Effects of recombinant human tumor necrosis factor (rhTNF) on normal human and mouse hemopoietic progenitor cells

We examined the effects of recombinant human tumor necrosis factor (rhTNF) on normal human and murine granulocyte-macrophage (CFU-gm) and erythroid (CFU-e, BFU-e) progenitor cells. We suppressed in vitro colony formation by human marrow CFU-gm, CFU-e and BFU-e or peripheral blood BFU-e by adding rhTNF to the culture in a dose-related manner. A half-maximal inhibition was observed with 1-10 ng/ml. Leukemic cell line K562 cells were found to be sensitive to rhTNF in the clonogenic colony assay. However, the clonal growth of murine marrow CFU-e and BFU-e colonies was less than 50% inhibited and CFU-gm growth was unaffected even at a concentration of 1,000 ng/ml. We observed slight to moderate inhibition after 24 h pulse exposure of both human and murine-committed progenitors to rhTNF prior to the culture. Intravenous injection of 1 mg/kg of rhTNF caused a marked decrease in marrow erythroid progenitors and consequently caused anemia in the mice. Our data indicate that rhTNF has a suppressive effect on normal human and murine hemopoietic colony formation in vitro and murine erythropoiesis in vivo.     

Acquisition and clonal growth of human sternal bone marrow obtained incidental to open-heart surgery

The establishment of a convenient method of acquiring ample human bone marrow and the successful growth of hematopoietic precursor cells in standard methylcellulose assay is described. Human sternal marrow curettings were obtained at open-heart surgery. Growth of colonies derived from CFU-gm, BFU-e, and CFU-e was successful. Comparison of the CFU-gm colony production between this and prior studies indicates a consistent mean and range of growth. These results document this to be an efficient source of normal human marrow.     

Microcapillary clonogenic assays for human marrow hematopoietic progenitor cells

The capillary clonogenic cell assay was developed and adapted to culture myeloid and erythroid colonies from human bone marrow cells. The plating efficiencies for femoral bone marrow granulocyte-macrophage progenitors (CFU-gm), erythroid colony-forming units (CFU-e) and erythroid burst-forming units (BFU-e) were 0.143%, 0.229% and 0.141%, respectively. Standard bone marrow progenitor Petri dish assays require a total culture volume of 1 ml per dish, and as such are not suitable for the small numbers of cells often obtained from human bone marrow samples. The microcapillary assay as developed and standardized in our laboratory has the unique advantage of being able to utilize small numbers of cells. This technique is suitable for evaluating the myelotoxicity of investigational new anti-cancer and anti-HIV agents and for further investigation of the mechanisms underlying chemotherapy-induced bone marrow toxicity.     

Complement-dependent cytotoxic factor to megakaryocyte progenitors in sera from patients with idiopathic thrombocytopenic purpura

The influence of sera from patients with idiopathic thrombocytopenic purpura (ITP) was examined on colony formation from megakaryocyte (M) progenitors. Though incubation of marrow cells in Iscove's modified Dulbecco's medium (IMDM) containing 50% sera from several ITP patients stimulated M-colony formation in 8 of 13 cases, incubation in the sera from the patients and in baby rabbit serum as a source of complement significantly suppressed the colony formation. Experiments showed that sera of immunoglobulin G from ITP patients had significant complement-dependent cytotoxicity to M-progenitors in normal marrow cells or in the marrow cells from corresponding patients, but not to CFU-e, BFU-e or CFU-gm. Cytospin preparations of individually collected M-colonies from marrow cells treated with ITP patients' sera and complement revealed a reduction of megakaryocyte colonies containing cells of multilineages. These results indicate that autoantibodies detected in ITP patients can bind not only to platelets and megakaryocytes, but may also bind to M-progenitors.     

Characterization of the in vitro stromal microenvironment of human bone marrow

Utilizing long-term in vitro culture techniques, we characterized the cellular composition and functional attributes of the human in vitro bone marrow stromal microenvironment. Morphologic, specific cytochemical and immunologic methods demonstrated that the marrow stromal adherent layer (AL) reached confluency at two to three weeks, and was comprised of 60%-70% fibroblastic cells, 10%-20% endothelial cells, 10%-20% monocyte/macrophages and 5%-10% fat-laden adherent cells. These proportions of cell types persisted for at least three months concomitant with proliferation of CFU-gm and BFU-e. In contrast, umbilical cord blood cells did not form a stromal AL despite persistence of hemopoietic progenitor cell proliferation. These findings provide a basis for improved understanding of cellular interactions regulating hemopoiesis.     

