Radiosensitivity of pluripotent stem cells detectable by cloning in the spleen of irradiated mice

It was shown that the dose--effect curves describing the radiosensitivity of CFUc of the bone marrow irradiated in vitro (0.04-3.7 Gy) and treated with normal rabbit serum (NRS) and anti-mouse-brain serum (AMBS) has two differently sloping portions indicating that two CFUc populations differing in radiosensitivity are present in the bone marrow. D0 was 0.93 Gy after irradiation with doses of 0.04-0.75 Gy and treatment with NRS, and 0.33 Gy after incubation of the bone marrow with AMBS. The addition of thymus cells "straightened" the dose--effect curve for the bone marrow treated with AMBS: in this case D0 was 1.81 Gy exceeding considerably the values of D0 for intact bone marrow. The CFUc population is suggested to be heterogeneous in radiosensitivity.     

THE PROLIFERATIVE STATE OF GRANULOCYTIC PROGENITOR CELLS IN HUMAN BLOOD AND MARROW

A double layer agar technique was used to investigate the proliferative state of granulocytic progenitor cells (Colony Forming Units in Culture; CFUc) in human peripheral blood and bone marrow. The sensitivity of the progenitor cells to the S-phase specific agent, hydroxyurea, was used as an index of the proportion of cells engaged in DNA synthesis. In the presence of low concentrations of colony stimulating factor (CSF) the CFUc were found to be virtually insensitive to the drug. However, when cultured in the presence of increasing concentrations of CSF the proportion of CFUc apparently killed by hydroxyurea increased to a maximum of 23% for those cells in the blood and 39% for those in the marrow. The results indicate that CFUc which are slowly proliferating are sensitive to low concentrations of CSF. In contrast, those CFUc which are proliferating more rapidly require high concentrations of CSF before they will form colonies in culture. A model has been devised which suggests that as CFUc mature, their cell cycle time shortens and their sensitivity to CSF decreases.     

Effect of anti-mouse brain serum on the CFUc in actively proliferating bone marrow

With hydroxyurea injected to donor mice a greater inhibition of splenic colony growth occurred after incubation of a bone marrow suspension with the rabbit antimouse brain serum (RAMBS), and restoration of the colony-formation by thymocytes was less pronounced than in normal bone marrow treated with the antibrain serum. The incubation of the bone marrow cells containing CFUc, which actively proliferate after irradiation or stimulation by vinblastine, with the antibrain serum sharply suppressed the splenic colony growth. In this case however, in contrast to normal bone marrow, the administration of thymocytes failed to exert a favourable action on the colony formation. It is suggested that functioning of accessory cells is not associated with the defined cell cycle stage of CFUc and that, in addition to the previously discovered accessory cell population, some other factors, inactivated by the RAMBS serum, are present in the bone marrow the analogue of which is absent in the thymus.     

An In Vitro Model of Hematopoietic Injury In Chronic Hypoplastic Anemia

Mice treated with high-dose busulfan develop a ‘latent’ form of bone marrow failure characterized by near-normal peripheral blood counts and marrow cellularity, but marked reductions in marrow pluripotent stem cells (CFUs) and myeloid progenitor cells (CFUc). Spleen cell suspensions from control and ‘latent’ mice were placed in liquid culture in the presence of colony-stimulating activity. Cells were harvested at intervals up to 14 days and sub-cultured in agar to assay for CFUc. Baseline splenic CFUc did not differ significantly between control and ‘latent’ mice. Splenic CFUc from control mice increased 50-fold and reached a peak at day 10 in liquid culture. In contrast, splenic CFUc from ‘latent’ mice increased only 7-fold and reached a peak at day 3. Our results indicate that although splenic CFUc are present in normal numbers in ‘latent’ mice, their proliferative capacity is markedly reduced, either as the result of defective CFUc self-renewal or defective feed-in from CFUs or both.     

