The effects of the Smad3- knockout on the hematopoiesis of mouse were investigated in this work. Five pairs of wild type and Smad3- null mice were studied. White blood cell(WBC), red blood cell(RBC) and platelet (PLT) counting of peripheral blood cells were performed with blood obtained from tails. And white blood cells were classified by their morphology. Bone marrow nucleated cells (BMNCs) were counted and classified. The CFU-GM, BFU-E, CFU-GEMM yields were measured in each pair of mice. CFU-S yield of each mouse was measured by injecting bone marrow cells into lethally irradiated 8-10 weeks old wild type female mice. And the pathomorphism of their bone marrows, spleens and livers were observed. As a result, WBC and PLT of Smad3- null mice were significantly higher than those in wild type mice. Smad3- null mice had much more proportion of granulocytes in classification. There wasn't any difference in RBC counting and BFU-E measurement. The yield of CFU-GM increased, while the yields of CFU-GEMM and CFU-S markedly reduced. Bone marrows are actively proliferative, with granulocytosis. The granulocyte/erythrocyte ratio increased. There were no obviously alterative in spleen and liver. Thus Smad3- knockout results in a decreased number of stem and progenitor cells. Moreover hematopoietic differentiation is abnormal with a tendency to forming more granulocytes and platelets. The effect of Smad3 on hematopoiesis is correlative to that of TGF-beta.
Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Erythrocytes ; cytology ; metabolism ; Erythroid Precursor Cells ; cytology ; metabolism ; Female ; Granulocyte-Macrophage Progenitor Cells ; cytology ; metabolism ; Granulocytes ; cytology ; metabolism ; Hematopoiesis ; genetics ; Mice ; Mice, Knockout ; Myeloid Progenitor Cells ; cytology ; metabolism ; Smad3 Protein ; genetics

BACKGROUND: Because there are lower incidence of graft versus host disease in HLA mismatched cord blood transplantation compared to bone marrow transplantation, development of smaller scale cord blood bank could be possible. So we analysed the content of hematopoietic stem cell in cord blood and the distribution of HLA as a basic study for cord blood bank. METHODS: Seventy eight cord bloods were collected in heparinized bottle immediately after caesarian section. After expulsion of placenta, additional cord blood and placental blood were collected with heparinized syringe. Fifteen mL was sent to the laboratory for analysis and the rest was cryopreserved. RESULTS: The mean collected cord blood volume was 96.8mL (range, 55~163mL). And mean 81.8mL (range, 40~148mL) was cryopreserved. It contained mean 7.4x108 (range, 2.8x108~12.2x108) nucleated cells. In 2x105 mononuclear cells, 85 +/- 48 BFU-E, 19 +/- 17 CFU-E, 107 +/- 73 CFU-GM and 124 +/- 113 CFU-GEMM were present. With dextran/albumin thawing media, the viability of cryopreserved cord blood mononuclear cell was better than usual washing method with IMDM (82.3% vs. 74.6% P=0.004). Each cord blood could findHLA full matched, 5 loci matched and 4 loci matched cord blood in the remainders with the probability of 0, 11.9% and 58.4%. CONCLUSION: Development of more smaller scale cord blood bank could be possible compared to bone marrow bank.
Bone Marrow ; Bone Marrow Transplantation ; Erythroid Precursor Cells ; Fetal Blood* ; Graft vs Host Disease ; Granulocyte-Macrophage Progenitor Cells ; Hematopoietic Stem Cells* ; Heparin ; Humans* ; Incidence ; Myeloid Progenitor Cells ; Placenta ; Syringes

