Production of nitric oxide by murine bone marrow cells. Inverse correlation with cellular proliferation.

The present studies were designed to assess the ability of primary cultures of bone marrow cells to produce nitric oxide. We found that two inflammatory stimuli, IFN-gamma and LPS, were potent inducers of nitric oxide production by bone marrow cells. In addition, the CSF granulocyte-macrophage (GM)-CSF and IL-3 as well as TNF-alpha, while inactive by themselves, were synergistic with LPS and IFN-gamma in inducing nitric oxide production. Maximal effects were observed with combinations of GM-CSF and LPS. Nitric oxide production by bone marrow cells was found to be dependent on the presence of L-arginine in the culture medium and inhibitable by NG-monomethyl-L-arginine and L-canavanine, two nitric oxide synthase inhibitors. Nitric oxide produced by the cells was also suppressed by TGF-beta 1 and the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Separation of bone marrow cells by density gradient centrifugation and flow cytometry revealed that the granulocyte-containing fraction was largely responsible for nitric oxide production. In additional experiments we found that treatment of bone marrow cells with GM-CSF significantly stimulated bone marrow cell growth. In contrast, the combination of GM-CSF and LPS or IFN-gamma markedly suppressed cellular proliferation. This suppression was completely reversed by treatment of the cells with NG-monomethyl-L-arginine. Taken together, these data demonstrate that various inflammatory stimuli and cytokines induce nitric oxide production by primary cultures of bone marrow cells and that this mediator may play a role in the regulation of bone marrow cell growth and development.     

RANKL from bone marrow adipose lineage cells promotes osteoclast formation and bone loss

Receptor activator of NF‐κB ligand (RANKL) is essential for osteoclast formation and bone remodeling. Nevertheless, the cellular source of RANKL for osteoclastogenesis has not been fully uncovered. Different from peripheral adipose tissue, bone marrow (BM) adipose lineage cells originate from bone marrow mesenchymal stromal cells (BMSCs). Here, we demonstrate that adiponectin promoter‐driven Cre expression (Adipoq^Cre ) can target bone marrow adipose lineage cells. We cross the Adipoq^Cre mice with rankl^fl/fl mice to conditionally delete RANKL from BM adipose lineage cells. Conditional deletion of RANKL increases cancellous bone mass of long bones in mice by reducing the formation of trabecular osteoclasts and inhibiting bone resorption but does not affect cortical bone thickness or resorption of calcified cartilage. Adipoq^Cre; rankl^fl/fl mice exhibit resistance to estrogen deficiency and rosiglitazone (ROS)‐induced trabecular bone loss but show bone loss induced by unloading. BM adipose lineage cells therefore represent an essential source of RANKL for the formation of trabecula osteoclasts and resorption of cancellous bone during remodeling under physiological and pathological conditions. Targeting bone marrow adiposity is a promising way of preventing pathological bone loss.     

A spectrophotometric approach for determining sporangium and zoospore viability of Plasmopara viticola

A spectrophotometric method for determining the viability of sporangia and zoospores of the oomycete Plasmopara viticola (causal agent of grapevine downy mildew) is described and evaluated to overcome the limitations of currently available methods for assessing propagule viability. Sporangia produced on leaf discs in the laboratory were harvested at different days after the initiation of sporulation (DAS) to yield differences in sporangium viability. Sporangia were suspended in sterile water, the suspensions were placed in a cuvette, and sporangium germination was monitored in a spectrophotometer (λ = 600 nm) at 2- to 3-min intervals for 5 hr. Absorbance started to increase after sporangia were suspended in water for ~30–60 min followed by major peak(s) for younger sporangia (1–3 DAS), whereas low to no increase in absorbance was observed for senescent sporangia (>7 DAS). Microscopic observation confirmed that the increase in absorbance corresponded to the release and active swimming of zoospores, whereas absorbance decreased when zoospores encysted and settled. A positive correlation (r = .839, p = .0365) was observed when the time to the initial increase in absorbance was plotted against the age of sporangia. The time to the absorbance peak (marking the time of maximum zoospore movement) was shortest for immature sporangia (0 DAS), longest for young sporangia (2 DAS) and decreased for mature and senescent sporangia. A similar pattern was observed for the standardized area under the absorbance curve (indicating the overall quantity of zoospores released), for which values were lowest for immature and senescent sporangia, highest for young sporangia and intermediate for mature sporangia. Consistent patterns obtained across two independent experiments suggest that the method is reproducible and may be further developed for other zoospore-releasing pathogens.     

