Survival and repopulation of irradiated 'pre-CFU-c' in mice

Supernatants of murine bone-marrow cultures contain a colony-promoting factor (CPF) which increases the number of granulocyte and macrophage colonies in semi-solid agar cultures in the presence of colony-stimulating factor (CSF). Incubation of bone-marrow cells with CPF results in an increase in the number of granulocyte/macrophage progenitor cells (CFU-c) and the CPF-responsive cells may be younger than the CFU-c. We have investigated the radiosensitivity and the pattern of the recovery after irradiation of CPF-responsive cells. We found that the radiosensitivity of CPF-responsive cells was significantly lower than those of CFU-c, burst-forming units-erythroid (BFU-e) and pluripotent stem cells in vivo (CFU-s) and in vitro (CFU-mix). The CPF-responsive cells remained subnormal even at 28 days after irradiation of the mice, a time when the CFU-s and CFU-c had recovered completely. Therefore the CPF-responsive cells may constitute a separate compartment, namely 'pre-CFU-c', in the maturation sequence of granulopoiesis, and this maturation of the 'pre-CFU-c' to CFU-c seems to be highly stimulated after irradiation to counterbalance the influx from CFU-s.     

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.     

THE INFLUENCE OF A GRANULOCYTIC INHIBITOR(S) ON HEMATOPOIESIS IN AN IN VIVO CULTURE SYSTEM

The in vivo diffusion chamber (DC) technique for mouse marrow culture was used to determine the effect of a granulocyte inhibitor on the proliferation of the pluri-potent stem cell (CFU-s) and the granulocyte progenitor cell (CFU-c). Granulocyte conditioned medium was injected intraperitoneally into mice bearing DCs during the initial 48 hr of culture. The early injection of inhibitor resulted in a significantly reduced number of granulocytic progeny formed within the DCs while there was no growth inhibition of mouse fibroblasts cultured under identical conditions. The reduced cell production was due in part to a significant reduction in the self-renewal rate of the CFU-c while no apparent direct effect was observed upon the growth of the CFU-s within the same cultures. These data suggest that the granulocytic inhibitor(s) acted to reduce proliferation within the CFU-c population and thereby diminished the amplification potential inherent in the initial cell inoculum.     

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.     

The influence of a granulocytic inhibitor(s) on hematopoiesis in an in vivo culture system.

The in vivo diffusion chamber (DC) technique for mouse marrow culture was used to determine the effect of a granulocyte inhibitor on the proliferation of the pluripotent stem cell(CFU-s) and the granulocyte progenitor cell (CFU-c). Granulocyte conditioned medium was injected intraperitoneally into mice bearing DCs during the initial 48 hr of culture. The early injections of inhibitor resulted in a significantly reduced number of granulocytic progeny formed within the DCs while there was no growth inhibition of mouse fibroblasts cultured under identical conditions. The reduced cell production was due in part to a significant reduction in the self-renewal rate of the CFU-c while no apparent direct effect was observed upon the growth of the CFU-s within the same cultures. These data suggest that the granulocytic inhibitor(s) acted to reduce the proliferation within the CFU-c population and thereby diminished the amplification potential inherent in the initial cell inoculum.     

Cells Forming Spleen Colonies At 7 Or 11 Days After Injection Have Different Proliferation Rates

Abstract In the early periods (7–9 days) after haemopoietic cell injection, colonies produced by CFU-s and by their progeny are identified in the spleen, while at later periods (11 days after injection) only spleen nodules produced by CFU-s persist. the increase in the suicide values of CFU-s after sublethal (2 Gy) irradiation of mice is associated with a higher proliferation rate of precursors of transitory spleen colonies, but not of CFU-s, as measured by different suicide techniques. During the log-phase of cell growth in a lethally irradiated recipient, the injected CFU-s and CFU-tr proliferate at a higher rate. Active proliferation of CFU-s and CFU-tr has been demonstrated in long-term bone-marrow cultures by the hydroxyurea in-vitro suicide assay. CFU-tr may be the cause of artifactual effects during measurement of haemopoietic stem-cell cycling by CFU-s suicide methods.     

