Wnt信号系统对骨髓间充质干细胞向心肌细胞分化的作用

骨髓间充质干细胞（BMSCs）移植治疗冠状动脉粥样硬化性心脏病、慢性心力衰竭等已经成为心脏病治疗学的热门课题,但细胞疗法依赖于干细胞向心肌细胞的定向分化,目前对BMSCs分化为心肌细胞的分子机制了解不多。Wnt信号系统与器官的分化和形成密切相关,大量研究表明,Wnt信号系统对干细胞向心肌细胞的定向分化有重要作用,对该信号系统的控制,在心脏病的细胞疗法中显得尤其重要。本文将就Wnt信号系统在BMSCs的增殖、迁移及心肌定向分化中的调控作用展开论述。     

Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro

Most studies on stem cell transplantation therapy on myocardially infarcted animal models and phase-I human clinical trials have focused on the use of undifferentiated stem cells. There is a strong possibility that some degree of cardiomyogenic differentiation of stem cells in vitro prior to transplantation would result in higher engraftment efficiency, as well as enhanced myocardial regeneration and recovery of heart function. Additionally, this may also alleviate the probability of spontaneous differentiation of stem cells into undesired lineages and reduces the risk of teratoma formation, in the case of embryonic stem cells. The development of efficient protocols for directing the cardiomyogenic differentiation of stem cells in vitro will also provide a useful model for molecular studies and genetic manipulation. This review therefore critically examines the various techniques that could possibly be used to direct and control the cardiomyogenic differentiation of stem cells in vitro.     

骨髓间充质干细胞向肝细胞的诱导分化

目的:探索肝细胞生长因子(HGF)、制瘤素M(OSM)在体外诱导骨髓间充质干细胞(MSCs)向肝细胞分化的能力及效果.方法:分离、培养大鼠MSCs,取第3代按以下分组诱导其向肝细胞分化:A组:低糖杜氏改良培养基(DMEM-LG) 100 mL/L胎牛血清(fetal calf,serum,FCS);B组:肝细胞生长培养基(HGM);C组:HGM 20μg/L HGF;D组:HGM 20μg/L OSM;E组:HGM 20μg/L HGF 20μg/L OSM,于不同时间点用免疫细胞化学检测甲胎蛋白(AFP)、细胞角蛋白18(CK18)表达,高碘酸-希夫氏(PAS)染色检测糖原表达,谷氨酰胺脱氢酶法检测上清液尿素含量.结果:C,E组于诱导第7天出现AFP阳性表达,以后其阳性表达率随诱导时间延长逐渐降低,诱导第7天E组阳性表达率高于C组(x~2=6.322,P<0.05).C组、E组分别于诱导第7、14天出现CK18阳性表达,于诱导第7天开始出现糖原阳性表达,随诱导时间延长表达率逐渐增高,同一时间点E组CK18(14 d:x~2=4.811,P<0.05;21 d:x~2=6.902,P<0.01;28 d:x~2=5.771,P<0.05)及糖原(14 d:x~2=6.902,P<0.01;21 d:x~2=6.818,P<0.01;28 d:x~2=6.818,P<0.01)阳性表达率高于C组.C,E组培养上清液中尿素浓度随诱导时间延长逐渐增高,E组增长幅度比C组高.A,B,D组各时间点均未见AFP、CK18、糖原表达及尿素浓度变化.结论:MSCs具有向肝细胞分化的能力,HGF能够诱导MSCs分化肝样细胞,OSM与HGF联合使用,能促进MSCs的分化,明显提高分化率.     

Dynamics of podosome stiffness revealed by atomic force microscopy

Podosomes are unique cellular entities specifically found in macrophages and involved in cell-matrix interactions, matrix degradation, and 3D migration. They correspond to a core of F-actin surrounded at its base by matrix receptors. To investigate the structure/function relationships of podosomes, soft lithography, atomic force microscopy (AFM), and correlative fluorescence microscopy were used to characterize podosome physical properties in macrophages differentiated from human blood monocytes. Podosome formation was restricted to delineated areas with micropatterned fibrinogen to facilitate AFM analyses. Podosome height and stiffness were measured with great accuracy in living macrophages (578 ± 209 nm and 43.8 ± 9.3 kPa) and these physical properties were independent of the nature of the underlying matrix. In addition, time-lapse AFM revealed that podosomes harbor two types of overlapping periodic stiffness variations throughout their lifespan, which depend on F-actin and myosin II activity. This report shows that podosome biophysical properties are amenable to AFM, allowing the study of podosomes in living macrophages at nanoscale resolution and the analysis of their intimate dynamics. Such an approach opens up perspectives to better understand the mechanical functionality of podosomes under physiological and pathological contexts.     

