PROLIFERATION OF ERYTHROID AND GRANULOCYTE PROGENITORS IN THE SPLEEN AS A FUNCTION OF STEM CELL DOSE

A study of the kinetics of cellular proliferation, in the morphologically unrecognizable haemopoietic progenitor cell compartment, as a function of injected CFU-S dose has been carried out in the spleens of lethally X-irradiated mice using ³H-TdR labelling. Amplification in this proliferating cell compartment was observed to decline as CFU-S dose increased. The number of divisions in the differentiated line arising from CFU-S up to the first appearance of recognizable erythroid precursors were calculated to be 9.2, 12.5, 15 and 17 for the 2, 0.35, 0.05 and 0.007 femur equivalent doses respectively. The growth of cell populations arising from CFU-S was biphasic, with a rapid initial phase having a doubling time of about 6.3 hr, and a slow phase of doubling time around 1 day. Analysis of the rapid phase by the FLM method gave a cycle time of 5.6 hr. Recognizable labelled erythroid precursors were detected at the same time as, or just after, the change in slope of the growth curve. Significant numbers of proliferating (labelled) granulocytes only appeared in the spleens of animals receiving the higher marrow doses (2 and 0.35 femur). The erythroid to granulocyte ratio was also a decreasing function of marrow dose.     

Radioprotection of mice with interleukin-1: relationship to the number of spleen colony-forming units

Compared to saline-injected mice 9 days after 6.5 Gy irradiation, there were twofold more Day 8 spleen colony-forming units (CFU-S) per femur and per spleen from B6D2F1 mice administered a radioprotective dose of human recombinant interleukin-1-alpha (rIL-1) 20 h prior to their irradiation. Studies in the present report compared the numbers of CFU-S in nonirradiated mice 20 h after saline or rIL-1 injection. Prior to irradiation, the number of Day 8 CFU-S was not significantly different in the bone marrow or spleens from saline-injected mice and rIL-1-injected mice. Also, in the bone marrow, the number of Day 12 CFU-S was similar for both groups of mice. Similar seeding efficiencies for CFU-S and percentage of CFU-S in S phase of the cell cycle provided further evidence that rIL-1 injection did not increase the number of CFU-S prior to irradiation. In a marrow repopulation assay, cellularity as well as the number of erythroid colony-forming units, erythroid burst-forming units, and granulocyte-macrophage colony-forming cells per femur of lethally irradiated mice were not increased in recipient mice of donor cells from rIL-1-injected mice. These results demonstrated that a twofold increase in the number of CFU-S at the time of irradiation was not necessary for the earlier recovery of CFU-S observed in mice irradiated with sublethal doses of radiation 20 h after rIL-1 injection.     

RECOVERY OF PROLIFERATING HAEMOPOIETIC PROGENITOR CELLS AFTER KILLING BY HYDROXYUREA

Two doses of 1 mg/g of hydroxyurea (HU), injected 7 hr apart into irradiated mice in which CFU-S were proliferating during marrow regeneration, killed about 90% of CFU-S. This same dose regime injected into normal female mice, with non-proliferating CFU-S killed 92 % of CFU-C, 99 % of ESC and only 30 % of CFU-S. One day after the treatment CFU-S had decreased to 50 % and remained at about this level for a further day then returned to normal values. In spleen the increase in CFU-S was delayed by a day and showed a marked overshoot. During the period that CFU-S were decreased in number they were actively proliferating. Marrow CFU-C recovered in an exponential manner with a doubling time of 16 hr. Spleen CFU-C recovered 1 day later than marrow and showed a pronounced overshoot. ESC recovered very rapidly with doubling time of 5 hr. The changes in ⁵⁹Fe incorporation into RBC, and the peripheral blood picture, were a delayed reflection of the changes in ESC and CFU-C.     

