Regulation of murine granulocyte-macrophage progenitor cell and haemopoietic stem cell proliferation by factors produced in human fetal liver

At early stages (11–14 weeks) of gestation in human fetal liver few granulocyte-macrophage colony-forming cells (GM-CFC) are in DNA synthesis, whereas later in gestation (> 14 weeks) a large proportion of GM-CFC are in S-phase [Moore M.A.S. & Williams N. (1973) Cell Tissue Kinet.6, 461].Incubation of normal murine bone marrow GM-CFC (approx. 40% in DNA synthesis) with a supernatant from an early human fetal liver (11–14 weeks), reduced the proportion synthesizing DNA to <5%. In contrast, the proportion of murine GM-CFC synthesizing DNA was not affected by incubation with a supernatant from a late fetal liver (> 14 weeks).GM-CFC that had been switched out of cycle by incubation with a supernatant from an early gestation human fetal liver were switched back into cycle following incubation with a late human fetal liver supernatant.It is likely that changes in the relative levels of a proliferation inhibitor and stimulator throughout gestation might control the proportion of GM-CFC in cycle. In normal murine bone marrow (NMBM) approx. 10% of the haematopoietic stem cells (CFU-S) are synthesizing DNA. The proportion of CFU-S synthesizing DNA was increased to approx. 40% by incubation with a human fetal liver supernatant from all gestational ages tested (11–18 weeks).The specificity of these CFU-S and GM-CFC proliferation regulators is well demonstrated by an early gestation human fetal liver supernatant which will stimulate CFU-S proliferation but inhibit GM-CFC proliferation.The inhibitor and stimulator of GM-CFC proliferation are both produced by non-adherent human fetal liver cells. The GM-CFC proliferation inhibitor has a mol. wt of > 100,000 and the stimulator a mol. wt of 30,000–50,000. In contradistinction, the CFU-S proliferation stimulator is produced by adherent human fetal liver cells and has a mol. wt of 30,000–50,000.     

A cellular analysis of residual hemopoietic deficiencies in mice after 4 repeated doses of 4.5 gray X rays

A sub-optimal plateau in numbers of femoral stem-cells (CFU-S) in mice after 4 doses of 4.5 Gray X rays (each separated by 21 days), was shown to persist at 20–30 % of control up to 1 year after the last dose, when about 50 % of the mice had survived. The concentration of white cells in the blood was maintained persistently at about 70% of control, whereas the concentration of red cells was normal up to 4 months and then it declined to about 75% of control at 10 months after irradiation. Concentrations of some committed progenitor cells in the marrow (GM-CFC and ERC), which are capable of amplification cell divisions, were intermediate between the concentrations of marrow stem cells and mature blood cells in both the granuloid and the erythroid cell lineages, respectively. Hence increased amplification was a mechanism operating for a prolonged period in the production of numbers of mature cells. The numbers were subnormal, however, and this corresponded to only 1 extra amplification division on average.There was a slow decline after 6 months in the numbers of CFU-S, BFU-E and GM-CFC, and in the hematocrit, with reference to age-matched controls. The decline was due partly to a prevention of the natural increase in cell numbers in the marrow with the age of the mice, which was also seen with the femoral content of a stromal progenitor cell (CFU-F). A defect in the repeatedly-irradiated CFU-S population was detected as a persistent inability to produce colonies containing the same number of daughter CFU-S as contained in colonies derived from unirradiated marrow and assayed at the same time.     