BACKGROUND: The possibility of cord blood transplantation in adults was limited by the amount of cord blood that could be collected. Cord blood transplantation after ex vivo expansion with cytokines have already been tried in adults. Amifostine is a phosphorylated aminothiol that affords broad cytoprotection from the myelosuppressive effects of antineoplastic agents. The purposes of this study were to investigate expansion of progenitor and myeloid cells after ex vivo culture of mononuclear cells (MNCs) in umbilical cord blood with growth factor and characterize hematopoietic activities of amifostine. METHODS: MNCs were cultured and ex vivo expanded into myeloid progenitors by using hematopoietic growth factors (IL-1beta, IL-3, IL-6, G-CSF, GM-CSF, SCF, EPO) which are known to stimulate differentiation and proliferation of myeloid progenitors. MNCs exposed to the appropriate amount of amifostine for 15 min were cultured in semisolid media and harvested at 24h intervals, and then apoptosis was assessed by propidium iodide staining. RESULTS: Myeloid colonies were successfully produced from MNCs. Maximal expansion was obtained with the combination of IL-3+SCF+G-CSF+GM-CSF. SCF was thought to be the most important growth factor for expansion of myeloid progenitor. Pretreatment with amifostine for 15 min stimulated formation of hematopoietic colonies at clinically relevant concentrations ranging from 1 to 100 micrometer. Increase in colony number compare to control were comparable after pretreatment with amifostine (10micrometer), and CFU-GEMM and BFU-E were highly responsive. Further enhancement of colony was not observed after prolonging the duration of pre- incubation exposure to 1, 8 and 24 hours. Amifostine enhanced IL-1 and IL-3 induced formation of CFU-GEMM and BFU-E. Incubation of MNCs with amifostine in suspension culture increased recovery of secondary colonies. Treatment with amifostine retarded cell loss and apoptosis, and promoted cell survival at 24, 48 and 72 hours in cytokine-deficient medium. CONCLUSION: Cord blood MNCs can be successfully expanded into myeloid progenitors by using hematopoietic growth factors. This investigation extend the previously recognized hematologic effects of amifostine, and indicate that in addition to its cytoprotective properties, amifostine is a stimulant of hematopoietic progenitor growth.
Adult ; Amifostine* ; Antineoplastic Agents ; Apoptosis ; Cell Survival ; Cytokines ; Cytoprotection ; Erythroid Precursor Cells ; Fetal Blood ; Granulocyte Colony-Stimulating Factor ; Granulocyte-Macrophage Colony-Stimulating Factor ; Humans ; Intercellular Signaling Peptides and Proteins ; Interleukin-1 ; Interleukin-3 ; Interleukin-6 ; Myeloid Cells ; Myeloid Progenitor Cells ; Propidium

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

BACKGROUND: During ex vivo expansion of cord blood (CB) CD34+ cells, differentiation of the expanded cells happened and hematopoietic potential of the progenitor cells decreased. In this study, we evaluate the effect of the expression of Fas antigen, Bcl-2, and Bax on CD34+ or AC133+ hematopoietic progenitor cells during ex vivo expansion. METHODS: CD34+ and AC133+ cells isolated from human CB were cultured in serum free medium supplemented with several cytokines for 7 days. After expansion culture, we re isolated CD34+ and AC133+ cells and compared the numbers of granulocyte-macrophage colony-forming units (CFU-GM) and granulocyte, erythrocyte, monocyte, and macrophage colony-forming units (CFU-GEMM), and expression of Fas antigen, Bcl-2, and Bax with unexpanded cells. RESULTS: CFU-GM was expanded 23.94 fold in CD34+ cells and 15.22 fold in AC133+ cells at day 7 of culture but CFU-GEMM was not expanded. The expression of Fas antigen and Bax was 7.44% and 2.75%, respectively, in fresh isolated CD34+ cells and increased to 19.71 % and 33.67%, respectively, in expanded CD34+ cells at day 7 culture, but Bcl-2 was not changed. In case of AC133+ cells, the expression of Fas antigen and Bax were also increased from 5.87% and 6.19% to 24.85% and 22.83%, respectively, and Bcl-2 was slightly decreased. Apoptosis was not changed in CD34+ cells and AC133+ cells during ex vivo expansion. CONCLUSION: These results indicate that the nature of expansion was similar between CD34+ and AC133+ cells, and expression of Fas antigen and Bax increased on CD34+ and AC133+ cells during ex vivo expansion. Selection of the expanded progenitor cells without apoptosis may be useful for the hematopoietic stem cell transplantation.
Antigens, CD95* ; Apoptosis* ; Cytokines ; Erythrocytes ; Fetal Blood ; Granulocyte-Macrophage Progenitor Cells ; Granulocytes ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells* ; Humans ; Macrophages ; Monocytes ; Myeloid Progenitor Cells ; Stem Cells

