Formation of osteoblast-like cells from human mononuclear bone marrow cultures

Osteoblast-like cells are commonly found in the vicinity of osteoclasts formed in long-term human bone marrow cultures, and they are believed to be derived from osteogenic cell precursors belonging to the stromal cell system. This paper describes a new culture method for human osteoblasts from the adherent cell population of long-term human mononuclear bone marrow cultures. The cells obtained exhibited all the classic characteristics of osteoblasts. They contained high intracellular concentrations of alkaline phosphatase and they secreted the osteoblast-specific marker bone Gla protein. Collagen production was mainly (95-98%) procollagen type I propeptide and only minute quantities of procollagen type III propeptide were detectable by radioimmunoassay in the conditioned medium. After eight weeks the cells formed a mineralized matrix on exposure to beta-glycerophosphate and ascorbic acid. This system provides a model for the study of osteoblast differentiation in vitro and may form the basis for the use of defined media in bone cell cultures due to the presence of high concentrations of osteoblast precursors.     

Human rib bone marrow mononuclear cells spontaneously synthesize and secrete IgE in vitro.

We have examined spontaneous secretion of IgE by human rib bone marrow mononuclear cells (MNC). Bone marrow MNC from nine out of 12 rib specimens synthesized and secreted substantial amounts of IgE during 14 days of in vitro culture. The 14-day supernatants from these bone marrow MNC contained a mean of 2589 pg/ml of IgE (n = 12) with a maximum production of 15,408 pg/ml of IgE compared with small amounts of IgE (80-200 pg/ml) produced by similarly cultured normal and inflammatory bowel disease intestinal lamina propria MNC. Using two rib specimens, time-course studies revealed spontaneous secretion of IgE to be minimal during the first 2 days of culture (152 pg/ml), followed by a steady increase between days 4 (517 pg/ml) and 14 (3588 pg/ml). The addition of pokeweed mitogen resulted in 72% suppression of spontaneous IgE production by bone marrow MNC. The bone marrow MNC isolated from the ribs consisted of 22% Leu12+ (B) cells of which 3.2% were surface IgE positive. Staining for cytoplasmic immunoglobulin revealed 1% of the bone marrow MNC to be cytoplasmic IgE+. The presence of IgE-bearing and IgE-secreting MNC in human bone marrow is consistent with the observation that allergen-specific IgE-mediated hypersensitivity is adoptively transferred by human bone marrow transplantation and demonstrates the usefulness of human bone marrow MNC for examination of IgE secretory and regulatory events.     

Cytotoxic effector capacity of bone marrow mononuclear cells.

Purified mononuclear cells from guinea pig bone marrow possess highly efficient cytotoxic effector cell capabilities in the phytohemagglutinin-induced cellular cytotoxity and antibody-dependent cellular cytotoxicity and antibody-dependent cellular cytotoxicity assays against chicken red blood cell targets. This cytotoxic activity is not removed by depletion of adherent cells by passage through rayon wool columns. Thus bone marrow lymphocytes themselves, depleted of adherent monocytes, possess this killer cell capacity. These effector cells may either arise de novo within the bone marrow parenchyma or arrive there as part of the recirculating lymphocyte pool. These studies lend further understanding of the development of functional capabilities of bone marrow lymphoid cells.     

CML患者骨髓单个核细胞基因表达谱分析

目的 探讨慢性髓细胞性白血病(CML)的基因表达变化。方法 分别从正常人和CML患者骨髓样品中抽取骨髓单个核细胞,分离出cDNA片段,然后再逆转录成cRNA并标记后,与人的全基因组表达谱芯片杂交,ABI 1700芯片分析系统进行分析基因表达谱的差异。结果 总共发现差异表达的基因有6706个,其中与CML相关的差异基因有75个(上调19个,下调56个)。结论 全基因组寡核苷酸基因芯片在分析基因表达谱差异研究中具有广泛的应用价值。     

Separation and characterization of human bone marrow mononuclear cells from aspirates and ribs

An immunoprecipitation-inhibition procedure is described for assessing whether different antigenic determinants are carried on the same molecule or on different molecules on cells. The procedure entails (a) exposing NP-40 lysates of cells to antibody against one antigenic speficity; (b) removing free antibody and immune complexes by absorption with protein A-bearing S. aureus bacteria; (c) adsorption of the NP-40 with a detergent binding resin; and (d) measuring the inhibitory activity of the lysates for antibody against another specificity by a rosetting assay. This method has several advantages over the widely used sequential immunoprecipitation procedure.     

