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1.
Article in Chinese | WPRIM | ID: wpr-272478

ABSTRACT

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>


Subject(s)
Animals , Cell Differentiation , Culture Media , Chemistry , Erythroid Precursor Cells , Cell Biology , Granulocyte-Macrophage Progenitor Cells , Cell Biology , Hematopoietic Stem Cells , Cell Biology , Metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Oxygen , Chemistry , Reactive Oxygen Species , Metabolism
2.
Article in Chinese | WPRIM | ID: wpr-278927

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the impact of cryopreservation duration of umbilical cord blood (UCB) on quality of hematopoietic stem cell and outcome of clinical transplantation.</p><p><b>METHODS</b>605 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.</p><p><b>RESULTS</b>No 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.</p><p><b>CONCLUSION</b>Cryopreservation 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.</p>


Subject(s)
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
3.
Article in Korean | WPRIM | ID: wpr-114285

ABSTRACT

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.


Subject(s)
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
4.
Article in Chinese | WPRIM | ID: wpr-349687

ABSTRACT

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.


Subject(s)
Adult , Animals , Bone Marrow Cells , Cell Line, Tumor , Colony-Forming Units Assay , Female , Granulocyte Colony-Stimulating Factor , Pharmacology , Granulocyte-Macrophage Progenitor Cells , Cell Biology , Granulocytes , Humans , Macaca mulatta
5.
Chinese Medical Journal ; (24): 475-482, 2014.
Article in English | WPRIM | ID: wpr-317959

ABSTRACT

<p><b>BACKGROUND</b>Radiation-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.</p><p><b>METHODS</b>BALB/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.</p><p><b>RESULTS</b>After 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.</p><p><b>CONCLUSION</b>Allogeneic 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.</p>


Subject(s)
Animals , Bone and Bones , Cell Biology , Cytokines , Metabolism , Female , Granulocyte-Macrophage Progenitor Cells , Cell Biology , Male , Mesenchymal Stem Cell Transplantation , Mice, Inbred BALB C , Mice, Inbred C57BL , Whole-Body Irradiation
8.
Chinese Journal of Hematology ; (12): 516-521, 2012.
Article in Chinese | WPRIM | ID: wpr-359434

ABSTRACT

<p><b>OBJECTIVE</b>To 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).</p><p><b>METHODS</b>Data 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.</p><p><b>RESULTS</b>Median 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).</p><p><b>CONCLUSIONS</b>Early 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.</p>


Subject(s)
Adolescent , Adult , Aged , Anemia, Aplastic , Pathology , Bone Marrow Cells , Pathology , Cells, Cultured , Child , Child, Preschool , Female , Granulocyte-Macrophage Progenitor Cells , Cell Biology , Humans , Male , Middle Aged , Myelodysplastic Syndromes , Pathology , Retrospective Studies , Stem Cells , Young Adult
9.
Article in Chinese | WPRIM | ID: wpr-324266

ABSTRACT

<p><b>OBJECTIVE</b>To 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).</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>The 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.</p>


Subject(s)
Benzoquinones , Toxicity , Bone Marrow Cells , Cell Biology , Cell Proliferation , Cells, Cultured , Erythroid Precursor Cells , Granulocyte-Macrophage Progenitor Cells , Cell Biology , Humans , Mesenchymal Stem Cells , Cell Biology
10.
Article in Chinese | WPRIM | ID: wpr-313901

ABSTRACT

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.


Subject(s)
Adolescent , Antigens, CD34 , Blood , Blood Banks , Child , Child, Preschool , Cord Blood Stem Cell Transplantation , Methods , Female , Fetal Blood , Cell Biology , Graft Survival , Granulocyte-Macrophage Progenitor Cells , Humans , Infant , Male , Retrospective Studies , Tissue Donors
11.
Chinese Journal of Cancer ; (12): 969-979, 2010.
Article in English | WPRIM | ID: wpr-296329

ABSTRACT

<p><b>BACKGROUND AND OBJECTIVE</b>Leukemic 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>Long-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.</p><p><b>CONCLUSIONS</b>Stromal 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.</p>


Subject(s)
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 , 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
12.
Acta Pharmaceutica Sinica ; (12): 38-41, 2009.
Article in Chinese | WPRIM | ID: wpr-232601

ABSTRACT

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.


