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PubMed1977

T cell function detected in murine bone marrow cells.

Mouse bone marrow cells were fractionated by BSA discontinuous density gradient centrifugation, and a small lymphocyte rich fraction was obtained at the high density. Cells of this fraction were shown to respond in vitro to T cell mitogens and alloantigens. Furthermore, they were able to mount a graft-versus-host reaction when assessed by spleen weight assay and by the method of inhibiting erythroid cell growth by allogeneic lymphoid cells. The results indicate that these lymphocytes possess T cell functions. On the other hand they were found to carry only little theta antigen assessed by the cytotoxic test and by the absorption test. It is presumed, therefore, that the amount of theta antigen on a cell might not correlate with T cell functions, and these lymphocytes might be mature ones in the course of postthymic maturation. Hemopoietic stem cells were determined by spleen colony formation and the peak of colony-forming efficiency was seen at the low density. These observations imply that immunocompetent cells causing GVHR can be separated from hemopoietic stem cells. This procedure may be applied for prevention and reduction of GVHR in allogeneic bone marrow transplantation in human.

Animals↗
PubMed2006

Efficient differentiation into skin cells of bone marrow cells recovered in a pellet after density gradient fractionation.

We had previously demonstrated the participation of whole bone marrow cells from adult mice in the reconstitution of skin, including the epidermis and hair follicles. To get an insight into cell populations that give rise to the epithelial components of the reconstituted skin, we fractionated bone marrow cells derived from green fluorescent protein-transgenic mice by density gradient. Unexpectedly, we found that a substantial amount of mononucleated cells (approximately 30%) was recovered in the pellet fraction and that the cells in the pellet fraction preferentially differentiated into epithelial components of skin, rather than the cells in the mononuclear cell fraction. The pellet fraction contained more CD45-negative (thus uncommitted to the hematopoietic cell lineage) cells than the mononuclear cell fraction. These results indicate that density gradient fractionation results in significant loss of specific progenitor cells into the usually discarded pellet fraction.

Animals↗
PubMed1988

Characterization of natural killer cells cultured from human bone marrow cells.

Human bone marrow (BM) cells, depleted of nylon wool-adherent cells, T cells, and natural killer (NK) cells, were cultured in medium containing recombinant interleukin 2 (rIL2). After 21 or 24 days in culture, numerous lymphoid cells with multiple azurophilic granules and a morphology similar to large granular lymphocytes (LGL) were found. Two-color analysis of surface phenotype showed many of these cells to be NKH1-positive and a limited number of cells had other NK markers such as CD16, CD2, or CD8. The CD3 antigen was not coexpressed with NKH1. The cultured BM cells were cytotoxic for K562, Daudi, and Raji cell lines. The NKH1+, CD2-, CD3-, CD16- cells were sorted and, in addition to having the LGL morphology, were found to be cytotoxic for K562 cells (NK [K562]). The generation of NK(K562) activity was significantly suppressed by 5-bromodeoxyuridine plus ultraviolet light treatment, indicating that DNA synthesis is required. These experiments suggest that the described culture conditions allow differentiation of progenitor cells, into immature, but functionally active, NK cells.

Antigens, Surface↗
PubMed1974

Remission of acute myeloid leukemia: studies of in vitro colony formation by bone marrow cells.

Human bone marrow contains cells which form leukocyte colonies in semisolid culture media. Each leukocyte colony arises from a single colony-forming cell which is thought to be a unipotential stem cell, and which is subject to regulation in vitro by colony-stimulating factor. In acute myelogenous leukemia variable abnormalities in colony formation by marrow cells occur. Usually colony formation either fails to occur or the colonies that are formed are small and contain fewer than 50 cells. Similar abnormalities have been described in bone marrow dysfunction preceding overt leukemia. Usually remission of leukemia is accompanied by improved cloning by marrow cells. In this study three patients are reported in whom remission was associated with impaired cloning, and one of these patients has remained in continuous remission for a further 18 months. These observations suggest that remission status is not necessarily associated with repopulation of the bone marrow by normal hematopoietic cells.

Adult↗
PubMed1978

Isolation and characterization of a virus-specific ribonucleoprotein complex from reticuloendotheliosis virus-transformed chicken bone marrow cells.

