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.     

Spontaneous mutagenesis in bone marrow cells in the CBA and CBA/H-T6 mouse strains

Comparative analysis of cytogenetic characteristics in bone marrow cells of the mouse lines CBA and CBA/H-T6 has been carried out. It was shown that translocation T6 effects the apparatus of cell division and can cause additional cytogenetic abnormalities.     

Stromal precursor cell count in the bone marrow of young and old CBA mice

The content of stromal precursor cells in the bone marrow of young (1-4-month-old) and old (24-30-month-old) CBA mice was measured by cloning in primary monolayer cultures. It was found that both the concentration (fibroblast colony forming efficiency) and the total number of stromal precursors in femoral bone marrow markedly increased with aging.     

Heterogeneity of stromal precursor cells isolated from rat bone marrow

Bone marrow stroma contains mesenchymal stem cells (MSC) which are precursor for at least mesenchyma-derived cells. Recent investigations revealed a lot of questions concerning MSC biology that should be further refined. The aim of this study was the comparative analysis of rat bone marrow stroma cells cultures. Mesenchymal precursor cells isolated from rat bone marrow were passed up to 50 times. Comparative morphological and immunophenotypical analysis of these cultures was carried out as well as their ability to osteogenic differentiation was studied. The isolated cultures contained morphologically different types of cells and thus showed a high heterogenity level. Morphology of these cell types was described. The heterogeneity level was reported to decrease over time. It was found out that subcultures isolated from different rats shared the same immunophenotype characteristics (CD90+, CD44+, CD54+, CD 106+, CD45-, CD11b-), but differed in their morphology as well as in ability to osteogenic differentiation. Thus MSC identification requires more specific marker and functional tests to be used.     

The reduced trabecular bone mass of adult ARKO male mice results from the decreased osteogenic differentiation of bone marrow stroma cells

Male mice with androgen receptor knock-out (ARKO) show significant bone loss at a young age. However, the lasting effect of AR inactivation on bone in aging male mice remains unclear. We designed this study to evaluate the effect of AR on bone quality in aging male mice and to find the possible causes of AR inactivation contributing to the bone loss. The mice were grouped according to their ages and AR status and their trabecular bones were examined by micro-CT analysis at 6, 12, 18, and 30 weeks old. We found that bone mass consistently decreased and the bone microarchitectures continuously deteriorated in male ARKO mice at designated time points. To determine the cause of the bone loss in ARKO mice, we further examined the role of AR in bone cell fate decision and differentiation and we conducted experiments on bone marrow stromal cells (BMSC) obtained from wild type (WT) and AR knockout (KO) mice. We found that ARKO mice had higher numbers of colony formation unit-fibroblast (CFU-F), and CD44 and CD34 positive cells in bone marrow than WT mice. Our Q-RT-PCR results showed lower expression of genes linked to osteogenesis in BMSCs isolated from ARKO mice. In conclusion, AR nullification disrupted bone microarchitecture and caused trabecular bone mass loss in male ARKO mice. And the fate of BMSCs was impacted by the loss of AR. Therefore, these findings suggest that AR may accelerate the use of progenitor cells and direct them into osteogenic differentiation to affect bone metabolism.     

Spindle damaging effect of salbutamol sulphate on bone marrow cells of mice

In vivo assessment and identification of aneuploidy are important phases of genotoxicity evaluation. Considerable effort has been devoted to assess the utility of the existing bioassays and to develop simpler techniques for identifying environmental aneugens. Salbutamol sulphate--an antiasthmatic drug was tested for its spindle damaging effects in bone marrow cells of mice using an in vivo technique, for the evaluation of mitotic index, C-mitotic effects, anaphase reduction and hyperdiploidy. Doses of 0.12, 0.24, 1.2, 2.4, mg/kg body weight were dissolved in bidistilled water and administered intraperitoneally to the mice. Colchicine was taken as positive control for its known aneuploidy-inducing effects. The drug showed positive C-mitotic effects accompanied with increases of mitotic index and decreased frequencies of anaphase in higher doses. Significant levels of hypodiploidy also noted at higher doses. The preliminary results indicated that Salbutamol is capable of inducing C-mitotic effects in mouse bone marrow cells, which is suggestive of possible induction of aneuploidy.     

Clastogenicity effect of ruthenium red in vivo, in bone marrow cells in mice

The ability of ruthenium red to induce chromosome aberrations in vivo in mammalian somatic cells has been evaluated by the micronucleus test on mouse bone marrow cells. Our results show that this compound, when administered i.p. at a sublethal level, can be considered as a weak clastogen.     

