THYMIDINE SUICIDE IN VIVO AND IN VITRO OF SPLEEN COLONY FORMING AND AGAR COLONY FORMING CELLS OF MOUSE BONE MARROW

The ‘thymidine suicide’technique for indicating differences in the proliferation rate of early haemopoietic progenitor cells (spleen colony forming and agar colony forming cells) in C57BL mice has been evaluated. Special care was taken to use the same bone marrow cell suspension for the two progenitor cell assays. Both the in vivo and the in vitro techniques were employed. Following ³H-TdR in vivo, about 20% of both types of progenitor cell are killed in normal mice; however, after incubation in vitro with ³H-TdR, 35% of agar colony forming cells but only 4% of spleen colony forming cells are killed. Reasons for the difference between the in vivo and the in vitro results are discussed. With bone marrow from continuously irradiated animals, the thymidine suicide for both agar colony forming and spleen colony forming cells is in the range 42–50%, and there is no difference between in vivo and in vitro suicide. The in vivo results support the conclusion, based on the effect of proliferation dependent cytotoxic agents, that in C57BL mice agar colony forming and spleen colony forming cells are proliferating at the same rate in normal animals, and are speeded up to the same extent by continuous γ-irradiation. It is considered that in normal C57BL mice the in vitro method does not give a correct estimate of the proliferation rate of these progenitor cells. It would seem that the similarity in the proliferation rate of agar colony forming and spleen colony forming cells in C57BL mice is not true for other strains of mice: indeed using normal CBA and in vivo suicide, we have shown a significantly greater thymidine suicide for agar colony forming cells compared to spleen colony forming 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 reconstitutive role during the refeeding process.     

Factors influencing hematopoietic spleen colony formation in irradiated mice. V. Effect of foreign plasma upon colony forming cell kinetics

Foreign plasma injection induces a profound and somewhat complex change in the size and location of the colony forming unit (CFU) cell compartment. Injection of foreign plasma before irradiation induces an increase in CFU cells as judged by endogenous colonies as well as by a modification of the endogenous method which excludes spleen colony formation from in situ spleen cells. However, the enlargement does not take place in the most populous CFU cell areas, the spleen and marrow. The concentration and/or total number of CFU cells in spleen and marrow was not increased by plasma injection whether judged by the number of transplantable cells or by the number of migrating endogenous cells. These studies emphasize the complexity of this cellular system and suggest that the use of but one type of stem cell assay may yield results which do not reflect changes within the total compartment. Evidence for cell damage in vitro as a factor influencing results in studies involving transplantation was searched for but was not forthcoming.     

A comparison of the effect of cytotoxic agents on agar colony forming cells, spleen colony forming cells, and the erythrocytic repopulating ability of mouse bone marrow

Three assays for bone marrow progenitor cells have been used to determine the effect of single doses of two cytotoxic agents, cyclophosphamide and vinblastine. The assays employed were the agar colony forming and spleen colony forming assays and the crythroid repopulating ability. In normal mice, there was little difference between the response of the progenitor cells assayed by the three methods, following cyclophosphamide: and no detectable difference following vinblastine. Bone marrow from continuously irradiated mice and bone marrow regenerating seven days following transplantation was also studied: in both these situations the proliferation rate of the progenitor cells is increased. Cyclophosphamide was found to be only slightly proliferation dependent with each assay. However, vinblastine was strikingly proliferation dependent. In irradiated mice and also in regenerating marrow the agar colony forming cells were many times more sensitive to this agent than were the other progenitor cells. These results show that under some but not all circumstances the agar colony forming and spleen colony forming cells behave similarly in C57BL mice, but are not a single population of cells.     

Adherence column and buoyant density separation of bone marrow stem cells and more differentiated cells

Mouse bone marrow cells were separated by adherence column and albumin density gradient procedures, assaying for spleen colony forming units (in vivo CFU's), agar colony forming cells (in vitro CFC's) and cluster forming cells. Column filtrates were enriched for CFU's whereas in vitro CFC's and cluster-forming cells were enriched in adherent fractions. Gradient separation of these column fractions gave density distribution profiles indicating the non-identity and heterogeneity of CFU's and in vitro CFC's.     