BACKGROUND: Classically, bone marrow (BM) has been the sole source of hematopoietic stem cell transplantation, but limitations of conventional bone marrow transplantation have stimulated a search for alternative sources of stem cells. METHODS: We compared hematopoietic stem cell activity of normal bone marrow (BM), in vivo G-CSF-stimulated bone marrow (G-CSF BM), and G-CSF-mobilized peripheral blood (G-CSF PB) by immunophenotyping, clonogeneicity, and long-term culture-initiating cell (LTC-IC) analysis. RESLUTS: The average numbers of CD34+/HLA-DR- cells after CD34+ cells isolation from each stem cell source were 59.64 +/- 8.70%, 91.39 +/- 1.98%, and 95.75 +/- 2.08% in normal BM, G-CSF BM, and G-CSF PB, respectively (normal BM vs. G-CSF BM, normal BM vs. G-CSF PB, p<0.0001). And the average numbers of CD34+/CD38- cells were 66.23 +/- 9.33%, 95.08+/- 2.09%, and 91.76 +/- 4.59% in normal BM, G-CSF BM, and G-CSF PB, respectively (normal BM vs. G-CSF BM, normal BM vs. G-CSF PB, p<0.0001). The numbers of CFU-GM was significantly higher in G-CSF PB (53.2 +/- 4.05) and G-CSF BM (52.5 +/- 3.63) than that of normal BM (31.3+/- 5.50) (p<0.0001). Also the numbers of CFU-GEMM and CFU-Mk were also significantly higher in G-CSF PB (110.3 +/- 8.79 and 13.3 +/- 1.49) and G-CSF BM (109.7 +/- 10.78 and 11.2 +/- 1.69) than that of normal BM (48.8 +/- 1.48 and 8.5 +/- 1.72) (p<0.05). Comparison of LTC-IC in the three sources of stem cells showed that G-CSF PB and G-CSF BM were superior to normal BM at five weeks of culture (p<0.05). CONCLUSIONS: These data suggest that the amount of both early progenitor cells and late progenitor cells in G-CSF PB and G-CSF BM are higher than that of normal BM. And our results further support that the higher stem cell transplantation using G-CSF-mobilized PB and in vivo G-CSF-stimulated BM can lead to more rapid and sustained engraftment even in cases of high risk of rejection.
Bone Marrow Transplantation ; Bone Marrow* ; Granulocyte Colony-Stimulating Factor* ; Granulocyte-Macrophage Progenitor Cells ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells* ; Immunophenotyping ; Myeloid Progenitor Cells ; Stem Cell Transplantation ; Stem Cells

PURPOSE: Umbilical cord blood transplantation is a alternative method as new hematopoietic stem cell transplantation and has been performed clinically in indicated disease. However, it have the problems for long-term storage of cord blood in liquid nitrogen and for limited application to adult due to small amount of hematopoietic stem cell. Therefore, several centers have carried out active research for ex vivo expansion of cord blood stem cell. We investigated the hematopoietic function of cord blood plasma for development of new techniques. METHODS: We acquired the nucleated cells of cord blood from healthy infant and bone marrow from healthy donor received granulocyte-colony stimulating factor. We evaluated hematopoietic colony formation according to source of stem cell and plasma by semisolid culture medium. Three experimental groups were divided as source of plasma: group for cord plasma, group for bone marrow plasma, group for mixture of cord plasma and bone marrow plasma. RESULTS: The results were as follows: 1) The colony formation according to source of stem cell in commercialized standard semisolid culture medium showed that cord blood in the number of CFU-GM was less than bone marrow, but not significantly different in CFU-GEMM. 2) The colony formation according to source of stem cell in semisolid culture medium using experimental plasma showed that cord blood in the number of CFU-GM was more than bone marrow. There were no cytotoxic effect of plasma to experimental cells. 3) The colony formation in semisolid culture medium contained plasma according to experimental group showed that the number of CFU-GM in cord blood plasma was significantly more than bone marrow plasma in spite of different source of stem cell. Conclusions: These results suggested that cord blood might contain enough hematopoiesis to enable to perform transplantation compared with bone marrow and, also, cord blood plasma might be contributed more effective colony formation than bone marrow plasma. Therefore, we propose that it may be good to store cord blood cells with cord blood plasma in long-term storage. We will investigate the composition of hematopoietic growth factors and cytokines in cord blood plasma and the effect of cord blood plasma for ex vivo expansion of cord blood cells.
Adult ; Bone Marrow ; Bone Marrow Cells ; Cytokines ; Fetal Blood* ; Granulocyte-Macrophage Progenitor Cells ; Hematopoiesis ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Humans ; Infant ; Intercellular Signaling Peptides and Proteins ; Myeloid Progenitor Cells ; Nitrogen ; Plasma* ; Stem Cells ; Tissue Donors