Some effects of chemotherapeutic drugs

Summary cis-Platinum is a relatively new active anticancer drug. In the study described in this paper, its toxicity was tested in the hematopoietic and renal systems of mice after six injections of 3 mg per kg body weight at 10-day intervals.Acute hematopoietic toxicity was studied by determining the survival of pluripotent (CFU-S) and granulo-macrophagic unipotent (GM-CFC) stem cells. The number of nucleated cells in the bone marrow and in the spleen and the number of granulocytes in the blood were determined.Renal toxicity was studied by histological examination of kidneys from treated mice compared with control animals.The number of stem cells in the bone marrow and in the spleen decreased during the treatment. One year after treatment, the autorepopulating ability of CFU-S was still diminished in spite of normal numbers of these cells.No renal damage could be demonstrated by light microscopy when the protocol described was used.Abbreviations used in this paper CFU-S pluripotent hemopoietic stem cells assessed by the spleen colony technique - GM-CFC granulo-macrophagic progenitor cells - BFU-E erythroid progenitor cells - E/G ratio ratio of erythroid and granulocytic colonies in the recipient spleen and assessed by histological examination - Ara-C cytosine arabinoside     

Further studies on the mechanism of CFU-S determination--II. Feedback regulation

In previous papers, we have demonstrated that the determination of CFU-S differentiation is under the control of humoral factors, which we have called pluripoietins. The activity of these regulators varies according to the experimental protocol. The aim of this work was to determine the mechanisms involved in the regulation of pluripoietin secretion after each treatment. The results suggest that the production of pluripoietin(s) is under the control of a feedback mechanism, originating, at least in part, in the progenitor compartments. After one dose of 20 mg of Ara-C, CFU-S determination is channelled towards erythropoiesis when the BFU-E compartment is more depleted than the GM-CFC compartment. During fractionated doses of Ara-C, at the time of the third injection, GM-CFC survival is lower than BFU-E survival and CFU-S orient their differentiation towards granulopoiesis. After bleeding, there is no difference of survival between the two compartments and CFU-S determination is not changed. These results seem to indicate that CFU-S determination is modified in order to restore normal homeostasis of the hemopoietic tissues by preferentially replenishing the more depleted compartment. This is most likely achieved by the secretion of pluripoietin(s) that vary in either their nature or their concentration according to the events occurring in the more mature compartments.     

Thermal sensitivity of haemopoietic and stromal progenitor cells in different proliferative states

Stromal progenitor cells (CFU-F) in normal mouse bone marrow were more sensitive to heat at 43 degrees C than haemopoietic progenitor cells (CFU-S and GM-CFC) by a factor of approximately 1.2. In marrow regenerating after 4.5 Gy X-rays, the changes in sensitivity were by less than a factor of 1.4 and the sensitivity of CFU-F changed slightly to become intermediate between that of CFU-S and GM-CFC. A comparison of sensitivities reported in the literature revealed an inexplicable large variation of up to a factor of 6 in the thermal sensitivities of CFU-S and GM-CFC.     

Synergistic interactions allow colony formation in vitro by murine haemopoietic stem cells

A clonogenic assay for cells that give rise to macroscopic colonies in agar or methyl cellulose cultures using untreated, normal murine bone marrow as a source of stem cells is described. We have characterized the clonogenic cell, which has been designated CFU-A, by comparing its properties with those of multipotential stem cells (assayed as CFU-S) and lineage-restricted progenitor cells (assayed as GM-CFC). The investigations have included assessment of proliferative status and response to CFU-S proliferation regulators, response to 5-fluorouracil, buoyant cell density, radial distribution in the femur and response to ionizing radiation. We conclude that the CFU-A has properties in common with CFU-S that differ from those of GM-CFC. The data are consistent with the CFU-A assay detecting part of the multipotential stem cell population also detected by spleen colony formation.     

Leucovorin antagonizes the effects of trimetrexate on mouse hemopoietic progenitors.

Trimetrexate (2, 4, diamino -5- methyl - 6 [3, 4, 5, trimethoxyanilino) methyl] quinazoline) (TMQ) is a non-classic folate antagonist that is used as an antineoplastic and antipneumocystis agent with promising results. TMQ and methotrexate (MTX) toxicities are comparable. Leucovorin (N-5-formyltetrahydrofolate) (LV) is used to prevent the toxic effects of MTX. In this study the effects of LV on TMQ induced hemopoietic progenitor damage are studied in a murine model. Changes of pluripotent stem cells (colony forming units spleen, CFU-S), granulocyte-macrophage committed progenitors (GM-CFC), erythroid committed progenitor (BFU-E) levels in the bone marrow were followed after administration to mice of a single dose of TMQ or of simultaneous injection of TMQ and LV. Results show that the latter significantly reduces the effects of the former on peripheral blood cells and on hemopoietic progenitors.     

