Development of a model system to analyze chondrogenic differentiation of mesenchymal stem cells

High-density cell culture is widely used for the analysis of cartilage development of human mesenchymal stem cells (HMSCs) in vitro. Several cell culture systems, as micromass, pellet culture and alginate culture, are applied by groups in the field to induce chondrogenic differentiation of HMSCs. A draw back of all model systems is the high amount of cells necessary for the experiments. Further, handling of large experimental approaches is difficult due to culturing e.g. in 15 ml tubes. Therefore, we aimed to develop a new model system based on “hanging drop” cultures using 10 to 100 fold less cells. Here, we demonstrate that differentiation of chondrogenic cells was induced as previously shown in other model systems. Real time RT-PCR analysis demonstrated that Collagen type II and MIA/CD-RAP were upregulated during culturing whereas for induction of hypertrophic markers like Collagen type X and AP-2 epsilon treatment with TGF beta was needed. To further test the system, siRNA against Sox9 was used and effects on chondrogenic gene expression were evaluated. In summary, the hanging drop culture system was determined to be a promising tool for in vitro chondrogenic studies.     

骨髓间充质干细胞成骨分化调控的研究进展

骨髓间充质干细胞具有较强的自我更新能力和多向分化潜能 ,其应用是目前国际上组织工程领域中重要的研究内容之一。近年来 ,许多实验室从分子、生化、物理等水平对其成骨分化调控进行了深入研究 ,取得了较大的进展     

视网膜干细胞培养上清液诱导骨髓间充质干细胞的分化

背景：骨髓间充质干细胞向神经细胞诱导分化可为神经系统受损伤后的修复和再生带来了新的希望。 目的：探讨骨髓间充质干细胞在视网膜干细胞培养上清液诱导条件下向神经元细胞分化。 方法：采用全骨髓培养方法,用视网膜干细胞培养上清液诱导骨髓间充质干细胞,通过免疫荧光染色鉴定其分化的结果。 结果与结论：诱导72 h,骨髓间充质干细胞表达神经干细胞的特异性抗体巢蛋白和神经元中的标志性微管相关蛋白微管相关蛋白2。视网膜干细胞培养上清液能够促进骨髓间充质干细胞向神经元样细胞分化,提示视网膜干细胞可能分泌神经生长因子。     

体外诱导骨髓间充质干细胞向心肌细胞分化的实验研究

目的:采用5-氮胞苷(5-aza)诱导骨髓间充质干细胞(M SCs)为心肌样细胞,对比分析诱导细胞与正常心肌细胞表型的差异,从而分析5-aza的诱导效应。方法:分离培养大鼠骨髓M SCs,通过检测不同浓度的5-aza诱导后M SCs表面CD 45、CD 54、CD 90分子的表达并与正常心肌细胞表型进行比较,以观察5-aza对间充质干细胞体外分化诱导的影响和作用。结果:5-氮胞苷能有效地诱导间充质干细胞分化为心肌样细胞,诱导后的细胞表面均表达M H C和desm in分子。浓度为10μm ol/L的5-aza诱导组细胞表型更接近于心肌细胞。结论:M SCs在体外条件下经5-aza诱导可以分化为心肌样细胞,10μm ol/L的诱导浓度为最优浓度。     