Effect of Myleran On Murine Hemopoiesis

Time- and dose-dependent patterns of depletion and regeneration of hemopoietic progenitor cells in mouse femora and spleens following treatment with the antileukemic agent Myleran (Busulphan, MY) were studied using the murine spleen colony system and the agar gel in vitro colony system. MY was found to depress granulopoiesis selectively, as manifested by the development of marked prolonged neutropenia, hypoplasia of the bone marrow and (to a lesser degree) of the spleen, reduction of the incidence of multipotential hemopoietic progenitor cells (CFU-S) and of granulocytic progenitor cells (CFU-C) in both femora and spleens, and impairment of the capacity of CFU-S from either tissue to generate granulocytic colonies in the spleens of irradiated hosts. the severity and duration was greatest at high dose levels of MY (800 μ). the action of MY on CFU-S was more pronounced than that on CFU-C, suggesting that MY is a cycle-independent agent. Repopulation of the CFU-C pool preceded that of the CFU-S pool. Development of neutropenia and maximal marrow hypoplasia followed the onset of depression of CFU-S and CFU-C incidence, while recovery of normal nucleated cellularity in the blood, femur and spleen preceded repopulation of the CFU-S and CFU-C pools. MY treatment resulted in transitory stimulation of colony stimulating factor (CSF) generation by the femur but had no effect on serum CSF levels. the peak of femoral CSF generation coincided with the nadir of CFU-C depression. These findings indicated that the prolonged neutropenia following MY treatment was secondary to depletion of the progenitor cell pools, that during recovery granulopoietic repopulation took precedence over self-maintenance of the hemopoietic progenitor cell pools, and that increased generation of CSF may play a role in the early phase of granulopoietic recovery.     

Effect of myleran on murine hemopoiesis. I. Granulocytic cell line specificity of action on progenitor cells.

Time- and dose-dependent patterns of depletion and regeneration of hemopoietic progenitor cells in mouse femora and spleens following treatment with the antileukemic agent Myleran (Busulphan, MY) were studied using the murine spleen colony system and the agar gel in vitro colony system. MY was found to depress granulopoiesis selectively, as manifested by the development of marked prolonged neutropenia, hypoplasia of the bone marrow and (to a lesser degree) of the spleen, reduction of the incidence of multipotential hemopoietic progenitor cells (CFU-S) and of granulocytic progenitor cells (CFU-C) in both femora and spleens, and impairment of the capacity of CFU-S from either tissue to generate granulocytic colonies in the spleens of irradiated hosts. The severity and duration was greatest at high dose levels of MY (800 microgram). The action of MY on CFU-S was more pronounced than that on CFU-C, suggesting that MY is a cycle-independent agent. Repopulation of the CFU-C pool preceded that of the CFU-S pool. Development of neutropenia and maximal marrow hypoplasia followed the onset of depression of CFU-S and CFU-C incidence, while recovery of normal nucleated cellularity in the blood, femur and spleen preceded repopulation of the CFU-S and CFU-C pools. MY treatment resulted in transitory stimulation of colony stimulating factor (CSF) generation by the femur but had no effect on serum CSF levels. The peak of femoral CSF generation coincided with the nadir of CFU-C depression. These findings indicated that the prolonged neutropenia following MY treatment was secondary to depletion of the progenitor cell pools, that during recovery granulopoietic repopulation took precedence over self-maintenance of the hemopoietic progenitor cell pools, and that increased generation of CSF may play a role in the early phase of granulopoietic recovery.     

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.     

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.     

Pluripotent (Cfu-S) and Granulocyte-Committed (Cfu-C) Stem Cells In Intact and 89Sr Marrow-Ablated S1/S1D Mice

Peripheral blood values, femur cell counts, spleen weights, pluripotent (CFU-S) and granulocyte progenitor cell (CFU-C) concentrations and total content of spleens and femurs have been evaluated in intact (non-marrow-ablated) and ⁸⁹Sr marrow-ablated S1/S1^d and +/+ mice. ⁸⁹Sr-irradiated mice were studied 6 and 11 days after the administration of ⁸⁹Sr. In intact S1/S1^d mice the femur CFU-S concentration, total femur CFU-S, femur CFU-C concentration and total femur CFU-C were 84, 54, 105 and 68% that of +/+ mice femurs respectively; the respective values for the spleens of S1/S1^d mice were 40,46,61 and 69%. These are the first simultaneous determinations of CFU-S and CFU-C concentrations, and content of spleens and marrows, of S1/S1^d and +/+ mice. In ⁸⁹Sr marrow-ablated mice, 11 days after injection of the radionuclide: (a) the total content of marrow CFU-C and CFU-S was about 1% of that found in the marrows of intact mice for both +/+ and S1/S1^d groups; (b) the spleens of +/+ mice increased in weight to 162% of the control, but the spleens of S1/S1^d mice did not increase in weight; and (c) the spleens of +/+ mice had a total content of CFU-C and CFU-S of 800% and 260% of the control, respectively, whereas the respective values for the S1/S1^d mice were 120% and 76% of the control. Thus the S1/S1^d spleen fails to compensate for marrow ablation by housing additional CFU-S and has an impaired ability to compensate by housing additional CFU-C.     