Interaction of inhibitor and stimulator in the regulation of CFU-S proliferation

An inhibitor and stimulator of CFU-S proliferation can be obtained from haemopoietic tissue containing, respectively, relatively quiescent CFU-S (e.g. normal bone marrow) and proliferating CFU-S (e.g. regenerating bone marrow). In this paper we explore the capacity for inhibitor and stimulator production in relation to changes in the proliferative status of the CFU-S and their mode of interaction. The increase in proliferative activity of CFU-S following a concentrated regime of phenylhydrazine injections is paralleled by an increase in the capacity for stimulator production and a decrease in the capacity for inhibitor production. These processes are reversed as the normal low proliferative activity returns. Using density fractionated marrow populations, dose-response measurements show that while inhibitor- and stimulator-producing cells persist throughout the changes in CFU-S proliferation, their magnitude of factor production moderates in relation to those changes. The inhibitor and stimulator are not mutually destructive: the two factors retain their activities after co-incubation and re-separation. On the other hand, the presence of either factor blocks the synthesis of the other by the appropriate producer cells. A model for the regulation of CFU-S proliferation by the inhibitor and stimulator is thus suggested in which the relative spatial distributions of CFU-S in a microenvironment containing inhibitor- and stimulator-producing cells are an important feature. It also implies the existence of a signal from the CFU-S compartment which determines the appropriate inhibitor or stimulator production.     

A stimulator of mouse stem cell proliferation produced by human regenerating bone marrow

We examined CFU-S proliferation stimulator, which recruits stem cells in DNA synthesis, in conditioned media prepared from bone marrow cells of patients with regeneration hemopoiesis after chemotherapy induced hypoplasia. This activity was estimated by hydroxyurea sensitivity of CFU-S in mice, under conditions of incubation with human bone marrow conditioned medium (BMCM). We found that CFU-S proliferation stimulator was present to a considerable extent in human regenerating BMCM, but less so in normal BMCM and that the production fluctuated with change of hemopoietic states, in the same patient. This stimulator was heat-labile, trypsin-sensitive and mainly produced by adherent cells. This factor may possibly be involved in regulation of proliferation of stem cells in regenerating bone marrow in humans.     

Effect of adult thymectomy and splenectomy on pluripotent stem cells and progenitors of the myeloid and B-lymphoid lineages

In order to investigate the regulatory interactions which occur between the bone marrow, the thymus and the spleen during hemopoiesis, the numbers of granulo-macrophage and B-lymphocyte precursors (GM-CFC and BL-CFC) and the kinetics of pluripotent stem cells (CFU-S) have been studied in the bone marrow and the spleen of normal adult thymectomized and/or adult splenectomized mice with or without T-dependent antigen stimulation. The results suggest that (1) medullary and splenic CFU-S may be two different populations of the stem cell compartment; (2) the thymus is involved in medullary but not in splenic CFU-S proliferation in response to T-antigen challenge; (3) the spleen influences GM-CFC and BL-CFC numbers and (4) antigenic stimulation modifies medullary BL-CFC.     

Effects of cisplatin on different haemopoietic progenitor cells in mice.

