The Expression of Fas antigen and Bax and Apoptosis in Ex Vivo Expanded Hematopoietic Progenitor Cells / 대한혈액학회지

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.

In Vitro Effect of Interleukin-11 (IL-11) on Megakaryopoiesis from Umbilical Cord Blood Cells

BACKGROUND: The megakaryopoiesis and platelet production is regulated by several hematopoietc factors such as thrombopoietin (TPO), interleukin-11 (IL-11) and interleukin-3 (IL-3). IL-11 is a potent stimulator of megakaryopoiesis in vivo, and acts primarily as a megakaryocyte maturation factor in vitro and it can act synergistically with IL-3 and TPO. We performed this study to investigate the effects of recombinant human IL-11 (rhIL-11) with other hematopoietic factors on megakaryocyte colony formation in vitro. METHODS: CD34+ cells were separated from umbilical cord blood and megakaryocyte colonies using MegaCult Assay Kit were cultured with rhIL-11, recombinant human IL-3 (rhIL-3), and recombinant human TPO (rhTPO) for 7 and 14 days. The number and percentage of CD34+ and CD41a+ cells were determined by flowcytometry. RESULTS: The number of CD41a+ cells were 0.54+/-0.05x10(4) (rhIL-11 100 ng/ml), 5.32+.-0.23x10(4) (rhIL-3 100 ng/ml), and 8.76+/-0.15x10(4) (rhTPO 50 ng/ml) of total expanded cells during the culture of the purified CD34+ cells in liquid phase for 7 days. The number of CD41a+ cells were increased to 7.47+/-0.69x10(4) (rhIL-3 rhIL-11), 11.92+/-0.19x10(4) (rhTPO rhIL-11) of total expanded cells, respectively, during the culture of the purified CD34+ cells in liquid phase for 7 days in the presence of rhIL-11 (100 ng/ml). When the purified CD34+ cells were cultured in semisolid media including various concentration of rhIL-11, the megakaryocyte colonies were not formed. When the purified CD34+ cells were cultured with rhIL-11 and rhTPO or with rhIL-11 and rhIL-3, the number of megakaryocyte colonies were increased compared with rhTPO or rhIL-3 alone. CONCLUSION: These results indicate that IL-11 exerts a potent proliferative activity to colony forming unit-megakaryocyte from human umbilical cord blood, and it acts with other hematopoietic factors synergistically

Atypical Hairy Cell Leukemia / 대한혈액학회지

We experienced a case of atypical hairy cell leukemia in a 42-year-old woman. She showed marked splenomegaly without palpable lymphadenopathy. Complete blood cell count revealed leukocytosis at 44,000/micro L with lymphocytes 74% and peripheral blood smear showed abnormal lymphoid cells with cytoplasmic projections. The bone marrow was easily aspirated and also revealed the abnormal lymphocytes in up to 95%. Tartrate-resistant acid phosphatase reactivity was negative in the hairy cells. Immunophenotyping results of lymphoid cells were CD5(-), CD7(-), CD10(-), CD19(+), and HLA-DR(+). She was treated with an adenosine analogue, fludarabine at a daily dose of 30mg/m2 for 5 consecutive days, every four weeks. Immediately after treatment, the size of the spleen was normalized. Correct diagnosis was difficult due to insufficient laboratory and pathologic data. The differential diagnosis of mature B-cell neoplasms with cytoplasmic projections in patients with splenomegaly includes hairy cell leukemia and splenic lymphoma with villous lymphocytes. We herein described the present case with a brief review of the literature.

Pathophysiologic Study of Aplastic Anemia by Long-term Bone Marrow Cultures / 대한혈액학회지

BACKGROUND: Several mechanisms have been proposed to account for bone marrow failure in aplastic anemia (AA), including deficiency in hematopoietic stem cells, a secondary stem cell defect involving immune regulation and defective marrow stroma, or microenvironment. We investigated the pathophysiology of AA through long-term bone marrow cultures (LTBMCs) using bone marrrow of AA patients before treatment and of patients responded to immunosuppressive therapy with anti-thymocyte globulin (ATG) and/or cyclosporine. METHODS: We investigated the hematopoietic defect in severe aplastic anemia (SAA) patients by using long-term bone marrow cultures (LTBMCs). Twenty patients with SAA have been studied. In these patients, 10 had been treated with ATG plus cyclosporine and the remainders were studied before therapy was begun. Subsequent assays of the production of negative-acting hematopoietic cytokines (TNF-alpha, IFN-gamma, MIP-1alpha and TGF-beta) by AA stroma in LTBMCs were performed. RESULTS: Initial assessment of CD34+ cells, CFU-GM and CFU-MK from LTBMCs in AA demonstrated severely reduced or absent in patients with SAA, even following hematologic recovery with immunosuppressive therapy,when compared with normal controls. Significant difference in concentrations of TNF-alpha, INF-gamma, and MIP-1alpha between the AA and control groups were apparent. Interestingly, the levels of those negative-acting hematopoietic cytokines were decreased in SAA patients receiving immunosuppressive therapy, but not the levels of controls. However, the mean TGF-beta concentrations in the AA patients and normal controls were not significantly different. The percent of CD34+ cells and CFU-MK in bone marrow was lower in SAA patients before immunosuppressive therapy was begun than that in SAA patients receiving immunosuppressive therapy and that in normal controls (mean 0.54+/-0.32% vs 0.96+/-0.32% vs 1.94+/-0.61%). CONCLUSIONS: These results indicate the presence of a in vitro functional deficiency in the hematopoietic system of patients with AA, including those that achieved partial or complete remission after immunosuppressive treatment, and add to the available evidence for defect of microenvironment with hematopoeitic stem cell in some cases of AA.