Subject(s)
Animals , Colony-Forming Units Assay , Drug Combinations , Drugs, Chinese Herbal , Pharmacology , Erythroid Precursor Cells , Fuzzy Logic , Granulocyte-Macrophage Progenitor Cells , Leukocytes , Mathematics , Medicine, Chinese Traditional , Mice , Plants, Medicinal , Chemistry , Principal Component Analysis , Methods , Radiation Injuries, Experimental , Blood
13.
Article in Korean | WPRIM | ID: wpr-218063

ABSTRACT

BACKGROUND: Umbilical cord blood (UCB) is generally stored overnight and it undergoes a CD34 positive selection process the next day for reducing the cost and due to the convenience. We intended to determine whether overnight storage of cord blood cells affects the short and long-term repopulating capacity. METHODS: Five individuals' UCB samples were analyzed by colony assay, apoptotic cell counts and long term bone marrow culture. All the samples were divided to four groups, which were the fresh group (immediate use of harvest), the overnight storage group (overnight storage at room temperature after harvest), the immediate cryopreservation group (immediate cryopreservation after harvest) and the overnight cryo group (cryopreservaton after overnight storage at room temperature after harvest). RESULTS: The number of colony forming units-granulocyte macrophage (CFU-GM) was 116.2+/-20.1 in the fresh group and 90.8+/-15.8 in the overnight storage group (P=0.07). The number of CFUs-GM was similar between the immediate and overnight cryo groups (P=0.79). The immediate cryo group showed a significantly lower number of CFUs-GM as compared to that of the fresh group (P=0.03). The apoptotic cells were detected at 21+/-6.8% in the fresh group and this was 24.2+/-2.4% in the overnight storage group (P=0.32), and this was similar between immediate and overnight cryo groups (P=0.80). The fresh group had a significantly lower number of apoptotic cells compared to that of the immediate cryo group (P=0.02). After long term stromal-based culture, the mean production of CFU-GM colonies was similar between all the groups (P>0.05). CONCLUSION: These results support the continue use of overnight storage of UCB before cryopreservation as a convenient, cost reducing measure.


Subject(s)
Bone Marrow , Cell Count , Cryopreservation , Fetal Blood , Granulocyte-Macrophage Progenitor Cells , Macrophages
14.
Article in Chinese | WPRIM | ID: wpr-318712

ABSTRACT

This study was aimed to investigate the biological characteristics of osteoblasts from patients with myelodysplastic syndrome (MDS) and their supportive capacity for hematopoiesis in vitro. A two-dimensional culture system was constructed by using osteoblasts derived from human marrow mesenchymal stem cells (MSC); MSCs were isolated from bone marrow of MDS patients and normal individuals and were cultured; the third passage of MSCs were induced into osteoblasts which were treated with mitomycin C and confluenced into a feeder layer. Ficolled bone marrow mononuclear cells were obtained from normal individuals and seeded into the two-dimensional culture system to culture in vitro without exogenous cytokines. By using colony-forming assay, the ability of the two-dimensional system to culture HPCs was observed. The cytokine expression of osteoblasts from MDS patient bone marrows in mRNA level was detected by RT-PCR and was compared with human osteoblast cell line hFOB1.19. The results showed that the osteoblasts from MDS patients could support short-term survival of GM-CFC in condition without exogenous cytokines, that is, osteoblasts played a crucial role in regulation of HPC growth. The results of RT-PCR clearly demonstrated that the osteoblast cell line hFOB1.19 expressed SCF, IL-6, SDF-1alpha, G-CSF and GM-CSF. The same expression patterns of above cytokines were also seen in osteoblasts derived from BM-MSCs of MDS patients and normal individuals, but these cells did not express GM-CSF. It is concluded that the biological characteristics of osteoblasts from bone marrow of MDS patients are generally not different from those of osteoblasts from normal bone marrow. Both of them can support GM -CFC to form colonies in vitro, it may be associated with expressing important related cytokines by osteoblasts.