The effects of Cisplatin on marrow haemopoietic progenitor cells, WBC and RBC were measured and compared in F1 (CBA x C57BL) female mice. Dose/survival curves of Cisplatin for CFU-S, CFU-C and BFU-E were found to be simply exponential, indicating that the effect of the drug has no cell-cycle dependency. BFU-E also appeared significantly (P less than 0.001) more sensitive to Cisplatin than CFU-S and CFU-C. After a single dose of 12 mg/kg of Cisplatin, WBC, MNC and CFU-E were seen to be markedly less reduced and to recover much earlier than CFU-C, and particularly BFU-E and CFU-S. Results suggest that the drug is more toxic for earlier haemopoietic progenitor cells than for the more mature cells, and that the latter are not reliable parameters for complete haemopoietic recovery in mice after treatment with this agent. In the animals treated, there was also a subsequent significant decrease of the RBC count, accompanied by a marked increase of the marrow CFU-E concentration. Possible underlying mechanisms (e.g. alterations of RBC after exposure to Cisplatin) were discussed.     

Spatial organisation of CFU-S proliferation regulators in the mouse femur

CFU-S proliferation can be regulated by an inhibitor and a stimulator which are produced locally in the bone marrow. The spatial distribution of cells elaborating these factors has been studied by separating marrow, zonally, into axial, marginal and bone-associated fractions and further separating the inhibitor and stimulator producing cells from these fractions by means of density cuts in bovine serum albumin. The inhibitor- and stimulator-producing cells are found respectively in bands of density < 1.064 g/cm³ and 1.064–1.072 g/cm³. Factors synthesised by these individual fractions were assayed for their ability to inhibit or stimulate CFU-S proliferation by thymidine suicide measurements. Axial cells contain a high concentration of inhibitor-producing cells but the marginal and bone fractions contain relatively few. Stimulator-producing cells have a very sharp gradient in the opposite direction with an extremely high concentration in the vicinity of the bone surface. These distributions fit with those of CFU-S proliferation and of fibroblastoid-CFC suggesting that the inhibitor-producing cell is an integral part of the micro-environment which maintains the stem cell's capacities while the stimulator-producing cell may be more closely associated with the differentiation processes which take place closer to the bone surface.     

Enhancing hemopoietic drug resistance: a rationale for reconsidering the clinical use of mitozolomide

Retroviral gene transfer was used to achieve expression in mouse bone marrow of a mutant form of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA), which exhibits resistance to inactivation by O6-benzylguanine (O6-beG). After reconstitution of mice with transduced bone marrow, approximately 50% of the bipotent granulocyte-macrophage colony-forming cell (GM-CFC) and multipotent spleen colony-forming unit (CFU-S) hemopoietic populations showed expression of the transgene; this expression was associated with resistance to either mitozolomide or to a combination of O6-beG and mitozolomide, relative to mock-transduced controls. Thus, at a dose of mitozolomide in vivo that allowed only 70% and 62% survival of mock-transduced GM-CFC and CFU-S, respectively, the hATPA/GA CFC were totally resistant to the same dose of mitozolomide (P < .05 and .001, respectively). In the presence of O6-beG, the toxicity of mitozolomide was greatly potentiated. Only 24% and 18%, respectively, of mock-transduced GM-CFC and CFU-S survived combination treatment, whereas 45% (P < .05) and 37% (P < .01) of GM-CFC and CFU-S, respectively, from hATPA/GA mice survived the same combination of doses. Furthermore, as a result of transgene expression, the number of micronucleated polychromatic erythrocytes induced by mitozolomide was significantly reduced (P < .05) by 40% relative to mock-transduced controls, indicating the potential of this approach to reduce the frequency of mutation associated with chemotherapy exposure. The protection against the toxic and clastogenic effects of mitozolomide in both primitive and more mature hemopoietic cells suggests that the severe myelosuppression that halted further clinical investigation of this drug could be substantially ameliorated by the exogenous expression of O6-alkylguanine-DNA alkyltransferase. Therefore, these data raise the prospect for the reinvestigation of mitozolomide and other proscribed drugs in the context of genetically protected hemopoiesis.     

Physical, phenotypic and cytochemical characterisation of stroma-adherent blast colony-forming cells.

