Protective effect of bone marrow mesenchymal stem cells on PC12 cells apoptosis mediated by TAG1

Objective: This study aims to explore the protection effect of bone marrow mesenchymal stem cells (BMSCs) on PC12 cells apoptosis mediated by transient axonal glycoprotein 1 (TAG1). Methods: PC12 cells were divided into control group, Aβ_25-35 group and BMSCs + Aβ_25-35 group. The effects of BMSCs on PC12 cells treated by Aβ_25-35 were detected using MTT, Hoechst 33258 and Annexin V-FITC/PI staining methods. The expression levels of TAG1, β-amyloid precursor protein (APP), AICD and p53 were determined by RT-PCR and Western blotting methods. The expression levels of Bax and Bcl-2 were determined by Western blotting method. The activity of Caspase 3 was detected by spectrophotometric method. Results: MTT results showed that cell activity decreased after the treatment of 20 μM Aβ_25-35 for 48 h (P<0.01) while it increased in BMSCs + Aβ_25-35 group (P<0.01). Hoechst 33258 and Annexin V-FITC/PI staining results showed that Aβ_25-35 could induce the apoptosis of PC12 cells while the apoptosis of PC12 cells was inhibited in BMSCs + Aβ_25-35 group. RT-PCR and Western blotting methods showed that 20 μM Aβ_25-35 could increase the expression levels of TAG1, APP, AICD and p53 (P<0.01) while they decreased in BMSCs + Aβ_25-35 group (P<0.01). 20 μM Aβ_25-35 could increase the expression levels of Bax and decrease the expression levels of Bcl-2 (P<0.01), while the expression levels of Bax decreased and the expression levels of Bcl-2 increase in BMSCs + Aβ_25-35 group (P<0.01). 20 μM Aβ_25-35 could enhance Caspase 3 activity while it decreased in BMSCs + Aβ_25-35 group (P<0.01). Conclusions BMSCs with Aβ_25-35 could inhibit the apoptosis of PC12 cells, which maybe related with TAG1/APP/AICD signal pathway.     

Inhibitory effect of CGRP on osteoclast formation by mouse bone marrow cells treated with isoproterenol

The present study was designed to elucidate the mode of action of isoproterenol (Isp; adrenergic beta-agonist) and to characterize the effect of the calcitonin gene-related peptide (CGRP; sensory neuropeptide) on osteoclast formation induced by Isp in a mouse bone marrow culture system. Treatment of mouse bone marrow cells with Isp generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) capable of excavating resorptive pits on dentine slices, and caused an increase in receptor activator of NF-kappaB ligand (RANKL) and a decrease in osteoprotegerin (OPG) production by the marrow cells. The osteoclast formation was significantly inhibited by OPG, suggesting the involvement of the RANKL-RANK system. CGRP inhibited the osteoclast formation caused by Isp or soluble RANKL (s-RANKL) but had no influence on RANKL or OPG production by the bone marrow cells treated with Isp, suggesting that CGRP inhibited the osteoclast formation by interfering with the action of RANKL produced by the Isp-treated bone marrow cells without affecting RANKL or OPG production. This in vitro data suggest the physiological interaction of sympathetic and sensory nerves in osteoclastogenesis in vivo.     

Induction of apoptosis in bone marrow cells is mediated via purinergic receptors

ATP activity in mouse bone marrow cells wasin vitro estimated by expression of phosphatidylserine on the outer membrane surface using FITC-labeled annexin. ATP induced apoptosis in bone marrow cells. Purinergic receptor antagonists PPADS and suramin modulated the apoptotic effect of ATP on hemopoietic cells. Acute and subacute administration of doxorubicin, an inductor of oxidative burst, decreased cell sensitivity to ATP and abolished its apoptotic effect. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 8, pp. 135–138, August, 2004     

Hurler disease bone marrow stromal cells exhibit altered ability to support osteoclast formation

