Separation of osteoblast-like cells from bone marrow by fluorescence-activated cell sorting

The purification of the osteoblast-like cells (2-3%) among the bone marrow cells (BMC) of C57BL/6 mice using a specific anti-osteoblast serum and a fluorescence-activated cell sorter is described. The antiserum was raised against osteoblast cells isolated from calvaria from neonatal mice. The majority of the cells of the osteoblast-enriched fraction from bone marrow showed a parathormone-induced increase in cyclic adenine monophosphate but no response to calcitonin. This is similar to the response of osteoblast cells obtained from the calvaria. Electron microscopic studies of the extracellular matrix of cultured osteoblast-like cells purified from bone marrow showed the deposition of apatite crystals within and in close apposition to the vesicles. These findings suggest that the isolated cell population was enriched in osteoblasts. Such a cell system from bone marrow might provide an experimental system for investigating the mechanism of bone formation.     

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.     

Functional heterogeneity of murine macrophage precursor cells from spleen and bone marrow

We previously reported that highly purified bone marrow-derived macrophage precursors can exert strong spontaneous cytotoxicity against YAC-1 tumor cells, Candida albicans, and protozoa of the genus Leishmania. In the present paper, evidence is shown that macrophage precursors in normal untreated mice are not confined to the bone marrow compartment but can also be found in the spleen. These organ-associated cells, which have the same buoyant density as large granular lymphocytes, have been positively sorted by means of an indirect rosetting technique employing the macrophage-specific monoclonal antibodies F4/80 and M143. The rosetting fractions represented an extremely homogeneous population of macrophage precursors characterized by high candidacidal and natural killer activity and by a strong proliferative response to the macrophage-specific colony-stimulating factor CSF-1. Spleen- and bone marrow-derived macrophage precursors differed in their target selectivity. In addition, the mature macrophages derived in vitro from these two precursor populations displayed striking differences in their candidacidal activity. The implications of these findings in relationship to heterogeneity in the macrophage differentiation line are discussed.     

Hybrid resistance to precursor cells of bone marrow stroma]

A focusl of hemopoiesis appearing after the transplantation of a bone marrow fragment of C57BL mice to syngeneic mice (under the kidney capsule) contained more hemopoietic cells than in transplantation to the semisyngeneic (CBA X C57BL) FI recipient. Experiments were conducted with a secondary seeding by intravenous injection of hemopoietic cells of the C57BL transplant genotype into the transplant depopulated by irradiation; it was shown that these differences were caused by lesser dimensions of the hemopoietic microenvironment in the focus in the hybrid organism in comparison with such in the syngeneic system. Thus, the hybrid resistance was expressed not only to the hemopoietic cells, but also to the stromal precursors transferring the hemopoietic microenvironment.     

IL-3 induces differentiation of bone marrow precursor cells to osteoclast-like cells

IL-3, a cytokine with hematopoietic differentiating capability, induced murine bone marrow cells to differentiate into cells resembling osteoclasts. The cells resulting from treatment with IL-3 were multi-nucleated and demonstrated tartrate-resistant acid-phosphatase activity, as do resident osteoclasts found in bone. IL-3-induced osteoclast-like cell development in the absence of serum-derived vitamin D metabolites, and a mAb that inhibited IL-3-induced proliferation of an addicted cell line also inhibited the development of osteoclasts in the presence of IL-3. The same Ab had no effect on 1 alpha, 25-dihydroxyvitamin D3-induced differentiation of osteoclasts. This newly described function of IL-3 may indicate a role for activated T cells in the bone resorption seen with rheumatoid arthritis.     

Effect of bone marrow suppression on granulocyte opsonin levels.

Levels of serum opsonin for neutrophilic granulocytes were measured in dogs made neutropenic by cyclophosphamide administration. Heat-labile opsonin became elevated within 24 hr following cyclophosphamide (P less than 0.005) and remained elevated over the 4-5 day period of observation (P less than 0.005). In contrast, heat stable opsonin was not significantly effected. Bone marrow suppression by X-ray and busulfan also caused serum opsonin levels to increase. Changes in the levels of IgG, C3, and total hemolytic complement during the course of bone marrow suppression did not correlate with the granulocyte opsonin levels. These findings suggest that serum granulocyte opsonin levels respond to bone marrow suppression and may provide an improved environment for the function of transfused granulocytes.     