Regulation of haemopoietic stem-cell proliferation in mice carrying the Slj allele

We investigated a haemopoietic stromal defect, in mice heterozygous for the Sl^j allele, during haemopoietic stress induced by treatment with bacterial lipopolysaccharides (LPS) or lethal total body irradiation (TBI) and bone-marrow cell (BMC) reconstitution. Both treatments resulted in a comparable haemopoietic stem cell (CFU-s) proliferation in Sl^j/+ and +/+ haemopoietic organs. There was no difference in committed haemopoietic progenitor cell (BFU-e and CFU-G/M) kinetics after TBI and +/+ bone-marrow transplantation in Sl^j/+ and +/+ mice. the Sl^j/+ mice were deficient in their ability to support macroscopic spleen colony formation (65% of +/+ controls) as measured at 7 and 10 days after BMC transplantation. However, the Sl^j/+ spleen colonies contained the same number of BFU-E and CFU-G/M as colonies from +/+ spleens, while their CFU-s content was increased. On day 10 post-transplantation, the macroscopic ‘missing’ colonies could be detected at the microscopic level. These small colonies contained far fewer CFU-s than the macroscopic detectable colonies. Analysis of CFU-s proliferation-inducing activities in control and post-LPS sera revealed that Sl^j/+ mice are normal in their ability to produce and to respond to humoral stem-cell regulators. We postulate that Sl^j/+ mice have a normal number of splenic stromal ‘niches’ for colony formation. However, 35% of these niches is defective in its proliferative support.     

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.     

Effect of lithium on diffusion chamber granulopoiesis

We studied the effect of lithium on diffusion chamber (DC) granulopoiesis. When DC loaded with bone marrow cells were implanted into the peritoneal cavity of mice previously injected with lithium carbonate, more proliferative and nonproliferative granulocytes were produced as compared to DC implanted into control hosts. The number of DC CFU-c was increased significantly in the lithium-treated group, but there was no difference in the number of DC CFU-s. Levels of DC fluid CSF showed no evident correlation with DC myelopoiesis. These data suggest that a humoral factor other than CSF mediates the action of lithium in DC granulopoiesis, and that lithium's influence on DC hematopoietic stem cell proliferation occurs mainly at the CFU-c level.     

Characteristics of the Cfu-S Population In Mice Carrying the SlJ Allele

Abstract Abstract. A tentative characterization of haemopoietic stem cells with respect to their organ distribution, seeding fraction and colony formation in the spleen, radiosen-sitivity and humoral regulation was attempted in mice heterozygous for the mutant allele Sl^J and in their normal littermates. Sl^J/+ mice were characterized by a deficient CFU-s content of the blood and spleen and had slightly lower femoral CFU-s numbers. This CFU-s distribution could not be explained by differences in seeding efficiency ‘f’ between CFU-s of Sl^J/+ and +/+ origin in lethally irradiated recipients used in the CFU-s assay. the seeding fraction of CFU-s of +/+ origin did not differ in +/+ and Sl^J/+ recipients. However, in irradiated SI^J/+ recipient mice a 30% decrease was observed in the number of the colonies derived from splenic and femoral CFU-s of both +/+ and Sl^J/+ origin. the serum level of SHSF (splenic haemopoiesis stimulating factor) was decreased in Sl^J/+ mice, but significantly increased in Sl/Sl^d mice, as compared to their respective normal +/+ littermates. Endogenous colony formation in Sl^J/+ spleens was deficient in comparison to that observed in +/+ spleens, and distinct sex differences were observed. However, mutant and normal CFU-s from spleen and bone marrow had a similar survival following in-vitro y irradiation. Femurs and spleens of both Sl^J/+ and +/+ origin were implanted into both Sl^J/+ and +/+ hosts. Six weeks later the Sl^J/+ grafts contained less CFU-s than the +/+ grafts. These data show that the splenic stroma of Sl^J/+ mice is not defective in its capacity to lodge injected CFU-s but is deficient in its ability to maintain CFU-s under ‘steady-state’ conditions and stimulate their colony formation in a ‘perturbed state’. Some of the characteristics of Sl^J/+ mice segregate them from Sl/Sl^d mice, i.e. a deficient splenic CFU-s content, normal seeding fractions ‘f’ of CFU-s from spleen and bone marrow in the presence of an almost compensated anemia, and decreased serum levels of SHSF. the study of the Sl^J trait may be a useful extension of the current Sl/Sl^d model for exploration of hereditary defects in haematopoietic stroma.     

