The effect of internal exposure to 90Sr on hematopoietic stem cells in CBA mice

After acute intake of 90Sr the changes of d-9 CFUs number in mice (CBA) bone marrow, spleen and peripheral blood were investigated. The obtained results indicated similar quantitative changes in bone marrow and spleen CFUs on exposure to the 90Sr when radiation doses did not cause the decrease in life-time (1.11 kBq/g). Sarcomogeneous doses of 90Sr (29.6 kBq/g) resulted in drastic changes of hemopoietic system: spleen haematopoiesis activation and suppression of bone marrow functions. On the first day after 90Sr injection (29.6 kBq/g) the increase in number of peripheral blood CFUs (circulating pool) was observed.     

Compensation mechanisms in hemopoietic stem cell pool (CFUs) under the conditions of experimental chronic gamma-irradiation

The kinetics, proliferation and differentiation potentials of hemopoietic stem cells (CFUs) of bone marrow and spleen were investigated in CBA-line mice in the early period (1-30 days) of chronic gamma-irradiation at a dose rate of 0.16 Gy/day to attain a cumulative dose of 4.8 Gy. The results of the experimental study showed the prevalent maintenance of productivity of granulocytic and erythrocytic hemopoietic cell series within the range of reference values, persistent inhibition of the megakaryocytic series (in terms of all hemopoiesis parameters of interest), more marked suppression of the population of polypotential CFUs in the bone marrow as compared with that in the spleen. The obtained results indicated that the mechanisms of hemopoiesis compensation at stem cell pool level were as follows: the increase in proliferation potency of erythrocytic and in polypotential precursors, the rise in the proportion of granulocytic precursors in the real differentiation potential of CFUs, and the processes of repopulation manifested with different intensity in all stem cell populations under study. For maintenance of the necessary productivity of CFUs in each of hemopoietic cell series, consecutively or simultaneously, several compensatory-adaptive mechanisms are started, which allows the avoidance of a sharp competition between hemopoietic cell series under the conditions of stem cell pool depopulation, and preservation of the hemopoiesis as a whole.     

Effect of 5'-deoxy-5'-S-isobutyladenosine on hematopoietic stem cells

Unlike known cytostatics, 5'-deoxy-5'-S-isobutyladenosine (SIBA) does not inhibit proliferative activity of hemopoietic stem cells of intact bone marrow. On the contrary, it raises CFUs number of 15-28% in vitro and in vivo. SIBA inhibits by 40% the 3',5'-cAMP-induced increase in CFUs proliferation.     

Effect of Hyperthyroidism On Haemopoietic Stem Cell Kinetics In Mice

Changes in the pool of haemopoietic colony-forming units (CFUs) of bone marrow and spleen were studied in experiments with mice fed dried thyroid gland (TH) for 21 days, and during the 13 days that followed feeding. After HU treatment, the number of CFUs in DNA synthesis was estimated. As early as the second day of TH treatment, the pool of CFUs is gradually increased, leading to an increase in the total number of splenic and bone marrow CFUs persisting after TH treatment for the period examined. Simultaneously, the numbers of nucleated cells in the bone marrow and spleen are increased. During TH feeding and following its termination, the total number of erythrocytes and the haematocrit values did not change significantly, whereas an increased number of leucocytes was observed in the peripheral blood after TH treatment. Elevation of the proliferative activity of CFUs occurred early in the period of TH treatment, with the maximum attained by end of the first week of TH feeding. This suggests a rapid response of the haemopoietic stem cell compartment to the administration of TH hormones. the participation of humoral factors controlling CFUs compartments in the mechanism of the stimulatory effect of TH hormones on haemopoietic stem cells is discussed.     

Capacity of hematopoietic colony-forming cells to maintain their own population in the late periods after prolonged radiation exposure

The content of multipotent CFUs in bone marrow and their self-maintenance capacity were studied for 15 months following protracted external radiation of CBA mice at the total dose 10 Gy (0.5 Gy per day). The mean life shortening was 16% in the irradiated mice. The proliferating, maturating and functional pools returned to normal within 1-3 months after exposure. The stem cell pool did not return to the values seen in the same age controls till the end of the life of experimental animals and averaged 55% of normal. The self-maintenance capacity of bone marrow CFUs was 2.5-4.5-fold as decreased in the irradiated mice. The failure of this unique property of multipotent CFUs was principally due to the foregoing increase in their proliferative activity.     