Chicken bone marrow cells transformed by reticuloendotheliosis virus (REV) produce in the cytoplasm a ribonucleoprotein (RNP) complex which has a sedimentation value of approximately 80 to 100S and a density of 1.23 g/cm3. This RNP complex is not derived from the mature virion. An endogenous RNA-directed DNA polymerase activity is associated with the RNP complex. The enzyme activity was completely neutralized by anti-REV DNA polymerase antibody but not by anti-avian myeloblastosis virus DNA polymerase antibody. The DNA product from the endogenous RNA-directed DNA polymerase reaction of the RNP complex hybridized to REV RNA but not to avian leukosis virus RNA. The RNA extracted from the RNP hybridized only to REV-specific complementary DNA synthesized from an endogenous DNA polymerase reaction of purified REV. The size of the RNA in the RNP is 30 to 35S, which represents the subunit size of the genomic RNA. No 60S mature genomic RNA was found within the RNP complex. The significance of finding the endogenous DNA polymerase activity in the viral RNP in infected cells and the maturation process of 60S virion RNA of REV are discussed.

Animals↗
PubMed1976

Effects of negative pi mesons on mouse bone marrow cells.

Mouse bone marrow cells exposed in vitro to graded doses of negative pi mesons at 3.5-6.0 rads/min. gave Do values of 160 rads in the plateau (0.6 and 12.0 cm), and 130 rads in the peak (16.5 and 18.4 cm). Survival curve shoulders were narrow, with Dq values of 25 and 15 rads for plateau and peak positions. Gamma rays from 60Co at 4.5 rads/min. produced a curve with wider shoulder but steeper slope such that the RBE was essentially 1 for single doses between 100-400 rads.

Animals↗
PubMed2003

Generation of a chimeric mouse reconstituted with green fluorescent protein-positive bone marrow cells: a useful model for studying the behavior of bone marrow cells in regeneration in vivo.

Studies have indicated that bone marrow contains both hematopoietic stem cells and mesenchymal stem cells that can differentiate into a variety of mesenchymal tissues, such as bone, cartilage, muscle, and adipose tissue. Therefore, bone marrow cells are thought to be very useful for cell and gene therapy for various diseases. However, the multipotentiality of these cells remains unclear. To address this issue, we established a chimeric model mouse stably reconstituted with green fluorescent protein (GFP)-marked bone marrow cells. We injected bone marrow cells from GFP-transgenic C57BL/6 mice into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10 Gy from a cesium source. Microscopic examination and fluorescence-assisted cell sorter (FACS) analysis showed that bone marrow cells, including mesenchymal cells, were almost completely reconstituted with GFP+ cells 5 weeks after transplantation. FACS analysis with lineage-specific antibodies confirmed that the GFP+ cells could differentiate into all types of blood cells. To confirm the usefulness of this mouse model, we studied the role of circulating bone marrow-derived cells in healing of damaged intestine. We performed amputation and anastomosis of the jejunum 10 cm from the pyloric region of the stomach. On the third day after operation, a large number of GFP+ cells were infiltrated in the area of anastomosis, and these cells were positive for CD45 and F4/80 antigens. In 7 days, several cells became negative for CD45 and F4/80 and positive for alpha smooth muscle actin antigen, which is specific for smooth muscle. This finding suggested that bone marrow-derived cells had differentiated into smooth muscle. Because reconstituted bone marrow cells as opposed to injected bone marrow cells, behave naturally, this model is ideal for studying the multipotentiality of bone marrow cells in vivo.

Animals↗
PubMed1999

Stimulation of bone marrow cells and bone formation by nacre: in vivo and in vitro studies.

