Differentiation of stem cells from different parts of the hematopoietic system of adult thymectomized CBA mice stimulated with thymarin or cortexine

Essential differences were detected in differentiation of GFUs from bone marrow and peripheral blood. It was shown that as a result of thymectomy of adult animals the ability of bone marrow CFUs to form granulocytic colonies decreased and that of splenic CFUs to form erythroid colonies increased. The immunostimulating low-molecular-weight polypeptides, thymarin and cortexine , normalized the differentiation of CFUs from bone marrow and spleen but interfered with the formation of erythroid colonies from CFUs of peripheral blood of thymectomized mice.     

Differentiation characteristics of circulating and bone marrow hematopoietic stem cells and the effect of thymus factors

Circulating hemopoietic stem cells (HSC) considerably differ from bone marrow HSC in active erythroid differentiation. After thymectomy of adult animals the number and differentiation of blood HSC remain unchanged, whereas during the cloning of bone marrow cells, a decrease in the number of granulocytic colonies is revealed. In in-vitro experiments, thymalin does not influence the number or differentiation of circulating HSC. On the contrary, in experiments made in vivo, it dramatically lowers erythroid specialization of blood HSC in thymectomized and sham-operated mice, which is followed by the diminution of the total number of circulating HSC. Differentiation of thymectomized mice bone marrow stem cells is completely normalized after thymalin injection. Sham-operated and thymectomized animals' HSC stimulated by thymalin injection become similar to bone marrow cells of normal mice as regards the trend of differentiation. Thymalin injection is likely to change the bone marrow HSC differentiation profile, thereby preventing the release of the cells with erythroid-oriented differentiation from the bone marrow to blood. The influence of thymalin on HSC is mediated by the environmental component which is present in the bone marrow and absent from the peripheral blood.     

Effect of the U-2 fraction of splenic extract from Horsfield's terrapin on the hematopoiesis of mammals

Intraperitoneal administration of a spleen extract from Testudo horsfieldi and its U-2 fraction increases the number of endogenous splenic haemopoietic colonies. The U-2 fraction administered to irradiated (4 Gy) mice increases the number of bone marrow CFUs. Bone marrow cells of exposed (4 Gy) mice preincubated in vitro with the U-2 fraction also increase the number of exogenous colonies in the recipient's spleen.     

The influence of dietary protein deficiency on haemopoietic cells in the mouse.

These experiments examined the effect of a diet limited only in protein (4% by weight) on haemopoietic stem cells in mice. This diet places severe restrictions on growth and cell proliferation and this was reflected in lower numbers of colony forming units (CFUs) and in vitro colony forming cells (CFCs). Differences were apparent in the response of different organs to this stress; for instance, the incidence of spleen CFUs fell sharply from around 40/mg spleen tissue to 1-4/mg spleen tissue after 3 weeks on a low protein diet. This selective loss did not occur in bone marrow where total CFUs remained proportional to cellular content. Yet a third pattern was shown by thymus CFUs--although the numbers were low these increased from 16/thymus in normal mice to 132/thymus in deprived mice. This was the only organ examined which showed an increase. The effects of a return to a high protein (18%) diet showed that the spleen was the most responsive organ. By day 5 after the return to 18% protein the spleen contained as many CFUs per million cells as the bone marrow. During this time the content of CFU in the spleen had increased some 50-fold whereas bone marrow CFUs only doubled. The spleen assumes the major reconstructive role during the refeeding process.     

DISTRIBUTION AND MULTIPLICATION OF COLONY FORMING UNITS FROM BONE MARROW AND SPLEEN AFTER INJECTION IN IRRADIATED MICE

The distribution and proliferation of CFUs from bone marrow and spleen cell suspensions were followed after injection in lethally irradiated isogeneic mice. It was found that a larger proportion of the injected bone marrow CFUs than of the spleen derived CFUs could be recovered from the recipient's spleen and femur. This consistently higher recovery points to the conclusion that a larger fraction of bone marrow-derived CFUs than of spleen-derived CFUs is capable of producing daughter CFUs, most likely due to a commitment to early differentiation of many spleen CFUs.     

Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.     

Effect of tissue macrophage destruction products on hematopoietic stem cells]

Products of mouse peritoneal macrophage destruction (PMD) obtained by aseptic freezing-thawing of the cells, repeated thrice, were found to elicit in syngeneic mice injected with PMD intraperitoneally an increase of CFUs count in the hemopoietic bone marrow tissue and the spleen, as demonstrated by the Till and McCullooch technique. This proved to be a true increase since the transplatned stem cell fraction sorbed by the recipient's spleen was relatively lower in donor mice given PMD than in the control. Although PMD caused an increase of both erythropoietic (E) and granulocytopoietic-monocytic (G) colonies number, the E/G ratio was decreased; one of the mechanisms of the described effect could be the influence of PMD on the hemopoiesis-inducing microenvironment, as the same effects were obtained in mice injected repeatedly with PMD prior to the transplantation of bone marrow tissue of normal donors. Other possible mechanisms of these effects were analyzed, with consideration to the fact that in experiments with preincubation of bone marrow tissue with PMD prior to injection to the lethally irradiated mice no direct stimulating influence of PMD on the stem cell could be revealed.     

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.     

Possible role of the T-cells in regulating in situ hematopoiesis

The aim of the study was to reveal the possible role of T cells in the negative regulation of hematopoiesis. The main experimental approach included incubation of bone marrow cells obtained from mice of different strains with the anti-serum against a specific marker of suppressor T cells--antigen I-J. Anti-I-Jk serum-treated cells and cells treated with nontoxic normal mouse serum or non-treated cells (controls) were further incubated with complement and tested for their CFUs content, using Till & McCulloch exocolonization technique. Treatment with anti-I-Jk serum had a stimulating effect on the CFUs colony formation in mice of the appropriate haplotype (CBA, AKR, A/Sn) bearing I-Jk, but not I-Jb (CC57Br) allele. The same results were obtained in transfer experiments using spleen cells; only in this case stimulating effect was observed in 7-8-day CFUs, while with the marrow transplant augmentation it was seen both 7-8 and 11-12 days following grafting. The seeding efficiency of CFUs was not changed after incubation with anti-I-J serum. The data prove that indigenous for the spleen and bone marrow of mice cells expressing I-J determinants are involved in the negative regulation of hematopoiesis in situ.     

Modification of the proliferative capacity of transplanted bone marrow colony forming units by changes in the host environment

Regulation of the proliferation of transplanted colony forming units (CFUs) was investigated in lethally irradiated mice, pretreated by methods known to accelerate hemopoietic recovery after sublethal irradiation. Prospective recipients were exposed to either hypoxia, vinblastine or priming irradiation and at different intervals thereafter lethally irradiated and transplanted with bone marrow. Repopulation of CFUs was determined by counting the number of splenic colonies in primary recipients or by retransplantation. Regeneration of grafted CFUs was greatly accelerated and their self-renewal capacity increased in mice grafted within two days after hypoxia. Also the number of splenic colonies formed by grafted syngeneic CFUs as well as by C57BL parent CFUs growing in BC3F1 hosts was significantly increased. The effect was not dependent on the seeding efficiency of CFUs and apparently resulted from hypoxia induced changes in the hosts physiological environment. Proliferative capacity of grafted CFUs increased remarkably in hosts receiving vinblastine two or four days prior to irradiation. Priming irradiation given six days before main irradiation accelerated, given two days before impaired regeneration of CFUs. The increased rate of regeneration was not related to the cellularity of hemopoietic organs at the time of transplantation. The growth of CFUs in diffusion chambers implanted into posthypoxic mice was only slightly improved which does indicate that the accelerated regeneration of CFUs in posthypoxic mice is mainly due to the changes in the hemopoietic microenvironment. A short conditioning of transplanted CFUs by host factor(s) was sufficient to improve regeneration. The results might suggest that the speed of hemopoietic regeneration depends on the number of CFUs being induced to proliferate shordy after irradiation, rather than on the absolute numbers of CFUs available to the organism.     