Improvement of culture conditions for human megakaryocytic and pluripotent progenitor cells by low oxygen tension

The effect of low oxygen tension on the growth of human hemopoietic progenitor cells in bone marrow was investigated using the semisolid methylcellulose colony assay. The clonal growth of granulocyte-macrophage progenitors (CFU-gm), early (BFU-e) and late (CFU-e) erythroid progenitors, megakaryocyte progenitors (CFU-meg) and pluripotent progenitors (CFU-mix) improved more markedly incubation at the low oxygen tension (5%) than in conventional air (20%). The thiol compound 2-mercaptoethanol had a strong additive effect on colony growth in conventional air, but little or no effect in the low oxygen tension. These results suggest that enhancement of colony growth in the low oxygen tension may be due to a decrease in the production of oxygen intermediates.     

Effect of L-phenylalanine methyl ester on the colony formation of hematopoietic progenitor cells from human bone marrow

We have previously shown that L-phenylalanine methyl ester (PME) is capable of removing monocytes and enhancing the growth of hematopoietic colonies from human peripheral blood (PB) mononuclear cells (MNC). In the present study, we further compared the effect of PME on the colony formation of bone marrow (BM) and PB. Low density (less than or equal to 1.077 g/ml) MNC were obtained by Ficoll-diatrizoate density gradient centrifugation. Granulocyte/macrophage colony-forming units (CFU-gm) and erythroid burst-forming units (BFU-e) were cultured in agarose with conditioned media (CM) and/or interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF) and granulocyte/macrophage-CSF (GM-CSF). Treatment of BM MNC with 5 mM PME for 15 min at room temperature yielded a nucleated cell recovery of 44.8 +/- 5.0% (mean +/- SE; N = 8). CFU-gm were enriched 2.7-fold (range 2.0 to 4.8). Using CM or CM supplemented with G-CSF or GM-CSF has minimal effect on the enrichment. Leukocyte differentials revealed that 94.3 +/- 3.05% of the monocytes, as well as 91.2 +/- 1.60% of the cells in the neutrophilic maturation series were removed by PME. Incubation for 40 min in PME abolished CFU-gm formation. BFU-e were not enriched by the PME treatment. In contrast, 40 min incubation of PB MNC produced higher enrichment of CFU-gm than that obtained from 15 min of treatment, although lower cell recovery was obtained with the longer treatment time. In conclusion, we have demonstrated that phagocytic cells can be removed from BM or PB MNC by PME treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

CFU-gm colony formation of peripheral blood and bone marrow in adult acute leukemia at presentation, during remission, and at relapse

Granulocyte/macrophage colony-forming unit (CFU-gm) formation was studied simultaneously in bone marrow and peripheral blood of 52 previously untreated adult patients with acute non-lymphocytic (ANLL) and 36 with acute lymphoblastic leukemia (ALL). They were followed during induction therapy at monthly intervals while in remission and in 19 ANLL and 22 ALL cases, until relapse. Patients showing a decreased colony number in the marrow but normal or increased colony numbers in the peripheral blood had a high probability of entering remission. Non-responding patients displayed an opposite pattern. The higher the degree of marrow repopulation with granulocytic progenitor cells after induction treatment, the longer remission duration and survival for ANLL patients and the longer survival for ALL patients. CFU-gm formation returned to normal in the early stages of complete remission, but then declined progressively. At ANLL and ALL relapse, colony growth was reduced markedly while cluster formation remained normal. The number of marrow colonies and clusters in ANLL were significantly higher at first and second relapse compared to the growth pattern at first presentation. A similar trend had been observed in ALL, suggesting a selection advantage.     

Altered colony-forming activities of bone marrow hematopoietic stem cells in mice following short-term in vivo exposure to parathion

This paper describes a study of hematopoiesis in parathion-treated mice. Adult mice (48 C57B1/6) were given a daily dose of parathion (4 mg/kg p.o.) or corn oil vehicle (5 ml/kg p.o.) for 14 days. During the pesticide and the examination period, treated animals showed no signs of poisoning and had normal body weights. On days 2, 5, 7, 9, 12 and 14 following parathion or corn oil, femoral marrow cells were assayed in vitro for granulocyte/monocyte (CFU-gm), erythroid (CFU-e and BFU-e), megakaryocyte (CFU-meg), stromal (CFU-str) and multipotential (CFU-mix) hematopoietic stem cells. Leukocyte counts were elevated on days 2 and 5, while platelet counts were not increased until day 12. No change was observed in either hematocrits or numbers of marrow cells. BFU-e were reduced (23% of control) by day 7, then increased to 137% of control by day 14. CFU-e were reduced (41% of control) on day 9, then increased to 71% of control by day 14. CFU-mix were 130% of control (day 2), then declined to control values by day 5. On days 12 and 14, CFU-mix colonies decreased to 40% of control. CFU-str were reduced at all time points examined. CFU-gm were 123%, 136% and 130% of control on days 7, 12 and 14, respectively, while CFU-meg were increased (145% of control) on day 7. The data suggest that parathion alters the cloning potential of bone marrow precursor stem cells.     