The development of catatonic reactions in male mice of CBA/Lac strain: the effect of repeated experience of aggression and submission

The development of catatonic reactions with rigid muscle tension due to stimulation of the skin at the scruff (catatonia-"pinch" test) and wax muscle plasticity (repeated pinch-induced catalepsy displayed on the parallel bars--BAR-test) was investigated in aggressive and submissive CBA/Lac male mice with repeated experiences of social victories (winners) or defeats (losers), accordingly. The expression of catatonic-like state in "pinch" test was significantly more in the losers after 20 daily agonistic confrontations in comparison with the winners. The catalepsy in the BAR-test was increased in animals with experience of agonistic confrontation in comparison with the controls, however expression of catalepsy reaction depended on kind and duration of agonistic interactions. The pronounced freezing predominated in the free behavior of the losers and, on the contrary, the winners demonstrated the abnormal undirected jumping. It was suggested that two contrast forms of catatonic syndrome accompanying by development of akinesia- or hiperkinesia-like states, are developed in the defeated and victorious (accordingly) mice of cataleptic CBA/Lac strain.     

Sl/Sld mice have an increased number of gap junctions in their bone marrow stromal cells

To study the defect of the hematopoietic inductive microenvironment (HIM) in Sl/Sld mice, femoral bone marrow tissue of 10 of each mutant, (Sl/Sld and W/Wv) their normal littermates (Sl+/Sl+ and W+/W+), and 20 normal C57BL mice were examined by electron microscopy using morphometric and statistical methods. Gap junctions were observed in all strains of mice, in the following stromal cell types: 1) reticular cells, 2) between reticular cells and periarterial adventitial cells, and 3) between periarterial adventitial cells. The frequency of gap junctions in bone marrow stromal cells of Sl/Sld mice (mean = 2.2/9.4 X 10(-3) mm2) was significantly higher than in control mice. It is suggested that there is a relationship between the increased numbers of gap junctions in bone marrow stromal cells of Sl/Sld mice and the defect in HIM function in these genetically anemic animals.     

Characteristics of tumors that have developed in mice injected with syngenic irradiated mesenchymal stem cells of bone marrow

Mesenchymal stem cells (MSCs) are found in virtually all organs and tissues. These cells can presumably be transformed into tumor stem cells by genotoxic factors and, subsequently, initiate tumor growth. The aim of the present work consisted in analysis of the possibility of malignant transformation of cultured MSCs from the bone marrow (BM) of mice after in vitro exposure to γ-radiation and in the characterization of biochemical and histological features of tumors that developed after the transplantation of BM MSCs to syngenic mice. Two of five mice developed tumors 3 to 4 months after the subcutaneous injection of BM MSCs irradiated at a dose of 1 Gy, five of five animals developed tumors after the administration of BM MSCs irradiated at a dose of 6 Gy, and only one of five mice injected with nonirradiated BM MSCs developed a tumor 6 months after cell transplantation. Telomerase activity in a tumor that developed from BM MSCs irradiated at a dose of 6 Gy was twice as high as that in the tumor that developed from BM MSCs irradiated at a dose of 1 Gy. The histological structure of the neoplasms corresponded to that of multicomponent mesenchymoma, a malignant tumor also termed “a mix of sarcomas.” The tumors consisted of tissue fragments of different histological types. Thus, BM MSCs exposed to 1 or 6 Gy of radiation can be transformed into tumor cells and give rise to multicomponent mesenchymomas, whereas malignant transformation of control BM MSCs occurs much less often.     

A comparative study on the genotoxic effect of pyrimethamine in bone marrow and spermatogonial mice cells

Pyrimethamine is an antimalarial agent widely used in clinical therapy. We aimed to compare its mutagenic potential in mammalian spermatogonial and bone marrow cells. For studying chromosomal aberrations mice were treated acutely (single treatment) with 4 dose levels of pyrimethamine (5, 10, 20 and 40 mg/kg). Pyrimethamine was found to produce a significant increase in structural chromosomal aberrations after acute treatment in bone marrow cells of mice (p < 0.001). It also induced chromosome abnormalities in spermatogonial cells (p < 0.05) at the highest dose.     

