Estrogen modulation of osteoblastic cell-to-cell communication

Two osteoblastic cell populations, calvarial and marrow stromal cells, were exposed to estrogen derivatives in vitro. The hormonal effect was monitored by following intracellular Ca⁺² levels [Ca⁺²]_i and gap-junction communication. We measured fast changes in intracellular Ca⁺² levels in response, of these cells, to the steroid hormones. The changes were dose dependent revealing maximal activity at 100 pM by 17-β-Estradiol and 1 nM by estradiol-CMO. Additionally, the effect of estrogen, on functional coupling of the cells, was measured using fluorescence dye migration and counting the number of neighboring cells coupled by gap junctions. An uncoupling effect was demonstrated in response of these cells to estrogen treatment. The quick stereospecific effect was achieved in the presence of 17-β-estradiol but not in the presence of 17-α-estradiol. These results suggest the involvement of plasma membrane receptors in addition to the already known nuclear receptors in transducing the hormone effects in the osteoblastic cells. J. Cell. Biochem. 69:282–290, 1998. © 1998 Wiley-Liss, Inc.     

Dexamethasone regulation of marrow stromal-derived osteoblastic cells

The clonal subtypes of cells in the osteogenic family represented by fibroblastoid MBA-15.33, preosteoblast MBA-15.4, and mature osteoblastic MBA-15.6 cells were used to study the effects of glucocorticoid (dexamethasone). The role of dexamethasone was monitored on cell attachment when plated on various protein substrata (BSA, collagen I, and Matrigel). A 24 h exposure of the cells to 10^-6 M or 10^-7 M dexamethasone differential affects their attachment preference. MBA-15.33 and MBA-15.4 cells increased their attachment capability on collagen I, while MBA-15.6 cells' attachment was inhibited. Pretreatment with (10^-6 M) dexamethasone caused an increase in attachment on Matrigel by MBA-15.33 cells and to less extent by MBA-15.4 cells. Additionally, measurements of two enzymatic activities were monitored; one is alkaline phosphatase (ALK-P), and the second is neutral endopeptidase (CD10/NEP). MBA-15.33, MBA-15.4, and MBA-15.6 cells were exposed to dexamethasone or to various growth factors (bone morphogenic protein (BMP-2 and BMP-3), TGFβ, and IGF-I). In some experiments, pretreatment of cells by dexamethasone was followed by exposure to the growth factors. The cells' challenged cellular responses were not uniform and revealed a differential pattern when their ALK-P and CD10/NEP enzymatic activities were measured. © 1996 Wiley-Liss, Inc.     

Identification and activation of mitogen-activated protein (MAP) kinase in normal human osteoblastic and bone marrow stromal cells: Attenuation of MAP kinase activation by cAMP,parathyroid hormone and forskolin

The mitogen-activated protein (MAP) kinases (p44^mapk and p42^mapk), also known as extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), are activated in response to a variety of extracellular signals, including growth factors, hormones and, neurotransmitters. We have investigated MAP kinase signal transduction pathways in normal human osteoblastic cells. Normal human bone marrow stromal (HBMS), osteoblastic (HOB), and human (TE85, MG-63, SaOS-2), rat (ROS 17/2.8, UMR-106) and mouse (MC3T3-E1) osteoblastic cell lines contained immunodetectable p44^mapk/ERK1 and p42^mapk/ERK2. MAP kinase activity was measured by 'in-gel' assay myelin basic protein as the substrate. Mainly ERK2 was rapidly activated (within 10 min) by bFGF, IGF-I and PDGF-BB in normal HOB, HBMS and human osteosarcoma cells, whereas both ERK1 and ERK2 were activated by growth factors in rat osteoblast-like cell lines, ROS 17/2.8 and UMR-106. The ERK1 activation was greater than the ERK2 in ROS 17/2.8 cells. Furthermore, ERK2 was also activated by bFGF and PDGF-BB in the mouse osteoblastic cell line, MC3T3-E1. This is the first demonstration of inter-species differences in the activation of MAP kinases in osteoblastic cells. Cyclic AMP derivatives or cAMP generating agents such as PTH and forskolin inhibited ERK2 activation by bFGF and PDGF-BB suggesting a 'cross-talk' between the two different signalling pathways activated by receptor tyrosine kinases and cAMP-dependent protein kinase. The accumulated results also suggest that the MAP kinases may be involved in mediating mitogenic and other biological actions of bFGF, IGF-I and PDGF-BB in normal human osteoblastic and bone marrow stromal cells.     

