Dexamethasone inhibition of confluence‐induced apoptosis in human mesenchymal stem cells

Human mesenchymal stem cells (MSCs) have been extensively characterized with respect to their in vitro expansion and differentiation potential, especially with respect to osteogenesis. Dexamethasone (Dex) is a well‐known inducer of osteogenic differentiation of MSCs, but little is known about the effect of Dex treatment on apoptosis in MSCs. In this study, apoptosis in MSCs was examined with respect to cell density and Dex supplementation, using DAPI staining and DNA fragmentation ELISA Assay. In MSC cultures initiated at 1.0, 3.0, and 9.0 × 10³ cells per cm², it was found that higher MSC density correlated with increased apoptosis and that this apoptotic effect was diminished in cultures containing 100 nM Dex. MSCs and fibroblasts were co‐cultured, along with empty insert controls, and assayed for apoptosis by ELISA and DAPI counts to determine if soluble factors accounted for the cell density‐related apoptosis. No difference was seen between MSCs cultured with inserts containing either MSCs, fibroblasts, or empty control. To determine cell contact effects, BrdU‐labeled MSCs were cultured alone or with unlabeled chondrocytes at 2× and 8× the number of MSCs, with and without Dex, and apoptosis levels quantified. The results showed increased apoptosis at greater cell densities, and that the amount of apoptosis was greatly diminished in cultures containing Dex. These results show that apoptosis in MSCs is cell density‐related, requires direct cell contact, and that Dex treatment reduces or eliminates this density‐related apoptosis. These results may impact how MSCs should be cultured for clinical applications. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:216–221, 2009     

Dexamethasone modulates BMP‐2 effects on mesenchymal stem cells in vitro

Dexamethasone/ascorbic acid/glycerolphosphate (DAG) and bone morphogenic protein (BMP)‐2 are potent agents in cell proliferation and differentiation pathways. This study investigates the in vitro interactions between dexamethasone and BMP‐2 for an osteoblastic differentiation of mesenchymal stem cells (MSCs). Bone marrow‐derived human MSCs were cultured with DAG (group A), BMP‐2 + DAG (group B), and DAG + BMP‐2 combined with a porous collagen I/III scaffold (group C). RT‐PCR, ELISA, immuncytochemical stainings and flow cytometry analysis served to evaluate the osteogenic‐promoting potency of each of the above conditions in terms of cell morphology/viability, antigen presentation, and gene expression. DAG induced collagen I secretion from MSCs, which was further increased by the combination of DAG + BMP‐2. In comparison, the collagen scaffold and the control samples showed no significant influence on collagen I secretion of MSCs. DAG stimulation of MSCs led also to a steady but not significant increase of BMP‐2 level. A DAG and more, a DAG + BMP‐2, stimulation increased the number of mesenchymal cells (CD105+/CD73+). All samples showed mRNA of ALP, osteopontin, Runx2, Twist 1 and 2, Notch‐1/2, osteonectin, osteocalcin, BSP, and collagen‐A1 after 28 days of in vitro culture. Culture media of all samples showed a decrease in Ca²⁺ and PO concentration, whereas a collagen‐I‐peak only occurred at day 28 in DAG‐ and DAG + BMP‐2‐stimulated bone marrow cells. In conclusion, BMP‐2 enhances DAG‐induced osteogenic differentiation in mesenchymal bone marrow cells. Both agents interact in various ways and can modify osteoblastic bone formation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1440–1448, 2008     

Comparison of the osteogenic capacity of minipig and human bone marrow‐derived mesenchymal stem cells

