Effect of different temperature fractions of RNA on allogenic inhibition of hematopoietic stem cells of mouse bone marrow and embryonal liver]

In the system of non-syngeneic transfer of stem hemopoietic cells, the preliminary incubation of the cells of bone marrow or embryonic liver of the C57BL mice with different temperature RNA fractions isolated from the spleen of (CBAXC57BL) F1 was shown to lead to the complete or partial restoration of the colony forming ability of the donor cells. The 63 degrees RNA fraction was shown to have the greatest restorative effect.     

Kinetics of hematopoietic stem cells in the bone marrow of hepatectomized mice

Two-thirds of the liver was removed from (CBA X C57BL/6j) F1 female mice. A significant increase of the number of endogenous colonies count in the spleen of partially hepatectomized mice was observed on the 5-th day after the operation. This increase was not associated with the changes in the number of stem cells in the bone marrow as partial hepatectomy at different times after the operation exerted no effect on the number of colony-forming units (CF1) in the bone marrow.     

Cloning stem cells in the bone marrow of irradiated mice]

Different amount of intact or irradiated bone marrow from syngenous donors was administered to mice irradiated with a lethal dose. There was revealed a linear dependence of the number of the 8-9-day colonies grown in the bone marrow of the femur on the amount of the administered cells, and an exponential dependence on the irradiation dose. Regularity of the stem cell cloning in the bone marrow was analogous to such in the spleen. Radiosensitivity of the colony-forming units (CFU) differed depending on the site (the spleen, the bone marrow) of their colony formation. The CFU settling in the marrow proved to be more radioresistant (D(0) equalled 160-200 P) in comparison with the CFU settling in the spleen (D(0) constituted 80-100 P). It is supposed that a different radiosensitivity of the CFU was caused by the presence of heterogenic population of the stem cells and also by specific peculiarities of the organ (the spleen, the bone marrow) in which the colonies formed.     

The effect of serotonin on the hematopoietic stem cells of the bone marrow]

Haemopoietic stem cells content and proliferative activity were studied in the bone marrow of female F1 (CBA x C57Bl6) mice after single (50 mg/kg) and chronic (0.5 mg/kg daily for 7 days) serotonin (S) injections. It is shown that 9-day and 12-day COEs contents in the bone marrow of experimental mice has been increasing for 24 h after single S injection. After chronic S injections twofold increase of 12-day COEs is observed without any increasing of 9-day COEs. Total myelokaryocyte number is increased too. The study of proliferative status by in vitro incubation of bone marrow cells with ARA-C has shown that the numbers of 9-day and 12-day COEs in S-phase have increased both after single and chronic S injections. Possible mechanisms of stimulating effect of S on bone marrow stem cells are discussed.     

Proliferative and differentiation properties of bone marrow hematopoietic stem cells in CBA mice of various ages

Hemopoietic bone marrow stem cells (CFCs) of young (3-5 months) and old (23-24 months) were studied according to their ability to form colonies in spleens of the lethally irradiated animals. The number, morphology and volume of CFCs were microscopically examined. The content of CFCs remained unchanged during aging. The number of erythroid colonies decreased in old mice, while that of granulocyte-macrophage and mixed colonies did not change. The megakaryocyte colonies showed even an increase with age. Volumes of all the colony types, except megakaryocyte, reduced. The results obtained thus reflect some age-related features of early stages of the stem-cell differentiation.     

Thiophosphamide-induced chromosome aberrations in embryonal liver and bone marrow cells of A/He and C57BL/L mice]

Chromosomal aberrations were studied in cells of embryonic liver and bone marrow of A/He and C57BL/6 mice following mutagenic treatment by the alkylating agent thiophosphamide in g1-S periods of the cell cycle. Higher sensitivity of chromosomes to aberration induction was found in A/He mice.     

Gene expression profile in bone marrow and hematopoietic stem cells in mice exposed to inhaled benzene

Acute myeloid leukemia and chronic lymphocytic leukemia are associated with benzene exposure. In mice, benzene induces chromosomal breaks as a primary mode of genotoxicity in the bone marrow (BM). Benzene-induced DNA lesions can lead to changes in hematopoietic stem cells (HSC) that give rise to leukemic clones. To gain insight into the mechanism of benzene-induced leukemia, we investigated the DNA damage repair and response pathways in total bone marrow and bone marrow fractions enriched for HSC from male 129/SvJ mice exposed to benzene by inhalation. Mice exposed to 100 ppm benzene for 6h per day, 5 days per week for 2 week showed significant hematotoxicity and genotoxicity compared to air-exposed control mice. Benzene exposure did not alter the level of apoptosis in BM or the percentage of HSC in BM. RNA isolated from total BM cells and the enriched HSC fractions from benzene-exposed and air-exposed mice was used for microarray analysis and quantitative real-time RT-PCR. Interestingly, mRNA levels of DNA repair genes representing distinct repair pathways were largely unaffected by benzene exposure, whereas altered mRNA expression of various apoptosis, cell cycle, and growth control genes was observed in samples from benzene-exposed mice. Differences in gene expression profiles were observed between total BM and HSC. Notably, p21 mRNA was highly induced in BM but was not altered in HSC following benzene exposure. The gene expression pattern suggests that HSC isolated immediately following a 2 weeks exposure to 100 ppm benzene were not actively proliferating. Understanding the toxicogenomic profile of the specific target cell population involved in the development of benzene-associated diseases may lead to a better understanding of the mechanism of benzene-induced leukemia and may identify important interindividual and tissue susceptibility factors.     