The precursor hematopoietic cell: its origin in ontogeny, proliferative activity and proliferative potential

Cells responsible for repopulation of irradiated longterm cultures of murine bone marrow and capable of generating CFUs for at least 4-5 weeks after seeding referred here to as primitive hemopoietic stem cells (P-HSC) were assayed by limiting dilution analysis. During development of mice P-HSC can be detected for the first time in the liver of 12-13-day-old embryos and their number is about 10 per organ. At day 17-18 of gestation the number of P-HSC increases ten-fold; however, we could not detect the proliferation of these cells using the technique of hydroxyurea suicide. In the adult mouse P-HSC content is about 100 precursors per femur and their concentration is one P-HSC per 1-2 x 10(5) bone marrow cells. P-HSC content in the spleen is 0.5 per 10(6) cells. In vivo treatment with 5-fluorouracil or hydroxyurea (six injections every 6 h) does not alter significantly the number of P-HSC, although either treatment kills about 99% of CFUs. Several months after reconstitution of lethally irradiated mice with a "small" inoculum of bone marrow cells (0.20-0.35 x 10(6)) the number of bone marrow P-HSC was reduced as compared to that in animals reconstituted by injection of a "large" cell dose (20-35 x 10(6)). These data suggest that P-HSC have limited proliferative potential and are incapable of self-maintenance.     

Murine hematopoietic stem cells after acute and chronic administration of vincristine

The effects of vincristine on bone marrow hemopoietic stem cells were studied in mice. After the administration of a single dose of the drug there is a sharp decline in the content of CFUs. When the drug is given in small, weekly doses for 16 weeks, after an initial drop, the stem cell content rises at a normal level where it remains, although with wide oscillations. On the whole the effects of vincristine on b.m. CFUs are comparable with that observed after vinblastine administration.     

Effect of239Pu on mouse hemopoietic stem cells in different types of bone marrow cavities

Summary Repopulative activity of hemopoietic stem cells of mice given i.v. 5 kBq²³⁹Pu/mouse (166.5 kBq/kg) was followed. The activity retained was measured in the whole mouse, the skeleton and the liver. Simultaneously average cumulative skeletal dose was calculated. Quantitative parameters of the stem cell compartment and the marrow cellularity were studied in variously arranged bones (femur, pelvis, lumbar vertebra) using the exocolonizing test and cytological techniques. The effects of radiation were most marked in lumbar vertebra, less serious changes were found in pelvis and only a moderate response was present in femur. The bone marrow hemopoiesis is damaged in various bone sites to different degrees and the percentage of cells at risk appears higher in trabecular than in cortical bone.     

RESPONSE OF THE PREOSTEOBLAST AND STEM CELL OF RAT BONE MARROW TO A LETHAL DOSE OF X-IRRADIATION OR CYCLOPHOSPHAMIDE

Following syngeneic or autotransplantation of hemopoietic tissue to a heterotopic location, bone formation has been observed to occur in the implanted tissue. the characteristics of the cell residing in hemopoietic tissue with bone forming potential (preosteoblast) are unknown. to define some properties of this cell, its response to X-irradiation and cyclophosphamide (CTX) was compared to the response of the hemopoietic stem cell. Adult, male rats were exposed to 900 R whole body X-irradiation or 220 mg/kg of intraperitoneal CTX. With either treatment the dose was sufficient to kill the animals by bone marrow failure. At intervals following the X-irradiation or CTX, hemopoietic tissue was examined for the presence of viable hemopoietic stem cells and preosteoblasts. Following X-irradiation, viable hemopoietic stem cells and preosteoblasts could not be detected. Following CTX these cells could be detected. It is suggested that in the rat CTX at 220 mg/kg, although causing death by bone marrow failure, does not reduce the population of the preosteoblast or hemopoietic stem cell as effectively as 900 R X-irradiation.     

