Purification of murine bone-marrow-derived granulocyte-macrophage colony-forming cells

Previous attempts to purify progenitor cells that form colonies and clusters of granulocytes and/or macrophages (CFU-GM) from adult murine bone marrow have had limited success because of the paucity of these cells. In the present paper we report studies with a rapid, reproducible method involving pretreatment of mice, three days prior to sacrifice, with 200 mg/kg of Cytoxan (cyclophosphamide), density separation on Ficoll-Hypaque, and counterflow centrifugal elutriation, that yielded highly enriched populations of CFU-GM. The peak CFU-GM-containing fraction (FR-28) eluted at a flow rate of 28 ml/min and contained very little contamination by other in vitro colony-forming cells (BFU-E, CFU-GEMM, CFU-MK). FR-28 contained 0.54% +/- 0.30% (16 experiments) of the unfractionated post-Cytoxan bone marrow nucleated cells and lacked significant contamination by lymphocytes and monocytes. The mean CFU-GM cloning efficiency of FR-28 was 44% +/- 9% in agar (11 experiments) and 75% +/- 10% in agarose (nine experiments). CFU-GM from FR-28 demonstrated linear plating characteristics even at very low cell density (25 cells), and formed colonies and clusters of granulocytes, macrophages, or both in the same proportions as did unfractionated post-Cytoxan or untreated bone marrow. Approximately 10% (assuming a seeding efficiency of 10%) of FR-28 cells were in vivo spleen colony-forming cells (CFU-S) measured at day 12. These results represent the highest degree of purity (up to 94%) of CFU-GM thus far reported and should prove useful in studies of this cell population.     

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

S ummary . 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 benzene on fibroblastoid colony-forming units in mice.

After exposure of C57BL6 x DBA/2 mice to benzene in air their number of bone marrow fibroblastoid precursor cells, CFU-F, was determined. The CFU-F exhibited an increasing plating efficiency, giving rise to a larger number of colonies and to colonies of greater size. This effect was dose dependent. When the mice were exposed for 16 weeks and were then allowed to rest, their CFU-F plating efficiency returned to normal within 6 weeks, but then increased again. Hematopoietic stem cells, such as CFU-S and CFU-C exhibited a dose-dependent depression. The in vitro exposure of bone marrow cells to benzene metabolites resulted in a dose-dependent depression of CFU-F numbers.     

Studies on the presence and possible oscillations of granulocytic progenitor cells (CFU-C) in human blood

The presence of CFU-C in the peripheral blood of 3 healthy volunteers was monitored over a period of 70 d. Blood samples were taken 3 times a week. Mononuclear cells (MNC) were separated from blood leucocytes by dextran sedimentation and the iron ingestion method. After cryopreservation the cells were cultured in soft agar. One sample of mononuclear cells was cultured using a leucocyte feeder layer as a source of colony stimulating activity (CSA) and another sample using human placental conditioned medium as CSA. In cultures using a leucocyte feeder layer, the average number of CFU-C was 29, 43, 118 per ml blood. No systematic oscillations were observed. When human placental conditioned medium was used the average number of CFU-C was 255, 125, 222 per ml blood, respectively. Significant oscillations with periods of 23, 19 and 25 d were observed.     

Expression of Ia-antigens on normal and chronic myeloid leukemic human granulocyte-macrophage colony-forming cells (CFU-GM) is associated with the regulation of cell proliferation by prostaglandin E

The presence of la-antigens and their relationship to the inhibitory effect of prostaglandin E on the proliferation of human CFU-GM was studied in animals and patients with chronic myeloid leukemia. Consistent reduction of normal colony formation to approximately 50% of baseline levels was observed using a monoclonal anti-human la antibody in a complement-dependent cytotoxicity assay titrated over serial dilutions. ELimination of the la-antigen-bearing CFU-GM population was associated with virtually a complete loss of responsiveness to the inhibitory effects of prostaglandin E. Maintenance of bone marrow cells in short-term suspension culture at 37 degrees C prior to agar culture resulted in the loss of detectable la-antigen on the CFU-GM and, similarly, loss of response to prostaglandin. In contrast, most patients with chronic myeloid leukemia showed greatly reduced levels of la-antigens on their CFU-GM in fresh marrow together with lack of prostaglandin sensitivity, suggesting a correlation with the abnormal growth regulation observed in these patients. In two chronic myeloid leukemia patients, levels of la-antigen higher than that observed in the majority of patients could be detected and correlated with a residual response to prostaglandin E. These results suggest a relationship in normals between the expression of la-antigens on CFU-GM and the physiologic response to regulation by prostaglandin E, and a possible mechanism for the aberrant regulatory response in patients with chronic myeloid leukemia.     

