Shushi Kaneko

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.

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.

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.

Bone Marrow Stromal Cells in Myeloproliferative Disorders

The number of bone marrow-derived fibroblastoid colony-forming cells (CFU-F) and the production of colony-stimulating activity (CSA) by bone marrow stromal cells were studied in 71 patients with myeloproliferative disorders (MPD). The numbers of CFU-F in chronic-phase chronic myelogenous leukemia (CML), polycythemia vera (PV) and essential thrombocythemia (ET) were not different from those in normal subjects. However, the number of CFU-F in acute-phase CML was markedly decreased. Bone marrow adipocyte colony-forming capacity (adipo-CFC), which was previously shown to reflect both the number of preadipocytes and the stromal cell function in vivo, was increased in patients with chronic-phase CML, PV and ET, but was absent in acute-phase CML patients. The production of CSA by marrow stromal cells of MPD patients, however, was not different from that of normal subjects. These results suggest that the characteristics of marrow stromal and its precursor cells of chronic-phase MPD patients were not different from those of normal subjects, however, they became changed in acute-phase CML patients.

Progression and prognosis of chronic myelogenous leukemia in the acute stage: hematologic and cytogenetic aspects.

Ten patients with Ph1 chromosome-positive chronic myelogenous leukemia entering the acute stage were divided hematologically into two different groups. One was characterized by a predominance of myeloblasts in marrow and was cytogenetic either by diploid or hypodiploid, whereas the other had generally low myeloblast counts without significant differences between the peripheral blood and the bone marrow, and was characterized by hyperdiploidy. It is suggested that an extramedullary acute transformation in the spleen occurs primarily in most cases of the latter group.

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.

Difference of bone marrow adipocyte colony-forming capacity between aplastic anemia and iron deficiency anemia

The bone marrow adipocyte colony-forming capacity (Adipo-CFC) in patients with aplastic anemia (AA), myelodysplastic syndrome (MDS) and iron deficiency anemia (IDA) was studied. Before treatment, Adipo-CFC in IDA was higher than that in AA and MDS. After treatment, Adipo-CFC decreased in IDA, but it increased in AA and MDS only at the responsive stage. In this context, it is suggested that increase of Adipo-CFC occurs during not only regenerating hemopoiesis but also accelerated erythropoiesis such as severe IDA. Colony-stimulating activity (CSA) production by marrow stromal cells (MSC) in AA was lower than that in normal subjects. Low Adipo-CFC and defective CSA production by MSC may explain in part the pathogenesis of microenvironmental defect in AA.

AUTOIMMUNE NEUTROPENIA ASSOCIATED WITH MONOCLONAL GAMMOPATHY

好中球減少症(35才,男性)において,副腎皮質ステロイド使用後に増加してきた自己成熟好中球に対する凝集素をLalezariの方法によつて検索し,本症が自己免疫性好中球抗体に起因することを証明した.本例の成熟好中球は末梢血および骨髄中に著減を示したが,骨髄芽球,好中球系の前骨髄球および骨髄球は減少を認めず, Robinson & Pike法による骨髄細胞の軟寒天培養法でコロニー形成細胞(colony forming cell in culture, CFU-C)が, 506/2×10 cellsと正常骨髄細胞(110±46/2×105cells)より著増していた.高γ-グロブリン血症(血清総蛋白6.9g/dl, γ-グロブリン22.8%, IgG 1800mg/dl, IgA 333mg/d1, IgM 516mg/dl, TTT 13.5, ZTT 21.2)を合併し,セルローズアセテート膜電気泳動で尖鋭なピークが認められ,免疫電気泳動でIgM(κ)のM成分と同定された.従つて,好中球自己抗体が単クローン性IgM(κ)にあるのか否かを追究する目的で,患者血清を薄層等電点分画法によつてIgGとIgMに分離した後, Lalezariの方法によつて白血球凝集素を検索したところ,好中球抗体はIgGにあることが判明した.副腎皮質ステロイドに極めて良く反応し,プレドニゾロン40mg/日の投与で1週間後には好中球数396/mm3から2840/mm3と著増した.血清の白血球凝集素は2048倍以上から,ステロイド使用後急速に低下して陰性化した.以上,本邦第1例の自己免疫性好中球減少症を報告すると共に,好中球抗体の検索法,好中球特異抗原,同種新生児好中球減少症, Felty症候群, SLEなどの鑑別診断などについて考察した.