ERYTHROID AND GRANULOCYTE-MACROPHAGE PROGENITOR CELLS IN PRE-NATAL 'STEEL' (SlJ/SlJ) ANAEMIC MICE

The erythropietin sensitivities of dissociated cell cultures and explanted fragments of fetal livers of congenitally anaemic Sl^J/Sl^J mice, and their normal littermates, have been compared. The erythropoietin responsiveness of Sl^J/Sl^J foetal liver cells is deficient in both types of culture. The maximum liver complement of erythroid colony forming cells (CFUe) occurs on the 16th day of development when ‘normal’ livers contain approximately 6 × 10⁵ erythroid colony forming cells/liver. In Sl^J/Sl^J fetuses the maximum reached is only 1 × 10⁵. Granulocyte-macrophage colony forming cells (CFU_C) in Sl^J/Sl^J fetal livers are also reduced to approximately 60% of normal numbers. Erythroid colony forming cells are also reduced in the spleen and femoral bone marrow of Sl^J/Sl^J mice in the 2–3 days preceding birth. Granulocyte-macrophage colony forming cells are rare in the femoral marrow of pre-natal Sl^J/Sl^J mice, but their production in the Sl^J/Sl^J pre-natal spleen appears unaffected.     

THE RELATIONSHIP BETWEEN STEM CELL SEEDING EFFICIENCY AND POSITION IN CELL CYCLE

The seeding efficiency of colony-forming cells from normal, regenerating and velocity-sedimented cycling and non-cycling narrow preparations was compared. Colony-forming cells in cycle were found to exhibit a 50% reduction in splenic seeding when compared to normal marrow or sedimented non-cycling cells. The results of this study indicate that the spleen colony assay underestimates the total number of colony-forming cells by a fraction which is directly related to the number of cells in cycle.     

CFU-C YIELDS FROM THE HAEMOPOIETIC TISSUES OF NORMAL AND LEUKAEMIC RFM MICE

Spleen and bone marrow cells from normal and leukaemic RFM mice have been assayed for numbers of colony forming cells in soft agar (CFU-C). The fluctuations in CFU-C yield observed during the development of myeloid leukaemia are similar to the results from in vitro experiments set up to test a model, and are not incompatible with the idea that interaction between normal and leukaemic cells may modify the yield of CFU-C under the present conditions of culture. Colonies grown from leukaemic spleen and bone marrow cells appear to be derived from the residual population of normal haemopoietic cells within the leukaemic mouse.     

Different recovery patterns of mouse haemopoietic stem cells in response to cytotoxic agents

The response and subsequent recovery of mouse haemopoietic progenitor cells (spleen colony forming cells and agar colony forming cells) has been studied following two cytotoxic agents. Busulphan was administered to normal mice and vinblastine to mice where the progenitor cell proliferation rate had been increased by a period of continuous γ-irradiation. With both these agents there is a difference between the response of the spleen colony forming cells and the agar colony forming cells during the first five days. They then recover together, but much more slowly after busulphan than after vinblastine even though their proliferation rate is increased. The rate of progenitor cell recovery after busulphan is increased if the progenitor cells are depleted further by vinblastine. However, methotrexate, which severely depletes the peripheral blood count and bone marrow cellularity but not the progenitor cells, has no effect on the recovery following busulphan. These results suggest that following cytotoxic agents the agar colony forming cells (“committed” stem cells) are not self-maintaining but are dependent on a supply of cells from the pluripotential spleen colony forming cells. In addition it appears that the depletion of the progenitor cells of the bone marrow and not the depletion of the maturing cells, provides a stimulus for stem cell recovery.     

Expression of congenital defects in the haemopoietic micro-environment. Erythroid and granulocyte-macrophage progenitor cells in pre-natal 'steel' (Slj/Slj) anaemic mice.

The erythropietin sensitivities of dissociated cell cultures and explanted fragments of fetal livers of congenitally anaemic Slj/Slj mice, and their normal littermates, have been compared. The erythropoietin responsiveness of Slj/Slj foetal liver cells is deficient in both types of culture. The maximum liver complement of erythroid colony forming cells (CFUe) occurs on the 16th day of development when 'normal' livers contain approximately 6 X 10(5) erythroid colony forming cells/liver. In Slj/Slj fetuses the maximum reached is only 1 X 10(5). Granulocyte-macrophage colony forming cells (CFUc) in Slj/Slj fetal livers are also reduced to approximately 60% of normal numbers. Erythroid colony forming cells are also reduced in the spleen and femoral bone marrow of Slj/Slj mice in the 2-3 days preceding birth. Granulocyte-macrophage colony forming cells are rare in the femoral marrow of pre-natal Slj/Slj mice, but their production in the Slj/Slj pre-natal spleen appears unaffected.     

