Seiji Motomura

Differentiation of human bone marrow-derived fibroblastoid colony forming cells (CFU-F) and their roles in haemopoiesis in vitro

Summary. Human bone marrow‐derived fibroblastoid colonies have been quantitatively developed in a liquid culture. A linear relationship between cell number plated and colony number formed supports their clonal origin and hydroxyurea killing indicates that the fibroblastoid colony forming cell (CFU‐F) is not in cell cycle in normal bone marrow. Adipose cells were induced in the fibroblastoid colonies by the addition of hydrocortisone (optimal concentration: 10−6 M). Furthermore, adherent layers with adipocytes provided a more favourable condition for maintaining haemopoiesis in Dexter‐system cultures. These results indicate that CFU‐F belongs to stromal precursor cells intimately involved in the formation of the haemopoietic microenvironment. Colony incidence of CFU‐F was almost normal in most patients with aplastic anaemia, haemopoietic dysplasia and chronic myelogenous leukaemia. However, in acute myelogenous leukaemia, it varied with the stage of the disease. It is concluded that the colony assay is useful for investigating stroma/haemopoietic cell interactions.

Effect of Platelet-Derived Growth Factor and Bone Marrow-Conditioned Medium on the Proliferation of Human Bone Marrow-Derived Fibroblastoid Colony-Forming Cells

The effects of various human sera, platelet lysates and platelet-derived growth factor (PDGF) on the proliferation of human bone marrow-derived fibroblastoid colony-forming cells (CFU-F) were examined. We obtained nearly identical growth curves of fibroblastoid colonies with sera, platelet lysates and PDGF as stimulants and concluded that PDGF was a main growth factor for CFU-F in human serum. In contrast to colony size, CFU-F number was irrelevant to the concentration of PDGF. Removal of culture medium containing hemopoietic cells after short-term incubation of bone marrow cells reduced both colony number and size in CFU-F cultures. When each of bone marrow-conditioned medium (BMCM), peripheral blood mononuclear cells (MNC) and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM) was added to the cultures, CFU-F number and colony size recovered. The role of PDGF and the factors present in BMCM, MNC and PHA-LCM in the growth of CFU-F and their precursor cells were discussed.

Human erythroid cell lines derived from a patient with acute erythremia

SummaryThree continuous human cell lines, designated KMOE, derived from a patient with acute erythremia (Di Guglielmos disease) are reported. The cell lines are the cultures of (1) bone marrow cells, (2) peripheral blood cells, and (3) cells from a tumor developed into an athymic nude mouse after transplantation of the cultured bone marrow cells. Cells of all three lines show morphology of immature erythroblast and have i(17q) marker chromosome. They are negative for both Philadelphia chromosome and Epstein-Barr virus nuclear antigen. Although all KMOE cells in suspension culture are benzidine-negative, benzidine-positive cells are found within colonies formed in semi-solid culture media. The relative number of colonies with benzidine-positive cells is increased when sodium butyrate is added to the culture.The KMOE cell lines are human erythroid cell lines with erythroblastic morphology and still retain their tendency for differentiation.

Clinical significance of human bone marrow stromal cell colonies in acute leukemias.

Human bone marrow-derived fibroblastoid colony-forming cells (CFU-F) and adipocyte colonies in 36 patients with acute leukemia were studied, and were serially analysed at different clinical stages. At untreated stage, CFU-F number in acute lymphoblastic leukemia (ALL) was lower than that in acute non-lymphoblastic leukemia (ANL). In ANLs, CFU-F number in M1 was lower than that in M2. Adipocyte colonies were frequently developed at regenerating and relapsing stages, but rarely at untreated and remission stages. The adipocyte colony formation did not correlate with any of CFU-F number, marrow cellularity nor number of leukemic cells, but might be associated with hemopoietic regeneration. The favorable prognosis was associated with normal CFU-F number and with adipocyte colony formation at regenerating or bottom stage. As adipocytes in marrow samples were completely removed before cultures, adipocyte colony was probably originated from preadipocytes. Thus, our results suggest that adipocyte precursor cells increase in regenerating marrow and that they are essential in active hemopoiesis.

Regulation of erythropoietin and burst-promoting activity production in patients with aplastic anemia and iron deficiency anemia.

To clarify the control mechanism of production of erythropoietic growth factors in anemic states, we compared erythropoietin (Epo) and burst-promoting activity (BPA) in patients with aplastic anemia and iron deficiency anemia, using in vitro erythroid progenitor assays. Although serum levels of Epo activity increased in the presence of anemia, the rise was more marked in patients with aplastic anemia. BPA was high only in the sera of aplastic anemia patients. Serum levels of BPA of patients with aplastic anemia negatively correlated with hemoglobin concentrations, while those of patients with iron deficiency anemia did not correlate. In 2 patients with aplastic anemia who responded well to androgen therapy, serum levels of Epo activity and BPA decreased after the hemopoiesis had recovered. These results suggest that serum levels of BPA do not rise in response to anemia only. The elevated BPA levels in sera in cases of aplastic anemia are probably related to a reduction in the number of hemopoietic stem cells. Moreover, we observed that BPA in bone-marrow-conditioned medium (BMCM) from patients with severe aplastic anemia increased more than in the BMCM from patients with severe iron deficiency anemia. Therefore, our findings suggest that the enhanced BPA production depends on a decrease in hemopoietic precursors rather than the anemic state.