Mucopolysaccharidosis type I (MPS IH; Hurler syndrome) is a rare genetic disorder that is caused by mutations in the α-L-iduronidase (IDUA) gene, resulting in the deficiency of IDUA enzyme activity and intra-cellular accumulation of glycosaminoglycans. A characteristic skeletal phenotype is one of the many clinical manifestations in Hurler disease. Since the mechanism(s) underlying these skeletal defects are not completely understood, and bone and cartilage are mesenchymal lineages, we focused on the characterization of mesenchymal cells isolated from the bone marrow (BM) of 5 Hurler patients. IDUA-mutated BM stromal cells (BMSC) derived from MPS IH patients exhibited decreased IDUA activity, consistent with the disease genotype. The expansion rate, phenotype, telomerase activity, and differentiation capacity toward adipocytes, osteoblasts, chondrocytes, and smooth muscle cells in vitro of the MPS I BMSC lines were similar to those of BMSC from age-matched normal control donors. MPS I BMSC also had a similar in vivo osteogenic capacity as normal BMSC. However, MPS I BMSC displayed an increased capacity to support osteoclastogenesis, which may correlate with the up-regulation of the RANKL/RANK/OPG molecular pathway in MPS I BMSC compared with normal BMSC.     

Sol-gel bioactive glasses support both osteoblast and osteoclast formation from human bone marrow cells

We have examined the ability of bioactive sol-gel glass ceramics to support both osteoblast and osteoclast differentiation from human bone marrow cells (HBMC). Nucleated cells from human bone marrow were cultured on tissue culture plastic and on two sol-gel coatings: A2 glass-ceramic containing 54 mol % CaO/40 mol % SiO(2) and S2 glass-ceramic containing 16 mol % CaO/80 mol % SiO(2). Osteoblast differentiation was followed by measuring alkaline phosphatase (ALP) activity, mRNA levels for ALP, osteopontin, RANK ligand (RANKL), and immunofluorescent co-localization of ALP and RANKL. Osteoclasts were identified by morphology and positive staining for tartrate-resistant acid phosphatase (TRAP). ALP activity and mRNA levels were similar for cells on A2 coatings and on tissue culture plastic, but mRNA levels of osteopontin and RANKL were tenfold higher on A2 than on plastic. Cultures on A2 coatings also contained multinucleated osteoclasts staining positively for TRAP. In contrast, cells cultured on S2 coatings had the characteristics of more differentiated osteoblasts as measured by higher ALP expression. However, the levels of osteopontin and RANKL mRNA on S2 glass were lower than on A2 glass and there were fewer, weakly staining TRAP-positive multinucleate cells. Thus, sol-gel glass-ceramic materials differing in CaO/SiO(2) ratios can produce markedly different effects on the osteoblast and osteoclast differentiation from HBMC.     

Granulocytic progenitor cells in the adherent layer of human long-term bone marrow cultures

The importance of the adherent layer in long-term mouse bone marrow cultures as a reservoir of the most primitive stem cells is known. The role of the adherent cell layer in long-term human cultures (LTC) is examined from this point of view. Confluent adherent layers developed after about 3 weeks of culture. At that time and weekly thereafter the cellularity and granulocytic-macrophage progenitor (CFU-GM) content of the adherent fraction were determined after tripsinisation. We have documented that CFU-GM were present in the adherent layer of human cultures for at least 8 weeks. These findings emphasize the importance of assessing the progenitor cell content of the adherent layer in long-term human cultures.     

Deflazacort increases osteoclast formation in mouse bone marrow culture and the ratio of RANKL/OPG mRNA expression in marrow stromal cells.

