Cytodifferentiation in the acute myeloblastic leukemias of man.

Correlation analysis of numbers of colony-forming progenitor cells was used as an approach to the quantitation of human pluripotent stem cells. Marrow specimens were obtained from 24 patients with untreated acute myeloblastic leukemia, 22 patients under treatment, and 29 patients with no hematologic malignant disease. Three classes of progenitor cells were assayed: burst-forming units dependent on erythropoietin (BFU-E), colony-forming units dependent on erythropoietin (CFU-E), and granulopoietic progenitors (CFU-C). Significant positive correlations between numbers of BFU-E, CFU-E, and CFU-C were found in all 3 groups of patients. In contrast, no such positive correlations were seen between marrow blasts and any of the classes of colony-forming progenitors. These results were compatible with a shared relationship of the colony-forming progenitors to a pluripotent cell of origin and raised the possibility that the immediate progenitors of the blasts may not be any of the myelopoietic progenitor cells monitored in these studies.     

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.     

Haematopoietic late effects of prolonged bleomycin treatment in mice

Summary In two studies, haematopoietic late effects of prolonged bleomycin treatment were evaluated in mice given serial injections of 21 mg bleomycin/m² weekly for 31 and 44 weeks, respectively. Femoral bone marrow cellularity measured at 43, 45, and 49 weeks after discontinuation of the drug in the first and after 20 weeks in the second study was found to be significantly (P<0.05) lower in the treated mice than in the controls. CFU-S, BFU-E, and CFU-C contents were also reduced in the treated bone marrow, but with the exception of CFU-S in the second study, differences from control values were not significant. Additional long-term bone marrow cultures performed in the second study revealed no marked changes in the marrow proliferative activity and the self-renewal of stem cells to explain the reduced marrow cellularity and stem cell content. These last findings might, therefore, be due to a decrease in femoral size with less marrow content in the treated mice, since measurements of the tibial weights in both groups showed that the bones in the treated animals were significantly (P<0.05) lighter than those in the controls.     

Effects of S-phase-specific agents on granulocyte-macrophage and erythroid progenitor cells obtained from normal individuals and from patients with chronic myelogenous leukemia

The effects of three S-phase-specific agents, [3H]thymidine, hydroxyurea, and 1-beta-D-arabinofuranosylcytosine, on granulocyte-macrophage colony-forming units (CFU-C) and erythroid progenitor cells (erythroid burst-forming units) (BFU-E) from the bone marrow or peripheral blood obtained from 23 normal individuals and 12 patients with chronic myelogenous leukemia were investigated. CFU-C, regardless of their source, showed comparable degrees of sensitivity to each of the S-phase-specific agents, with perhaps a slightly greater level of sensitivity to [3H]thymidine. In contrast, the sensitivities of chronic myelogenous leukemia and normal marrow BFU-E to the 3 agents were quite different, with essentially all BFU-E being killed by [3H]thymidine, 50 to 70% being killed by 1-beta-D-arabinofuranosylcytosine, and only 15 to 20% being killed by hydroxyurea. BFU-E present in normal peripheral blood were insensitive to [3H]thymidine or hydroxyurea but were sensitive to 1-beta-D-arabinofuranosylcytosine. These studies demonstrated similarities between the CFU-C and BFU-E of CML patients and the CFU-C and BFU-E present in normal bone marrow. On the other hand, the sensitivities of normal peripheral blood progenitor cells to "S-phase-specific" agents differed from that of CML progenitor cells or the progenitor cells present in normal bone marrow. Additionally, these studies have demonstrated the limitations inherent in suicide techniques as methods for estimating the cell cycle characteristics of clonogenic cells.     

Effects of tilorone on hemopoietic stem cells and on the development of Friend leukemia

Summary Hematological effects of tilorone, an interferon inducer, on the hematopoietic cell system of normal CBA/Ca mice and on the development of Friend virus (FV-P)-induced polycythemia in DBA/2 mice were studied. In normal mice 80 mg/kg IP had a marked depressive effect on pluripotent (CFU-S), granuloid committed (CFU-C), and erythroid committed (CFU-E) stem cells with regeneration between days 5 and 12. In bone marrow smears only lymphopenia was detected. Treatment of mice before FV-P infection caused a slight retardation in the development of the splenomegaly and the transformation of bone marrow cells to Ep independence. Repeated treatment after FV-P infection also reduced the increase in spleen weight and the development of reticulocytosis, but the Ep independence of bone marrow and spleen cells was not influenced. In vitro exposure of normal cells and cells from FV-P-infected animals to the drug showed the same sensitivity of colony growth in normal as well as in Ep-independent CFU-E. The action of the drug on Friend leukemia is at least in part considered a toxic effect on the hematopoietic stem cell system.     