ADAPTATION OF HEMOPOIETIC TISSUE RESULTING FROM ESTRONE-INDUCED OSTEOSCLEROSIS IN MICE

The redistribution of hemopoietic tissue resulting from estrone-induced osteosclerosis in the mouse was studied. As the marrow was gradually replaced by bone, extramedullary hematopoiesis in the spleen increased at a rate sufficient to maintain hemopoietic homeostasis. The total numbers of colony forming units (CFU) in the tibia and spleen as well as the proportion of CFU in cycle was assessed. After five injections of estrone, tibial CFUs decreased to 2% of control values whereas splenic CFUs increased approximately nine-fold. The proliferative capacity of the splenic CFU was also increased in the estrone-treated animals. The increased numbers of splenic CFUs as well as the increased proliferative capacity of this compartment are probably related to the ability of extramedullary hematopoiesis in the spleen to compensate for a marrow that has been replaced by bone.     

Il-17 mobilizes peripheral blood stem cells with short- and long-term repopulating ability in mice

Autologous and allogeneic bone marrow transplantations have evolved as important cancer therapy modalities. For both indications, peripheral blood has been shown to have distinct advantages over bone marrow as the stem cell source. Cytokine combinations for mobilization have enhanced stem cell yield and accelerated engraftment. However, novel mobilizing agents and strategies are needed to further improve clinical outcomes. Within the donor graft, the dynamic equilibrium between T cells and stem cells critically influences engraftment and transplantation results. IL-17 is a cytokine produced almost exclusively from activated T cells. IL-17 was expressed in vivo with adenovirus technology. Here, proof-of-principle studies demonstrate that IL-17 effectively mobilizes hemopoietic precursor cells (CFU-granulocyte-erythrocyte-macrophage-monocyte, CFU-high proliferative potential) and primitive hemopoietic stem cells (Lin(-/low)c-kit(+)Sca1(+)). Moreover, mouse IL-17 adenovirus-mobilized peripheral blood stem cells rescued lethally irradiated mice. Bone marrow was found to be 45-75% of donor origin at 1 year. In secondary recipients, donor-derived bone marrow cells ranged from 45 to 95%. These data show that IL-17 mobilizes stem cells in mice with short- and long-term reconstituting capacity. Additional comparative studies are needed as well as studies in tumor models to refine distinct potential clinical applications for IL-17-mobilized peripheral blood stem cells.     

The study of mechanisms of radioprotective action of indometofen n hematopoietic stem cells in mouse long-term bone marrow cultures

The influence of indometophen (an analog of tamoxiphen) on the dynamic content and the proliferative activity of CFUs (colony-forming units) and CFU-GM (granulocyto-macrophages precursors) and the level of colony-stimulating factor (GM-CSF) in mouse long-term bone marrow cultures were studied for 4 weeks after administration. Five days after indometophen injection the long-term cultures were exposed to irradiation with a dose of 2 Gy and on the time course of postirradiation recovery haemopoietic precursors cells and dynamic release of GM-CSF in the culture supernatants were examined. The data of this report suggest that the mechanisms responsible for the radioprotective action of indometophen may be associated both with its direct effects on the proliferation and differentiation of hemopoietic cellular precursors and with the stimulation of release of growth-differential factors by hemopoietic microenvironmental elements.     

Study of hematopoietic stem cell pool maintenance in successive transplantations using a quantitative method of assessment]

The ability of transplantable hemopoietic stem cells (HSC) to maintain their pool was studied using successive bone marrow transplantations with quantitative evaluation of hemopoiesis restoring units (HRU) in each transfer. The number of injected HRU increased (3.6-48.6--fold) upon each transfer; however, the normal level could not be attained. The ability fo HRU for further multiplication was exhausted after five transfers. HRU lost totipotentiality after four transfers. The data obtained support the concertion of Kay (1965) that HSC department is a pool of heterogeneous cells, and the property of "stemness" is inversely related to the number of divisions of ancestral cells. Transplantation, being a proliferative stress for the dormant HSCs, thus lowers the stem potential of the whole pool. The experimental data suggest that while dividing stem cell does not have a choice to self-renew or to differentiate into maturing cells, but it really differentiates into HSCs of lower rank.     

