Stem cell kinetics in spleen and bone marrow after single and fractionated irradiation of infant mice

Summary The number and type of stem cells in spleen and bone marrow of mice were determined after exposure to a single dose of 150 R on day 6, to a single dose of 500 R on day 6 or day 9 or to a fractionated dose of 150 R + 350 R on day 6 and 9. The stem cells were assayed on the basis of colony forming units (CFU) in spleen and of incorporation of iododeoxyuridine in spleen and bone marrow of lethally irradiated host mice. During the first month of life, the number of stem cells in non-irradiated mice increases markedly in bone marrow and slightly in spleen. Irradiation causes a long-lasting depression in stem cells, particularly in bone marrow and affecting preferentially erythropoietic precursor cells. Following a dose of only 150 R, the number of CFU in bone marrow is still below control levels 24 days later. An exposure to 500 R fractionated between day 6 and 9 has a markedly greater effect on stem cells in the spleen than the same dose given in a single application either at day 6 or 9.Supported by the Schutzkommission am Ministerium des Innern der BRD and contract B232-76-1BIOB of the Biology Division of the Commission of the European Community (Publikation No. 1727)     

Effects of chorionic gonadotropin on the hematopoietic stem cells

It was shown on the exogenic colony-forming unit (CFU) assay that the chorionic gonadotropin (CG) administration to female mice CBA in doses correlating with its concentration in different stages of woman pregnancy stimulated (depending upon the doses) the CFU formation of bone marrow, but not spleen origin. Injections of CG to the ovariectomized mice has the opposite (inhibited) effect on the CFU contents in bone marrow and spleen. CG-administration in the dose of 40 U1 to the ovariectomized and non-castrated irradiated recipients bone marrow cells stimulates (statistically significant) colonies formation. As for 200 U1 dose hormone has the similar effect only on the non-castrated animals.     

Effect of erythrocyte breakdown products on mast cells and erythropoietin formation]

Experiments were conducted on CBA mice and albino rats. A study was made of the effect of erythrocyte destruction products (EDP) on the content of hemopoietic colony-forming units (CFU), differentiation of stem cells and the erythropoietin production. It was shown that 3 or 4 EDP injections to normal mice or to lethally irradiated (1000 rad) mice after the transplantation of bone marrow cells caused no changes in the CFU level of stem cells differentiation. In case of a daily (for 3 days) administration of EDP to mice before the irradiation (1000 rad) and bone marrow transplantation there was observed an increase of the colonies count in the recipients' spleen on account of the erythroid colonies. EDP injection caused no changes in the erythropoietic activity of the blood serum. A possible role of erythrocyte destruction products in the mechanism of erythropoiesis autoregulation is discussed.     

Diffusion chamber and spleen colony assay of murine haematopoietic stem cells

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

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

SELF-RENEWAL OF COLONY FORMING UNITS (CFU) IN SERIAL BONE MARROW TRANSPLANTATION EXPERIMENTS

The capacity of stem cells (CFU) for self-renewal was tested by transplanting normal bone marrow (primary transplantation) and bone marrow which had been subjected to one or two earlier transplantations (secondary and tertiary transplantation) into lethally irradiated syngeneic recipients. It was found that the capacity for self-renewal is diminished within the first weeks after one or more previous transplantations. This ability of stem cells recovered after a longer interval after the previous transplantation. The time required for this recovery depended upon the number of previous transplantations and amounted to more than 1 or 2 months after one or two transplantations respectively. Shortly after transplantation the CFU/nucleated cell ratio in bone marrow was below normal and its decrease was more pronounced when the bone marrow had been transplanted more often. An increase of the ratio towards normal values was observed in the course of one month after the last transplantation. Measurements of the spleen colony size after transplantation of normal and re-transplanted bone marrow indicated that CFUs from re-transplanted marrow gave slightly smaller spleen colonies than those of normal marrow.
It is concluded that the decreased self-renewal of stem cells shortly after previous transplantations is probably not due to a limitation in the number of normal mitoses they can perform, but to a loss of stem cells by transfer to the compartment of differentiating cells.     

CFU-S and haematopoietic microenvironment in mice after fractionated irradiation. A study on spleen colonies.

