The in vitro differentiation of density sub-populations of colony-forming cells under the influence of different types of colony-stimulating factor.

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density subpopulations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSFMLCM), post-endotoxin mouse serum (CSFES) and from human urine (CSFHu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macrophage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

THE IN VITRO DIFFERENTIATION OF DENSITY SUB-POPULATIONS OF COLONY-FORMING CELLS UNDER THE INFLUENCE OF DIFFERENT TYPES OF COLONY-STIMULATING FACTOR

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density sub-populations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSF_MLCM), post-endotoxin mouse serum (CSF_ES) and from human urine (CSF_Hu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macro-phage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

Effects of human leukocyte conditioned medium on mouse haemopoietic progenitor cells.

Medium conditioned by human peripheral blood leukocytes (HLCM) was studied for its in vitro effects on haemopoietic progenitor cells (CFU-s and CFU-c) present in mouse bone marrow. HLCM has poor colony stimulating activity in semi-solid cultures of mouse bone marrow cells, but invariably increases the number of colonies obtained in the presence of plateau levels of semi-purified colony stimulating factor (CSF). In liquid cultures, HLCM appears to contain a potent initiator of DNA synthesis in CFU-s, an activity which coincides with an increased CFU-s maintenance and causes a three- to four-fold increase in CFU-c number. It is apparent from this study that HLCM, in addition to stimulating colony formation in cultures of human bone marrow cells, has a profound in vitro effect on primitive haemopoietic progenitor cells of the mouse, which cannot be attributed to CSF.     

EFFECTS OF HUMAN LEUKOCYTE CONDITIONED MEDIUM ON MOUSE HAEMOPOIETIC PROGENITOR CELLS

Medium conditioned by human peripheral blood leukocytes (HLCM) was studied for its in vitro effects on haemopoietic progenitor cells (CFU-s and CFU-c) present in mouse bone marrow. HLCM has poor colony stimulating activity in semi-solid cultures of mouse bone marrow cells. but invariably increases the number of colonies obtained in the presence of plateau levels of semi-purified colony stimulating factor (CSF). In liquid cultures, HLCM appears to contain a potent initiator of DNA synthesis in CFU-s. an activity which coincides with an increased CFU-s maintenance and causes a three- to four-fold increase in CFU-c number. It is apparent from this study that HLCM, in addition to stimulating colony formation in cultures of human bone marrow cells, has a profound in vitro effect on primitive haemopoietic progenitor cells of the mouse, which cannot be attributed to CSF.     

Formation of eosinophilic-like granulocytic colonies by mouse bone marrow cells in vitro

Formation of granulocytic and macrophage colonies in agar cultures of mouse marrow or spleen cells was stimulated by the addition of medium from pokeweed mitogen-stimulated cultures of mouse spleen cells (PKW-CM). Approximately 5% of the colonies developing were large, dispersed granulocytic colonies (DG-colonies) composed of cells with eosinophilic cytoplasmic granules. The capacity to stimulate DG-colonies was shown by media conditioned by PKW-treated lymphoid and peritoneal cells but not by other cells or organ fragments. Velocity sedimentation studies indicated that cells generating DG-colonies were separable from cells generating regular granulocytic or macrophage colonies. DG-colonies did not survive if transfered to cultures containing other forms of CSF. The active colony stimulating factor in pokeweed mitogen-conditioned medium which stimulates DG-colony formation was antigenically distinct from the factor stimulating granulocytic and macrophage colony formation, was separable electrophoretically from the latter factor and on gel filtration had an apparent molecular weight of 50,000. Although the cells in DG-colonies have not been established to be eosinophils, DG-colonies represent an interesting new system for analysing further aspects of the control of growth and differentiation in hemopoietic populations.     

An in vitro assay for T lymphocyte progenitors (CFU-preT)

Thy-1.2 negative progenitors give rise to Thy-1.2 positive colony cells when mouse bone marrow is cultured in vitro. The bone marrow cells are immobilized in a viscous medium containing methyl cellulose; discrete colonies are identifiable at 2 days and contain 30–60 cells by day 3 of culture. Colonies are tightly packed spheres (raspberries) and grow suspended in the gel. Growth of the raspberry colonies is absolutely dependent upon the presence of the appropriate serum (horse or human; not fetal calf) and conditioned medium from pokeweed mitogen-stimulated mouse spleen cells. As little as 0.1% of the conditioned medium is sufficient to promote raspberry colony growth. Under these conditions, nude mouse bone marrow yields as many colonies (1 per 1,000 nucleated cells plated) as normal marrow. Thymus, lymph node; and spleen (normal or nude) do not form colonies. Colony precursors are predominantly in S phase of the cell cycle, as determined by tritiated thymidine suicide of fresh bone marrow. Their numbers fall with age. Because the cells in colonies are Thy-1 positive, peanut agglutinin-positive, and active in a pre-T cell synergy assay, we conclude that their precursors are early committed T cell progenitors, and propose that they be called CFU-preT.     

