Ultrastructural localization of peroxidase activity in normal human bone marrow cells

Summary The ultrastructural localization of peroxidase activity has been studied in the cells of normal human bone marrow using the diaminobenzidine peroxidase technique. Peroxidase activity has been localized within the primary (azurophil) granules of the neutrophilic series as well as in the cytoplasmic granules of eosinophils, basophils and monocytes. Peroxidase activity appears within the cisternal system (nuclear envelope, Golgi complex and rough endoplasmic reticulum) of these cells during the period of peroxidase-containing lysosome production. With the cessation of granulogenesis, peroxidase activity disappears from the cisternal system and does not reappear in subsequent developmental stages. In cells incubated in peroxide-free media, staining of granular components, but not of cisternae, is reduced. The inclusion of catalase in peroxide-free media eliminates all staining. This indicates that an endogenous peroxide is present within the cisternae and granules of these cell types.Supported by Grant No. AM-HE-12084-12 from the National Institutes of Health, Bethesda, Maryland.Appreciation is expressed to Anita Topson and Barbara Jordan for their technical assistance and to Dr. Arthur Sagone who performed the marrow aspirations.     

Ultrastructural localization of fibronectin in bone marrow of the embryonic chick and its relationship to granulopoiesis

Summary Fibronectin was immunolocated in embryonic chick bone marrow by the use of both a direct peroxidase conjugated antiserum and an indirect Streptavidin bridge technique. Fibronectin is located in the extravascular granulopoietic compartment and, to a lesser extent, in the vascular, erythropoietic compartment. There is no evidence of fibronectin being associated with blood-stromal cell interactions involving either erythropoiesis or thrombopoiesis. However, mature thrombocytes display a substantial surface coat containing fibronectin. Much of the fibronectin appears to be situated on surfaces of those fibroblastic stromal cells which support granulopoiesis. Fibronectin containing extracellular material connects surfaces of developing granulocytes with surfaces of stromal cells. Fibronectin is a surface component of granulocytes as well as nearby stromal cells. However, there appear to be fewer ferritin particles per unit of surface on granulocytic cells. Many of the ferritin particles are not clearly associated with amorphous matrix material at cell surfaces. Immunocytochemical attempts to identify laminin were unsuccessful. These studies indicate that fibronectin is situated at sites where it could mediate adhesive interaction between granulopoietic cells and their stromal cells. Furthermore, cell surface-matrix interaction involving fibronectin could mediate migration of blood cells within the extravascular spaces.     

Immunochemical characterization and ultrastructural localization of chondroitin sulfates and keratan sulfate in embryonic chick bone marrow

Summary Monoclonal antibodies directed against specific carbohydrate epitopes on chondroitin 4-/dermatan sulfate, chondroitin 6-sulfate, keratan sulfate, and a monoclonal antibody directed against the hyaluronate binding region were used to characterize proteoglycans extracted from embryonic chick bone marrow. About half of the proteoglycans separate into the high density fraction on a CsCl gradient. Glycosaminoglycan-specific antibodies recognize proteoglycans from all fractions; this includes an antibody directed against keratan sulfate. Some proteoglycans, principally in the high buoyant density fraction, contain sites recognized by the antibody specific for the hyaluronate binding region. Within limits of detection, all core proteins belong to the high-molecular-weight category, with weights in excess of 212 kD. Antibodies directed against chondroitin 4-/dermatan sulfate and against keratan sulfate primarily bind to extracellular matrix material located in the extracellular spaces and to matrix elements in the pericellular regions of fibroblastic stromal cells. The antibody that recognizes chondroitin 6-sulfate binds to sites on surfaces of fibroblastic stromal cells and also to extracellular matrix material. Little or no antibody binding is detected on surfaces of granulocytic cells. These studies indicate that chondroitin sulfate and keratan sulfate chains are both present in the proteoglycan extract.     