Proliferation inhibition of murine pluripotent haemopoietic stem cells by interferon or poly I:C

The effect of mouse serum interferon (IF) in vitro and an inducer in vivo on the proliferation of a pluripotent stem cell population with high turnover rate was studied. Proliferation rate was characterized by the number of CFUs in the S phase of the cell cycle. Increased proliferation of bone marrow stem cell populations was produced either by irradiating the donor mice with 3.36 Gy (336 rad) 60Co-gamma rays 7 days before the experiment or by incubating normal bone marrow cells with 10(-11) M concentration of isoproterenol. IF considerably reduced the number of CFUs in S phase in both cases without reducing the CFUs content of the samples. Injection of IF inducer (4 mg/kg poly I:C) into regenerating mice also inhibited the proliferation of CFUs without decreasing the femoral CFUs level. Regeneration kinetics of CFUs from irradiated poly I:C-treated mice ran parallel with that of irradiated untreated animals but showed a characteristic delay corresponding to approximately one CFUs doubling. A transient, non-cytotoxic proliferation inhibitory effect of IF or IF inducer is, therefore, proposed.     

The proliferative potential of CFUs from the bone marrow of thymectomized mice]

Proliferative potential of CFUs in bone marrow of young and adult mice (1.5-25 months) and thymus influence on this property were studied. It has been shown on the model of adult thymectomized mice that during "steady state" hematopoiesis, proliferative potential of bone marrow CFUs does not depend on the animals age and on thymic factors.     

Analysis of DNA status in bone marrow cells of mice internally irradiated with 90Sr

The response of bone marrow cells of CBA mice injected with 22.2, 222 and 592 kBq/animal to additional gamma-irradiation (3 Gy) for testing purposes was evaluated using SCG (Comet assay). A decrease in induction of DNA damage right after additional gamma-irradiation was determined. It correlated with bone marrow cell quantity and the tail length before additional gamma-irradiation. The results support the suggestion about the activation of DNA repair in bone marrow cells under exposure to 90Sr in vivo.     

Effect of alkylating derivatives of cyclic adenosine-3' ,5'-monophosphate on proliferation of mouse bone marrow stem cells

The effect of the physiological concentration of cyclic adenosine-3' ,5'-monophosphate (cAMP) analogues on the proliferation of mouse bone marrow stem hemopoietic cells (CFUs) was examined. The stimulating effect was estimated from the decrease in CFUs expressed in the percentage derived from comparing the number of spleen colonies in the control and experimental groups treated with hydroxyurea 10(-3) M (incubation with hydroxyurea resuted in the cell death in S-phase). Cyclic AMP stimulted the proliferation of CFUs by 60%, while its analogues such as 8-(N-chloroacetylaminoethylamino)-cAMP, 1-(N-chloroacetylaminoethyoxy)-cAMP and 1-(N-(p-fluorosulfonyl)-benzoylaminoethoxy)-cAMP stimulated the proliferation by 39.2%, 32.4% and 21.9%, respectively. Therefore, the synthetic analogues of cAMP were not only far from inhibiting the proliferation of CFUs but, on the contrary, exerted a stimualting effect unlike most antineoplastic alkylating drugs that depress hemopoiesis up to its total aplasia.     

The regulation of hematopoietic stem cell proliferation and differentiation (CFU-S) during antigenic exposure

Hemopoietic stem cell (CFUs) proliferation is controlled by regulatory activities (stimulator and inhibitor) produced by bone marrow macrophages. Previously it has been shown that antigen administration stimulates CFUs proliferation. The data obtained in this study show the possible mechanism of antigen-induced stimulation of CFUs proliferation. 3-4 days after antigen injection bone marrow cells of BDF1 mice cease to produce inhibitory activity in contrast to similar cells of control animals. Therefore, increased CFUs proliferation in immunized mice can be due to decreased production of inhibitory activity and resulting abundance of stimulating factors. In BAlB/c mice CFUs proliferation is not changed after antigen injection and their bone marrow cells continue to synthesize inhibitory substances. Differentiation of CFUs into committed blood precursor cells may depend on the proliferation level in CFUs population since activation of CFUs proliferation in immunized BDF1 mice is accompanied by a decreased number of CFU-GM and CFU-M but an increased number of BFU-E. It should be noted that intact BAlB/c mice show a high level of CFUs proliferation similar to that of immunized BDF1 mice.     

