Differentiation of the mouse hepatic primordium. II. Extrinsic origin of the haemopoietic cell line

The whole hepatic primordium (endoderm + mesenchyme of the septum transversum) was isolated from mouse embryos at various developmental stages, from 8 to 10 days of gestation, and was either grafted into chick or quail embryo or cultivated in vitro. Haemopoiesis developed only if the liver rudiment had been explanted after the 28- to 30-somite stage, but not if explanted prior to this stage, despite normal differentiation of the hepatocytes.However, when the liver rudiment, isolated before the 28-somite stage in in vitro culture, was supplied with exogenous haemopoietic stem cells, haemopoiesis developed in the hepatic tissue.These data show that foetal hepatic haemopoiesis depends on migration of haemopoietic cells which home the liver rudiment at the 28- to 30-somite stage.     

Haemopoiesis in mice genetically lacking granulocyte-macrophage colony stimulating factor during chronic infection with Mycobacterium avium

In order to test the role of granulocyte-macrophage colony stimulating factor (GM-CSF) in haemopoiesis during chronic infection, mice with a targeted disruption of the gene for GM-CSF were infected intraperitoneally with the facultative intracellular pathogen, Mycobacterium avium. The bacteria spread to lungs, liver and spleen and persisted for more than 10 weeks at levels between 105 and 106 CFU. Bacterial numbers did not differ significantly between infected GM-CSF-/- and wild-type mice, making this an excellent model in which to study the effects of GM-CSF deficiency on haemopoietic cells without complications of interpretation relating to differences in bacterial load. Haemopoietic colony forming cells (CFC) in the bone marrow of GM-CSF-/- mice before infection were not different from wild-type. However, whereas CFC in wild-type mice increased 1.5-fold with infection, GM-CSF-/- mice were unable to increase their CFC and numbers were significantly lower than in infected wild-type mice. Cells attracted to the peritoneal cavity of the GM-CSF-/- mice following i.p. injection of bacteria were notably lacking in the large, granular macrophages of activated appearance, which were a feature in wild-type mice. Nitric oxide production by peritoneal cells from GM-CSF-/- mice was deficient. Thus, GM-CSF is not critical for haemopoiesis during chronic infection, but in its absence the mice are unable to increase their output of haemopoietic cells and there are deficiencies in macrophage activation.     

Ontogeny-derived hematopoietic stem cells divide successively producing clones of differentiated hematopoietic cells

Forty seven individual haemopoietic cell clones bearing unique radiation markers were studied in long-term bone marrow cultures. Throughout cultivation, clones appeared at different times, 1 to 12 weeks after explanation, survived during 1-10 weeks and markedly varied in size. Usually, the number of metaphases characteristic of an individual clone rapidly increased, achieved a maximum and declined. The cells of disappeared clones were never seen again. The experimental results provide further evidence for the model of haemopoiesis by clonal succession. The data obtained are discussed with respect to the functioning of haemopoietic stem cell population.     

The mechanisms of lymphoid regulation and the radiosensitivity of the hematopoietic system]

On the basis of the authors' own data and the literature it has been inferred that the key principles of the haemopoietic system regulation are similar to those of the immune system. The cells of a lymphoid origin are found, which implement helper and suppressor functions with respect to early haemopoiesis precursors; the influence of lymphokines on this compartment under the effect of radiation is described. Disturbances in the haemopoiesis system regulation, that result from various damaging effects, might be corrected by T-lymphocytes and lymphokines. The data obtained suggest that the formation of splenic colonies is the result of the interaction of some cell populations. That is why many radiobiological characteristics of CFUs may be attributed to partner cells (for instance, T-lymphocytes).     

Estimation of the critical level of an arrest in differentiation of human hematopoietic stem cells

On the basis of the analysis of postirradiation changes in the specific number human peripheral blood neutrophils the authors have estimated the level of the "arrest of differentiation" (AD) of their precursors (haemopoietic stem cells (HSC) which amounted to 0.2-0.9% of their normal number at a local functional site of haemopoiesis. The time of the onset of AD, with subcritical doses (to 6.4-8.8 Gy) leading to HSC pool depletion, which does not reach a critical level, decreases linearly with dose, and its duration is practically independent of the dose and makes up 10 (6 to 16) days on an average.     