Effects of daunorubicin and doxorubicin, free and associated with DNA, on hemopoietic stem cells.

We have compared daunorubicin (DNR)-DNA with free DNR and doxorubicin (DOX)-DNA with free DOX for their effects in vivo in mice on pluripotent stem cells and granulocytic committed stem cells. Dose-survival, time-survival, and recovery curves were obtained after one i.v. injection of either drug. The dose-survival curves of colony-forming units-spleen (CFU-S) and colony-forming units-committed stem cells (CFU-C) were exponential in shape with both agents. DNR-DNA appeared more toxic to the hemopoietic precursor cells than did free DNR. In contrast, DOX-DNA was less toxic toward CFU-S and as toxic as DOX toward CFU-C. Time-survival curves indicated a minimum level of CFU-S and CFU-C at about 33 hr. After that, the recovery of CFU-S was rapid for DNR-treated mice but remained below 50% of the controls on Day 12 for the DNR-DNA-treated group. In mice previously given injections of DOX or DOX-DNA, the recovery of the CFU-S was more protracted in time with a better recovery in mice treated with DOX-DNA. Both DNR and DNR-DNA induced an initial CFU-C decrease followed by a rapid but transient rise with a maximum on Day 4 after chemotherapy. On Day 12, the CFU-C recovery was still incomplete in both DNR- and DNR-DNA-treated mice. In the groups treated with DOX, the CFU-C recovery was more important after DOX-DNA complex than after free DOX. The results are discussed in view of the "lysosomotropic chemotherapy" hypothesis.     

Mechanisms of abnormal erythropoiesis in malignancy

In order the investigate mechanisms of diminished red cell production in malignancy, we assayed erythroid progenitor cell proliferative responses to erythropoietin in plasma clot cultures of bone marrow cells from 34 cancer patients. Erythroid colony growth by marrow cells of 11 healthy donors (means of 58 CFU-E and 19 BFU-E derived colonies/6 X 10(4) cells) was similar to that in cultures of cells from patients either with (means of 44 CFU-E and 22 BFU-E derived colonies/6 X 10(4) cells) or without (means of 50 CFU-E and 19 BFU-E derived colonies/6 X 10(4) cells) myelophthisis. Colony formation was normal at all erythropoietin concentrations tested, indicating that both the CFU-E and BFU-E retain normal erythropoietin sensitivity in vitro. CFU-E proliferation correlated negatively (r = -0.56; P less than 0.001) with the level of hemoglobin. In contrast to marrow cell proliferative responses to erythropoietin, serum erythropoietin levels were inappropriately reduced in all 19 patients in whom they were measured, a finding which may be important in the pathogenesis of anemia in patients with cancer.     

Hemopoietic regeneration in murine spleen following transfusion of normal and irradiated marrow: different response of granulocyte/macrophage and erythroid precursors

To investigate cell proliferation in regenerating spleen, bone marrow of normal and gamma-irradiated donor mice (3 weeks after 5 Gy) was transfused into lethally irradiated recipients. In the donors and in the recipient spleens numbers of CFU-S and progenitor cells were determined. In the irradiated donors the progenitors were at control level after 3 weeks of recovery although CFU-S were still at 50% of control. Recipients of the irradiated marrow received therefore an increased proportion of progenitors. CFU-C appeared to be self-renewing and/or increased in number due to enhanced CFU-S differentiation, but not the erythroid progenitors. CFU-S self-renewal was reduced after 5 Gy. The data suggest that cell differentiation and maturation proceed during early splenic regeneration. The quantity of CFU-C does not necessarily mirror the situation in the stem cell compartment.     

