Early and late effects in the bone marrow of mice following 2 Gy (6 MeV) neutron irradiation

Summary Following 5 Gy gamma irradiation, residual damage in bone marrow persisted up to one year and was ascribed to genetic defects in hemopoietic stem cells (von Wangenheim et al. 1986). To see whether high LET radiation is more efficient in inducing late effects, mice were whole-body irradiated with a single dose of 2 Gy neutrons ( = 6 MeV) and femoral cellularity, CFU-S number, proliferation ability of bone marrow cells (PF) and the compartment ratio (CR), i.e. the splenic 125-iodo-deoxyuridine incorporation per transfused CFU-S were measured up to one year after the radiation insult. Within 12 weeks, femoral cellularity, PF and CR recovered to control or near-control level, whereas CFU-S numbers remained significantly below control. No further recovery was observed. On the contrary, PF and CR deteriorated again after 12 and 26 weeks, respectively. CFU-S per femur tended to decrease as well. Thus it is demonstrated that a single dose of 2 Gy 6 MeV neutrons causes significant injury in function (PF) and structure (CFU-S numbers, CR) of bone marrow which persisted up to one year. While this residual injury can be attributed to genetic defects in hemopoietic stem cells, its increasing expression is probably due to late evolving damage in microenvironmental cells. The RBE of 6 MeV neutrons for the introduction of late effects in the bone marrow is in the range of 3.     

辐射对造血微环境损伤在放疗中的意义

本文观察了500～3000rad、局部照射后一年内骨髓中CFU-S数的变化动态,同时了解造血微环境支持造血的功能之演变过程。实验发现,500rad照射后局部骨髓中CFU-S含量明显减少,恢复不稳定,同时造血微环境支持造血的功能亦有相类似的波形起伏的损伤修复过程。1000rad局部照射的骨髓中CFU-S有更显著的降低,恢复缓慢而不稳定,造血微环境支持造血的功能早期明显受损,以后虽有修复但不能恢复到正常水平。2000rad以上的X线照射可导致局部骨髓长期再生不良,造血微环境亦见剧烈而持久的功能缺陷,这一结果表明:局部照射后,屏蔽区正常造血干细胞不能在照射部位骨髓中正常种植增殖,其原因与局部造血微环境的功能障碍密切相关。     

The effect of low dose rate on recovery of hemopoietic and stromal progenitor cells in gamma-irradiated mouse bone marrow

Long-term recovery of mouse hemopoietic stem cells (CFU-S and CFU-S per colony), granulocyte-macrophage precursor cells (GM-CFC), and stromal colony-forming units (CFU-F) after doses up to 12.5 Gy was almost complete by 1 year when the dose rate was reduced to 0.0005 Gy/min compared to incomplete recovery after doses up to only 6.5 Gy given at greater than 0.7 Gy/min. This sparing effect of dose rate on long-term hemopoietic recovery is in contrast to the generally reported lack of dependence on dose rate for acute survival of hemopoietic progenitors after doses up to 5 Gy. The present results are compatible with the hypothesis that good recovery of the stroma should be reflected in the long-term recovery of hemopoiesis.     

Effect of Continuous γ-Irradiation at Low Doses on Clonogenic Hemopoietic (CFU-S) and Stromal (CFU-F) Cells of Bone Marrow

We studied the effects of low doses of continuous -irradiation (Co⁶⁰, 10 days, mean daily dose power 1.5-2.0 mGy, total dose 15 mGy) on hemopoietic and stromal progenitor cells of murine bone marrow. The content of hemopoietic clonogenic cells representing a younger (CFU-S-11) and more mature (CFU-S-7) categories in the compartment of stem cells was determined in the bone marrow. The state of bone marrow stroma was estimated by the method of in vitro cloning according to the number of progenitor cells that form colonies of fibroblasts (CFU-F) and by the method of ectopic transplantation according to the capacity of stroma of organizing and building new hemopoietic territories. Continuous -irradiation at low doses, that were by one order of magnitude lower than those inducing hermesis, exerted a stimulating effect on both hemopoietic (CFU-S) and stromal (CFU-F) progenitor cells. The number of CFU-S in the compartment of stem cells of the bone marrow markedly increased and they formed larger hemopoietic territories but these cells appeared to create a qualitatively different microenvironment, which stimulated the proliferation of CFU-S.     

