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.     

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)     

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.     

E838对CTX化疗损伤小鼠的保护作用

 目的 观察E838对环磷酰胺所致小鼠损伤的保护作用。 方法 对小鼠采用连续5天腹腔注射E838药物,从第3天同时给予环磷酰胺（CTX）,阳性对照为茜草双酯,观察外周血白细胞、骨髓有核细胞数、内源性脾结节形成（CFU-S）、脾脏指数及胸腺指数的变化。 结果 E838可明显减轻化疗所致小鼠免疫功能的抑制,并可使化疗后小鼠的体重恢复增长, 白细胞、骨髓有核细胞和CFU-S明显高于对照组,差异有统计学意义（P<0.05）。中、高剂量组白细胞与茜草双酯组比较,差异有统计学意义（P<0.05）。 结论 提示E838具有对抗化疗损伤的作用。     

Residual toxicity in hematopoietic cells following a single dose of methylnitrosourea

The residual injury to the proliferation capability of hemopoietic stem cells (CFU-S) which results from their exposure to leukemogenic agents was evaluated in mice given a single leukemogenic dose of methol nitrosourea (MNU 50 mg/kg body weight, i.v.). Bone marrow cellularity, splenic weight, number of CFU-S and the proportion of cycling to noncycling CFU-S were measured in an effort to detect acute and residual injury to the CFU-S from mice given MNU 21 and 3 days earlier. Marrow cells were also transferred into lethally irradiated mice to observe the self-renewal capability of the CFU-S in the recipient spleen and bone marrow. The results of these measurements show that the CFU-S in marrow from mice given 50 mg/kg of MNU 21 days earlier still have a defective ability for self-renewal, although the total cellularity, number of CFU-S and proportion of cycling and noncycling CFU-S in the donor have returned to the normal range. The relationship of this self-renewal defect to the development of leukemia after this leukemogenic dose of MNU is not known.     

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.     

Kinetics of normal hemopoietic stem cells during leukemia growth before and after induction of a complete remission. Studies in a rat model for acute myelocytic leukemia (BNML)

During the invasion of leukemic cells of the rat acute myelocytic leukemia model BNML in the bone marrow, the number of normal bone marrow stem cells (CFU-S) decreased while simultaneously an increase of CFU-S in the leukemic spleen was observed. A small reduction in the tumor load by low dose cyclophosphamide treatment (10 mg/kg) caused a temporary CFU-S recovery in the bone marrow. After a therapeutic dose of cyclophosphamide (100 mg/kg), the CFU-S numbers in femur and spleen decreased to low levels but they rapidly increased immediately thereafter. In the spleen, however, the CFU-S increase halted when femoral CFU-S numbers reached normal levels. Splenectomy following cyclophosphamide treatment revealed that the splenic CFU-S population does not play a role in regeneration of hemopoiesis. During the subsequent leukemia relapse, CFU-S in the femur decreased again while spleen CFU-S tended to rise. It is concluded that the bone marrow CFU-S, which survive both the leukemia and the remission-induction treatment, and not the migrated, extramedullary localized stem cells are the major source for the restoration of normal hemopoiesis.     

Effects of CFU-S inhibitors on murine bone marrow during Ara-C treatment—I. Effects on stem cells

In order to determine whether the dialysable extract of fetal calf bone marrow, which has previously been shown to inhibit CFU-S entry into DNA synthesis, was also capable of protecting bone marrow CFU-S during chemotherapy, experiments were performed in which the dialysate was injected to mice simultaneously with Ara-C. The number of modullary CFU-S was significantly higher in mice receiving the dialysate with the drug than in mice receiving the drug alone, while the numbers of nucleated cells and of GM-CFC were not different in the two groups. These results suggest a specific protective effect on bone marrow CFU-S survival. By chromatography on Sephadex G10 it was possible to isolate a low molecular weight fraction which inhibits significantly the percentage of CFU-S in DNA synthesis but does not increase CFU-S number when assayed in the same protocol. These experiments seem to imply that there is little, if any, correlation between the inhibition of CFU-S entry into cycle and an increase in survival of these cells after Ara-C treatment. These observations as well as the mechanism of the protective effect (increase of the number of CFU-S) of the dialysate will be discussed.     