Differentiation of embryonic stem cells in adult bone marrow

Embryonic stem cells (ESCs) are a potential source of generating transplantable hematopoietic stem and progenitor cells, which in turn can serve as "seed" cells for hematopoietic regeneration. In this study, we aimed to gauge the ability of mouse ESCs directly differentiating into hematopoietic cells in adult bone marrow (BM). To this end, we first derived a new mouse ESC line that constitutively expressed the green fluorescent protein (GFP) and then injected the ESCs into syngeneic BM via intra-tibia. The progeny of the transplanted ESCs were then analyzed at different time points after transplantation. Notably, however, most injected ESCs differentiated into non-hematopoietic cells in the BM whereas only a minority of the cells acquired hematopoietic cell surface markers. This study provides a strategy for evaluating the differentiation potential of ESCs in the BM micro-environment, thereby having important implications for the physiological maintenance and potential therapeutic applications of ESCs.     

Characteristics of the differentiation of hematopoietic stem cells of the bone marrow in experimental lesions of the liver

The liver lesion in the CBA mice has been induced by administration of one of three agents five times every day; gamma-globulin fraction of antihepatocytotoxic serum in doses of 4.8 and 7.7 mg of protein per 100 g of body mass; gamma-globulin fraction of normal rabbit serum and bovine serum albumin in a dose of 4.8 mg of protein; three- four- or five-fold introduction of carbon tetrachloride in a dose of 0.5 ml per 100 g of body mass with oil (1:1) each three days; calibrated stenosis of the portal vein was produced. The total number of hemopoietic stem cells in the bone marrow was estimated by the colony-forming unit/spleen assay. Histological analysis of the colony-forming units was applied. The liver lesion was accompanied by a decrease in the ratio of the erythroid/granulocytic colonies.     

Cellular repair of sublethal radiation damage in 2 subpopulations of the CFUs from embryonal liver and bone marrow of adult mice

The authors studied the ability of the CFU-s, forming colonies on the 8 and 11 day after transplantation of cells from fetal liver (FL) of 14-18 day gestation and adult mouse bone marrow (BM), to repair the sublethal radiation damages (SRD), according to Elkind's model. The ability to repair the SRD of 11-day CFU-s (both EL- and BM-derived) was lower than the ability of 8-day CFU-s. Both subpopulations of CFU-s (as 8-, as 11-day) from FL have a reduced index of SRD reparation as compared with the corresponding meanings for BM.     

Proliferation of hemopoietic stem cells in culture of embryonal liver of mice

The vinblastine technique was used to demonstrate that the proliferative capacity of colony-forming cells in organ culture of embryonal liver of mice between the first and seventeenth day of cultivation is adequately high. The relative content of CFU increases in culture with a maximum between one and two weeks.     

2 populations of bone marrow cells transfering the hematopoietic environment]

A bone marrow fragment transplanted under the kidney capsule created a focus of ectopic hemopoiesis, whose isze, measured by the number of hemopoietic cells, was proportional to the implant size. Dimensions of the focus proved to be 11/2--21/2 greater in the irradiated than in the intact recipients. Cells building up the focus of heterotopic hemopoiesis had a different radiosensitivity in the intact and irradiated recipients--their Do constituted about 160 and 350 rad, respectively. In this connection it is supposed that two cell populations of precursors took part in the creation of the focus. Their possible relations with the determined and inducible osteogenic precursor cells are discussed.     

Effect of syngenic lymphocytes on allogenic inhibition of hematopoietic stem cells of embryonic liver]

The transplantation of liver from the embryos and newborn C57BL-6 mice to the lethally irradiated hybrids (CBA X C57BL/6) F1resulted in 90% allogenic inhibition of the colony-forming activity of the donor elements. The degree of allogenic inhibition of liver cells of 19 days old embryos and newborn mice may be changed with the help of syngenic lymphocytes of adult mice or delayed transplantation of cells 72 hrs following the irradiation of recipients but these procedures proved to be ineffective with the liver cells of 13 and 16 days old embryos. A suggestion is put forward to the effect that the allogenic inhibition is based on the active reaction of recipient hybrids (CBAXXC57BL/6) F1 to the stem hemopoietic cells of C57BL/6 mice.     

