Enhanced triggering of stem cell proliferation after two doses of hydroxyurea

Abstract. Hydroxyurea (HU) injected into experimental mice increases the proliferation rate of the pluripotent haemopoietic stem cells (CFUs). This effect can be enhanced if hydroxyurea is administered in two doses, separated by 2 hr. The effect does not seem to be caused by more extensive bone marrow damage.     

Effect of Danggui Buxue Tang on immune-mediated aplastic anemia bone marrow proliferation mice

To investigate the pharmacological effects of Danggui Buxue Tang (DBT) on immune-mediated aplasia anemia mice. The model of immune-mediated aplasia anemia mice was induced by means of ⁶⁰Co γ-ray irradiation and mixed cells of thymus and lymphnode of DBA/2 mice infusion through tail vein, the parameters tested indices were as following: blood picture, bone marrow nucleated cell count (BMNC), murine colony-forming unit-megakaryocytes (CFU-GM) of bone marrow cells, murine colony-forming unit-erthroid (CFU-E) and burst forming unit-erythroid (BFU-E). The results showed that DBT could not only withstand significantly decreation of blood cells by immune-mediated, but also stimulate on the growth of bone marrow colony cell and increase the weight of hemopoietic progenitor of bone marrow. Therefore, DBT had an obvious treat effect on immune-mediated aplasia anemia models mice.     

The Additive Effect of Mesenchymal Stem Cells and Bone Morphogenetic Protein 2 on γ-Irradiated Bone Marrow in Mice

Irradiation from γ-rays can cause severe damage to bone marrow and hematopoietic tissues. Presently, the most effective method available to treat severe hematopoietic injury is a bone marrow transplant (BMT). Allogeneic BMT is a difficult technique to perform due to the differences in human leukocyte antigen proteins between the donor and recipient, with acute graft-versus-host disease being a major complication of the technique. This limits the widespread applicability of allogeneic BMT. To develop a novel treatment for acute hematopoietic damage, we transplanted bone marrow derived mesenchymal stem cells (MSCs) into recipient mice and treated them with recombinant human bone morphogenetic protein 2 (rhBMP2) to investigate whether MSCs and rhBMP2 could additively promote the restoration of hematopoietic function. MSCs are vital components of the hematopoietic microenvironment that supports hematopoiesis, and bone morphogenic protein is a key factor in hematopoiesis. The 30-day survival rate as well as the numbers of nucleated cells, bone marrow colony-forming unit-granulocyte macrophages, spleen colony-forming units and peripheral blood cells were enumerated. The results showed that, after γ-irradiation and transplantation, MSCs and rhBMP2 additively promoted and improved hematopoietic restoration and function in vivo and in vitro. This additive effect of MSCs and rhBMP2 may one day provide a novel means of treating acute hematopoietic damage.     

Effects of cyclophosphamide on murine bone marrow and splenic megakaryocyte-CFC,granulocyte-macrophage-CFC,and peripheral blood cell levels

The effect of cyclophosphamide (CY) on megakaryocytopoiesis in mice was examined with assays of megakaryocyte colony-forming cells (Meg-CFC) in bone marrow and spleen and simultaneous determinations of peripheral blood counts, after a single intraperitoneal dose (200 mg/kg) of CY. Significant rebound thrombocytosis (170% of normal) occurred at day 11 after injection with CY, although only modest preceding thrombocytopenia (70% of normal) was observed. After an initial 3–5-day period of suppression, total megakaryocyte colony-forming cells (Meg-CFC) in both bone marrow and spleen of CY-treated mice demonstrated rebound increases at 5 and 7 days, respectively, after administration of the drug. Granulocyte-macrophage colony-forming cells (GM-CFC) exhibited alterations which were similar to those of Meg-CFC, suggesting similar sensitivities of Meg-CFC and GM-CFC to CY. The increase in Meg-CFC in both bone marrow and spleen preceded development of thrombocytosis by 4–6 days. This suggests that increased platelet counts in CY-treated mice are attributable, at least in part, to alterations in feedback mechanisms which control megakaryocytopoiesis, with resultant stimulation of the megakaryocyte progenitor compartment.     