AN EARLY RESPONSE OF THE MORPHOLOGICALLY RECOGNIZABLE ERYTHROID PRECURSORS TO BLEEDING

⁵⁵Fe autoradiography of the peripheral red blood cells has been used to study the proliferation of the recognizable erythroid precursors in bled animals. The transit time of the recognizable erythroid precursors present in the bone marrow and labelled with ⁵⁵Fe 6 hr before bleeding, remains unchanged, but the number of red cells produced by these precursors is significantly greater than normal. It is deduced that the increased red cell production is brought about by an increase in the number of divisions that the cells undergo during maturation and that a shortening in the red cell cycle time is implied. The possibility that the transit time of the progeny of cells differentiating into pro-erythroblasts after bleeding may be shorter than the transit time of the precursors already differentiated before bleeding, is briefly discussed.     

New role for histamine in interleukin-3-induced proliferation of hematopoietic stem cells

This study investigated the effect of histamine generated by murine bone marrow cells in response to IL-3 on one particular biological activity of this growth factor, i.e., triggering of cells forming colonies in spleen (CFU-S) into S phase. Evidence is provided that i) IL-3-induced day-8 CFU-S cell cycling, evaluated by hydroxy-urea suicide, is completely abrogated when the binding of histamine to its H2 receptors is blocked by the specific antagonist oxmetidine, whereas cetirizine, a H1 receptor antagonist, is ineffective; and ii) the entry of day-8 CFU-S into S phase in response to IL-3 is likewise abolished when the histamine synthesis promoted by the growth factor is prevented by alpha-fluoromethylhistidine, a specific inhibitor of the histamine-forming enzyme, histidine decarboxylase. Similar results are obtained with both drugs, when a progenitor-enriched bone marrow population is used instead of total cells. Furthermore, i.v. injection of recombinant (r)IL-3 results within 2 hr in a substantial increase in bone marrow cell histamine synthesis together with triggering of day-8 CFU-S into cycle, the latter being completely abolished by a simultaneous injection of the H2 histamine receptor antagonist oxmetidine. Thus, our findings support the notion that both in vitro and in vivo the proliferation of early CFU-S in response to IL-3 is modulated by histamine via its H2 receptors. This conclusion is also consistent with the observation that dimaprit, a specific agonist of these receptors not only enhances the sensitivity of day-8 CFU-S to HU after a 2 hr incubation with bone marrow cells but also increases, to the same extent as IL-3, the number of colonies formed in irradiated spleens after a 5 hr pretreatment.     

Local regulation of haemopoietic stem cell proliferation in mice following irradiation

Changes in the kinetic state of pluripotent haemopoietic spleen colony forming cells (CFU-S) and of the CFU-S proliferation stimulator have been studied following whole-body X-irradiation. Rapid recruitment of CFU-S into cell cycle by 30 min after irradiation was observed following low doses (0.5 Gy) but a delay of 6 h occurred after higher doses (1.5 and 4.5 Gy). These changes in proliferative state correlated with the presence of the CFU-S proliferation stimulator. CFU-S irradiated in vitro in bone marrow plugs were also recruited into cycle illustrating directly the local nature of the feedback mechanism. CFU-S removed from 1.5 Gy irradiated recipients at a time when they were not in cycle were not responsive to the CFU-S proliferation stimulator. The CFU-S proliferation stimulator was produced by Ia positive cells in the irradiated bone marrow. The regulation changes occurring shortly after irradiation cannot simply be controlled by the size of the CFU-S compartment.     

Local regulation of haemopoietic stem cell proliferation in mice following irradiation

Abstract Changes in the kinetic state of pluripotent haemopoietic spleen colony forming cells (CFU-S) and of the CFU-S proliferation stimulator have been studied following whole-body X-irradiation. Rapid recruitment of CFU-S into cell cycle by 30 min after irradiation was observed following low doses (0.5 Gy) but a delay of 6 h occurred after higher doses (1.5 and 4.5 Gy). These changes in proliferative state correlated with the presence of the CFU-S proliferation stimulator. CFU-S irradiated in vitro in bone marrow plugs were also recruited into cycle illustrating directly the local nature of the feedback mechanism. CFU-S removed from 1.5 Gy irradiated recipients at a time when they were not in cycle were not responsive to the CFU-S proliferation stimulator. The CFU-S proliferation stimulator was produced by Ia positive cells in the irradiated bone marrow. The regulation changes occurring shortly after irradiation cannot simply be controlled by the size of the CFU-S compartment.     

