It is discussed that traditional Chinese medicine will be consummate medical science when its theory can be described by mathematic language.
Animals ; Colony-Forming Units Assay ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Erythroid Precursor Cells ; drug effects ; Fuzzy Logic ; Granulocyte-Macrophage Progenitor Cells ; drug effects ; Leukocytes ; drug effects ; Mathematics ; Medicine, Chinese Traditional ; Mice ; Plants, Medicinal ; chemistry ; Principal Component Analysis ; methods ; Radiation Injuries, Experimental ; blood