灌注型生物反应器中流速对人骨髓间充质干细胞增殖及成骨分化的影响

目的探讨在灌注型生物反应器中,大段磷酸三钙(β-TCP)载体内不同流速对人骨髓间充质干细胞(humanmesenchymal stem cells,hMSCs)增殖及成骨分化的影响。方法利用灌注型生物反应器对复合了hMSCs的大段β-TCP载体分别以3、6、9 mL/min的流速培养15 d,通过葡萄糖消耗量、细胞活力(MTT比色法)以及扫描电镜(SEM)观测细胞在载体内的增殖情况;以Real-time PCR检测成骨分化相关基因的表达。结果各组葡萄糖的日消耗量随培养时间的延长而增加,初期9 mL/min组细胞增殖明显快于3、6 mL/min组(P<0.001),但是灌注培养后期6 mL/min组的细胞增殖快于9、3 mL/min组(P<0.05)。灌注培养15 d后,6 mL/min组细胞活力显著高于3、9 mL/min组(P<0.001)。SEM观察发现3组β-TCP载体内均形成复合细胞层,3 mL/min组细胞层呈疏松的簇状,6 mL/min组复合细胞层部分呈膜片状,9 mL/min组复合细胞层多数呈膜片状。在进行成骨诱导灌注培养15 d后,6、9 mL/min组碱性磷酸酶(ALP)及骨桥蛋白(OP)的表达均显著高于3 mL/min组(P<0.01);3、6 mL/min组骨钙素(OC)的表达基本相同(P>0.05),而9 mL/min组OC的表达量则显著高于另外两组(P<0.001)。结论在利用灌注型生物反应器对hMSCs进行灌注培养的早期,9 mL/min的流速最有利于hMSCs的增殖,而晚期6 mL/min的流速最有利于hMSCs的增殖。β-TCP载体内流体剪切应力(flow shear stress,FSS)随灌注流速的增加而增加,适当的FSS可促进细胞外基质的合成和分布,并增加成骨相关基因的表达。     

microRNA-130b downregulation potentiates chondrogenic differentiation of bone marrow mesenchymal stem cells by targeting SOX9

Osteoarthritis (OA) is a chronic health condition. MicroRNAs (miRs) are critical in chondrocyte apoptosis in OA. We aimed to investigate the mechanism of miR-130b in OA progression. Bone marrow mesenchymal stem cells (BMSCs) and chondrocytes were first extracted. Chondrogenic differentiation of BMSCs was carried out and verified. Chondrocytes were stimulated with interleukin (IL)-1β to imitate OA condition in vitro. The effect of miR-130b on the viability, inflammation, apoptosis, and extracellular matrix of OA chondrocytes was studied. The target gene of miR-130b was predicted and verified. Rescue experiments were performed to further study the underlying downstream mechanism of miR-130b in OA. miR-130b first increased and drastically reduced during chondrogenic differentiation of BMSCs and in OA chondrocytes, respectively, while IL-1β stimulation resulted in increased miR-130b expression in chondrocytes. miR-130b inhibitor promoted chondrogenic differentiation of BMSCs and chondrocyte growth and inhibited the levels of inflammatory factors. miR-130b targeted SOX9. Overexpression of SOX9 facilitated BMSC chondrogenic differentiation and chondrocyte growth, while siRNA-SOX9 contributed to the opposite trends. Silencing of SOX9 significantly attenuated the pro-chondrogenic effects of miR-130b inhibitor on BMSCs. Overall, miR-130b inhibitor induced chondrogenic differentiation of BMSCs and chondrocyte growth by targeting SOX9.     

骨髓间充质干细胞向软骨细胞分化过程中miR130α的表达

目的诱导骨髓间充质干细胞向软骨细胞分化,初步探索miR130a在骨髓间充质干细胞向软骨细胞分化过程中的调控作用。方法体外TGF-β1诱导骨髓间充质干细胞向软骨细胞分化,免疫荧光法和免疫组化染色鉴定Ⅱ型胶原,阿辛兰染色鉴定氨基葡聚糖。用Real-time PCR法检测细胞miR130a的表达。结果骨髓间充质干细胞在TGF-β1诱导下可分化为软骨细胞。培养7 d后,诱导培养组细胞miR130a表达的水平显著低于培养前的水平(P<0.05)。结论骨髓间充质干细胞体外诱导可以分化为软骨细胞,在向软骨细胞分化的早期,miR130a表达降低伴随着软骨细胞的分化,提示与软骨形成的过程有关。     

人骨髓间充质干细胞体外内皮细胞诱导分化的研究(英)