Neovascularization and bone regeneration by implantation of autologous bone marrow mononuclear cells

We examined whether transplantation of autologous bone marrow mononuclear cells (BM-MNCs) can augment neovascularization and bone regeneration of bone marrow in femoral bone defects of rabbits. Gelatin microspheres containing basic fibroblast growth factor (bFGF) were prepared for the controlled release of bFGF. To evaluate the in vivo effect of implanted BM-MNCs, we created bone defects in the rabbit medial femoral condyle, and implanted into them 5 x 10(6) fluorescent-labeled autologous BM-MNCs together with gelatin microspheres containing 10 microg bFGF on an atelocollagen gel scaffold. The four experimental groups, which were Atelocollagen gel (Col), Col + 5 x 10(6) BM-MNCs, Col + 10 microg bFGF, and Col + 5 x 10(6) BM-MNCs + 10 microg bFGF, were implanted into the sites of the prepared defects using Atelocollagen gel as a scaffold. The autologous BM-MNCs expressed CD31, an endothelial lineage cell marker, and induced efficient neovascularization at the implanted site 2 weeks after implantation. Capillary density in Col + BM-MNCs + bFGF was significantly large compared with other groups. This combination also enhanced regeneration of the bone defect after 8 weeks to a significantly greater extent than either BM-MNCs or bFGF on their own. In summary, these findings demonstrate that a combination of BM-MNCs and bFGF gelatin hydrogel enhance the neovascularization and the osteoinductive ability, resulting in bone regeneration.     

Generation of neuroprogenitor-like cells from adult mammalian bone marrow stromal cells in vitro

Recently, it has been proposed that bone marrow stromal cells (BMSCs) have a broader capacity for differentiation than previously contemplated. In vitro studies have indicated that BMSCs may have the capacity to differentiate into neuroectodermal-like cells in response to various growth conditions, including those commonly used to maintain and differentiate cultures of primary neural stem cells (NSCs). Interpreting the wealth of data on this subject has been difficult because of variation in the starting cell population and the differences between the methods used to induce their differentiation. Here we evaluate how cultures of expanded BMSCs with a consistent immunophenotype respond to a variety of growth conditions and induction agents and review their ability to form neural-like derivatives. In addition, we report on some modifications to previously published techniques for the generation of neural-like cells from BMSCs in vitro.     

Ultrastructure of mouse mononuclear phagocytes in bone marrow colonies grown in vitro.

Recently a new method was developed to culture bone marrow cells in a liquid medium on a glass surface. Two kinds of colonies develop in these cultures, namely mononuclear phagocyte and granulocyte colonies. The study of the ultrastructure of the cells in the mononuclear phagocyte colonies was the primary aim of the present study. The architecture of the mononuclear phagocyte colonies appeared to be quite different from that of the granulocyte colonies, since in the latter, the cells lie close together in dense clusters, whereas in mononuclear phagocyte colonies the cells are more loosely dispersed with the highest cell density at the center and stellate orientation of the cells at the periphery. However, both kind of colonies grow entirely separate from each other and mixed colonies are not observed. Electron microscopy showed that there are three types of cell in the mononuclear phagocyte colonies, i.e., monoblasts, promonocytes, and macrophages. The ultrastructure of the promonocytes and macrophages of the colonies is similar to that of the same types of cell isolated directly from the mouse. The monoblast, the most immature cell of mononuclear phagocyte colonies has not been characterized before. The ultrastructure of this cell is clearly distinct from that of the promonocyte in having a round contour without pseudopods, a nuclear to cytoplasmic ratio greater than one, a cytoplasm that contains many polyribosomes, a few small granules, and a small Golgi complex surrounded by a few short strips of rough endoplasmic reticulum.     