Changes in trabecular bone turnover and bone marrow cell development in tail-suspended mice

Skeletal unloading induces trabecular bone loss in loaded bones. The tail-suspended mouse model simulates conditions associated with lack of mechanical stress such as space flight for the loaded bones. In such a model, the tail supports the body weight. The forelimbs are normally loaded and the movement of its hindlimbs is free without weight bearing. Histomorphometric analyses of the murine tibiae of the elevated hindlimbs show that trabecular bone volume rapidly diminishes within one week and stabilizes at that level in the subsequent week of tail suspension. Two-week reloading after one-week unloading completely restores trabecular bone volume, but this does not happen after two-week unloading. Unloading for one or two weeks significantly reduces bone formation rate and increases both the osteoclast surface and number compared with age-matched ground control mice. Subsequent reloading restores reduced bone formation and suppresses increased bone resorption. In bone marrow cell cultures, the numbers of alkaline phosphatase (ALP)-positive colony-forming units-fibroblastic (CFU-f) and mineralized nodules are significantly reduced, but the numbers of adherent marrow cells and total CFU-f are unaltered after tail suspension. On the other hand, subsequent reloading increases the number of adherent marrow cells. Unloading for one week significantly increases the number of tartrate-resistant acid phosphatase (TRAP)- positive multinucleated cells compared with the control level. Our data demonstrate that tail suspension in mice reduces trabecular bone formation, enhances bone resorption, and is closely associated with the formation of mineralized nodules and TRAP-positive multinucleated cells in bone marrow cultures obtained from tibiae. Two-week reloading restores bone volume reduced after one-week unloading, but does not after two-week unloading. The tail-suspended model provides a unique opportunity to evaluate the physiological and cellular mechanisms of the skeletal response to unloading and reloading.     

Colony-forming cells and colony-stimulating activity during listeriosis in genetically resistant or susceptible mice

Serum colony-forming activity (CSA) and colony-forming cells (CFC) of resistant (C57BL/10 ScSn) and susceptible (BALB/cJ) mice were studied during Listeria monocytogenes infection. Key findings were also checked in susceptible CBA/H mice. Prompt, bacterial dose-dependent increases in serum CSA were observed in all mice following infection. In response to the same challenge dose, serum CSA increased more in susceptible mice, possibly because rapid bacterial proliferation lead to high bacterial numbers. Thus CSA is not a limiting factor which accounts for the differences in Listeria resistance, but is produced in response to bacterial load. In uninfected mice, there were higher numbers of colony-forming cells in the bone marrow and spleen of resistant mice than in susceptible mice. By 24 hr postinfection there was a sharp drop in total cell numbers including CFC, in the bone marrow of resistant C57BL/10 ScSn mice. This coincides with the time when monocytes have been first observed in the blood of infected mice and when differences in bacterial growth between the mouse strains were first observable. Since the superior resistance of C57BL/10 mice has been shown to be radiosensitive, it is probable that this larger, readily mobilized reserve of monocyte/granulocyte precursors in the resistant mice plays an important role in early control of infection. The significance of this is discussed.     

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.     

Effects of purified recombinant human interleukin-3 on the growth of human hematopoietic progenitor cells in "long-term" bone marrow culture

Recombinant human interleukin-3 (rhuIL-3) was assessed for its effects on the growth of normal human hematopoietic bone marrow nucleated cells, and on granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells in a liquid culture system which allows for the prolonged growth of these cells in vitro. RhuIL-3, at concentrations of 100 and 500 units/mL, significantly enhanced the numbers of nucleated cells, as well as the numbers of supernatant and adherent CFU-GM and BFU-E growing in tissue culture flasks or dishes over a period of 4 to 6 weeks. The results demonstrated the rhuIL-3 has a stimulating effect on the growth of human marrow cells in prolonged culture. This information is consistent with the effects of rhuIL-3 in short-term marrow colony assays in vitro and with the in vivo actions of recombinant murine IL-3 in mice, and may be of relevance to clinical trials that will be assessing the hematopoietic effects of rhuIL-3 in humans.     

Conservation of bone marrow CFUc in different phases of the cell cycle during cryopreservation

The effect of low temperature (-196 degrees C) preservation on the recovery of colon-forming units (CFUs) of bone marrow at different phases of the cell cycle before cryopreservation is dealt with. The intact bone marrow "enriched" with CFUs in S phase of the cell cycle and the bone marrow without colony-forming units in S phase were exposed to cryopreservation. After cryopreservation of the bone marrow enriched with CFUs in S phase and th bone marrow without colony-forming units in S phase the number of CFUs decreases by the same value as in the cryopreserved bone marrow obtained from intact mice.     

Effects of chorionic gonadotropin on the hematopoietic stem cells

It was shown on the exogenic colony-forming unit (CFU) assay that the chorionic gonadotropin (CG) administration to female mice CBA in doses correlating with its concentration in different stages of woman pregnancy stimulated (depending upon the doses) the CFU formation of bone marrow, but not spleen origin. Injections of CG to the ovariectomized mice has the opposite (inhibited) effect on the CFU contents in bone marrow and spleen. CG-administration in the dose of 40 U1 to the ovariectomized and non-castrated irradiated recipients bone marrow cells stimulates (statistically significant) colonies formation. As for 200 U1 dose hormone has the similar effect only on the non-castrated animals.     