Subject(s)
Cytokines , Metabolism , Granulocyte-Macrophage Colony-Stimulating Factor , Metabolism , Granulocyte-Macrophage Progenitor Cells , Cell Biology , Hematopoietic Stem Cells , Cell Biology , Humans , Interleukin-6 , Metabolism , Myelodysplastic Syndromes , Metabolism , Pathology , Osteoblasts , Metabolism , Physiology , RNA, Messenger , Metabolism , Stem Cell Factor , Metabolism
15.
Article in Chinese | WPRIM | ID: wpr-253315

ABSTRACT

This study was purpose to examine the effect of dimethyl sulfoxide (DMSO) and Tween 80 on the growth and viability of stromal cells (BMSC), colony-forming units for granulocytes and macrophages (CFU-GM) and bone marrow endothelial cell line (BMEC) from murine bone marrow in vitro, and to analyze the concentration-effect relationship. The colony yields of colony-forming units fibroblastic (CFU-F) and CFU-GM were assessed in the murine bone marrow cell cultures at various concentrations of DMSO or Tween 80 and in the control groups. The MTT assay and trypan blue exclusion were used to determine the cell viability and percentage of survival in BMSC and BMEC cultures with or without either of these organic solvents. The results showed that the colony yields of both CFU-F and CFU-GM were decreased significantly (p<0.05 or <0.01) at the concentrations (v/v final) of 2% DMSO or 0.005%-0.01% Tween 80 respectively, as compared with control. The cell viability and percentage of survival of BMSC and BMEC cultures were significantly reduced (p<0.05 or <0.01) at 0.5%-1.0% DMSO or 0.002%-0.005% Tween 80, as compared with control. With the increase of volume fractions of these solvents, the decreased percentages of corresponding measurements were increased by degrees. It is concluded that when the concentration of DMSO or Tween 80 goes to a certain level in cell culture medium, either of the organic solvents has an inhibitory action or/and cytotoxicity on the growth and viability of BMSCs, CFU-GM and BMECs. The growth inhibition and cytotoxic response are more significant at higher concentrations of these solvents.


Subject(s)
Animals , Bone Marrow Cells , Cell Biology , Cell Line , Cell Proliferation , Cell Survival , Cells, Cultured , Dimethyl Sulfoxide , Pharmacology , Dose-Response Relationship, Drug , Endothelial Cells , Cell Biology , Female , Granulocyte-Macrophage Progenitor Cells , Cell Biology , Male , Mice , Polysorbates , Pharmacology , Solvents , Pharmacology , Stromal Cells , Cell Biology
16.
Article in Korean | WPRIM | ID: wpr-187480

ABSTRACT

BACKGROUND: The efficient collection of peripheral blood stem cells (PBSC) from donors who donate for allogeneic transplants as well as from patients undergoing autologous transplants is essential for a successful transplant. Recently, the Amicus cell separator and the associated MNC collection computer software program for PBSC collection were introduced in Korea. METHODS: Two apheresis machines (Amicus, Baxter Healthcare; and CS-3000 plus, Baxter Healthcare) were compared retrospectively. A total number of 144 procedures were performed on 14 donors and 28 patients. The pre- and post-apheresis complete blood cell (CBC) counts and the number of hematopoietic progenitor cells (HPC) were determined in the peripheral blood from the subjects. The CBC, HPC, CD34+ cell counts and the level of colony-forming unit-granulocyte-macrophages (CFU-GM) were measured in the PBSC product collected from both machines. RESULTS: Both machines collected a similar number of CD34+ cells from the donors and patients. On the other hand, the Amicus collected significantly more nucleated cells, MNCs, HPCs and CFU-GM in the patients with significantly less RBC contamination than those with CS-3000 plus. The decrease in the peripheral blood platelet counts in the donors and patients was more prominent after apheresis using the CS-3000 plus (117.00+/-42.75 x 10(3)/microliter, 61.22+/-43.62 x 10(3)/microliter) than Amicus (26.04+/-18.68 x 10(3)/microliter, 22.15+/-28.66 x 10(3)/microliter)(p<0.05). CONCLUSION: PBSC collection can be performed successfully using CS-3000 plus and Amicus. Amicus is superior to CS-3000 plus in avoiding apheresis-induced thrombocytopenia, and is expected to prevent unnecessary platelet transfusion.


