The Spleen Colony Technique. Iii. Comparison of the Overlap Effect and of Errors In Cfu-S Determination and the [3H]-Thymidine Suicide Data For Several Strains of Mice

A mathematical model of errors of the spleen colony technique is applied to data obtained from four mouse strains and F₁ hybrids. the variance of the colony counts was close to the Poisson distribution in inbred mice and F¹ hybrids. However, it should be checked regularly. the magnitude of the error in CFU-s determination and of the estimations of the S phase fraction was derived, and is presented relative to the mean colony counts for all mouse strains studied. the optimum spleen colony counts are generally higher than those which are commonly used. However, the utilization of the optimum spleen colony counts requires a correction for the effect of colony overlap.     

The Spleen Colony Technique. I. Correction For the Overlap Effect and Sources of Error In Cfu-S Determination

Abstract. A linear model for the errors of the 'spleen colony' assay for haemopoietic stem cells has been derived. the components emerging from the model are interpreted and practical recommendations given for interpreting measurements made with this assay. the model permits correction for the effect of overlapping colonies and gives average errors for single measurements of the number of CFU-s. More reliable and more precise information can be obtained using this model.     

Comparative studies on the characteristics of proliferation and differentiation of spleen colony-forming cells

Using a single spleen colony transplantation technique and sex chromosome typing as a natural cytogenetic marker, most spleen colony-forming cells (CFC) in adult bone marrow or fetal livers of inbred LACA or C57 mice re-established hemopoiesis in lethally irradiated mice when the spleen colonies were sampled at 13 days after transplantation. However, most of the spleen colony-forming cells in the peripheral blood of normal mice possess little potential for proliferation and are less efficient in the re-establishment of hemopoiesis in lethally irradiated mice. The CFC population is heterogeneous in the mice. From the subsequent retransplantation of colonies from colony-forming cells in the peripheral blood, the simple assessment of spleen colony-forming units (CFU-s) content, based on the number of splenic colonies, does not reliably represent the content of hemopoietic stem cells.     

Characteristics of the Cfu-S Population In Mice Carrying the SlJ Allele

Abstract Abstract. A tentative characterization of haemopoietic stem cells with respect to their organ distribution, seeding fraction and colony formation in the spleen, radiosen-sitivity and humoral regulation was attempted in mice heterozygous for the mutant allele Sl^J and in their normal littermates. Sl^J/+ mice were characterized by a deficient CFU-s content of the blood and spleen and had slightly lower femoral CFU-s numbers. This CFU-s distribution could not be explained by differences in seeding efficiency ‘f’ between CFU-s of Sl^J/+ and +/+ origin in lethally irradiated recipients used in the CFU-s assay. the seeding fraction of CFU-s of +/+ origin did not differ in +/+ and Sl^J/+ recipients. However, in irradiated SI^J/+ recipient mice a 30% decrease was observed in the number of the colonies derived from splenic and femoral CFU-s of both +/+ and Sl^J/+ origin. the serum level of SHSF (splenic haemopoiesis stimulating factor) was decreased in Sl^J/+ mice, but significantly increased in Sl/Sl^d mice, as compared to their respective normal +/+ littermates. Endogenous colony formation in Sl^J/+ spleens was deficient in comparison to that observed in +/+ spleens, and distinct sex differences were observed. However, mutant and normal CFU-s from spleen and bone marrow had a similar survival following in-vitro y irradiation. Femurs and spleens of both Sl^J/+ and +/+ origin were implanted into both Sl^J/+ and +/+ hosts. Six weeks later the Sl^J/+ grafts contained less CFU-s than the +/+ grafts. These data show that the splenic stroma of Sl^J/+ mice is not defective in its capacity to lodge injected CFU-s but is deficient in its ability to maintain CFU-s under ‘steady-state’ conditions and stimulate their colony formation in a ‘perturbed state’. Some of the characteristics of Sl^J/+ mice segregate them from Sl/Sl^d mice, i.e. a deficient splenic CFU-s content, normal seeding fractions ‘f’ of CFU-s from spleen and bone marrow in the presence of an almost compensated anemia, and decreased serum levels of SHSF. the study of the Sl^J trait may be a useful extension of the current Sl/Sl^d model for exploration of hereditary defects in haematopoietic stroma.     