Mechanism of action of the stem-cell inhibition factor on the formation of exogenous hemopoietic colonies in the spleen of mice

It was established by previous works that thymocytes treated with antilymphocyte serum secrete soluble factor capable of inhibiting exogenous colony formation in the spleen of lethally irradiated mice injected with bone marrow cells treated with the stem cell inhibition factor (SCIF). The purpose of the present investigation was to explore possible mechanisms of SCIF action. Regeneration of erythropoiesis (measured by 59Fe incorporation) in the spleen and bone marrow of mice injected with SCIF-treated bone marrow cells was inhibited as compared with control, while CFUs started proliferating with a 3-day delay. Two hours after SCIF treatment 60% of CFUs entered S phase as judged by hydroxyurea cell kill. The CFUs fraction treated with the SCIF was found to be diminished 3-4-fold as compared with control. The data obtained suggest that SCIF treatment makes CFUs enter 3 phase, which may account for the reduced capacity of CFUs to populate the spleen and to proliferate with a 3-day delay.     

Elimination from bone marrow of a supplementary population participating in the proliferation of pluripotent hematopoietic stem cells using mouse brain antiserum

Changes in the number of spleen exo-colonies and post-radiation repopulation of hematopoietic organs were studied in recipients upon injection of bone marrow treated with anti-brain serum (ABS) with and without thymocytes on days 9-14. It was shown that on days 9-11 colony formation in mice injected bone marrow treated with ABS was much lower than the control level. However, by day 14 the number of colonies increased drastically as compared to the control. Thymocyte supplementation normalized colony formation at any time of observation. Similar pattern is noted in post-radiation repopulation of spleen and bone marrow of mice injected bone marrow pretreated with ABS with or without thymocytes. It is assumed that ABS inactivates bone marrow cells participating in the regulation of CFUs proliferation.     

Proliferation and differentiation of hematopoietic stem cells in hypokinesia]

Using the method of exogenous cloning in vivo of the hemopoietic stem cells of the bone marrow and spleen in the femur and the spleen of mice it was shown that during hypokinesia the kinetics of the stem cells differed in both organs (the spleen and the bone marrow). Differentiation of transplanted stem cells from different sources was unchanged in the spleen, but stem cells of the bone marrow seeding in the femur changed the character of their differentiation in the direction of increase of the erythopoietic function, whereas stem cells of the spleen failed to alter the direction of differentiation.     

THE INFLUENCE OF DIETARY PROTEIN DEFICIENCY ON HAEMOPOIETIC CELLS IN THE MOUSE

These experiments examined the effect of a diet limited only in protein (4% by weight) on haemopoietic stem cells in mice. This diet places severe restrictions on growth and cell proliferation and this was reflected in lower numbers of colony forming units (CFUs) and in vitro colony forming cells (CFCs). Differences were apparent in the response of different organs to this stress; for instance, the incidence of spleen CFUs fell sharply from around 40/mg spleen tissue to 1 -4/mg spleen tissue after 3 weeks on a low protein diet. This selective loss did not occur in bone marrow where total CFUs remained proportional to cellular content. Yet a third pattern was shown by thymus CFUs–although the numbers were low these increased from 16/thymus in normal mice to 132/thymus in deprived mice. This was the only organ examined which showed an increase. The effects of a return to a high protein (18 %) diet showed that the spleen was the most responsive organ. By day 5 after the return to 18% protein the spleen contained as many CFUs per million cells as the bone marrow. During this time the content of CFU in the spleen had increased some 50-fold whereas bone marrow CFUs only doubled. The spleen assumes the major reconstitutive role during the refeeding process.     