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.     

Blood colony-forming cells (CFU-C) and leucocyte colony-stimulating activity (CSA) in patients with neutrophilic leucocytosis.

The incidence of circulating colony-forming cells (CFU-C) and the ability of peripheral leucocytes to stimulate the colony formation (CSA) have been studied through a double layer agar culture system in 26 patients with neutrophilic leucocytosis and compared to the values obtained in 26 normal subjects. Both mean CFU-C incidence and mean leucocyte CSA of the whole group of patients were found significantly higher than normal, but considerable variation was observed among singular patients. The different patterns of blood CFU-C and leucocyte CSA are discussed. The combined evaluation of blood CFU-C and leucocyte CSA is found a useful tool to investigate the pathogenetic mechanisms of neutrophilic leucocytosis.     

Characteristics of bone marrow fibroblast colony-forming cells (CFU-F) and their progeny in patients with myeloproliferative disorders

Chronic myeloproliferative disorders (MPD) are clonal diseases of the pluripotent hematopoietic stem cell frequently associated with myelofibrosis (MF). There is only indirect evidence indicating that the increased deposition of collagen in bone marrow matrix is a secondary phenomenon. A liquid culture system for cloning and growing bone marrow fibroblasts has permitted us to approach more directly the understanding of the pathogenesis of myelofibrosis by comparing the biophysical, growth, and functional characteristics of fibroblasts from normals, MPD patients without MF, and those with MF. In patients with MF, marrow fibroblast colony (CFU-F) formation could not be studied; fibroblasts were grown from marrow explants. CFU-E from normals and MPD patients exhibited similar cell density distribution and similar cell sedimentation rates. These similarities contrasted sharply with the differences seen when the erythroid and granulocyte-macrophage progenitors were studied by the same methods. There was a marked light density shift and a rapidly sedimenting shift of MPD hematopoietic colony-forming cells. Marrow fibroblasts from MPD patients with and without MF displayed the same in vitro growth characteristics as fibroblasts from normals. Both types of fibroblasts exhibited anchorage and serum dependence, and contact inhibition of growth. Marrow fibroblasts were also characterized for the presence and distribution of fibronectin and collagen types by immunofluorescent staining using monospecific antibodies. Extracellular matrix, membrane-, and cytoplasm- associated fibronectin, type I, type III, and type V collagen showed a similar staining pattern in both normal and myelofibrotic marrow fibroblasts. Plasminogen-dependent fibrinolytic activity elicited from normal and myelofibrotic marrow fibroblasts were equivalent. Chromosomal analysis of hematopoietic cells and marrow fibroblasts from Philadelphia chromosome positive chronic myelocytic leukemia patients with and without MF showed that the Philadelphia chromosome was present only in hematopoietic cells. The results of these studies taken together demonstrate that bone marrow collagen-producing cells from MPD patients with and without MF behave in vitro as do those from normals. These findings support the hypothesis that that the marrow fibrosis observed in patients with MPD results from a reactive process rather than from a primary disorder affecting the marrow collagen-producing cells.     

Application of hyperthermia to the treatment of human acute leukemia: purging human leukemic progenitor cells by heat

The application of hyperthermia to the treatment of neoplastic disease has focused on solid tumors. Since the hyperthermic sensitivity of human acute leukemia cells is not known, we have studied the in vitro response of human leukemic progenitor cells (L-CFU) to hyperthermia using a quantitative assay system for L-CFU. Human L-CFU were found to be more sensitive than committed normal myeloid progenitor cells to hyperthermic killing (41 to 42 degrees C). In addition, in the five acute myelogenous leukemic patients studied, it was shown that their leukemic progenitor cells--all types were studied according to the French-American-British diagnosis--were unable to form colonies when exposed to a temperature of 42 degrees C for 60 minutes, whereas the residual normal clones suppressed by the leukemic cell population were found to recover and to form more colonies in vitro as compared with untreated leukemic marrows. This strongly suggests that in vitro hyperthermia may selectively purge residual leukemic cells, especially L-CFU in stored remission bone marrow before autologous bone marrow transplantation.     