Primitive cells defined as long-term culture initiating cells (LTCIC) and blast colony-forming cells (Bl-CFC) bind to cultured stromal layers, but cells at later stages of maturation [granulocyte-erythroid-macrophage-monocyte colony-forming cells (GEMM-CFC) granulocyte-macrophage (CM-CFC) and erythroid burst-forming units (BFU-E)] do not. The precise relationship between the LTCIC and Bl-CFC is not known and this study was undertaken to determine their relative positions in the haemopoietic hierarchy. We have defined the Bl-CFC population in terms of its density profile and antigenic phenotype and compared these characteristics with GM-CFC and BFU-E. The progenitor cell populations did not differ in density. The major phenotypic difference was seen using the myeloid monoclonal antibody S17-25 which reacted with fewer Bl-CFC than GM-CFC. Also, we have cytochemically analysed the cells in colonies derived from Bl-CFC. Our studies indicate that the Bl-CFC precede BFU-E and GM-CFC but not the LTCIC.     

Effect of a hyperthermic treatment on the pluripotent haemopoietic stem cell in normal and anaemic mice

Up to now, the hyperthermic sensitivity of pluripotent haemopoietic stem cells is unknown, and the few existing data from reports in the literature are conflicting. There are two main drawbacks in the set-up of those studies: (1) only CFU-S day 9 results were presented, whereas it is questionable if this assay gives a true reflection of the pluripotent stem cell, and (2) no attention has been paid to heat effects on the seeding efficiency, i.e. the amount of stem cells which will lodge in the spleen. The present study focused on the procedural differences and compared the results of a hyperthermic treatment (60 min, 42°C) on the stem cells, assayed with the CFU-S day 9 and the CFU-S day 12 method, using the following three stem cell suspensions, all differing in their proliferative activity: bone marrow from normal mice and bone marrow and spleen cells from anaemic mice. Furthermore, we investigated the seeding efficiency before and after heat treatment. Resting stem cells, assayed with the CFU-S day 12 method, turned out to be resistant to hyperthermia as compared with the active cycling stem cells, while with the CFU-S day 9 assay the stem showed the same thermosensitivity in the two bone marrow suspensions. The active cycling stem cells do not significantly differ in thermosensitivity, in CFU-S day 9 and day 12 assays, although there is a difference between bone marrow and spleen. Hyperthermia appears to influence the seeding efficiency for spleen CFU-S; an increase of 1?73 was observed. The difference in heat sensitivity between the resting and the active cycling stem cells, assayed with both in vivo methods, however, cannot be explained by a change in seeding efficiency only. Comparing the amount of cycling cells in the three stem cell suspensions and their thermosensitivity leads to the conclusion that the differences in heat sensitivity might be fully explained by the cycling status of the stem cell.     

Haemopoietic colony formation (BFU-E, GM-CFC) during the development of pure red cell hypoplasia induced in the cat by feline leukaemia virus

The GM-CFC assay for granulocyte-macrophage progenitors and the BFU-E and CFU-E assay for early and late erythroid progenitors from cat bone marrow were characterized. GM-CFC gave 59 +/- 4 to 118 +/- 6 colonies per 10(5) bone marrow cells using colony stimulating factors (CSF) from cat, mouse or human sources. The CFU-E and BFU-E assays gave 114 +/- 7 and 58 +/- 7 colonies respectively with optimum doses of erythropoietin. Irradiated cat bone marrow cells were good sources of CSF and of burst promoting activity for these assays. Kittens infected with feline leukaemia virus, subgroup C (FeLV-C), which induces pure red cell hypoplasia, showed the incidence of BFU-E decreased to 25-35% of controls as early as one week postinfection, and even lower values at later times. In contrast, the incidence of GM-CFC remained normal for several weeks. No evidence of inhibitory cells or of lack of stimulatory cells in the infected marrows was seen when they were cultured together with normal marrow in the BFU-E assay. Conversely, normal marrow cells were not able to restore BFU-E growth from infected marrow. This suggests a direct action of FeLV-C on early erythroid precursors. Infection with FeLV, subgroup A, which induces only a mild transitory anaemia, produces only a moderate decrease in the incidence of BFU-E.     

