Exogenous Colony-stimulating Factor Research Articles

Inhibition of normal human macrophage progenitors by retinoic acid.

The effects of retinoic acid on the clonal growth in nutrient agar of neutrophil and macrophage progenitors from normal bone marrow and fetal liver were studied. The technique of whole plate fixation and staining with luxol fast blue and haematoxylin was employed to distinguish colonies and clusters into morphological groups. Retinoic acid at a concentration of 3 X 10(-7) M resulted in 97% inhibition of macrophage clonal growth and a mean increase in neutrophil clonal growth from bone marrow of 150 +/- 14%. These effects of retinoic acid were most obvious on bone marrow cluster forming cells. The inhibition of macrophage cluster growth was independent of exogenous colony stimulating factor. This dual action of retinoic acid is likely to be a direct effect on the progenitor cells since it is (1) independent of cell concentration, (2) not mediated by phagocytic cells or other bone marrow cells, and (3) was maximal in the first 2 d of incubation before the appearance of clusters.

Radiosensitivity of haematopoietic stem cells (DCPC and CFU-GM) from cord blood.

The radiosensitivity of human cord blood haematopoietic stem cells was measured by assessing colony formation in methylcellulose cultures and diffusion chambers. The results show that fetal colony forming units, granulocytic, monocytic (CFU-GM), are radiosensitive: colonies formed by electron-irradiated CFU-GM, both in spontaneous and in colony stimulating factor (CSF) supplemented cultures decrease in number in an exponential fashion with increasing doses of irradiation. Do values up to 800 rads were, for highly purified fetal lymphoid cells, 160 +/- 88 rads; for whole buffy coat cells, 155 +/- 75 rads; and for buffy coat cells plus exogenous CSF: 155 +/- 77 rads. Extrapolation numbers (N) approached the value of 1. Among cord blood diffusion chambers progenitor cells (DCPC), radiosensitive and radioresistant subpopulations of myelopoietic stem cells exist, as tested over a range up to 1500 rads. Monocytopoietic CFU-D are radioresistant.

Glucocorticoids and prostaglandins inhibit the induction of macrophage DNA synthesis by macrophage growth factor and phorbol ester.

The tumor-promoting phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), stimulates starch-elicited mouse peritoneal macrophages to undergo DNA synthesis in vitro, apparently without the generation of an endogenous macrophage growth factor (MGF). No evidence was found for any synergistic interaction between TPA and exogenous colony stimulating factors (CSFs) for macrophage DNA synthesis. Low concentrations of glucocorticoids and also prostaglandins E1 and E2 suppress both the CSF-1-stimulated and the TPA-stimulated macrophage DNA synthesis; these same drugs inhibit the CSF-1-mediated and TPA-mediated enhancement of macrophage plasminogen activator (PA) activity. Thus glucocorticoids and prostaglandins E1 and E2 oppose the action of growth factors and the tumor promoter on macrophage and precursor cell function.

Cellular aggregates in bone marrow cultures of patients with acute myeloid leukemia.

We describe the incidence and morphology of cellular aggregates which may develop in 8 day bone marrow (BM) cultures of patients with acute myeloid leukemia (AML). Aggregates formed in at least one BM culture from 50% (20/39) of the AML group. They developed irrespective of the patient's status (i.e. stages M1-M4), FAB type, and presence of colony stimulating factor (CSF). All aggregates were composed of macrophages, plasma cells, and cells of the myelocyte series surrounding a core of adipocytes and collagen fibrils. The percentage of blasts and promyelocytes in the plated BM aspirate governed the final composition of the aggregate. Patients in Stages M3 or M4 with FAB types M1 or M2 formed aggregates with a high proportion of myelocytic cells; aggregates of all other AML patients were composed predominantly of macrophages and plasmacytes. Aggregates appeared to form as a result of attraction of cells in the medium toward the stroma cell core. Furthermore, the development of aggregates in the absence of exogenous CSF, suggested that stromal cells excreted a factor with CSF-like activity. The results indicate that cellular aggregates in AML-BM cultures reflect the important role of BM stroma in creating microenvironments which enhance the development of hemopoietic stem cells.