Effect of Hyperthyroidism On Haemopoietic Stem Cell Kinetics In Mice

Changes in the pool of haemopoietic colony-forming units (CFUs) of bone marrow and spleen were studied in experiments with mice fed dried thyroid gland (TH) for 21 days, and during the 13 days that followed feeding. After HU treatment, the number of CFUs in DNA synthesis was estimated. As early as the second day of TH treatment, the pool of CFUs is gradually increased, leading to an increase in the total number of splenic and bone marrow CFUs persisting after TH treatment for the period examined. Simultaneously, the numbers of nucleated cells in the bone marrow and spleen are increased. During TH feeding and following its termination, the total number of erythrocytes and the haematocrit values did not change significantly, whereas an increased number of leucocytes was observed in the peripheral blood after TH treatment. Elevation of the proliferative activity of CFUs occurred early in the period of TH treatment, with the maximum attained by end of the first week of TH feeding. This suggests a rapid response of the haemopoietic stem cell compartment to the administration of TH hormones. the participation of humoral factors controlling CFUs compartments in the mechanism of the stimulatory effect of TH hormones on haemopoietic stem cells is discussed.     

B lymphocyte differentiation in lethally irradiated and reconstituted mice. I. The effect of Strontium-89 induced bone marrow aplasia on the recovery of the B cell compartment in the spleen.

The influence of ⁸⁹Sr-treatment on the recovery of the B cell compartment in lethally irradiated, fetal liver reconstituted mice was studied by means of membrane fluorescence. ⁸⁹Sr is a bone-seeking radio-isotope which causes in a dose of 3 μCi ⁸⁹Sr/g body weight a depletion of all nucleated cells, including immunoglobulin-bearing (B) cells, of the bone marrow.Treatment of irradiated and fetal liver reconstituted mice with 3 μCi ⁸⁹Sr/g body weight immediately and at 17 days after irradiation and reconstitution prevented recovery of the nucleated cell population, including B cells, in the bone marrow. In the spleen of such mice both nucleated cells and B cells reappeared at day 7 and 14 respectively. The B cell population in the spleen did not recover up to normal values during the experimental period of 45 days. It is concluded that B cell differentiation in lethally irradiated, fetal liver reconstituted mice can take place outside the bone marrow. The efficiency of this extra-medullary differentiation is discussed. The conclusion was drawn that mice with a ⁸⁹Sr-induced bone marrow aplasia are able to generate B lymphocytes. Consequently the bone marrow microenvironment seems not to be obligate to the differentiation of B lymphocytes. The peripheral lymphoid organs of such mice were found to be unable to compensate completely for the absence of B lymphocyte production in the bone marrow.     

Effects of Trichinella spiralis infections upon bone marrow stem cells in BALB/c mice

Trichinella spiralis infections provoke a variety of responses in the host, some of which involve stem cell proliferation and myeloid cell maturation, increases in the mast cell precursor cell populations, and maturation and eosinopoiesis. Very little is known about the influence of T. spiralis upon bone marrow stem cells and splenic colony formation. In the present communication we report that T. spiralis infection in mice stimulates the generation of colony-forming units in the spleen (CFU-S). Passive transfer of bone marrow cells from uninfected BALB/c mice to X-irradiated (650 R) T. spiralis-infected recipients resulted in a significant increase of CFU-S at 14 and 24 days postinfection. Passive transfer of bone marrow cells from T. spiralis-infected mice to X-irradiated uninfected mice also resulted in increased numbers of CFU-S in the donor mice at 24 days postinfection. These findings strongly suggest that T. spiralis infection conditions the microenvironment in the spleen which stimulates CFU-S.     

小鼠造血干细胞增殖与分化性能的研究

本文选择Ｙ染色体特异的性别决定基因作为新的细胞遗传标志,采用ＰＣＲ技术研究了小鼠造血干细胞的增殖与分化性能。将雄鼠骨髓细胞输注给经致死剂量射线照射的雌性受体小鼠、ＰＣＲ测试结果表明,所有ＣＦＵ－Ｓ均为供体起源。供体来源的ＣＦＵ－Ｓ在其输入体内后,可通过增殖,分化形成各系造血细胞,但ＣＦＵ－Ｓ中的纤维母细胞和ＣＦＵ－Ｓ重建造血后受体小鼠骨髓中的纤维母细胞均为受体起源。由此可见,小鼠骨髓中的ＣＦＣ－Ｓ     

Haemopoietic progenitors in different parts of one femur perform different functions during regeneration