The maturation state of three types of granulocyte/macrophage progenitor cells from mouse bone marrow

The progenitor cells of neutrophil granulocytes and macrophages which are able to proliferate and differentiate in vitro (CFU-c) form a heterogeneous population. By the use of specific colony stimulating activities and cell separation by equilibrium density centrifugation, three subpopulations of CFU-c can be detected. These three CFU-c are characterized by buoyant densities of 1.070, 1.075 and 1.080 g.cm^?3 and by their proliferative response to 18 h postendotoxin serum, colony stimulating factor from extracts of mouse embryos and uteri (CSF-pmue) and erythrocyte lysate, respectively. The three CFU-c are compared with respect to their differentiation potential, the maturation rate of their progeny cells and their proliferation capacity. It is shown that with increasing density of the CFU-c the maturation rate increases (sequential maturation of colonies derived from CFU-c with densities of 1.080, 1.075, 1.070 g.cm^?3) and the proliferation capacity decreases (colony size decreases in the sequence of CFU-c with densities 1.070, 1.075, 1.080 g.cm^?3). Concerning the differentiation potential it is shown that all three CFU-c detected have the capacity to form granulocytes as well as macrophages. On the basis of these results it is concluded that the CFU-c with densities of 1.070, 1.075 and 1.080 g.cm^?3 represent a maturation sequence.     

Hematopoietic precursor cells in radiation chimeras restored by bone marrow from thymectomized mice]

Radioprotective capacity of bone marrow CFUs of adult thymectomized mice was studied. Lethally irradiated mice were inoculated with bone marrow of mice thymectomized 8-11 months before. The colony forming capacity and proliferative rate of CFUs were studied 1-7.5 months after obtaining the radiation chimeras. It has been shown that proliferative capacity of bone marrow of adult thymectomized mice was reduced in comparison with that of normal animals. It is related to the decrease (4-fold) of the proliferative rate of bone marrow of thymectomized mice which was inoculated into lethally irradiated recipients 1 month before. We also found that the content of CFUs in bone of those chimeras was reduced later--after 7.5 months. In this period (1-7.5 months) the cellularity of bone marrow did not change.     

Detection of murine bone marrow granulocyte/macrophage progenitor cells (GM-CFU) in serum-free cultures stimulated with purified M-CSF or GM-CSF

Colony formation by mouse granulocyte/macrophage progenitors (GM-CFU) responding to purified colony-stimulating factors (CSF) in serum-free cultures is described. Analysis of the lipid requirements for colony growth stimulated by purified macrophage CSF (M-CSF) demonstrated that cholesterol is essential. Linoleic acid further promoted colony growth only if cholesterol was present, but phospholipid was inhibitory. More colonies were obtained in serum-free cultures, than in serum-supplemented controls. This difference could not be attributed to a change in the range of sensitivity to M-CSF. Stimulation of GM-CFU with granulocyte/macrophage CSF (GM-CSF) required further supplementation with hydrocortisone for optimal expression of colony-forming capacity in serum-free cultures. Hydrocortisone slightly inhibited colony growth stimulated with M-CSF. Under these culture conditions, the number of GM-CFU responding to GM-CSF was twice that obtained with M-CSF.     

In vitro immune response by murine bone marrow cells stimulated against soluble immune complexes.

Synchronized nonadherent bone marrow lymphocytes were stimulated with soluble immune complexes, in antigen excess formed by C3H/HeJ antibodies and various noncross-reacting protein antigens, in a suspension culture which allowed longterm cultivation. On binding of these complexes, lymphocytes underwent blast transformation with mitosis and formation of plasma cells which secreted specific antibodies to the antigen; a cyclic sequence of lymphocytes, blasts, and plasma cells was observed until the majority of the cell population appeared to be plasma cells. The relative percentage of mature plasma cells then decreased leaving mostly small lymphoid cells among which evidence suggests the presence of memory cells. Complexes at equivalence stimulated for the first few days, whereas antibody excess caused stimulation only initially followed by inhibition of the response. Antibodies passively added to the cultures inhibited the proliferative reaction; free antigen induced a typical secondary-type response.     

Pre-B cells in mouse bone marrow: in vitro maturation of peanut agglutinin binding B lymphocyte precursors separated from bone marrow by fluorescence-activated cell sorting

Peanut agglutinin (PNA) binding by mouse bone marrow cells and fractionation by the fluorescence-activated cell sorter have previously been shown to separate high concentrations of pre-B cells, as identified by cytoplasmic mu-chains (c mu). PNA+ and PNA- marrow cell fractions have now been assayed for the presence of functional pre-B cells able to generate mature B cells in culture, as defined by three criteria, the appearance of cell surface mu-chains (s mu), immunoglobulin secretion in response to bacterial lipopolysaccharides, and B cell colony formation. Small PNA+ cell fractions contained pre-B cells that developed into mature B lymphocytes in 1/2 to 1 day but did not sustain B cell production. Large PNA+ cells included pre-B cells that gave rise to mature B lymphocytes after an interval of 1 1/2 to 3 days and were able to sustain B cell genesis in vitro for at least 3 to 5 days thereafter. PNA- cell fractions contained mature B cells but lacked pre-B cell activity. The results demonstrate that PNA binding allows the separation of functional subsets of pre-B cells from bone marrow and that the three in vitro assays used in this study are closely comparable with one another as functional pre-B cell criteria. The findings suggest correlations between functional assays, c mu expression, PNA receptors, and cell size in characterizing stages of pre-B cell development.     