THE EFFECT OF E TYPE PROSTAGLANDINS ON THE PROLIFERATION OF HAEMOPOIETIC STEM CELLS IN VIVO

The ability of protaglandins E₁ and E₂ to stimulate the proliferation of haemopoietic stem cells (CFU_s) was studied in vivo. PGE₂, in a dose range of 10^-4 to 10^-1μg/g body weight and PGE₁ in a dose range of 10^-5 to 10^-1μg/g body weight, produced a rapid cycling wave of CFU_s. The increase in the number of CFU_s in S phase was not followed by a rise in the femoral CFU_s content, and except for a transient increase in femoral CFU_c level, no increase in differentiation was found either. Therefore, it is proposed that haemopoiesis after PG-induced CFU stimulation is ineffective. PGE₂ did not stimulate regeneration of CFU_s in a perturbed state (after sublethal irradiation). All these findings support the idea that PGEs might represent potent stimulators of the haemopoietic stem cells acting in physiological doses. However, if acting concurrently with physiological control systems PGs lead to ineffective haemopoiesis (under normal conditions) or do not exert any measurable effect (after sublethal irradiation).     

The development of radiation late effects to the bone marrow after single and chronic exposure

The marrow is a tissue distributed in numerous skeletal parts and works as an organ which is composed of a haemopoietic cell parenchyma and a supporting stroma. The pathophysiological mechanisms involved in the radiation-induced late effects depend mainly on the damage produced to each of these elements. Parenchymal cell damage ends with a failure of the stem cell pool to supply an adequate number of highly differentiated functional blood cells and is clinically manifested as aplastic anaemia or leukaemia. The effects of radiation on the haemopoietic stem cell can be measured by means of spleen colony forming units (CFU-S) in rodents. The self-maintaining capacity of the CFU-S was found to be lower than normal 16 weeks after a dose of 0.64 Gy. In larger animals it is only possible to measure the activity of some of the progenitor cells, estimating the number of granulocyte-macrophage colonies in culture (CFU-GM) as an indicator of stem cell changes. Their number in the blood is about 50 per cent of normal even 160 days after about 0.78 Gy. The stromal cells are also radiosensitive if measured with respect to their capacity to support long-term cell replication in vitro. Marrow fibrosis develops after single, repeated and chronic radiation exposure, and a dose of 40 Gy impairs the capacity of the marrow to support haemopoiesis.     

Characteristics of the dynamics of hematopoietic precursor cells cloned in diffusion chambers and recorded in experiments on mice and dogs irradiated in median lethal doses]

In experiments with mice and dogs irradiated with LD50, it was shown the postirradiation depopulation of haemopoietic polypotent (CFUs) cell-precursors in mouse bone marrow was more pronounced than that of granulocytic and macrophagal cells (CFUdc). The rate of repopulation of CFUs during the first week was higher than that of CFUdc (T1/2 was 2.5 and 8.8 days respectively). In dogs, one could notice a partial change in the colony formation, a prolonged plateau period in the postirradiation CFUdc dynamics, and a coincidence in time with cellularity restoration in the bone marrow and peripheral blood leukocytes. It is suggested that in conditions of heterogeneous incubation in diffuse chambers, the haemopoietic cell-precursors are more mature than in the syngeneic system. The method of CFUdc determination has proved to be ineffective in estimating the onset and intensity of the postirradiation haemopoiesis recovery in dogs. The study of the bone marrow CFUdc population may, however, be used in intact animals to predict the probability of their death after irradiation within the median lethal dose range.     

Stem cell maintenance and commitment to differentiation in long-term cultures of murine marrow obtained from different spatial locations in the femur

Abstract. Murine bone marrow was separated into axial and marginal fractions in order to investigate the ability of cells from different spatial locations in the marrow to establish long-term cultures. The maintenance of haemopoiesis was significantly poor in long-term cultures of marginal marrow compared with axial or control (unfractionated marrow) cultures. Using techniques to further fractionate the axial or marginal marrow by depleting either stromal or haemopoietic cells, it was possible to investigate the relative importance of stromal and haemopoietic cell components. In the combinations studied, the more important determinant of effective in vitro haemopoiesis was the source of the haemopoietic cells rather than the stroma. The most effective stem cell maintenance and commitment to differentiation was observed when the source of the haemopoietic population was axial marrow. The data are consistent with axial marrow being a source of 'high quality' stem cells and this quality being an intrinsic property of the cells rather than one imposed by the stromal environment.     