Systematic analysis of the ability of stromal cell lines derived from different murine adult tissues to support maintenance of hematopoietic stem cells in vitro.

Hematopoietic stem cells interact with a complex microenvironment both in vivo and in vitro. In association with this microenvironment, murine stem cells are maintained in vitro for several months. Fibroblast-like stromal cells appear to be important components of the microenvironment, since several laboratories have demonstrated that cloned stromal cell lines support hematopoiesis in vitro. The importance of the tissue of origin of such cell lines remains unknown, since systematic generation of stromal cell lines from adult tissues has never been accomplished. In addition, the capacity of stromal cell lines to support reconstituting stem cell has not been examined. We have previously described an efficient and rapid method for the immortalization of primary bone marrow stromal cell lines (Williams et al., Mol. Cell. Biol. 8:3864-3871, 1988) which can be used to systematically derive cell lines from multiple tissues of the adult mouse. Here we report the immortalization of primary murine lung, kidney, skin, and bone marrow stromal cells using a recombinant retrovirus vector (U19-5) containing the simian virus large T antigen (SV40 LT) and the neophosphotransferase gene. The interaction of these stromal cells with factor-dependent cells Patterson-Mix (FDCP-Mix), colony forming units-spleen (CFU-S), and reconstituting hematopoietic stem cells was studied in order to analyze the ability of such lines to support multipotent stem cells in vitro. These studies revealed that stromal cell lines from these diverse tissues were morphologically and phenotypically similar and that they quantitatively bound CFU-S and FDCP-Mix cells equally well. However, only those cell lines derived from bone marrow-supported maintenance of day 12 CFU-S in vitro. One lung-derived stromal cell line, ULU-3, supported the survival of day 8 CFU-S, but not the more primitive CFU-S12. A bone marrow-derived stromal cell line, U2, supported the survival of long-term reconstituting stem cells for up to 3 weeks in vitro as assayed by reconstitution 1 year post-transplant. These studies suggest that adherence of HSC to stromal cells is necessary but not sufficient for maintenance of these stem cell populations and that bone marrow provides specific signals relating to hematopoietic stem cell survival and proliferation.     

Isolation of murine pluripotent hemopoietic stem cells in the Go phase

A method to purify pluripotent hemopoietic stem cells in the Go phase from mouse bone marrow was established. Bone marrow cells from 5-fluorouracil (5-FU)-treated mice were fractionated by Percoll density gradient. The cells with density between 1.063 and 1.075 were further separated into wheat germ agglutinin (WGA)-positive and -negative cells using fluorescent-activated cell sorter (FACS) after staining with fluorescein isothiocyanate-conjugated WGA (FITC-WGA). An assay for spleen colony-forming units (CFU-S) revealed that the WGA-positive cells (1 X 10(6)) produced 1380 CFU-S (about 150 times of the number in the original bone marrow cells) on day 12 (but no CFU-S on day 8), whereas the WGA-negative cells produced no CFU-S. Thus, the stem cells in the Go phase are found to be enriched 150 times in 5-FU-treated WGA-positive cells.     

5-Fluorouracil treatment after irradiation impairs recovery of bone marrow functions

Summary In mice, persisting radiation-induced growth retardation of hematopoietic tissue suggested that at least part of the surviving stem cells are genetically injured. Additional mitotic stress some time after the radiation insult might remove injured stem cells, thus improving the overall recovery of the irradiated bone marrow.Mice were treated with 5 Gy whole-body gamma irradiation. Two weeks later half of the animals were injected i.v. with 150 mg/kg 5-fluorouracil (5-FU), the other half remained untreated (5 Gy-controls). 2 or 10 weeks later, femoral cellularity and CFU-S content, proliferation ability of transplanted bone marrow and the compartment ratio (CR; ratio of splenic IUdR incorporation at day 3 and number of CFU-S transfused) were determined.Four weeks after 5 Gy and 2 weeks after 5-FU treatment all parameters showed significant impairment of recovery. 12 weeks after 5 Gy and 10 weeks after 5-FU CFU-S and CR were still reduced compared to the 5 Gy-controls. 5-FU treatment of unirradiated mice did not produce permanent effects on the quality of stem cells or the hematopoietic microenvironment. It is concluded, therefore, that an increased proliferation stimulus does not aid in the removal of injured CFU-S and may even impair recovery of bone marrow functions by increasing the proportion of genetically injured stem cells which continue proliferation.Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthday     