目的：探讨人骨髓间充质干细胞hMSCs(human mesenchymal stem cells,hMSCs)体外内皮分化基础及其诱导条件。方法：〖HTSS〗采用密度分离与贴壁筛选结合法分离hMSCs，体外联合应用生长因子VEGF165和不同细胞外基质纤维连接蛋白(FN)与Ⅰ型胶原(Col)，对其进行内皮诱导分化。通过免疫细胞化学、细胞化学、流式细胞分析法及透射电镜对其分化后细胞进行鉴定。结果：hMSCs表达早期内皮分化标志之一KDR；PAS反应阳性及超微结构显示，hMSCs胞浆内含有大量糖原形成糖原池，分化后细胞胞浆外质内糖原明显减少或消失，暗示细胞发生分化；细胞诱导后CE34、β1整合素和KDR表达均增强。结论：诱导后细胞为过渡细胞群类型(transit population,TP)，可向内皮前体细胞(endothelial progenitor cells,EPC)EPC方向分化。推测由此类型细胞再分化为内皮细胞(endothelial cells,ECs)，即hMSCs→TP→EPC→ECs。     

微重力对骨髓间充质干细胞成骨分化的影响

骨髓间充质干细胞（BMSCs）是一种多能成体于细胞,是组织工程重要的种子细胞来源之一.微重力对BMSCs成骨分化具有抑制作用,可使骨量减少和骨微结构改变,从而导致骨质疏松症.这一过程受到多条信号通路的调控,如MAPK信号通路、Notch信号通路和Wnt/β-catenin信号通路等,它们协同调节微重力下BMSCs向成骨细胞方向的分化.研究微重力对BMSCs成骨分化的影响,可以阐明骨质流失机理,为相关疾病的治疗提供新的靶点,促进我国太空宇航事业的发展.     

切应力诱导间充质干细胞分化成内皮细胞的研究

目的研究绿色荧光蛋白（GFP）小鼠骨髓间充质干细胞（MSCs）体外分离培养及扩增及其在流体剪切力条件下向内皮细胞（EC）定向分化能力。方法选用GFP小鼠作为研究对象,分离培养骨髓间充质干细胞,并在体外进行细胞的扩增和传代。应用流体剪切力刺激方法对骨髓间充质干细胞进行诱导,并观测其对GFP小鼠骨髓间充质干细胞分化为内皮细胞的积极作用,对其结果进行免疫荧光检测,计算阳性细胞比例,统计分析。结果GFP小鼠骨髓间充质干细胞每扩增一代,细胞数量增加5-8倍。检测诱导后MSCs,结果显示在诱导3 d时出现vWF表达阳性细胞。结论GFP小鼠MSCs在体外的培养扩增能力强,MSCs在流体剪切应力的作用下可以转化成内皮细胞。     

晚期骨关节炎患者膝关节滑膜间充质干细胞的体外成骨分化

背景：间充质干细胞是组织工程中常用的种子细胞,而来源于人膝关节滑膜组织的间充质干细胞能否作为骨组织修复与再生中合适的种子细胞还需要进一步验证。 目的：观察晚期骨关节炎患者膝关节滑膜组织来源的间充质干细胞体外向成骨细胞定向分化的潜能,对所诱导细胞的成骨特性进行鉴定。 方法：无菌条件下从行全膝关节置换的晚期骨关节炎患者膝关节腔内获取滑膜组织,Ⅰ型胶原酶消化分离获得有核细胞。以最适密度接种(200个有核细胞/cm2),挑选单细胞克隆,筛选出滑膜间充质干细胞。基础培养基培养,传至第3代时改换为含地塞米松、β-甘油磷酸钠和抗坏血酸的成骨诱导培养基诱导培养。 结果与结论：体外分离培养的滑膜间充质干细胞早期可形成葵花样细胞集落,克隆样生长。传代至第3代,细胞呈成纤维细胞样生长,形态均一。滑膜间充质干细胞成骨诱导后呈典型的“铺路石样”成骨细胞形态,诱导至第7天碱性磷酸酶染色呈强阳性,碱性磷酸酶活性表达也在诱导7 d时达最高峰;诱导21 d茜素红染色可见大量钙结节形成;同时反转录PCR检测到Ⅰ型胶原、Runx2、骨结合蛋白和骨桥蛋白的表达在诱导后增加,21 d时表达最高。从晚期骨关节炎患者膝关节滑膜组织分离获得的滑膜间充质干细胞,在体外可成功诱导分化为成骨细胞,所诱导生成的细胞具有典型的成骨细胞特性。提示滑膜间充质干细胞有望成为骨组织工程理想的种子细胞来源,在骨组织的再生修复中发挥重要作用。     