Effects of okadaic acid on mouse hemopoietic cells

Effects of okadaic acid, a potent non-12-O-tetradecanoyl-phorbol-13-acetate(TPA)-type tumor promoter, on mouse hemopoietic cells were investigated. Okadaic acid stimulated mouse bone marrow cells to form granulocyte-macrophage colony-forming unit (CFU-GM) colonies without added colony stimulating factors(CSFs). At the concentration of 1.82 x 10(-8) M, colony formation of 77 +/- 14 colonies/1 x 10(5) bone marrow cells was observed. Observations on the effects of other cells on the CSF induction suggested that okadaic acid primarily stimulated the functions of macrophages, and the CSF production from macrophages might be attributed to the CFU-GM colony formation. On the other hand, the erythroid colony-forming unit(CFU-E) colony formation stimulated by     

Heterogeneity of in vitro colony- and cluster-forming cells in the mouse marrow: segregation by velocity sedimentation.

C57BL bone marrow cells were separated on the basis of their sedimentation velocity at unit gravity and cell fractions cultured in agar using three types of colony stimulating factor (CSF). Colony-forming cells separated as a single peak (s equal 4.4 mm/hr) in cultures stimulated by mouse lung conditioned medium (CSFMLCM) or endotoxin serum (CSFES). Cluster-forming cells were separable into two peaks and the majority were larger than colony-forming cells (s equal 5.7 mm/hr). Partial segregation of colony-forming cells was observed according to the morphological types of colonies generated, large cells tending to generate macrophage colonies and small cells, granulocytic colonies. Large colony-forming cells were more responsive to stimulation by CSF than small cells. Human urine (CSFHU) appeared unable to proliferation of most small colony-forming cells. Colony-forming cells appear to be a highly heterogeneous population with intrinsic differences in responsiveness to CSF and with differing capacities to generate colonies whose cells differentiate to granulocytes of macrophages.     

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

Using a modification of the agar gel method for bone marrow culture, serum from various strains of mice has been tested for colony stimulating activity. Ninety percent of sera from AKR mice with spontaneous or transplanted lymphoid leukemia and 40–50% of sera from normal or preleukemic AKR mice stimulated colony formation by C57B1 bone marrow cells. Sera from 6% of C3H and 30% of C57B1 mice stimulated similar colony formation. The incidence of sera with colony stimulating activity rose with increasing age. All colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells. Bone marrow cells exhibited considerable variation in their responsiveness to stimulation by mouse serum. Increasing the serum dose increased the number and size of bone marrow cell colonies and with optimal serum doses, 1 in 1000 bone marrow cells formed a cell colony. Preincubation of cells with active serum did not stimulate colony formation by washed bone marrow cells. The active factor in serum was filterable, non-dialysable and heat and ether labile.     

Colony-stimulating factors and regulation of macrophage tumoricidal and microbicidal activities

Conditioned medium from antigen- or mitogen-stimulated spleen cells, lymphokines, contained factors that induced formation of granulocyte and macrophage colonies in cultures of bone marrow cells (CSF). Lymphokines also contained factors that induced macrophage non-specific tumoricidal activity against fibrosarcoma 1023, antibody-dependent tumoricidal activity against lymphoma 18-8, and antimicrobial activities against amastigotes of the protozoan parasite, Leishmania tropica. The factors that regulated macrophage effector functions, however, were different from those that induced colony formation, and could be distinguished from CSF by Sephadex gel chromatography or heat sensitivity. To further analyze a role for CSF in induction of macrophage effector activities, conditioned medium from several nonlymphoid cell sources (L-929, WEHI-3, and endotoxin-treated lung cells) were assayed for CSF activities and capacity to induce tumoricidal and microbicidal activities. Conditioned medium that contained either macrophages CSF (CSF-1) or the factor that induced formation of both macrophage and granulocyte colonies failed to activate macrophages for effector activities against fibrosarcoma 1023, lymphoma 18-8, and L. tropica amastigotes (either resistance to infection or intracellular destruction). These data suggest that CSF has no direct role in activation of macrophages for tumoricidal and microbicidal activities against these targets.     