Thermic and pH modulation of phosphofructokinase during trout (Salmo gairdneri R.) haemopoiesis: influence of fructose 6-phosphate

Thermic and pH modulation of phosphofructokinase (EC 2.7.1.11) activity with respect to fructose 6-phosphate has been studied comparatively in trout (Salmo gairdneri R.) haemopoietic cells and erythrocytes. Phosphofructokinase of both cellular populations displays a biphasic kinetic behaviour with respect to fructose 6-phosphate at two values of pH and temperature. In haemopoietic cells, when pH decreases the enzyme-substrate affinity increase while an opposite effect is found in erythrocytes. Decreases in temperature act as a positive modulator in haemopoietic cells while in erythrocytes this effect is observed only at low fructose 6-phosphate concentrations. Therefore a different pH and temperature modulation of phosphofructokinase during trout haemopoiesis has been established.     

Macrophages in haemopoietic and other tissues of the developing mouse detected by the monoclonal antibody F4/80.

Macrophages are widely distributed in lymphohaemopoietic and other tissues of the normal and diseased adult, where they play an important role in host defence and repair. Although the development of haemopoiesis has been well studied in several species, the ontogeny of the mononuclear phagocyte system remains poorly understood. We have used a highly specific mAb, F4/80, to examine the distribution of mature macrophages in the developing mouse, with special reference to their presence in the haemopoietic microenvironment. Monocytes and macrophages were first seen in embryos on day 10 in the yolk sac and liver as well as in mesenchyme. In liver, spleen and bone marrow, there was expansion of this population associated with the initiation of haemopoiesis on days 11, 15 and 17, respectively. Macrophages in these sites formed part of the haemopoietic stroma and their extensively spread plasma membrane processes could be seen making intimate contacts with clusters of differentiating haemopoietic cells. F4/80+ cells were widely dispersed in undifferentiated mesenchymal tissue in organs such as lung, kidney and gut. Numbers of F4/80-labelled cells increased concomitantly with organ growth and local mitoses were evident, as well as actively phagocytic macrophages. Our studies establish that macrophages are among the earliest haemopoietic cells to be produced during development and that they are relatively abundant in fetal tissues in the absence of overt inflammatory stimuli. Their distribution is correlated with the sequential migration of haemopoiesis and they constitute a prominent component of the stroma in fetal liver, spleen red pulp and bone marrow. Apart from a role in haemopoietic cellular interactions, their highly developed endocytic and biosynthetic activities suggest that macrophages contribute major undefined functions during growth, turnover and modelling of fetal tissues.     

Stem cell maintenance and commitment to differentiation in long-term cultures of murine marrow obtained from different spatial locations in the femur

Murine bone marrow was separated into axial and marginal fractions in order to investigate the ability of cells from different spatial locations in the marrow to establish long-term cultures. The maintenance of haemopoiesis was significantly poor in long-term cultures of marginal marrow compared with axial or control (unfractionated marrow) cultures. Using techniques to further fractionate the axial or marginal marrow by depleting either stromal or haemopoietic cells, it was possible to investigate the relative importance of stromal and haemopoietic cell components. In the combinations studied, the more important determinant of effective in vitro haemopoiesis was the source of the haemopoietic cells rather than the stroma. The most effective stem cell maintenance and commitment to differentiation was observed when the source of the haemopoietic population was axial marrow. The data are consistent with axial marrow being a source of 'high quality' stem cells and this quality being an intrinsic property of the cells rather than one imposed by the stromal environment.     

The enigma of the haemogram "left-shift" in periwinkles infected with trematodes

To make further progress in the understanding of digenean immune evasive tactics in their snail host, we compared the haemopoietic parameters and haemocyte functional potencies in the prosobranch Littorina littorea, which were either healthy or infected with echinostome trematode Himasthla elongata. The haemocyte concentration in the circulation of infected individuals was significantly higher than in uninfected ones, 3300 microl(-1) and 1882 microl(-1), respectively. Intense haemopoiesis in haemolymph of infected snails was evidenced by 4-fold higher BrdU incorporation into nuclei of haemocytes as well as elevated level of cyclin D expression in these cells. Evident skewing of the haemocyte population toward a higher frequency of immature, undifferentiated haemocytes in infected L. littorea was found. Haemocytes in infected snails had a much lower functional potency in production of reactive oxygen intermediates (ROI) and cell-mediated cytotoxicity. Correlation analysis shows that both cytotoxic and ROI generation values were significantly and negatively correlated with proportion of juvenile cells in circulation. Experimental injection of H. elongata excretory/secretory products modulated haemopoiesis toward increasing a juvenile cells proportion by 7th day post-injection. This can be considered a haemogram "left-shift" by analogy to that seen in the human neutrophil compartment, when more immature bandforms are found in blood during acute inflammation or bone marrow disorders. We hypothesize that echinostomatide trematodes may interfere in the normal neuroendocrine management of haemopoiesis in the host and cause a haemopoietic signal to initiate multiplication to near neoplastic levels.     

