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.     

Conservation of bone marrow CFUc in different phases of the cell cycle during cryopreservation

The effect of low temperature (-196 degrees C) preservation on the recovery of colon-forming units (CFUs) of bone marrow at different phases of the cell cycle before cryopreservation is dealt with. The intact bone marrow "enriched" with CFUs in S phase of the cell cycle and the bone marrow without colony-forming units in S phase were exposed to cryopreservation. After cryopreservation of the bone marrow enriched with CFUs in S phase and th bone marrow without colony-forming units in S phase the number of CFUs decreases by the same value as in the cryopreserved bone marrow obtained from intact mice.     

Mechanism of action of the stem-cell inhibition factor on the formation of exogenous hemopoietic colonies in the spleen of mice

It was established by previous works that thymocytes treated with antilymphocyte serum secrete soluble factor capable of inhibiting exogenous colony formation in the spleen of lethally irradiated mice injected with bone marrow cells treated with the stem cell inhibition factor (SCIF). The purpose of the present investigation was to explore possible mechanisms of SCIF action. Regeneration of erythropoiesis (measured by 59Fe incorporation) in the spleen and bone marrow of mice injected with SCIF-treated bone marrow cells was inhibited as compared with control, while CFUs started proliferating with a 3-day delay. Two hours after SCIF treatment 60% of CFUs entered S phase as judged by hydroxyurea cell kill. The CFUs fraction treated with the SCIF was found to be diminished 3-4-fold as compared with control. The data obtained suggest that SCIF treatment makes CFUs enter 3 phase, which may account for the reduced capacity of CFUs to populate the spleen and to proliferate with a 3-day delay.     

Bone Marrow Response to Damage Induced By Hydroxyurea Or Colchicine

The extent of bone marrow damage caused by the administration of single or repeated doses of either hydroxyurea (1000 mg/kg b.w.) or colchicine (1 mg/kg b.w.) are comparable. This conclusion is based on serial studies of bone marrow cellularity and of the CFUc numbers in the bone marrow. the proliferation response of the pluripotential haemopoietic stem cells, determined by the cells forming colonies in the spleen of lethally irradiated mice (CFUs) markedly differs if the bone marrow damage is caused by hydroxyurea or colchicine. While hydroxyurea administration stimulates a large proportion of the resting G₀ cells into the cell cycle, the damage induced by colchicine is followed by only a mild increase in the CFUs proliferation rate. The seeding efficiency of the spleen colony technique has been determined after both hydroxyurea and colchicine administration. This parameter, important for the estimation of the number of the pluripotential haemopoietic stem cells in blood forming organs, is significantly affected by hydroxyurea administration, but also by repeated injections of colchicine. Following a single dose of hydroxyurea, the time-course of the CFUs numbers, which were corrected for the change in the seeding efficiency, shows an overshoot occurring after 18–20 hr. At the other time periods, the number of pluripotential haemopoietic stem cells is little affected by a single hydroxyurea injection. This poses a question about the nature of the stimulus, which after hydroxyurea administration triggers the CFUs from the resting G₀ state into the cell cycle. There is evidence that this stimulus is probably not represented by the damage caused to the various intensively proliferating cell populations of the bone marrow. This evidence is based on experiments which show that colchicine induced damage, of a degree similar to that after hydroxyurea, does not stimulate the CFUs proliferation rate to an extent comparable to hydroxyurea. The possibility that colchicine could block CFUs in the G₀ state or that it could interfere with the progress of CFUs through the G₁ and S phases of the cell cycle have been ruled out by experiments which demonstrated that colchicine (1 mg/kg b.w.), administered 10 min before hydroxyurea, does not reduce the number of CFUs triggered into the cell cycle as the consequence of hydroxyurea administration.     

Study on the proliferative state of haemopoietic stem cells (CFU).

Hydroxyurea was used to study the proliferation rate of haemopoietic stem cells (CFUs) in normal mice, after irradiation or transplantation into irradiated recipients. It was demonstrated that the proliferation rated of endogenous CFUs (endo-CFUs) and exogenous CFUs (exo-CFUs) are identical. After irradiation (650 R) the surviving endo-CFUs begin to proliferate immediately. By contrast exo-CFUs transplanted into the irradiated recipient mouse (850 R), begin to proliferate only after about 30 hr. However, injection of isoproterenol (which stimulates adenyl cyclase) or dibutyryl cyclic adenosin 3',5'-monophosphate shortly after marrow cell graft, triggers the transplanted CFUs into cell cycle as shown by an almost immediately increased sensitivity to hydroxyurea. Isoproterenol is capable of inducing DNA synthesis also in stem cells of normal mice but it takes about 20 hr before CFUs become to be increasingly sensitive to hydroxyurea.     