Molecular heterogeneity of β-thalassaemia in the Japanese: identification of two novel mutations

Five unrelated Japanese β‐thalassaemia genes, from one homozygote and four heterozygotes, have been systematically characterized using DNA polymorphism analysis, polymerase chain reaction, dot‐blot hybridization and direct sequencing of amplified genomic DNA. Four different molecular defects were observed on three different β‐globin gene frameworks. One of these, the A→G mutation in the TATA box, a previously described mutation, was detected by dot‐blot hybridization in one homozygote and one heterozygote with the β‐globin gene of framework 2. The second mutation is a C→T substitution at position 654 of IVS‐2, the mutation commonly found in Chinese, which was associated with the framework 1 gene. Another two mutations, both associated with framework 3 genes, are novel ones; an amber mutation in codon 90 (GAG to TAG) and a frameshift (+G) insertion in codon 54, both of which cause a β0‐thalassaemia phenotype by premature termination of the β‐globin chain synthesis.

Serial Studies of Bone Marrow-Derived Fibroblastoid Colony-Forming Cells and Granulocyte/Macrophage Precursor Cells in Patients with Acute Leukemia

Bone marrow-derived fibroblastoid colony-forming cells (CFU-F) and granulocyte/macrophage precursor cells (CFU-GM) were studied in patients with acute leukemia. The numbers of CFU-F and CFU-GM were significantly lower in patients with acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL) at diagnosis than in normal subjects, although patients with AML had a very wide range of CFU-F colony-forming efficiency. However, a suppressive effect of leukemic cells on normal CFU-F colony formation was not observed. CFU-F and CFU-GM in patients with acute leukemia recovered to normal levels when complete remission (CR) was achieved and decreased again at relapse. Serial studies showed that the increase in CFU-F preceded the recovery of CFU-GM. In AML, furthermore, patients who achieved CR had a higher number of CFU-F than patients without CR, suggesting that the CFU-F level at diagnosis may contribute to the prediction of the likelihood of remission induction in patients with AML.

Periodical Appearance of Erythropoietin-Independent Erythropoiesis in Chronic Myelogenous Leukemia with Cyclic Oscillation1

A patient with Ph1-positive chronic myelogenous leukemia (chronic phase) had a cyclic oscillation in white blood cells, platelets and percent saturation of transferrin. The cycle comprised about 70 days. The number of circulating granulocyte-macrophage colony-forming units (CFU-GM) oscillated with the same phase, while that of bone marrow CFU-GM and erythroid colony-forming units (CFU-E) oscillated in a reverse phase. At the nadir, we observed an abnormal increase in bone marrow endogenous CFU-E (e-CFU-E). An erythropoietin (Epo) dose-response curve of CFU-E showed a high Epo-sensitivity. Anti-Epo rabbit serum did not inhibit the e-CFU-E colony formation. This indicates that Epo-independent erythropoiesis occurs periodically at the nadir. It is suggested that the interactions between the abnormal stem cell and the hematopoietic regulating system cause cyclic oscillation.

Characterization of new non-T, non-B acute lymphoblastic leukemia cell lines: Analysis of surface antigens by quantitative cellular radioimmunoassay and flow cytometry☆

Two novel acute lymphoblastic leukemia (ALL) cell lines, HOON and HYON, have been established from patients with non-T, non-B ALL. The cell lines have been characterized and shown to express phenotypic markers on non-T, non-B ALL. By indirect immunofluorescence and flow cytometry they express Ia and common ALL (CALLA) antigens and are reactive with monoclonal antibodies BA-1, BA-2 and OKT-9. However, the cells do not express detectable amounts of B1 antigen or of cytoplasmic mu chain, which are markers of pre-B cells. Quantitation of Ia and CALLA antigens on HOON and HYON cell lines using a cellular radioimmunoassay revealed that both cells bind high levels of anti-Ia antibodies, 110 X 10(4) molecules per cell, and low levels of anti-CALLA antibodies, 7 X 10(4) molecules per cell. Although both HOON and HYON carry equivalent amounts of Ia on their surface, only the former is a good stimulator of the one-way mixed lymphocyte reaction.

Hydrocortisone modulates colony‐stimulating activity produced by human bone marrow‐derived adherent cells

In an attempt to clarify the significance of hydrocortisone (HC) in human long‐term bone marrow cultures, the production of colony‐stimulating activity (CSA) and colony‐enhancing activity (CEA) by human bone marrow‐derived adherent cells (MDAC) and the modulation by HC were examined. The CSA production by MDAC was demonstrated using bilayer agar cultures. After treatment of MDAC with 10−6 mol/l HC, the CSA production was markedly enhanced, and after treatment of macrophages with 10−6 mol/l HC, the CSA production was inhibited. When added to a granulocyte‐macrophage precursor cells (CFU‐GM) assay system, HC inhibited colony formation. These results suggest that HC treatment directly stimulates CSA production by non‐macrophage cells of MDAC. The conditioned medium of the confluent layer of MDAC contained CEA, which was not influenced by the HC treatment of MDAC. Thus, HC plays an essential role in granulopoiesis in vitro, enhancing the CSA production by MDAC and inhibiting the differentiation of CFU‐GM.