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

PURPOSE: Many studies for hematopoietic stem cell have investigated CD133, instead of CD34, as a new surrogate stem cell marker. Counterflow centrifugal elutriation (CCE) is a physical separation of a homogeneous cell population through cell sedimentation characteristics. We evaluated the stem cell distribution and hematopoietic function from cord blood (CB) and bone marrow (BM) through CCE. METHODS: We obtained total nucleated cells from CB and BM, and separated the cell fractions according to media infusion flow rates (17 mL/min (FR 17), 24 mL/min (FR 24), 29 mL/min (FR 29), and rotor off (R/O) ) by CCE. We analyzed the proportion of CD34+ and CD133+ cells in each fraction, and performed methylcellulose-based colony assay. RESULTS: In CB, the cell recovery rates after CCE were 5.9+/-4.3% in FR 17, 4.2+/-2.1% in FR 24, 19.4+/-11.9% in FR 29, and 61.9+/-11.7% in R/O. In BM, they were 14.9+/-8.2% in FR 17, 17.4+/-13.4% in FR 24, 23.6+/-6.11% in FR 29, and 27.1+/-8.9% in R/O. The distributions of CD133+ and CD34+ cells in CB were more abundant in R/O (2.91%, 1.85%) than in other fractions. In BM, CD133+ and CD34+ cell rates in R/O (5.40%, 2.75%) were similar with those in unmanipulated BM (5.48%, 2.78%). In both CB and BM, there was more CFU-GM and BFU-E in R/O than in other fractions. CONCLUSION: We suggested that the distribution of CD34+ and CD133+ cells might be different between CB and BM. However, the R/O containing relatively large cells could have an effective clonogenicity compared with the unmanipulated sample in both CB and BM.
Bone Marrow* ; Erythroid Precursor Cells ; Fetal Blood* ; Granulocyte-Macrophage Progenitor Cells ; Hematopoietic Stem Cells ; Stem Cells

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

PURPOSE: Umbilical cord blood is increasingly being used in the setting of allogeneic marrow transplantation. However, while neutrophil engraftment is comparable to that of marrow transplants, delayed platelet engraftment is often a concern for cord blood transplant recipients. This delay may be due to relative weakness of the megakaryocyte lineage in cord blood. We evaluated the potential of ex vivo expansion and clonality from different stem cell sources. METHODS: The CD34 cells from bone marrow (BM), umbilical cord blood (CB), and mobilized peripheral blood (PB) were cultured for burst-forming unit of erythrocyte (BFU-E), colony-forming unit of granulocyte and monocyte (CFU- GM) and colony-forming unit of megakaryocyte (CFU-MK) at day 0, day 4, day 7, and day 14 under the combination of growth factors, with cell counts. Cytokines included recombinant human megakaryocyte growth and development factors (100 ng/mL), interleukin-3 (10 ng/mL), stem cell factor (100 ng/mL), and flt-3 ligand (50 ng/mL). RESULTS: CB-derived CD34 cells had significantly higher total cell proliferation than either BM or PB at day 7 (1.6 to 18.2 fold) and day 14 (1.2 to 17.2 fold). The colony count of BFU-E was in general more plentiful in CB than in BM and PB at day 4, day 7 and day 14, among which the difference was the most distinct at day 7 culture. Also, CB CD34 cells produced more CFU-Mk colonies than did BM or PB at day 4 and day 7. There were no differences in colonies count of BFU-E and CFU-Mk between BM and PB. CONCLUSION: Ex vivo expansion of CB cells may be most promising in producing total cellular expansion, CFU-Mk and BFU-E compared with BM and PB, especially at day 7, because the former was the most productive hematopoietic source on a per volume basis.
Blood Platelets ; Bone Marrow* ; Cell Count ; Cell Proliferation ; Culture Media, Serum-Free* ; Cytokines ; Erythrocytes ; Erythroid Precursor Cells ; Fetal Blood* ; Granulocytes ; Hematopoietic Stem Cells ; Humans ; Intercellular Signaling Peptides and Proteins ; Interleukin-3 ; Megakaryocytes ; Monocytes ; Neutrophils ; Stem Cell Factor ; Stem Cells ; Thrombopoietin ; Transplantation ; Umbilical Cord*