Influence of the spleen on the blood distribution of the colony-forming cells (CFU-C) in man

The incidence of the blood committed granulocyte progenitor cells (CFU-C) before and after epinephrine administration has been studied in 10 normal, 16 splenomegalic and 8 splenectomized subjects through a double-layer agar culture system. A significant increase of the mean values of CFU-C per milliliter of blood has been observed after epinephrine administration in normal and in splenomegalic subjects. In splenectomized patients the baseline mean values of CFU-C per milliliter of blood were higher than those observed in the other groups of subjects, but they did not increase after epinephrine infusion. The concentration of CFU-C per 10(6) total blood leukocytes was the same in all three groups of subjects and it was not modified by epinephrine administration. Our results seem to indicate that the CFU-C are distributed in two blood compartments, the spleen representing an important section of the marginal compartment of the blood CFU-C.     

Purification and characterization of granulocytic progenitor cells (CFU-C) from human peripheral blood using immunologic surface markers.

The concentration of committed granulocytic progenitor cells (CFU-C) in functionally unique subpopulations of human peripheral blood mononuclear cells has been determined by the in vitro methyl-cellulose assay. Using immunoabsorbent column chromatography and rosette-depletion techniques, we have demonstrated that CFU-C, although not present in either purified T or B lymphocyte populations, are highly concentrated in the "null" cell population, which lacks sheep erythrocyte receptors and surface immunoglobulin. Further fractionation of this null subset has demonstrated that CFU-C do not bear complement receptors, but require the presence of peripheral blood mononuclear cell feeder layers for maximum proliferation.     

Prediction of chemotherapy response in human leukemia using an in vitro chemotherapy sensitivity test on the leukemic colony-forming cells

An in vitro test system to quantitatively assess the chemotherapy sensitivity of human acute leukemic colony-forming cells (L-CFU) in relation to normal granulocytic precursor cells (CFU-C) has been developed. After simultaneous exposure of leukemic and normal bone marrow cells to individual drugs in vitro, cells were grown using an improved agar culture method with daily feeding. A sensitivity index (SI) was determined as the ratio of survival fraction of CFU-C to that of L-CFU, L-CFU being more (or less) sensitive than CFU-C if the SI were higher (or lower) than unity. Thirty SI were determined for 6 single drugs actually given in various combinations to a total of 9 patients (8 with acute nonlymphocytic leukemia and 1 with chronic myelomonocytic leukemia). A highly significant correlation was observed between high (or low) SI and achievement of (or failure to achieve) complete remission, with only 6 false correlations (p = 0.0013). Also, the mean of these SI (MSI) for the multiple single drugs given to each patient as components of a combination chemotherapy was used to indicate an overall sensitivity for each trial of the chemotherapy. Among the 10 chemotherapy trials (1 trial each for 8 patients and 2 trials for 1 patient), 4 trials resulting in complete remission had MSI higher than 1.0, and 6 trials not resulting in complete remission had MSI lower than 1.0 (p = 0.0048). This assay system appears useful in predicting the response of patients to chemotherapy and in the selection of the most effective drugs for use in individual patients.     

Staging of leukemic progenitor cells in acute myeloid leukemia by phenotyping with myeloid monoclonal antibodies

We studied the immune phenotype of leukemic progenitor cells (AML-CFU-L) in 16 cases of acute myeloid leukemia (AML) using 12 myeloid monoclonal antibodies (McAbs) in a complement-mediated cytotoxicity assay. On the basis of their reactivities with normal day-14 (D14) and day-7 (D7) myeloid progenitor cells (CFU-GM), these McAbs could be classified into three groups: six McAbs reacted strongly with "early" antigens on D14-CFU-GM, two McAbs reacted only with "late" antigens on D7-CFU-GM, while four McAbs formed an "intermediate" group that reacted both with the "late" antigens on D7-CFU-GM and to some extent with the "early" antigens of D14-CFU-GM. The McAbs all reacted with antigens on AML-CFU-L:McAbs that reacted with "early" antigens reacted consistently strongly with AML-CFU-L, in contrast to McAbs in the two other groups, which displayed greater heterogeneity. On the basis of antigenic phenotype, the predominating CFU-L in AML could thus be placed in one of three "stages." This phenotypic "staging" of AML correlated with the French-American-British (FAB) morphological classification of AML. The AML-CFU-L of patients with FAB morphological types M2, M4, and M5 expressed more differentiated antigenic phenotypes than those of the three patients with M7. The latter reacted poorly with the "intermediate" and "late" McAbs. Our data and those of others suggest that classification based on AML-CFU-L antigenic profile may complement the FAB classification of AML.     