Radiosensitivity of vertebral bone marrow CFU-S surviving in mice internally contaminated with239Pu or241Am

Summary The radiosensitivity of pluripotent hemopoietic stem cells was studied in ICR Swiss mice (28 g/mouse) given i.v. 198.6 kBq²³⁹Pu/kg as citrate complex or 208.6 kBq²⁴¹Am/kg as nitrate at the age of 10 weeks. The bone marrow cells were examined at the early and late phases of radionuclide contamination. To obtain data for survival curves andD ₀ of stem cells the CFU-S assay was used and the donor vertebral marrow cells were exposed to the complementary X-irradiation either early after injection to the heavily irradiated recipients or to the in vitro irradiation given before the transplantation. To determine the iron uptake in splenic erythroid progeny the recipients given marrow cells unexposed to the X-rays received 37 kBq⁵⁹Fe 6 h before they were killed and the relative activity per colony was calculated. The radiation effect of the used actinides on the bone marrow cells resulted in decreased cellularity and seriously altered both relative and absolute CFU-S numbers. The radiosensitivity of CFU-S increased in all intervals examined (D ₀ from 0.60 to 0.86 Gy, in controls 0.97 to 1.06 Gy) and was more expressed when the CFU-S were exposed to the X-rays immediately after the bone marrow cell transplantation to the heavily irradiated hosts. The stem cell pool appeared, especially at older age, to be affected also in its ability to produce erythrocytic progeny.     

A comparative study of the kinetic changes of hemopoietic stem cells in mice infected with lethal and non-lethal malaria.

The kinetic changes of hemopoietic stem cells in bone marrow and spleen were compared between lethal Plasmodium berghei- and non-lethal P. yoelii 17x-infected mice. P. yoelii 17x-infected mice showed more severe splenomegaly than those infected with P. berghei. P. yoelii 17x-infected mice also showed a greater degree of sustained increase in number of multipotent hemopoietic stem cells (colony-forming units in spleen: CFU-S) and committed stem cells for granulocytes and macrophages (CFU-GM) and for erythrocytes (CFU-E) than P. berghei-infected mice. Such an increase was predominantly seen in the spleen of P. yoelii 17x-infected mice. In P. berghei-infected mice, the number of CFU-S, CFU-GM and also CFU-E only transiently increased and then decreased to a subnormal level at the late stage of infection. The proportion of cycling CFU-S was higher in P. berghei-infected mice than in P. yoelii 17x-infected mice. The IL-3 producing activity per spleen was much higher in P. yoelii 17x-infected than in P. berghei-infected mice at any point in time during the infection. Thus, hemopoietic changes seen after malaria infection seem to be closely related to the pathogenicity of the malaria parasite.     

Characterization of stem cells and progenitors of hemopoiesis by cell sorting.

Cell sorting has been used as a method for characterizing hemopoietic stem cells and progenitors. Fluorescent antibody-surface labels and changes in fluorescence polarization induced by in vitro stimulation with potential hemopoietic regulators were used. As detected by significant enrichment of CFU-S (pluripotent stem cells) in fluorescence-activated cell sorting, some CFU-S bear 'unique antigens' recognized by rabbit anti-human brain sera, human anti-human sperm sera, and 129 anti-F9 serum, but not A . TH anti-A . TL (Ia) ascites. Significant changes in fluorescence polarization induced by in vitro stimulation of mouse bone marrow with potential hemopoietic regulators were also observed; further, progenitors of human T-lymphocyte colonies were observed to exhibit a significantly decreased mean polarization value after short-term stimulation with PHA-LCM (phytohemagglutinin-stimulated leukocyte conditioned medium).     