脂肪基质干细胞的分离培养及其作为软骨种子细胞的研究

目的研究人体脂肪基质干细胞(ADSC)分离培养的方法,探讨其在体外向成软骨细胞诱导分化的能力。方法取临床吸脂所得脂肪组织,胶原酶消化分离后,接种于培养基中进行原代培养,传至一代时换软骨诱导培养液进行成软骨诱导分化,定期通过阿尔辛蓝染色、天狼猩红染色及免疫组化等方法进行证实。结果ADSC在体外成软骨诱导后,可向软骨细胞分化,经诱导的细胞可分泌软骨细胞特异性的Ⅱ型胶原以及硫酸蛋白多糖。结论在适当诱导条件下,ADSCs有向软骨细胞分化的潜能,为软骨组织工程种子细胞来源的研究提供了新方法。     

红景天苷与淤胆血清诱导骨髓间充质干细胞向肝样细胞分化

背景：大量实验证实骨髓间充质干细胞在诱导因子及特定微环境下可诱导分化为肝细胞,并已广泛用于终末肝病的临床替代治疗,而其最佳诱导条件目前尚不清楚。 目的：初步探讨中草药红景天苷联合淤胆大鼠血清体外诱导大鼠骨髓间充质干细胞向肝细胞分化的可行性和有效性。　 方法：采用全骨髓贴壁培养法从大鼠骨髓中获取骨髓间充质干细胞,流式法检测干细胞表型;胆总管结扎切断法制备大鼠淤胆血清。取第3代骨髓间充质干细胞分3组体外诱导培养：空白对照组,基础培养基+5%淤胆血清;红景天苷组：基础培养基+5%淤胆血清+30 µmol/L红景天苷;阳性对照组：基础培养基+5%淤胆血清+20 µg/L肝细胞生长因子;观察各组诱导培养过程中细胞形态变化,RT-PCR法、Western-Blot法检测各诱导组肝细胞特异性蛋白表达水平。 结果与结论：骨髓间充质干细胞高表达CD90、CD105,不表达CD45、CD14、CD34、CD79a;空白对照组、红景天苷组、阳性对照组细胞在诱导培养中均出现多角及双核细胞;空白对照组、红景天苷组、阳性对照组在诱导培养7 d开始出现甲胎蛋白、白蛋白的mRNA及蛋白表达;在同一时间点空白对照组表达率最低(P < 0.05),红景天苷组、阳性对照组间比较,差异无显著性意义(P > 0.05)。与传统淤胆血清体外诱导相比,红景天苷联合淤胆血清能更有效诱导骨髓间充质干细胞向肝样细胞分化。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;组织工程全文链接：     

Mechanical regulation of mesenchymal stem cell differentiation

Biophysical cues play a key role in directing the lineage commitment of mesenchymal stem cells or multipotent stromal cells (MSCs), but the mechanotransductive mechanisms at play are still not fully understood. This review article first describes the roles of both substrate mechanics (e.g. stiffness and topography) and extrinsic mechanical cues (e.g. fluid flow, compression, hydrostatic pressure, tension) on the differentiation of MSCs. A specific focus is placed on the role of such factors in regulating the osteogenic, chondrogenic, myogenic and adipogenic differentiation of MSCs. Next, the article focuses on the cellular components, specifically integrins, ion channels, focal adhesions and the cytoskeleton, hypothesized to be involved in MSC mechanotransduction. This review aims to illustrate the strides that have been made in elucidating how MSCs sense and respond to their mechanical environment, and also to identify areas where further research is needed.     