Inflammatory T cells rapidly induce differentiation of human bone marrow stromal cells into mature osteoblasts

Activated T cells secrete multiple osteoclastogenic cytokines which play a major role in the bone destruction associated with rheumatoid arthritis. While the role of T cells in osteoclastogenesis has received much attention recently, the effect of T cells on osteoblast formation and activity is poorly defined. In this study, we investigated the hypothesis that in chronic inflammation activated T cells contribute to enhanced bone turnover by promoting osteoblastic differentiation. We show that T cells produce soluble factors that induce alkaline phosphatase activity in bone marrow stromal cells and elevated expression of mRNA for Runx2 and osteocalcin. This data indicate that T cell derived factors have the capacity to stimulate the differentiation of bone marrow stromal cells into the osteoblast phenotype. RANKL mRNA was undetectable under any conditions in highly purified bone marrow stromal cells. In contrast, RANKL was constitutively expressed in primary osteoblasts and only moderately up-regulated by activated T cell conditioned medium. Interestingly, both bone marrow stromal cells and osteoblasts expressed mRNA for RANK, which was strongly up-regulated in both cell types by activated T cell conditioned medium. Although, mRNA for the RANKL decoy receptor, osteoprotegerin, was also up-regulated by activated T cell conditioned medium, it's inhibitory effects may be mitigated by a simultaneous rise in the osteoprotegerin competitor TNF-related apoptosis-inducing ligand. Based on our data we propose that during chronic inflammation, T cells regulate bone loss by a dual mechanism involving both direct stimulation of osteoclastogenesis, by production of osteoclastogenic cytokines, and indirectly by induction of osteoblast differentiation and up-regulation of bone turnover via coupling.     

Parathyroid hormone promotes the disassembly of cytoskeletal actin and myosin in cultured osteoblastic cells: mediation by cyclic AMP.

Parathyroid hormone (PTH) alters the shape of osteoblastic cells both in vivo and in vitro. In this study, we examined the effect of PTH on cytoskeletal actin and myosin, estimated by polyacrylamide gel electrophoresis of Triton X-100 (1%) nonextractable proteins. After 2-5 minutes, PTH caused a rapid and transient decrease of 50-60% in polymerized actin and myosin associated with the Triton X-100 nonextractable cytoskeleton. Polymerized actin returned to control levels by 30 min. The PTH effect was dose-dependent with an IC50 of about 1 nM, and was partially inhibited by the (3-34) PTH antagonist. PTH caused a rapid transient rise in cyclic AMP (cAMP) in these cells that peaked at 4 min, while the nadir in cytoskeletal actin and myosin was recorded around 5 min. The intracellular calcium chelator Quin-2/AM (10 microM) also decreased cytoskeletal actin and myosin, to the same extent as did PTH (100 nM). To distinguish between cAMP elevation and Ca++ reduction as mediators of PTH action, we measured the phosphorylation of the 20 kD (PI 4.9) myosin light chain in cells preincubated with [32P]-orthophosphate. The phosphorylation of this protein decreased within 2-3 min after PTH addition and returned to control levels after 5 min. The calcium ionophore A-23187 did not antagonize this PTH effect. Visualization of microfilaments with rhodamine-conjugated phalloidin showed that PTH altered the cytoskeleton by decreasing the number of stress fibers. These changes in the cytoskeleton paralleled changes in the shape of the cells from a spread configuration to a stellate form with retracting processes. The above findings indicate that the alteration in osteoblast shape produced by PTH involve relatively rapid and transient changes in cytoskeletal organization that appear to be mediated by cAMP.     

Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells

Dexamethasone is capable of directing osteoblastic differentiation of bone marrow stromal cells (BMSCs) in vitro, but its effects are not lineage-specific, and sustained exposure has been shown to down-regulate collagen synthesis and induce maturation of an adipocyte subpopulation within BMSC cultures. Such side effects might be reduced if dexamethasone is applied in a regimented manner, but the discrete steps in osteoblastic maturation that are stimulated by dexamethasone are not known. To examine this, dexamethasone was added to medium to initiate differentiation of rat BMSCs cultures and then removed after a varying number of days. Cell layers were analyzed for cell number, rate of collagen synthesis, expression of osteocalcin (OC), bone sialoprotein (BSP) and lipoprotein lipase (LpL), and matrix mineralization. Withdrawal of dexamethasone at 3 and 10 days was found to enhance cell number relative to continuous exposure, but did not affect to decrease collagen synthesis slightly. Late markers of osteoblastic differentiation, BSP expression and matrix mineralization, were also sensitive to dexamethasone and increased systematically with exposure while LpL systematically decreased. These results indicate that dexamethasone acts at both early and late stages to direct proliferative osteoprogenitor cells toward terminal maturation.     

