THE PROLIFERATIVE CAPACITY OF HAEMOPOIETIC COLONY FORMING UNITS IN THE RAT

After transplantation into rats lethally treated with cytotoxic chemicals both bone marrow and spleen CFU in the spleen and spleen derived CFU in the bone marrow expand with doubling times ( T _d) of approximately 18 hr. However, bone marrow derived CFU in the bone marrow have a T _d of 36 hr. Evidence obtained using tritiated thymidine in vitro and methotrexate in vivo show that the proliferation rate of bone marrow derived CFU is similar in both the bone marrow and spleen and calculations suggest that the different T _d between these two sites is due to the higher loss of CFU through differentiation in the bone marrow compared to the spleen. These findings further support the hypothesis of an environment in the spleen which favours CFU self-maintenance over differentiation with the opposite situation occurring in the bone marrow.     

THE INFLUENCE OF GENETIC RESISTANCE ON CFU GROWTH KINETICS IN SPLEEN AND FEMUR

An impaired colony formation of C57BL marrow cells transplanted into F_t (C57BL x CBA) mice was observed. In accordance with the literature this phenomenon has been designated as ‘genetic resistance’. Studies to elucidate the mechanism of the genetic resistance demonstrated that the multiplication phase of the CFU growth curve started in the semi-isogeneic combination about 48 hr later than in the isogeneic combination. In the spleen this resulted in a lower ‘dip’. For the spleen as well as for the femur similar CFU doubling times were found during the multiplication phase when both transplantation combinations were compared. Furthermore the percentage of CFU in S-phase (assessed with the ³H-TdR suicide technique) during the first days after transplantation were similar in both combinations. When the spleen was removed 5–6 months before irradiation and bone marrow transplantation was performed the growth curve of parental CFU in the femur was identical with the growth curve of isogeneic CFU (no delay was observed). These results are discussed and a few theories explaining the observations are proposed.     

THE ROLE OF THE SPLEEN IN THE REPOPULATION OF THE HAEMOPOIETIC SYSTEM OF HEAVILY-IRRADIATED MICE

The respective role of the spleen or of the bone marrow in the regeneration of the haemopoietic progenitor compartment of heavily-irradiated mice has been investigated. Splenectomy was used to this end in animals injected with exogenous isogenic cells or regenerating from endogenous spleen or marrow cells. Analysis of the data as a function of time shows that the presence of the spleen affects marrow CFU repopulation only at the early post-irradiation stages. The expansion of the marrow progenitor pool proceeds, however, rather independently of the spleen and marrow CFU remain eventually as the main source of haemopoietic cells in the surviving mice. Thus the reaction of the spleen may be envisaged as a fast, important but transient contribution to the overall haemopoietic function of heavily-irradiated animals.     

Conditions Required For the Inhibition of In Vitro Growth of A Mouse Myeloma Cell Line By Adherent Bone-Marrow Cells

The in vitro growth of the MPC-11 myeloma cell line was inhibited when these cells were co-cultured with adherent cells from mouse bone marrow. This growth inhibition involved prolongation of the specific population doubling time of the MPC-11 cell line. Control cultures of MPC-11 cells exhibited an average doubling time of 14–15 hr, whereas in the presence of adherent layers the length of the doubling time was up to 28 hr. This prolongation in the doubling time did not depend on the duration of incubation, but on the relative proportions of tumour cells and adherent cells employed. MPC-11 cells seeded in relatively high starting cell concentrations partially overcame the growth inhibition. the inhibitory activity of adherent cells from the bone marrow did not appear to be due to production of soluble factor(s), since media conditioned by adherent cells did not affect cell growth. Moreover, in modified co-cultures in which MPC-11 cells grew physically separated from the adherent layers, only marginal growth inhibition activity was observed. the possibility that cell-to-cell interactions lead to the inhibition of growth of MPC-11 cells by adherent cells from the bone marrow, and the implications of these findings to the control of cell growth by the haemopoietic microenvironment, are discussed.     