Characterization of erythropoietin-like activity from mouse spleen cells

Supernatants from mouse spleen cell cultures contain a factor which acts in a similar manner to erythropoietin (Ep) to stimulate the formation of 2-day erythroid (CFU-E) colonies in vitro from bone marrow or fetal liver cells. Analysis of conditioned media by high performance liquid chromatography (HPLC) on anion exchange, reverse phase, molecular size exclusion, and hydroxyapatite columns demonstrated that the erythropoietin-like activity (EpLA) has different biochemical characteristics to mouse Ep from anemic mouse serum. In addition, EpLA has a molecular weight (Mr), of 20,000 daltons determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), compared to 42,000 for mouse Ep. Partially purified EpLA was found to be active in vivo as well as in vitro. Highly purified preparations of gamma-interferon, Multilineage hemopoietic growth factor (Multi HGF), Interleukin-2 (IL-2), IL-1, and colony stimulating factor 1 (CSF-1) did not support CFU-E colony formation. Thus, it was established that EpLA could not be attributed to other known components of spleen cell conditioned medium. Titration of mouse Ep and EpLA suggests that only a portion of the Ep-responsive CFU-E population in fetal liver is sensitive to EpLA.     

人骨髓细胞培养液中干细胞刺激物的分析研究

人骨髓细胞体外培养液中含有高活力的 CSF,在长期培养过程中,CSF 活力的变化,与 CFU-C 数量的变化有大致平行的趋势。这种 CSF 对狗和小鼠也同样有效。人骨體条件液中的 CSF 对培养中的 CFU-S 也有明显的激发作用。这一结论可以从几个方面获得证据:第一,小鼠骨髓细胞与人骨髓条件液保温六小时后,再测定其中 CFU-S 数,结果是增加了。第二,经亚致死剂量照射的小鼠,腹腔注射适量的人骨髓条件液,其内源性脾结节也明显增多。第三,采用阿糖胞苷自杀的方法,测定小鼠骨髓经与人骨髓条件液保温后,其中 CFU-S 的自杀率也有增高的趋势。上述几方面的实验,说明人骨髓长期培养中存在着某种活性物质,调节体外造血。至于这种物质的来源,以及在体外造血中所起的作用,还需要做很多工作,逐步予以澄清。     

Serum of lipopolysacharide-treated mice contains two types of colony-stimulating factor,separable by affinity chromatography

Serum from mice traated with bacterial lipopolysaccharide (LPS) was fractionated by Con A-Sepharose affinity chromatography, and assayed in vitro for colony-stimulating factor (CSF) using mouse bone marrow cells. The CSF failing to bind to concanavalin A-Sepharose (pool A) had similar biological properties to the unfractionated serum, i.e., it stimulated the formation of about equal numbers of granulocytic, mixed granulocyte-macrophage and macrophage colonies. The fraction eluted from the Con A-Sepharose column with α-methyl-D-glucopyranoside (pool B) had a steeper dose-response curve than either the unfractionated serum or the pool A CSF and most of the colonies were composed of macrophages. A mixture of the pool A and pool B CSFs stimulated colonies in a similar way as unfractionated serum and pool A. The apparent molecular weights of the two types of CSF were determined by two different gel-filtration procedures. Sephacryl S-200 gel-filtration suggested an apparent molecular weight of 85,000 for pool A CSF and 180,000 for pool B CSF. Gel-filtration on Sepharose CL-6B in the presence of guanidine hydrochloride (6M) yielded an apparent molecular weight of approximately 23,000 for pool A CSF and 33,000 for pool B CSF. The colony-forming cells (CFC) responding to pool B CSF were found to have a relatively high sedimentation velocity (peak sedimentation velocity 5.6–6.2 mm/hr) compared to the CFC responding to mouse-lung conditioned medium (MLCM) whose peak sedimentation velocity was between 4.0–4.5 mm/hour. The CFC responding to pool A CSF had an intermediate sedimentation velocity (peak 4.6–5.2 mm/hour). A time-course analysis of the morphology of clones or colonies in cultures stimulated with either MLCM or pool B CSF showed that the proporation of different colony types depends significantly on the incubation period and suggested that pool B CSF induced an early commitment of CFC towards macrophage differentiation.     

Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar

Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%–10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony-forming cells from the peritoneal exudate are similar to bone marrow colony-forming cells in vitro in that they both require a substance(s) present in conditioned medium from L-cells or mouse embryo fibroblasts or the serum from endotoxin-treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony-forming cells have a much longer initial lag period (10–14 days) and can survive longer in the absence of L-cell conditioned medium than bone marrow colony-forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.     

The suppressive influences of human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: synergism of tumor necrosis factor and interferon-gamma

The influences of human tumor necrosis factor (TNF) (LuKII), recombinant human TNF-alpha, natural human interferon-gamma (HuIFN-gamma), recombinant HuIFN-gamma, and natural HuIFN-alpha were evaluated alone or in combination for their effects in vitro on colony formation by human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells incubated at 5% CO2 in lowered (5%) O2 tension. TNF (LuKII) and recombinant TNF-alpha caused a similar dose-dependent inhibition of colony formation from CFU-GM, BFU-E, and CFU-GEMM. Day 7 CFU-GM colonies were more sensitive than both day 14 CFU-GM colonies and day 7 CFU-GM clusters to inhibition by TNF. BFU-E colonies and CFU-GEMM colonies were least sensitive to inhibition with TNF. The suppressive effects of TNF (LuKII) and recombinant TNF-alpha were inactivated respectively with hetero-anti-human TNF (LuKII) and monoclonal anti-recombinant human TNF-alpha. The hetero-anti-TNF (LuKII) did not inactivate the suppressive effects of TNF-alpha and the monoclonal anti-recombinant TNF-alpha did not inactivate TNF (LuKII). The suppressive effects of TNF did not appear to be mediated via endogenous T lymphocytes and/or monocytes in the bone marrow preparation, and a pulse exposure of marrow cells with TNF for 60 min resulted in maximal or near maximal inhibition when compared with cells left with TNF for the full culture incubation period. A degree of species specificity was noted in that human TNF were more active against human marrow CFU-GM colonies than against mouse marrow CFU-GM colonies. Samples of bone marrow from patients with non-remission myeloid leukemia were set up in the CFU-GM assay and formed the characteristic abnormal growth pattern of large numbers of small sized clusters. These cluster-forming cells were more sensitive to inhibition by TNF than were the CFU-GM colonies and clusters grown from the bone marrow of normal donors. The sensitivity to TNF of colony formation by CFU-GM of patients with acute myelogenous leukemia in partial or complete remission was comparable with that of normal donors. When combinations of TNF and HuIFN were evaluated together, it was noted that TNF (LuKII) or recombinant TNF synergized with natural or recombinant HuIFN-gamma, but not with HuIFN-alpha, to suppress colony formation of CFU-GM, BFU-E, and CFU-GEMM from bone marrow of normal donors at concentrations that had no suppressive effects when molecules were used alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

The nature of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated hemopoiesis, colony stimulating factor (CSF) requirement for colony formation, and the effect of TPA on [125I]CSF-1 binding to macrophages

The tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) was found to act both independently of and synergistically with the mononuclear phagocyte specific colony stimulating factor (CSF-1) to stimulate the formation of macrophage colonies in cultures of mouse bone marrow cells. In contrast, TPA did not synergize with other CSF subclasses that stimulate the formation of eosinophil, eosinophil-neutrophil, neutrophil, neutrophil-macrophage, and macrophage colonies, nor with either of the two factors required for megakaryocyte colony formation, megakaryocyte CSF, and megakaryocyte colony potentiator. In serum-free mouse bone marrow cell cultures TPA retained the ability to independently stimulate macrophage colony formation. However, TPA-stimulated colony formation was suboptimal and delayed in serum-free cultures that could support optimal colony formation in the presence of CSF-1. In addition, TPA did not directly compete with [125I]CSF-1 at 4 degrees C for its specific, high-affinity receptor on mouse peritoneal exudate macrophages. However, a 2-hour preincubation of the cells with TPA at 37 degrees caused almost complete loss of the receptor. Thus, TPA is able to mimic CSF-1 in its effects on CSF-1 responsive cells in some aspects (the spectrum of target cells, the morphology of resulting colonies, and the ability to down-regulate the CSF-1 receptor) but it is not able to mimic CSF-1 in other ways (TPA alone cannot stimulate the full CSF-1 response, TPA does not stimulate the most primitive CSF-1 responsive cells, and TPA does not bind to the CSF-1 receptor).     

