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.     

The radiosensitivity of hematopoietic stem cells from mice forming splenic colonies after 8 and 12 days following bone marrow cell transplantation (CFU-S-8 and CFU-S-12)]

The method of "macro-" and "microcolonies" was used to study the radiosensitivity of CFU-S that form "early" (8 days) and "late" (12 days) splenic colonies after transplantation of syngeneic bone marrow to fatally exposed mice: no significant differences were found. Median lethal doses (D0) for CFU-S-8 and CFU-S-12 were 1.03 and 1.13 Gy for "microtest" and 0.99 and 1.16 Gy for "microtest" respectively.     

Comparative radiosensitivity of CFUs of the mouse bone marrow and embryonal liver forming 7- and 11-day colonies

The authors studied the radiosensitivity of CFU-s, forming 7- and 11-day colonies from fetal liver (FL) of 14 and 17 day gestation and bone marrow (BM) of adult mice. The index of D0 for 7-day colonies, formed by CFU-s from 14-day, 17-day FL and BM was 2.02; 1.57 and 0.78 Gy, accordingly. 11-day CFU-s both from FL, and from BM did not distinguish statistically at their radiosensitivity (their D0 was 1.25 Gy).     

Antibody-mediated suppression of bone marrow colony formation in vitro.

Antisera to mouse brain reacts with hematopoietic stem cells in the mouse bone marrow. We have examined the effect of anti-mouse brain serum (AMBS) on the development of in vitro colonies from mouse bone marrow cells. The addition of 5% AMBS to the cultures markedly decreased the numbers of colonies formed to an average of 10% of the number obtained with normal rabbit serum. AMBS suppressed formation induced by colony stimulating factors (CSF) derived from three different sources; serum from endotoxin treated mice, mouse L-cell conditioned media, and human peripheral mononuclear cell conditioned media. The suppressive activity was quantitatively recovered in the IgG fraction of AMBS. Divalent F(ab')2 fragments were as effective as the intact IgG in decreasing colony formation. Fab fragments were not suppressive. These results suggest that colony formation is induced via a dynamic interaction between CSF and the progenitor cell membrane, and that antibody directed at cell membrane antigen(s) interferes with the generation of the induction signal.     

Effect of anti-mouse brain serum on the CFUc in actively proliferating bone marrow

With hydroxyurea injected to donor mice a greater inhibition of splenic colony growth occurred after incubation of a bone marrow suspension with the rabbit antimouse brain serum (RAMBS), and restoration of the colony-formation by thymocytes was less pronounced than in normal bone marrow treated with the antibrain serum. The incubation of the bone marrow cells containing CFUc, which actively proliferate after irradiation or stimulation by vinblastine, with the antibrain serum sharply suppressed the splenic colony growth. In this case however, in contrast to normal bone marrow, the administration of thymocytes failed to exert a favourable action on the colony formation. It is suggested that functioning of accessory cells is not associated with the defined cell cycle stage of CFUc and that, in addition to the previously discovered accessory cell population, some other factors, inactivated by the RAMBS serum, are present in the bone marrow the analogue of which is absent in the thymus.     

Radiosensitivity of mouse bone marrow and spleen CFUdc during gamma-irradiation in different oxygenation conditions

After exposure in vitro and in situ CFUdc of CBA mouse bone marrow and spleen were characterized by the comparable parameters of radiosensitivity and oxygen-dependent modification: the values of D0 for bone marrow and spleen cells were 1.31 and 1.35 Gy (in vitro) and 1.36 and 1.37 Gy (in situ), and the values of the oxygen effect were 2.3 and 2.5, respectively.     

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

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

Radiosensitivity increases with differentiation status of murine hemopoietic progenitor cells selected using enriched marrow subpopulations and recombinant growth factors

The radiosensitivity of populations of colony-forming cells (CFC) in murine bone marrow was investigated using different recombinant colony-stimulating factors (CSFs; murine IL-3 and granulocyte-macrophage CSF and human granulocyte CSF), or purified murine macrophage CSF. With unfractionated normal bone marrow the CFC increased in radiosensitivity as they progressed through the granulocyte lineage. The D0 values ranged from 129 +/- 12 cGy for CFC stimulated with GM-CSF down to 42 +/- 2 cGy after stimulation with G-CSF. IL-3 stimulated a CFC population which gave the only survival curve with a shoulder (n = 1.9 +/- 0.3). With semipurified populations of primitive or bipotential CFC, D0 values were generally lower with respect to the equivalent values for unpurified bone marrow (range 62 +/- 7 cGy to 135 +/- 7 cGy). Changes in cluster/colony ratio and colony morphology together possibly with products of accessory cells influence the interpretation of the radiosensitivity parameters.     