Alcohol suppresses the granulopoietic response to pulmonary Streptococcus pneumoniae infection with enhancement of STAT3 signaling

Enhanced granulopoietic activity is crucial for host defense against bacterial pneumonia. Alcohol impairs this response. The underlying mechanisms remain obscure. G-CSF produced by infected lung tissue plays a key role in stimulating bone marrow granulopoiesis. This study investigated the effects of alcohol on G-CSF signaling in the regulation of marrow myeloid progenitor cell proliferation in mice with Streptococcus pneumoniae pneumonia. Chronic alcohol consumption plus acute alcohol intoxication suppressed the increase in blood granulocyte counts following intrapulmonary challenge with S. pneumoniae. This suppression was associated with a significant decrease in bone marrow granulopoietic progenitor cell proliferation. Alcohol treatment significantly enhanced STAT3 phosphorylation in bone marrow cells of animals challenged with S. pneumoniae. In vitro experiments showed that G-CSF-induced activation of STAT3-p27(Kip1) pathway in murine myeloid progenitor cell line 32D-G-CSFR cells was markedly enhanced by alcohol exposure. Alcohol dose dependently inhibited G-CSF-stimulated 32D-G-CSFR cell proliferation. This impairment of myeloid progenitor cell proliferation was not attenuated by inhibition of alcohol metabolism through either the alcohol dehydrogenase pathway or the cytochrome P450 system. These data suggest that alcohol enhances G-CSF-associated STAT3-p27(Kip1) signaling, which impairs granulopoietic progenitor cell proliferation by inducing cell cycling arrest and facilitating their terminal differentiation during the granulopoietic response to pulmonary infection.     

Granule targeting of soluble tumor necrosis factor (TNF) receptor expressed during granulopoietic maturation in murine bone marrow cells

In this experiment, we explored the potential of secretory lysosomes of hematopoietic cells to act as vehicles for immunomodulatory protein delivery at an inflammation site. We investigated whether exogenous soluble TNF-receptor 1 (sTNFR1) could be expressed in primary hematopoietic progenitor cells and become targeted for storage and secretion during granulopoietic differentiation. An sTNFR1 construct with a transmembrane domain (tm) and a cytosol sorting signal (Y) taken from CD63, was retrovirally transduced to lineage-negative murine hematopoietic bone marrow stem/progenitor cells. This process was followed by cytokine-driven granulopoietic maturation. The sTNFR1-tm-Y was found to be synthesized in precursor cells and to persist in mature granulocytes and monocytes/macrophages. Immunofluorescence-localization studies showed a granule pattern of sTNFR1-tm-Y in both precursor and mature granulocytes and secretion to phagosomes after ingestion of bacteria. Immunoelectron microscopy revealed co-localization between the sTNFR1-tm-Y and the primary (azurophil) granule marker myeloperoxidase. Collectively, our results demonstrated granule targeting, storage, and secretion of exogenous sTNFR1-tm-Y constitutively expressed during normal granulopoietic differentiation. These findings support the concept of using storage organelles of circulating hematopoietic cells as vehicles for targeting sites of inflammation with immunoregulatory agents.     

Human cells in suspension. 1 Human lymphoid and granulopoietic cells in primary and secondary cultures: effects of in vitro induction and prolonged methanol acetic acid fixation on metaphase chromosome structure.

Modifications of Hungerford's method (1965) for production of chromosomal slides from human lymphoid cells in culture have been developed. Modified in vitro induction of banding and uncoiling has been used to produce chromosomal slides from human neoplastic cells of granulopoietic origin. The chromosomes are well spread and appear either long, thin and segmented or uncoiled. It is suggested that it is the combined action of the prolonged fixation used, and the in vitro induction, which leads to the observed structural alteration of the chromosomes. A method for increasing the yield of metaphase cells when working with bone marrow has been developed on the basis of culturing the granulopoietic cells in medium containing colony stimulating factor (CSF). Comparative analysis of metaphases from primary and secondary cultures of bone marrow cells showed that the culturing conditions for the secondary cultures do not induce chromosome abnormalities in the cells during the growth period.     