Minipigs are a recommended large animal model for preclinical testing of human orthopedic implants. Mesenchymal stem cells (MSCs) are the key repair cells in bone healing and implant osseointegration, but the osteogenic capacity of minipig MSCs is incompletely known. The aim of this study was to isolate and characterize minipig bone marrow (BM) and peripheral blood (PB) MSCs in comparison to human BM‐MSCs. BM sample was aspirated from posterior iliac crest of five male Göttingen minipigs (age 15 ± 1 months). PB sample was drawn for isolation of circulating MSCs. MSCs were selected by plastic‐adherence as originally described by Friedenstein. Cell morphology, colony formation, proliferation, surface marker expression, and differentiation were examined. Human BM‐MSCs were isolated and cultured from adult fracture patients (n = 13, age 19–60 years) using identical techniques. MSCs were found in all minipig BM samples, but no circulating MSCs could be detected. Minipig BM‐MSCs had similar morphology, proliferation, and colony formation capacities as human BM‐MSCs. Unexpectedly, minipig BM‐MSCs had a significantly lower ability than human BM‐MSCs to form differentiated and functional osteoblasts. This observation emphasizes the need for species‐specific optimization of MSC culture protocol before direct systematic comparison of MSCs between human and various preclinical large animal models can be made. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1019–1025, 2012     

Matrix stiffness regulation of integrin‐mediated mechanotransduction during osteogenic differentiation of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) cultured on extracellular matrices with different stiffness have been shown to possess diverse lineage commitment owing to the extracellular mechanical stimuli sensed by the cells. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through the mechanotransducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MSCs. MSCs were cultured in osteogenic medium on tunable polyacrylamide hydrogels coated with type I collagen with elasticities corresponding to Young's modulus of 7.0 ± 1.2 and 42.1 ± 3.2 kPa. Osteogenic differentiation was increased on stiffer matrices, as evident by type I collagen, osteocalcin, and Runx2 gene expressions and alizarin red S staining for mineralization. Western blot analysis demonstrated an increase in kinase activities of ROCK, FAK, and ERK1/2 on stiffer matrices. Inhibition of FAK, an important mediator of osteogenic differentiation, and inhibition of ROCK, a known mechanotransducer of matrix stiffness during osteogenesis, resulted in decreased expression of osteogenic markers during osteogenic induction. In addition, FAK affects osteogenic differentiation through ERK1/2, whereas ROCK regulates both FAK and ERK1/2. Furthermore, α₂‐integrin was upregulated on stiffer matrices during osteogenic induction, and its knockdown by siRNA downregulated the osteogenic phenotype through ROCK, FAK, and ERK1/2. Taken together, our results provide evidence that the matrix rigidity affects the osteogenic outcome of MSCs through mechanotransduction events that are mediated by α₂‐integrin. © 2011 American Society for Bone and Mineral Research.     

重组人促红细胞生成素在体外抑制H2O2诱导的骨髓间充质干细胞凋亡

目的 探讨重组人促红细胞生成素(rhEPO)对H2O2诱导的人骨髓间充质干细胞(MSC)凋亡的影响及其机制.方法 体外培养MSC,培养至第6代,分为5组.rhEPO+ Ly294002组、rhEPO+U0126组和rhEPO+ anisomycin组分别使用Ly294002、U0126和anisomycin处理后,换用含rhEPO的培养基培养;rhEPO组始终用含rhEPO的培养基培养;对照组使用普通培养基培养.一部分MSC在培养1h后,用H2O2处理,然后在显微镜下观察细胞形态,用流式细胞术检测细胞凋亡情况;一部分MSC继续用含rhEPO的完全培养基培养,用蛋白质印迹法检测各组rhEPO作用前后MSC中细胞外调节蛋白激酶(ERK1/2)、丝裂原活化蛋白激酶p38(p38MAPK)及磷脂酰肌醇3-激酶、蛋白激酶B(PI3K/Akt)通路蛋白的表达情况.结果 rhEPO可使MSC的PI3K/Akt通路磷酸化水平升高,抑制p38MAPK通路磷酸化水平,但对MSC的ERK通路和总Akt、总p38MAPK水平无明显影响.rhEPO组中凋亡细胞所占百分率低于对照组(P＜0.01),rhEPO+ anisomycin组凋亡细胞所占百分率也低于对照组(P＜0.01);但rhEPO+ Ly294002组凋亡细胞所占百分率高于rhEPO组(P＜0.01).结论 rhEPO可激活人骨髓间充质干细胞的PI3K/Akt通路,并抑制p38MAPK通路,具有抑制H2O2诱导的人骨髓间充质干细胞凋亡作用.PI3K/Akt通路的激活参与了其抗凋亡作用的调控.     