MULTIPLICATION OF HAEMOPOIETIC COLONY FORMING UNITS (CFU) IN MICE AFTER X-IRRADIATION AND BONE MARROW TRANSPLANTATION

Kinetics of the multiplication of haemopoietic CFUs was studied in lethally irradiated mice receiving various numbers of syngeneic bone marrow cells. After transplantation of a small number of bone marrow cells, the growth rate of CFU in femoral bone marrow appeared to decrease after about 10 days after transplantation, before the normal level of CFU in the femur was attained. In the spleen it was found that the overshoot which was observed about 10 days after transplantation of a large number of bone marrow cells is smaller or absent when a small number of cells is transplanted. Experiments dealing with transplantation of 50 x 10⁶ bone marrow cells 0, 4 or 10 days after a lethal irradiation indicated that the decline in growth rate of CFUs about 10 days after irradiation could not be attributed to environmental changes in the host.
The results are explained by the hypothesis that a previous excessive proliferation of CFUs diminishes the growth rate thereafter. This hypothesis is supported by experiments in which 50 x 10⁶ bone marrow cells derived from normal mice or from syngeneic chimaeras were transplanted. The slowest growth rate was observed when bone marrow that had been subjected to the most excessive proliferation in the weeks preceding the experiment was transplanted.     

MOBILIZATION OF HAEMOPOIETIC STEM CELLS (CFU) INTO THE PERIPHERAL BLOOD OF THE MOUSE; EFFECTS OF ENDOTOXIN AND OTHER COMPOUNDS

Factors affecting the circulation of haemopoietic stem cells (CFU) in the peripheral blood of mice were investigated. I.v. injection of sublethal doses of endotoxin, trypsin and proteinase appeared to raise the number of CFU per ml blood from about 30–40 to about 300–400 or more within 10 min. The effect was smaller when smaller doses of the substances were injected. After this initial rise the number of circulating cells returned to normal in a few hours. Following endotoxin there was a second rise which started 2–3 days after injection and attained a peak on the 6th–7th day. The first rise is explained as a mobilization of stem cells from their normal microenvironments into the blood stream; the second rise is considered to reflect proliferation of CFUs in the haemopoietic tissues. The spleen seems to be acting as an organ capturing CFUs from the blood and not as a source adding stem cells to the blood.
The early mobilization of CFU after endotoxin injection did not coincide with a mobilization of neutrophils. The number of circulating band cells was increased during the first hours.
The importance of 'open sites'in the haemopoietic tissue for capturing CFUs was studied by emptying these sites through a lethal X-irradiation and injecting normal bone marrow cells. When a greater number of syngeneic bone marrow cells was injected intravenously, the level of circulating CFU in irradiated mice was slightly lower than the level in unirradiated mice during the first hours.     

GRAFT SIZE CONSIDERATIONS IN THE KINETICS OF SPLEEN COLONY DEVELOPMENT

The kinetics of spleen colony development has been studied after the injection of 10⁶, 10⁵ and 3 × 10⁴ bone marrow cells. The results indicate that:

• 1 The CFU population growth rate is independent of cell dose until the logarithmic growth phase is passed. Slowing of growth was seen by day 12 after the highest dose, by day 15 after the median dose, but was not observed during the period of observation after the low dose.
• 2 The growth rate of CFU per colony is independent of cell dose, but the curves are not identical. The differences between the curves leads to the conclusion that there is a dose-dependent delay in the commencement of CFU proliferation. The delay is roughly equal to one cell cycle time between the medium and high inoculum groups and also between the medium and low inoculum groups.
• 3 The number of cells per colony is graft size dependent, the doubling times, where these can be roughly assessed, being inversely related to the graft size. From the average number of cells per colony on day 6 it is calculated that the mean doubling time in the early stages of colony development is less than 7 hr.
• 4 The proportion CFU:colony cells is dose dependent with the highest inoculum having the highest proportion and the low inoculum group having the lowest proportion.

     

THE INFLUENCE OF DIETARY PROTEIN DEFICIENCY ON HAEMOPOIETIC CELLS IN THE MOUSE

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

Effect of age of mouse recipients on the functional activity of transplanted hematopoietic and lymphoid cells]

When transplanting the bone marrow cells from adult C57BL mice to the lethally irradiated (CBA X C57BL) F1 hybrids of different age, the decrease of the colony forming activity of the stem haemopoietic cells was observed in the spleen of the older recipients, as compared with the 3 months old ones. The joint transplantation of the bone marrow and thymus cells resulted in both the cases in the stimulation of the growth of colonies. The number of endogenous colonies of haemopoietic cells arising in the spleen of animals following the sublethal irradiation was greater in younger hybrids. After the induction of the "transplant versus host" reaction by the lymph node or spleen cells from the CBA mice, the relative weight of spleen and regional lymph node, respectively, in the older recipients exceeded those in the younger ones.     