Effects of low‐level 50 Hz magnetic fields on the level of host defense and on spleen colony formation

The results of 3 sets of experiments on the effects of 22 μT sinusoidal 50 Hz magnetic fields (MF), applied for 1 h on 5 successive days (1 h/5 days), on the level of host defense and on spleen colony formation are reported. The first set of experiments shows the effects on the number of colony‐forming units (CFUs) on the spleen and on the cellularity of the thymus in mice. The MF exposures resulted in an increase in CFUs which was statistically significant with respect to the controls, but not with respect to the shams. Statistically significant changes in the thymic weight and thymic index with respect to both the controls and the shams were measured 1 h after the last MF exposure. In the second set of experiments, the mice were given a sublethal dose of X‐rays (6 Gy), which was followed by exposure 2 h later to the MF. The MF exposure was repeated at the same time of day for 5 days. The number of colonies per spleen showed a consistent, statistically significant increase with MF exposure and the number of CFUs per femur was decreased. In the third set of experiments, bone marrow was taken from mice which had been exposed to 22 μT fields and injected into mice which had been exposed to a lethal dose of X‐rays (9 Gy). The number of CFUs per femur in the recipient mice was shown to be reduced by a statistically significant amount at 1 and 4 days after injection. Bioelectromagnetics 20:57–63, 1999. © 1999 Wiley‐Liss, Inc.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Organ interactions in the regulation of hematopoiesis: in vitro interactions of bone, thymus, and spleen with bone marrow stem cells in normal, Sl/Sld and W/Wv mice.

Hematopoietic cell differentiation is influenced by organ-dependent microenvironmental factors as well as humoral regulators. A technique is described for examining certain aspects of the hemopoietic inductive microenvironment in vitro. Suspension and agar cultures of mouse bone marrow were used to study the effects of organ stromal factors on cellular proliferation and differentiation. Bone, spleen, and thymus fragments from irradiated mice were placed in direct contact with or separated by a Nuclepore membrane from syngeneic marrow cells growing in suspension cultures. Normal adult mouse bone and spleen influenced granulocytic differentiation as well as cell proliferation. In this system, bone marrow and organ fragments from W/Wv and SlSld mice behaved like those of their non-anemic littermates. The most prominent difference between W/Wv and Sl/Sla mice and their normal counterparts was observed in the inductionof CFU-C from splenic precursors un-er the influence of CSA. In both types of anemic mice, in vitro generation of CFU-C from spleen was abnormal in young animals but was corrected by four months of age.     

Colony-forming activity of hematopoietic stem cells in tolerant mice as affected by antigens

The effect of sheep red blood cells (SRBC) and human red blood cells (HRBC) on the amount of CFUs in the bone marrow and spleen of (CBA X C57BL/6) FI SRBC-tolerant mice was studied. The increase in the number of bone marrow and spleen CFUs was demonstrated in SRBC-tolerant mice injected with HRBC. Using SRBC test injection the increase in CFUs amount was observed in the spleen, but not the bone marrow, where the amount of CFUs remained unchanged.     

Cramp过表达对小鼠骨髓造血干细胞功能的影响

目的研究Cramp蛋白过表达对小鼠骨髓造血干细胞自我更新和分化能力的影响。方法应用流式细胞仪分析Cramp过表达转基因小鼠及同龄野生型小鼠的骨髓、脾脏、胸腺等组织器官中各种细胞的比例;分选骨髓造血干细胞,体外培养,观察其克隆形成能力。结果与野生型小鼠相比,Cramp过表达转基因小鼠的骨髓、脾脏、胸腺等组织器官中各种细胞的比例、骨髓造血干细胞的克隆形成能力等均无明显变化。结论本研究中,Cramp过表达转基因小鼠骨髓造血干细胞的分化能力、克隆形成能力无明显变化。     

The role of the hemopoietic microenvironment on the hemopoiesis-stimulating effect of cystamine

The influence of cystamine delivered in a radioprotective dose before and after irradiation of mouse-recipients (8 Gy) on the effectiveness of exogenous bone marrow cloning has been investigated. Cystamine administered prior to irradiation exerts a protective effect on CFUs and also causes an increase in the number of splenic colonies grown from CFUs of the transplanted bone marrow. With cystamine administered after irradiation the protective effect is absent, but the CFUs number in the femur increases in recipients transplanted with intact bone marrow in comparison with those transplanted without cystamine. It is believed, that in addition to the specific protective mechanism of action of radioprotectors, there is a nonspecific mechanism of increasing the proliferation of protected stem cells that is connected with the stimulatory effect of radioprotective agents on the haemopoietic stroma elements.