Kinetics of the basic sections of the hemopoietic system in radiation chimeras]

During first 3 days after mice irradiation and syngeneic bone marrow transplantation in them the number of CFUs (about 0,5% of the injected cells) was stable, although the proliferation induction began 24 hours after transplantation. As it was shown by the method of "thymidine self-distruction". Twenty four hours later all the CFUs entered the mitotic cycle. On the contrary, the commited cells (granulopoesis precursors) compartment (CFUc) enters the logarithmic growth phase since the first day. The exponential growth of the CFUs number was observed from the 4th day simultaneously with the increasing of the proliferation rate of CFUc and the beginning of the recovery of the bone marrow cells total number. In late radiation chimeras (1 month after radiation and reconstitution) the total number of CFUs was 50--70% of the initial. The other hemopoetic parameters were in the normal limits.     

Enrichment and cryopreservation of bone marrow progenitor cells for autologous reinfusion

From 20 patients with solid tumors or acute nonlymphocytic leukemia in remission, hemopoietic progenitor cells were taken and stored in liquid nitrogen, for use in autologous bone marrow transplantation. Bone marrow aspiration resulted in a volume of 920(+/- 170) ml containing 16.8(+/-6.0) x 10(9) nucleated bone marrow cells and 7.2(+/-4.4) x 10(6) myeloid progenitor cells (CFUc). With use of the Haemonetics blood cell separator a progenitor cell-enriched fraction is obtained. This fraction is depleted of 90(+/-6)% of the erythrocytes and 59(+/-15)% of the neutrophils contained in the original. The original aspirate volume is reduced to one-fifth (21 +/- 3%) while containing 88(+/-38)% of the original CFUc's and 52(+/-11)% of the nucleated bone marrow cells. This technique of bone marrow enrichment has the advantage of a minimum of open-air contact, being independent of extensive laboratory facilities and manpower. The enriched fraction is frozen in autologous plasma and a final concentration of 10% (v/v) DMSO, using a program-controlled freezer (L'Air Liquide). Materials are stored at liquid nitrogen temperature in bags (Gambro) and test vials. Total CFUc recovery in test vials after thawing was 81(+/-32)%.     

Diffusion chamber and spleen colony assay of murine haematopoietic stem cells

Mouse bone marrow cells have been cultured in diffusion chambers and their capacity to form spleen colonies in irradiated mice investigated after different culture periods. The number of spleen colony-forming units (CFU) in the chambers decreased during the first day of culture. The number then increased rapidly to a level significantly above the original chamber value on the third to fifth day of culture. By that time large numbers of granulocytes and macrophages had also appeared. Histological examination of spleen colonies showed that prior culturing did not alter the ratio between the different types of colonies. Cultured bone marrow cells which were transferred to new chambers retained granulopoietic capacity. This capacity increased between the first and second day of primary culturing. At this time hydroxyurea injections to chamber hosts revealed that the progenitor cells were proliferating. The results show that the granulopoietic progenitor cells of the chambers are stem cells, and that one progenitor cell type is identical with the CFU.     

Circadian variations in the committed stem cell responsiveness to granulopoiesis inhibitory factor (chalone) in vitro

The effect of inhibitory extracts of human granulocytes (chalone) on the circadian rhythm of myelopoietic colony formation in soft agar (CFUc) was studied over one year. Normally, the number of bone marrow CFUc shows a strong circadian rhythm. Preincubation of the cells with the extracts before plating modulated this rhythm with phase shifts and modifications of amplitude. The effects of the extracts oscillated between significant inhibition and stimulation over a 24 h period. No significant seasonal variations in the inhibitory effects were found.     