Subject(s)
Autografts , Blood Cells , Blood Component Removal , Cell Count , Delivery of Health Care , Granulocyte-Macrophage Progenitor Cells , Hand , Hematopoietic Stem Cells , Humans , Korea , Platelet Count , Platelet Transfusion , Retrospective Studies , Stem Cells , Thrombocytopenia , Tissue Donors
17.
Chinese Journal of Pediatrics ; (12): 494-498, 2005.
Article in Chinese | WPRIM | ID: wpr-312147

ABSTRACT

<p><b>OBJECTIVE</b>The previous studies indicated that mesenchymal stem cells (MSCs) either from umbilical cord blood (UCB) or from bone marrow (BM) had the same biological characteristics and the function of secreting hematopoietic growth factors (HGFs). The present study aimed to understand the effects of human UCB MSCs on the expansion of CD(34)(+) cells from UCB.</p><p><b>METHODS</b>1. Human UCB CD(34)(+) cells were incubated in the system containing UCB MSCs, HGFs and serum free medium. 2. The surface markers (CD(34)(+), CD(34)(+)CD(38)(-), CD(34)(+)CD(3)(+), CD(34)(+)CD(19)(+), CD(34)(+)CD(33)(+), CD(34)(+)CD(41a)(+)) on expanded UCB cells were examined by flow cytometry on the 6th and 12th days. 3. The expanded and unexpanded cells were cultured in semi-solid culturing system and checked for colony forming units of granulocyte and macrophage (CFU-GM), erythroid burst-forming unit (BFU-E), colony forming units of granulocyte- erythrocyte-megakaryocyte-macrophage (CFU-Mix) and colony forming units of high-proliferative potential (CFU-HPP).</p><p><b>RESULTS</b>1. The expansion folds of CD(34)(+)CD(38)(-) cells from UCB MSCs + HGFs groups on the 6th and 12th days were 159.43 and 436.68, respectively. Interestingly, the percentage of CD(34)(+)CD(38)(-) cells declined in HGFs group after expanding for 12 days, but it rose to 9.98% in the UCB MSCs + HGFs group. 2. Colony forming capacity of expanded UCB cells showed that the folds of CFU-Mix and CFU-HPP of UCB MSCs + HGFs group increased from day 6 to day 12, but the folds decreased in the HGFs group. 3. From day 0 to day 12, CD(34)(+)CD(33)(+) cells and CD(34)(+)CD(41a)(+) cells were amplified gradually, but CD(34)(+)CD(19)(+) and CD(34)(+)CD(3)(+) cells decreased gradually, and in UCB MSCs + HGFs group this phenomenon was more significant than that in HGFs group.</p><p><b>CONCLUSION</b>1. UCB MSCs containing system not only has the ability to expand the primitive HSCs but also has the ability to sustain the proliferation of HSCs. 2. UCB MSCs containing system amplified mainly myeloid and megakaryocytoid progenitor subsets. These may have clinical significance in reducing infection and hemorrhage.</p>


Subject(s)
Antigens, CD34 , Metabolism , Cell Culture Techniques , Cell Differentiation , Cell Proliferation , Culture Media, Conditioned , Culture Media, Serum-Free , Erythroid Precursor Cells , Fetal Blood , Cell Biology , Flow Cytometry , Granulocyte-Macrophage Progenitor Cells , Hematopoietic Cell Growth Factors , Pharmacology , Hematopoietic Stem Cells , Metabolism , Humans , Infant, Newborn , Mesenchymal Stem Cells , Allergy and Immunology , Metabolism
18.
Article in Korean | WPRIM | ID: wpr-181768

ABSTRACT

PURPOSE: Valproic acid (VPA) has been used as an anticonvulsant for a long time. Recently, there are many reports on VPA activity with regards to intracellular signal transduction, including differentiation, proliferation, and apoptosis. We experienced several hematologic toxicities during the long-term use of VPA. Therefore, we investigated whether VPA has effects on short-term or long-term hematopoiesis with respect to differing concentrations. METHODS: We obtained bone marrow mononuclear cells (BMMNC) from a 5 week old female C3H/He strain mouse. The BMMNC were cultured in semi-solid media mixed with VPA according to the concentrations of colony forming unit for granulocyte-monocytes (CFU-GM). The concentrations of VPA were used as follows: 0.01 mM, 0.1 mM, 1 mM, and 10 mM (therapeutic level: 0.07~1.1 mM). We performed long-term liquid culture under VPA to compare the frequency of long-term culture initiating cells (LTC-IC) according to various VPA levels. RESULTS: The number of CFU-GM was highest with 1 mM of VPA (45.2+/-13.5), with higher therapeutic level than control (25.7+/-11.9), in 0.01 mM of VPA (26.5+/-12.1) and in 0.1 mM of VPA (26.6+/-12.2). In 10 mM of VPA, a toxic level of VPA, was the lowest at 1.6+/-1.1 (P< 0.01). In long-term culture, the frequency of LTC-IC was increased in 0.1 mM of VPA (67.7+/-16.3%), lower therapeutic level than in control (5.5+/-10.6%). In 1 mM of VPA, the high therapeutic level decreased to 81.6+/-9.3%. With toxic levels of VPA, 10 mM, there was no hematopoiesis. CONCLUSION: The VPA might enhance short-term hematopoiesis at high therapeutic levels, while preserving LTC-IC in long-term hematopoiesis under low therapeutic concentrations. Therefore, we suggest that VPA to be used within a low therapeutic level to escape from hematopoietic suppression when using VPA as long-term medication for seizure control.