Chronobiologic approach to beat-to-beat variations of cultured murine myocardial cells.

An earlier demonstration of a circadian rhythm in rat atria by others is complemented herein by observations in culture: A single murine myocardial cell and two sets of grouped cells beating in culture for several days reveal several features of an anticipated, presumably built-in spectrum of multifrequency rhythms and trends, the chronome. Circadian and about 12-h (circasemidian) components are modulated by an approximately 84-h (circasemiseptan) component, which cannot be separated from trends in view of the brevity of the series. The circumstance under which the culture is aging and in which fibroblasts proliferate is a further complication that limits the findings to a single cycle reproduced in three separate cultures. Whether it is a rhythm that repeats itself of a response to placement into culture, an approximately 3.5-d component in the beating of myocardial cells in culture is to be aligned with a very prominent similar component found in the incidence of 85,819 human myocardial infarctions.     

Presence of cells in B-cell lineage in mixed (GEMM) colonies from murine marrow cells

Recent progress in clonal cell culture techniques makes it possible to detect pluripotent hemopoietic precursors from murine marrow cells. The precursors can proliferate, differentiate and form mixed colonies containing erythroblasts, granulocytes, macrophages and often megakaryocytes in viscid culture medium. In the present investigation, the presence of cells of B-cell lineage in mixed colonies was investigated. Experiments on colonies containing cIgM, cIgG, sIgM and sIgG bearing cells using goat IgG fluorescein-conjugated anti-mouse IgM, goat F(ab')2 fraction fluorescein-conjugated anti-mouse IgG and immunobeads revealed the presence of cytoplasmic IgM bearing cells in 47% of the colonies and surface IgM bearing cells in 74-84% of the colonies. Mixed colonies, however, did not contain either cIgG bearing cells or sIgG bearing cells. The results may indicate that some CFU-MIX proliferate and differentiate along B-cell lineage to sIgM or cIgM bearing cells in vitro.     

Isolated cilia and crystalloid inclusions in murine bone marrow stromal cells

Bone marrow stromal cells, especially reticular cells and macrophages, are thought to have important functions in the hematopoietic inductive microenvironment (HIM). In order to define the details of their fine structure, the femoral bone marrow of 50 C 57 BL mice was examined by electron microscopy after fixation by vascular perfusion. Almost all the structures of the murine bone marrow stromal cells were similar to those previously reported. Murine bone marrow reticular cells had some isolated cilia (with a frequency of 6.0%) of 9 + 0 pattern in their axoneme. The murine bone marrow macrophages had crystalloid inclusions (CI) that revealed characteristic periodic substructure. These periodic structures could be classified into four types (types A-D). Type A inclusions had a rather amorphous structure and were the most common (with a frequency of 61.9%). Type B inclusions had a periodic lamellar structure oblique to the long axis of the CI itself at intervals of about 3.5 nm. Type C inclusions had a periodic lattice structure at intervals of about 3.7 nm, and were the least frequent (with a frequency of 6.0%). Type D inclusions had periodic laminated structure parallel to the long axis of CI at intervals of about 3.5 nm. Although the true significance of isolated cilia of murine bone marrow reticular cells and the periodic internal structures of CI in murine bone marrow macrophages remains unknown, they must not be ignored as only incidental findings: they may be useful to the understanding of the relationship between function and structure in future research on HIM.     