大鼠脂肪干细胞向雪旺细胞诱导分化能力的研究

目的：研究脂肪干细胞（ADSCs）向雪旺细胞的诱导分化,为神经组织工程提供新的种子细胞。方法：取SD大鼠项背处的皮下脂肪,分离出脂肪干细胞并培养传代,流式细胞仪检测细胞表面特异标记CD29,CD34,CD44,CD45,CD90,以评价干细胞的生物学特性;采用b-FGF和forskolin等诱导脂肪干细胞向雪旺细胞分化,光镜观察诱导后细胞形态的变化;免疫荧光染色鉴定雪旺细胞特异性标记物S100、P75和GFAP的表达;PCR检测诱导前后雪旺细胞特异性标记物S100、P75的表达。结果：分离培养的鼠脂肪干细胞CD29、CB90表达呈阳性,而CD34、CD44和CD45表达呈阴性,具有脂肪干细胞的生物学特性;脂肪干细胞经过胶质细胞生长因子的作用,光镜下发现诱导的细胞形态与雪旺细胞相似;免疫荧光染色S100、P75和GFAP阳性;RT-PCR结果显示诱导的雪旺细胞标记物S100和P75表达上调。结论：脂肪干细胞可诱导分化成雪旺细胞,其表型和分子特征与雪旺细胞相似,诱导分化的脂肪干细胞是一种理想的神经组织工程的种子细胞。     

Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells

To clarify the mechanism of the stimulatory effect of statins on bone formation, we investigated the effect of simvastatin, a widely used statin, on osteoblastic and adipocytic differentiation in primary cultured mouse bone marrow stromal cells (BMSCs). Simvastatin treatment enhanced the expression level of mRNA for osteocalcin and protein for osteocalcin and osteopontin, and increased alkaline phosphatase activity significantly (p<0.05). After BMSCs were exposed to an adipocyte differentiation agonist, Oil Red O staining, fluorescence activated cell sorting, and decreased expression level of lipoprotein lipase mRNA showed that treatment with simvastatin significantly inhibits adipocytic differentiation compared to controls that did not receive simvastatin (p<0.05). Lastly, we found that simvastatin induces high expression of BMP(2) in BMSCs. These observations suggested that simvastatin acts on BMSCs to enhance osteoblastic differentiation and inhibits adipocytic differentiation; this effect is at least partially mediated by inducing BMP(2) expression in BMSCs.     

Adult rat bone marrow stromal cells differentiate into Schwann cell-like cells in vitro

Bone marrow stromal cells (MSCs) have the capability of differentiating into mesenchymal and non-mesenchymal lineages. In this study, MSCs isolated from adult Sprague-Dawley rats were cultured to proliferation, followed by in vitro induction under specific conditions. The results demonstrated that MSCs were transdifferentiated into cells with the Schwann cell (SC) phenotypes according to their morphology and immunoreactivities to SC surface markers including S-100, glial fibrillary acidic protein (GFAP) and low-affinity nerve growth factor receptor (p75). Consequently, rat adult MSCs can be induced in vitro to differentiate into SC-like cells, thus developing an abundant and accessible SC reservoir to meet the requirements of constructing tissue engineered nerve grafts for peripheral nerve repair.     

Anti-inflammatory roles of mesenchymal stromal cells during acute Streptococcus pneumoniae pulmonary infection in mice

Background

Pneumonia is the fourth leading cause of death worldwide, and Streptococcus pneumoniae is the most commonly associated pathogen. Increasing evidence suggests that mesenchymal stromal cells (MSCs) have anti-inflammatory roles during innate immune responses such as sepsis. However, little is known about the effect of MSCs on pneumococcal pneumonia.

骨髓基质细胞的特征及其在细胞和基因治疗中的应用

骨髓基质细胞是一类独特的间质干细胞,可分化为多种非造血系的组织。骨髓基质细胞具有贴壁生长的特性,因而易于在体外分离和扩增;另外骨髓基质细胞可在体内外表达多种治疗性的外湖目的基因。因此,骨髓基质细胞被认为是一种理想的治疗性细胞的基因治疗中的靶细胞。本文对骨髓基质细胞的研究进展及其在细胞和基因治疗中的应用作一综述。     

Cell-cycle phases and genetic profile of bone marrow-derived mesenchymal stromal cells expanded in vitro from healthy donors

Human mesenchymal stromal cells (MSCs) expanded in vitro for cell therapy approaches need to be carefully investigated for genetic stability, by employing both molecular and conventional karyotyping. Reliability of cytogenetic analysis may be hampered in some MSC samples by the difficulty of obtaining an adequate number of metaphases. In an attempt to overcome this problem, a methodology apt to evaluate the cell‐cycle structure on synchronous MSCs was optimised. Results obtained in five independent experiments by comparing cell‐cycle analysis of synchronous and asynchronous MSC populations evaluated at early and late culture passages documented that in synchronous MSCs, 30% of cells entered G2/M phase after about 27–28 h of culture, while in asynchronous MSCs only 8% of cells in G2/M phase could be observed at the same time point. Cytogenetic analysis on synchronous MSCs allowed us to obtain 20–25 valuable metaphases/slide, whereas only 0–4 metaphases/slide were detectable in asynchronous preparations. J. Cell. Biochem. 112: 1817–1821, 2011. © 2011 Wiley‐Liss, Inc.     

