Effect of adult thymectomy and splenectomy on pluripotent stem cells and progenitors of the myeloid and B-lymphoid lineages

In order to investigate the regulatory interactions which occur between the bone marrow, the thymus and the spleen during hemopoiesis, the numbers of granulo-macrophage and B-lymphocyte precursors (GM-CFC and BL-CFC) and the kinetics of pluripotent stem cells (CFU-S) have been studied in the bone marrow and the spleen of normal adult thymectomized and/or adult splenectomized mice with or without T-dependent antigen stimulation. The results suggest that (1) medullary and splenic CFU-S may be two different populations of the stem cell compartment; (2) the thymus is involved in medullary but not in splenic CFU-S proliferation in response to T-antigen challenge; (3) the spleen influences GM-CFC and BL-CFC numbers and (4) antigenic stimulation modifies medullary BL-CFC.     

Effect of a hyperthermic treatment on the pluripotent haemopoietic stem cell in normal and anaemic mice

Up to now, the hyperthermic sensitivity of pluripotent haemopoietic stem cells is unknown, and the few existing data from reports in the literature are conflicting. There are two main drawbacks in the set-up of those studies: (1) only CFU-S day 9 results were presented, whereas it is questionable if this assay gives a true reflection of the pluripotent stem cell, and (2) no attention has been paid to heat effects on the seeding efficiency, i.e. the amount of stem cells which will lodge in the spleen. The present study focused on the procedural differences and compared the results of a hyperthermic treatment (60 min, 42°C) on the stem cells, assayed with the CFU-S day 9 and the CFU-S day 12 method, using the following three stem cell suspensions, all differing in their proliferative activity: bone marrow from normal mice and bone marrow and spleen cells from anaemic mice. Furthermore, we investigated the seeding efficiency before and after heat treatment. Resting stem cells, assayed with the CFU-S day 12 method, turned out to be resistant to hyperthermia as compared with the active cycling stem cells, while with the CFU-S day 9 assay the stem showed the same thermosensitivity in the two bone marrow suspensions. The active cycling stem cells do not significantly differ in thermosensitivity, in CFU-S day 9 and day 12 assays, although there is a difference between bone marrow and spleen. Hyperthermia appears to influence the seeding efficiency for spleen CFU-S; an increase of 1?73 was observed. The difference in heat sensitivity between the resting and the active cycling stem cells, assayed with both in vivo methods, however, cannot be explained by a change in seeding efficiency only. Comparing the amount of cycling cells in the three stem cell suspensions and their thermosensitivity leads to the conclusion that the differences in heat sensitivity might be fully explained by the cycling status of the stem cell.     

Modifications of pluripotent stem cell differentiation after Ara-C treatment: Clonal analyses of CFU-S progeny

The nature of the mechanisms controlling CFU-S differentiation is a crucial problem in haematology and, thus far, little is known concerning these phenomena. Work done in our laboratory has shown that the distribution of the histologic cell types represented in spleen colonies (CFU-S) differ depending on whether normal bone marrow or marrow from Ara-C treated mice is injected into the irradiated recipients. As measured by the mean of the absolute number of colonies per spleen, bone marrow from Ara-C treated mice gives more erythroid colonies and fewer granulocytic colonies than do cells from normal bone marrow. We have demonstrated that these modifications are under the control of humoral factors. Two significant questions arise from these observations. First, are the colonies after Ara-C treatment derived from a single multipotential cell rather than from already committed progenitors and, second, is this shift in granulocytic-erythroid representation a reflection of modifications at the CFU-S level introduced by our Ara-C system? To answer these questions, we analysed the progeny of each individual spleen nodule either by reinjecting each colony unit into a secondary recipient or by cloning these cells in methyl cellulose with appropriate stimulating factors. We thus determined the number of retransplantable stem cells, as well as the number of committed precursors present in each spleen nodule. Our results demonstrate that most spleen colonies are transplantable and give rise to secondary colonies. These secondary colonies are of all haematological types, therefore proving that the nodules contain CFU-S and that these CFU-S are pluripotent. All spleen colonies contain GM-CFC, even in the nodules that were histologically erythroid. We thus conclude that modifications in the E/G ratio of spleen colonies after injection of bone marrow from Ara-C treated mice are a reflection of changes in CFU-S differentiation pathways.     