FLT3/FLK2 ligand promotes the growth of murine stem cells and the expansion of colony-forming cells and spleen colony-forming units

The effect of FLT3/FLK2 ligand (FL) on the growth of primitive hematopoietic cells was investigated using ThyloSca1+ stem cells. FL was observed to interact with a variety of factors to initiate colony formation by stem cells. When stem cells were stimulated in liquid culture with FL plus interleukin (IL)-3, IL-6, granulocyte colony- stimulating factor (G-CSF), or stem cell factor (SCF), cells capable of forming colonies in secondary methylcellulose cultures (CFU-c) were produced in high numbers. However, only FL plus IL-6 supported an increase in the number of cells capable of forming colonies in the spleens of irradiated mice (CFU-s). Experiments with accessory cell- depleted bone marrow (Lin- BM) showed that FL alone lacks significant colony-stimulating activity for progenitor cells. Nevertheless, FL enhanced the growth of granulocyte-macrophage progenitors (CFU-GM) in cultures containing SCF, G-CSF, IL-6, or IL-11. In these assays, FL increased the number of CFU-GM initiating colony formation (recruitment), as well as the number of cells per colony (synergy). Many of the colonies were macroscopic and contained greater than 2 x 10(4) granulocytes and macrophages. Therefore, FL appears to function as a potent costimulus for primitive cells of high proliferative potential (HPP). FL was also observed to costimulate the expansion of CFU-GM in liquid cultures of Lin- BM. In contrast, FL had no growth- promoting affects on progenitors committed to the erythrocyte, megakaryocyte, eosinophil, or mast cell lineages.     

A murine cytokine fusion toxin specifically targeting the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on normal committed bone marrow progenitor cells and GM-CSF-dependent tumor cells

A fusion protein was synthesized consisting of the murine granulocyte- macrophage colony-stimulating factor (mGM-CSF) gene spliced to a truncated form of the diphtheria toxin (DT390) gene coding for a molecule that retained full enzymatic activity, but excluded the native binding domain. The DT390-mGM-CSF hybrid gene was cloned into a vector under the control of an inducible promoter and the protein expressed in Escherichia coli. After induction, a protein was purified from inclusion bodies in accord with the deduced molecular weight of DT390 mGM-CSF. Cell-free studies of the adenosine diphosphate-ribosylating activity of DT390 mGM-CSF showed results that were similar to those of native DT. The DT390 mGM-CSF immunotoxin inhibited FDCP2.1d, a murine myelomonocytic tumor line expressing the GM-CSF receptor with an IC50 (concentration inhibiting 50% activity) of 5 x 10(-11) mol/L. The fusion toxin was specifically cytotoxic and directed by the GM-CSF portion of the molecule because addition of a monoclonal antibody directed against GM-CSF inhibited its ability to kill the cell line. Cell lines that do not express GM-CSF receptor were not inhibited by the fusion toxin. DT390 mGM-CSF was also able to specifically inhibit normal committed bone marrow (BM) progenitor cells as measured in clonal colony-forming unit granulocyte-macrophage assays. Together, these findings indicate that DT390 mGM-CSF may be useful as a novel tool for purging BM of contaminating leukemia cells or in vivo for eliminating residual leukemia cells. Also, it can be used to determine whether committed and/or noncommitted BM progenitor cells express the GM-CSF receptor.     

Preferential growth inhibition of human leukemic versus normal myeloid colony-forming cells by 12-O-tetradecanoylphorbol-13-acetate

Myeloid leukemic cells can be induced to differentiate by various compounds, suggesting the possibility of controlling leukemia through induced differentiation. For this to be feasible, the growth of leukemic progenitor cells should be inhibited by these compounds, with the inhibition preferentially affecting leukemic over normal hemopoietic progenitor cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was chosen as a differentiation inducer and was studied for its effect on the growth of leukemic colony-forming cells (L-CFU) in ten patients with acute nonlymphocytic leukemia in comparison with normal myeloid colony-forming cells (CFU-C). Growth inhibition of both L-CFU and CFU-C was observed with TPA concentrations as low as 10(-10) M. With increasing concentrations of TPA, survival of L-CFU tended to decline more precipitously than that of CFU-C. In eight of ten patients, inhibition of L-CFU was significantly greater (P less than 0.01) than CFU-C with TPA concentrations of 10(-9) M or higher. This study indicates that a compound capable of inducing differentiation of leukemic cells can inhibit growth of leukemic progenitor cells and that this growth inhibition applies preferentially to leukemic cells as compared with normal hemopoietic cells.     