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.     

The kinetics of hematopoietic stem cells during and after hypoxia. A model analysis

A previously described mathematical model of the hematopoietic stem cell system has been extended to permit a detailed understanding of the data during and after hypoxia. The model includes stem cells, erythroid and granuloid progenitors and precursors. Concerning the intramedullary feedback mechanisms two basic assumptions are made: 1) The fraction "a" of CFU-S in active cell cycle is regulated. Reduced cell densities of CFU-S, progenitors or precursors lead to an accelerated stem cell cycling. Enlarged cell densities suppress cycling. 2) The self renewal probability "p" of CFU-S is also regulated. The normal steady state is described by p = 0.5, indicating that on statistical average each dividing mother stem cell is replaced by one daughter stem cell, while the second differentiates. Diminished cell densities of CFU-S or enlarged densities of progenitors and precursors induce a more intensive self renewal (p greater than 0.5), such that the stem cell number increases. The self renewal probability declines (p less than 0.5) if too many CFU-S or too few progenitors and precursors are present. The model reproduces bone marrow data for CFU-S, BFU-E, CFU-C, CFU-E, 59 Fe-uptake and nucleated cells in hypoxia and posthypoxia. Although the ratio of differentiation into the erythroid and granuloid cell lines is kept constant in the model, a changing ratio of CFU-E and CFU-C results. The model suggests that stem cells and progenitor cells are regulated by a regulatory interference of erythropoiesis and granulopoiesis.     

Hemopoietic precursor cell defects in nonanemic but stem cell-deficient W44/W44 mice

Hematopoietic stem cell deficiencies cause a severe macrocytic anemia in W/Wv mice. W44/W44 mice, on the other hand, are not anemic, but, since they accept marrow implants without prior total body irradiation, they have inherited a stem cell lesion. In an attempt to identify the aberrant stem cell(s), we have determined the concentration in W44/W44 marrow of hematopoietic precursors known to be deficient in W/Wv marrow. The in vitro erythroid burst-forming units (BFU-E), the in vivo spleen colony-forming units (CFU-S), and the cells that repopulate the erythroid compartment of stem cell-deficient mice were examined. The progenitors of 7-day bursts are dramatically reduced in W/Wv marrow but are present in normal concentrations in W44/W44 marrow. W44/W44 marrow CFU-S, unlike W/Wv, generate visible spleen colonies 10 days after injection into lethally irradiated recipients. The colonies are, however, smaller and at least 2 times less numerous than those produced from equivalent numbers of +/+ marrow. An additional defect was the inability of W44/W44 stem cells to compete with genetically marked +/+ cells during erythroid repopulation. An estimate of the number of W44/W44 stem cells needed to compete with +/+ cells was provided by enriching W44/W44 progenitors fivefold. Twice as many enriched W44/W44 marrow cells as unfractionated +/+ cells were required to replace competitor cells. This suggests that there are up to 10 times fewer stem cells somewhere in the W44/W44 erythrogenerative pathway. The data support the conclusion that an erythroid progenitor less mature than the BFU-E is one of the cells most severely affected by expression of the mutant gene.     

THE EFFECT OF FETUIN AND OF SPLEEN EXTRACTS ON HEMATOPOIETIC PRECURSORS AND ON DIFFERENTIATED HEMATOPOIETIC CELLS IN IRRADIATED MICE

Injection of extracts from normal mouse spleen tissue into irradiated mice enhance the rate of regeneration of colony forming units (CFU-S) in the femoral marrow. This effect was most pronounced when spleen extract was injected between 24 hr before and 24 hr after the time of irradiation, and was observed only during the first week after a single injection of extract. Another result of injecting spleen extract was an immediate and transient decrease in the marrow cellularity and particularly in the number of mature myeloid cells in the marrow. Fetuin produced comparable effects on the rate of regeneration of CFU-S and on the numbers of mature myeloid cells in the marrow. On the basis of these results it is tempting to speculate that injection of spleen extracts and of fetuin primarily cause a rapid depletion of the marrow's granulocyte reserve. This in turn releases the precursor cell compartment from the inhibitory effects of cell–cell interaction and results in an acceleration of the rate of CFU-S regeneration. It is equally plausible that factors present in spleen extract and in fetuin cause a depletion of the marrow granulocyte reserve and, by an unrelated mechanism, directly accelerate the rate of regeneration of CFU-S.     