Tolerogenic effect of mesenchymal stromal cells on gliadin-specific T lymphocytes in celiac disease

Background aimsCeliac disease is caused by a dysregulated immune response toward dietary gluten, whose only treatment is a lifelong gluten-free diet. We investigated the effects of mesenchymal stromal cells (MSCs) on gliadin-specific T cells, which are known to induce intestinal lesions, in view of a possible use as new therapy.MethodsBone marrow–derived MSCs and gliadin-specific T-cell lines were obtained from allogeneic donors and mucosal specimens of celiac patients, respectively. The immunosuppressant effect of MSCs was evaluated in terms of proliferative response and interferon (IFN)-γ production upon gliadin stimulation of long-term T-cell lines; the immunomodulant effect was assessed in terms of apoptotic rate, immunophenotype and cytokine profile of short-term T-cell lines generated in the presence of MSCs. Different MSC:T-cell ratios were applied, and statistics were performed as appropriate.ResultsMSCs inhibited both proliferative response and IFN-γ production of long-term T-cell lines in a dose-dependent manner while limiting the expansion of short-term T-cell lines by increasing the apoptotic rate. Moreover, a reduction of the CD4⁺ population and expansion of the regulatory FoxP3⁺ subset were found in T-cell lines cultured with MSCs, in which a significant decrease of interleukin (IL)-21, IFN-γ and IL-10 paralleled by an upregulation of transforming growth factor-β1, IL-6 and IL-8 were observed. Finally, an increase of the indoleamine 2,3-dioxygenase activity was found, possibly playing a key role in mediating these effects.ConclusionsMSCs exert potent immunomodulant effects on gliadin-specific T cells, which may be exploited for future therapeutic application in celiac disease.     

Culture and behavior of osteoblastic cells isolated from normal trabecular bone surfaces

Summary We report the characterization of human osteoblastic cells that were derived from the surface of trabecular bone fragments. After removal of bone marrow cells, the bone lining osteoblastic cells lining the bone surface were obtained by migration and proliferation from the trabecular surface onto a nylon mesh. The isolated population proliferated in culture and exhibited osteoblastic phenotype. Cultured cells show a regular arrangment in vitro and exhibited multiple interconnecting junctions on scanning electron microscopic examination. Immunocytochemical staining showed that the cells produced almost exclusively type I collagen. Bone-surface-derived cells responded to 1–34 human parathyroid hormone by increasing intracellular cyclic AMP. Cell cultures exhibited high alkaline phosphatase activity, which was unaffected by 1,25 (OH)₂ vitamin D. Untreated cells produced high levels of osteocalcin, a bone-specific protein, and they responded to 1,25(OH) vitamin D by increasing osteocalcin synthesis in a dose-dependent manner. Although cells cultured for up to 5 mo. still produced osteocalcin, the response to 1,25(OH)₂D decreased after multiple passages. This study shows that the bone cell populations isolated from trabecular bone surface are enriched in osteoblast precursors and mature osteoblstic cells.     

Parathyroid hormone (1-34) regulates integrin expression in vivo in rat osteoblasts.

Intermittent administration of parathyroid hormone (PTH) activates new sites of bone formation by stimulating osteoblast differentiation and function resulting in an increase in bone mass. Because integrins have been shown to play a crucial role in osteoblast differentiation and bone formation, in the present study, we evaluated whether human PTH (1-34) upon administration to rats, influenced integrin expression in osteoblastic cells isolated from the metaphysis and the diaphysis of rat long bones. Initial immunohistochemical evaluation of bone sections demonstrated that the osteoblasts expressed at least alphav, alpha2, alpha3, and alpha5beta1 integrins. Immunocolocalization studies for integrins and vinculin established that alphav, alpha2, and alpha5beta1, but not alpha3 integrins were present in the focal adhesion sites of osteoblasts attached to FN coated surfaces. Osteoprogenitor cells isolated from metaphyseal (but not diaphyseal) marrow of rats injected with intermittent PTH (1-34) exhibited greater alphav and reduced alpha2 levels, with no apparent changes in alpha3, and alpha5beta1 integrin levels, as assessed by immunohistochemistry, Northern, and Western blot analyses. However, these changes were not observed on the same cells treated with PTH in vitro. These observations suggest that integrin modulation by PTH is likely to be indirect and that selective phenotypic expression of integrin subtypes is part of the cascade of events that lead to PTH (1-34) mediated osteoblast differentiation.