Similarities in long-term cultures of blood and bone marrow from patients with acute myelogenous leukemia

Dexter-type long-term cultures (LTC) were initiated with peripheral blood (PB) and/or bone marrow cells from 11 patients with acute myelogenous leukemia (AML), and 2 with myelodysplastic syndrome in blastic transformation. Marrow and PB cells from normal subjects served as controls. Assessment of nucleated cells and clonogenic progenitors in the adherent and nonadherent fractions of LTC revealed active hemopoiesis for greater than 5 wks in 4 of 8 cultures of AML blood, and 4 of 7 of AML marrow. The morphology and kinetics of nucleated cells and progenitors with putative normal (granulocyte-macrophage colony-forming units or CFU-gm), and abnormal (blast) phenotype in LTC from AML blood were similar to those from AML marrow, and adherent cells positive for collagen I and III and vimentin were found in both types of LTC. Growth of CFU-gm colonies ceased by wk 5-8 in AML cultures, significantly earlier than in LTC of normal marrow cells (survival of greater than 10 wks), which may indicate derivation of the CFU-gm from a transformed clone or deficiency of stromal function in the leukemic state. In most AML blood and AML marrow LTCs, growth of abnormal (blast) colonies continued until wk 4-6. This study demonstrates certain similarities of morphology and function between LTC of AML blood and AML marrow cells. LTC may provide a useful model for further analysis of circulating primitive hemopoietic progenitor cells in leukemic states.     

The effect of carbamylcholine on CFU-s differentiation

The proportion of spleen colony-forming units (CFU-s) killed by hydroxyurea was greatly increased after bone marrow cells (BMCs) from LACA mice were exposed to carbamylcholine (Cach; 1 X 10(-13) to 1 X 10(-9) in vitro and there was a marked change in the proportion of spleen colony types. Following treatment with Cach, granulocytic and mixed erythroid-type colonies increased from 20 to 26.3% and 16.1 to 29.6% in 9-day colonies and from 8.3 to 28.2% and 21.7 to 39.4% in 13-day colonies, respectively. Single cell suspensions of spleen colonies were made for granulocyte-macrophage progenitor (CFU-gm) and late erythroid progenitor (CFU-e) assays. The number of CFU-gm from Cach-treated BMC was about twice that from control BMC for both day 9 and day 13 groups; the number of CFU-e decreased relatively. The results suggest that cholinergic receptors on CFU-s may increase the tendency to differentiate into the granulocytic/monocytic line.     

Velocity sedimentation profiles of normal and regenerating primitive erythroid burst-forming units: relation to phase of cell cycle and growth in vitro

Abstract. The primitive burst-forming unit-erythroid (BFU-e) derived from normal and regenerating murine bone marrow was examined by velocity sedimentation at unit gravity. An increase in the modal sedimentation velocity and the percentage of rapidly sedimenting BFU-e was found in regenerating marrow as compared to normal marrow. Neither hypertransfusion-induced plethora nor administration of erythropoietin (Ep) during regeneration altered the changes from normal in the velocity sedimentation profile observed during regeneration. Separated marrow cells were pooled as rapidly sedimenting and slowly sedimenting and then examined for percentage of BFU-e in DNA synthesis and growth response in vitro to increasing concentrations of a partially purified Ep preparation. The percentage of BFU-e in DNA synthesis as determined by tritiated thymidine killing does not correspond to the BFU-e growth response to Ep in vitro . No difference in growth was noted between BFU-e from rapidly and slowly sedimenting normal marrow cells despite an increased percentage in DNA synthesis of normal BFU-e which sedimented rapidly. No significant difference in the percentage of BFU-e in DNA synthesis was found between the rapidly and slowly sedimenting subpopulations of regenerating BFU-e, but the latter had a reduced growth response to low concentrations of Ep.     