There is frequently a loss of vertebral bone due to disease or aging. Nacre (mother of pearl from the oyster Pinctada maxima) stimulates bone cell differentiation and bone formation in vitro and in vivo. Experimental bone defects were prepared in the vertebrae of sheep and used to test the suitability of nacre as an injectable osteogenic biomaterial for treating vertebral bone loss. Twenty-one cavities were prepared in the first four upper lumbar vertebrae of 11 sheep and filled with nacre powder. The lumbar vertebrae were removed after 1 to 12 weeks, embedded undecalcified in methacrylate, and processed for histological studies. The nacre slowly dissolved and the experimental cavities contained a large active cell population. By 12 weeks, the experimental cavity was occupied by newly matured bone trabeculae in contact with or adjacent to the dissolving nacre. The functional new bone trabeculae were covered with osteoid lined with osteoblasts, indicating continuing bone formation. The in vitro study on rat bone marrow explants cultured with a water-soluble extract of the nacre organic matrix also resulted in the stimulation of osteogenic bone marrow cells with enhanced alkaline phosphatase activity. Thus, both the in vivo and in vitro findings suggest that nacre contains one or more signal molecules capable of activating osteogenic bone marrow cells.

Alkaline Phosphatase↗
PubMed1999

Post-liver transplantation lymphoproliferative disorders with and without infusions of donor bone marrow cells.

Donor bone marrow cells (DBMC) infusions have been used in an attempt to decrease the untoward effects of immunosuppressive treatment and to improve immunocompetence in the post-liver transplantation (PLT) period. Between March 1987 and July 1996, 558 orthotopic liver transplantations were performed at Jackson Memorial Hospital/University of Miami. Of these, 164 patients (29%) received 10 x 10(8) DBMC/Kg using various schedules. All patients received similar immunosuppressive therapy. After a minimum follow up of 1 year, five cases of Posttransplant Lymphoproliferative Disorder (PTLD) were diagnosed in patients without DMBC (1.3%, 5/394) when compared with none (0/164) in patients who received DBMC (p = 0.15, Fisher). Four patients had malignant lymphoma and one a diffuse atypical lymphoproliferative disorder. All lymphomas were non-Hodgkin's B-cell type, three diffuse large cell lymphoma, and one mixed cell lymphoma. All PTLD tested positive for EBV by in situ hybridization. Lymphomas occurred at 2, 4, 6 months and 4 years PLT. The outcome was poor with one patient diagnosed at autopsy while two patients died a few days after diagnosis. An 8-year-old girl is the only long-term survivor (> 5 years) after a partial response to combination chemotherapy and radiation therapy. The patient with diffuse atypical lymphoproliferative disorder died 3 months later. All patients with PTLD had histologic evidence of liver rejection. Although there is no statistical significant difference between the two groups, a larger cohort of patients will determine the significance of DBMC in preventing PTLD. We believe that the infusion of cytotoxic donor T cells found in the DBMC might suppress EBV-related lymphomagenesis.

Adolescent↗
PubMedDate not supplied

Phenotype and functional identity of GM-CSF-independent dendritic cells generated by long-term propagation of DC progenitor cells in bone marrow cells and skin Langerhans cells.

Evidence is provided that dendritic cells (DC) generated by either long-term bone marrow cell (BMC) culture with Flt3L and interleukin-6 (IL-6), or after short-term BMC culture with granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), contain heterogeneous cell populations of admixed DC and Mphi, regardless of the cytokine source. By employing GM-CSF-independent culture systems with the aid of Flt3/Flk-2 ligand and IL-6 and phenotypic characterization of BMC-derived DC and skin Langerhans cells (LC), revealed similar phenotypes. Furthermore, CD103 (OX62), which is widely used for rat DC separation, was found to be insufficient to enrich DC, due to downregulation of the marker. In this regard, the most efficient selection of rat DC, was obtained by CD161a (NKR-P1A), a member of the C-type lectin family. Despite the phenotypic similarity with BMC-derived DC, the nucleus of LC showed a distinct morphology. A large population of DC generated by Flt3L/IL-6 from GM-CSF receptor-deficient mice by do not express NK1.1 (NKR-P1B and NKR-P1C). The profiles for BMC-derived DC were the same as for skin Langerhans cells.

Animals↗
PubMed2002

Comparison of bone marrow cells harvested from various bones of cynomolgus monkeys at various ages by perfusion or aspiration methods: a preclinical study for human BMT.