滑膜间充质干细胞及其在组织工程中的应用

目的对滑膜间充质干细胞(synovium-derived mesenchymal stem cells,SMSCs)的研究进展及在组织工程中的应用进行综述。方法查阅近年SMSCs相关文献,从分离培养方法、基本特性及在组织工程中的应用三方面进行综述。结果 SMSCs具有分离方法简便、增殖能力及多分化能力较强的特点,已有研究将其应用于软骨、肌腱、韧带及骨组织工程领域。结论 SMSCs是MSCs家族的新成员,可能会成为组织工程领域新的种子细胞,还需要进一步深入研究。     

Amine‐surface‐modified superparamagnetic iron oxide nanoparticles interfere with differentiation of human mesenchymal stem cells

Superparamagnetic iron oxide (SPIO) nanoparticles have been widely used for stem cell labeling and tracking. Surface modification has been known to improve biocompatibility, biodistribution, and labeling efficiency of SPIO nanoparticles. However, the effects of amine (NH)‐surface‐modified SPIO nanoparticles on proliferation and differentiation of human mesenchymal stem cells (hMSCs) remain unclear. The purpose of this study is to investigate how amine‐surface‐modified SPIO nanoparticles affected hMSCs. In this study, intracellular uptake and the contiguous presence of amine‐surface‐modified SPIO nanoparticles in hMSCs were demonstrated by Prussian blue staining, transmission electron microscopy and magnetic resonance imaging. Moreover, accelerated cell proliferation was found to be associated with cellular internalization of amine‐surface‐modified SPIO nanoparticles. The osteogenic and chondrogenic differentiation potentials of hMSCs were impaired after treating with SPIO, while adipogenic potential was relatively unaffected. Altered cytokine production profile in hMSCs caused by amine‐surface‐modified SPIO nanoparticles may account for the increased proliferation and impaired differentiation potentials; concentrations of the growth factors in the SPIO‐labeled condition medium including amphiregulin, glial cell‐derived neurotrophic factor, heparin‐binding EGF‐like growth factor and vascular endothelial growth factor, as well as soluble form of macrophage colony‐stimulating factor receptor and SCF receptor, were higher than in the unlabeled‐condition medium. In summary, although amine‐surface‐modified SPIO labeling is effective for cell tracking, properties of hMSCs may alter as a consequence and this needs to be taken into account when evaluating therapeutic efficacies of SPIO‐labeled stem cells in vivo. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1499–1506, 2012     

Three‐dimensional high‐density co‐culture with primary tenocytes induces tenogenic differentiation in mesenchymal stem cells

Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and tissue engineering and may represent an attractive option for tendon repair and regeneration. Thus far the ability of MSCs to differentiate into tenocytes in vitro has not been investigated. Experiments were performed with and without growth factors (IGF‐1, TGF‐β1, IGF‐1/TGF‐β1, PDGF‐BB, and BMP‐12), in co‐cultures of tenocytes and MSCs mixed in different ratios and by culturing MSCs with spent media obtained from primary tenocytes. Tenogenesis was induced in MSCs through a combination of treatment with IGF‐1 and TGF‐β1, in high‐density co‐cultures and through cultivation with the spent media from primary tenocytes. Electron microscopy and immunoblotting were used to demonstrate up‐regulation of collagen I/III, decorin, tenomodulin, β1‐Integrin, MAPKinase pathway (Shc, Erk1/2), and scleraxis in the co‐cultures and provide simultaneous evidence for the inhibition of apoptosis. In monolayer co‐cultures extensive intercellular contacts between MSCs and tenocytes were observed. Cells actively exchanged vesicles, which were labeled by using immunofluorescence and immunogold techniques, suggesting the uptake and interchange of soluble factors produced by the MSCs and/or tenocytes. We conclude that MSCs possess tenogenic differentiation potential when provided with relevant stimuli and a suitable microenvironment. This approach may prove to be of practical benefit in future tissue engineering and tendon regenerative medicine research. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1351–1360, 2011     