The paper is aimed at evaluating the quantity and quality of the haematopoietic stem cells, CFU-S, in the bone marrow and the functional effectiveness of the haematopoietic microenvironment of the spleen in two time intervals after repeated exposure of mice to doses of 0.5 Gy gamma-rays once a week (total doses of 12 and 24 Gy). After irradiation, bone marrow was cross-transplanted between fractionatedly irradiated and control mice. The parameter evaluated were numbers of spleen colonies classified into size categories. The data obtained provide evidence for a significant damage to the CFU-S, concerning both their number and proliferation ability, after both total doses used. The functional effectiveness of the haematopoietic microenvironment of the spleen was impaired only in bone marrow recipients receiving a transplant after having been exposed to a total dose of 24 Gy; this dose combined with subsequent pre-transplantation irradiation resulted in a marked suppression of cell production within the spleen colonies formed from a normal bone marrow on the spleens of fractionatedly irradiated mice.     

Radiosensitivity of human bone marrow granulocyte-macrophage progenitor cells and stromal colony-forming cells: effect of dose rate

Study of the radiation biology of human bone marrow hematopoietic cells has been difficult since unseparated bone marrow cell preparations also contain other nonhematopoietic stromal cells. We tested the clonogenic survival after 0.05 or 2 Gy/min X irradiation using as target cells either fresh human bone marrow or nonadherent hematopoietic cells separated from stromal cells by the method of long-term bone marrow culture (LTBMC). Sequential nonadherent cell populations removed from LTBMC were enriched for hematopoietic progenitors forming granulocyte-macrophage colony-forming unit culture (GM-CFUc) that form colonies at Day 7, termed GM-CFUc7, or Day 14 termed GM-CFUc14. The results demonstrated no effect of dose rate on the D0 or n of fresh marrow GM-CFUc (colonies greater than or equal to 50 cells) after plating in a source of their obligatory growth factor, colony-stimulating factor (CSF) (GM-CFUc7 irradiated at 2 Gy/min, D0 = 1.02 +/- 0.05, n = 1.59 +/- 0.21; at 0.05 Gy/min, D0 = 1.07 +/- 0.03, n = 1.50 +/- 0.04; GM-CFUc14 at 2 Gy/min, D0 = 1.13 +/- 0.03, n = 1.43 +/- 0.03; at 0.05 Gy/min, D0 = 1.16 +/- 0.04, n = 1.34 +/- 0.05). There was a decrease in the radiosensitivity of GM-CFUc7 and GM-CFUc14 derived from nonadherent cells of long-term bone marrow cultures compared to fresh marrow that was observed at both dose rates. In contrast, adherent stromal cells irradiated at low compared to high dose rate showed a significantly greater radioresistance (Day 19 colonies of greater than or equal to 50 cells; at 2 Gy/min, D0 = 0.99 Gy, n = 1.03; at 0.05 Gy/min D0 = 1.46 Gy, n = 2.00). These data provide strong evidence for a difference in the radiosensitivity of human marrow hematopoietic progenitor compared to adherent stromal cells.     

FETAL HEMOPOIESIS IN DIFFUSION CHAMBER CULTURES

The growth pattern of fetal liver (FL), normal adult bone marrow (NABM) and regenerating (post Velban treatment) adult bone marrow (RABM) colony forming units (CFU) cultured in diffusion chambers (DC) was studied. When twenty CFU were implanted into DC the recovery of CFU after 4 days with FL, NABM or RABM was 133 ± 7, 19 + 2 and 34 ± 2 CFU, respectively. The transplantation fraction of CFU from NABM decreased from 10-4% on day 0 to 6–9 % on day 4; that of FL did not change from the initial 6-2%. The growth rate of CFU derived from FL was substantially greater than that from NABM. The relative growth of FL and RABM CFU was clearly inhibited when the concentration of cells cultured was increased. Spleen colonies from FL cells before culture were larger (P < 0–005) than colonies from NABM but after 7 days of culture there was no difference between the two groups. Histological examination of spleen colonies showed that after DC culture FL and NABM CFU were differentiating along the three normal pathways. These data suggest that intrinsic differences exist between fetal and adult stem cells in the in vivo diffusion chamber culture system.     

Effect of syngeneic lymphocytes, T immunodeficiency and the genotype of bone marrow donors on the differentiation of early and late splenic colonies

The formation of "early" (5-8 days) and "late" (12-14 days) colonies in spleen of lethally irradiated syngeneic or hybrid recipients after transplantation of bone marrow cells has been studied. The differentiation pattern did not depend on bone marrow cell donor's genotype and the donor-recipient combination. Erythroid to granulocyte colonies ratio (E/G) equals 2. Change of direction of bone marrow colony-forming units (CFU) differentiation has the same pattern at different stages of colony-formation. Under the influence of antigen-stimulated lymphocytes the granulopoiesis (E/G 0.3-0.5) dominanted. The thymectomy of adult animals leads to a predominant formation of erythroid colonies (E/G 3.5-5.1). When T-immunodeficiency is reversed with syngeneic lymphocytes, the differentiation of CFU is normalized at all stages of colony-formation. The process of differentiation of haemopoietic precursors, that form "early" and "late" colonies, is under T-lymphocyte control.     