Granulopoiesis in long-term culture by marrow from mice with busulfan-induced chronic latent aplasia

Mice given high-dose busulfan therapy develop a chronic latent marrow aplasia characterized by normal peripheral blood neutrophil numbers, hematocrits and marrow cellularity but reduced numbers of pluripotent hemopoietic stem cells (CFU-s) and granulocyte-monocyte progenitor cells (CFU-gm). To study the pathogenesis of this lesion, bone marrow was propagated in long-term marrow cultures (LTMC). Small amounts of normal marrow readily established and sustained long-term granulopoiesis in vitro. In contrast, inocula of marrow from busulfan-treated animals containing three to five times as many stem and progenitor cells failed to establish long-term granulopoiesis in vitro. These results suggest that high-dose busulfan therapy produces a qualitative defect in either the hemopoietic stem cells, the stromal-forming elements, or both, rendering them incapable of establishing long-term granulopoiesis in vitro. Furthermore, mixing experiments employing normal and busulfan-damaged marrow demonstrate that this qualitative defect is not due to the emergence of a suppressor cell population. LTMC can show types of marrow damage not detectable by other techniques currently available and represent a powerful tool for studying latent bone marrow failure.     

Comparative distribution of marrow CFU-e and CFU-gm progenitors in different anatomic sites in the dog

The interpretation of marrow cloning activity, particularly in serial cultures, is greatly influenced by the reproducibility of the collected marrow samples. In order to establish whether bone marrow cloning activities and precision of the cloning assays are influenced by the site of bone marrow collection in the dog, we studied the incidence of marrow erythroid (CFU-e) and granulocyte-macrophage (CFU-gm) progenitor cells in the iliac crest, sternum, vertebrae, femur, and humerus, using microplasma clot and soft agar culture systems. Marrow samples obtained from the femur and humerus revealed consistently higher cell concentrations than those from the iliac crest, vertebrae, or sternum. Those aspirated from the sternum and vertebrae had lower cell concentrations and were less reproducible. Statistical analysis revealed no significant differences in the incidence of marrow CFU-e and CFU-gm progenitor cells between the femur, humerus, iliac crest or vertebrae. With multiple sampling, the marrow cloning efficiency was consistent and reproducible within the individual dogs. We conclude that the distribution of CFU-e and CFU-gm is comparable throughout the active marrow in the dog and that these sites may be used interchangeably for multiple quantitative analysis of marrow hematopoietic progenitor cells.     

Failure of bone marrow cryopreservation in chronic granulocytic leukemia: relation to excessive granulo-macrophagic progenitor pool

Autologous bone marrow transplantation (ABMT) in chronic granulocytic leukemia (CGL) aims at reversing the acute or acceleration phases by injection of stem cells collected during the chronic phase. This study was designed to explain an unusual rate of delayed engraftment (50%) in our experience of ABMT in CGL patients. We investigated all the factors possibly responsible for abnormal perpetuation of aplasia following infusion of cryopreserved marrow stem cells. The study of CFU-gm recovery in 41 bags of frozen marrow from 25 patients revealed an overall deficiency with a mean CFU-gm recovery of 55 +/- 38% in CGL patients versus 73 +/- 15% in the control group (p less than 0.001). Our data also showed an inverse linear relation (r = -0.40, p less than 0.05) between CFU-gm concentration and recovery after freezing. A good CFU-gm recovery (greater than or equal to = 50%) was observed in 70% of cases when the concentration was less than 3700 CFU-gm/ml as compared to 30% of cases when the concentration was over 3700 CFU-gm/ml (p less than 0.001). The lack of improvement by diluting rich CFU-gm marrows to reduce CFU-gm concentration/ml, as well as the absence of relationship between CFU-gm recovery after freezing and nucleated cells concentration, suggest a particular fragility of CGL stem cells to freezing, probably related to their excessive amplification. At the present time, we strongly recommend that the highest possible dose of progenitor cells be cryopreserved, preferably at a low concentration, in patients with CGL, and particular attention devoted to the freezing procedure in each individual patient, with numerous appropriate efficiency tests.     

Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria

The complement system provides critical immunoprotective and immunoregulatory functions but uncontrolled complement activation can lead to severe pathology. In the rare hemolytic disease paroxysmal nocturnal hemoglobinuria (PNH), somatic mutations result in a deficiency of glycosylphosphatidylinositol-linked surface proteins, including the terminal complement inhibitor CD59, on hematopoietic stem cells. In a dysfunctional bone marrow background, these mutated progenitor blood cells expand and populate the periphery. Deficiency of CD59 on PNH red blood cells results in chronic complement-mediated intravascular hemolysis, a process central to the morbidity and mortality of PNH. A recently developed, humanized monoclonal antibody directed against complement component C5, eculizumab (Soliris; Alexion Pharmaceuticals Inc., Cheshire, CT, USA), blocks the proinflammatory and cytolytic effects of terminal complement activation. The recent approval of eculizumab as a first-in-class complement inhibitor for the treatment of PNH validates the concept of complement inhibition as an effective therapy and provides rationale for investigation of other indications in which complement plays a role.     