In vitro induction of CFU-S proliferation by a non viral splenic activity from myeloproliferative sarcoma virus infected mice

The myeloproliferative sarcoma virus (MPSV) induces a myeloproliferative syndrome in DBA/2 mice. It is characterized by a considerable increase in the number (100-fold) and in the concentration (10-fold) of pluripotent hematopoietic stem cells detected in vivo (CFU-S) in the spleens of infected animals. Prior studies have shown the presence of a mixed-colony promoting activity (MPA) in neoplastic spleens. In the presence of a small quantity of erythropoietin, MPA induces the proliferation and differentiation of pluripotent hematopoietic stem cells, detected in vitro (Mix-CFU). We tested the effect of factors produced by neoplastic spleen cells on the proliferation of day 10 CFU-S and their entry into the cell cycle. This was done by comparing the number of day 10 CFU-S present in suspensions of normal bone marrow cells incubated for 2 days on agar underlays containing cells from either normal or neoplastic spleens. Our results show the existence of an activity secreted by cells from the spleens of MPSV-infected animals which starts CFU-S cycling and which is physically distinct from MPSV. The presence of this activity, whose identity with MPA remains to be proven, would enable us to explain the proliferation of CFU-S in the course of the disease.     

Regulators of stem cell proliferation in the haemopoietic tissues of W/Wv and SI/SId mice

The presence of CFU-S proliferation stimulatory and inhibitory activities in the bone marrow and spleens of normal mice (+/+) and mice with mutations affecting the proliferative behaviour of stem cells (S1/S1^d and W/W^v) has been investigated.S1/S1^d and +/+ bone marrow and spleen contain virtually no detectable stimulator but the corresponding tissues from W/W^v mice are both strongly stimulatory. SI/SI^d marrow in particular, but also +/+ marrow are strongly inhibitory whilst +/+ spleen, S1/SI^d spleen, W/W^v marrow and W/W^v spleen all contain inhibitory activity but at a lower specific activity.The data are compatible with studies of cell and tissue grafts that have indicated an intrinsic haemopoietic stem cell defect in W/W^v mice and an extrinsic, microenvironmental defect in SI/SI^d mice. It is suggested that they are also compatible with defective regulatory interactions between stem cells and regulator-producing cells and that the W and S1 loci may code for products involved in the production of, or response to, CFU-S proliferation regulators.     

Pluripoietin effects on CFU-S lineage determination: possible mechanisms

Regulation of pluripotent stem cell (CFU-S) proliferation kinetics by humoral factors is now well documented. However, the mechanism of choice of CFU-S differentiation pathways is still a controversial issue. We suggest that long-range humoral factors (pluripoietins) are capable of preferentially channelling CFU-S towards one of the cell lineages after various perturbations such as irradiation and drug treatment. The differentiation pathway depends on the treatment protocol. We present data concerning one of the protocols: injection of 20 mg of cytosine arabinoside (Ara-C). When conditioned medium from bone marrow of treated mice is incubated with normal marrow, CFU-S of the latter generate spleen colonies with an E/G ratio above normal values. Clonal analyses of spleen colonies demonstrate that they are generated by pluripotent stem cells. Therefore, any modification in the E/G ratio is due to modifications of CFU-S channelling and not to the variations of committed cells. It was of interest to determine the mechanism of pluripoietin activity. This was studied at three levels: membrane receptors, gene activation and protein synthesis. These studies suggest that CFU-S have receptors for pluripoietins which activate genes responsible for specific mRNA synthesis. De novo synthesis of proteins is a necessary prerequisite for pluripoietin activity to be expressed. Hypotheses for these mechanisms are presented.     

Effects of myeloproliferative sarcoma virus on the pluripotential stem cell and granulocyte precursor cell populations of DBA/2 mice

The hematopoietic stem cell (CFU-S) and granulocyte precursor cell (CFU-C) populations have been assayed in the spleen, blood, and bone marrow of DBA/2 mice at various times after infection with the myeloproliferative sarcoma virus (MPSV). Beginning between 7 and 19 days after virus infection, the number of CFU-S showed a steady, parallel increase in the blood and spleen, reaching a maximum at both sites by days 25-30. At the maximum, in the spleen the concentration of CFU-S was 10 times greater than that in the blood, and the total number of CFU-S was over 100 times greater than that of normal animals. During the same period, in the bone marrow the number of CFU-S decreased to one-half of normal. Nevertheless, the CFU-S from MPSV-infected animals differentiated normally in the spleens of irradiated, normal recipient mice (except for some hyperplasia of the erythroid component of spleen colonies). The CFU-C content of the bone marrow, spleen, and blood paralleled the CFU-S content of these organs: The CFU-S and CFU-C populations changed almost synchronously after MPSV infection. In the terminal stage of the MPSV-induced disease, a variable proportion of the CFU-C population acquired the ability to differentiate in the absence of added colony-stimulating factor.     