Self-renewal capacity of murine hemopoietic stem cells under internal contamination with239Pu and241Am

Summary The self-renewal capacity of murine pluripotent hemopoietic stem cells (CFU-S) of vertebral bone marrow was studied under conditions of short-term and long-term internal contamination with²³⁹Pu or²⁴¹Am in female mice. Measurement of the CFU-S self-renewal capacity was carried out using double transplantation assay. To evaluate the production of differentiated progeny of stem cells average erythroblast numbers/visible spleen colony and⁵⁹Fe-uptake/colony were computed. The marrow cellularity/vertebra and the number of CFU-S/vertebra were decreased and affected more by²³⁹Pu than by²⁴¹Am. The production of erythroblasts per a single CFU-S and the⁵⁹Fe-uptake/colony were reduced, similarly the numbers of secondary spleen colonies and of secondary CFU-S in primary colonies. The above changes resulting from impaired functions of surviving CFU-S were more serious with²⁴¹Am than with²³⁹Pu. The biological effects of plutonium and americium appeared independent of the phase of contamination.     

Kinetics of reparation of sublethal radiation damage in early hematopoietic precursors

The authors studied the ability of the CFU-S, forming colonies on the 8th and 11th days after bone marrow cells transplantation, to repair the sublethal radiation damages (SRD), according to Elkind's model. Special attention was given to the kinetics fo reparation for SRD for two subpopulations of CFU (8th- and 11th-days' CFU-S). the 1-6 hour intervals between two equivalent doses of irradiation were made. The ability to repair the SRD of the 11th-days' CFU-S was lower than that of the 8th-days' CFU-S at all time intervals. The maximum reparation of the 8th-days' CFU-S was observed at 5-hour period; and that was twice as high as the maximum reparation of the 11th-days' CFU-S, which was determined at 3-hour interval between the two irradiation doses.     

Developments in modern hematology.

In the past 40 years our concepts about hemopoiesis have been changed dramatically. The results of bone marrow transplantation into lethally irradiated mice since the mid-fifties suggested the existence of a hemopoietic stem cell, which was initially identified as a spleen colony forming cell (CFU-S). Later experiments showed that the stem cell compartment is rather heterogeneous and that the most primitive stem cell, unlike the CFU-S, has the ability for long-term engraftment of an irradiated recipient. Daughter cells of such primitive quiescent stem cells lose their capacity for self-generation gradually with each mitosis and become more and more committed to a specific differentiation lineage. In vitro culture techniques in a serum-free semi-solid medium enabled the establishment and analysis of specific hemopoietic growth factors. Such factors, which are essential for the maintenance, proliferation and differentiation of progenitor cells and the functional activity of mature cells can now be produced with recombinant DNA techniques in pure form and large quantities. Hemopoiesis requires an appropriate microenvironment, consisting of various stromal cell types and an extracellular matrix. Intercellular contacts, adhesion of cells and growth factors to the matrix molecules seem essential in the regulating action of this hemopoietic microenvironment. In long-term bone marrow cultures the development of a stromal hemopoietic microenvironment can facilitate long-term maintenance of stem cells and hemopoietic differentiation. For bone marrow transplantation and infusion of hemopoietic growth factors many clinical indications are well established and our possibilities to interfere in the regulation of hemopoiesis are still growing.     

小鼠造血干细胞增殖与分化性能的研究

本文选择Ｙ染色体特异的性别决定基因作为新的细胞遗传标志,采用ＰＣＲ技术研究了小鼠造血干细胞的增殖与分化性能。将雄鼠骨髓细胞输注给经致死剂量射线照射的雌性受体小鼠、ＰＣＲ测试结果表明,所有ＣＦＵ－Ｓ均为供体起源。供体来源的ＣＦＵ－Ｓ在其输入体内后,可通过增殖,分化形成各系造血细胞,但ＣＦＵ－Ｓ中的纤维母细胞和ＣＦＵ－Ｓ重建造血后受体小鼠骨髓中的纤维母细胞均为受体起源。由此可见,小鼠骨髓中的ＣＦＣ－Ｓ     

Transplantation of murine bone marrow without prior host irradiation.