Proliferative potential of subpopulations of granulocyte-macrophage progenitor cells in normal subjects and chronic myelogenous leukemia patients

The studies described compare the subpopulations of granulocyte- macrophage progenitor cells present in normal marrow with those derived from the marrow of patients with Ph1-positive chronic myelogenous leukemia (CML). The subpopulations were separated on the basis of size by velocity sedimentation and measured for their proliferative capacity by the colony formation technique. A pattern of development of colonies in the individual fractions was obtained by assaying the absolute number of colonies present at time intervals from 3 to 21 days. The number of colonies present at 3 days was taken as 100%, and the percentage of increase or decrease from this value was determined on subsequent days. In the fractions containing the most rapidly sedimenting large cells, the pattern of development of colonies derived from normal and CML marrow was similar. The CML colony-forming units in culture (CFU-C) began to show a deviation from the normal CFU-C pattern of development in the fractions containing CFU-C intermediate in size, and this deviation became progressively more pronounced in the slowest sedimenting small cell fractions. In these latter fractions, the CFU-C derived from CML marrow decreased in number at a rate similar to those arising from the more rapidly sedimenting fractions. This is in contrast to CFU-C derived from normal marrow, which increased in number in the more slowly sedimenting fractions and in the intermediate fractions, remained constant in number, or decreased at a rate slower than those arising from the more rapidly sedimenting fractions. The most likely explanation for these findings is accelerated maturation of the early small granulocyte-macrophage progenitor cells in CML so that these cells show the same limited proliferative capacity as do the later larger progenitor cells.     

Interactions between lymphocytes and hematopoietic progenitor cells in normal and leukemic human bone marrow (phase contrast observations)

Summary Single cell observations of normal and of leukemic human bone marrow cells demonstrated cell-cell interactions of lymphocytes with hematopoietic progenitor cells. In all cases lymphocytes and target cells were from the same individual. Lymphocyte-target cell interactions occurred more frequently with normal committed progenitor cells and leukemic blast cells from acute myeloid leukemia than with precursor cells of the proliferative cell pool, including granuloblasts, promonocytes, erythroblasts and megakaryocytes. Both induction of mitosis and degeneration of the progenitor cells occurred after cell-cell interaction with almost the same frequency. Acute myeloid leukemic blast cells degenerated after contact with lymphocytes with the same frequency as normal progenitor cells (i.e. in 16% of cell contacts), but especially during mitosis. In contrast, normal and regenerating bone marrow progenitor cells from myeloproliferative diseases demonstrated no degeneration after cell-cell interaction with lymphocytes during mitosis. Otherwise the induction of mitoses by lymphocyte-target cell interactions was more frequently observed in normal progenitor cells than in leukemic blasts.With technical assistance of Miss C. DomeyerSupported by Deutsche Forschungsgemeinschaft     

Recombinant alpha-interferon inhibits colony formation of bone marrow fibroblast progenitor cells (CFU-F).

Alpha-Interferon (IFN-alpha) has been shown to inhibit colony formation of hematopoietic progenitor cells, including colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), day 7 colony-forming unit granulocyte-macrophage (CFU-GM), day 14 CFU-GM, burst-forming unit erythroid (BFU-E), pluripotent stem cells (CFU-S), and colony-forming unit megakaryocyte (CFU-MK). The present study was designed to see whether IFN-alpha also has inhibitory effects on bone marrow fibroblast progenitors (CFU-F). We found that IFN-alpha exerted a significant inhibitory effect on both rabbit and human CFU-F formation. Inhibition of human marrow CFU-F formation by alpha interferon was unaffected by removal of 98% of monocytes/macrophages and T lymphocytes from light density marrow cells. This finding suggests that IFN-alpha probably exerts a direct inhibitory effects.