John E. Brandt

Characterization of a human hematopoietic progenitor cell capable of forming blast cell containing colonies in vitro.

A hematopoietic cell (CFU-B1) capable of producing blast cell containing colonies in vitro was detected using a semisolid culture system. The CFU-B1 has the capacity for self-renewal and commitment to a number of hematopoietic lineages. Monoclonal antibody to the human progenitor cell antigen-1 (HPCA-1) and a monoclonal antibody against the major histocompatibility class II antigen (HLA-DR) were used with fluorescence activated cell sorting to phenotype the CFU-B1. The CFU-B1 was found to express My10 but not HLA-DR antigen; experiments using complement-dependent cytotoxicity to eliminate DR positive cells confirmed this finding. Pretreatment of marrow cells with two chemotherapeutic agents, 5-fluorouracil and 4-hydroperoxycyclophosphamide facilitated detection of CFU-B1 derived colonies, while diminishing or totally inhibiting colony formation by other hematopoietic progenitor cells. CFU-B1-derived colony formation was dependent upon the addition of exogenous hematopoietic growth factors. Media conditioned either by the human bladder carcinoma cell line 5637 or lectin stimulated leukocytes, as well as recombinant granulocyte-macrophage colony stimulating factor, interleukin 3 or interleukin 1 alpha promoted blast cell colony formation. By contrast, neither recombinant erythropoietin, recombinant interleukin 4, purified macrophage colony stimulating factor or recombinant granulocyte colony-stimulating factor alone promoted blast cell colony formation.

Cytokine-dependent long-term culture of highly enriched precursors of hematopoietic progenitor cells from human bone marrow

Human marrow cells positive for the CD34 antigen but not expressing HLA-DR, CD15, or CD71 antigens were isolated. In a liquid culture system supplemented with 48-hourly additions of recombinant interleukins IL-1 alpha, IL-3, IL-6, or granulocyte/macrophage colony-stimulating factor (GM-CSF), these cells were capable of sustaining in vitro hematopoiesis for up to eight weeks. The establishment of an adherent cell layer was never observed. Cultures containing no exogenous cytokine produced clonogenic cells for only 1 wk. IL-1 alpha and IL-6 were alone able to support hematopoiesis for 2 or 3 wk. Cells maintained with GM-CSF proliferated and contained assayable colony-forming cells for 3 or 4 wk, while maximal cellular expansion and generation of assayable progenitor cells occurred in the presence of IL-3 for 4-5 wk. When IL-3 was combined with IL-1 alpha or IL-6, hematopoiesis was sustained for 8 wks. Basophil numbers were markedly increased in the presence of IL-3. These studies indicate that marrow subpopulations can sustain hematopoiesis in vitro in the presence of repeated additions of cytokines. We conclude that a major function of marrow adherent cells in long-term cultures is that of providing cytokines which promote the proliferation and differentiation of primitive hematopoietic cells.

Effect of recombinant and purified human haematopoietic growth factors on in vitro colony formation by enriched populations of human megakaryocyte progenitor cells