Regulation of haemopoietic stem-cell proliferation in mice carrying the Slj allele

We investigated a haemopoietic stromal defect, in mice heterozygous for the Slj allele, during haemopoietic stress induced by treatment with bacterial lipopolysaccharides (LPS) or lethal total body irradiation (TBI) and bone-marrow cell (BMC) reconstitution. Both treatments resulted in a comparable haemopoietic stem cell (CFU-s) proliferation in Slj/+ and +/+ haemopoietic organs. There was no difference in committed haemopoietic progenitor cell (BFU-e and CFU-G/M) kinetics after TBI and +/+ bone-marrow transplantation in Slj/+ and +/+ mice. The Slj/+ mice were deficient in their ability to support macroscopic spleen colony formation (65% of +/+ controls) as measured at 7 and 10 days after BMC transplantation. However, the Slj/+ spleen colonies contained the same number of BFU-E and CFU-G/M as colonies from +/+ spleens, while their CFU-s content was increased. On day 10 post-transplantation, the macroscopic 'missing' colonies could be detected at the microscopic level. These small colonies contained far fewer CFU-s than the macroscopic detectable colonies. Analysis of CFU-s proliferation-inducing activities in control and post-LPS sera revealed that Slj/+ mice are normal in their ability to produce and to respond to humoral stem-cell regulators. We postulate that Slj/+ mice have a normal number of splenic stromal 'niches' for colony formation. However, 35% of these niches is defective in its proliferative support.     

Simulation of CFU-s kinetics after irradiation

A simulation model of the CFU-s population is used to interpret data from experimental studies of bone marrow recovery after irradiation. The model includes an original hypothesis that the proliferation rate in the CFU-s population depends on the number of DNA-synthesizing CFU-s. It is assumed that the DNA-synthesizing CFU-s produce a factor in the presence of which CFU-s enter the resting state G0 after mitosis and remain there for prolonged periods of time. The model can adequately reproduce complex CFU-s kinetics observed after severe damage caused by irradiation with a unique set of parameters.     

Expression of tumor-associated surface membrane antigens on marrow CFU-s, CFC-gm, BFU-e, and brain CFU-s

Antiserum raised against a mouse mast cell line (FMP1) reacts with 90% to 100% of spleen colony-forming units (CFU-s), granulocyte/macrophage colony-forming cells (CFC-gm), erythroid burst-forming units (BFU-e), and 15% of nucleated marrow cells, using a complement-dependent cytotoxicity assay. We demonstrated that bone marrow, spleen, or thymus cells are able to absorb this activity from the antiserum. Although mouse brain cells have low reactivity with anti-FMP1 serum, the cytolysis level was reduced to background when antiserum was absorbed with brain cells. In addition, colony formation by marrow CFU-s, CFC-gm, and BFU-e was no longer prevented when the cells were incubated with brain-absorbed anti-FMP1 serum and complement. These findings suggest the presence of brain-associated antigens on CFU-s, CFC-gm, and BFU-e. To test whether a CFU-s accessory cell population in marrow is affected by treatment with anti-FMP1 serum and complement, antibody-treated marrow cells were mixed with large numbers of thymocytes and injected into recipient mice. Colony formation was not altered, indicating that the antiserum reacted directly with antigens on CFU-s and not on CFU-s accessory cells.     