Ultrastructural localization of glycogen in erythrocytes and developing erythrocytic cells in normal human bone marrow

Summary The periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) reaction was employed for the ultrastructural cytochemical localization of saliva-labile glycogen in the erythrocytic cells in normal human blood and bone marrow. Particulate glycogen was demonstrated in the cytoplasm of all developmental forms of erythrocytic cells from the proerythroblast through the reticulocyte; a few particles of glycogen also were present in mature erythrocytes even in the peripheral blood. Statistical evaluation of the number of glycogen particles in mid-plane cell sections at each morphological stage of development indicated a significant and stepwise decrease during cellular maturation. This change in glycogen content may reflect both cellular utilization and mitosis during the maturational sequence.Supported by Grant No. SR01AM 12084-15 from the National Institutes of Health, Bethesda, Maryland.Appreciation is expressed to Anita Topson, Barbara Speakmon and Marjorie Griffith for their technical assistance and to Dr. Gerald King for performing the bone marrow aspirations.     

Human CD133-derived bone marrow stromal cells establish ectopic hematopoietic microenvironments in immunodeficient mice

Cultured adherent bone marrow stromal cells (BMSCs) are capable of forming ectopic hematopoietic microenvironments (HMEs) in immunodeficient mice. However, the cell surface phenotype of the native bone marrow stem/progenitor cell that gives rise to BMSCs that support hematopoiesis remains poorly defined. We recently reported the derivation of human BMSC-like cells (CD133BMSCs) by magnetic cell sorting against Prominin-1 (CD133), an epitope expressed by embryonic, fetal, and adult stem cells. Here we demonstrate that CD133BMSCs are capable of forming ectopic HMEs. Cultured adherent CD133BMSCs derived from sorted CD133-positive cells lacked CD133 expression, but were uniformly positive for CD146, an epitope recently described to identify self-renewing osteoprogenitor cells that could transfer the HME. CD133BMSCs were genetically-tagged by lentivirus, expanded, and seeded into HA/TCP/fibrin constructs that were implanted subcutaneously. After 60 days, CD133BMSCs produced human osteocytes, osteoblasts, adipocytes, and reticular cells that supported murine hematopoiesis. CD133BMSCs that were not transduced with lentivirus also formed HMEs. Control constructs seeded with human dermal fibroblasts formed connective tissue, but failed to form HMEs. Our data indicate that CD133 expression identifies a native human bone marrow stem/progenitor cell that gives rise to BMSCs capable of forming the HME.     

Visualization of the secretory canaliculi of human parietal cells with a peroxidase-labelled peanut lectin

Summary Peanut lectin reactivity was examined in normal fundic glands from human gastric samples, both at light- and electron-microscopic levels, using a peroxidase conjugate. Positive reaction was observed in the glycocalyx of parietal cell secretory canaliculi as well as in the mucous globules of mucous cells and in the luminal cell coat of chief cells. The presence of terminal galactose in the canalicular glycocalyx may be connected with the peculiar function of hydrochloric acid secretion. Peroxidase-labelled peanut lectin is proposed as a marker for visualizing the secretory canaliculus of parietal cells.     

Characterization of sinusoidal endothelial cells of the liver and bone marrow using an intravital lectin injection method

The vascular endothelia express a variety of structural and biological phenotypes. We used an intravital injection method of plant derived lectins (Lycopersicon esculentum lectin (LEL), Ricinus communis Agglutinin-I (RCA-I), Ulex europaeus Agglutinin-I (UEA-I) and Concanavalin A (ConA)) to elucidate the morphology and function of the sinusoidal endothelium of the liver and bone marrow. All four lectins stained the sinusoidal endothelia of the liver and bone marrow in a patchy granular pattern which differed from the uniform and smooth staining pattern of non-sinusoidal vessels in other organs. By transmission electron microscopy, the granular pattern was identified as internalization of these lectins and subsequent accumulation within the endothelial cells, suggesting their active endocytosis. The endocytosis of these lectins emphasizes the fact that sinusoidal endothelial cells of the liver and bone marrow belong to the reticuloendothelial system (RES), a cell system characterized by internalization of foreign material. We introduce this intravital lectin injection as a useful technique to discriminate sinusoidal endothelial of the liver and bone marrow from other vascular endothelia.     

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.     