人骨髓间充质干细胞向汗腺样细胞诱导分化的研究

目的：观察体外热休克汗腺细胞（SGCs）和人骨髓间充质干细胞（BM-MSCs）共培养体系中 BM-MSCs的形态和表型变化,为进一步表观遗传学表达谱的检测及汗腺诱导关键转录因子的研究提供实验基础。方法：体外分离、培养、扩增人BM-MSCs和 SGCs,成骨和成脂诱导分化以鉴定BM-MSCs 的分化功能。在 Tran-swell间接共培养体系中,培养的BM-MSCs和经47℃高温处理造成热休克的 SGCs 在 Transwell 板中间接共培养;在Transwell＋诱导因子共培养体系中,上室的BM-MSCs培养基中添加了汗腺诱导因子（无汗性外胚叶发育不良蛋白、重组人表皮生长因子和胰岛素-转铁蛋白-亚硒酸钠）。监测共培养过程中BM-MSCs的细胞形态变化,免疫荧光法检测诱导后BM-MSCs的表型改变。结果：经与热休克 SGCs 共培养诱导10 d后,部分 BM-MSCs 有由长梭形变为扁平状多边形的趋势,且局部细胞间连接紧密成片。BM-MSCs 诱导前不表达 CEA 和 CK19;BM-MSCs诱导后,Transwell间接共培养体系部分细胞 CEA 和 CK19表达阳性,Transwell＋诱导因子共培养体系CEA和CK19阳性细胞数明显多于 Transwell 间接共培养体系。结论：热休克汗腺细胞与 BM-MSCs 在 Tran-swell间接培养以及相关汗腺诱导因子的共培养体系下,BM-MSCs呈现向 SGCs诱导分化趋势。     

成人间充质干细胞体外诱导分化为成骨细胞的研究

目的 研究成人骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)体外培养定向诱导分化为成骨细胞,探讨其作为骨组织工程种子细胞的可行性和应用价值.方法 抽取健康成年骨髓,Ficoll密度梯度离心结合贴壁培养法经连续传代后改用含10 nmol/L地塞米松、10 mmol/Lβ-甘油磷酸钠、50 μmol/L维生素C的条件培养基培养,在相差显微镜下观察细胞形态变化,流式细胞仪细胞表面分子标志鉴定,免疫组化和RT-PCR检测Ⅰ型胶原蛋门的表达,同时测定细胞内碱件磷酸酶的含量.结果 BMSCs原代和传代培养的细胞具有活跃的增殖能力,诱导培养后细胞形态向成骨细胞转化,经RT-PCR、流式细胞仪、细胞碱性磷酸酶活性、糖原染色鉴定为成骨细胞.结论 BMSCs经合理的体外诱导培养后符合成骨细胞的形态特征和生物学特性,有望成为理想的骨组织工程种子细胞来源.     

血管内皮生长因子对骨髓间充质干细胞向软骨细胞分化的影响

[目的]探讨外源性血管内皮生长因子（VEGF）对人骨髓来源间充质干细胞（MSCs）增殖、向成软骨方向定向分化等生物学行为的影响及其机理，为MSCs构建组织工程化软骨奠定基础．[方法]取人骨髓来源间充质干细胞体外培养，将传代后的细胞分别置于含有800ng／ml和1600ng／ml VEGF的诱导培养基中进行培养，将具有促进细胞增殖分化及抑制多种炎性介质活性等多重生物学效应的TGF-β1以10ng／ml浓度配置无血清培养基诱导同组MSCs作阳性对照，观察细胞的增殖，分化。用Ⅱ型胶原免疫组化染色评价不同诱导因素下的软骨基质合成情况。[结果]所有细胞显示了良好的增殖能力，暴露于外源性血管内皮生长因子下的MSCs保持了良好的生长活性，并开始向软骨表型分化，与未添加诱导因子组细胞相比差异有显著性意义，不同浓度VEGF组的细胞合成软骨基质的能力无差异，但均不如阳性对照组。[结论]外源性VEGF具有诱导体外培养的人MSCs向软骨表型分化的能力，但与TGF-β1的诱导能力相比有差距。提示VEGF在MSCs来源的软骨修复过程中充当诱导信息提供者的角色。     