Different Sensitivities of Granulocyte-Macrophage and Erythroid Progenitor Cells of Normal Human Bone Marrow to S-Phase-Specific Agents

The extraordinary sensitivity of early erythroid progenitor cells (BFU-e) of normal human bone marrow to tritiated thymidine ([³H]TdR) was studied. While exposure of bone-marrow cells to [³H]TdR for 1 hr resulted in the death of only 40% of the granulocyte-macrophage progenitor cells (CFU-c), 90% of BFU-e were killed. Experiments in which normal bone-marrow cells were mixed with bone-marrow cells which had been exposed to [³H]TdR demonstrated that the excessive killing of BFU-e by [³H]TdR reflected carry-over of the [³H]TdR by the exposed cells. A carry-over effect was not observed for CFU-c, suggesting the presence of a fundamental difference in the metabolism of TdR between CFU-c and BFU-e. There was a suggestion of a carry-over effect regarding two other S-phase-specific agents, hydroxyurea and 1-β-D-arabinofuranosylcytosine.     

The maturation state of three types of granulocyte/macrophage progenitor cells from mouse bone marrow

The progenitor cells of neutrophil granulocytes and macrophages which are able to proliferate and differentiate in vitro (CFU-c) form a heterogeneous population. By the use of specific colony stimulating activities and cell separation by equilibrium density centrifugation, three subpopulations of CFU-c can be detected. These three CFU-c are characterized by buoyant densities of 1.070, 1.075 and 1.080 g.cm^?3 and by their proliferative response to 18 h postendotoxin serum, colony stimulating factor from extracts of mouse embryos and uteri (CSF-pmue) and erythrocyte lysate, respectively. The three CFU-c are compared with respect to their differentiation potential, the maturation rate of their progeny cells and their proliferation capacity. It is shown that with increasing density of the CFU-c the maturation rate increases (sequential maturation of colonies derived from CFU-c with densities of 1.080, 1.075, 1.070 g.cm^?3) and the proliferation capacity decreases (colony size decreases in the sequence of CFU-c with densities 1.070, 1.075, 1.080 g.cm^?3). Concerning the differentiation potential it is shown that all three CFU-c detected have the capacity to form granulocytes as well as macrophages. On the basis of these results it is concluded that the CFU-c with densities of 1.070, 1.075 and 1.080 g.cm^?3 represent a maturation sequence.     

Thymic dependency of the humoral regulation of CFU-s proliferation in mice bearing a CSF-producing tumor

A better understanding of the mechanisms involved in the proliferation of splenic colony-forming units (CFU-s) during tumor growth is important for the prevention of bone marrow aplasia during chemotherapy. The in vivo growth of EMT6 cells, a colony-stimulating factor-secreting mammary tumor, in BALB/c and nude mice resulted in splenomegaly and an increase in the number of splenic granulocyte/macrophage colony-forming cells (GM-CFC). Proliferation of CFU-s, observed in BALB/c mice but not in nude mice, most likely resulted from combined direct and indirect actions of factors secreted by tumor and host cells (in particular helper T cells). These factors were detectable in the serum immediately following tumor cell injection. Thus, the GM-CFC response to factors secreted by the EMT6 tumors is thymus-independent while the CFU-s response is dependent upon the presence of T cells. Finally, we show that EMT6 tumor growth had no effect on the determination of CFU-s differentiation toward the various myeloid cell lineages.     

Stem cell (CFU-s) proliferation in sublethally irradiated mice

Abstract. The proliferation rate of haemopoietic stem cells (CFU-s) was followed after sublethal total body irradiation with 1.5 Gy. The [³H]-thymidine suicide technique was used to measure the CFU-s proliferation rate. The measurements extended from 10 min after irradiation up to 21 days. The CFU-s did not enter the DNA synthesis period (S-phase) shortly after irradiation, as had been previously suggested, but did so only with a delay of 14–16 hr. A large scatter of results was explained by an oscillatory pattern in CFU-s proliferation. The CFU-s prepared for cell division in synchronized waves, with a period of 20–22 hr.     