定向诱导骨髓间充质干细胞向心肌细胞分化

目的:采用骨髓间充质干细胞体外转化为心肌样细胞,为心衰的干细胞移植治疗提供基础。方法:分离培养大鼠骨髓间充质干细胞,5-氮杂胞苷诱导24h后正常培养,通过形态学、PAS反应、磷钨酸苏木素染色(PTAH)、免疫细胞化学染色作鉴定。结果:诱导细胞以梭形、柱状为主,部分细胞有分支,单个核、卵圆形、居中,似心肌细胞,PAS及PTAH染色均为阳性。诱导后培养2周的细胞表达Sarcomeric Actin和Connexin-43较弱,以后表达逐渐增强,而且1、3、5代细胞均稳定表达。结论:骨髓MSCs可在体外被诱导分化为心肌样细胞,有望成为心衰干细胞移植治疗的理想细胞材料。     

流体静压力及雌激素共同作用对骨髓间充质干细胞成骨及成软骨分化的影响

目的观察流体静压力联合雌激素对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖、细胞骨架及成骨、成软骨向分化的影响,检测这两种刺激共同作用是否具有生物叠加效应。方法采用全骨髓贴壁分离法分离培养BMSCs,流式细胞仪进行BMSCs表面标志物分子鉴定。细胞分为对照组(C组)、1 nmol/L17β-雌二醇作用组(E组)、1 nmol/L雌激素受体拮抗剂TAM作用组(T组)、90 kPa压力加载1 h组(P组)、17β-雌二醇预处理12 h后再加压1 h组(P+E组)以及TAM预处理12 h后再加压1 h组(P+T组)。流式细胞仪检测各组细胞的细胞周期变化;FITC-鬼笔环肽染色后激光共聚焦显微镜下观察F-actin细胞骨架表达与重组情况;各组细胞经成骨诱导7 d和14 d后茜素红染色观察钙结节形成量、Real-time PCR检测成骨细胞标志基因Col I、ON、OPN及BSP的mRNA表达;成软骨诱导14 d和28 d后甲苯胺蓝染色检测蛋白多糖表达、Real-time PCR检测成软骨细胞标志基因Sox9、Aggrecan及ColⅡ的mRNA表达。采用SPSS 16.0对数据进行单因素方差分析,后续Dunnett t检验进行各时间点上实验组与空白对照组数据之间及各实验组之间的组间比较。结果流体静压力(90 kPa,1 h)及1 nmol/L 17β-雌二醇作用下细胞增殖能力以及细胞骨架的活性均增强,但并不具有生物整合效应。成骨诱导14 d后,钙结节形成。Real-time PCR结果显示,17β-雌二醇作用组中成骨标志基因Col I、ON、OPN及BSP的表达水平显著增加。两种刺激在成骨诱导分化7 d时均可提高标志基因的表达,具有生物协同效应,但成骨诱导14 d时两者则表现出相互拮抗的作用。成软骨诱导28 d后,甲苯胺蓝染色阳性。成软骨诱导过程中,单纯压力刺激组成软骨标志基因Sox9、Aggrecan及ColⅡ的表达显著升高,雌激素预调则削弱压力对间充质干细胞的促成软骨分化作用。结论流体静压力及雌激素仅在骨髓间充质干细胞成骨分化早期表现生物整合效应,而在增殖、细胞骨架活性方面则无此效应。在成骨分化晚期及成软骨分化中两种刺激因素表现出拮抗作用,雌激素可促成骨分化,而压力则表现为促成软骨分化的作用。     

Influence of mechanical fluid shear stress on the osteogenic differentiation protocols for Equine adipose tissue-derived mesenchymal stem cells