Compensatory adjustment of hemopoietic stem cell pool of CBA mice after acute intake of 90Sr

It was investigated the functional status of stem cell pool (CFUs) of bone marrow, spleen and peripheral blood in mice (CBA) in early (1-30 days) and late (180-360 days) period after acute intake of 90Sr (29.6 kBq/g). Cumulative dose in red bone marrow due to incorporated 90Sr was 0.98-87.7 Gy. The kinetics, proliferative and differentiative potential of stem hemopoietic cells (CFUs) and productivity of hemopoietic tissues were significantly influenced by dose rate, absorbed dose and degree of suppresssion of bone marrow functions.The obtained results indicated that the sarcomogenous doses of 90Sr (29.6 kBq/g) resulted in realization of compensatory reactions in hemopoietic stem cell pool to support the life ability of irradiated animals: higher proliferative potential of CFUs and its repopulation, redistribution of cell subpopulations during differentiation and activation of spleens hemopoiesis.     

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.     

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.     

MOBILIZATION OF HAEMOPOIETIC STEM CELLS (CFU) INTO THE PERIPHERAL BLOOD OF THE MOUSE; EFFECTS OF ENDOTOXIN AND OTHER COMPOUNDS

Factors affecting the circulation of haemopoietic stem cells (CFU) in the peripheral blood of mice were investigated. I.v. injection of sublethal doses of endotoxin, trypsin and proteinase appeared to raise the number of CFU per ml blood from about 30–40 to about 300–400 or more within 10 min. The effect was smaller when smaller doses of the substances were injected. After this initial rise the number of circulating cells returned to normal in a few hours. Following endotoxin there was a second rise which started 2–3 days after injection and attained a peak on the 6th–7th day. The first rise is explained as a mobilization of stem cells from their normal microenvironments into the blood stream; the second rise is considered to reflect proliferation of CFUs in the haemopoietic tissues. The spleen seems to be acting as an organ capturing CFUs from the blood and not as a source adding stem cells to the blood.
The early mobilization of CFU after endotoxin injection did not coincide with a mobilization of neutrophils. The number of circulating band cells was increased during the first hours.
The importance of 'open sites'in the haemopoietic tissue for capturing CFUs was studied by emptying these sites through a lethal X-irradiation and injecting normal bone marrow cells. When a greater number of syngeneic bone marrow cells was injected intravenously, the level of circulating CFU in irradiated mice was slightly lower than the level in unirradiated mice during the first hours.     

MULTIPLICATION OF HAEMOPOIETIC COLONY FORMING UNITS (CFU) IN MICE AFTER X-IRRADIATION AND BONE MARROW TRANSPLANTATION

Kinetics of the multiplication of haemopoietic CFUs was studied in lethally irradiated mice receiving various numbers of syngeneic bone marrow cells. After transplantation of a small number of bone marrow cells, the growth rate of CFU in femoral bone marrow appeared to decrease after about 10 days after transplantation, before the normal level of CFU in the femur was attained. In the spleen it was found that the overshoot which was observed about 10 days after transplantation of a large number of bone marrow cells is smaller or absent when a small number of cells is transplanted. Experiments dealing with transplantation of 50 x 10⁶ bone marrow cells 0, 4 or 10 days after a lethal irradiation indicated that the decline in growth rate of CFUs about 10 days after irradiation could not be attributed to environmental changes in the host.
The results are explained by the hypothesis that a previous excessive proliferation of CFUs diminishes the growth rate thereafter. This hypothesis is supported by experiments in which 50 x 10⁶ bone marrow cells derived from normal mice or from syngeneic chimaeras were transplanted. The slowest growth rate was observed when bone marrow that had been subjected to the most excessive proliferation in the weeks preceding the experiment was transplanted.     

Hematopoietic precursor cells in radiation chimeras restored by bone marrow from thymectomized mice]

Radioprotective capacity of bone marrow CFUs of adult thymectomized mice was studied. Lethally irradiated mice were inoculated with bone marrow of mice thymectomized 8-11 months before. The colony forming capacity and proliferative rate of CFUs were studied 1-7.5 months after obtaining the radiation chimeras. It has been shown that proliferative capacity of bone marrow of adult thymectomized mice was reduced in comparison with that of normal animals. It is related to the decrease (4-fold) of the proliferative rate of bone marrow of thymectomized mice which was inoculated into lethally irradiated recipients 1 month before. We also found that the content of CFUs in bone of those chimeras was reduced later--after 7.5 months. In this period (1-7.5 months) the cellularity of bone marrow did not change.     

DISTRIBUTION AND MULTIPLICATION OF COLONY FORMING UNITS FROM BONE MARROW AND SPLEEN AFTER INJECTION IN IRRADIATED MICE

The distribution and proliferation of CFUs from bone marrow and spleen cell suspensions were followed after injection in lethally irradiated isogeneic mice. It was found that a larger proportion of the injected bone marrow CFUs than of the spleen derived CFUs could be recovered from the recipient's spleen and femur. This consistently higher recovery points to the conclusion that a larger fraction of bone marrow-derived CFUs than of spleen-derived CFUs is capable of producing daughter CFUs, most likely due to a commitment to early differentiation of many spleen CFUs.     

