CFUs of the normal and regenerating liver in the sexually mature mouse

Functional properties of CFUs have been studied in intact and regenerating liver of mice. According to a number of properties (proliferative activity, character of colony growth) CFUs in the liver are similar to CFUs in the peripheral blood and, probably, make the same population. In the regenerating liver relative contents of CFUs 3-5 days after a partial resection is substantially increasing. Concentration of CFUs (endogenic) increases significantly also in a locally injured and regenerating lobe, comparing to the intact lobe that is in the same organ. The transplanted bone marrow CFUs prevail in number in the regenerating liver over the intact liver. It is concluded that increasing contents of CFUs in the regenerating liver depend mainly on its creased property to invade and/or to hold the extrahepatic CFUs.     

Colony-forming units of the bone marrow and spleen of immunodeficient mice after stimulation of the cells with thymalin

It has been previously demonstrated by the authors that histological characteristics of colony-forming units (CFUs) in normal mice prove a certain shift in their differentiation in erythroid direction comparing to the bone marrow CFUs. Thymectomy of mature animals is accompanied with weakening growth of granular colonies at cloning of the bone marrow CFUs and with loss of stability in direction of splenic CFUs differentiation. Polypeptide preparation of the thymus--thymalin stimulates growth of the granulocytic colonies from the splenic CFUs in thymectomized mice both in in vivo and in vitro experiments. Differentiation of the bone marrow CFUs is normalized under the effect of thymalin in in vivo experiment only. The data obtained confirm the suggestion made by R. V. Petrov on existence of T-cell clone, enhancing CFUs differentiation in granulocytic direction. Activation of this clone in the spleen is revealed at thymectomy and stimulation of the cells with thymalin both in in vivo and in vitro experiments. Thus, affirmations are obtained on differences of clonic T-cell regulation of the CFUs differentiation in the bone marrow and in the spleen.     

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.     

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.     

Differentiation of stem cells from different parts of the hematopoietic system of adult thymectomized CBA mice stimulated with thymarin or cortexine

Essential differences were detected in differentiation of GFUs from bone marrow and peripheral blood. It was shown that as a result of thymectomy of adult animals the ability of bone marrow CFUs to form granulocytic colonies decreased and that of splenic CFUs to form erythroid colonies increased. The immunostimulating low-molecular-weight polypeptides, thymarin and cortexine , normalized the differentiation of CFUs from bone marrow and spleen but interfered with the formation of erythroid colonies from CFUs of peripheral blood of thymectomized mice.     

Comparison of the reparative property of early and late CFUs after sublethal radiation injury

Restoration ability of early and late CFUs has been studied using bone marrow sublethal fractional irradiation. It has been shown that CFUs capacity for early postirradiation repair on the 11th day is sharply reduced, as compared to that of CFUs on the 8th day.     

Regeneration of CFUs in the marrow of mice exposed to 300 rads after having recovered from 950 rads.

Exposure to 950 rads 60Co radiation has been reported to cause long-lasting damage to the hematopoietic stroma (HS), although the size of the CFUs population recovers to pre-irradiation levels. In these studies HS damage was detected only after subcutaneously implanting the femurs of the irradiated mice into syngeneic hosts. To exclude the possibility that what was considered to be HS damage was merely caused by artifacts due to the process of implantation in a new host, we compared the rate of regeneration of CFUs in mice which had recovered from 950 rads prior to receiving 300 rads 60Co radiation (950 + 300 rads group) with that of mice which received only 300 rads (0 + 300 rads group). The CFUs population in the 950 + 300 rads group grew exponentially for 2 weeks at a rate which did not differ significantly from that of CFUs in the 0 + 300 rads group. However, the rate of CFUs growth reached a plateau before full recovery was achieved in contrast to that in the 0 + 300 rads mice. We therefore conclude that the incomplete regeneration of CFUs in the marrows of 950 + 300 rads mice was most likely caused by X-irradiation-induced damage to the HS rather than damage to the inherent repopulating potential of the CFUs per se.     

Studies on the mechanism of haemopoietic stem cell (CFUs) mobilization. A role of the complement system.

A variety of substances can mobilize haemopoietic stem cells (CFUs) into the peripheral blood. In this study the involvement of the complement system in the mobilization process was investigated. Pretreatment of mice with the complement-activating factor of cobra venom (CoF), which lowered the serum C3 levels to 10-25% of the normal value, could completely prevent CFUs mobilization induced by high doses of CoF, endotoxin (ET) from Salmonella typhosa, inulin, zymosan and the proteolytic enzymes proteinase and trypsin. On the other hand, mobilization induced by the polyanions dextran sulphate and the copolymer of polymethacrylic acid and styrene could not be prevented, or at least affected only slightly. There appears to be a relationship between the extent of decomplementation by CoF and the extent of CFUs mobilization induced by ET. The results indicate that certain agents mobilize CFUs via the complement system, whereas other agents induce CFUs mobilization independent of the availability of complement components.     

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.     

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.     

Hematopoietic stem cells in mice during embryonic development preceding the establishment of liver hematopoiesis]

We studied the time course of appearance of CFUs (7-8 days old) in embryos of (C57B1/6 x CBA)F1 mice from the 8th day of embryonic development. Significant amounts of CFUs could be detected from the 10th day of development, initially in the body of the embryo from the stage of 30-33 pairs of somites, then in the yolk sac and still later, from the stage of about 40 pairs of somites, in liver anlage. CFUs could not be reliably detected until the 9th day of development either in the embryo itself or in the yolk sac. However, after incubation of nine day old embryos for four days in organ culture, such cultures contained CFUs. CFUs could be found in significant levels in embryos explanted from the embryos at the stage no earlier than 24 pairs of somites. When the yolk sac and the embryo were cultivated separately, CFUs could also be detected, however, the removal of liver primordium from the embryo did not influence the amount of CFUs in its body. CFUs were not found in cultures of liver primordium from nine day old embryos. Thus, we can detect pre-CFUs in 9 day old embryos at the stage 25-28 pairs of somites using the system of organ culture; at the same time CFUs cannot be found in intact embryos of the same age. These data provide evidence that before the establishment of liver hemopoiesis precursors of CFUs are located both in the yolk sac and in the embryo outside rudimentary liver. However, our results do not provide any data for the conclusion about the primary source of pre-CFUs in the mouse embryo.     