The effect of food and water deprivation on the peripheral blood parameters of the mouse

Various peripheral blood and bone marrow parameters were determined during food and water deprivation and during food deprivation alone in order to obtain base lines that may be used to make comparisons with similar data from irradiated mice. The peripheral blood parameters following food and water deprivation were similar to those following food deprivation alone. The mean survival time was about 5 days and the weight loss 40% of the control weight. There was an absolute decrease in the total circulating lymphocyte and platelet counts, while the total granulocyte count remained unchanged or increased. The blood volume decreased, while the hematocrit and specific gravity of the blood increased. The bone marrow parameters following food and water deprivation showed that erythropoiesis was more markedly depressed than myelopoiesis. The tritiated thymidine labeling index for granulopoietic cells and megakaryocytes decreased progressively during starvation. The variations in the white blood count and the bone marrow parameters are not comparable with those found in irradiated mice having the G.I. syndrome; the changes in mean survival time, weight loss, hematocrit, and blood volume are similar.     

Buoyant density analysis of myeloid colony-forming cells in germfree and conventional mice.

Granulocyte-macrophage colony-forming cells (CFUc), in the bone marrow of germfree and conventioal CBA mice, were compared quantitatively and qualitatively. Cells were separated on the basis of their buoyant density by equilibrium centrifugation in continuous albumin density gradients. CFUc in the density subpopulations were detected by culture in agar containing three different types of colony stimulating factor (CSF). The sources of the CSF were post-endotoxin mouse serum (CSFES), mouse lung conditioned medium (CSFMLCM) and human urine (CSFHU). Mice were removed from the germfree environment and the buoyant density status of their CFUc was examined 1, 4 and 8 weeks later. No difference was found between germfree and conventional mice in the number of nucleated cells per femur or in their modal density. Neither was the number of CFUc per femur different. The cell cycle status of CFUc, as determined by the thymidine suicide technique was not significantly different. Functional heterogeneity was found among the density subpopulations for both groups of mice. This depended on the type of CSF. The density distribution of CFUc was significantly different in germfree mice. There were proportionately more low density CFUc. The mean modal density of CFUc under CSFES stimulation was less by 0.0045 g/cm3 in germfree mice. The removal of mice from the germfree environment resulted in a shift of the distribution to higher densities. The trend was towards the conventional situation. The significance of the buoyant density status of CFUc is discussed.     

Damage and repair of bone marrow CFUf and CFUc at different times after whole body sublethal gamma-irradiation of animals

Cellularity and colony-forming ability of stromal (CFUf) and haemopoietic (CFUc) bone marrow stem cells have been studied at different times after irradiation. The results obtained are indicative of the similarity between the kinetics of the damage and repair of stromal and haemopoietic stem cells.     

BUOYANT DENSITY ANALYSIS OF MYELOID COLONY-FORMING CELLS IN GERMFREE AND CONVENTIONAL MICE

Granulocyte-macrophage colony-forming cells (CFUc), in the bone marrow of germfree and conventional CBA mice, were compared quantitatively and qualitatively. Cells were separated on the basis of their buoyant density by equilibrium centrifugation in continuous albumin density gradients. CFUc in the density subpopulations were detected by culture in agar containing three different types of colony stimulating factor (CSF). The sources of the CSF were post-endotoxin mouse serum (CSF_ES), mouse lung conditioned medium (CSF_MLCM) and human urine (CSF_HU). Mice were removed from the germfree environment and the buoyant density status of their CFUc was examined 1, 4 and 8 weeks later. No difference was found between germfree and conventional mice in the number of nucleated cells per femur or in their modal density. Neither was the number of CFUc per femur different. The cell cycle status of CFUc, as determined by the thymidine suicide technique was not significantly different. Functional heterogeneity was found among the density subpopulations for both groups of mice. This depended on the type of CSF. The density distribution of CFUc was significantly different in germfree mice. There were proportionately more low density CFUc. The mean modal density of CFUc under CSF_ES stimulation was less by 0.0045 g/cm³ in germfree mice. The removal of mice from the germfree environment resulted in a shift of the distribution to higher densities. The trend was towards the conventional situation. The significance of the buoyant density status of CFUc is discussed.     

Human lung tissue as a source of colony stimulating activity.