AGE DEPENDENCE OF THE NUMBER OF THE STEM CELLS IN HAEMOPOIETIC TISSUES OF RATS

The number and concentration of haemopoietic stem cells in the femoral bone marrow and spleen of Wistar rats of different ages were investigated. Stem cells were assayed by the spleen colony technique in irradiated rat recipients. The ability of the recipient spleen to harvest transplanted tissue as a macroscopic colony was found to be dependent on the recipient's age. Changes with senescence were observed also in the concentration and the size of the stem cell compartment both in the marrow and spleen. No differences were demonstrated in the seeding of transplanted colony-forming units into the spleen of recipients of 1 and 4 months of age. A rats-mice strain difference in the effect of senescence on the haemopoietic stem cells is discussed.     

Macrophages in haemopoietic and other tissues of the developing mouse detected by the monoclonal antibody F4/80.

Macrophages are widely distributed in lymphohaemopoietic and other tissues of the normal and diseased adult, where they play an important role in host defence and repair. Although the development of haemopoiesis has been well studied in several species, the ontogeny of the mononuclear phagocyte system remains poorly understood. We have used a highly specific mAb, F4/80, to examine the distribution of mature macrophages in the developing mouse, with special reference to their presence in the haemopoietic microenvironment. Monocytes and macrophages were first seen in embryos on day 10 in the yolk sac and liver as well as in mesenchyme. In liver, spleen and bone marrow, there was expansion of this population associated with the initiation of haemopoiesis on days 11, 15 and 17, respectively. Macrophages in these sites formed part of the haemopoietic stroma and their extensively spread plasma membrane processes could be seen making intimate contacts with clusters of differentiating haemopoietic cells. F4/80+ cells were widely dispersed in undifferentiated mesenchymal tissue in organs such as lung, kidney and gut. Numbers of F4/80-labelled cells increased concomitantly with organ growth and local mitoses were evident, as well as actively phagocytic macrophages. Our studies establish that macrophages are among the earliest haemopoietic cells to be produced during development and that they are relatively abundant in fetal tissues in the absence of overt inflammatory stimuli. Their distribution is correlated with the sequential migration of haemopoiesis and they constitute a prominent component of the stroma in fetal liver, spleen red pulp and bone marrow. Apart from a role in haemopoietic cellular interactions, their highly developed endocytic and biosynthetic activities suggest that macrophages contribute major undefined functions during growth, turnover and modelling of fetal tissues.     

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

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

Compartments and cell flows within the mouse haemopoietic system. I. Restricted interchange between haemopoietic sites.

Two chromosomally distinguishable haemopoietic cell populations were injected into lethally irradiated syngeneic recipients. The presence or absence of the T(14;15)6Ca reciprocal translocation (indicated by T6 marker chromosomes) did not affect the proliferation of a population. Wide disparities were found in the proportions of the two donor cell populations between animals and between the right and left femora of individual animals. This suggest (i) that there is, at most, a very limited interchange of proliferating cells and their precursors between the marrow of different bones; and (ii) that the number of clones proliferating in the bone marrow at any one time must be rather small; there was evidence that this number depended in part on the number of haemopoietic cells injected. Exchange between the mitotically active cell populations of spleen, thymus, lymph nodes and bone marrow was also limited, as shown by significant disparities in the proportions of the two donor populations proliferating in the different tissues of individual mice.     

COMPARTMENTS AND CELL FLOWS WITHIN THE MOUSE HAEMOPOIETIC SYSTEM

Two chromosomally distinguishable haemopoietic cell populations were injected into lethally irradiated syngeneic recipients. The presence or absence of the T(14;15)6Ca reciprocal translocation (indicated by T6 marker chromosomes) did not affect the proliferation of a population. Wide disparities were found in the proportions of the two donor cell populations between animals and between the right and left femora of individual animals. This suggests (i) that there is, at most, a very limited interchange of proliferating cells and their precursors between the marrow of different bones; and (ii) that the number of clones proliferating in the bone marrow at any one time must be rather small; there was evidence that this number depended in part on the number of haemopoietic cells injected. Exchange between the mitotically active cell populations of spleen, thymus, lymph nodes and bone marrow was also limited, as shown by significant disparities in the proportions of the two donor populations proliferating in the different tissues of individual mice.     

Effect of radiation and radioprotectors on the properties of hematopoietic stem cells

Sublethal irradiation of donors leads to a change in some properties of bone marrow haemopoietic stem cells (HSC) during the exponential growth (days 1-8) of the syngeneic recipients in the spleen. They are: an increase in the rate of proliferation, a slight reduction in time of the population doubling, and a tendency toward an increase in the percentage of cells settled in the spleen after transplantation. These changes in the properties of HSC provide a more rapid repopulation thereof as compared to HSC of intact mice. In all appearance, a pretreatment of donors with AET and 2ADT does not influence the HSC changes induced by radiation, and, at the same time, retains the number of HSC at a high level.     