EFFECTS OF BACTERIAL ENDOTOXIN ON HEMOPOIETIC COLONY-FORMING CELLS IN THE SPLEENS OF NORMAL MICE AND MICE OF GENOTYPE S1/S1

Multiple doses of S. typhosa endotoxin caused an increase in the number of hemopoietic stem cells present in mouse marrow and spleen that could be detected using the spleen-colony assay. This increase was inhibited by Colcemid, and by the genetically-determined defect in hemopoiesis in mice of genotype S1/S1^d. However, the defect in S1/S1^d hosts did not prevent an endotoxin-induced increase in the number of cells capable of forming colonies in cell culture. The results support the view that bacterial endotoxin acts, via a genetically-controlled regulatory mechanism, to stimulate the proliferation of hemopoietic stem cells in the spleen.     

THE IN VITRO DIFFERENTIATION OF DENSITY SUB-POPULATIONS OF COLONY-FORMING CELLS UNDER THE INFLUENCE OF DIFFERENT TYPES OF COLONY-STIMULATING FACTOR

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density sub-populations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSF_MLCM), post-endotoxin mouse serum (CSF_ES) and from human urine (CSF_Hu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macro-phage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

Physical separation of colony stimulating cells from in vitro colony forming cells in hemopoietic tissue

Hemopoietic colony formation in agar occurred spontaneously in mass cultures of marrow cells obtained from a number of species (guinea pig, rat, lamb, rabbit, pig, calf, human and Rhesus monkey). This contrasted with the observation that colony formation by mouse bone marrow exhibited an absolute requirement for an exogenous source of a colony stimulating factor. Analysis of spontaneous colony formation in Rhesus monkey marrow cultures revealed the presence of a cell type in hemopoietic tissue, capable of elaborating colony stimulating factor when used to condition media or as feeder layers. Equilibrium density gradient centrifugation separated colony stimulating cells from in vitro colony forming cells in monkey bone marrow. Separation studies on spleen, blood and marrow characterized the stimulating cells as of intermediate density, depleted or absent in fractions enriched for cells of the granulocytic series and localized in regions containing lymphocytes and monocytes. Adherence column separation of peripheral blood leukocytes showed the stimulating cells to be actively adherent, unlike the majority of lymphocytes, and combined adherence column and density separation indicated that stimulating cells were present in hemopoietic tissue within the population of adherent lymphocytes or monocytes.     

THE EFFECT OF CYTOSINE ARABINOSIDE IN VITRO ON AGAR COLONY FORMING CELLS AND SPLEEN COLONY FORMING CELLS OF C57BL MOUSE BONE MARROW

This study reports the effect of cytosine arabinoside in culture on two classes of bone marrow progenitor cells in C57BL mice, agar colony forming cells (ACU) and spleen colony forming cells (CFU). Both normal cells and rapidly proliferating cells were studied. The results show that in normal mice, 23 % of ACU but only 7 % of CFU are killed following 1 hr incubation with the drug. With longer periods of incubation, the survival of ACU in the controls is poor, and the results for the drug-treated cultures suggest that the cells are held up in cycle. In continuously irradiated mice, the proportion of ACU and CFU killed after 1 hr incubation with drug is increased to 43–54%, confirming previous results that these cells are proliferating more rapidly than in normal mice. In mice treated with myerlan, 54 % of ACU are killed by 1 hr in vitro exposure to cytosine arabinoside, again confirming that ACU are rapidly proliferating. However, the proportion of CFU killed is lower (23 %). These results are compared with other studies of the effect of cytosine arabinoside in vivo and also with thymidine suicide in the same strain of mice. The results show that cytosine arabinoside has the same effect as tritiated thymidine, and also that the proportion of CFU killed by these agents in vitro is lower than when the agents are injected in vivo. It is suggested that the conditions in culture have an adverse effect on CFU, which cease DNA synthesis, and are protected from the killing effect of cytosine arabinoside and tritiated thymidine. Since cytosine arabinoside in vitro has an effect similar to tritiated thymidine in vitro on bone marrow progenitor cells in C57BL mice, in vitro incubation with cytosine arabinoside could be an alternative method to thymidine suicide for measuring differences in cell proliferation rate.     