COLONY-FORMING UNITS IN DIFFUSION CHAMBERS (CFU-d) AND COLONY-FORMING UNITS IN AGAR CULTURE (CFU-c) OBTAINED FROM NORMAL HUMAN BONE MARROW: A POSSIBLE PARENT–PROGENY RELATIONSHIP

A series of experiments was performed to elucidate the relationship between cells that form granulocytic colonies in fibrin clot diffusion chambers implanted into the peritoneum (i.p.) of irradiated mice (CFU-d) and day 7 and day 14 CFU-c which give rise to colonies after 7 and 14 days in agar cultures in vitro, respectively. Normal human bone marrow cells were cultured in suspension in vitro or in diffusion chambers implanted into irradiated or non-irradiated mice. During these culture conditions there was an initial decrease in the number of CFU-c per culture. This was followed by an increase between day 2 and day 7 of culture. No similar increase of neutrophilic CFU-d was observed. When CFU-d, day 14 and day 7 CFU-c in normal marrow were separated by velocity sedimentation and cultured in suspension culture or in diffusion chambers for 7 days, the maximum increase of day 7 and day 14 CFU-c was observed in slowly sedimenting cell fractions which contained the majority of CFU-d. After 3 days in suspension culture, the maximum increase of day 14 CFU-c was found in fractions which also gave rise to maximum numbers of CFU-c after 7 days. However, day 7 CFU-c were found in fractions which initially contained the majority of day 14 CFU-c. No increase in CFU-d was found in fractions initially containing peak numbers of CFU-c. Between 53 and 71% of CFU-c harvested from diffusion chambers in irradiated mice or from suspension cultures were sensitive to pulse incubation with tritiated thymidine, suggesting that the cells were proliferating during these culture conditions. In diffusion chambers implanted into non-irradiated mice, however, CFU-c were found to be relatively resistant to this treatment (3–11% sensitive to tritiated thymidine). Thus marked increases in CFU-c were also observed during experimental conditions, where no significant DNA synthesis was detected. A reproducible time sequence of increase in CFU-c populations in culture was observed. Day 14 CFU-c and cells that gave rise to clusters on day 7 in agar increased between day 2 and day 4, whereas day 7 CFU-c increased between day 4 and day 7. The results suggested that CFU-d gave rise to CFU-c in culture and that day 14 CFU-c were precursors of day 7 CFU-c.     

Comparison of the effects of IL-3, granulocyte-macrophage colony-stimulating factor, and macrophage colony-stimulating factor in supporting monocyte differentiation in culture. Analysis of macrophage antibody-dependent cellular cytotoxicity

Cultured human monocytes undergo a process of differentiation and maturation lasting 5 to 10 days that ultimately leads to the appearance of large macrophage-like cells. This differentiation is growth factor dependent: of all the cytokines tested, only macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF (GM-CSF), and IL-3 proved capable of supporting the differentiation and the long term survival of the macrophage-like cells. Although all three cytokines yield cells with macrophage characteristics, cells developed in M-CSF have features distinct from those matured in either IL-3 or GM-CSF. At the morphologic level, the M-CSF-supported monocyte cultures yield elongated, spindle-shaped cells whereas those supported with IL-3 or GM-CSF yielded round cells with distinct nuclei. All three macrophage populations expressed similar levels of HLA-DR, CD11b, and CD11c, but the M-CSF-treated cultures yielded more CD14+ and CD16+ (Fc gamma RIII) cells. All three cell populations developed capacity for antibody-dependent cellular cytotoxicity (ADCC) as well as antibody-independent cytotoxicity with peak activity achieved after 8 to 12 days in culture. ADCC capacity developed earliest and the level of activity was usually greatest in the M-CSF-treated cultures, possibly correlating with the higher level of expression of CD16. Our findings indicate that any of these cytokines, but particularly M-CSF, may be useful clinically in enhancing the tumoricidal capacity of tumor-specific mAb through augmentation of macrophage capacity for ADCC.