Adoptive cellular immunotherapy: NK cells and bone marrow transplantation

Allogeneic bone marrow transplantation (BMT) has been increasingly used for the treatment of both neoplastic and non-neoplastic disorders. However, serious obstacles currently limit the efficacy and thus more extensive use of BMT. These obstacles include: graft-versus-host disease (GVHD), relapse from the original tumor, and susceptibility of patients to opportunistic infections due to the immunosuppressive effects of the conditioning regimen.Overcoming these obstacles is complicated by dual outcome of existing regimens; attempts to reduce GVHD by depleting T cells from the graft, result in increased rates of tumor relapse and failure of engraftment. On the other hand, efforts to increase graft-versus-tumor (GVT) effects of the transplant also promote GVHD. In this review, the use of natural killer (NK) cells to overcome some of these obstacles of allogeneic BMT is evaluated. Adoptive immunotherapy using NK cells after allogeneic BMT has several potential advantages. First, NK cells can promote hematopoiesis and therefore engraftment by production of hematopoietic growth factors. Second, NK cells have been shown to prevent the incidence and severity of GVHD. This has been shown to be at least partially due to TGF-beta, an immunosuppressive cytokine. Third, NK cells have been shown to augment numerous anti-tumor effects in animals after BMT suggesting a vital role of NK cells in mediating GVT effects. Finally, NK cells have been demonstrated to affect B cell recovery and function in mice. Therefore, understanding the mechanisms of beneficial effects of NK cells after BMT may lead to significant increases in the efficacy of this procedure.     

Human bone marrow stromal cells express an osteoblastic phenotype in culture

Summary This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two “clonings” and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [³H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D₃ by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of β-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by “budding” structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with⁴⁵Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.     

Impaired mitochondrial function and reduced viability in bone marrow cells of obese mice

Bone marrow cells (BMCs) are the main type of cells used for transplantation therapies. Obesity, a major world health problem, has been demonstrated to affect various tissues, including bone marrow. This could compromise the success of such therapies. One of the main mechanisms underlying the pathogenesis of obesity is mitochondrial dysfunction, and recent data have suggested an important role for mitochondrial metabolism in the regulation of stem cell proliferation and differentiation. Since the potential use of BMCs for clinical therapies depends on their viability and capacity to proliferate and/or differentiate properly, the analysis of mitochondrial function and cell viability could be important approaches for evaluating BMC quality in the context of obesity. We therefore compared BMCs from a control group (CG) and an obese group (OG) of mice and evaluated their mitochondrial function, proliferation capacity, apoptosis, and levels of proteins involved in energy metabolism. BMCs from OG had increased apoptosis and decreased proliferation rates compared with CG. Mitochondrial respiratory capacity, biogenesis, and the coupling between oxidative phosphorylation and ATP synthesis were significantly decreased in OG compared with CG, in correlation with increased levels of uncoupling protein 2 and reduced peroxisome proliferator-activated receptor-coactivator 1α content. OG also had decreased amounts of the glucose transporter GLUT-1 and insulin receptor (IRβ). Thus, Western-diet-induced obesity leads to mitochondrial dysfunction and reduced proliferative capacity in BMCs, changes that, in turn, might compromise the success of therapies utilizing these cells.     

Expression of functional human EGF receptor on murine bone marrow cells.

The human epidermal growth factor-receptor (EGF-R) was introduced into primary mouse bone marrow cells (BMC), utilizing retrovirus mediated gene transfer. Cultivation of infected BMC in the presence of interleukin-3 (IL-3) led to the outgrowth of IL-3 dependent myeloid cells, which efficiently expressed functional EGF-R, exhibiting its two characteristic affinity states. EGF acts on these cells synergistically with IL-3 in stimulating DNA synthesis and cell proliferation even under IL-3 saturation conditions. However, EGF was not sufficient to replace the requirement for IL-3. In contrast, EGF was able to maintain proliferation of a factor-dependent hemopoietic cell line (FDC-P1) infected with the EGF-R retrovirus in the absence of IL-3, but these cells did not respond to EGF in the presence of IL-3. No influence of EGF on IL-3 induced mast cell differentiation of BMC expressing the EGF-R could be observed by histological criteria. These data show that the expression of EGF-R alone is not sufficient to induce or maintain cell proliferation in IL-3 dependent bone marrow derived cells, although it can do so in established hemopoietic cell lines.     