Ultraviolet difference spectroscopy study of peanut lectin binding to mono- and disaccharides

Ultraviolet difference spectroscopy was used to study the interaction of peanut (Arachis hypogaea) lectin with complementary carbohydrates. A correlation was observed between variations of ultraviolet spectra during the binding of sugars to the lectin and the specificity and the strength of the binding. The association constant, free energy, enthalpy and entropy for peanut lectin-lactose interactions were calculated over the temperature range 10–30°C. The binding constants for 10 mono- and disaccharides containing a D-galactopyranosyl or a D-talopyranosyl residue were calculated. Comparing their effectiveness to interact with peanut lectin, methyl $\text{α-}\text{D}\text{-galactopyranoside}$ appeared to have a more marked affinity than lactose; D-galacturonic acid and methyl $\text{7-}\text{deoxy-}\text{D}\text{-glycero}\text{-β-}\text{D}\text{-galacto-heptopyranoside}$ had no measurable affinity; the other sugars showed a lower affinity than lactose. The correlations between these differences and the conformations of the sugars obtained by X-ray analysis are discussed.     

骨髓间充质干细胞向神经细胞分化的研究

无论是在体外实验、还是在体内实验,MSCs都可以向中枢神经系统(CNS)神经细胞分化,但争议颇多。因为功能性神经元不仅要具有典型神经元的形态、特异性标记,还要求具有可兴奋性、能和其他神经元形成突触联系、产生突触电位等,所以对于骨髓间充质干细胞是否能诱导出真正具有功能的神经元存在很大分歧。在此对MSCs向神经细胞诱导分化研究的现况、存在的问题及发展前景给以综述。     

CCN2 (Cellular Communication Network factor 2) in the bone marrow microenvironment,normal and malignant hematopoiesis

CCN2, formerly termed Connective Tissue Growth Factor, is a protein belonging to the Cellular Communication Network (CCN)-family of secreted extracellular matrix-associated proteins. As a matricellular protein it is mainly considered to be active as a modifier of signaling activity of several different signaling pathways and as an orchestrator of their cross-talk. Furthermore, CCN2 and its fragments have been implicated in the regulation of a multitude of biological processes, including cell proliferation, differentiation, adhesion, migration, cell survival, apoptosis and the production of extracellular matrix products, as well as in more complex processes such as embryonic development, angiogenesis, chondrogenesis, osteogenesis, fibrosis, mechanotransduction and inflammation. Its function is complex and context dependent, depending on cell type, state of differentiation and microenvironmental context. CCN2 plays a role in many diseases, especially those associated with fibrosis, but has also been implicated in many different forms of cancer. In the bone marrow (BM), CCN2 is highly expressed in mesenchymal stem/stromal cells (MSCs). CCN2 is important for MSC function, supporting its proliferation, migration and differentiation. In addition, stromal CCN2 supports the maintenance and longtime survival of hematopoietic stem cells, and in the presence of interleukin 7, stimulates the differentiation of pro-B lymphocytes into pre-B lymphocytes. Overexpression of CCN2 is seen in the majority of B-acute lymphoblastic leukemias, especially in certain cytogenetic subgroups associated with poor outcome. In acute myeloid leukemia, CCN2 expression is increased in MSCs, which has been associated with leukemic engraftment in vivo. In this review, the complex function of CCN2 in the BM microenvironment and in normal as well as malignant hematopoiesis is discussed. In addition, an overview is given of data on the remaining CCN family members regarding normal and malignant hematopoiesis, having many similarities and some differences in their function.     

Identification of parathyroid hormone-regulated proteins in mouse bone marrow cells by proteomics

The ability of parathyroid hormone (PTH) to enhance bone formation has recently been exploited in the treatment of osteoporosis. Several studies have suggested that the activation of bone marrow stromal cells could be preceded to show the anabolic effect of PTH on bone formation, but little is known of PTH-regulated proteins in bone marrow cells. Therefore, protein profiling in the intermittent PTH-treated bone marrow cells was evaluated using proteomics. Daily treatment for 5 days consisting of subcutaneous injection of either 150 microg/kg per day of mouse PTH (1-84) or vehicle (0.9% normal saline) was performed on the ICR mouse. At the end of the treatment period, bone marrow cells were separated and used in proteomics. The expression levels of seven proteins including vimentin were decreased, but those of four proteins including calreticulin and thioredoxin domain containing 7 protein (Txnde7) were increased. Among these, the decrease of vimentin and the increase of both calreticulin Txnde7 in mRNA levels were confirmed by semi-quantitative RT-PCR. In PTH-treated mouse MC3T3-E1 osteoblast cells, mRNA expression levels were not totally consistent with the results observed in proteomics. In conclusion, the differentially expressed proteins in bone marrow cells depending on PTH could be highly linked to the differentiation of osteoprogenitor cells in the bone marrow into preosteoblast cells.     