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     

Regulation of the growth fraction of CFU-S by an inhibitor produced by bone marrow

Humoral factors were extracted from cell suspensions obtained from rat bone marrow and thymus and fractionated using Amicon filters to obtain crude molecular weight bands of 50–100 K and 30–50 K. These fractions were incubated in vitro with haematopoietic stem cells (CFU-S) obtained from drug-treated mice to evaluate their effects on the proliferation of stem cells. An inhibitor was found in the 50–100 K fraction from normal rat bone marrow that would decrease the proportion of stem cells synthesising DNA in the regenerating bone marrow from ～ 50 to ～ 10%. This inhibitor was not found in the 30–50 K fraction extracted from rat bone marrow or in the 50–100 K fraction extracted from rat thymus and had no effect on the proportion of committed progenitors (CFU-C) synthesis DNA or the proliferation of mitogen-stimulated lymphocytes. The inhibitor has the same properties as that described by Lord et al. [11].     

Characteristics of bone marrow and blood cells in human leukemia that produce leukemia inhibitory activity (LIA)

Cells in the bone marrow and blood of patients with acute and chronic myeloid and lymphoid leukemia which produce leukemia inhibitory activity (LIA) specific for normal granulocyte-macrophage progenitor cells (CFU-c) have been characterized as belonging to the third population of lymphoid-like cells which are neither T nor B but which have Fe receptors. The cells are non-adherent, non-phagocytic, of low density (<1.070 g/cm³), slowly sedimenting (2–6 mm/h) and present in the sheep red blood cell rosetting populations which are E^?, EAC^?, Ig^?, EA⁺, and are Ia^? as determined by a complement cytotoxicity test using rabbit anti-human Ia-like antibody. The LIA-producing cell has been further characterized by its response to certain agents with known immunoregulatory activity. Bacterial lipopolysaccharide, tuberculin purified protein derivative, Bacillus Calmette-Guérin, dextran sulphate and pokeweed mitogen suppressed the production of LIA. Total suppression of LIA production by LPS required the constant presence of this agent; but hydrocortisone and dexamethasone abrogated LPS suppression of LIA production. LIA could not be found in normal human adult bone marrow and blood cells or fetal bone marrow, spleen, or liver cells. Further evidence for the specificity of LIA action was obtained since LIA did not inhibit erythropoietin dependent human erythroid progenitor cell (BFU-e, CFU-e) proliferation.     

Differentiation pathways of pluripotent hemopoietic stem cells in long-term mouse bone marrow culture

Differentiation pathways of CFU-S were examined in mouse bone marrow long-term culture system. E/G ratios of non-adherent CFU-S in this system increased from 2 to 7-9 by 2-3 weeks of culture and then fluctuated during the culture period. On the other hand, E/G ratios of adherent CFU-S were lower than that of non-adherent CFU-S, and always remained under 2. Some of the supernatants collected from culture flasks at weekly intervals increased E/G ratios of normal bone marrow CFU-S after 18-20 h of incubation. These results suggest that the differentiation of CFU-S is controlled by humoral factors, secreted from yet unknown cells in bone marrow, in this system.     

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.     

Regeneration of spleen colony-forming cells and granulocyte-monocyte progenitors in T-cell deprived mice treated with cyclophosphamide

The role of T cells in regeneration of CFU-S(d8) and GM-CFU was investigated in adult Cy treated mice. CBA mice were deprived of T cells by adult thymectomy and irradiation (TIR) and treated with 200 mg/kg Cy. The type of CFU-S(d8) and GM-CFU regeneration curve after Cy was not different from that found in nonthymectomized, irradiated (NIR) mice. The activity of a stimulator of CFU-S(d8) proliferation was increased to the same degree in the bone marrow of both TIR and NIR mice. However CFU-S(d8) recovered to a higher level in TIR than in NIR mice. The results presented demonstrate that T-cell depletion in adult mice influences CFU-S(d8) growth.     

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 sensitivity to hyperthermia of mouse normal haemopoietic stem cells related to proliferation activity and organ source

Thermosensitivity of haemopoietic stem cells was studied in relation to the organ source and the proliferative state of the cells. Heat treatment was carried out at 41, 42, 43, 44 and 45 degrees C until about 1 per cent survival was reached. Treatments at 42 degrees C and below appear to be critical in revealing thermosensitivity differences between haemopoietic stem cells, characterized by the time T(o) at a given temperature to induce a lethal event. At these temperatures, foetal liver CFU-S (about 35 per cent in S phase) were more thermosensitive than steady-state bone marrow and spleen CFU-S (less than 10 per cent in S). We consider that these thermosensitivity differences cannot be attributed exclusively to differences in proliferation rates of the CFU-S, since exponentially-proliferating marrow CFU-S (48 per cent in S) does not significantly differ in sensitivity compared with steady-state CFU-S, while regenerating spleen CFU-S does (34 per cent in S). An Arrhenius analysis of heat survival curves of the different CFU-S allowed us to estimate only one activation energy (Ea) in the inactivation process of foetal liver and regenerating spleen CFU-S, and two Ea in the case of steady-state marrow and spleen CFU-S and regenerating marrow CFU-S.