Self-maintenance of migrating hematopoietic stem cells]

Decreased self-maintenance ability of the migrating stem cells (CFU) from the peripheral blood or ectopic focus of hemopoiesis in comparison to the settled bone marrow CFU, as measured by the spleen colony method or by means of chromosomal markers, has been studied. The competence for myeloid and lymphoid differentiation was essentially the same for migrating and settled stem cells.     

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.     

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.     

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.     

Characteristics of the T-lymphocytes interacting with hematopoietic stem cells: the expression of Lyt-3 antigen

The lymph node cells were treated with monoclonal anti-Lyt-3 antibodies (anti-Lyt-3) and complement. Their interaction with colony-forming units (CFU) of bone marrow was studied. Anti-Lyt-3 did not affect the activity of T-lymphocytes, which changed the pattern of syngenic CFU differentiation in spleen of irradiated mice. On the contrary, the activity of T-lymphocytes, which inactivated proliferation and differentiation of non-syngenic CFU in spleen, was decreased. In the peritoneal cavity of irradiated mice antibodies did not affect the activity of T-lymphocytes which suppressed the colony-formation of non-syngenic stem cells and switched off the activity of T-lymphocytes that stimulated syngenic CFU colony-formation. The subpopulation characteristics of regulatory and effector T-cells has been analysed.     

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

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

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.     

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

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

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

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

DISSECTING THE HEMATOPOIETIC MICROENVIRONMENT. I. STEM CELL LODGMENT AND COMMITMENT, AND THE PROLIFERATION AND DIFFERENTIATION OF ERYTHROPOIETIC DESCENDANTS IN THE Sl/Sld MOUSE

The anemic Sl/Sl^d mouse and its normal (+/+) congenic control were used to explore the possibility of stromal control over four phases of erythropoiesis: CFU lodgment, commitment of multipotent stem cells to the erythropoietic line, proliferation of stem cells and their descendants, and the differentiation of those descendants into successively more mature forms. Lodgment was found to be the same in the Sl/Sl^d as in the normal mouse, but commitment, although characteristically different for spleen compared to the bone marrow, was subnormal. The stimulus to proliferate, as measured by spleen colony size and cell type content, was even more reduced. It is suggested that the direct control of differentiation into more mature cells may not be under stromal control.     

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.     

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.     

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.     

PROPERTIES OF HAEMOPOIETIC STEM CELLS IN PHENYLHYDRAZINE TREATED MICE

Recovery of erythropoiesis was fast in Balb/c mice irradiated 700 R 5 days after initiation of phenylhydrazine treatment and took place predominantly in the spleen, which showed numerous large frequently confluent endogenous colonies. Post irradiation phenylhydrazine induced anaemia did not accelerate recovery of erythropoiesis; it did, however, produce a slight but significant rise in endogenous colony formation.
Radiosensitivity of spleen CFU-S from phenylhydrazine treated mice was similar to that of CFU-S in normal mouse spleen.
Spleen CFU-S in mice 5 days after initiation of phenylhydrazine treatment were sensitive to the lethal action of Hydroxyurea, while bone marrow CFU-S were not.
The self-renewal capacity of CFU-S in the endogenously repopulated spleen of phenylhydrazine pretreated 700 R X-irradiated mice was low when compared to that of spleen exogenously repopulated by cells from normal mouse bone marrow, normal and phenylhydrazine treated mouse spleen. CFU circulating in blood of phenylhydrazine treated mice had a low self-renewal capacity.
The marked strain differences in self-renewal capacity of spleen CFU-S, and of the capacity of spleen CFU-S to increase by proliferation are discussed.     

The radiosensitivity and direction of differentiation of splenic colony forming cells]

Radiosensitivity of spleen CFU localized in the bone marrow and in the spleen proved to be the same. Do is about 105--120r for the CFU-forming colonies in the spleen and 120--135r for the CFU-forming colonies in the bone marrow. As suggested, there are two CFU fractions in the bone marrow. One of them is radiosensitive and another--radio-resistant. Chiefly the radiosensitive CFU fraction is the one localized in the spleen.     

Temporary characteristics of the process of stem cell inactivation by allogenic lymphocytes]

The time during which transplated lymphocytes block proliferation and differentiation of non-syngeic stem cells has been determined by retrasplantation of immuno-competent cells from one lethally irradiated recipient to another one. It was established that process of inactivation of CFU by allogeneic lymphocytes proceeds itwo stages. At the first stage, the colonization of recipient's tissues takes place. The colonization of tissues and processes of early recognition are completed during the first hours after transplantation of cell mixtures. At the second stage, the processes of redistribution of injected cells occur and a complete inactivation of stem cells take place. These events are completed in bone marrow and spleen 4-5 days after transplantation of cells mixture, possibly with the participation of lymphocytes sensibilized with the target-cells.     

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

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

Cardiac pressure overload initiates a systemic stem cell response

Background aims. Acute cardiac injury results in the activation and recruitment of resident and non-cardiac stem cells. In this study we sought to define the pattern of peripheral stem cells and resident cardiac stem cell (CSC) activation induced acutely by cardiac pressure overload (PO). Methods. PO was induced in mice by transaortic constriction (TAC). CSC, endothelial progenitor cells (EPC), hematopoietic stem cells (HSC) and stage-specific embryonic antigen (SSEA)-1(+) cells were profiled in the heart, spleen and bone marrow after TAC by flow cytometry. Results. The combination of a systemic and local stem cell response resulted in increases in SSEA-1 (+) cells and EPC in the heart 7 and 14 days post-TAC, respectively. Locally, modest SSEA-1(+) proliferation at 4 days preceded the elevated myocardial stem cell number. We observed no significant proliferation of EPC and CSC in the heart. The systemic stem cell response was characterized by a biphasic loss of splenic SSEA-1(+) cells at 2 and 7 days post-TAC and loss of bone marrow and spleen EPC at 4 and 7 days, respectively. Spleen size changed dynamically after TAC. A negligible response of HSC to TAC was observed. Significant EPC and SSEA-1(+) proliferation in the bone marrow and spleen occurred only after their local levels were decreased. Conclusions. Our results demonstrate that an orchestrated systemic stem cell response (EPC and SSEA-1 (+) ) takes place in response to TAC. The increase of SSEA-1(+) cells and EPC in the heart in response to pressure is likely to be because of a combination of local proliferation and stem cell recruitment.     

Age-related changes in hematopoietic stem cell populations of a long-lived hybrid mouse

Age-related changes in the number and concentration of pluripotential and unipotential hematopoietic stem cells in the femoral bone marrow and spleen of BC3F₁ mice were investigated. Pluripotential stem cells were assayed by the spleen colony technique, and unipotential stem cells were determined by an agar cloning method and by erythropoietin responsiveness in polycythemic mice. Changes with senescence were observed in the concentration of both uni- and pluripotential stem cells in the bone marrow; the size of the stem cell compartment in the marrow did not change significantly with age. Also, a reduction in the seeding of transplanted spleen colony-forming units into the spleens of aged recipients was demonstrated. The implications of these findings for the kinetics of hematopoietic stem cell proliferation in aged animals are discussed.