急性淋巴细胞白血病患者骨髓单个核细胞TAp63基因的表达

背景：据作者查新检索,国内外有关急性白血病患者骨髓单个核细胞TAp63基因表达的报道罕见。 目的：观察急性淋巴细胞白血病患者骨髓单个核细胞TAp63基因的表达及其意义。 方法：50例急性淋巴细胞白血病患者,其中32例急性B淋巴细胞白血病,18例急性T淋巴细胞白血病。同期选择27例非恶性血液病患者作为对照。取肝素抗凝的骨髓液2~4 mL,用Ficoll液分离骨髓单个核细胞,半定量反转录聚合酶链反应法检测TAp63的表达。 结果与结论：50例急性淋巴细胞白血病患者有49例表达TAp63,急性淋巴细胞白血病组明显高于非恶性血液病组(P < 0.05),急性B淋巴细胞白血病表达水平显著高于急性T淋巴细胞白血病(P < 0.05)。动态观察了5例初治急性淋巴细胞白血病化疗后不同阶段TAp63的表达变化,发现初治时TAp63表达,缓解后低表达或不表达,复发后又表达。结果表明TAp63在急性淋巴细胞白血病患者骨髓单个核细胞的表达明显高于非恶性血液病患者,尤其在急性B淋巴细胞白血病患者中高表达。     

Generation of natural killer cells from bone marrow precursors in vitro.

A simple and highly reproducible bone marrow culture system for the generation of cytolytically active NK cells from immature precursors in the bone marrow is described. The NK cells can be generated with various sources of IL-2, including Con A-conditioned medium, supernatants from IL-2-producing cell lines and recombinant IL-2. Neither IL-1, IL-3 nor alpha/beta interferon induced significant cytotoxicity in bone marrow cells. Identification of the cytotoxic cells as NK cells was based on their phenotypic characteristics (aGM1+, Thy 1 +/-, Ly 1-2- X Ia-, RIL-2 +/-, H2+), as well as their spectrum of target specificity. The deliberate addition of peripheral blood mononuclear cells as a source of mature NK cells and elimination of cells expressing markers specific for mature NK cells indicated that the generated NK cells were descendants of precursors of NK cells harboured in the bone marrow and not derived from mature NK cells contaminating the bone marrow preparations. Thus, it was shown that not only functionally active NK cells but also their precursors are highly dependent on IL-2 for differentiation and growth. This culture system should be helpful in studying the origin of NK cells in relation to other cell lineages as well as the regulation of the maturation of NK cells from their precursors.     

小儿骨髓间质干细胞体外诱导分化为神经元样细胞

BACKGROUND:In the past, the culture and differentiation of bone marrow mesenchymal stem cells in vitro were mostly reported in the adult or animal rather than in children. OBJECTIVE:To explore the ability of bone marrow mesenchymal stem cells from children differentiating into neural stem cells and nerve cells. METHODS:Bone marrow mesenchymal stem cells from children were isolated and cultured, and passage 12 cells were cultured in the pre-induction medium (DMEM culture medium containing 10% fetal bovine serum and 1 mmol/L β-mercapto ethanol) and induction medium (DMEM containing 2% dimethyl sulfoxide and 150 μmol/L butylated hydroxyanisole). Expression of nestin and β-tublin III was detected using immunocytochemistry method at 30 minutes and 7 days after induction, while RT-PCR was used to detect nestin mRNA expression at 0, 5.5, 6 days after induction. RESULTS AND CONCLUSION: After combined induction, the cells shrank from round shape to tapered, polygonal or oval shape, and cell processes extended gradually and became filament-like shape. Interconnected cells formed a network at 6 days after combined induction. The expression of nestin antigen was positive at 30 minutes after induction, while the expression of β-tublin was positive at 7 days. RT-PCR findings showed that positive expression of nestin mRNA was detected at 5.5 hours of induction, and then disappeared at 6 days. These findings show that the combined use of dimethyl sulfoxide and butylated hydroxyanisole can induce bone marrow mesenchymal stem cells from children to differentiate into neural stem cells and nerve cells in vitro.     