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.     

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.     

The stimulating effect of preliminary irradiation of the hematopoietic stroma with small doses on the content of hematopoietic stem cells in the bone marrow in vivo and in a long-term culture system

Preirradiation of mouse recipients with a dose of 1-2 Gy 24 and 48 h before lethal irradiation (8 Gy) made CFUs content of femur increase upon transplantation of bone marrow from exposed and intact donors. The same was with the long-term bone marrow culture: preirradiation of a stromal sublayer increased the number of CFUs in the transplanted bone marrow preirradiated with 6 Gy radiation. Retransplantation of bone marrow to irradiated donors after 5 day cultivation, a sublayer being activated, increased the number of CFUs in the femur in comparison with donors which were injected with the bone marrow from the culture without activation of the sublayer by low-level radiation.     

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.     

The role of the hemopoietic microenvironment on the hemopoiesis-stimulating effect of cystamine

The influence of cystamine delivered in a radioprotective dose before and after irradiation of mouse-recipients (8 Gy) on the effectiveness of exogenous bone marrow cloning has been investigated. Cystamine administered prior to irradiation exerts a protective effect on CFUs and also causes an increase in the number of splenic colonies grown from CFUs of the transplanted bone marrow. With cystamine administered after irradiation the protective effect is absent, but the CFUs number in the femur increases in recipients transplanted with intact bone marrow in comparison with those transplanted without cystamine. It is believed, that in addition to the specific protective mechanism of action of radioprotectors, there is a nonspecific mechanism of increasing the proliferation of protected stem cells that is connected with the stimulatory effect of radioprotective agents on the haemopoietic stroma elements.     

Protective effect of bone marrow and spleen suspensions on radiation-induced leukemogenesis in C57BL/6 mice

Summary The present experiments are an attempt to precise the type and localization of the cells involved in the protective effect of hemopoietic suspensions against the radiation-induced thymic lymphosarcoma (TLS) of C57BL/6 mice. Inocula containing variable numbers of BM or spleen CFUs from 60-day-old and 360-day-old donors were tested. According to their origin, the suspensions differed with respect to the CFU replication rate, the CFU ability to differentiate towards the T lineage and the content of the suspensions in thymic precursors. Two levels of inhibition were observed: BM suspensions from 60-day-old donors containing 1,500 CFUs had the best protective effect: 14.5% of TLS; 1,500 CFUs from 360-day-old donors were slightly but not significantly less efficient (28.5%). The second level of inhibition (36–46% of TLS) was obtained with all the following inocula:a) 1,200 and 300 spleen CFUs or 300 and 95 BM CFUs from 60-day-old donors,b) 1,500 spleen CFUs from aged donors. Seventy-six spleen CFUs from 60-day-old donors, 120 BM or 175 spleen CFUs from aged donors had no effect. These results suggest that in addition to the high replication rate of the BM CFUs as compared with spleen CFUs, cells endowed with an optimal protective effect are present in BM suspensions and are either absent or present in very small amount in spleen suspensions. These cells which induce an early repopulation of the thymus might correspond to thymic precursors.     

Stimulation of the suppressor activity in relation to hematopoietic cells in the mouse thymus as affected by pertussis vaccine

After a single intraperitoneal injection of formalinized whole-cell pertussis vaccine 5374 into CBA mice a drastic depletion of cells in the thymus accompanied by the enhancement of suppressor T-cells production is observed. T-cells inhibit the endogenous and exogenous colony-formation in the spleen by hematopoietic stem cells (CFUs). Treatment of thymus cells with the anti-I-Jk alloantiserum against a specific marker of suppressor T-cells in the presence of complement removes the inhibitory effect on CFUs proliferation and has a stimulatory action on the capacity of the marrow CFUs to form macroscopic hematopoietic colonies in the spleen compared with control ones. These results suggest probable involvement of I-J-bearing suppressor T-cells in the hematopoietic disorders induced by pertussis vaccine.     

The influence of dietary protein deficiency on haemopoietic cells in the mouse.

These experiments examined the effect of a diet limited only in protein (4% by weight) on haemopoietic stem cells in mice. This diet places severe restrictions on growth and cell proliferation and this was reflected in lower numbers of colony forming units (CFUs) and in vitro colony forming cells (CFCs). Differences were apparent in the response of different organs to this stress; for instance, the incidence of spleen CFUs fell sharply from around 40/mg spleen tissue to 1-4/mg spleen tissue after 3 weeks on a low protein diet. This selective loss did not occur in bone marrow where total CFUs remained proportional to cellular content. Yet a third pattern was shown by thymus CFUs--although the numbers were low these increased from 16/thymus in normal mice to 132/thymus in deprived mice. This was the only organ examined which showed an increase. The effects of a return to a high protein (18%) diet showed that the spleen was the most responsive organ. By day 5 after the return to 18% protein the spleen contained as many CFUs per million cells as the bone marrow. During this time the content of CFU in the spleen had increased some 50-fold whereas bone marrow CFUs only doubled. The spleen assumes the major reconstructive role during the refeeding process.     

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.