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

PURPOSE: Because of the unavailability of marrow transplantation, umbilical cord blood (CB) is increasingly being used. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from cord blood source and mobilized peripheral blood (PB) in a serum-free media. METHODS: The CD34+ cells, selected from CB and mobilized PB, were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at culture days 0, day 4, day 7, and day 14 with various growth factors. RESULTS: The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the PB at day 7 (2 fold increase than PB). The CB-selected CD34+ cells produced more BFU-E colonies than did the PB on culture at days 7 and at day 14. Also, the CB-selected CD34+ cells produced more CFU-Mk colonies than did the PB on culture at day 4 and at day 7. CONCLUSION: The ex vivo expansion of the CB cells may be promising in producing total cellular expansion, CFU-Mk and BFU-E compared with PB for 7 to 14 days. The growth factors combination including megakaryocyte growth and development, flt3-ligand and interleukin-3 showed more expansion in the view of total cells and clonal maintenance compared with less combination.
Bone Marrow ; Culture Media, Serum-Free ; Erythrocytes ; Erythroid Precursor Cells ; Fetal Blood* ; Granulocytes ; Growth and Development ; Intercellular Signaling Peptides and Proteins ; Interleukin-3 ; Megakaryocytes ; Monocytes ; 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

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

Hematopoiesis is the process that generates blood cells in an organism from the pluripotent stem cells. Hematopoietic stem cells are characterized by their ability to undergo self-renewal and differentiation. The self-renewing ability ensures that these pluripotent cells are not depleted from the bone marrow niche. A proper balance between cell death and cell survival is necessary to maintain a homeostatic condition, hence, apoptosis, or programmed cell death, is an essential step in hematopoiesis. Recent studies, however, have introduced a new aspect to this process, citing the significance of the apoptosis mediator, caspase, in cell development and differentiation. Extensive research has been carried out to study the possible role of caspases and other apoptosis related factors in the developmental processes. This review focuses on the various apoptotic factors involved in the development and differentiation of myeloid lineage cells: erythrocytes, megakaryocytes, and macrophages.
Apoptosis* ; Blood Cells ; Bone Marrow ; Caspases ; Cell Death ; Cell Survival ; Erythrocytes ; Hematopoiesis ; Hematopoietic Stem Cells ; Macrophages ; Megakaryocytes ; Monocytes ; Myeloid Cells* ; Pluripotent Stem Cells

Hematopoiesis involves a series of lineage differentiation programs initiated in hematopoietic stem cells (HSCs) found in bone marrow (BM). To ensure lifelong hematopoiesis, various molecular mechanisms are needed to maintain the HSC pool. CCCTC-binding factor (CTCF) is a DNA-binding, zinc-finger protein that regulates the expression of its target gene by organizing higher order chromatin structures. Currently, the role of CTCF in controlling HSC homeostasis is unknown. Using a tamoxifen-inducible CTCF conditional knockout mouse system, we aimed to determine whether CTCF regulates the homeostatic maintenance of HSCs. In adult mice, acute systemic CTCF ablation led to severe BM failure and the rapid shrinkage of multiple c-Kit(hi) progenitor populations, including Sca-1⁺ HSCs. Similarly, hematopoietic system-confined CTCF depletion caused an acute loss of HSCs and highly increased mortality. Mixed BM chimeras reconstituted with supporting BM demonstrated that CTCF deficiency-mediated HSC depletion has both cell-extrinsic and cell-intrinsic effects. Although c-Kit(hi) myeloid progenitor cell populations were severely reduced after ablating Ctcf, c-Kit(int) common lymphoid progenitors and their progenies were less affected by the lack of CTCF. Whole-transcriptome microarray and cell cycle analyses indicated that CTCF deficiency results in the enhanced expression of the cell cycle-promoting program, and that CTCF-depleted HSCs express higher levels of reactive oxygen species (ROS). Importantly, in vivo treatment with an antioxidant partially rescued c-Kit(hi) cell populations and their quiescence. Altogether, our results suggest that CTCF is indispensable for maintaining adult HSC pools, likely by regulating ROS-dependent HSC quiescence.
Adult ; Animals ; Bone Marrow ; Cell Cycle ; Chimera ; Chromatin ; Fibrinogen* ; Hematopoiesis ; Hematopoietic Stem Cells* ; Homeostasis ; Humans ; Lymphoid Progenitor Cells ; Mice* ; Mice, Knockout ; Mortality ; Myeloid Progenitor Cells ; Reactive Oxygen Species