Effect of low-dose whole-body irradiation on granulopoietic progenitor cell subpopulations: implications for CFUc release

Abstract. The reaction of the granulopoietic system to whole body irradiation with 0.80 and 1.60 Gy was studied in dogs by means of colony formation assays in combination with velocity sedimentation and tritiated thymidine cytocidal techniques. Depression of circulating CFUc was associated with a marked shift in the size distribution of granulopoietic progenitors in the bone marrow. This effect lasted much longer in those animals receiving the lower X-ray dose. We conclude that circulating CFUc are not a random proportion of the bone marrow but a subpopulation of cells which are smaller in size. These cells are in an equilibrium with the larger marrow CFUc, which is very sensitive to perturbations.     

Radiosensitivity and postirradiation dynamics of granulocyte-macrophage precursors (CFU-DC) in the bone marrow of 2 lines of mice--CBA and BALB/c

CFU-DC in the bone marrow of CBA and BALB/c mice, which are contrast in total radiosensitivity, have close characteristics: D0 is 1.35 and 1.32 Gy, respectively. The proliferation rate of CFU-DC after single exposure to a non-lethal dose of 4 Gy is higher in CBA than in BALB/c mice. The time of doubling the CFU-DC population during the period of exponential growth after irradiation is 40 and 72 h for CBA and BALB/c mice, respectively.     

Survival of erythroid burst-forming units and erythroid colony-forming units in canine bone marrow cells exposed in vitro to 1 MeV neutron radiation or X rays

Conditioned media (CM) from allogeneic stimulated cultures of light density cells (less than 1.08 g/cm3) from the peripheral blood of normal dogs were used to stimulate the growth of erythroid burst-forming units (BFU-E) in bone marrow from normal dogs. Maximum numbers of BFU-E were obtained when 5% (vol/vol) 3 X CM and 2 U/ml erythropoietin were added to plasma clot cultures of bone marrow cells. In addition, the radiation sensitivity (D0 value) was determined for CFU-E and for BFU-E in bone marrow cells exposed in vitro to 1 MeV fission neutron radiation or 250 kVp X rays. BFU-E were more sensitive than CFU-E to the lethal effects of both types of radiation. For bone marrow cells exposed to 1 MeV neutron radiation, the D0 for CFU-E was 0.27 +/- 0.01 Gy, and the D0 for BFU-E was 0.16 +/- 0.03 Gy. D0 values for CFU-E and BFU-E were, respectively, 0.61 +/- 0.05 Gy and 0.26 +/- 0.09 Gy for cells exposed to X rays. The neutron RBE values for the culture conditions described were 2.3 +/- 0.01 for CFU-E and 1.6 +/- 0.40 for BFU-E.     

The postradiation radiosensitivity of rats following uniform external irradiation

In experiments with mongrel male rats exposed to double uniform gamma-irradiation (60Co), the postirradiation radiosensitivity was studied by the criterion of a median lethal dose; clinical and laboratory indices were investigated after preirradiation with doses of 1-5 Gy. A function was proposed to link the dynamics of the postirradiation radiosensitivity of rats to the proliferative activity of bone marrow and its total cellularity.     

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.     

Long-term effects of low-level 239Pu contamination on murine bone-marrow stem cells and their progeny

The effects of long-term internal contamination with 13.3 kBq kg-1 239Pu injected intravenously were studied in 10-week-old ICR (SPF) female mice. Radiosensitivity of spleen colony-forming units (CFU-S) and 125IUdR incorporating into proliferating cells of vertebral bone marrow and spleens were determined in plutonium-treated and control animals one year after nuclide injection. The CFU-S in 239Pu-treated mice were more sensitive to X-rays (D0 = 0.52 +/- 0.01 Gy) than in controls (D0 = 0.84 +/- 0.02 Gy). 125IUdR incorporation into bone marrow and spleen cells was reduced after plutonium contamination. At one year following plutonium injection, the occurrence of chromosome aberrations was evaluated in metaphase figures of femoral bone marrow cells. The frequency of aberrations increased early after plutonium treatment, at later intervals it tended to decrease but not below the control level. While the relative numbers of vertebral marrow CFU-S decreased significantly, but only to 86 per cent of normal, cellularity of vertebral bone marrow, peripheral blood counts and survival of 239Pu-treated mice did not differ from the control data.     

