Takehisa Kikuta

Mobilization of hematopoietic primitive and committed progenitor cells into blood in mice by anti-vascular adhesion molecule-1 antibody alone or in combination with granulocyte colony-stimulating factor

OBJECTIVE One of the mechanisms for mobilization of hematopoietic stem cells and progenitor cells is alternation of adhesion molecules. We investigated the mobilization of hematopoietic progenitor cells in blood by administration of anti-vascular cell adhesion molecule (VCAM)-1 antibody (Ab) in mice. MATERIALS AND METHODS Twelve- to 14-week old C57BL/6J mice were injected intravenously with anti-VCAM-1 Ab and anti-very late antigen (VLA)-4 Ab at a dose of 5 mg/kg for 2 days. RESULTS The number of colony-forming cells (CFCs) in blood was increased 11.4-fold after anti-VCAM-1 Ab treatment, but the number of CFCs was not increased after treatment with anti-VLA-4 Ab. The number of colony-forming unit spleen (CFU-S) also was increased 21.6-fold in the peripheral blood by administration of anti-VCAM-1 Ab. The number of CFCs and CFU-S in the bone marrow of mice treated with anti-VCAM-1 Ab was decreased and that in the spleen also was decreased. On administration of recombinant human granulocyte colony-stimulating factor (125 microg/kg twice daily) with anti-VCAM-1 Ab, the numbers of CFCs and CFU-S were increased 141.8-fold and 439-fold, respectively. CONCLUSIONS These observations demonstrated that administration of anti-VCAM-1 Ab induced mobilization of hematopoietic progenitor cells into blood from bone marrow and spleen and that granulocyte colony-stimulating factor has synergistic effects on anti-VCAM-1 Ab-induced mobilization.

CD34 + /CD41a + cells best predict platelet recovery after autologous peripheral blood stem cell transplantation

Reliable markers for megakaryocytic reconstitution after peripheral blood stem cell transplantation (PBSCT) have not been established. To determine a convenient and reliable predictor, we measured the number of megakaryocyte progenitor cells in PBSC grafts by clonogenic and flow cytometric assays. Seventeen patients with hematological and solid malignancies were included in this study. For the clonogenic assay, we used thrombopoietin (TPO) as a growth factor to evaluate the maximum number of megakaryocyte progenitor cells. Using a flow cytometric assay, we examined the expression of platelet glycoproteins on CD34+ cells to count the number of megakaryocyte progenitor cells. We used buffer containing EDTA to prevent platelet adhesion to CD34+ cells and selected CD34+ cells by immunomagnetic beads. The best correlation was observed between the number of CD34+/CD41a+ cells and the time to platelet recovery (P = 0.0205), rather than the total number of CD34+ cells. In addition, a close correlation was observed between the number of CD34+/CD41a+ cells and colony-forming unit megakaryocyte (CFU-MK) (P = 0.0018). These observations suggest that the number of CD34+/CD41a+ cells is the best predictor for platelet reconstitution after PBSCT.

Effects of thrombopoietin (c-mpl ligand) on growth of blast cells from patients with transient abnormal myelopoiesis and acute myeloblastic leukemia

Abstract: Thrombopoietin (TPO) is a ligand for c‐mpl that promotes both proliferation and differentiation of megakaryocytes in vivo and in vitro. We investigated the expression of c‐mpl transcripts and the effects of recombinant human TPO (rhTPO) on the proliferation and differentiation of human leukemic cell lines or fresh samples obtained from 32 patients with transient abnormal myelopoiesis (TAM) or acute myeloblastic leukemia (AML). Cells were cultured with TPO alone or combined with rh interleukin‐3 (IL‐3) or stem cell factor (SCF). Expression of c‐mpl was verified in 6 of 13 cases tested. All but one of the cases that showed c‐mpl expression responded to TPO. Blasts from all cases of TAM or French–American–British (FAB) subtype M7 showed growth responses to TPO with higher sensitivity than cells of other FAB subtypes and these responses were increased by addition of rhIL‐3 or rhSCF in some cases. Responses of cells of other FAB subtypes varied. In addition, increased expression of platelet‐specific surface antigens on MO7E cells after incubation with rhTPO was observed. These data suggest that TPO may be involved in the abnormal proliferation and differentiation of human leukemic cells, especially of M7 and TAM cells, considered to be of megakaryocytic lineage.

Serum thrombopoietin levels in patients undergoing autologous peripheral blood stem cell transplantation

Recently, the ligand for c-mpl has been cloned and initial studies have shown it to be the platelet regulatory factor, thrombopoietin (TPO). To elucidate the role of TPO in the reconstitution of megakaryopoiesis and platelet production after stem cell transplantation, we measured serum TPO levels in nine patients undergoing autologous peripheral blood stem cell transplantation (PBSCT) and in healthy volunteers. Serum TPO levels significantly correlated with the degree of peripheral thrombocytopenia and a strong inverse correlation between serum TPO level and platelet count was observed (r = −0.700, P < 0.001). serum tpo levels began to rise as the platelet count decreased after chemotherapy. tpo levels peaked at over 25.00 fmoles/ml between days 0 and 10; tpo levels then decreased gradually as the platelet count began to rise. one patient with multiple myeloma received purified cd34+ peripheral blood stem cells. No difference was observed in the kinetics of serum TPO levels between unfractionated and purified PBSCT. These observations suggest that TPO plays a critical role in the reconstitution of megakaryopoiesis and platelet production after PBSCT.