In vivo studies on the regeneration kinetics of enriched populations of haemopoietic spleen colony-forming cells from normal bone marrow

Functional properties of mouse haemopoietic spleen colony-forming cells, enriched 40- to 80-fold, from normal bone marrow were studied. It was found that: (1) the number of partially purified CFU-s (colony forming unit-spleen) required to rescue lethally irradiated mice was similar to the number of normal unfractionated bone marrow CFU-s giving the same level of protection; (2) the homing of partially purified CFU-s was similar to that of CFU-s from unfractionated bone marrow; (3) the regeneration of CFU-s in spleen was similar for enriched and unfractionated cell populations between 4 and 11 days after transplantation. In contrast, the rate of regeneration of CFU-s in femur was slower with enriched progenitor cells than with unfractionated bone marrow. The growth rate in femur, however, could be restored to normal by injecting freshly isolated syngeneic thymocytes with the enriched CFU-s population. The results indicate that the partially purified CFU-s are by themselves functionally normal and show that the rate of CFU-s repopulation in bone marrow can be affected by cell types other than spleen colony-forming cells.     

Cells Forming Spleen Colonies At 7 Or 11 Days After Injection Have Different Proliferation Rates

Abstract In the early periods (7–9 days) after haemopoietic cell injection, colonies produced by CFU-s and by their progeny are identified in the spleen, while at later periods (11 days after injection) only spleen nodules produced by CFU-s persist. the increase in the suicide values of CFU-s after sublethal (2 Gy) irradiation of mice is associated with a higher proliferation rate of precursors of transitory spleen colonies, but not of CFU-s, as measured by different suicide techniques. During the log-phase of cell growth in a lethally irradiated recipient, the injected CFU-s and CFU-tr proliferate at a higher rate. Active proliferation of CFU-s and CFU-tr has been demonstrated in long-term bone-marrow cultures by the hydroxyurea in-vitro suicide assay. CFU-tr may be the cause of artifactual effects during measurement of haemopoietic stem-cell cycling by CFU-s suicide methods.     

Expression of tumor-associated surface membrane antigens on marrow CFU-s, CFC-gm, BFU-e, and brain CFU-s

Antiserum raised against a mouse mast cell line (FMP1) reacts with 90% to 100% of spleen colony-forming units (CFU-s), granulocyte/macrophage colony-forming cells (CFC-gm), erythroid burst-forming units (BFU-e), and 15% of nucleated marrow cells, using a complement-dependent cytotoxicity assay. We demonstrated that bone marrow, spleen, or thymus cells are able to absorb this activity from the antiserum. Although mouse brain cells have low reactivity with anti-FMP1 serum, the cytolysis level was reduced to background when antiserum was absorbed with brain cells. In addition, colony formation by marrow CFU-s, CFC-gm, and BFU-e was no longer prevented when the cells were incubated with brain-absorbed anti-FMP1 serum and complement. These findings suggest the presence of brain-associated antigens on CFU-s, CFC-gm, and BFU-e. To test whether a CFU-s accessory cell population in marrow is affected by treatment with anti-FMP1 serum and complement, antibody-treated marrow cells were mixed with large numbers of thymocytes and injected into recipient mice. Colony formation was not altered, indicating that the antiserum reacted directly with antigens on CFU-s and not on CFU-s accessory cells.     

Embryonic characteristics in superovulated mouse strains. Comparative analyses of the incidence of chromosomal aberrations, morphological malformations, and mortality of embryos from two strains of superovulated mice

Superovulation was induced in an inbred (C3HeB/FeJ) and a random-bred (CD-1) strain of mice and pregnant females were serially sacrificed from 6-15 days of gestation. All embryos were examined chromosomally and by external morphology. A G-banding technique was used to detect specific chromosomal constitutions. The common types of chromosomal aberrations were monosomies and trisomies. Our results showed that the proportion of chromosomally abnormal embryos was significantly greater in the superovulated group of inbred mice, than in the spontaneously ovulating inbred group, an observation not made in the random-bred mice. In each strain, superovulation yielded a significantly higher proportion of morphologically malformed embryos, but no significant difference between strains with respect to this criterion was noted after superovulation. There was a significant increase in the post-implantation embryonic mortality in the random-bred strain but not in the inbred strain following superovulation. This study indicates that each strain has its intrinsic pattern with regard to the fate of embryos following superovulation.     