Information on precise effects of deflazacort on bone cell function, especially osteoclasts, is quite limited. Therefore, the present study was undertaken to test effects of deflazacort on osteoclast-like cell formation in mouse bone marrow cultures and on the regulation of osteoprotegerin (OPG) and its ligand (RANKL) mRNA expressions by RT-PCR in the ST2 marrow stromal cells. TRAP-positive mononuclear cells increased after the treatment of deflazacort at 10(-9) to 10(-7) M alone for 6 days in a dose-dependent manner. Number of TRAP-positive multi-nucleated cells (MNCs) increased significantly with combined treatment of deflazacort at 10(-7) M and 1,25-(OH)2D3 at 10(-9) M compared to that of cultures treated with 1,25-(OH)2D3 alone (p<0.05). Exposure to deflazacort at 10(-7) M in the presence of 1,25-(OH)2D3 at 10(-9) M in the last 3-day culture had greater stimulatory effect on osteoclast-like cell formation than that of the first 3-day culture did. Deflazacort at 10(-10) -10(-6) M downregulated OPG and upregulated RANKL in mRNA levels in a dose-dependent manner. These observations suggest that deflazacort stimulate osteoclast precursor in the absence of 1,25-(OH)2D3 and enhance differentiation of osteoclasts in the presence of 1,25-(OH)2D3. These effects are, in part, thought to be mediated by the regulation of the expression of OPG and RANKL mRNA in marrow stromal cells.     

全骨髓直接贴壁分离人骨髓间充质干细胞的生物学特性****☆

背景：体外分离培养骨髓间充质干细胞成为实验研究和临床应用的关键环节。 目的：采用全骨髓培养法分离人骨髓间充质干细胞,建立一种简单、有效的分离培养骨髓间充质干细胞的方法。 方法：通过全骨髓培养法分离培养人骨髓间充质干细胞,并通过传代进行纯化和扩增培养。分别在特定诱导体系中诱导人骨髓间充质干细胞向脂肪、成骨及软骨细胞分化。 结果与结论：采用全骨髓培养分离法能获得90%以上纯度的骨髓间充质干细胞;流式细胞仪检测结果显示,贴壁细胞均表达CD73、CD90、CD105,不表达造血细胞表型CD34、CD45和HLA-DR;细胞倍增时间为(24.04±0.49) h;细胞周期分析表明：G0~G1期和S+G2+M期所占比例分别为71.63%和28.37%;诱导分化结果显示,人骨髓间充质干细胞能够向脂肪、骨和软骨细胞分化。说明全骨髓分离培养法是一种较好的分离人骨髓间充质干细胞的方法。     

Interleukin-4 directly inhibits tumor necrosis factor-alpha-mediated osteoclast formation in mouse bone marrow macrophages

Recently it has been found that osteoclast differentiation is induced by tumor necrosis factor (TNF)-alpha. Interleukin (IL)-4 was reported to suppress osteoclast differentiation and bone resorption. However, no study has investigated the effect of IL-4 on TNF-alpha-induced osteoclast formation. In this study, we investigated whether IL-4 inhibits TNF-alpha-mediated osteoclast formation in mouse bone marrow derived macrophages (BMM). First, IL-4 suppresses RANKL-induced osteoclast formation and bone resorption. Next, when BMM were cultured with TNF-alpha, osteoclast-like cells were formed. When they were cultured with both TNF-alpha and IL-4, osteoclast formation and bone resorption was suppressed by IL-4 in a dose-dependent manner. It has been recently found that TNF-alpha and RANKL synergistically promote osteoclastogenesis. Finally, we investigated whether IL-4 had the ability to inhibit synergistic TNF-alpha and RANKL-induced osteoclastogenesis, with the result that it effectively inhibited the synergistic osteoclast formation in a dose-dependent manner. We conclude that IL-4 can strongly inhibit osteoclast formation that is related to both physiological bone resorption induced by RANKL and pathological bone resorption induced by TNF-alpha.     