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.     

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.     

PROLIFERATION RATE OF HAEMOPOIETIC STEM CELLS AFTER DAMAGE BY SEVERAL CYTOSTATIC AGENTS

The haemopoietic tissue of mice was damaged by different cell-cycle-stage specific and cell-cycle-stage non-specific cytostatic agents. The proliferation rate among the surviving pluripotential stem cells, i.e. those cells forming colonies in spleens of lethally irradiated mice (CFUs), was then investigated. The results suggest that, at least in the CFUs population, the cells which synthesize DNA in the S phase of the cell cycle inhibit the entry of the non-proliferating G₀ cells into cell cycle. This evidence was based on the ability of three cytostatic agents, hydroxyurea, cytosine arabinoside and methotrexate, which are toxic specifically to the S phase cells to increase the proliferation in the CFUs population. This increase was quite out of proportion to the small amount of damage they caused to the population. Colchicine, which kills cells in mitosis, and ionizing irradiation, damaging cells in all stages, proved to be much weaker stimulators of proliferation. It has been suggested that a mechanism for the control of cellular proliferation might be based on the negative feedback in the cell cycle. In this feedback control loop the cells which are preparing for cell division in the S phase of the cell cycle inhibit the entry of the non-proliferating G₀ cells into cell cycle.     

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 fractionated thymocytes from intact and irradiated mice on CFU recovery in the bone marrow after sublethal irradiation

In studying the influence of thymocytes fractionated by their size in the ficoll density gradient on the CFUs content of the irradiated mouse bone marrow, two subpopulations of T-cells were isolated: the administration of the first thymocyte subpopulation decreased the CFUs content during the postirradiation recovery period while thymocytes of the second subpopulation increased the content of CFUs in the bone marrow. When thymocytes of mice exposed to low-level radiation were separated a considerable stimulatory effect was produced by certain thymus cell fractions on the number of CFUs in the bone marrow of exposed recipients; no inhibitory effect was registered.     

Post-irradiation thymocyte regeneration after bone marrow transplantation. II. Physical characteristics of thymocyte progenitor cells

Bone marrow cells were separated according to buoyant density, velocity sedimentation and cell surface charge. Fractionated (C3H x AKR)F1 bone marrow cells were transplanted into lethally-irradiated C3H recipients. In all fractions, the CFUs content and the capacity to restore the thymus cell population were determined. For all the physical parameters tested, the thymocyte progenitor cells show the same distribution as CFUs. The relationship between number of thymocyte progenitor cells and number of CFUs is dependent on density. Bone marrow progenitors of PHA responsive cells are of low buoyant density and show a distribution which resembles the distribution of the progenitors of Thy 1 positive cells. After transplantation of large numbers of bone marrow cells into irradiated mice, no significant change in the CFUs content of the thymus was observed.     

Modification of the proliferative capacity of transplanted bone marrow colony forming units by changes in the host environment

Regulation of the proliferation of transplanted colony forming units (CFUs) was investigated in lethally irradiated mice, pretreated by methods known to accelerate hemopoietic recovery after sublethal irradiation. Prospective recipients were exposed to either hypoxia, vinblastine or priming irradiation and at different intervals thereafter lethally irradiated and transplanted with bone marrow. Repopulation of CFUs was determined by counting the number of splenic colonies in primary recipients or by retransplantation. Regeneration of grafted CFUs was greatly accelerated and their self-renewal capacity increased in mice grafted within two days after hypoxia. Also the number of splenic colonies formed by grafted syngeneic CFUs as well as by C57BL parent CFUs growing in BC3F1 hosts was significantly increased. The effect was not dependent on the seeding efficiency of CFUs and apparently resulted from hypoxia induced changes in the hosts physiological environment. Proliferative capacity of grafted CFUs increased remarkably in hosts receiving vinblastine two or four days prior to irradiation. Priming irradiation given six days before main irradiation accelerated, given two days before impaired regeneration of CFUs. The increased rate of regeneration was not related to the cellularity of hemopoietic organs at the time of transplantation. The growth of CFUs in diffusion chambers implanted into posthypoxic mice was only slightly improved which does indicate that the accelerated regeneration of CFUs in posthypoxic mice is mainly due to the changes in the hemopoietic microenvironment. A short conditioning of transplanted CFUs by host factor(s) was sufficient to improve regeneration. The results might suggest that the speed of hemopoietic regeneration depends on the number of CFUs being induced to proliferate shordy after irradiation, rather than on the absolute numbers of CFUs available to the organism.