Activation of bone marrow cells treated with Canova in vitro

Canova is a Brazilian complex homeopathic medication produced from Aconitum, Thuya, Bryonia, Lachesis and Arsenicum. Previous studies demonstrated that Canova induces up-regulation in numbers of leukocytes. The bone marrow microenvironment is composed of growth factors, stromal cells, extracellular matrix, and progenitor cells that differentiate into mature blood cells. As it is the major site of blood cell formation, we studied in vitro Canova effects on bone marrow cells of mice. Swiss mouse femurs were dissected, cleaned, and the marrow was flushed. The cells were plated, treated or not, incubated for different times and processed for light, scanning electron, and confocal microscopy, and also flow cytometry. The treatment did not modify the expression of the analyzed surface markers or cytokine production. All microscopy techniques showed that a monocytic lineage (CD11b(+)) and stromal cells (adherent cells) were activated by treatment. Canova also increased cell clusters over adherent cells, suggesting proliferation areas.     

小型猪骨髓间充质干细胞的分离和培养

目的建立小型猪骨髓间充质干细胞（mesenchymal stem cells,MSCs）的体外分离和培养方法。方法穿刺小型猪髂后上嵴抽取骨髓,经密度梯度法离心得到骨髓单个核细胞,接种后形成单层贴壁细胞。用形态学方法鉴定培养的MSCs。结果经培养存活的MSCs原代和一代呈纺锤型、多边型或星型。至二代起呈均一纺锤型,似成纤维细胞样,长宽比例约为（2～3）？1。体外培养的原代MSCs8～10d达到融合,传代后仍具有较强的增殖能力。结论小型猪MSCs可在体外长期、稳定培养,其分离、培养体系的建立为基础研究和组织工程技术提供了一个有价值的动物模型。     

骨髓单个核细胞在周围神经修复中的应用

基础研究证实,多种细胞移植可以促进周围神经修复,其中来源丰富的骨髓单个核细胞,因具有取材过程简单、无交叉感染风险、无免疫排斥、可以自体移植等诸多优点,是目前重要的候选细胞之一。本文就近期有关骨髓单个核细胞的神经修复作用机制的研究、细胞植入修复受损周围神经的文献、以及与各种生物材料复合应用构建的组织工程化神经等方面最新进展进行综述,以期促进该领域基础向临床应用的转化。     

TWEAK promotes the production of Interleukin-17 in rheumatoid arthritis

An understanding of anthrax toxins on the emerging immune system and blood production are significant to medicine. This study examined the effects of anthrax toxin on hematopoiesis and determined roles for cytokines. Anthrax holotoxin toxin is three components: protective antigen (PA) binds to the target cell and mediates the entry of lethal factor (LF) and edema factor (EF). Anthrax toxin dramatically inhibits signaling in immune cells. We first identified the cell subsets that interacted with the protective antigen (PA) and then studied the effects on hematopoietic progenitors in clonogenic assays: granulocytic-monocytic (CFU-GM) and late erythroid (CFU-E). Multi-color immunofluorescence with FITC-PA indicated its interaction with early and late myeloid cells. Clonogenic assays, in the presence or absence of holotoxin and individual toxin proteins resulted in significant suppression by hologenic toxic alone, despite the presence of growth-promoting cytokines. Antibodies to anthrax receptor (ATR1) reversed the suppressive effects, indicating specificity. Monomeric proteins showed different effects on myeloid and erythroid progenitors. Suppression was not due to cell death, based on undetectable active caspase 3. Cytokine array analyses with supernatants from toxin-stimulated stroma showed an increase in the hematopoietic suppressor, MIP-1α. This finding, in addition to our previous studies, showing an increase in IL-10, suggested indirect roles for cytokines in toxin-mediated hematopoietic suppression. The chemokine, SDF-1α was increased. Since SDF-1 is involved in the mobilization of hematopoietic cells, it is likely that anthrax holotoxin could induce cell exit from BM. In summary, anthrax holotoxin, but not individual toxins, exerted hematopoietic effects on myeloid and erythroid progenitors via specific receptor, partly through the induction of cytokines.     