THE ROLE OF BONE MARROW OF X-IRRADIATED MICE IN THYMIC RECOVERY

The influence of the bone marrow on the repopulation of the thymus in X-irradiated mice has been investigated.
It was observed that the thymus and a certain population of bone marrow lymphocytic cells were repopulated in parallel in a cyclic fashion. This occurred either after a single exposure of mice to 400 R or after serial weekly X-ray treatments with 170 R. Lethally irradiated recipients which were grafted with bone marrow cells obtained 12-24 days after four weekly irradiations of donor mice with 170 R also exhibited a cyclic repopulation of both the thymus and the bone marrow lymphocytic population. In contrast, mice which were transplanted with bone marrow cells from unirradiated donors, containing an equal number of stem cells (CFU), exhibited a continuous rather than a cyclic recovery of both cell populations. the bone marrow stem cells of mice recovering from X-irradiation were found to have a decreased proliferative activity, since they produced significantly smaller spleen colonies in lethally irradiated recipients than marrow cells from unirradiated mice.
The results were interpreted as indicating that the bone marrow lymphocytic cells may act as thymic precursor cells and that thymic lymphopoiesis is dependent on the presence of such cells. Evidently, the production of lymphocytic cells will decrease when the stimulus for granulocyte production increases due to the limited proliferative activity of the surviving bone marrow stem cells after irradiation. This may result in a cyclic variation of the production of bone marrow lymphocytic cells and it follows that thymic lymphopoiesis will run parallel.     

Genetic control of allogenic inhibition of hematopoietic stem cells]

Adult mice of C57BL/6, CBA (CBA X C57BL/6) F1, (CBA X C57BL/6) F2, F1 X CBA and F1 X C57BL/6 strains were lethally irradiated and reconstituted with a constant dose of 3-10(5) C57BL/6 bone marrow cells. At the 9th day after the bone marrow transplantation the colony count was performed in spleen of irradiated recipients. In the spleen of F1, CBA and C57BL/6 mice were registered low (0--8, intermediate (6--18) and high (22-40) numbers of colonies respectively. The segregation ratios in F2 progeny were close to 2 (low): 1(intermediate): 1(high). The segregation ratios in backcross (F1 X CBA) were close to 1(low): 1(intermediate)numbers of colonies. Backcrosses (F1 X C57BL/6) were distributed to low and high numbers of colonies with the ratio 1:1. The number of spleen colonies of males and females was the same in all segregating progeny. The results of hybrid analysis suggest that a single pair of allelic genes is involved in genetic control of allogenic inhibition, and that the resistance (manifestation of inhibition) to C57BL/6 stem cells is conferred by the dominant allele.     

Influence of poly-A:U, dextran sulfate and yeast RNA on the colony forming ability of bone marrow in irradiated mice]

Poly-A:U, dextran sulfate and yeast RNA were shown to increase the number of endogenous (CFU) in sublethally (525 r.) irradiated mouse spleens seemingly as a result of their mutagenic effect on proliferating CFU. The preparations had no effect on the number of exogenous colonies when injected together with bone marrow syngeneic cells transfer from intact donors. Dextran sulfate led to a 2.7 time increase in the number of endogenous colonies in unevenly irradiated mouse spleens mostly due to the CFU migration from the protected sites of the bone marrow. Poly-A:U and yeast RNA complex was ineffective in such an experiment. It is quite possible that the ability of dextran sulfate to increase the migrational potencies of the stem hematogenic cells served as one of the essential factors in the mechanism of its adjuvant activity.     

Bone marrow colony-forming capacity in mice subjected to burn trauma]

A method of exogenous splenic colonies was applied to the study of the dynamics of the content of the colony-forming units (CFU) in the bone marrow of CBA mice to which thermal burn of the III degree of 15% of the body surface was inflicted. On the 4th and 16th days after the burn the CFU content in the bone marrow of mice decreased 1.7-2.1 times. The thymus cells of the intact mice administered simultaneously with the bone marrow of the burned mice increased, the amount of the splenic exogenous colonies formed in the recipients. The data obtained permitted to make a suggestion that not only the CFU count diminished in the bone marrow in the burned animals, but also the thymus-dependent cells necessary for normal colony formation.     

Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.     

Hemopoietic colony studies. VI. Increased eosinophil-containing colonies obtained by antigen pre-treatment of irradiated mice reconstituted with bone marrow cells

The cellular response to an intraperitoneal injection of antigen (tetanus toxoid) was studied in reconstituted animals in order to determine the mechanism of control of eosinophil granulocytopoiesis. Antigen treatment of the marrow cell donors did not consistently increase the number of spleen and bone marrow colonies in recipient animals or change the percentage of eosinophil or other hemopoietic colony types. Antigen pre-treatment of the irradiated recipients increased the percentage of eosinophil-containing colonies in the spleen and femoral bone marrow without significantly changing the total number of either spleen or marrow colonies. Antigen treatment of both the bone marrow cell donor and recipient produced a further increase in the percentage of eosinophil-containing colonies in the marrow cavity, but not in the spleen. Antigen treatment of the irradiated recipient increased the number of eosinophilic cells (but not the total number of cells) in both the peritoneal cavity and the bone marrow. Antigen treatment of both the marrow donor and recipient produced a further increase in the number of eosinophilic cells in the peritoneal cavity, but not in a single femur. Since antigen treatment of the marrow recipient, or recipient and donor, but not of the marrow donor alone, results in increased eosinophilic cell and colony numbers, the effect of antigen appears to be mediated through some host factor(s), perhaps the eosinophilic hemopoietic inducing microenvironment (HIM), rather than directly on the hemopoietic stem cells.     

Genome mutation frequency in the cells of spleen colonies of different origins in NIH mice

Genome mutation frequencies (GMF) were determined in cells of endogenous (from bone marrow) and exogenous (from bone marrow, spleen and embryonic liver) spleen colonies on the basis of variations in DNA contents of interphase nuclei. In cells of the former GMF varied from 1.1 X 10(-2) to 10.8 X 10(-2), and in the latter these were equal to 8.9 X 10(-2). In the cells of exogenous colonies derived from X-irradiated precursors (1.8 and 5.9 Gy) GMF were 10.1 X 10(-2) and 11.9 X 10(-2), resp. The mode of transplantation influenced greatly on the GMF: after an additional short transplantation (4-6 days) the number of GMF increased by 1.5-2 times. It is concluded that the increased number of GMF may be responsible for the limited life-span of bone marrow stem cells in the course of their serial transplantations in the irradiated syngenic mice.     

Effect of prolonged exposure to a magnetic field on the haematopoietic stem cell.

The authors studied the effect of prolonged exposure (3, 4 and 5 months) to the action of a magnetic field of 180-200 gauss formed by the poles of a rotating permanent magnet on the haematopoietic stem cells of mouse bone marrow donors. The effect of the field was evaluated from the ability of the donors' bone marrow cells to form haematopoietic colonies in the spleen of lethally irradiated mice. It was found that the number of stem cells was not reduced by the action of the above magnetic field and that proliferative capacity was likewide unimpaired.     

Chemical radioprotection of hemopoietic colony-forming cells: comparative effect of AET, anoxia, and urethan

The bone marrow colony-forming unit (CFU) technique of Till and McCulloch was employed to test the radioprotective effect of AET, anoxia, urethan on marrow cells irradiated in vivo. For AET and anoxia, a dose-reduction factor of 1.9 to 2.1 was found. Since the marrow cells were assayed for CFU content immediately after irradiation of the donor, the observed effect can be interpreted as a "true" radiation dose reduction. By contrast, urethan injection did not increase the survival of marrow CFU assayed immediately after whole-body x-irradiation. However, both urethan and AET afforded radioprotection of endogenous CFU content of spleen and bone marrow, but not of endogenous spleen colony count. It is concluded that the mechanism of radioprotection by urethan is fundamentally different from that of AET or anoxia.     

Effect of herbal preparation, brahma rasayana, in amelioration of radiation induced damage

Oral administration of brahma rasayana (BR; 10 and 50 mg/dose/animal) for 15 days increased significantly total leukocyte count and percentage of polymorphonuclear cells in irradiated mice. Bone marrow cellularity and alpha-esterase positive cells also increased significantly in radiation-treated animals after BR administration. Number of nodular colonies on the surface of spleen on day seven increased significantly in lethally irradiated recipients receiving bone marrow cells from animals treated with BR. Oral administration of BR also enhanced in serum level of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and granulocyte macrophage-colony stimulating factor(GM-CSF) in normal and irradiated mice. These results indicated that proliferation of stem cells induced by BR in irradiated mice may be related to its stimulation of cytokine production.