Comparison of direct and indirect radiation effects on osteoclast formation from progenitor cells derived from different hemopoietic sources

Hemopoietic stem and progenitor cells from different sources differ in radiosensitivity. Recently, we have demonstrated that the multinucleated cell responsible for bone resorption and marrow cavity formation, the osteoclast, is in fact of hemopoietic lineage. In this investigation we have studied the radiosensitivity of osteoclast formation from two different hemopoietic tissues: fetal liver and adult bone marrow. Development of osteoclasts from hemopoietic progenitors was induced by coculture of hemopoietic cell populations with fetal mouse long bones depleted of their own osteoclast precursor pool. During culture, osteoclasts developed from the exogenous cell population and invaded the calcified hypertrophic cartilage of the long bone model, thereby giving rise to the formation of a primitive marrow cavity. To analyze the radiosensitivity of osteoclast formation, either the hemopoietic cells or the bone rudiments were irradiated before coculture. Fetal liver cells were found to be less radiosensitive than bone marrow cells. The D0, Dq values and extrapolation numbers were 1.69 Gy, 5.30 Gy, and 24.40 for fetal liver cells and 1.01 Gy, 1.85 Gy, and 6.02 for bone marrow cells. Irradiation of the (pre)osteoclast-free long bone rudiments instead of the hemopoietic sources resulted in a significant inhibition of osteoclast formation at doses of 4 Gy or more. This indirect effect appeared to be more prominent in the cocultures with fetal than with adult hemopoietic cells. Furthermore, radiation doses of 8.0-10.0 Gy indirectly affected the appearance of other cell types (e.g., granulocytes) in the newly formed but underdeveloped marrow cavity. The results indicate that osteoclast progenitors from different hemopoietic sources exhibit a distinct sensitivity to ionizing irradiation. Radiation injury to long bone rudiments disturbs the osteoclast-forming capacity as well as the hemopoietic microenvironment.     

Mechanisms of lymphocytic choriomeningitis virus-induced hemopoietic dysfunction.

Results of this study showed that lymphocytic choriomeningitis virus infection causes a marked activation of natural killer (NK) cells not only in the spleen but also in the bone marrow. This activity reached its peak at about day 3 of infection and declined after days 6 to 7. Enhanced NK cell activity was found to correlate with decreased receptivity for syngeneic stem cells in bone marrow and spleen, with the notable exception that decreased receptivity persisted longer in bone marrow. Treatment of infected recipients with anti-asialo GM1 (ganglio-N-tetraosylceramide) significantly increased the receptivity for syngeneic hemopoietic cells. These findings are consistent with the hypothesis that NK cell activation causes rejection of syngeneic stem cells, thus resulting in hemopoietic depression. To understand the mechanisms behind the prolonged decrease in bone marrow receptivity (and bone marrow function in the intact mouse) mentioned above, we followed the changes in the number of pluripotential stem cells (CFU-S) circulating in the peripheral blood and in endogenous spleen colonies in irradiated mice, the limbs of which were partially shielded. It was found that following a marked early decline, both parameters increased to normal or supranormal levels at about day 9 after infection. Because the bone marrow pool of CFU-S is only about 20% of normal at this time after infection, a marked tendency for CFU-S at this stage in the infection to migrate from the bone marrow to the spleen is suggested. It seems, therefore, that as NK cell activity declines, the spleen regains the ability to support growth of hemopoietic cells and the bone marrow resumes an elevated export of stem cells to the spleen. This diversion of hemopoiesis could explain both the long-standing deficiencies of the bone marrow compartment and the prolonged decrease in the receptivity of this organ.