Effect of a hyperthermic treatment on the pluripotent haemopoietic stem cell in normal and anaemic mice

Up to now, the hyperthermic sensitivity of pluripotent haemopoietic stem cells is unknown, and the few existing data from reports in the literature are conflicting. There are two main drawbacks in the set-up of those studies: (1) only CFU-S day 9 results were presented, whereas it is questionable if this assay gives a true reflection of the pluripotent stem cell, and (2) no attention has been paid to heat effects on the seeding efficiency, i.e. the amount of stem cells which will lodge in the spleen. The present study focused on the procedural differences and compared the results of a hyperthermic treatment (60 min, 42 degrees C) on the stem cells, assayed with the CFU-S day 9 and the CFU-S day 12 method, using the following three stem cell suspensions, all differing in their proliferative activity: bone marrow from normal mice and bone marrow and spleen cells from anaemic mice. Furthermore, we investigated the seeding efficiency before and after heat treatment. Resting stem cells, assayed with the CFU-S day 12 method, turned out to be resistant to hyperthermia as compared with the active cycling stem cells, while with the CFU-S day 9 assay the stem showed the same thermosensitivity in the two bone marrow suspensions. The active cycling stem cells do not significantly differ in thermosensitivity, in CFU-S day 9 and day 12 assays, although there is a difference between bone marrow and spleen. Hyperthermia appears to influence the seeding efficiency for spleen CFU-S; an increase of 1.73 was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cellular specificity of haemopoietic stem cell proliferation regulators

The range of specificity of the CFU-S proliferation inhibitor and stimulator which are produced endogenously in the bone marrow has been investigated by measuring their effects on the proportion of cells killed by tritiated thymidine in mixed colony- (CFC-mix), erythroid burst- (BFU-E) and granulocyte/macrophage colony- (GM-CFC) forming cells as well as spleen colony forming units (CFU-S). Both CFU-S and CFC-mix were triggered by the stimulator into DNA-synthesis but BFU-E and GM-CFC were unaffected. The range of activity of the inhibitor was confined solely to the CFU-S population. This defined the specificity of both inhibitor and stimulator for the multipotent cells. The differential sensitivity of CFU-S and CFC-mix to the inhibitor and the lack of it for the stimulator suggested (a) that the CFC-mix is a relatively mature subpopulation of the CFU-S compartment and (b) that the relative sensitivity of a CFU-S to these factors changes as it matures from the early stem cell stage (Inhibitor-sensitive) to the more mature stages (Stimulator-sensitive) before becoming committed to a specific line of differentiation. The specificity of the inhibitor for haemopoietic stem cells suggests its potential value during chemotherapeutic procedures.     

Differential seeding of injected hemopoietic precursor cells in the bone marrow and spleen of irradiated mice

The seeding efficiency was determined of syngeneic granulocyte, macrophage, erythroid/mixed and megakaryocyte colony-forming cells (G-GFC, GM-CFC, M-CFC, E/Mix-CFC, MEG-CFC) in the femoral bone marrow and spleen of lethally-irradiated C57BL mice. The overall seeding efficiency of CFC's was similar to that for multipotential stem cells (CFU-S) in the marrow but in the spleen CFC seeding efficiency was ten-fold lower than for CFU-S. Two and a half hours after transplantation of 10⁷ bone marrow cells, the relative frequencies of E/Mix-CFC's and M-CFC's recovered from the recipient marrow were higher than in the injected marrow population. However the relative frequencies of CFC's recovered from the spleen corresponded closely to those of the injected marrow population.     

A cellular analysis of residual hemopoietic deficiencies in mice after 4 repeated doses of 4.5 gray X rays

A sub-optimal plateau in numbers of femoral stem-cells (CFU-S) in mice after 4 doses of 4.5 Gray X rays (each separated by 21 days), was shown to persist at 20–30 % of control up to 1 year after the last dose, when about 50 % of the mice had survived. The concentration of white cells in the blood was maintained persistently at about 70% of control, whereas the concentration of red cells was normal up to 4 months and then it declined to about 75% of control at 10 months after irradiation. Concentrations of some committed progenitor cells in the marrow (GM-CFC and ERC), which are capable of amplification cell divisions, were intermediate between the concentrations of marrow stem cells and mature blood cells in both the granuloid and the erythroid cell lineages, respectively. Hence increased amplification was a mechanism operating for a prolonged period in the production of numbers of mature cells. The numbers were subnormal, however, and this corresponded to only 1 extra amplification division on average.There was a slow decline after 6 months in the numbers of CFU-S, BFU-E and GM-CFC, and in the hematocrit, with reference to age-matched controls. The decline was due partly to a prevention of the natural increase in cell numbers in the marrow with the age of the mice, which was also seen with the femoral content of a stromal progenitor cell (CFU-F). A defect in the repeatedly-irradiated CFU-S population was detected as a persistent inability to produce colonies containing the same number of daughter CFU-S as contained in colonies derived from unirradiated marrow and assayed at the same time.     