In vitro hemopoiesis within a microenvironment created by MC3T3-G2/PA6 preadipocytes

The clonal preadipose cell line, MC3T3-G2/PA6, has the capacity to differentiate into adipocytes in response to glucocorticoids and to support in vitro growth of hemopoietic stem cells (CFU-S). To study the relationship between these capacities, we precultured the MC3T3-G2/PA6 cells for varying days in the presence or absence of dexamethasone and then cocultured them with mouse bone marrow cells. Logarithmically growing cultures contained no detectable adipocytes and showed the highest growth-supporting activity for CFU-S, whereas cultures containing the largest number of adipocytes showed the lowest activity. When bone marrow cells were seeded onto 3-day-old MC3T3-G2/PA6 preadipocyte layers at 1 X 10(5) cells/35-mm dish, day 12 CFU-S grew with a population doubling time of about 37 hr, and at least 75% of them were associated with the cell layer between days 2 and 7. In the absence of the preadipocytes, CFU-S were not detected in the adherent cell fraction and decreased with a half-life of about 18 hr. More than 80% of CFU-C were also found to be associated with the preadipocyte layer, and they increased about 24-fold in number during 7 days in culture. Morphologically, hemopoietic cells developing into mature granulocytes and macrophages were distributed between the layers of preadipocytes. Dendritic processes of preadipocytes were frequently in close alignment with the hemopoietic cells. However, adipocytes failed to show such an intimate association with hemopoietic cells. These results indicate that MC3T3-G2/PA6 cells in the preadipocyte stage, but not in the adipocyte stage, have the capacity to support CFU-S growth, and that hemopoiesis in our cocultivation system proceed within the microenvironmental milieu provided by MC3T3-G2/PA6 preadipocytes.     

The role of cells sensitive to anti-mouse brain serum in the regulation of CFU-S proliferation

CFU-S differentiation and regeneration kinetics in the spleen and femur was studied after treatment of bone marrow cells with RAMB serum. The effect of thymocytes on the rate of CFU-S regeneration was also investigated. It was found that CFU-S regeneration in the spleen was similar in RAMBS-treated and intact cell populations on days 4-14 after transplantation. On the contrary, the rate of CFU-S regeneration in the femur was slower in RAMBS-treated than in intact bone marrow cells. However, the growth rate in the femur could be restored to the normal level by the administration of freshly isolated syngeneic thymocytes to mice pre-injected with RAMBS-treated CFU-S population. The treatment of bone marrow suspension with RAMB serum did not affect the differentiation of spleen colonies. It is suggested that RAMBS eliminates cell population regulating CFU-S proliferation, without affecting its differentiation.     

Haemopoietic modulation in tumour-bearing animals: enhanced progenitor-cell production in femoral marrow

Altered haematopoiesis in the femoral marrow was observed in mice bearing the Lewis lung carcinoma (LLca). During tumour growth, a marked reduction was observed in the myeloperoxidase-positive cells (granulocytes) of the marrow 7 days after inoculation of the LLca tumour reaching a nadir (17% of control) by day 28. Accompanying this suppression of mature white cells was a gradual expansion of the CFUc-GM compartment followed by an increase in the number of femoral CFUs. Humoral-stimulating activity (HSA) increased through day 14 in the serum of these animals; then returned to control levels by day 28. During this same interval, the more primitive erythroid progenitor (BFUe) compartment expanded to 168% of control, while the more differentiated (CFUe) compartment was reduced (45% of control at day 28). Reductions in both 59Fe-incorporation and erythroblasts/femur confirmed the suppression of erythroid differentiation in marrow during tumour growth. Similar results were observed following the daily injection (188 mg equivalent dose; q 24 hr X 10) of the supernatant prepared from LLca tissue. Marked differences were observed between the response of the spleen and the marrow to the supernatant. The data suggest that the growth of the LLca tumour results in a dissociation of the normal continuity of haematopoietic steady-state differentiation in the marrow of tumour-bearing animals.     

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.