Regulation of haemopoietic stem-cell proliferation in mice carrying the Slj allele

We investigated a haemopoietic stromal defect, in mice heterozygous for the Slj allele, during haemopoietic stress induced by treatment with bacterial lipopolysaccharides (LPS) or lethal total body irradiation (TBI) and bone-marrow cell (BMC) reconstitution. Both treatments resulted in a comparable haemopoietic stem cell (CFU-s) proliferation in Slj/+ and +/+ haemopoietic organs. There was no difference in committed haemopoietic progenitor cell (BFU-e and CFU-G/M) kinetics after TBI and +/+ bone-marrow transplantation in Slj/+ and +/+ mice. The Slj/+ mice were deficient in their ability to support macroscopic spleen colony formation (65% of +/+ controls) as measured at 7 and 10 days after BMC transplantation. However, the Slj/+ spleen colonies contained the same number of BFU-E and CFU-G/M as colonies from +/+ spleens, while their CFU-s content was increased. On day 10 post-transplantation, the macroscopic 'missing' colonies could be detected at the microscopic level. These small colonies contained far fewer CFU-s than the macroscopic detectable colonies. Analysis of CFU-s proliferation-inducing activities in control and post-LPS sera revealed that Slj/+ mice are normal in their ability to produce and to respond to humoral stem-cell regulators. We postulate that Slj/+ mice have a normal number of splenic stromal 'niches' for colony formation. However, 35% of these niches is defective in its proliferative support.     

Infection of Haematopoietic Stem Cells In Mice With Friend Virus Induced Erythroleukaemia

Abstract. Animals infected with conventional anaemia (FVA) or polycythemia-inducing (FVP) strains of the Friend virus develop lethal erythroleukaemia. A variant strain, RFV, induces an initially identical disease except that it spontaneously regresses in 50% of infected mice. to determine whether pluripotent stem cells as measured by spleen colony forming units (CFU-s) in leukaemic mice are productively infected with virus and whether their infection influences the outcome of the disease, we tested CFU-s from leukaemic mice for susceptibility to cytotoxicity by monospecific antiviral gp70 antiserum. Spleen CFU-s from progressively leukaemic (FVP, FVA and RFV) mice were productively infected with virus. CFU-s in RFV progressors became infected by 40 days post-virus inoculation. FVA and FVP progressors became infected between 15 and 21 days post virus. Infection of CFU-s was accompanied by an increase in the proportion of replicating (S phase) CFU-s in these populations. Spleen CFU-s from fully regressed RFV regressor mice were uninfected and remained so throughout the course of their disease. Bone marrow CFU-s in both regressors and progressors remained uninfected and were not induced to increased cell cycling.     

Infection of haematopoietic stem cells in mice with Friend virus induced erythroleukaemia

Animals infected with conventional anaemia (FVA) or polycythemia-inducing (FVP) strains of the Friend virus develop lethal erythroleukaemia. A variant strain, RFV, induces an initially identical disease except that it spontaneously regresses in 50% of infected mice. To determine whether pluripotent stem cells as measured by spleen colony forming units (CFU-s) in leukaemic mice are productively infected with virus and whether their infection influences the outcome of the disease, we tested CFU-s from leukaemic mice for susceptibility to cytotoxicity by monospecific antiviral gp70 antiserum. Spleen CFU-s from progressively leukaemic (FVP, FVA and RFV) mice were productively infected with virus. CFU-s in RFV progressors became infected by 40 days post-virus inoculation. FVA and FVP progressors became infected between 15 and 21 days post virus. Infection of CFU-s was accompanied by an increase in the proportion of replicating (S phase) CFU-s in these populations. Spleen CFU-s from fully regressed RFV regressor mice were uninfected and remained so throughout the course of their disease. Bone marrow CFU-s in both regressors and progressors remained uninfected and were not induced to increased cell cycling.     

Stem and stromal cell reconstitution of lethally irradiated mice following transplantation of hematopoietic tissue from donors of various ages

If the limited life span of hematopoietic tissues in vitro is due to a finite proliferative capacity of individual stem cells, one might expect tissues of young donors to possess a greater proliferative capacity and to contain a larger population of primitive stem cells than those of older donors. To test this hypothesis, we used 12- and 8-day spleen colony formation (CFU-s) to assay more and less primitive stem cell subpopulations of three murine hematopoietic tissues: fetal liver (FL) and weanling (WBM) and adult (ABM) bone marrow. Subsequently, the same assays and a stromal cell assay were performed on the bone marrow from groups of lethally irradiated mice reconstituted with these tissues. Comparison of the CFU-s content of the donor tissues revealed that FL contained a significantly greater proportion of primitive stem cells as evidenced by a (Day 12):(Day 8) CFU-s ratio of 3.0 +/- 1.0 as compared to 0.9 +/- 0.1 for WBM and ABM. In addition, at 21 weeks post-transplantation the CFU-s/femur values of the FL reconstituted group were significantly greater than those of the ABM and WBM reconstituted groups. These results suggest that fetal hematopoietic tissue contains a greater proportion of primitive stem cells and has a greater proliferative potential than hematopoietic tissue from older donors. No differences were seen in stromal cell reconstitution of the three experimental groups. In all cases, assayable fibroblast colony forming cells (CFU-f) remained at 20-40% of control values, even at 21 weeks postreconstitution.     