Cryopreservation of autologous marrow grafts in acute leukemia: survival of in vivo clonogenic leukemic cells and normal hemopoietic stem cells

The survival of pluripotent hemopoietic stem cells and in vivo clonogenic leukemic cells after cryopreservation was determined in a rat model for human acute myelocytic leukemia (BNML). These stem cell populations can be selectively quantified with modified spleen colony assays (day 8 and day 12 CFU-S; LCFU-S). It appeared that the most primitive rat hemopoietic stem cell (day 12 CFU-S) was significantly less vulnerable to the freezing and thawing procedure as compared with the clonogenic leukemic cell (30% and 1.4% survival, respectively; p = 0.0026). Survival of the day 8 CFU-S population fell between those percentages (8.6%). In view of autologous bone marrow transplantation (ABMT), an attempt was made to extrapolate these and previously reported BNML rat data to man. Taking into account that a) only 1% of the clonogenic leukemic cells survive cryopreservation; b) the fraction of clonogenic leukemic cells in man is approximately 0.001; c) leukemic cells reinfused with the autologous marrow graft may lodge at sites unfavourable for growth; and d) supralethal high-dose chemoradiotherapy significantly hampers the regrowth of leukemia, it becomes rather unlikely that leukemic cells in the autologous marrow graft significantly contribute to a leukemia relapse after ABMT. Therefore, residual leukemia in the host surviving high-dose chemoradiotherapy is the most crucial factor as regards the final outcome of ABMT in acute leukemia.     

Regulation of murine granulocyte-macrophage progenitor cell and haemopoietic stem cell proliferation by factors produced in human fetal liver

At early stages (11–14 weeks) of gestation in human fetal liver few granulocyte-macrophage colony-forming cells (GM-CFC) are in DNA synthesis, whereas later in gestation (> 14 weeks) a large proportion of GM-CFC are in S-phase [Moore M.A.S. & Williams N. (1973) Cell Tissue Kinet.6, 461].Incubation of normal murine bone marrow GM-CFC (approx. 40% in DNA synthesis) with a supernatant from an early human fetal liver (11–14 weeks), reduced the proportion synthesizing DNA to <5%. In contrast, the proportion of murine GM-CFC synthesizing DNA was not affected by incubation with a supernatant from a late fetal liver (> 14 weeks).GM-CFC that had been switched out of cycle by incubation with a supernatant from an early gestation human fetal liver were switched back into cycle following incubation with a late human fetal liver supernatant.It is likely that changes in the relative levels of a proliferation inhibitor and stimulator throughout gestation might control the proportion of GM-CFC in cycle. In normal murine bone marrow (NMBM) approx. 10% of the haematopoietic stem cells (CFU-S) are synthesizing DNA. The proportion of CFU-S synthesizing DNA was increased to approx. 40% by incubation with a human fetal liver supernatant from all gestational ages tested (11–18 weeks).The specificity of these CFU-S and GM-CFC proliferation regulators is well demonstrated by an early gestation human fetal liver supernatant which will stimulate CFU-S proliferation but inhibit GM-CFC proliferation.The inhibitor and stimulator of GM-CFC proliferation are both produced by non-adherent human fetal liver cells. The GM-CFC proliferation inhibitor has a mol. wt of > 100,000 and the stimulator a mol. wt of 30,000–50,000. In contradistinction, the CFU-S proliferation stimulator is produced by adherent human fetal liver cells and has a mol. wt of 30,000–50,000.     

A comparison of the response to hyperthermia of murine haemopoietic stem cells (CFU-S) and L1210 leukaemia cells: enhanced killing of leukaemic cells in presence of normal marrow cells.

When the clonogenic survival of mouse haemopoietic stem cells (CFU-S) and leukaemia L1210 cells growth as ascites tumours are compared after being heated in vitro and assayed in vivo by spleen-colony assay, there is no significant difference in the terminal slopes of the survival curves. The shoulders of the survival curves differ, but this may be explained by differences in cell kinetics. By contrast, L1210 leukaemic marrow cells are considerably more susceptible to the lethal effects of hyperthermia (43 degrees C) than either normal marrow stem cells or L1210 leukaemic cells grown as ascites tumours. Moreover, the killing of L1210 ascites cells by hyperthermia can be enhanced by heating L1210 ascites cells with an equal number of normal marrow cells, or as upernatant removed from heated marrow cells. Most cells in lukaemic marrow are normal, and it is postulated that the increased thermal sensitivity of L1210 cells in leukaemic marrow is caused by diffusible factors (e.g. lysosomal enzymes) released by heating normal marrow cells.     