EFFECTS OF PRE-IRRADIATION ON ISOGENEIC AND SEMI-ISOGENEIC CFU GROWTH: A STUDY ON GENETIC RESISTANCE

The genetic resistance to a parental bone marrow transplant as demonstrated, when transplantation was performed early after irradiation, failed to occur if the interval between irradiation and transplantation was increased to 4 days. A similar radiation induced weakening of genetic resistance to a parental bone marrow graft in spleen and bone marrow could be demonstrated in mice, which had been irradiated with a sublethal dose at 7 days prior to the lethal irradiation and transplantation. The pre-irradiation of the recipient with a sublethal dose induced an enhancement of the growth in spleen and bone marrow of isogeneic transplanted CFU. The pre-irradiation of a single tibia also resulted in a significant weakening of the resistance in the spleen. The experiments with partial body pre-irradiation suggested a local effect of the pre-irradiation, but it could be shown that the enhanced CFU growth is not caused by an enhanced seeding of CFU in pre-irradiated bone marrow. The role of microenvironment in the phenomenon of genetic resistance is discussed.     

MEASUREMENT OF THE DURATION OF DNA SYNTHESIS IN ERYTHROID CELLS OF THE BONE MARROW USING A DOUBLE LABELLING AUTORADIOGRAPHIC TECHNIQUE DESIGNED SPECIFICALLY FOR USE WITH HAEMOPOIETIC TISSUES

This paper describes a double labelling autoradiographic technique for use with haemopoietic tissues. In involves two photographic emulsions separated by a thin piece of mica on which the cells have been smeared. In this way the autoradiographic grains due to tritium and carbon-14 appear above and below the cells respectively. Applying the method to bone marrow normoblasts of young rats, the average duration of DNA synthesis (t_s) for the pro- and early normoblasts taken together is found to be 5.1 hr and the mean cell cycle time (t_c) to be 8.2 hr. For the intermediate normoblasts, the corresponding figures are 6.3 hr and 15.7 hr. Average values for all dividing normoblasts in the bone marrow are 5.8 hr and 12.8 hr respectively for t_s and t_c. The average duration of mitosis is 32 min.     

DEFECT OF ERYTHROPOIESIS IN NON-LEUKAEMIC AKR MICE

The kinetics of the CFU population and of erythropoiesis were investigated in the AKR strain mouse prior to the onset of thymic leukaemias: haemopoiesis was compared in syngeneic AKR, semi-allogenic C3H and (C3H x AKR) F, mice injected with AKR stem cells. These experiments demonstrate that the reduction in the number of spleen colonies previously described by Perkins et al. (1971) in syngeneic hosts, as compared to semi-allogenic C3H hosts, is actually related to defective erythropoiesis resulting from a dysfunction of the AKR haemopoietic inductive microenvironment (HIM). Erythropoietin secretion is normal in AKR mice. the early haemopoietic events related to the stem cell: lodgement of the CFU (‘f’ factor) and doubling time, are not disturbed, but the onset of CFU proliferation is markedly delayed in the AKR strain. the main expression of the AKR HIM dysfunction is a significant reduction in the number of erythroid (E) colonies and an impaired output of red blood cells per E-colony in the syngeneic host as compared to the allogenic one. In addition, data indicate that a weakly histo-incompatible system, such as that in C3H and hybrid hosts, does not interfere with the stages of haemopoiesis except by lengthening the doubling time of the CFU. the results, on the whole, emphasize the prevalent influence of HIM.     

EFFECTS OF ENVIRONMENTAL TEMPERATURE ON HAEMOPOIETIC STEM CELLS IN THE TAILS OF MICE

Using the endogenous spleen colony assay method of Till & McCulloch (1963), the numbers of haemopoietic stem cells present in the bone marrow in the tails of mice were estimated under different environmental temperatures. Compared to animals kept at 22–26°C, mice transferred to and kept at 36.5°C showed a doubling of colony-forming units in the tail in 1–4 weeks. Exposing them to 8°C caused a significant depopulation to approximately one-third in 3–4 weeks. By transferring the mice from one temperature extreme to another these changes could be reversed. Tail marrow depleted of viable stem cells by X-irradiation was repopulated within approximately 3 weeks in animals kept at room temperature or above but this process was inhibited in the cold.     

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.