人骨髓间充质干细胞复合不同载体的体内成骨

目的：探讨冻干松质骨与羟基磷灰石对骨髓间充质干细胞体内成骨的影响。方法：取人骨髓间充质干细胞,使其附着于冻干松质骨和羟基磷灰石,并植入裸鼠皮下。定期腹腔内注射四环素液。18只裸鼠分别在术后2、4、6周处死,取标本进行HE、甲苯胺蓝染色观察和四环素荧光观察。结果：2周时,可见到骨髓间充质干细胞贴附在细胞复合松质骨（MC）组的标本表面及较深部的骨小梁表面,而在细胞复合羟基磷灰石（MH）组,细胞仅贴附在标本的表面。4周时,MC组的贴附细胞呈规则排列的立方体形,而MH组的细胞为不规则的立方体,排列也比较紊乱。到6周时,仅MC组出现了类骨质和四环素荧光,在松质骨骨块的所有孔隙内都可见到来自宿主的纤维组织长入,而羟基磷灰石仅在邻近表面的孔隙内见到这种纤维组织。结论：作为间充质干细胞载体,目前冻干松质骨要优于羟基磷灰石。     

人髓核间充质干细胞的分离提纯方式及生物学活性鉴定

目的 :探索人髓核间充质干细胞(nucleus pulposus mesenchymal stem cells,NPMSCs)的提纯方法并鉴定其生物学活性。方法:收集3例腰椎间盘突出症患者的退变髓核组织(Pfirrmann分级均为Ⅳ级),利用酶消化法分离细胞。采用两种方法分离提纯NPMSCs,一组细胞采用贴壁法培养(贴壁组),另一组通过流式细胞分选技术利用NPMSCs表面阳性标志物CD73、CD90、CD105获得NPMSCs(流式组)。将两种方法获得的NPMSCs进行体外培养扩增,分别进行形态学观察,细胞计数试剂盒(Cell Counting Kit,CCK-8)检测增殖能力。贴壁组NPMSCs采用流式细胞分选仪在进行分选之前检测免疫表型,流式组NPMSCs在生长达80%~90%融合时进行免疫表型的检测。向成骨、成脂、成软骨诱导分化,诱导28d后分别进行茜素红染色观察其成骨能力、油红O染色观察其成脂能力、甲苯胺蓝染色观察其成软骨能力,利用Imag J软件计算染色区域所占的面积百分比。比较两组NPMSCs在形态学、免疫表型及增殖和分化能力的差异。结果:形态学观察发现,两组NPMSCs均呈漩涡状生长,贴壁组NPMSCs可见散在的单个细胞生长;流式组NPMSCs长梭形形态更长,排列更加紧密,少见散在的单个贴壁生长细胞。流式细胞分选后所得的NPMSCs占细胞总数的(89.67±2.52)%,可以进行体外培养扩增,细胞为典型的长梭形特征,漩涡状生长,在接种后12~15d达80%~90%融合,增殖能力在接种后5~13d明显高于贴壁组NPMSCs(P0.05)。流式组NPMSCs的CD73、CD90、CD105的表达率明显高于贴壁组NPMSCs(P0.05),并且低表达CD34、CD45及HLA-DR。两种方法获得的NPMSCs均能完成三系诱导分化,流式组成骨、成脂、成软骨染色区域百分比均明显高于贴壁组(P0.05)。结论:利用流式细胞分选技术从人退变髓核组织中可获得较高纯度的NPMSCs,并能进行后续培养扩增。与贴壁法获得的NPMSCs相比,流式细胞分选的NPMSCs具有更强的增殖与分化能力。     