In vitro growth of erythropoietic progenitor cells in long-term remission of acute leukemia

In vitro growth of CFU-e and BFU-e in bone marrow and of circulating BFU-e in a group of adult long-term survivors of acute leukemia has been evaluated. Six patients with acute nonlymphoblastic and three patients with acute lymphoblastic leukemia in first continuous remission for more than four years (range 4-12 years) and without maintenance therapy for at least one year were studied. BFU-e and CFU-e growth in patients' bone marrow was not statistically different from a control group of 12 healthy adult volunteers. However, proliferation of BFU-e in peripheral blood of patients was significantly reduced (p less than 0.001). This growth pattern was found in both lymphoblastic and myeloblastic leukemia.     

Use of gel-well culture chambers as a liquid culture system to measure responses of hematopoietic colony-forming cells to growth factors

This report presents the results of an investigation in which Gel-Well culture chambers were evaluated for their utility as a liquid culture assay system to measure the responses of hematopoietic colony-forming cells (CFC) to recombinant and cell-derived growth factors. Gel-Wells, designed for anchorage-independent cell growth and diffusion of media components, permitted the weekly replacement of media and growth factors without removing cells from the culture chambers. In these studies, changes in cellularity and CFC content in Gel-Well cultures of human umbilical cord blood cells induced by recombinant interleukin 3 (rIL-3) were quantified. After one week in culture without rIL-3, the number of erythroid burst-forming units (BFU-e) had decreased to 25 +/- 38% of pre-values. In contrast, addition of rIL-3 induced an increase in the number of BFU-e to 390 +/- 135% of pre-values. By three weeks with rIL-3, the number of granulocyte-macrophage colony-forming units (CFU-gm) had increased to 292 +/- 58% of pre-values. Also, the presence of a bone marrow stromal cell layer under the Gel-Well helped to maintain the survival of CFC in liquid culture. These studies demonstrated that Gel-Well culture chambers provide a useful liquid culture system for studying the responses of CFC to growth factors.     

Effects of recombinant interferons on the clonogenic growth of leukemic cells and normal hemopoietic progenitors

We have examined the effects of recombinant immune and leukocyte interferons (rIFN-gamma and rIFN-alpha) on the clonogenic growth of leukemic cells and normal hemopoietic progenitors using in vitro colony assays. Both interferons suppressed the colony formation by granulocyte-macrophage progenitors (CFU-gm) and erythroid progenitors (CFU-e and BFU-e) in a dose-dependent manner. Six myeloid leukemic cell lines were less sensitive to rIFN-gamma than CFU-gm. The colony formation of some myeloid leukemic cell lines was suppressed more potently by rIFN-alpha than by CFU-gm. Four lymphoid leukemic cell lines of the T-cell type were very resistant to both recombinant interferons. Reduced sensitivity of leukemic cells to rIFN-gamma, a possible hemopoietic regulator, may explain partially the unregulated proliferation of leukemic cells in vivo.     

The enhancement of committed hematopoietic stem cell colony formation by nandrolone decanoate after sublethal whole body irradiation

The ability of an anabolic steroid, nandrolone decanoate, to increase committed topoietic stem cell (CFU-gm, CFU-e, and BFU-e) colony formation after sublethal irradiation was evaluated. Immediately after receiving whole body irradiation and on the next two days, each mouse was injected intraperitoneally with nandrolone decanoate (1.25 mg) in propylene glycol. Irradiated control mice received only propylene glycol. Compared to controls, drug-treated mice showed marked peripheral blood leukocytosis and more stable packed red cell volume. Drug-treated mice also demonstrated increased erythropoiesis, as CFU-e/BFU-e concentrations from both marrow (9% to 581%) and spleen (15% to 797%) were elevated. Granulopoiesis was increased similarly, as CFU-gm concentrations from marrow (38% to 685%) and spleen (9% to 373%) were elevated. These results demonstrate that nandrolone decanoate enhances hematopoietic stem cell recovery after sublethal whole body irradiation. This suggests that following hematopoietic suppression, nandrolone decanoate may stimulate the recovery of hematopoiesis at the stem cell level and in peripheral blood.