脂质体介导骨保护素基因转染大鼠骨髓基质干细胞及其表达

背景：基因修饰骨组织工程种子细胞,可提高对骨缺损的修复能力。 目的：构建含有骨保护素的真核表达载体并检测转染骨髓基质干细胞后的表达。 方法：取4周龄SD大鼠胫骨和股骨行骨髓基质干细胞的分离和培养,分为载体组、空载体组、对照组。空载体组转染plRES2-EGFP载体,载体组转染plRES2-EGFP-OPG。 结果与结论：转染后激光扫描共聚焦显微镜观察可见骨髓基质干细胞内绿色荧光蛋白表达;RT-PCR检测结果可见质粒载体组在1 200 bp有明显条带,空载体组及对照组未见表达;免疫细胞化学检测骨髓基质干细胞内骨保护素蛋白呈阳性;MTT法检测各组细胞增殖活力无明显改变(P > 0.05)。证实应用plRES2-EGFP-骨保护素质粒转染大鼠骨髓基质干细胞,可获得外源性骨保护素的基因及蛋白表达。     

3D microtissue formation of undifferentiated bone marrow mesenchymal stem cells leads to elevated apoptosis

Current implantation formats to deliver bone marrow-derived mesenchymal stem cells (MSCs) to the site of myocardial injury resulted only in limited cell retention and integration. As an alternative concept to single cell transplantation, we investigated the fate of cell tracker-labeled syngenic rat MSC microtissue implants, injected into the scar area in a chronic rat myocardial infarction model. Analysis of the explants after 2 and 7 days revealed substantial amounts of the cell tracker within the infarct region. However, the signal was associated with the extracellular matrix rather than with viable implanted cells. Following these results, we systematically evaluated the behavior of MSCs derived from mouse, rat, and human origin in the microtissue format in vitro. We found that MSC-composed microtissues of all three species displayed highly elevated levels of apoptotic activity and cell death. This effect could be attenuated by initiating osteogenic differentiation during the tissue formation process. We conclude that MSCs used for tissue regeneration undergo apoptosis in their new environment unless they get appropriate signals for differentiation that permit sustained survival. These findings may explain the limited cellular regeneration potential in current MSC-based clinical trials and may change therapeutic strategies away from pure, unmodulated cell delivery concepts.     

TNF-α Induces Transient Resistance to Fas-Induced Apoptosis in Eosinophilic Acute Myeloid Leukemia Cells

Tumor necrosis factor α （TNF-α） has been recognized as an activator of nuclear factor kB （NF-kB）, a factor implicated in the protection of many cell types from apoptosis. We and others have presented evidence to suggest that Fas-induced apoptosis may be an important aspect of the resolution of inflammation, and that delayed resolution of inflammation may be directly associated with NF-kB-dependent resistance to Fas. Because TNF-α activates NF-kB in many cell types including inflammatory cells such as eosinophils, we examined effects of TNF-α signaling on the Fas-mediated killing of an eosinophilic cell line AML14. While agonist anti-Fas （CHII） treatment induced apoptosis in AML14 cells, no significant cell death occurred in response to TNF-α alone. Electrophoretic mobility shift assay （EMSA） revealed that TNF-α induced NF-kB transactivation in AMLI4 cells in a time- and dose-dependent fashion, and subsequent supershift assays indicated that the translocated NF-kB was the heterodimer p65 （RelA）/p50. Pre-treatment of cells with TNF-α dramatically decreased the CHll-induced cell death in a transient fashion, accompanied by suppression of activation of caspase-8 and caspase-3 activation. Inhibition of NF-kB transactivation by inhibitors, BAY 11-7085 and parthenolide, reversed the suppression of Fas-mediated apoptosis by TNF-α. Furthermore, TNF-α up-regulated X-linked inhibitor of apoptosis protein （XIAP） transiently and XIAP levels were correlated with the temporal pattern of TNF-α protection against Fas-mediated apoptosis. This finding suggested that TNF-α may contribute to the prolonged survival of inflammatory cells by suppression of Fas-mediated apoptosis, the process involved with NF-kB transactivation, anti-apoptotic XIAP up-regulation and caspase suppression. Cellular ＆ Molecular Immunology. 2007;4（1）：43-52.     