Regulation of haemopoietic stem-cell proliferation in mice carrying the Slj allele

We investigated a haemopoietic stromal defect, in mice heterozygous for the Sl^j allele, during haemopoietic stress induced by treatment with bacterial lipopolysaccharides (LPS) or lethal total body irradiation (TBI) and bone-marrow cell (BMC) reconstitution. Both treatments resulted in a comparable haemopoietic stem cell (CFU-s) proliferation in Sl^j/+ and +/+ haemopoietic organs. There was no difference in committed haemopoietic progenitor cell (BFU-e and CFU-G/M) kinetics after TBI and +/+ bone-marrow transplantation in Sl^j/+ and +/+ mice. the Sl^j/+ mice were deficient in their ability to support macroscopic spleen colony formation (65% of +/+ controls) as measured at 7 and 10 days after BMC transplantation. However, the Sl^j/+ spleen colonies contained the same number of BFU-E and CFU-G/M as colonies from +/+ spleens, while their CFU-s content was increased. On day 10 post-transplantation, the macroscopic ‘missing’ colonies could be detected at the microscopic level. These small colonies contained far fewer CFU-s than the macroscopic detectable colonies. Analysis of CFU-s proliferation-inducing activities in control and post-LPS sera revealed that Sl^j/+ mice are normal in their ability to produce and to respond to humoral stem-cell regulators. We postulate that Sl^j/+ mice have a normal number of splenic stromal ‘niches’ for colony formation. However, 35% of these niches is defective in its proliferative support.     

A comparison of low and high dose-rate radiation for recipient mice in spleen-colony studies

Over the last 15 years, endogenous spleen-colony formation in our mice, following lethal irradiation, has increased to an unacceptable level. It has been found necessary, therefore, to introduce a new method of preparing recipient mice for spleen-colony studies. Irradiation with low dose-rate 60Cobalt gamma rays has been compared with high dose-rate linear accelerator electrons, and their effects on endogenous spleen colony formation compared with earlier X and gamma ray dose-response data. It was found that a large dose (13.5 Gy) of gamma rays results in fewer endogenous colonies than 8.5 Gy of electrons, yet because of its low dose rate (14.1 X 10(-3) Gy/min) it has a marked sparing of the intestinal tissue as measured by the intestinal microcolony technique. This in turn permits better survival and, therefore, a 'healthier' animal for spleen-colony work. Exogenous colony formation is also lower in the low dose-rate, gamma-irradiated recipients and this is shown to be due to a reduced spleen-seeding efficiency. It is concluded that very low dose-rate radiation is preferable for haemopoietic ablation, that a mouse colony requires constant monitoring for changes of endogenous spleen-colony formation and that the spleen-seeding efficiency of CFU-s depends on the irradiation technique used--there is no absolute value for a given strain of mouse.     

A Comparison of Low and High Dose-Rate Radiation For Recipient Mice In Spleen-Colony Studies

Over the last 15 years, endogenous spleen-colony formation in our mice, following lethal irradiation, has increased to an unacceptable level. It has been found necessary, therefore, to introduce a new method of preparing recipient mice for spleen-colony studies. Irradiation with low dose-rate ⁶⁰Cobalt γ rays has been compared with high dose-rate linear accelerator electrons, and their effects on endogenous spleen colony formation compared with earlier X and γ ray dose-response data. It was found that a large dose (13.5 Gy) of γ rays results in fewer endogenous colonies than 8.5 Gy of electrons, yet because of its low dose rate (14.1 × 10^?3 Gy/min) it has a marked sparing of the intestinal tissue as measured by the intestinal microcolony technique. This in turn permits better survival and, therefore, a ‘healthier’ animal for spleen-colony work. Exogenous colony formation is also lower in the low dose-rate, γ-irradiated recipients and this is shown to be due to a reduced spleen-seeding efficiency. It is concluded that very low dose-rate radiation is preferable for haemopoietic ablation, that a mouse colony requires constant monitoring for changes of endogenous spleen-colony formation and that the spleen-seeding efficiency of CFU-s depends on the irradiation technique used-there is no absolute value for a given strain of mouse.     