The experiments presented test the hypothesis that pluripotential stem cells (assayed in the mouse as CFU-S) are normally not in cycle and that the failure of normal marrow transfusions to take in normal recipients is due to the absence of a stimulus to turn CFU-S into cycle. Following marrow transfusion from male donors into female isogeneic recipients, spleen, liver, and various parts of the skeleton were shielded to protect transfused donor cells from lethal doses of radiation gives to the rest of the body. Percentages of hemopoietic donor and host cells were subsequently determined by karyotyping C banded marrow and spleen metaphases and identifying of Y chromosome. The results support the notion that the failure of normal marrow to take in normal recipients is not due to inadequate numbers of transfused cells. Permanent colonization by donor cells, however, requires not only triggering CFU-S into cycle, but also emptying of 'niches' normally occupied by endogenous CFU-S. Partial body radiation meets both requirements. In addition, the results indicate that recently arrived donor cells, protected in the shielded portion of the body, seed more readily into the irradiated areas of the skeleton than do similarly protected host cells.     

Residual radiation effect in the murine hematopoietic stem cell compartment

Stem cells surviving radiation injury may carry defects which contribute to long-term effects. The ratio of 125-iododeoxyuridine (IUdR) uptake into spleens of lethally irradiated recipient mice between day 3 and day 5 after cell transfusion revealed reduced proliferative ability (PF) of spleen seeding cells in parallel with reduced CFU-S content of donors throughout the study period of one year after 5 Gy gamma irradiation. Additional data aided in evaluating possible mechanisms of PF reduction. Within the range of the graft sizes used, PF was independent of the numbers of cells or CFU-S transfused. Radiation-induced increase in loss of label between days 3 and 5 and prolonged doubling time of proliferating cells indicated enhancement of cell maturation and increase in mitotic cycle time. Increased IUdR uptake per transfused CFU-S suggested extra divisions of transit cells due to insufficiency in the stem cell compartment. It is concluded that persisting defects in surviving stem cells interfere in a complex way with cell proliferation in the hemopoietic system.     

Radiosensitivity and proliferative activity of vertebral CFU-S surviving in mice injected with oncogenic activities of 239Pu or 241Am

Survival, radiosensitivity and capability to produce differentiated progeny were followed in CFU-S from lumbar vertebrae of mice injected with 198.6 kBq 239Pu/kg or 208.6 kBq 241Am/kg. The CFU-S assay and 59Fe uptake into spleen colonies were used. The number of CFU-S from treated mice was significantly lower than in controls. Higher radiosensitivity of CFU-S from 239Pu- or 241Am-treated mice was demonstrated using additional exposure to 0.5 Gy X-rays 1, 24, 48, 72 hrs after cell transplantation and expressed more precisely by survival curves obtained 1 hr after the marrow cell injection. The effect of 239Pu on CFU-S was characterized by Do 0.58 Gy (n = 0.91) and that of 241Am by Do 0.64 Gy (n = 0.91); corresponding control values were Do 0.89 Gy, n = 1.11. Lower iron utilization due not only to the decreased CFU-S numbers, but also to the defective production of erythroid cells per one CFU-S was found. Complexity of radiation effect on hemopoietic stem cells was demonstrated by the present study.     

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.     

Production of humoral factors that stimulate spleen colony-forming units in mice irradiated with moderate doses of X rays

The production of humoral factors that stimulate spleen colony-forming units (CFU-S) has been studied in irradiated mice using an in vivo diffusion chamber assay. The experiments show that a significant release of factors that stimulate CFU-S takes place in the first few days after irradiation with moderate doses of 1.5 or 5 Gy. In contrast, the release of significant amounts of these humoral factors was not seen in animals irradiated with either low (0.75 Gy) or high (10 Gy) doses of X rays. The correlation observed between the production of factors that stimulate the CFU-S and the hemopoietic regeneration kinetics of the irradiated mice suggests that these factors represent part of the physiological regulators controlling the proliferation of CFU-S.     

Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10⁵ bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.