Studies on colony formation in vitro by mouse bone marrow cells. II. Action of colony stimulating factor

An analysis was made of some of the processes involved in the stimulation by colony stimulating factor (CSF) of cluster and colony formation by mouse bone marrow cells in agar cultures in vitro. Colony formation was shown to be related to the concentration and not the total amount of CSF. The concentration of CSF determined the rate of new cluster initiation in cultures and the rate of growth of individual clusters. Colony growth depleted the medium of CSF suggesting that colony cells may utilise CSF during proliferation. Bone marrow cells incubated in agar in the absence of CSF rapidly died or lost their capacity to proliferate and form clusters or colonies. CSF appears (a) to be necessary for survival of cluster-and colony-forming cells or for survival of their proliferative potential, (b) to shorten the lag period before individual cells commence proliferation and (c) to increase the growth rate of individual clusters and colonies.     

The role of humoral factors in the regression of leukemia in chickens as measured by in vitro colony formation

Leukemic myeloblasts induced by avian myeloblastosis virus in the chicken formed small compact (type II) colonies in semi-solid agar medium. Normal yolk sac cells from 12-day old embryos formed large diffuse (type I) colonies under the same conditions. Type I colony formation (but not type II) was strictly dependent upon the presence in the medium of a colony stimulating factor (CSF) present in fresh chicken serum or conditioned medium. Serum CSF levels were determined for normal, leukemic, and birds which had spontaneously regressed from myeloblastic leukemia. When type I colony formation was used as the assay, serum CSF levels of leukemic birds were found to be significantly lower than levels in either normal or regressed birds. When the same sera were tested for their ability to induce type II colonies, leukemic birds demonstrated a significantly higher CSF level than either normal or regressed sera. Regressed chickens had serum CSF levels similar to normal birds.     

DNA topoisomerase inhibitors block erythropoiesis and delay hemoglobinization in vitro

To examine the importance of topological constraints on DNA during erythroid development, we measured the effects of camptothecin and teniposide, two tumoricidal agents which are also specific inhibitors of type I and type II topoisomerases respectively, on the formation of hematopoietic colonies by cultured human bone marrow cells. When added to bone marrow culture, each inhibitor alone impairs the formation of early BFU-E-derived colonies, late CFU-E-derived colonies and mixed hematopoietic (CFU-GEMM-derived) colonies by up to 100%. Inhibition of colony formation is directly related to the time of inhibitor addition and the inhibitor concentration tested. Although either inhibitor alone reduces colony formation by 90%, when added together at a submaximal concentration, camptothecin and teniposide exert a synergistic suppressive effect. Furthermore, addition of topoisomerase inhibitors to culture impairs hemoglobinization of colony erythroblasts in a time-dependent fashion. In contrast to the effects of topoisomerase inhibitors, the antiproliferative agent aphidicolin reduces erythroid colony number and size without altering hemoglobinization of colony erythroblasts. Since neither topoisomerase inhibitor alters the morphology of cultured cells, the capacity of cells to exclude trypan blue or the potential to form erythroid colonies through the interval required for the first progenitor cell division, it is unlikely that camptothecin or teniposide are cytotoxic to hematopoietic cells. Human mononuclear cells enriched in bone marrow lymphocytes and nucleated erythroblasts from both human and mouse sources release DNA into the detergent soluble fraction. Release requires functional topoisomerases and is altered by acute exposure to topoisomerase inhibitors. Our results suggest that topoisomerases are critical not only to proliferation but also to differentiation of human marrow erythroid progenitor cells and stem cells in culture.     

The effect of DLE fractions on GM-progenitors of haematopoietic stem cells in vitro

Dialysable leucocyte extract (DLE) prepared from buffy coats of human blood, potentiates the effect of Colony-stimulating factor (CSF) on the growth of granulocyte-macrophage colony forming cell (GM-CFC) colonies in vitro. This relative increase of the number of colonies is apparent when diluted CSF (present in lung conditioning medium) as a control, and DLE, in a wide range of concentrations are added to the culture of mouse bone marrow cells. Fractionation of DLE on Amicon membranes revealed that the activity resides in molecules of 0-5kD. Molecules 5-10kD have no potentiating effect. DLE and its fractions (0-5kD, 0-1kD), except fractions 0-500 D and 5-10kD, when added undiluted i.e. at the initial concentration, exerted a suppressive effect: colonies are not formed despite the presence of CSF. In a pilot experiment, it was shown that DLE is able to stimulate colony-forming activity of earlier progenitors of erythroid cells (BFUe), under the influence of erythropoietin.