Establishment of myoid cells from bone marrow

A peculiar adherent cell clone (R613BM) was established under muscle tissue free conditions from bone marrow of a Wistar rat. The cloned cell line was able to form myofibrils and expressed nicotinic acetylcholine receptors specific for skeletal muscles. The muscle specific characteristics have been maintained consistently for more than five years. These results suggest that bone marrow contains a precursor cell which has the potency to differentiate into muscle cells.     

Seasonal fluctuations in the activity of stromal precursor cells of human bone marrow

The results of the cloning of fibroblastic colony-forming units (CFU-F) from the bone marrow of normal sites of the spongy bones were analysed in 250 orthopaedic patients. It has been shown that the activity of CFU-F was changing during a year. The number of negative results of CFU-F's cloning were 33%, 60% and 50% in March, April and October respectively. The absolute values of CFU-F cloning were lower in March and April than in other months. The seasonal changes in the activity of CFU-F in human bone marrow should be taken into consideration when studying the physiology and pathology of the bone and hemopoietic system, and in clinical practice.     

Development of osteogenic tissue in diffusion chambers from early precursor cells in bone marrow of adult rats

Diffusion chambers containing bone marrow cells from adult rats were implanted intraperitoneally into rat hosts and cultured in vivo for up to 64 days. Biochemical and histological analyses of the contents of the chambers demonstrate that a connective tissue consisting of bone, cartilage and fibrous tissues is formed by precursor cells present in marrow stroma. The amounts of osteogenic tissue and DNA are directly correlated with time of implantation and with number of cells inoculated. In the chambers there is initial formation of fibrous tissue which is strongly reactive to collagen type III, laminin and fibronectin. In areas of osteogenesis which appear later within this fibrous anlage, expression of collagen type III, laminin and fibronectin decrease and collagen types I and II increase in association with bone and cartilage respectively. Where osteogenesis does not develop, fibrous tissue continues to express collagen type III. The sequential expression of the different extracellular matrix components is similar to that previously observed during osteogenic differentiation in embryonic and adult developmental systems. It is concluded that the formation of fibrous and osteogenic tissues in diffusion chambers by precursor cells present in adult marrow, resembles the normal developmental process.     

Inhibition of the growth of IL-3-dependent mast cells from murine bone marrow by recombinant granulocyte macrophage-colony-stimulating factor

The mouse mast cell line PT-18 demonstrates [3H] thymidine uptake in the presence of either mouse IL-3 or mouse recombinant granulocyte-macrophage CSF (rGM-CSF). Experiments were thus undertaken to determine whether rGM-CSF would affect IL-3-dependent growth of mast cells from mouse bone marrow cells (BMC). BMC placed in liquid culture containing 50 U/ml of IL-3 gave rise to cultures containing up to 95% mast cells by 2 to 3 wk. The rise in percentage of mast cells was accompanied by an increase in total cell-associated histamine. In contrast, BMC grown in the presence of 50 U/ml of rGM-CSF gave rise to cultures containing primarily macrophages and granulocytes with less than 1% mast cells. The addition of increasing amounts of rGM-CSF to BMC cultures grown in the presence of IL-3 resulted in a decrease in the number of mast cells present in culture at 2 to 3 wk. Cells other than mast cells in these cultures consisted principally of granulocytes and macrophages. The rGM-CSF-related inhibition of mast cell growth was not abrogated by the addition of indomethacin to cultures. Granulocyte-macrophage cell populations added to IL-3-containing cultures did not inhibit mast cell growth. The suppressive effect of rGM-CSF on IL-3-dependent mast cell growth may indicate an important role for GM-CSF in the down-regulation of mast cell proliferation in tissues.     

Poly(I:C) induce bone marrow precursor cells into myeloid-derived suppressor cells

Dendritic cells (DC) and myeloid-derived suppressor cells (MDSC) are important cells involved in immune response. DC can be generated from mouse bone marrow (BM) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. Recent studies have revealed that combined treatment of bone marrow MDSC with LPS plus IFN-γ inhibited the DC development but enhanced MDSC functions, such as NO release and T cell suppression. In our study, bone marrow precursor cells cultures in GM-CSF and IL-4 were treated with poly(I:C) through the culture, Gr1(+)CD11b(+) cells with MDSC functions, such as NO release and T cell suppression were accumulated in the culture system. Then the similar phenomenon was observed in the vesicular stomatitis virus infection in vivo. In conclusion, we demonstrated that the bone marrow precursor cells in the presence of GM-CSF and IL-4 can differentiate into MDSC, which is dependent on the dynamic of interaction with poly(I:C).