4-Hydroperoxycyclophosphamide inhibits proliferation by human granulocyte-macrophage colony-forming cells (GM-CFC) but spares more primitive progenitor cells

Despite its considerable toxicity to haemopoietic colony-forming cells, 4-hydroperoxycyclophosphamide (4-HC) has successfully been used to purge marrow of leukaemic cells before it is used to rescue patients from high-dose chemoradiotherapy. These conflicting observations indicate that haemopoietic progenitor cells that are not detected by the established colony-forming assays survive exposure to 4-HC and repopulate the marrow. The recent finding that murine spleen colony-forming cells (CFU-S) are resistant to 4-HC [Porcellini A, et al. (1983) Expl Hemat. 11 (suppl 14) 331 (abstract)] [14] also indicates that sensitivity to 4-HC can be used to distinguish primitive progenitor cells from committed progenitor cells. As part of a study on the nature of a population of blast colony-forming cells in human bone marrow, we tested their sensitivity to 4-HC to see whether they also are spared by the drug. We found that 4-HC had much less effect on the blast colony-forming cells than on the granulocyte-macrophage colony-forming cells (GM-CFC). This result suggests that the blast-colony-forming cells may be early human haemopoietic progenitor cells.     

Haemopoietic stem cells in leukaemic AKR mice: the (AKR x C57BL/6)F1 mouse as an in vivo assay system.

Characteristics of the AKB6F1 mouse strain make it suitable as an assay animal for quantitating haemopoietic stem cells, or spleen colony-forming units (CFU-S), from leukaemic AKR mice. Marrow cells harvested from leukaemic mice were assayed for CFU-S in lethally irradiated AKR or AKB6F1 hosts. Survival times and numbers of leukaemic colony-forming units (L-CFU) in unirradiated recipients were used to detect proliferation of transplanted leukaemic cells. In contrast to AKR recipients, the proliferation of transplanted leukaemic cells was suppressed in F1 hosts. Injection of marrow cells from normal nonleukaemic AKR mice into AKR and F1 hosts yielded significantly more CFU-S and spleen 59Fe uptake in F1 than in AKR hosts. In marked contrast, injection of marrow from leukaemic AKR mice suppressed CFU-S proliferation in the F1 recipients. Thus it was possible to quantitate CFU-S in marrow containing L-CFU.     

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     

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.     

In vitro studies of erythropoietin-dependent regulation of erythropoiesis in myelodysplastic syndromes

Erythropoietin-dependent regulation of erythropoiesis in myelodysplastic syndromes (MDS) was evaluated by measuring the in vitro response of primitive (BFU-E) and relatively mature (CFU-E) erythroid progenitors from 12 patients and from eight healthy donors to recombinant human erythropoietin (rhEPO), and by quantifying relationships between circulating EPO levels and progenitor cell frequencies in MDS marrow. Half-maximal growth of MDS CFU-E and BFU-E was detected at a 4-fold higher rhEPO concentration than required by control erythroid progenitors. Nine of the patients evaluated exhibited maximal growth of erythroid colonies at 5- to 20-fold higher than control saturating rhEPO concentrations. Circulating EPO levels in MDS patients were elevated, with a mean value approximately 35-fold higher than that of controls. The frequency of MDS marrow CFU-E and BFU-E was 57 +/- 42% and 18 +/- 9% of the mean control values, respectively. Correlation analysis of the relationships between MDS EPO levels and erythroid progenitors indicated that the anemia in MDS is not attributable to an abnormality in the capacity of EPO to induce the generation of CFU-E, but may be influenced by the BFU-E population, whose severe deficiency results in insufficient influx of EPO-responsive cells. Our findings therefore suggest that treatment of MDS patients with rhEPO may be of limited benefit, since the generation of BFU-E from more primitive ancestors and the initial growth requirements of these cells are not under the regulatory influence of this hormone.     

rhIL-2对人骨髓CFU-GM与CFU-E和BFU-E的影响

目的:为了探索rhIL-2对人骨髓造血干祖细胞体外增殖的影响。方法:本文采用甲基纤维素体外半固体培养法。结果:证明了50～1000U/ml rhIL-2单用时对人骨髓造血祖细胞的CFU-GM、CFU-E及BFU-E无直接刺激增生作用,当与GM-CSF联合应用时,100～400U/ml rhIL-2其CFU-GM集落形成率明显高于单用GM-CSF(100U/ml)的对照组(P<0.05),当与EPO(2U/mi)联合应用时其CFU-E,及BFU-E集落形成率也明显高于单用EPO对照组(P<0.05),而1000U/ml rhIL-2其三种集落形成数目呈下降趋势,可能有抑制作用。结论:以上结果说明100～400U/ml rhIL-2可应用于协同GM-CSF及EPO体外扩增骨髓干/祖细胞来进行骨髓移植。     

Fatal polycythemia induced in mice by dysregulated erythropoietin production by hematopoietic cells.