Estimates of the efficacy of the local protection of dogs against ionizing radiation

The possibility of prognosis of survival of dogs subjected to acute nonuniform irradiation leading to haemopoietic radiation sickness has been demonstrated. The prognosis is based on the "equal dose" concept that is implemented together with the dose dependence of the bone marrow haemopoiesis activity. The estimates of the consequences of irradiation of dogs, with certain body parts being shielded, have shown a good correlation between the calculation results and the experimental data.     

Haemopoietic stem cells during development of mouse embryo.

Haemopoiesis in mammals takes place in yolk-sac and in mouse it can be detected on the 7th day of gestation. Erythropoietin (EPO) responsive cells can be detected from 7th day onwards. However, the cells committed to the myeloid lineage which can respond to the haemopoietic growth factor (viz. granulocyte macrophage colony stimulating factor; GM-CSF) can be demonstrated only on 10th day of gestation. At the same time, the 12-day spleen colony forming cells i.e. the late colony forming unit spleen (CFU-s) which are multipotent stem cells can also be detected. Data suggest that the stem cells seen in the embryo from 7-10 days of gestation may be a primitive population confined only to the yolk-sac. Liver haemopoiesis which begins in the liver of 13-day embryos is due to primitive haemopoietic pluripotent stem cells, arising de novo in the embryo and not in the yolk-sac, since no primitive pluripotent stem cells capable of repopulating lethally irradiated bone-marrow can be detected in the yolk-sac.     

The possible pathways of IL-2 stimulation of colony formation by bone marrow cells irradiated in vitro

The authors have made an attempt to find out the reasons of IL-2 stimulation of spleen colony growth by in vitro (1 Gy) irradiated bone marrow cells. It was shown that the effect of IL on haemopoiesis manifests itself with merely small radiation doses implying that the influence of the preparation makes the process of haemopoietic organ repopulation start at a higher level of cell survival, which is presumably related to a more active repair of radiation-induced CFUs damages: this leads, with other things being equal (e.g. proliferation rate and f factor), to a higher yield of colonies than it is observed with the recipients protected with the exposed bone marrow only.     

The effect of autoblood in a hyposmotic state on the colony-forming activity of hematopoietic stem cells]

It has been shown that hypoosmotic autoblood injected sub- or intracutaneously stimulates the colony-forming activity of haemopoietic stem cells in mice. Autoblood injected to animals immediately after their irradiation stimulates haemopoiesis even after a single dose. When mice are injected with autoblood prior to irradiation, the time between the first injection and the day of irradiation is critical for manifestation of the immunomodulating effect. Autoblood infusions immediately before, the day before, or two days before irradiation markedly deteriorate the clinical status of experimental animals and cause death in some of them. It is suggested that stimulation of haemopoiesis is associated with the appearance in the blood stream of a population of radiosensitive cells, apparently T-cell precursors.     

Presence of pluripotent haemopoietic precursors in vitro (CFU-mix) in haemopoietic tissues from mice of W/Wv genotype

Abstract. The presence or absence of haemopoietic precursors, which produce mixed colonies in vitro (CFU-mix) was examined in the bone marrow and spleen of (WB x C57BL/6) F1- W/W^v mice. Despite the failure of macroscopically evident colonyformation in the spleens of irradiated mice, haemopoietic cells of W/W^v mice did produce macroscopically-evident mixed colonies containing erythroid cells, macrophages, and often megakaryocytes, in culture medium. The size and constitution of mixed colonies derived from W/W^v mice were comparable to those of mixed colonies from congenic +/+ mice. The present results appear consistent with in vivo haemopoiesis in the W/W^v mice, which is obviously deficient, but sufficient for survival.