Nonadherent low density T‐lymphocyte depleted (NALT‐) marrow cells from normal donors were sorted on a Coulter Epics 753 Dye Laser System using Texas Red labelled My 10 and phycoerythrin conjugated anti HLA‐DR monoclonal antibodies in order to obtain enriched populations of colony forming unit‐megakaryocyte (CFU‐MK). The CFU‐MK cloning efficiency (CE) was 1.1 ± 0.5% for cells expressing both high densities of My 10 and low densities of HLA‐DR (My 10+++DR+). This procedure resulted in an 18‐fold increase in CE over NALT‐ cells. The effect of purified or recombinant human haematopoietic growth factors including erythropoietin (Epo), thrombocytopoiesis stimulating factor (TSF), interleukin 1α (IL‐1α), granulocyte colony stimulating factor (G‐CSF), granulocyte‐macrophage colony stimulating factor (GM‐CSF), macrophage colony stimulating factor (M‐CSF or CSF‐1) and interleukin‐3 (IL‐3) on MK colony formation by My10+++DR+ cells was determined utilizing a serum depleted assay system. Neither Epo, TSF, CSF‐1, IL‐lα nor G‐CSF alone augmented MK colony formation above baseline (2.5 ± 0.8/5 × 103 My 10+++DR+ cells plated). In contrast, the addition of GM‐CSF and IL‐3 each increased both CFU‐MK colony formation and the size of colonies with maximal stimulation occurring following the addition of 200 units/ml of IL‐3 and 25 units/ml of GM‐CSF. At maximal concentration, IL‐3 had a greater ability to promote megakaryocyte colony formation than GM‐CSF. The stimulatory effects of GM‐CSF and IL‐3 were also additive in that the effects of a combination of the two factors approximated the sum of colony formation in the presence of each factor alone. The CFU‐MK appears, therefore, to express HPCA‐1 and HLA‐DR antigens. These studies also indicate that GM‐CSF and IL‐3 are important in vitro regulators of megakaryocytopoiesis, and that these growth factors are not dependent on the presence of large numbers of macrophages or T cells for their activity since the My 10+++DR+ cells are largely devoid of these accessory cells.

Effect of perturbation of specific folate receptors during in vitro erythropoiesis.

Although antisera to specific placental folate receptors inhibits the uptake of 5-methyltetrahydrofolate into cultured malignant human cells, little is known of the functional significance of folate receptors in normal human cells. Human bone marrow cells were therefore assayed for erythropoietic burst-forming units in the presence of an antihuman placental folate receptor serum and preimmune serum to determine the role of such a receptor in erythroid differentiation. When marrow cells were assayed in the presence of anti-receptor antiserum, there was (i) a threefold increase in erythropoietic burst formation and a twofold increase in the number of cells per erythroid burst; (ii) morphological evidence for nuclear/cytoplasmic dissociation of orthochromatic normoblasts composing erythroid bursts (megaloblastic erythropoiesis); (iii) intracellular folate deficiency with a 70% reduction of intracellular folate in antiserum treated cells as compared with control cells; and (iv) complete reversal of antiserum-induced changes on preincubation of antiserum with purified human placental folate receptor. These data support the conclusion that folate receptors on marrow cells provide an important function in the cellular uptake of folates during in vitro erythropoiesis. This process of folate uptake also appears to play a pivotal role in the differentiation and proliferation of erythroid progenitor cells.

Megaloblastic hematopoiesis in vitro. Interaction of anti-folate receptor antibodies with hematopoietic progenitor cells leads to a proliferative response independent of megaloblastic changes.

We tested the hypothesis that anti-placental folate receptor (PFR) antiserum-mediated effects on hematopoietic progenitor cells in vitro of increased cell proliferation and megaloblastic morphology were independent responses. We determined that (a) purified IgG from anti-PFR antiserum reacted with purified apo- and holo-PFR and specifically immunoprecipitated a single (44-kD) iodinated moiety on cell surfaces of low density mononuclear cells (LDMNC); (b) when retained in culture during in vitro hematopoiesis, anti-PFR IgG (in contrast to PFR-neutralized anti-PFR IgG and nonimmune IgG) consistently led to increased cloning efficiency of colony forming unit-erythroid (CFU-E), burst forming unit-E (BFU-E), CFU-granulocyte macrophage (CFU-GM), and CFU-GEM megakaryocyte (CFU-GEMM), and objectively defined megaloblastic changes in orthochromatic normoblasts from CFU-E- and BFU-E-derived colonies; (c) when anti-PFR antiserum was removed after initial (less than 1 h) incubation with LDMNC, a cell proliferation response was induced, but megaloblastic changes were not evident. (d) Conversely, delay at 4 degrees C for 24 h before long-term plating with antiserum resulted in megaloblastosis without increased cell proliferation; (e) however, 500-fold molar excess extracellular folate concentrations completely abrogated the expected anti-PFR antiserum-induced megaloblastic changes, without altering cell proliferative responses. Thus, although cell proliferative and megaloblastic changes are induced after short-term and prolonged interaction of antibody with folate receptors on hematopoietic progenitors, respectively, they are independent effects.