Separate actions of different colony stimulating factors from human placental conditioned medium on human hemopoietic progenitor cell survival and proliferation

More than 20% of human granulocyte-macrophage and eosinophil colony-forming cells survived in agar culture for up to 4 days without the addition of exogenous colony stimulating factors (human placental-conditioned medium, HPCM). Survival was reduced slightly but not significantly, by the removal of adherent cell populations. Significant survival occurred even when only 100 cells enriched for colony-forming cells (CFCs) were cultured per dish. When individual colonies, initiated by stimulation with HPCM for 5 days, were transferred to dishes without HPCM, subsequent proliferation was significantly reduced compared with control cultures containing HPCM. Using the fluorescence-activated cell sorter and the fluoresceinated lectin from Lotus tetragonolobus, two populations of marrow cells were obtained, one enriched for day 7 and the other for day 14 colony-forming cells. Two colony-stimulating factors fractionated from HPLCM (CSF beta and CSF alpha) have been shown previously to stimulate the day 7 and day 14 colony-forming cell populations, respectively. Developing clones from cultures initiated with CSF beta died between the fifth and tenth day of culture after transfer to dishes with CSF alpha or CSF beta or to dishes with no stimulus. Cells in clusters initiated with CSF alpha proliferated significantly between the fifth and tenth day of culture when transferred to CSF alpha or CSF beta but not when transferred to dishes with not stimulus. These studies provide further evidence for the existence of two subtypes of human granulocyte-macrophage progenitor cells each under the primary control of a specific regulator and indicate that these two regulators can both act on some developing clones of cells.

Circulating haematopoietic stem cells (CFUc) in healthy and diseased pre-term infants.

During fetal life, haematopoiesis is in a physiologic state of rapid expansion. The activity of the pluripotent haematopoietic stem cell compartment of the bone marrow probably is reflected by the number and proliferative potentiality of CFUc in the blood circulation. We have studied, by the methylcellulose culture technique and without addition of exogenous colony stimulating factor (CSF), blood CFUc in pre-term and full-term newborn infants, in full-term infants 10 to 17 d of age, and in adults. Two groups of pre-term infants were studied: otherwise healthy infants and sick infants, including infants small-for-date and in perinatal distress. In healthy pre-term infants and in full-term infants 147 +/- 25 (SD) and 105 +/- 41 (SD) colonies per 100 000 lymphoid cells were formed, respectively. This is clear evidence for the presence in the blood of both. CFUc and CSF producing cells. By contrast, there was no or only slight spontaneous colony formation by sick pre-term infants, by older infants, and by adult.

Regulation of Myelopoiesis In Vitro: Partial Replacement of Colony-Stimulating Factors by Tumor-Promoting Phorbol Esters

AbstractTumor-promoting phorbol esters such as 12–0-tetradecanoyl-phorbol-13-acetate (TPA) or phorbol-12,13-didecanoate (PDD) stimulate freshly isolated murine bone marrow cells in vitro to form colonies of myeloid cells in semisolid medium without added colony-stimulating factors (CSF), while phorbol and its nonpromoting derivatives do not induce marrow colony formation. TPA-stimulated colonies are composed primarily of monocyte-macrophage cells with admixed granulocytes in some colonies, and cell dose-response curves for colony formation suggest clonal origin of the colonies. The cells forming colonies in the presence of TPA alone appear to be a subset of marrow granulocyte-macrophage colony-forming cells (GM-CFC) characterized by larger mean cell size (determined by velocity sedimentation), and TPA-stimulated colony formation is inhibited by dexamethasone and prostaglandin E1, agents that also inhibit GM-CFC colony formation stimulated by CSF. The active phorbol esters can apparently replace exogenous CSF in agar cultures of murine bone marrow and may do so by either (1) directly stimulating GM-CFC. (2) inducing endogenous CSF secretion, or (3) increasing GM-CFC’s responsiveness to CSF. We were unable to demonstrate induction of CSF secretion by marrow cells in liquid culture in the presence of TPA. We did find that TPA synergistically enhanced colony formation stimulated by suboptimal concentrations of CSF from two sources, and this effect of TPA was concentration dependent. PDD also enhanced colony formation stimulated by suboptimal CSF concentrations, while phorbol had no effect. In long-term flask cultures of normal murine marrow, TPA caused rapid, concentration-dependent, terminal differentiation (to macrophages), and decline in the progenitor cell compartment. Our data indicate that tumor-promoting phorbol esters may alter the function of cell surface receptors for CSF on GM-CFC, causing stimulation of proliferation and differentiation in the absence of CSF or at very low concentrations of CSF. These compounds should be important in the study of hematopoietic regulation.