Medium conditioned by human lung tissue was found to contain colony stimulating activity (CSA). This material was tested against mouse and human bone marrow as target system. Colony forming units (CFUc) from both species responded and gave rise to clonal growth in agar cultures. This colony formation was dose dependent and the relationship was a sigmoid one. Experiments to determine the molecular weight of human lung derived colony stimulating Factors brought evidence for four active molecular weight fractions with approximately 79000, 40000, 23000 and 2000 daltons. The 23000 dalton fraction activated human cells only, whereas the other fractions were active on both human and mouse bone marrow cells.     

Description of a suicide technique in vitro for granulocytic stem cells (CFUc) by hydroxyurea on normal human bone marrow

It has been possible, by studying 15 normal human bone marrow samples, to develop a technique for the suicide effect in vitro by hydroxyurea (H.U.), applied to human granulocytic stem cells (CFUc). This study suggests a contact time of medullary cells from 60 to 80 minutes in the presence of a hydroxyurea concentration 6 mM per 10(6) cells per ml in vitro. Preliminary results concerning the clusters are discussed.     

Effect of an anti-murine transferrin receptor-ricin A conjugate on bone marrow stem and progenitor cells treated in vitro

A monoclonal antibody with specificity for murine transferrin receptor was conjugated with the toxic A subunit of ricin. The dose range, specificity, and kinetics of inhibition of protein synthesis of the conjugate were determined on the murine T-lymphoma cell line, BW5147. When toxin was present throughout the period of culture, in vitro myeloid (CFUc) and erythroid (CFUe and BFUe) bone marrow colonies were inhibited by doses of conjugate comparable to those that inhibit protein synthesis in murine cell lines (IC50 of 5 X 10(-11)M). Bone marrow exposed briefly (30 min to 6 h) to anti-transferrin receptor antibody-ricin A conjugate was assayed for myeloid (CFUc) and erythroid (CFUe and BFUe) progenitors in vitro and for in vivo spleen colony formation (CFUs). Only CFUe were depleted by this pulse exposure, consistent with the higher frequency of proliferating cells and transferrin receptor expression in the CFUe population relative to other progenitors.     

Visualization of carbohydrate-binding molecules expressed by myelomono- and erythropoietic cells derived from human bone marrow: an immunoenzymatic double-staining study

Interactions between human haematopoietic and bone marrow stromal cells are governed by complex carbohydrate-mediated adhesion processes. In order to evaluate corresponding carbohydrate-binding sites on human myelo- and erythropoietic cells which were able to react with mono- or oligosaccharides, we established an immunocytochemical double-staining assay. In a first step, cell lineages were visualized using Lewis^x (CD15) or glycophorin C-specific monoclonal antibodies. The second step included polyacrylamide-conjugated carbohydrate structures. According to our results, the carbohydrate-binding potential of granulopoietic cells increased during the process of maturation, contrasting a reduction of carbohydrate-binding sites on erythroid precursor cells during differentiation. With respect to previous in vitro studies, these findings shed some light on certain aspects of bone marrow homing as well as on the trafficking of mature cellular elements into circulation. It is tempting to speculate that carbohydrate-mediated adhesion mechanisms may be involved in the various functional defects of progenitor cells in chronic myelogenic leukaemia, especially regarding their complex interactions with the marrow microenvironment.     

Reduced variance of bone-marrow transit time of granulopoiesis—a possible pathomechanism of human cyclic neutropenia

Abstract. Human cyclic neutropenia (CN) is a haematological disorder characterized by oscillations in the numbers of neutrophilic granulocytes and other blood cells with a stable period of approximately 21 days. In most cases the neutrophils oscillate well below normal values such that these patients are chronically neutropenic. A comprehensive concept of the origin of CN is proposed. It assumes an abnormally small variance of the transit time of bone marrow cells (compared to normal human granulopoiesis) for the origin of the characteristic cycles. Furthermore, a reduced responsiveness of the immature granulopoietic bone marrow cells to the mitotic feedback stimuli is assumed to account for the subnormal neutrophil peaks. Together with feedback control provided in a simulation model of normal human granulopoiesis these two abnormalities can explain experimental and clinical cell kinetic data for bone marrow and blood in CN.