The effect of carbamylcholine on CFU-s differentiation

The proportion of spleen colony-forming units (CFU-s) killed by hydroxyurea was greatly increased after bone marrow cells (BMCs) from LACA mice were exposed to carbamylcholine (Cach; 1 X 10(-13) to 1 X 10(-9) in vitro and there was a marked change in the proportion of spleen colony types. Following treatment with Cach, granulocytic and mixed erythroid-type colonies increased from 20 to 26.3% and 16.1 to 29.6% in 9-day colonies and from 8.3 to 28.2% and 21.7 to 39.4% in 13-day colonies, respectively. Single cell suspensions of spleen colonies were made for granulocyte-macrophage progenitor (CFU-gm) and late erythroid progenitor (CFU-e) assays. The number of CFU-gm from Cach-treated BMC was about twice that from control BMC for both day 9 and day 13 groups; the number of CFU-e decreased relatively. The results suggest that cholinergic receptors on CFU-s may increase the tendency to differentiate into the granulocytic/monocytic line.     

Effects of human leukocyte conditioned medium on mouse haemopoietic progenitor cells.

Medium conditioned by human peripheral blood leukocytes (HLCM) was studied for its in vitro effects on haemopoietic progenitor cells (CFU-s and CFU-c) present in mouse bone marrow. HLCM has poor colony stimulating activity in semi-solid cultures of mouse bone marrow cells, but invariably increases the number of colonies obtained in the presence of plateau levels of semi-purified colony stimulating factor (CSF). In liquid cultures, HLCM appears to contain a potent initiator of DNA synthesis in CFU-s, an activity which coincides with an increased CFU-s maintenance and causes a three- to four-fold increase in CFU-c number. It is apparent from this study that HLCM, in addition to stimulating colony formation in cultures of human bone marrow cells, has a profound in vitro effect on primitive haemopoietic progenitor cells of the mouse, which cannot be attributed to CSF.     

The transplantability of bone marrow and spleen cells after filtration through silon tissue]

Investigations were carried out on the separation of haematopoietic stem cells from suspensions of the bone-marrow and spleen by means of filtration with silon tissue. The presence of stem cells in the filtrates was determined by the spleen colony test according to the method of Till and McCulloch in irradiated mice. The investigations revealed that a selective separation of haematopoietic stem cells could not be achieved when proceeding in this way. From the results of further test series, in which suspensions were also used which had been gained from haematopoietic tissues of hypersplenic mice, the conclusion could be drawn that the haematopoietic stem cells obtained by filtrating the bone-marrow will have another affinity to the spleen tissue of irradiated mice than the haematopoietic stem cells gained by filtrating the spleen tissue.     

Effect of haemopoietic stem-cell proliferation regulators on early and late spleen colony-forming cells

An inhibitor and stimulator of CFU-s proliferation can be obtained from haemopoietic tissue containing, respectively, relatively quiescent CFU-s (e.g. normal bone marrow) and proliferating CFU-s (e.g. regenerating bone marrow). Their effects on the proliferative behaviour of steady-state and regenerating marrow CFU-s, which produce colonies 7, 10 and 12 days post-transplantation have been investigated. The results demonstrate changing sensitivities of CFU-s to inhibitor and stimulator as they progress through a developmental age structure, 'Older' CFU-s (producing early spleen colonies) are more sensitive to stimulator, 'Younger' CFU-s (producing late spleen colonies) are more sensitive to inhibitor.     

Effect of Haemopoietic Stem-Cell Proliferation Regulators On Early and Late Spleen Colony-Forming Cells

An inhibitor and stimulator of CFU-s proliferation can be obtained from haemopoietic tissue containing, respectively, relatively quiescent CFU-s (e.g. normal bone marrow) and proliferating CFU-s (e.g. regenerating bone marrow). Their effects on the proliferative behaviour of steady-state and regenerating marrow CFU-s, which produce colonies 7, 10 and 12 days post-transplantation have been investigated. The results demonstrate changing sensitivities of CFU-s to inhibitor and stimulator as they progress through a developmental age structure. ‘Older’ CFU-s (producing early spleen colonies) are more sensitive to stimulator, ‘Younger’ CFU-s (producing late spleen colonies) are more sensitive to inhibitor.