Using cynomolgus monkeys, we have previously established a new method for harvesting bone marrow cells (BMCs) with minimal contamination of the BMCs with T cells from the peripheral blood. We originally conducted this new "perfusion method" in the long bones (the humerus, femur, and tibia) of cynomolgus monkeys. Here, we apply the perfusion method to obtain BMCs from the ilium of cynomolgus monkeys, since BMCs are usually collected from the ilium by the conventional aspiration method in humans. The perfusion method consists of two approaches: transverse iliac perfusion and longitudinal iliac perfusion. BMCs harvested by the perfusion method from the long bones and ilium were compared with those collected from the ilium by the aspiration method. The contamination of BMCs with peripheral blood, determined by the frequencies of CD4+ and CD8+ T cells, was significantly lower in BMCs obtained from the ilium or long bones by the perfusion method (CD4+ plus CD8+ T cells <4%) than in those obtained by the iliac aspiration method (CD4+ plus CD8+ T cells >20%). However, the numbers of immature myeloid cells, such as myeloblasts, promyelocytes, myelocytes, and metamyelocytes, were higher in BMCs obtained by the iliac perfusion method than in those obtained by the iliac aspiration method. The assays for in vitro colony-forming unit in culture revealed that progenitor activity was significantly higher in BMCs obtained by the perfusion method than in those obtained by the aspiration method. These findings suggest that the contamination of BMCs with peripheral blood is much less when using the perfusion method than when using the aspiration method. To determine the best site for harvesting BMCs by the perfusion method, age-dependent changes in BMCs harvested by the perfusion method from the long bones and ilium were examined. The numbers of BMCs varied in the long bones (humerus > femur > tibia) and showed age-dependent decreases, whereas they remained similar in the ilium of cynomolgus monkeys from 3 years to 6 years of age. However, in cynomolgus monkeys, BMC harvesting by the perfusion method from the ilium (but not from the long bones) is found to involve the risk of fat emboli, particularly when the BMCs are quickly perfused under high pressure. These findings suggest, even in humans, that the perfusion method is better than the aspiration method, and that the best site for collection of BMCs is the humerus.

Aging↗
PubMed1999

[Biological effects of basic fibroblast growth factor (bFGF) and osteogenic protein-1 (OP-1/BMP-7) on bone marrow cells and development of cell-hybrid artificial bone].

In this study, the effects of bFGF and OP-1 on rat bone marrow cells and bone formation by the cultured bone marrow cells in porous hydroxyapatite (HA) in subcutaneous sites of syngeneic rats were investigated. The effects of bFGF and OP-1 on the proliferation and the differentiation of rat bone marrow cells were investigated by means of measurement of [3H] thymidine incorporation into DNA, ALP activity and bone nodule formation. This in vitro study showed that both bFGF and OP-1 increased [3H] thymidine incorporation, especially bFGF which stimulated it 3.5-fold at a dose of 30ng/ml. OP-1 stimulated ALP activity and bone nodule formation, whereas bFGF suppressed them. These data suggested that bFGF stimulates proliferation of rat bone marrow cells and OP-1 accelerates their differentiation. These growth factors were used to fabricate cell hybrid artificial bone, which consisted of the bone marrow cells. The ectopic bone formation was induced by the bone marrow cells cultured in HA, regardless of the treatment of these growth factors. However, the histological analysis revealed that the treatment with bFGF and/or OP-1 increased the area of new bone formation and accelerated the maturation of bone compared with the controls. The results suggested that the bone marrow cells cultured in HA three-dimensionally could be used clinically as a biomaterial. Furthermore, the use of bFGF and OP-1 to produce this cell-hybrid artificial bone increases its bone forming activity.

Animals↗
PubMed2004

Role of interaction between Ly49 inhibitory receptors and cognate MHC I molecules in IL2-induced development of NK cells in murine bone marrow cell cultures.