人骨髓间充质干细胞分离培养及体外诱导分化为成骨细胞

[目的]建立人骨髓来源的间充质干细胞( hBMSCs)分离、培养及传代的方法,观察成hBMSCs体外成骨潜能.[方法]采用全骨髓贴壁筛选法分离培养hBMSCs,流式细胞仪检测细胞表型;所得细胞第3代用含100 nmol/L地塞米松、5mmol/Lβ -甘油磷酸钠,50 μg/ml抗坏血酸的条件培养基进行骨诱导,茜素红染色鉴定.[结果]分离培养的细胞流式细胞术检测显示CD44、CD105阳性,而CD34、CD45阴性,符合MSCs特征;hBMSCs经骨诱导后可形成钙结节,茜素红染色显示阳性.[结论]全骨髓贴壁筛选法可分离获得高纯度的hBMSCs,其在体外具有成骨潜能.     

Tissue engineering‐based cartilage repair with mesenchymal stem cells in a porcine model

This in vivo pilot study explored the use of mesenchymal stem cell (MSC) containing tissue engineering constructs in repair of osteochondral defects. Osteochondral defects were created in the medial condyles of both knees of 16 miniature pigs. One joint received a cell/collagen tissue engineering construct with or without pretreatment with transforming growth factor β (TGF‐β) and the other joint from the same pig received no treatment or the gel scaffold only. Six months after surgery, in knees with no treatment, all defects showed contracted craters; in those treated with the gel scaffold alone, six showed a smooth gross surface, one a hypertrophic surface, and one a contracted crater; in those with undifferentiated MSCs, five defects had smooth, fully repaired surfaces or partially repaired surfaces, and one defect poor repair; in those with TGF‐β‐induced differentiated MSCs, seven defects had smooth, fully repaired surfaces or partially repaired surfaces, and three defects showed poor repair. In Pineda score grading, the group with undifferentiated MSC, but not the group with TGF‐β‐induced differentiated MSCs, had significantly lower subchondral, cell morphology, and total scores than the groups with no or gel‐only treatment. The compressive stiffness was larger in cartilage without surgical treatment than the treated area within each group. In conclusion, this preliminary pilot study suggests that using undifferentiated MSCs might be a better approach than using TGF‐β‐induced differentiated MSCs for in vivo tissue engineered treatment of osteochondral defects. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1874–1880, 2011     

脂肪组织来源干细胞与脂肪瘤间充质干细胞特性的比较研究

目的 比较体外培养的脂肪来源干细胞(adipose-derived stem cells,ASCs)与脂肪瘤间充质干细胞(lipoma-derived mesenchymal stem cells,LMSCs)的生物特性,以探讨ASCs移植的安全性.方法 对正常脂肪组织和脂肪瘤组织进行切片染色,分离培养ASCs和LMSCs,观察细胞形态;MTS比色法检测细胞活性并绘制细胞生长曲线;流式细胞仪测定细胞周期及表面分子表达;QRT-PCR检测高迁移率族蛋白2(HMGA2)表达水平;免疫组织化学染色法鉴定端粒酶逆转录酶(hTERT)的表达.结果 正常脂肪组织和脂肪瘤组织切片差异明显;ASCs细胞形态一致性好,而LMSCs具有不均质性;MTS活性测定ASCs增殖活性要远低于LMSCs细胞(P=0.000);流式细胞仪检测结果显示ASCs与LMSCs在干细胞标志CD29、CD44、CD105上表达类似,而在肿瘤干细胞标志CD133表达上,ASCs(5.35%)要远低于LMSCs(26.87%);细胞周期显示ASCs的增殖能力低于LMSCs;定量PR-PCR显示ASCs中HMGA2平均aQ值为1,远低于在LMSCs中的表达(1.79),两者差异具有统计学意义(P<0.01);免疫细胞化学结果:hTERT在ASCs和LMSCs中的累计吸光度A值分别为1 379.597±498.617和3 328.108±902.856,面积分别为132 390.27±35 568.945和238 000.53±49 264.289,平均吸光度A值分别为0.009±0.003和0.014±0.003,ASCs中hTERT表达远低于LMSCs.结论 体外培养的ASCs生物学特性与LMSCs存在显著差异,未发现其恶性转、化为肿瘤干细胞的证据.