Role of pluripoietins in murine bone marrow stem cell differentiation

The decision of the differentiation pathways taken by pluripotent stem cells seems to be under the influence, at least in part, of humoral factors acting at the CFU-S level. This differentiation is assessed by histological examinations of the recipient spleen colonies in order to determine the E/G ratios. In vitro cultures are made to determine GM-CFC and BFU_E concentrations. After AraC treatment, CFU-S differentiate preferentially towards erythropoiesis. After total body irradiation, preferential differentiation is toward granulopoiesis. In both cases, there is a competitive phenomenon between the two cell lineages. This is observed in vivo and also in the in vivo-in vitro experiments where the responder cell population is in contact only with the eventual diffusible factors and not with the secreting cells or with the drug. These factors do not seem to be EPO, in the case of AraC, nor GM-CSF in the case of irradiation. We therefore suggest that humoral mediators other than those acting at the progenitor cell level can modulate CFU-S differentiation in a specific way after various types of aggression to the bone marrow.     

Lack of differential sensitivity of normal hematopoietic stem cells and murine lymphoblastic leukemic cells to a lysosomotropic agent, N-dodecyl morpholine

The effect of N-dodecyl morpholine (NDM), a lysosomotropic compound, on the clonogenic capacity of GK15, Sp2.0, Hb131, and L1210 lymphoblastic tumor cells and CFU-GM and CFU-S progenitor cells from DBA/2 mice was measured in order to evaluate the potential use of this compound for the purging of tumor-contaminated bone marrow (BM) in autologous BM transplantation. The growth of clonogenic tumor cells from all of the tested cell lines was inhibited with doses of NDM that also killed 100% CFU-GM and CFU-S, and no optimal dose could be found in this animal model to purge marrow while sparing sufficient stem cells to ensure engraftment in syngeneic BM transplantation.     

Some effects of chemotherapeutic drugs

Summary cis-Platinum is a relatively new active anticancer drug. In the study described in this paper, its toxicity was tested in the hematopoietic and renal systems of mice after six injections of 3 mg per kg body weight at 10-day intervals.Acute hematopoietic toxicity was studied by determining the survival of pluripotent (CFU-S) and granulo-macrophagic unipotent (GM-CFC) stem cells. The number of nucleated cells in the bone marrow and in the spleen and the number of granulocytes in the blood were determined.Renal toxicity was studied by histological examination of kidneys from treated mice compared with control animals.The number of stem cells in the bone marrow and in the spleen decreased during the treatment. One year after treatment, the autorepopulating ability of CFU-S was still diminished in spite of normal numbers of these cells.No renal damage could be demonstrated by light microscopy when the protocol described was used.Abbreviations used in this paper CFU-S pluripotent hemopoietic stem cells assessed by the spleen colony technique - GM-CFC granulo-macrophagic progenitor cells - BFU-E erythroid progenitor cells - E/G ratio ratio of erythroid and granulocytic colonies in the recipient spleen and assessed by histological examination - Ara-C cytosine arabinoside     

Thermal sensitivity of haemopoietic and stromal progenitor cells in different proliferative states

Stromal progenitor cells (CFU-F) in normal mouse bone marrow were more sensitive to heat at 43 degrees C than haemopoietic progenitor cells (CFU-S and GM-CFC) by a factor of approximately 1.2. In marrow regenerating after 4.5 Gy X-rays, the changes in sensitivity were by less than a factor of 1.4 and the sensitivity of CFU-F changed slightly to become intermediate between that of CFU-S and GM-CFC. A comparison of sensitivities reported in the literature revealed an inexplicable large variation of up to a factor of 6 in the thermal sensitivities of CFU-S and GM-CFC.     

Detection of colonic growth factors using a human colonic carcinoma cell line (LIM1215)

Although the colonic mucosa is one of the most rapidly proliferating epithelial tissues in the body, little is known about the factors that direct this proliferation. In this report we have studied the parameters of both a mitogenic and a clonogenic assay for detecting potential colonic growth factors (CGF). Using a colon carcinoma cell line (LIM1215), which has retained a number of the properties of normal colonic mucosa, we have assayed a range of mitogenic factors for CGF activity. 3H-thymidine incorporation by the LIM1215 cell line was stimulated by low concentrations of epidermal growth factor and basic fibroblast growth factor and, to a lesser extent, by higher concentrations of interleukin-1 and insulin-like growth factor 1. The cells did not respond to a range of other mitogens and lymphokines. Optimal clonogenic response in a soft-agar assay was obtained using a primary pituitary extract.     