真性红细胞增多症患者骨髓单个核细胞植入再生障碍性贫血小鼠

背景：真性红细胞增多症患者骨髓的高增殖低凋亡特性使其造血干细胞植入NOD/SCID小鼠后成功分化出粒红系细胞,但其能否植入并改善再生障碍性贫血小鼠造血功能,目前国内外尚未有报道。 目的：探讨JAK2阳性真性红细胞增多症患者骨髓单个核细胞植入后对再生障碍性贫血小鼠造血重建的影响。 方法：应用注射用重组人γ-干扰素联合白消安的方法建立再生障碍性贫血小鼠模型,随机分为实验组(n=10)和对照组(n=10),给药结束后第5天实验组经尾静脉输注真性红细胞增多症患者骨髓单个核细胞悬液,对照组同法输注等容积生理盐水。输注后第14天检测小鼠外周血常规、骨髓细胞形态、骨髓组织病理变化以及小鼠外周血和骨髓内CD45+细胞的百分含量。 结果与结论：输注后第14天,实验组小鼠血细胞计数三系减少,骨髓涂片可见散在淋巴细胞和早期造血细胞,骨髓组织活检可见骨髓增生减低,少量粒系细胞及幼红细胞,未见巨核细胞,与对照组比较,造血功能无明显改善。对照组小鼠外周血及骨髓均未检测到CD45+细胞,实验组小鼠外周血及骨髓均可检测到人源化的CD45+细胞,且骨髓中CD45+细胞比外周血高,提示JAK2V617F阳性真性红细胞增多症患者骨髓单个核细胞能成功植入再生障碍性贫血小鼠体内,但血常规、骨髓涂片及活检结果显示未能明显改善骨髓造血功能。  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

兔骨髓基质细胞诱导成骨细胞复合同种异体生物衍生骨实验研究

目的探讨体外培养的骨髓基质细胞与自体来源的生物衍生骨复合后的生长特性，为进一步研究骨髓基质细胞作为种子细胞，以及探索一种良好的支架材料提供实验依据。方法分离纯化兔骨髓基质细胞并诱导成成骨细胞后与同种异体来源的生物衍生骨复合后体外培养，并在相差显微镜和扫描电镜下观察其生长情况。结果骨髓基质细胞在生物衍生骨上贴附生长、增殖良好。结论骨髓基质细胞可作为骨组织工程的理想种子细胞，与生物衍生骨复合后可作为骨组织工程的载体。     

The thiol-proteindisulfide oxidoreductase in human mononuclear cells of blood and bone marrow

The in vivo function of the thiol-proteindisulfide oxidoreductase (TPO, EC 1.8.4.2; proteindisulfide isomerase, EC 5.3.4.1) in biosynthesis of immunoglobulin was investigated by studying the enzyme content in human lymphoid and other cells by an immunocytochemical method. In contrast to peripheral blood, B lymphocytes which showed no or no demonstrable TPO, normal as well as malignant bone marrow plasma cells (all Ig classes) were found to contain abundant amounts of this enzyme. TPO containing plasma cells were identified by double-staining techniques. This finding suggests that TPO is involved in the terminal step of B cell differentiation and immunoglobulin biosynthesis. Besides plasma cells, approximately 10% of mononuclear marrow cells as yet unidentified medium-sized and large cells, exhibited also strong anti-TPO reactivity. Furthermore, using surface-cytoplasmic double staining methods, monocytes from human peripheral blood could be identified to represent the only cytoplasmic TPO-containing normal mononuclear blood cells.     

Reparation of the myocardium after transplantation of mononuclear bone marrow cells

Safety and efficiency of intracoronary transventricular transplantation of autologous mononuclear bone marrow cells in rats with postinfarction cardiosclerosis were studied. The cells migrated to the damaged area and were detected only in the cicatricial tissue; they have fibroblast-like phenotype and some of them were stained with Fapα (marker of reactive fibroblasts). More active proliferation of non-muscular cells and formation and maturation of collagen fibers in the cicatricial tissue were observed after transplantation of mononuclear cells. This led to thickening of the cicatricial wall, but the size of the scar and index of dilatation of the left ventricle remained unchanged. The number and volume density of newly formed blood vessels in the damaged area increased after transplantation, but no labeled cells were seen in the vascular walls. It can be hypothesized that stimulation of neoangiogenesis is mediated by paracrine mechanisms, which also explains improvement of global contractility of the left ventricle (increased contractility index in functional tests). Thus, transplantation of mononuclear bone marrow cells leads to thickening and strengthening of the cicatricial wall, stimulates angiogenesis, and improves global myocardial contractility. However, no morphological signs of reverse remodeling of the left-ventricular myocardium were revealed.