Cell-based therapies have become a promising approach to promote tissue regeneration and the treatment of musculoskeletal disorders. Bone regeneration maintains bone homeostasis, mechanical stability and physical performance. Mechanical stimulation showed to induce stem cell differentiation into the osteogenic fate. However, the effect of various osteogenic protocols on the osteogenic commitment of equine adipose-derived stem cells is not fully elucidated. Here we examined the influence of fluid-based shear stress (FSS) via mechanical rocking to assess whether mechanical stimulation promotes osteogenic differentiation of equine adipose-derived stem cells (ASCs). ASCs were induced into osteogenic fate using osteogenic differentiation medium (ODM) protocol or additional supplementation of 5?mM CaCl₂ and 7.5?mM CaCl₂ protocol compared to cells cultivated in basal medium (BM) up to 21 day. The ASCs proliferation pattern was evaluated using the sulforhodamine B (SRB) protein assay. Osteogenic differentiation examined via semi-quantification of alizarin red staining (ARS) and alkaline phosphatase activity (ALP) as well as, via quantification of osteocalcin (OC), alkaline phosphatase (ALP), osteopontin (OP), and collagen type-1 (COL1) gene expression using RT-qPCR. We show that mechanical FSS increased the proliferation pattern of ASCs compared to the static conditions. Mechanical FSS together with 5?mM CaCl₂ and 7.5?mM CaCl₂ promoted osteogenic nodule formation and increased ARS intensity compared to the standard osteogenic protocols. We observed that combined mechanical FSS with ODM protocol increase ALP activity compared to static culture conditions. We report that ALP and OC osteogenic markers expression were upregulated under mechanical FSS culture condition particularly with the ODM protocol. Taken together, it can be assumed that mechanical stress using FSS promotes the efficiency of the osteogenic differentiation protocols of ASCs through independent mechanisms.     

In vitro and in vivo differentiation of mesenchymal stem cells in the cardiomyocyte direction

The possibility of mesenchymal stem cell differentiation in the cardiomyocyte direction was studied on Wistar-Kyoto rats with myocardial infarction induced by ligation of the left coronary artery. In vitro treatment of mesenchymal stem cells with 5-azacitidine led to spontaneous contractions of about 15% cells in culture. Analysis of the expression of matrix RNA showed expression of fetal and functional markers of the myocardium in this cell culture. In vivo on day 21 after myocardial infarction and intravenous transplantation of mesenchymal stem cells into the periinfarction area, myocardial cells carrying donor label were detected. Immunohistochemical analysis showed that these cells were cardiomyocytes integrated into the myocardium. These cells can be a result of differentiation of transplanted mesenchymal stem cells or fusion of endogenous cardiomyocytes with exogenous mesenchymal stem cells. __________ Translated from Kletochnye Tekhnologii v Biologii i Medicine, No. 4, pp. 194–197, December, 2006     

骨髓基质干细胞体外诱导成软骨的研究进展

骨髓基质干细胞具有强大的体外扩增能力和多向分化潜能,在不同诱导因素诱导下可以分化为软骨细胞,已成为软骨组织工程理想种子细胞的研究热点。近十几年对于如何诱导骨髓基质干细胞成软骨已有不少研究,如诱导因子诱导、基因修饰诱导及与软骨细胞共培养诱导等,都可以将骨髓基质干细胞成功诱导为软骨细胞。就骨髓基质干细胞体外定向诱导成软骨的方法进行综述对其研究进展作一回顾.     

体外冲击波通过三磷酸腺苷诱导人骨髓间充质干细胞向成骨细胞分化

目的 研究体外冲击波是否通过三磷酸腺苷（ATP）激活P2X7受体,诱导人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)向成骨细胞分化。方法 培养hMSCs细胞,检测冲击波是否引起其向外释放ATP;通过检测碱性磷酸酶(ALP)活性、骨钙素表达和钙结节形成,判断骨化形成和钙质沉积;用实时定量PCR检测P2X7受体的mRNA表达;用ATP水解酶、P2X7受体的siRNA以及 P2受体的抑制剂评估ATP释放和P2X7受体在冲击波诱导hMSCs成骨分化中的作用。结果 冲击波可引起细胞内ATP向外释放,冲击波和细胞外ATP能够诱导hMSCs向成骨分化,采用ATP水解酶、P2X7受体的siRNA和抑制剂能够抑制冲击波引起的hMSCs成骨化作用。结论 冲击波通过引起细胞内ATP向外释放,激活P2X7受体传导信号通路,促进hMSCs向成骨细胞分化。本研究结果为冲击波促进骨折愈合和治疗骨不连疗法提供了理论依据。