Genetic control of allogenic inhibition of hematopoietic stem cells]

Adult mice of C57BL/6, CBA (CBA X C57BL/6) F1, (CBA X C57BL/6) F2, F1 X CBA and F1 X C57BL/6 strains were lethally irradiated and reconstituted with a constant dose of 3-10(5) C57BL/6 bone marrow cells. At the 9th day after the bone marrow transplantation the colony count was performed in spleen of irradiated recipients. In the spleen of F1, CBA and C57BL/6 mice were registered low (0--8, intermediate (6--18) and high (22-40) numbers of colonies respectively. The segregation ratios in F2 progeny were close to 2 (low): 1(intermediate): 1(high). The segregation ratios in backcross (F1 X CBA) were close to 1(low): 1(intermediate)numbers of colonies. Backcrosses (F1 X C57BL/6) were distributed to low and high numbers of colonies with the ratio 1:1. The number of spleen colonies of males and females was the same in all segregating progeny. The results of hybrid analysis suggest that a single pair of allelic genes is involved in genetic control of allogenic inhibition, and that the resistance (manifestation of inhibition) to C57BL/6 stem cells is conferred by the dominant allele.     

The repopulating potential and differentiation capacity of hematopoietic stem cells from the blood and bone marrow of normal mice

Using the hematopoietic colony technique, we have investigated the repopulating potential of bone marrow cells and leukocytes of blood from normal mice and have demonstrated that the frequency of hematopoietic stem cells in bone marrow is 50 to 150 times that of stem cells in the circulating blood. The differentiation capacity of these stem cells has also been examined. Results of comparative studies of the serial sections of hematopoietic colonies formed from marrow and blood leukocytes indicate that the differentiation capacity of stem cells from marrow and blood is similar, and that at least 80% of these cells differentiate along a single cell line. Thus, peripheral blood stem cells can effect a complete hematopoietic graft, establishing in the host, donor red cells, granulocytes, and platelets. The possibility that blood leukocytes may serve as a potential source of stem cells for hematopoietic transplants has been considered. Although blood contains stem cells, their frequency is so low as to make it unlikely that they would become a useful source of precursor cells for transplantation purposes.     

Function and malfunction of hematopoietic stem cells in primary bone marrow failure syndromes

Hematopoietic stem cells (HSCs) are responsible for the production of mature blood cells in bone marrow; peripheral pancytopenia is a common clinical presentation resulting from several different conditions, including hematological or extra-hematological diseases (mostly cancers) affecting the marrow function, as well as primary failure of hematopoiesis. Primary bone marrow failure syndromes are a heterogeneous group of diseases with specific pathogenic mechanisms, which share a profound impairment of the hematopoietic stem cell pool resulting in global or selective marrow aplasia. Constitutional marrow failure syndromes are conditions caused by intrinsic defects of HSCs; they are due to inherited germline mutations accounting for specific phenotypes, and often involve also organs and systems other than hematopoiesis. By contrast, in acquired marrow failure syndromes hematopoietic stem cells are thought to be intrinsically normal, but subjected to an extrinsic damage affecting their hematopoietic function. Direct toxicity by chemicals or radiation, as well as association with viruses and other infectious agents, can be sometimes demonstrated. In idiopathic Aplastic Anemia (AA) immunological mechanisms play a pivotal role in damaging the hematopoietic compartment, resulting in a depletion of the hematopoietic stem cell pool. Clinical and experimental evidences support the presence of a T cell-mediated immune attack, as confirmed by clonally expanded lymphocytes, even if the target antigens are still undefined. However, this simple model has to be integrated with recent data showing that, even in presence of an extrinsic damage, preexisting mutations or polymorphisms of genes may constitute a genetic propensity to develop marrow failure. Other recent data suggest that similar antigen-driven immune mechanisms may be involved in marrow failure associated with lymphoproliferative or autoimmune disorders characterized by clonal expansion of T lymphocytes, such as Large Granular Lymphocyte leukemia. In this wide spectrum, a unique and intriguing condition is Paroxysmal Nocturnal Hemoglobinuria (PNH); even in presence of a somatic mutation of the PIG-A gene carried by one or more HSCs and their progeny, the typical marrow failure in PNH is likely due to pathogenic mechanisms similar to those involved in AA, and not to the intrinsic abnormality conferred to the clonal population by the PIG-A mutation. The study of hematopoietic stem cell function in marrow failure syndromes provides hints for specific molecular pathways disturbed in many diseases of hematopoietic and non-hematopoietic stem cells. Beyond the specific interest of investigators involved in the field of these rare diseases, marrow failure syndromes represent a model that provides intriguing insight into quantity and function of normal hematopoietic stem cells, improving our knowledge on stem cell biology.     

Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche

Hematopoietic stem cells (HSCs) reside and self-renew in the bone marrow (BM) niche. Overall, the signaling that regulates stem cell dormancy in the HSC niche remains controversial. Here, we demonstrate that TGF-β type II receptor-deficient HSCs show low-level Smad activation and impaired long-term repopulating activity, underlining the critical role of TGF-β/Smad signaling in HSC maintenance. TGF-β is produced as a latent form by a variety of cells, so we searched for those that express activator molecules for latent TGF-β. Nonmyelinating Schwann cells in BM proved responsible for activation. These glial cells ensheathed autonomic nerves, expressed HSC niche factor genes, and were in contact with a substantial proportion of HSCs. Autonomic nerve denervation reduced the number of these active TGF-β-producing cells and led to rapid loss of HSCs from BM. We propose that glial cells are components of a BM niche and maintain HSC hibernation by regulating activation of latent TGF-β.