全骨髓贴壁法培养兔骨髓间充质干细胞体外定向成骨诱导分化及鉴定

背景：流式细胞仪分离法和免疫磁珠分离法对细胞活性影响较大,密度梯度离心法虽然能够获得纯度高的单核细胞,但由于多次离心可造成细胞的大量流失且对细胞活性有一定的影响使其应用值得商榷。 目的：采用全骨髓贴壁法分离兔骨髓间充质干细胞进行成骨诱导分化及鉴定。 方法：采用全骨髓贴壁法体外分离培养兔骨髓间充质干细胞,倒置显微镜下观察细胞形态学特征。在成骨诱导剂作用下,通过碱性磷酸酶染色试剂盒行碱性磷酸酶染色,Ⅰ型胶原免疫细胞化学染色,Von Kossa法及茜素红进行矿化结节染色以及电镜下检测兔骨髓间充质干细胞成骨诱导后的形态结构。 结果与结论：经诱导后细胞出现与成骨细胞相似的形态学特征,碱性磷酸酶染色阳性,Ⅰ型胶原免疫细胞化学染色,Von-Kossa法及茜素红矿化结节染色阳性。表明经成骨诱导剂诱导后全骨髓贴壁法体外分离纯化培养的兔骨髓间充质干细胞能向成骨细胞方向分化增殖。     

Constitutive expression of IL-18 and IL-18R in differentiated IEC-6 cells: effect of TNF-alpha and IFN-gamma treatment.

The multifunctional cytokine interleukin-18 (IL-18) is an important mediator in intestinal inflammatory processes. The aim of this study was to evaluate the constitutive expression of IL-18 and its receptors (IL-18Ralpha and IL-18Rbeta) in intestinal epithelial cells (IEC) stimulated by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In addition, cellular proliferation and evaluation of brush border enzymes as differentiation markers were studied. Nontransformed rat intestinal epithelial IEC-6 cells were grown on an extracellular matrix (ECM) in medium with or without TNF-alpha, IFN-gamma, or a combination of both. Gene expression of IL-18, its receptors and apoptotic markers was evaluated using real-time PCR. Expression of IL-18Ralpha protein was demonstrated by flow cytometry and Western blot. Enzymatic activities of brush border enzymes and caspase-1 were determined. The constitutive expression of IL-18, IL-18Ralpha and IL-18Rbeta mRNAs and proteins were detected in IEC-6 cells. The biologically active form of IL-18 was released in response to TNF-alpha and IFN-gamma treatment. Exogenous IL-18 had no effect on cellular proliferation, brush border enzyme activities, and gene expression of apoptotic markers. However, the addition of IL-18 stimulated production and release of the chemokine IL-8. These data suggest that IEC-6 cells may be not only a source of IL-18 but also a target for its action.     

IL-12 synergizes with IL-18 or IL-1beta for IFN-gamma production from human T cells

IL-18 is a proinflammatory cytokine that plays an important role in NK cell activation and T(h)1 response. IL-18 has a structural homology to IL-1, particularly IL-1beta. IL-18R, composed of IL-1R-related protein (IL-18Ralpha) and IL-1R accessory protein-like (IL-18Rbeta), belongs to the IL-1R family. Furthermore, IL-18R at least partly shares the signal transducing system with IL-1R. Thus, the IL-18-IL-18R system has a striking similarity to the IL-1-IL-1R system. For this reason, we regarded it important to investigate whether, like IL-18, IL-1beta synergizes with IL-12 in inducing IFN-gamma production from human T cells and plays an important role in the T(h)1 response. Here we show that IL-12 and IL-1beta synergistically induce T cells to proliferate and produce IFN-gamma without their TCR engagement. IL-12 stimulation induced an increase in the proportion of T cells positive for IL-18R. Then, IL-12-stimulated T cells responded to IL-18 or IL-1beta by their proliferation and IFN-gamma production, although levels of IL-1beta-induced responses were lower. CD4(+)CD45RA(+) T cells, although they constitutively expressed IL-18Rbeta mRNA, did not express IL-18Ralpha mRNA. Phytohemagglutinin (PHA) stimulation alone induced IL-18Ralpha mRNA without affecting the expression of IL-18Rbeta mRNA. T(h)1-inducing conditions (PHA, IL-12 and anti-IL-4) further increased this expression. We also show that T(h)1 cells but not T(h)2 cells have increased expression of IL-18R and IL-1R, and produce IFN-gamma in response to IL-18 and/or IL-1beta.     