Clastogenicity of carbazole in mouse bone marrow cells in vivo

Clastogenicity of carbazole was evaluated by employing mouse in vivo chromosomal aberration (CA) test. Carbazole administered intraperitoneally (i.p.) at the rate of 25, 50, 100, 150 and 200 mg/kg b.w. to Swiss albino mice in vivo resulted in mitotic depression and induction of chromosomal aberrations. Dose related decrease in mitotic index (MI) and increase in the frequencies of chromosomal aberrations per cell (CAs/cell) and percent abnormal cells were recorded in bone marrow cells. However, statistically significant reduction in MI and increase in CAs/cell and percent abnormal cells were found only for the two higher doses. The results obtained indicate that carbazole or its metabolite, if any, is moderately clastogenic in the bone marrow cells of Swiss albino mice.     

Effect of hypoxic treatment on bone marrow cells that are able to migrate to the injured liver

Restricted numbers and poor regenerative properties limit the use of adult stem cells. We tested the effect of hypoxic treatment as a method by which to increase cell migration. Bone marrow cells (BMCs) were cultured under oxygen saturations of 0.1, 3, and 20% for 24 h. After hypoxic treatment, BMCs of apoptotic fraction were decreased. The expression of CXCR4 was noticeably increased in the hypoxia-treated BMCs and their migration in response to SDF-1α was enhanced compared with cells cultured under normoxic condition. Hypoxic BMCs had a higher degree of engraftment to the CCl₄-injured liver than the normoxic cells. Hypoxic treatment of BMCs may have merits in decreasing apoptosis of those cells as well as in enhancing cellular migration to SDF-1α, the chemokine which binds to BMCs expressed CXCR4 and to the injured tissue, such as CCl₄ damaged liver.     

Development of liver regenerative therapy using glycoside-modified bone marrow cells

Several recent studies have reported that bone marrow cells (BMCs) have the ability to generate functional hepatocytes. However, the efficiency at which BMC transplantation generates functional hepatocytes is rather low. We assumed that if BMCs accumulated directly in liver, the functional BMC-derived hepatocytes should increase efficiently. We tried to increase the accumulation of BMCs directly in liver through the interaction between hepatic asialoglycoprotein receptor and desialylated BMCs. Desialylated BMCs were produced with treatment of neuraminidase. Desialylated BMCs that expressed green fluorescent protein (GFP) were injected into Long Evans Cinnamon (LEC) rats, a human Wilson's disease model, intravenously. At 3 and 5 months after transplantation, GFP-expressing hepatocyte nodules appeared in the liver of these BMC-transplanted LEC rats. These findings suggest that the functional BMC-derived hepatocytes can be generated by the direct accumulation of BMCs and that this strategy is new BMC therapy for liver regeneration.     

α-Smooth muscle actin is expressed in a subset of bone marrow stromal cells in normal and pathological conditions

A series of 217 trephine bone marrow biopsies from adult patients and specimens from 16 fetuses and 5 infants were examined for the presence of stromal myoid cells (MCs) using a monoclonal antibody recognizing α-smooth muscle actin. In the normal adult bone marrow, stromal cells did not contain α-smooth muscle actin, whereas during fetal life, many α-smooth muscle actin-containing MCs were connected with vascular sinusoids in the primitive bone marrow. This cell type reappeared in various characteristic distribution patterns in adult bone marrow during different neoplastic and non-neoplastic conditions including metastatic carcinoma, Hodgkin’s disease, multiple myeloma, hairy cell leukemia, acute myeloid leukemia (FAB M4, 5, 7) and chronic myeloproliferative diseases. In general, the appearance of MCs was associated with a slight to pronounced increase in the deposition of reticulin and collagen fibers. We propose that bone marrow MCs represent a distinct subpopulation of fiber-associated or adventitial reticular cells undergoing cytoskeletal remodeling in response to various stimuli.