Enrichment and cryopreservation of bone marrow progenitor cells for autologous reinfusion

From 20 patients with solid tumors or acute nonlymphocytic leukemia in remission, hemopoietic progenitor cells were taken and stored in liquid nitrogen, for use in autologous bone marrow transplantation. Bone marrow aspiration resulted in a volume of 920(+/- 170) ml containing 16.8(+/-6.0) x 10(9) nucleated bone marrow cells and 7.2(+/-4.4) x 10(6) myeloid progenitor cells (CFUc). With use of the Haemonetics blood cell separator a progenitor cell-enriched fraction is obtained. This fraction is depleted of 90(+/-6)% of the erythrocytes and 59(+/-15)% of the neutrophils contained in the original. The original aspirate volume is reduced to one-fifth (21 +/- 3%) while containing 88(+/-38)% of the original CFUc's and 52(+/-11)% of the nucleated bone marrow cells. This technique of bone marrow enrichment has the advantage of a minimum of open-air contact, being independent of extensive laboratory facilities and manpower. The enriched fraction is frozen in autologous plasma and a final concentration of 10% (v/v) DMSO, using a program-controlled freezer (L'Air Liquide). Materials are stored at liquid nitrogen temperature in bags (Gambro) and test vials. Total CFUc recovery in test vials after thawing was 81(+/-32)%.     

Age-related characteristics of postradiation regeneration of the blood system

This study was pursued on hybrid (CBA X C57Bl)F1 mice at the ages of 17 and 27 months irradiated at a dose of 4 Gy. It was shown that the postirradiation recovery of haemopoiesis in old mice was the same as in the young (2-3 month-old), and in some respects (for instance, granulocytic and erythroid cells of the bone marrow, and CFUc) the recovery was even more active exhibiting a period of a pronounced hyperregeneration.     

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.     

Radiosensitivity of clonogenic granulocytic macrophage cell precursors in the bone marrow and spleen of tumor-bearing mice

Clonogenic granulocytic macrophagal cells-precursors (CFU-DC) of bone marrow and spleen of intact C57Bl/6 mice and those inoculated subcutaneously with LLC tumor cells do not substantially differ in their radiosensitivity; the concentration of CFU-DC in the spleen markedly varies as tumor grows. The values of Do and extrapolation number n for CFU-DC of the bone marrow are 0.9-1.4 and 1.5-3.0 Gy, and of the spleen, 0.8-1.6 and 1.0-2.6 Gy, respectively.     

Cloning stem cells in the bone marrow of irradiated mice]

Different amount of intact or irradiated bone marrow from syngenous donors was administered to mice irradiated with a lethal dose. There was revealed a linear dependence of the number of the 8-9-day colonies grown in the bone marrow of the femur on the amount of the administered cells, and an exponential dependence on the irradiation dose. Regularity of the stem cell cloning in the bone marrow was analogous to such in the spleen. Radiosensitivity of the colony-forming units (CFU) differed depending on the site (the spleen, the bone marrow) of their colony formation. The CFU settling in the marrow proved to be more radioresistant (D(0) equalled 160-200 P) in comparison with the CFU settling in the spleen (D(0) constituted 80-100 P). It is supposed that a different radiosensitivity of the CFU was caused by the presence of heterogenic population of the stem cells and also by specific peculiarities of the organ (the spleen, the bone marrow) in which the colonies formed.     

An In Vitro Model of Hematopoietic Injury In Chronic Hypoplastic Anemia

Mice treated with high-dose busulfan develop a ‘latent’ form of bone marrow failure characterized by near-normal peripheral blood counts and marrow cellularity, but marked reductions in marrow pluripotent stem cells (CFUs) and myeloid progenitor cells (CFUc). Spleen cell suspensions from control and ‘latent’ mice were placed in liquid culture in the presence of colony-stimulating activity. Cells were harvested at intervals up to 14 days and sub-cultured in agar to assay for CFUc. Baseline splenic CFUc did not differ significantly between control and ‘latent’ mice. Splenic CFUc from control mice increased 50-fold and reached a peak at day 10 in liquid culture. In contrast, splenic CFUc from ‘latent’ mice increased only 7-fold and reached a peak at day 3. Our results indicate that although splenic CFUc are present in normal numbers in ‘latent’ mice, their proliferative capacity is markedly reduced, either as the result of defective CFUc self-renewal or defective feed-in from CFUs or both.