Granulocyte-Macrophage colony-stimulating factor(GM- CSF) is naturally generated protein that stimulates the survival, proliferation, and differentiation of myeloid progenitor cells. The determination of the molecular sequence of this protein by recombinant DNA technology enabled us to produce sufficient quantity for preclinical and clinical use. In the animal studies, rhGM-CSF to wounds has been reported to result in increased formation of granulation tissue, increased breaking strength, and reversal of wound contraction. A number of case reports have been published on the favorable effect of rhGM-CSF as a treatment for infected, nonhealing wounds, and ulcers. However, there are no clinical reports about the effect of GM-CSF on wound healing in normal patients. Therefore, in this report, we examined the effect of GM-CSF on the proliferation of human dermal fibroblasts which play a crucial role in wound healing process in vitro. To determine an optimal GM-CSF concentration for human dermal fibroblast proliferation, the cells were incubated with either one of 13 concentrations of GM-CSF(0 - 30mug/ml). The media used in this study was DMEM/F- 12(GIBCO, Grand Island, NY, USA). The fibroblasts were seeded at 1.5 x 104 cells/well in 500mul of medium including 10% fetal bovine serum and either one of 13 concentrations of GM-CSF in 24-well plates. The cells were incubated for 6 days at 5% CO2, 100% humidity at 37degrees C. On the 6th day of plating, fibroblast proliferation was determined by hematocytometer. Each concentration was tested 8 times.Low concentration of GM-CSF(below 5.0mug/ml) stimulated the proliferation of human dermal fibroblasts. How ever, high concentration of GM-CSF(over 10mug/ml) downregulated the proliferation of human dermal fibroblasts. The best fibroblast proliferation was seen at 1.0mug/ml of GM- CSF. These results demonstrated that GM-CSF influenced human dermal fibroblast proliferation and the GM-CSF concentration was critically important factor in vitro.
Animals ; DNA, Recombinant ; Fibroblasts* ; Granulation Tissue ; Granulocyte-Macrophage Colony-Stimulating Factor* ; Humans* ; Humidity ; Myeloid Progenitor Cells ; Ulcer ; Wound Healing ; Wounds and Injuries

Granulocyte-Macrophage colony-stimulating factor(GM- CSF) is naturally generated protein that stimulates the survival, proliferation, and differentiation of myeloid progenitor cells. The determination of the molecular sequence of this protein by recombinant DNA technology enabled us to produce sufficient quantity for preclinical and clinical use. In the animal studies, rhGM-CSF to wounds has been reported to result in increased formation of granulation tissue, increased breaking strength, and reversal of wound contraction. A number of case reports have been published on the favorable effect of rhGM-CSF as a treatment for infected, nonhealing wounds, and ulcers. However, there are no clinical reports about the effect of GM-CSF on wound healing in normal patients. Therefore, in this report, we examined the effect of GM-CSF on the proliferation of human dermal fibroblasts which play a crucial role in wound healing process in vitro. To determine an optimal GM-CSF concentration for human dermal fibroblast proliferation, the cells were incubated with either one of 13 concentrations of GM-CSF(0 - 30mug/ml). The media used in this study was DMEM/F- 12(GIBCO, Grand Island, NY, USA). The fibroblasts were seeded at 1.5 x 104 cells/well in 500mul of medium including 10% fetal bovine serum and either one of 13 concentrations of GM-CSF in 24-well plates. The cells were incubated for 6 days at 5% CO2, 100% humidity at 37degrees C. On the 6th day of plating, fibroblast proliferation was determined by hematocytometer. Each concentration was tested 8 times.Low concentration of GM-CSF(below 5.0mug/ml) stimulated the proliferation of human dermal fibroblasts. How ever, high concentration of GM-CSF(over 10mug/ml) downregulated the proliferation of human dermal fibroblasts. The best fibroblast proliferation was seen at 1.0mug/ml of GM- CSF. These results demonstrated that GM-CSF influenced human dermal fibroblast proliferation and the GM-CSF concentration was critically important factor in vitro.
Animals ; DNA, Recombinant ; Fibroblasts* ; Granulation Tissue ; Granulocyte-Macrophage Colony-Stimulating Factor* ; Humans* ; Humidity ; Myeloid Progenitor Cells ; Ulcer ; Wound Healing ; Wounds and Injuries