Toxicity of trimetrexate on hemopoietic progenitor cells in normal mice

Single increasing doses of methotrexate (MTX) and trimetrexate (TMQ) were administered to normal mice. Survival of hemopoietic progenitor cells assayed as CFU-S and GM-CFC was determined 24 hr after drug injection. The survival of each population in TMQ-treated animals was not statistically different from that observed in mice treated with MTX. No difference was observed in time-survival curves of hemopoietic progenitor cells comparing TMQ to MTX. TMQ toxicity at the hematological level thus seems comparable to that of MTX.     

The response of tumour cells to radiation and cytotoxic drugs--a comparison of clonogenic and isotope uptake assays

We have carried out a series of experiments to compare the response to radiation and drugs of cells disaggregated from solid tumours as assayed by clonogenic survival and by an isotope incorporation method. This latter assay consisted of measuring the 24 h uptake of tritium labelled thymidine into cells plated in liquid medium upon a layer of semi-solid agar. The isotope was administered 4 days after plating. For cells from the RIF-1 mouse tumour, good agreement was seen between response to radiation, adriamycin, vincristine and CCNU as measured by the two assays. The two curves for radiation response, for example, showed similar shoulders and subsequent exponential regions. For cells from xenografts of the NCI-H69 human small cell lung cancer line, the response to radiation was dose-related for both assays, but the curve for clonogenic assay was about twice as steep as that for isotope uptake. For a range of five cytotoxic drugs, good agreement was seen between the two assays over the first 1 1/2 decades of response but with a tendency for the isotope uptake curve to plateau with further increasing drug dose. It appears that, at least for these two well-defined experimental tumour systems, the isotope uptake assay can provide a rapid quantitative assessment of cellular drug and radiation sensitivity comparable to that provided by clonogenic assay but in a much shorter period of time.     

Residual haemopoietic damage in the mouse after fractionated gamma-irradiation, down to 0.1 Gy per fraction

Residual damage in haemopoietic progenitor cell populations, spleen and granulocyte-macrophage colony-forming cells (CFU-S and GM-CFC) was detected in mice after 15 daily fractions where the dose per fraction was as low as 0.1 Gy. The injury was dose-dependent and after higher total fractionated doses of 7.5-10 Gy the CFU-S population recovered to about 50% of control between 2 and 12 months after irradiation. Residual damage was also detected in the stroma, in the form of reduced numbers of fibroblastoid colony-forming cells and of CFU-S in ossicles under the kidney capsule. The response to a second course of 15 fractions, given 3 weeks after the end of the first course, was similar and additive to the response to the first course in the short term. However, in the long term, recovery levels were similar after either one or two courses.     

Sensitivity of normal mouse marrow and RIF-1 tumour to hyperthermia combined with cyclophosphamide or BCNU: a lack of therapeutic gain.

The effect of simultaneous whole-body heat (45 min 41 degrees C) on cyclophosphamide (CTX) and BCNU toxicity to normal mouse marrow stem cells and to the RIF-1 tumour in C3H/He mice has been studied. Marrow stem-cell survival was assayed by the spleen-colony technique at both 2 and 24 h after treatment, and also by following peripheral WBC count during the weeks after treatment. Heat potentiation of CTX toxicity to marrow stem cells was similar at both times and 24 h after treatment heat was dose-modifying with a DMF of 2.0. The heat potentiation of BCNU toxicity to stem cells was much greater at 24 h than at 2 h, and at 24 h had a DMF of 2.1. Peripheral WBC counts supported the results from 24 h assay for both drugs. RIF-1 tumour response was assayed by clonogenic cell survival measured 24 h after treatment, and by growth delay. For clonogenic tumour-cell survival after CTX, heated and unheated curves were parallel at doses above 75 mg/kg, yielding DMFs varying between 1.9 and 1.4 according to dose. DMFs for BCNU were also dose-dependent, lying between 2.0 and 1.6, the RIF-1 tumour being much less sensitive to BCNU than to CTX. Growth-delay data agreed with clonogenic cell survival. Therapeutic ratios for the combination of heat with CTX or BCNU fell in the range 0.91--0.69, according to dose, i.e. no gain or even therapeutic loss under the conditions of this study.