Effect of a hyperthermic treatment on the pluripotent haemopoietic stem cell in normal and anaemic mice

Up to now, the hyperthermic sensitivity of pluripotent haemopoietic stem cells is unknown, and the few existing data from reports in the literature are conflicting. There are two main drawbacks in the set-up of those studies: (1) only CFU-S day 9 results were presented, whereas it is questionable if this assay gives a true reflection of the pluripotent stem cell, and (2) no attention has been paid to heat effects on the seeding efficiency, i.e. the amount of stem cells which will lodge in the spleen. The present study focused on the procedural differences and compared the results of a hyperthermic treatment (60 min, 42 degrees C) on the stem cells, assayed with the CFU-S day 9 and the CFU-S day 12 method, using the following three stem cell suspensions, all differing in their proliferative activity: bone marrow from normal mice and bone marrow and spleen cells from anaemic mice. Furthermore, we investigated the seeding efficiency before and after heat treatment. Resting stem cells, assayed with the CFU-S day 12 method, turned out to be resistant to hyperthermia as compared with the active cycling stem cells, while with the CFU-S day 9 assay the stem showed the same thermosensitivity in the two bone marrow suspensions. The active cycling stem cells do not significantly differ in thermosensitivity, in CFU-S day 9 and day 12 assays, although there is a difference between bone marrow and spleen. Hyperthermia appears to influence the seeding efficiency for spleen CFU-S; an increase of 1.73 was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of hyperthermia on murine myeloid precursors

Murine bone marrow was exposed to hyperthermia temperatures of 41.5 to 45.5 degrees C. The proliferation capacity of myeloid progenitor and committed precursors was assayed in vivo utilizing spleen colony formation and diffusion chamber (DC) techniques. The survival of both pluripotential (CFU-S) and committed myeloid (CFU-DG) stem cells decreased exponentially with an increase in the heating period. Progression from CFU-S to CFU-DG significantly altered thermal sensitivity in the temperature range examined. Proliferation of mature granulocyte-monocytes (G-M) in DC is more thermostable than their stem cell precursors. Heat inactivation energies (enthalpies) of CFU-S and CFU-DG were derived from the slope of the heating time survival curves. Enthalpy of CFU-S is 300 kcal/mole below 43 degrees and 105 kcal/mole above 43 degrees. The enthalpy of CFU-DG is 250 and 145 kcal/mole below and above 43 degrees, respectively.     

Effect of H2 antagonists cimetidine and famotidine on the hemotoxicity of cyclophosphamide

A comparison between the effects of H2 antagonists Cimetidine and Famotidine on the hemotoxicity of Cyclophosphamide in vivo in DBA/2NCrBl mice is described. Hemotoxicity of anticancer drug was determined by peripheral blood leukocytes, bone marrow cells and bone marrow CFU-S, GM-CFC. Results show that Famotidine does not increase Cyclophosphamide hemotoxicity while Cimetidine enhances the toxicity of the anticancer drug only on normal pluripotent hemopoietic stem cells.     

Differential seeding of injected hemopoietic precursor cells in the bone marrow and spleen of irradiated mice

The seeding efficiency was determined of syngeneic granulocyte, macrophage, erythroid/mixed and megakaryocyte colony-forming cells (G-GFC, GM-CFC, M-CFC, E/Mix-CFC, MEG-CFC) in the femoral bone marrow and spleen of lethally-irradiated C57BL mice. The overall seeding efficiency of CFC's was similar to that for multipotential stem cells (CFU-S) in the marrow but in the spleen CFC seeding efficiency was ten-fold lower than for CFU-S. Two and a half hours after transplantation of 10⁷ bone marrow cells, the relative frequencies of E/Mix-CFC's and M-CFC's recovered from the recipient marrow were higher than in the injected marrow population. However the relative frequencies of CFC's recovered from the spleen corresponded closely to those of the injected marrow population.