Murine bone marrow (BM) cell preparations lack mature cytotoxic natural killer (NK) cells, but NK cells may be induced in these cell preparations by culturing with interleukin-2 (IL2). Present study was aimed at studying the role of interactions between Ly49 molecules and major histocompatibility complex (MHC) class I molecules during IL2-induced development of mature NK cells in BM cell cultures. Addition of monoclonal antibodies (mabs) specific to class I MHC molecules of H-2b haplotype, to block any interaction of MHC I molecules with their receptors, was found to inhibit NK cell development. Mouse NK cells express several types of Ly49 molecules including Ly49C, which is an inhibitory receptor specific to MHC I molecules of H-2b haplotype. Blocking Ly49-MHC I interaction by using anti-Ly49C mab inhibited the development of cytotoxic NK cells. Addition of anti-Ly49A (no specificity for H-2b MHC I molecules) or anti-Ly49D (activating receptor specific for MHC I molecules of many H-2 haplotypes including H-2b) mabs, however, had no effect on IL2-induced NK cell development in BM cells. Mabs specific to Ly49C molecule and MHC I molecules of H-2b haplotype inhibited the development of mature NK cells from highly purified NK precursor cell population. These results indicate that specific interaction between inhibitory self-reactive Ly49 molecules and MHC I molecules may be crucial for NK cell development. We propose a model in which Ly49-MHC I interaction may have a permissive role in allowing development of only such NK cell clones that expresses at least one self-reactive inhibitory Ly49 molecule so that lysis of autologous healthy cells by mature NK cells may be avoided.

Animals↗
PubMed1993

Prevention of autoimmune inflammatory polyarthritis in male New Zealand black/KN mice by transplantation of bone marrow cells plus bone (stromal cells).

OBJECTIVE: To prevent polyarthritis in male New Zealand black/KN (NZB/KN) mice by transplantation of both bone marrow cells (BMC) and bone (to recruit stromal cells) from normal mice. METHODS: Arthritic lesions in male NZB/KN mice injected intravenously with BMC plus bone from C57Bl/10 mice were compared with those in untreated male NZB/KN mice. RESULTS: Male NZB/KN mice engrafted with BMC plus bone were both radiologically and histopathologically normal, and had decreased production of anti-single-stranded DNA antibodies and rheumatoid factors at 8-12 months of age. CONCLUSION: Bone marrow transplantation prevented polyarthritis in male NZB/KN mice.

Animals↗
PubMedDate not supplied

The proliferative capacity of pure red cell aplasia bone marrow cells.

Pure red cell aplasia (PRCA) is a heterogeneous disorder. Immunologic abnormalities have recently been uncovered suggesting that both cell-mediated and humoral immune mechanisms may be of etiological importance in PRCA. Utilizing a technique for the cloning of bone marrow erythroid precursors, we determined the in vitro proliferative capacity of erythroid cells obtained from 21 patients with PRCA. Bone marrow cells from one group of patients produced normal or increased numbers of erythroid colonies while the in vitro proliferative capacity of bone marrow cells from a second group was characterized by subnormal erythroid colony formation. Sera obtained from the former group was frequently associated with demonstrable serum inhibitors of erythropoiesis, while PRCA in the latter group was probably the consequence of intrinsic erythroid stem cell defects or pathologic cellular interactions with nonerythroid regulatory cells. This survey of a relatively large population of patients with PRCA provides evidence for the multiple causative mechanisms that can be operative in the production of PRCA.

Adolescent↗
PubMed2006

Cardiac ischemia activates vascular endothelial cadherin promoter in both preexisting vascular cells and bone marrow cells involved in neovascularization.

Vascular endothelial cadherin (VE-cadherin) is expressed on vascular endothelial cells, which are involved in developmental vessel formation. However, it remains elusive how VE-cadherin-expressing cells function in postnatal neovascularization. To trace VE-cadherin-expressing cells, we developed mice expressing either green fluorescent protein or LacZ driven by VE-cadherin promoter using Cre-loxP system. Although VE-cadherin promoter is less active after birth than during embryogenesis in blood vessels, it is reactivated on cardiac ischemia. Both types of reporter-positive cells are found in the vasculature and in the infarcted myocardium. Those found in the vasculature were pre-existing endothelial cells and incorporated endothelial progenitor cells derived from extracardiac tissue. In addition to the vasculature, VE-cadherin promoter-activated cells were positive for CD45 in the bone marrow cells of the infarcted mice. VE-cadherin promoter-reactivated CD45-positive leukocytes were also found in the infarcted area. In addition, VE-cadherin promoter was activated in the bone marrow vessels of the infarcted mice. Collectively, our findings reveal a new ischemia-induced neovascularization mechanism involving VE-cadherin; the re-expressed VE-cadherin-mediated cell adhesion between cells may be involved not only in homing of bone marrow-derived cells to ischemic area but also mobilization from bone marrow.

Animals↗