Bursal origin of bone marrow cells with competence for antibody formation

The impaired antibody response of neonatally bursectomized chickens was restored by bone marrow cells from normal—but not from bursectomized—syngeneic donors. Chimaerism of donor cells in the recipients was demonstrated using the sex chromosomal marker. Attempts to substitute autologous bursal cell suspensions for the bursa in situ were unsuccessful. It is concluded that B lymphocytes in chicken bone marrow have matured in the bursa.     

IL-12 gene therapy for cancer: in synergy with other immunotherapies

In preclinical models of cancer, gene therapy with interleukin 12 (IL-12) has reached unprecedented levels of success when combined with immunotherapy approaches such as gene transfer of other cytokines and/or chemokines, costimulatory molecules or adoptive cell therapy. These combinations have been found to produce synergistic rather than additive effects. Meanwhile, IL-12 gene therapy is beginning clinical testing as a single agent, but combination strategies are at hand.     

Osteoclast formation in vitro from bone marrow mononuclear cells in osteoclast-free bone

Recent evidence points to the fact that osteoclasts are derived from mononuclear cells of hematopoietic bone marrow. In this study we have examined the formation of osteoclasts from mononuclear cells in vitro. The mononuclear cells were isolated after 7 days from cultures of mouse bone marrow cells. The isolated cells were co-cultured with osteoclast-free, fetal-mouse calvaria. After 10 to 14 days of co-culture, multinucleated cells which have all the characteristics of osteoclasts were found in juxtaposition to seams of woven bone. These data strongly suggest that bone marrow mononuclear cells, when suitably induced, can give rise to osteoclasts in vitro.     

Sphingosine-1-phosphate differently regulates the cytokine production of IL-12, IL-23 and IL-27 in activated murine bone marrow derived dendritic cells

Sphingosine-1-phosphate (S1P) modulates many cell functions such as lymphocyte trafficking and signaling as well as keratinocyte proliferation. However, less is known about the specific effects of S1P on cytokine production, particularly on the interaction between dendritic cells (DCs) and keratinocytes, cell types which are crucial for the initiation and maintenance of chronic inflammatory skin diseases like atopic dermatitis or psoriasis. Especially the cytokines of the IL-12 family play a dominant role in many inflammatory diseases as they have a significant impact on T-helper cell function. In the present study we show that S1P decreased the production of the pro-inflammatory cytokines IL-12 and IL-23 in LPS-stimulated DCs via the common subunit p40 as well as in the crosstalk with activated keratinocytes. By using specific S1P receptor agonists (SEW2871, FTY720-P) and antagonist (JTE013) we identified an important role for S1P receptor 1 in the modulation of the cytokine profile. While diminishing IL-12 and IL-23 secretion, S1P enhanced IL-27 production in DCs. To elucidate the mechanism of the different impact on the IL-12 family cytokine production, we investigated the mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase (PI3K) pathways in DCs. By using specific MAPK-Inhibitors (U0126, SB202190, SP600125) we demonstrated that ERK, p38 and JNK differently regulate each pathway of each cytokine. While p38 and JNK did not seem to play a role in the modulation properties of S1P on cytokine production, ERK is at least partially involved in the S1P mediated modulation of IL-12 and IL-27. The PI3K-Inhibitor abrogated the S1P-induced decrease of IL-12 and IL-23 secretion, while it had no influence on the S1P-induced increase of IL-27 production. These data implicate, that S1P has an anti-inflammatory impact on the production of IL-12 family cytokines, indicating therapeutic potential for S1P treatment of several inflammatory diseases like psoriasis.