Cell interactions in the bone marrow microenvironment: role of endogenous colony-stimulating activity.

Adherent stromal cells from mouse bone marrow inhibited the formation of granulocyte/monocyte (G/M) colonies induced in vitro by colony-stimulating factor (CSF). This inhibition occurred both when crude conditioned media obtained from various sources were used to induce colony formation or when a pure CSF preparation from mouse lung origin was tested. The inhibition did not appear to be toxic in nature since despite the lack of colony formation, progenitor CFU-C proliferated in the presence of stromal cells. Medium conditioned by adherent stromal cells was devoid of inhibitory activity when incorporated into the culture medium used for G/M colony formation, indicating that the inhibitory activity may not be present in a soluble form. Inhibitors of prostaglandins did not affect G/M colony formation. In contrast, D-glucose and a number of other free monosaccharides but not pyruvate lactate or glycerol induced formation of myeloid colonies in the presence of stromal cells. This did not require addition of exogenous CSF. Released factors concentrated from serum-free medium conditioned by stromal cells exhibited colony-stimulating activity provided that the medium contained a high glucose concentration during incubation. It is proposed that stromal cells produce a resident CSF that, in contrast to exogenous CSF species, is capable of inducing myelopoiesis within the bone and marrow stroma.

The effect of sonicated serum on growth of normal and leukaemic human cells in agar cultures.

The existence of circulating factors in blood of possible importance for granulopoiesis was investigated by examining the effect of sonicated serum on agar cultures of human haematopoietic cells. Ultrasound treatment of serum can activate enzymes normally bound to carrier proteins. In normal bone marrow cultures, growth was inhibited by sonicated serum when the cells were cultured in a single layer without exogenous colony-stimulating factor (CSF) included in the culture, while an enhancing effect with a 2--5 fold increase in the number of colonies was seen in feeder layer-stimulated cultures. Morphologically, in contrast to the normal change to eosinophils and monocytes-macrophages during the culture period, the cultures with sonicated serum added showed continuous growth of neutrophils and no increase of other cell types. Experiments using tritiated thymidine indicated that the enhancing effect of sonicated serum involved marrow cells which were more prone to thymidine S-phase kill. The effect of sonicated serum was further evaluated in cultures from patients with leukaemia. In AML in relapse, feeder-layer stimulated cultures of bone marrow cells were inhibited (11/14 cases) by sonicated serum, while reversion to the normal enhancing pattern was seen for patients in remission.

Hemopoietic colony forming cells in regenerating mouse liver.

Cells from regenerating mouse liver removed 2-25 days post 68% hepatic resection have been assayed for in vivo colony forming capacity in soft agar (CFU-C), proliferative capacity in liquid culture, and in vivo spleen colony forming capacity (CFU-S). These studies demonstrated low concentrations of CFU-C and CFU-S in normal and sham operated liver, with an appreciable increase of both in regenerating liver, reaching maximum values in tissue removed 5--7 days post hepatic resection. Colony formation in agar by regenerating liver cells occurred in the absence of exogenous colony stimulating factor. Separation of liver cells on the basis of adherent properties prior to culture indicated concentration of CFU-C in the non-adherent fraction, while cells producing colony stimulating factor were concentrated in the adherent fraction. Foci of actively dividing cells of themacrophage and granulocyte series arose in liquid culture from preparations of sham operated and regenerating liver, although total cell formation was greater with regenerating liver. A small proportion of the colonies formed in agar from regenerating liver consisted of cords of epithelioid cells, which resembled hepatocytes and differed from the macrophages or granulocytes found in the majority of colonies, raising the possibility that regenerating hepatocytes form colonies in agar culture.

Attempts to transform murine hemopoietic cells by Rauscher leukemia virus.

Primary Rauscher leukemia virus (RLV)-induced myeloid leukemias can produce many small clones in agar in the absence of a factor needed for the proliferation of normal myeloid cells. It seems that leukemic cells can more efficiently utilize the small amount of colony-stimulating factor (CSF) that is produced by them. At optimal stimulation by exogenous CSF, leukemic cells exhibit a poorer rate of proliferation than normal bone marrow cells. Hemopoietic cells can replicate the virus after infection in vitro. In some experiments, infection of normal bone marrow cells leads to the production of some cells with the same growth pattern as cells from primary leukemias.