FLT-3 ligand mobilizes hematopoietic primitive and committed progenitor cells into blood in mice

Abstract: We investigated the effects of the administration of FLT‐3 ligand (FL) on mobilization of primitive and committed progenitor cells in mice. C57bI/6J mice were injected subcutaneously with FL once a day for 5 d at doses of 20, 100 and 200 μg/kg. After the collection of peripheral blood, we determined the number of white blood cells (WBCs) with the differential counts. The formation of colony‐forming cells (CFCs) in peripheral blood, bone marrow and spleen was evaluated. Although the administration of FL, 20 μg/kg, did not stimulate leukocytosis, its administration at doses of 100 and 200 μg/kg increased the number of WBC up to 1.7‐ and 2.4‐fold, respectively. Committed progenitor cells were mobilized into the peripheral blood dose‐dependently and the number of CFCs was increased up to 5.5‐fold by the administration of FL at 200 μg/kg on d 5. The number of CFCs in the bone marrow increased, but not dose‐dependently. The number of CFCs in the spleen also increased up to 32‐fold at a dose of 200 μg/kg FL. Mobilized peripheral blood mononuclear cells were transplanted into lethally irradiated mice and the number of CFU–S (d 12) was scored. A dose‐dependent mobilization of CFU–S (d 12) into peripheral blood was also observed. These observations suggest that FL can mobilize hematopoietic primitive and committed progenitor cells into the peripheral blood of mice and those cells mobilized by FL may be applicable to peripheral blood stem cell transplantation.

A translocation between 3q21 and 12q24 in a patient with minimally differentiated acute myeloid leukemia (AML-MO)

Only a small number of reports have described the cytogenetic analysis of minimally differentiated acute myeloid leukemia (AML, M0). We performed a cytogenetic analysis on a patient with AML (M0) with a normal platelet count. It revealed a chromosomal translocation between chromosome bands 3q21 and 12q24. 3q. Abnormalities in AML are known to be associated with normal or elevated platelet counts. 3q21 and 12q24 are common translocation sites in AML patients, but this is the first report of translocation t(3;12)(q21;q24) in an AML patient.

t(7;11) and trilineage myelodysplasia in acute myelomonocytic leukemia

A 48-year-old Japanese man was admitted to our hospital because of general fatigue, nasal bleeding, and petechiae on his extremities. He was diagnosed with acute myelomonocytic leukemia with trilineage myelodysplasia (T-MDS). Chromosomal analysis of bone marrow cells revealed t(7;11)(p15;p15), which has been rarely reported but known to be characteristic of Japanese patients. Although t(7;11)(p15;p15) has been reported mainly in acute myelogenous leukemia (AML), it can be occasionally found in so-called stem cell diseases such as chronic myelogenous leukemia or chronic myeloproliferative disorders. Therefore, t(7;11)(p15;p15) might affect trilineage progenitors or stem cells as well as myeloid lineage cells, subsequently resulting in AML with T-MDS, as in our case reported here.

Hematopoietic Reconstitution after Peripheral Blood StemCell Transplantation: Effects of Granulocyte Colony-Stimulating Factor and Progenitor Cell Dose

The effect of granulocyte colony-stimulating factor (G-CSF) on accelerating the rate of neutrophil recovery after peripheral blood stem cell transplantation (PBSCT) remains controversial.We retrospectively analyzed 37 patients who received high-dose chemotherapy followed by PBSCT. Patients were divided into three groups: those who received no G-CSF (control), a low dose (50 mg/m2), or a high dose (150 mg/m2), subcutaneously. Seven patients who received increased numbers of colony forming units-granulocyte macrophage (CFU-GM) (>100x104/kg) and either low-or high-dose G-CSF were separately analyzed. Neutrophil recovery (0.5 x109/1) was accelerated in the G-CSF-treated patients. It occurred at a median of 10 and 9 days in the low-and high-dose G-CSF groups compared with 14 days in the control group (p< .0001). In patients receiving increased numbers of progenitor cells, the time until recovery was 9 days. No statistically significant differences were observed for platelet and reticulocyte recovery, transfusions, days febrile and parenteral antibiotic requirement. Thus, the administration of low-dose G-CSF is recommended in PBSCT.

In Vivo Effects of FLT-3 Ligand in Mice

We investigated the effects of the administration of FLT-3 ligand (FL) on mobilization of hematopoietic stem and progenitor cells in mice. C57b1/6J mice were injected subcutaneously with FL once a day for 5 days at doses of 20, 100, and 200 µg/kg. After the collection of peripheral blood, we determined the number of white blood cells (WBCs), and the formation of colony-forming cells (CFCs) in blood, bone marrow and spleen. The administration of FL at doses of 100 and 2001.1g/kg increased the number of WBC up to 1.7- and 2.4-fold, respectively. Hematopoietic progenitor cells (HPCs) were mobilized into the blood dose-dependently. On Day 5, the number of HPCs was increased up to 2.2-, 2.8-, and 5.5-fold by the administration of FL at 20, 100, and 200 µg/kg, respectively. The number of HPCs in the bone marrow increased up to 1.9- to 3.8- fold. The number of HPCs in the spleen also increased up to 32-fold at a dose of 200 µtg/kg FL. Mobilized peripheral blood mononuclear cells were transplanted into lethally irradiated mice and the number of CFU-S (Day 12) was scored. A dose-dependent mobilization of CFU-S (Day 12) into blood was observed. These observations suggest that FL can mobilize hematopoietic stem and progenitor cells into the blood of mice and those cells mobilized by FL may be applicable to peripheral blood stem cell transplantation.