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 stimulation of bone marrow granulopoiesis of normal CBA mice in vitro and in vivo by serum obtained from leucophoretic rats

Abstract. The effect of leucophoretic serum (LS), obtained from rats with polyvinylpyrrolidone (PVP)-induced inflammation, on granulopoiesis in the bone marrow of normal CBA mice was studied. The following test systems were used: short term cultures (4 hr), diffusion chambers (8, 24, 48 and 72 hr) and in vivo assays (12, 24 and 48 hr). The results indicate that LS stimulates the proliferations of granulocytic cells by increasing the number of proliferative granulocytes in mitosis, as well as increasing the total number of proliferative granulocytes. LS did not appear to effect monocytes and other cell lines. It is concluded that a factor present in LS specifically stimulates the proliferation of granulocytic cells, both in vitro and in vivo .     

Characteristics of human mesenchymal stem cells isolated from bone marrow and adipose tissue

Populations of human mesenchymal stem cells were derived from bone marrow and adipose tissue. Here analysis of six individuals is represented. Cells were isolated, expanded and evaluated by the expression of surface antigens using flow cytometry. These cells displayed similar characteristics for many markers. Cells isolated from bone marrow and adipose tissue were found to be homogeneously positive for CD13, CD44, CD90, CD105, and negative for CD45, CD34, CD31 and CD117. Besides, differences in surface antigene CD10 expression between narrow and adipose tissue-derived cells were detected. All these findings indicate that both bone marrow and adipose tissue are important sources of mesenchymal stem cells, which could be used in cell therapy protocols.     

Distribution of the bone marrow cells in the skeleton of mice]

The data on the content of nucleated bone marrow cells in 17 skeletal portions of the hybrid F1 (CBA X C57B1) laboratory mice weighing 18-21 g are presented. The bone marrow content in the bones of the spine, head, inferior limb, pelvis, upper limb, sternum and ribs constituted 33.7; 19.6; 17.6; 11.9; 8.2 and 9.0% of the total amount, respectively. The epxerimental results are compared with the literature data.     

Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow

The enormous plasticity of mesenchymal stem cells (MSCs) suggests an improvement of a standard protocol of isolation and ex vivo expansion for experimental and clinical use. We isolated and expanded MSCs from bone marrow (BM) of pediatric and young adult donors, to analyze the growth kinetic, immunophenotype, telomere length, karyotype during ex vivo expansion. Seventeen BM samples were collected from young adult donors and 8 from pediatric donors. MSCs isolated from two groups showed no morphological differences while their cell growth was strictly related to the donor's age. The MSCs isolated from pediatric donors reached a cumulative PD almost twice as high as MSCs isolated from young adult donors after 112 days (10.2 +/- 1.9 versus 5.5 +/- 3.7). Furthermore, we analyzed the modulation of antigen expression in the MSCs isolated from two groups until 10th passage (77 days) and there was no significant difference between the modulation of antigen expression. In particular, at the first passage, MSCs showed a low contamination of hemopoietic cells which became insignificant in the following passages. There was a high expression of CD90, CD29, CD44 and CD105 and variable and moderate expression of CD166 and CD106 at the start of MSC culture and at each passage during expansion. No chromosomal alteration or evidence of cellular senescence were observed in all analyzed samples. All these data suggest that MSCs can be isolated and expanded from most healthy donors, providing for an autologous source of stem cells.     

The origin of antibody-forming cells detected in the bone marrow after thymus-independent antigen-stimulation and abnormality in migration of B cells of X-linked immunodeficient CBA/N mice

The anti-TNP IgM plaque-forming cells (PFC) were generated in the spleen and bone marrow of non-immunodeficient normal mice after intraperitoneal administration of TNP-LPS. Irradiation of normal mice while shielding bone marrow completely abrogated the generation of bone marrow PFC, indicating that they are derived from extramedullary sites. The bone marrow PFC, response to TNP-LPS was low in X-linked immunodeficient CBA/N strain mice, while the spleen response was comparable to that seen in the normal mice. To further study the basis of the deficient bone marrow PFC response in CBA/N mice, spleen cells were adoptively transferred to irradiated syngeneic mice stimulated with TNP-LPS. While spleen cells from normal mice generated high numbers of PFC in recipient bone marrow and spleen, those from CBA/N strain mice could not generate bone marrow PFC. This result was obtained regardless of whether normal or CBA/N recipients were used. These results indicate that TNP-LPS administration normally results in the migration of B lymphocytes from the periphery into the bone marrow and that B cells from immunodeficient CBA/N strain mice bear an inherent defect in this migratory function. This migratory defect was shown to be X-linked, as are the other previously reported B cell defects in this inbred mouse strain. The possible relationship between this migratory defect and the maturational defects of B cell lineage as reported previously in CBA/N strain mice is discussed.