C57BL/6J murine bone marrow cells, infected with a retroviral vector (MP Zen) carrying a monkey erythropoietin cDNA, were transplanted into lethally irradiated syngeneic recipients to study the effect of erythropoietin production by hemopoietic cells. High levels of erythropoietin were recorded in the plasma (median value: 1.2 u/ml) and in media conditioned by peritoneal, spleen, and bone marrow cells from recipient mice. In transplanted mice, the hematocrit was elevated (90 +/- 5%) and the mice died at a mean of 71 days after transplantation. In the blood, platelet counts were usually low and nucleated blood cells slightly elevated. Spleen weight increased 5-fold and bone marrow cellularity decreased slightly. There was a 9.9-fold increase in erythroblast numbers, a 2-fold reduction of lymphocytes, and no variation of the myeloid cells when the total cellularity of bone marrow, spleen, peripheral blood, and peritoneal cells were considered. Calculation of the total numbers of progenitor cells in these organs revealed a 18-fold increase in erythroid colony-forming units (CFU-E) but no significant variation of the erythroid burst-forming units (BFU-E), and myeloid progenitor cell numbers. A variable proportion of CFU-E, (12% or 24% in bone marrow or spleen, respectively) was able to proliferate in unstimulated cultures. Erythropoietic amplification occurred in the spleen and there was a redistribution of the BFU-E and myeloid cells from the bone marrow to the spleen. No significant extramedullary erythropoiesis was seen. This study emphasizes the erythroid specificity of erythropoietin and shows that elevated dysregulated erythropoietin production by hemopoietic cells leads to a fatal polycythemia without erythroid neoplastic transformation.     

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.     

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.     

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.     

Survival and characteristics of murine leukaemic and normal stem cells after hyperthermia: a murine model for human bone marrow purging

The effect of hyperthermia in vitro on the survival and leukaemogenic effectiveness of WEHI 3-B cells and on the survival and transplantation efficiency of bone marrow cells was compared in a murine model system. Normal murine clonogenic haemopoietic cells (day 9 CFU-S and CFU-GM) proved to be significantly less sensitive to 42.5 degrees C hyperthermia (Do values: 54.3 and 41.1 min, respectively) than leukaemic clonogenic cells (CFU-L) derived from suspension culture or from bone marrow of leukaemic mice (Do: 17.8 min). Exposure for 120 min to 42.5 degrees C reduced the surviving fraction of CFU-L to 0.002 and that of CFU-S to 0.2. If comparable graft sizes were transplanted from normal or heat exposed bone marrow, 60-day survival of supralethally irradiated mice was similar. Surviving WEHI 3-B cells were capable of inducing leukaemia in vivo. The two log difference in the surviving fraction of CFU-L and CFU-S after 120 min exposure to 42.5 degrees C suggests that hyperthermia ex vivo may be a suitable purging method for autologous bone marrow transplantation.     

Effect of cyclophosphamide on the bone marrow hematopoiesis in the mouse

This paper reports the effect of cyclophosphamide on the bone marrow hematopoiesis in the mouse. Cyclophosphamide 0.12 mg/g body weight was injected into the mice once and the observation lasted for 2 weeks. After the injection, peripheral leukocytes were reduced to the lowest level on day 4 and then increased higher than the control on day 7 to 14. The number of nucleated cell in the bone marrow was the lowest at the 48th hour and gradually became normal within two weeks. The pluripotent hemopoietic stem cells--CFU-S (colony forming unit-spleen) were depleted abruptly in 24 hours, then reproliferated exponentially to a peak on day 3, followed by a second decrease and came back to normal level on day 11 to 14. The changes of granulocytic progenitor cell CFU-D (colony forming unit-diffusion chamber) and CFU-C (colony forming unit-culture) were quite similar to that of CFU-S but their proliferation peak was on day 4. The peripheral leukocyte drop was slower and the return to normal was earlier than the hemopoietic cells. So the recovery of leukocyte count does not mean a real reconstruction of hematopoiesis. The bone marrow stroma observed by CFU-F (colony forming unit-fibroblastoid) assay and marrow microcirculation were also damaged and did not recover to normal during the observation. The bone marrow stroma and microcirculation showed a more serious damage.