Subject(s)
Animals , Apoptosis , Bone Marrow , Female , Granulocyte-Macrophage Progenitor Cells , Hematopoiesis , Humans , Mice , Seizures , Signal Transduction , Stem Cells , United Nations , Valproic Acid
19.
Article in Korean | WPRIM | ID: wpr-181767

ABSTRACT

PURPOSE: Discordance with expectation there are a few differences in hematopoietic stem cells according to their source. The purpose of this study is to compare the cryopreservative potential of hematopoiectic stem cell containing fractions between cord blood (CB) and mobilized peripheral blood (mPB) during long term cryopreservation. METHODS: Nineteen CB and seven mPB were frozen with a programed freezer and stored in liquid phase of nitrogen from 1 to 4 years. After thawing the viability of mononuclear cells (MNCs) and the recovery rate of MNC, CD34 positive cell, colony form unit-granulocyte/monocyte was measured by CD34 flow cytometry and colony formation in semisolid methycellulose culture. CD34 positive cell was purified from cryopreserved-thawed or fresh mPB using magnetic associated cell sorting (MACS) CD34 selection kit. RESULTS: Though there is no difference in the viability and the recovery rate of CFU-GM between cryopreserved-thawed CB and mPB, the recovery rate of MNCs and CD34 positive cells is much higher in CB. Cell aggregation during the thawing process of long term cryopreserved mPB was effectively prevented by using high viscosity thawing buffer like as 10% dextran and 20% human serum albumin. We can also purify the CD34 positive cells from the long term cryopreserved mPB in the purity of more than 90%. CONCLUSION: Contrary to mPB long term cryopreserved CB can maintain the hematopoietic stem cell fraction without considerable loss, so it can be clinically used for hematopoietic stem cell transplantation.


Subject(s)
Cell Aggregation , Cryopreservation , Dextrans , Fetal Blood , Flow Cytometry , Granulocyte-Macrophage Progenitor Cells , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells , Humans , Nitrogen , Serum Albumin , Stem Cells , Viscosity
20.
Article in Korean | WPRIM | ID: wpr-77297

ABSTRACT

BACKGROUND: It is evident that inhalational anesthetics, such as nitrous oxide, possess a certain degree of myelodepressive effect in humans. However, unlike nitrous oxide, propofol is frequently recommended for the anesthesia of oncologic patients or for harvesting bone marrow from donors. To evaluate the possible toxicity of propofol on hematopoietic stem cells, the in vitro sensitivity of colony growth to propofol was assessed using murine clonogenic hematopoietic progenitor cells. METHODS: Femoral and tibial marrow cells were obtained from 4- to 6-week-old male BALB/c mice. Propofol was diluted in culture medium (30microM, 300microM and 1 mM) and added into methylcellulose semi-solid culture media. After 14 days of culturing, the numbers of colony-forming unit granulocyte/monocyte (CFU-GM) colonies were counted. An advance liquid culture (RPMI 1640) of 5 hours duration was also applied prior to culturing in semisolid media to assess the short term exposure toxicity. RESULTS: The colony counts were significantly decreased compared to the control at higher concentrations than 1 mM (P<0.05). The pre-exposure to propofol did not affect the number CFU-GM colony count at the concentrations of 30microM and 300microM or under conditions of co-culture. CONCLUSIONS: No myelodepressive effect was observed in mouse bone marrow cells with exposure of propofol at concentrations under 300microM.


Subject(s)
Anesthesia , Anesthetics , Animals , Bone Marrow Cells , Bone Marrow , Coculture Techniques , Culture Media , Granulocyte-Macrophage Progenitor Cells , Hematopoiesis , Hematopoietic Stem Cells , Humans , Male , Methylcellulose , Mice , Nitrous Oxide , Propofol , Stem Cells , Tissue Donors
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