BACKGROUND: Post remission therapy is one of the most important issues in the treatment of acute myelocytic leukemia (AML). Recently, autologous peripheral blood stem cell transplantation (PBSCT) has become an accepted procedure to support high dose chemotherapy in children with AML. But collection of PBSC from small pediatric patients provides many challenges not faced when collecting from adult patients. Therefore, the efficient procedures and optimal timing to perform the leukapheresis should be decided. The goal of the present study was to evaluate the practice of PBSC mobilization and collection and establish predictors of the leukapheresis in children with AML. METHODS: From November 1995 to February 1998, PBSC mobilizations were performed in 15 patients with AML. PBSCs were mobilized by high dose of cytosine arabinoside and etoposide plus G-CSF. CBC and peripheral blood smear were performed daily after WBC nadir. Leukapheresis was started when the WBC count recovered to 1,000/microliter from myelosuppression and monocytes appeared on the peripheral blood smear. Leukapheretic products were assayed for mononuclear cells, CD34+ cells and CFU-GM colonies. Correlations between the yields of leukapheresis and patients characteristics were evaluated by Wilcoxon rank sums test and Pearson correlation analysis. RESULTS: Eighteen mobilizations were done in 15 patients. The duration of absolute neutrophil count<0.5x103/microliter and platelet count<20x103/microliter were 6 days (0~10 days) and 8 days (5~21 days) after mobilization chemotherapy, respectively. Duration of fever was 1 day, but documented septicemia was not occurred in any of the patients. A median 5 leukaphereses (range : 3~6) were undergone per patient. The WBC on the first day of the leukapheresis was 1,640/microliter (850~16,840/microliter) and percentage of monocyte on the first day of the leukapheresis was 12% (4~36%). A median 5 leukaphereses yielded median of 11.02x108 (4.5~26.42x108) MNCs/kg, 7.63x106 (0.33~42.21x106) CD34+ cells/kg, and 8.46x104 (0.27~147.83x104) CFU-GM/ kg. The dose of 1x108 MNCs was harvested in 100% after 3 harvests and 1x106 CD34+ cells in 87% after 3 harvests. No serious adverse effects occurred in all patients during the leukapheresis procedures. A rapid rise in WBC count (> or = 3,000/microliter/day) during recovery was independent variable correlated to the peak MNCs, average MNCs, peak CD34+ cells and average CD34+ cells (P<0.01). CONCLUSIONS: Mobilization procedures using high dose cytosine arabinoside and etoposide plus G-CSF are tolerable and the leukapheresis can be initiated when WBC count recovers to 1,000/microliter from myelosuppression and monocytes appear on the peripheral blood smear. Sufficient numbers of PBSC can be obtained by three leukapheresis procedures without serious adverse effects in children with AML.
Adult ; Blood Platelets ; Child* ; Cytarabine ; Drug Therapy ; Etoposide ; Fever ; Granulocyte Colony-Stimulating Factor ; Granulocyte-Macrophage Progenitor Cells ; Humans ; Leukapheresis* ; Leukemia, Myeloid, Acute* ; Monocytes ; Neutrophils ; Peripheral Blood Stem Cell Transplantation ; Sepsis ; Stem Cells*