Femoral haemopoietic tissue was divided into cells released by flushing and cells released by grinding and washing flushed femora. The flushed femur contained 5 times more nucleated cells than the ground femur, 40 times more macrophage colony-forming cells and 6 times more developmentally late, day 8, and developmentally early, day 13, spleen colony-forming cells. However, the ground femur contained 2 times more developmentally early high proliferation potential colony-forming cells and 3 times more late ones. Haemopoietic regeneration of mice treated with fluorouracil was compared in samples obtained by flushing alone and grinding flushed femora. The number of nucleated cells recovered by flushing fell thirteen-fold by the sixth day after administration of the drug and the number recovered by grinding fell six-fold by the eighth day. Developmentally early high-proliferation-potential colony-forming cells which were recovered by grinding doubled their number in half the time taken by similar cells recovered by flushing. These observations are consistent with haemopoietic cells in different parts of the same bone performing different functions during regeneration. Large numbers of high-proliferation-potential colony-forming cells were not found in the circulation until 8 days after treatment with fluorouracil. Five days after mice had been treated with fluorouracil, when their blood forming systems were regenerating, early high-proliferation-potential colony-forming cells in one sample of marrow were derived from different founder cells than were late cells in the same sample. At the same time, early high-proliferation-potential colony-forming cells in the ground sample of a femur were derived from different founder cells than were cells at the same stage of development in the flushed sample of the femur. These observations are consistent with the view that haemopoietic regeneration after treatment with fluorouracil is due to the growth of few founder cells whose progeny have migrated little within 5 days of drug treatment.     

Age-related changes in hematopoietic stem cell populations of a long-lived hybrid mouse

Age-related changes in the number and concentration of pluripotential and unipotential hematopoietic stem cells in the femoral bone marrow and spleen of BC3F₁ mice were investigated. Pluripotential stem cells were assayed by the spleen colony technique, and unipotential stem cells were determined by an agar cloning method and by erythropoietin responsiveness in polycythemic mice. Changes with senescence were observed in the concentration of both uni- and pluripotential stem cells in the bone marrow; the size of the stem cell compartment in the marrow did not change significantly with age. Also, a reduction in the seeding of transplanted spleen colony-forming units into the spleens of aged recipients was demonstrated. The implications of these findings for the kinetics of hematopoietic stem cell proliferation in aged animals are discussed.     

Rapid method for improving slide quality in the bone marrow micronucleus assay; an adapted cellulose column procedure

Micronuclei are routinely scored in anucleate erythrocytes in bone marrow smears stained with acridine orange. Intense fluorescence from the many nucleated cells in the preparations can interfere with micronucleus detection and cause fatigue in the reader. A method for removing nucleated cells by filtering bone marrow through cellulose packed in syringes was developed by Romagna some ten years ago, but has not been used routinely because of the excessive time needed to prepare columns. We have modified the method very simply by filling chromatography columns by pipet with a cellulose suspension. We show here that column filtration of bone marrow does not affect the numbers of micronucleated polychromatic erythrocytes (MN-PCEs) scored from mice treated with the chromosome breaking agents mitomycin C and cyclophosphamide, or the aneuploidy-inducing spindle poisons, colchicine and vinblastine. The extra preparation time is only about half an hour for a full scale micronucleus assay, and results in better slides and faster scoring.     

Prethymic T cell precursors express receptors for antigen

An anti-idiotype serum raised in BALB/c mice against syngeneic lymph node T cells from 2,4-dinitrofluorobenzene (DNFB)-sensitized mice was used to study the early expression of antigen receptors on developing T cells. Normal BALB/c bone marrow cells were treated with either anti-Thy-1.2 plus complement or anti-Thy-1.2 and anti-idiotype plus complement before use in the reconstitution of lethally irradiated syngeneic mice. Five weeks after reconstitution, recipient mice were assayed for both contact sensitivity (CS) and in vitro proliferative responses to DNFB. Mice reconstituted with bone marrow cells treated with both anti-Thy and anti-idiotype sera showed a significant decrease in reactivity to DNFB in both assay systems when compared with mice reconstituted with marrow treated with anti-Thy only. CS response to the noncross-reacting hapten oxazolone was identical in both recipient groups. Bone marrow mixing experiments showed no evidence of anti-idiotype-induced suppressor cells in these experiments. These data provide strong evidence that at least some T cell precursors express receptors for antigen prethymically.     

Radiosensitivity of clonogenic granulocytic macrophage cell precursors in the bone marrow and spleen of tumor-bearing mice

Clonogenic granulocytic macrophagal cells-precursors (CFU-DC) of bone marrow and spleen of intact C57Bl/6 mice and those inoculated subcutaneously with LLC tumor cells do not substantially differ in their radiosensitivity; the concentration of CFU-DC in the spleen markedly varies as tumor grows. The values of Do and extrapolation number n for CFU-DC of the bone marrow are 0.9-1.4 and 1.5-3.0 Gy, and of the spleen, 0.8-1.6 and 1.0-2.6 Gy, respectively.     

Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10⁵ bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.