A role for the thymic epithelium in the selection of pre-T cells from murine bone marrow

A rat thymic epithelial cell line IT45-R1 has been previously described as secreting soluble molecules that in vitro chemoattract rat hemopoietic precursor cells. The development of such an in vitro migration assay was based on the ability of cells to migrate across polycarbonate filters in Boyden chambers. In the present paper, by using the same strategy, we studied murine bone marrow cells capable of migrating in vitro toward IT45-R1 conditioned medium. The responding cells were shown to represent a minor bone marrow subpopulation characterized by a low capacity to incorporate tritiated thymidine in vitro (less than 10% of control). Moreover, this cell subset was considerably impoverished with respect to granulocyte-macrophage CFU (less than 7% of control) and pluripotent hemopoietic stem cells (less than 12% of control). Potential generation of T cells of donor-type in the lymphoid organs of irradiated recipients was measured by using C57BL/Ka Thy-1.1 and Thy-1.2 congenic mice. Thy-1.1 irradiated mice were injected intrathymically or intravenously with the selectively migrated cell subset of Thy-1.2 donor-type bone marrow cells. The use of an i.v. transfer route allowed us to show that these cells possess thymus-homing and colonization abilities. In a time-course study after intrathymic cell transfer, these migrated cells were able to generate Thy-1.2+ donor-type thymocytes represented by all cortical and medullary cell subsets in a single wave of repopulation from day 20 to day 30 after transfer, with a peak around days 23 to 25. The degree of repopulation closely resembled that seen with unfractionated bone marrow cells in terms of absolute numbers of donor cells per thymus (82% of control, 22 x 10(6) Thy-1.2+ cells) as well as in percent donor cells per thymus (105% of control). Thy-1.2+ cells were also detected in the lymph nodes and the spleens of reconstituted recipient mice. Taken together, these results support the idea that the supernatant of the established thymic epithelium IT45-R1 induces the migration of a murine bone marrow subset that contains hemopoietic stem cells already committed to the lymphoid lineage (i.e., pre-T cells).     

An efficient method for isolation of murine bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been isolated based on the ability of adherence to plastic surfaces. The potential of these cells to differentiate along multiple lineages is the key to identifying stem cell populations in the absence of molecular markers. Here we describe a homogenous population of MSCs from mouse bone marrow isolated using a relatively straightforward and novel approach. This method is based on the combination of frequent medium change (FMC) and treatment of the primary cultures with trypsin. Cells isolated using this method demonstrated the MSCs characteristics including their ability to differentiate into mesenchymal lineages. MSCs retained the differentiation potentials in expanded cultures up to 10 passages. Isolated MSCs were reactive to the CD44, Sca-1, and CD90 cell surface markers. MSCs were negative for the hematopoietic surface markers such as CD34, CD11b, CD45, CD31, CD106, CD117 and CD135. The data presented in this report indicated that this method can result in efficient isolation of homogenous populations of MSCs from mouse bone marrow.     

Cyclophilin A produced by thymocytes regulates the migration of murine bone marrow cells

Supernatant obtained from high dose hydrocortisone resistant thymocytes can induce migration of the bone marrow cell precursors to the periphery. This biological activity depends on the presence of the 18 kDa protein, whose amino acid sequence fits with the sequence of the secretory form of murine cyclophilin A (SP-18). Cyclophilin A isolated from the supernatant of the cortisone-resistant thymoma EL-4 shows its characteristic functional features as it demonstrates isomerase activity and binds with cyclosporine A. The cyclophilin A obtained manifests chemotactic activity that regulates migration of bone marrow cell precursors of neutrophils, T-, B- and dendritic cells.     

Hemopoietic activities of cryopreserved murine fresh and postmortem bone marrow cells

The percentage of preservation of erythropoietic and granulopoietic precursor cells in the murine bone marrow was studied using in vitro methylcellulose clonal cell culture assays and in vivo murine spleen colony assays. This study clearly demonstrates

a. Type of Spleen Colonies Induced by 6-hr Postmortem Murine Bone Marrow Cells^a

	Mean (%)
Type of colonies	t Score	P Value	Unfrozen	Frozen
			Erythrocytic	26.283	14.100	2.09	0.059
Granulocytic	23.741	32.917	1.45	0.173
Mixed	49.321	52.700	0.55	0.59

a

$\text{N = 92}$. the presence of pluripotent hemopoietic precursor cells in cryopreserved 0-, 3-, 6-, 9-, and 12-hr postmortem murine bone marrow cells. Apparently, the erythropoietic precursor cells are more sensitive to freezing injury as compared to granulopoietic precursor cells.