Factors influencing hematopoietic spleen colony formation in irradiated mice. IV. The effect of erythropoietic stimuli

A variety of erythropoietic stimuli influenced the number of endogenous spleen colonies in irradiated mice and the number of transplantable colony forming cells in the spleen and marrow of unirradiated mice. Bleeding was the most effective stimulus. Bleeding before irradiation resulted in a 30-fold increase in endogenous spleen colonies and in increases in spleen weight, spleen iron and iododeoxyuridine uptake and volume of packed red cells ten days after irradiation. Bleeding unirradiated mice produced a 10-fold increase in the number of transplantable colony forming cells in the spleen and a slight decrease in the total number in the humerus. Bleeding before irradiation resulted in a significant reduction in 30-day post irradiation deaths, an effect abolished by splenectomy. Plasma from bled mice induced an increase in endogenous colonies when injected before irradiation into normal mice. Injection of erythropoietin, testosterone or testosterone plus cobalt induced effects which were, in general, qualitatively similar to those of bleeding, although they were less effective quantitatively. Except for a slight effect induced by ten injections of erythropoietin, post-irradiation stimulation in normal mice proved ineffective. Erythropoietin increased colony numbers and spleen iron uptake when given after irradiation to hypertransfused mice. The results of these studies do not support the concept that the colony forming cell and the erythropoietin sensitive cell are separate entities.     

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.     

The relationship between stem cell seeding efficiency and position in cell cycle.

The seeding efficiency of colony-forming cells from normal, regenerating and velocity-sedimented cycling and non-cycling narrow preparations was compared. Colony-forming cells in cycle were found to exhibit a 50% reduction in splenic seeding when compared to normal marrow or sedimented non-cycling cells. The results of this study indicate that the spleen colony assay underestimates the total number of colony-forming cells by a fraction which is directly related to the number of cells in cycle.     

Anchorage-independent growth of RSV-transformed rat (GCA, W 12 and XC) cells

Three RSV-transformed rat cell lines: GCA, W12 and XC were characterized as to their ability to anchorage-independent growth in comparison to normal rat kidney (NRK-49F) cells. Differences in the threshold density (TD) and colony forming efficiency (CFE) of the investigated cells are described. The ability of virally transformed cells to stimulation of soft agar colony formation of NRK cells in coculture assay was presented. The production of TGFs-like factors by GCA, W12 and XC cells was suggested.     

Radioprotection of hematopoietic tissues in mice by indomethacin

We recently reported that indomethacin, an inhibitor of prostaglandin (PG) synthesis, increased the radioresponse of PG-producing murine tumors, but it protected the hematopoietic system from radiation damage [Furuta et al., Cancer Res. 48, 3008-3013 (1988)]. Here we have investigated possible mechanisms responsible for the radioprotective effect of indomethacin. In the exogenous spleen colony assay, bone marrow cells from indomethacin-treated mice showed a similar radioresponse to those from mice not treated with indomethacin, thus excluding true radioprotection as a mechanism. Also, neither the total number of bone marrow cells nor the number of stem cells in bone marrow were affected by the treatment with indomethacin. However, indomethacin induced significant splenomegaly, which was associated with an increased number of both nucleated cells and hematopoietic stem cells in the spleen. The latter was determined by the exogenous spleen colony assay. Thus indomethacin protected hematopoietic tissue indirectly through stimulation of hematopoietic cells in the spleen. When indomethacin was combined with WR-2721, which is a true radioprotector, we obtained a greater radioprotective effect than with either used alone according to the endogenous spleen colony assay.     

Haemopoietic stem cells during development of mouse embryo.

Haemopoiesis in mammals takes place in yolk-sac and in mouse it can be detected on the 7th day of gestation. Erythropoietin (EPO) responsive cells can be detected from 7th day onwards. However, the cells committed to the myeloid lineage which can respond to the haemopoietic growth factor (viz. granulocyte macrophage colony stimulating factor; GM-CSF) can be demonstrated only on 10th day of gestation. At the same time, the 12-day spleen colony forming cells i.e. the late colony forming unit spleen (CFU-s) which are multipotent stem cells can also be detected. Data suggest that the stem cells seen in the embryo from 7-10 days of gestation may be a primitive population confined only to the yolk-sac. Liver haemopoiesis which begins in the liver of 13-day embryos is due to primitive haemopoietic pluripotent stem cells, arising de novo in the embryo and not in the yolk-sac, since no primitive pluripotent stem cells capable of repopulating lethally irradiated bone-marrow can be detected in the yolk-sac.