Melatonin regulates mesenchymal stem cell differentiation: a review

Among the numerous functions of melatonin, the control of survival and differentiation of mesenchymal stem cells (MSCs) has been recently proposed. MSCs are a heterogeneous population of multipotent elements resident in tissues such as bone marrow, muscle, and adipose tissue, which are primarily involved in developmental and regeneration processes, gaining thus increasing interest for tissue repair and restoration therapeutic protocols. Receptor‐dependent and receptor‐independent responses to melatonin are suggested to occur in these cells. These involve antioxidant or redox‐dependent functions of this indolamine as well as secondary effects resulting from autocrine and paracrine responses. Inflammatory cytokines and adipokines, proangiogenic/mitogenic stimuli, and other mediators that influence the differentiation processes may affect the survival and functional integrity of these mesenchymal precursor cells. In this scenario, melatonin seems to regulate signaling pathways that drive commitment and differentiation of MSC into osteogenic, chondrogenic, adipogenic, or myogenic lineages. Common pathways suggested to be involved as master regulators of these processes are the Wnt/β‐catenin pathway, the MAPKs and the, TGF‐β signaling. In this respect melatonin emerges a novel and potential modulator of MSC lineage commitment and adipogenic differentiation. These and other aspects of the physiological and pharmacological effects of melatonin as regulator of MSC are discussed in this review.     

间充质干细胞分化为肝样细胞的研究进展

肝炎后肝硬化等慢性进展性肝病、急性肝功能衰竭、肝脏代谢性疾病及肝脏恶性肿瘤等终末期肝病的发病率日益上升,美国贝塞斯达回忆宣布肝移植是目前治疗终末期肝病最有效的方法,但由于肝源有限、移植后的免疫排斥反应以及高额费用等限制了这种治疗方法的开展。近年来,随着对干细胞研究的深入,发现间充质干细胞(MSCs)具有向肝样细胞分化的潜能,且安全性、可行性及疗效均显示了较好的应用前景,为终末期肝病的治疗提供了新的途径。本文就MSCs不同组织来源分化特点、体内外分化为肝样细胞研究、分化后的肝样细胞的生物学特性及MSCs分化为肝样细胞的机制、MSCs应用的一些问题作一概述。     

骨髓间充质干细胞心肌样分化的微环境因素

目的:探讨促使骨髓间充质干细胞(MSCs)心肌样分化的微环境因素。方法:分离大鼠MSCs并传至第6代,随机分为混合培养组、条件组及对照组,分别将MSCs与大鼠心肌细胞共同培养(混合培养组),或将心肌细胞培养上清液加入MSCs培养体系(条件组)。1周后,检测MSCs的心肌特异性蛋白titin、Cx43及MHC表达情况。结果:MSCs能在心肌细胞培养上清液及与心肌细胞共同培养中正常生长;条件组MSCs表达titin、Cx43显著增加,但未观察到肌小节样结构;混合培养组MSCs可表达上述蛋白,且部分细胞中可观察到肌小节样结构,与心肌细胞交界面上有Cx43的聚集。3组MSCs均未检测到MHC表达。结论:心肌细胞自身、源于心肌的体液因素及MSCs分化过程中形成的感应器是MSCs心肌样分化的必要条件。     

In vitro hepatic differentiation of human mesenchymal stem cells

This study examined whether mesenchymal stem cells (MSCs), which are stem cells originated from embryonic mesoderm, are able to differentiate into functional hepatocyte-like cells in vitro. MSCs were isolated from human bone marrow and umbilical cord blood, and the surface phenotype and the mesodermal multilineage differentiation potentials of these cells were characterized and tested. To effectively induce hepatic differentiation, we designed a novel 2-step protocol with the use of hepatocyte growth factor and oncostatin M. After 4 weeks of induction, cuboidal morphology, which is characteristic of hepatocytes, was observed, and cells also expressed marker genes specific of liver cells in a time-dependent manner. Differentiated cells further demonstrated in vitro functions characteristic of liver cells, including albumin production, glycogen storage, urea secretion, uptake of low-density lipoprotein, and phenobarbital-inducible cytochrome P450 activity. In conclusion, human MSCs from different sources are able to differentiate into functional hepatocyte-like cells and, hence, may serve as a cell source for tissue engineering and cell therapy of hepatic tissues. Furthermore, the broad differentiation potential of MSCs indicates that a revision of the definition may be required.     

Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells

INTRODUCTIONMost liver diseases lead to hepatocyte dysfunction with the possibility of eventual organ failure. The replacement of diseased hepatocytes and the stimulation of endogenous or exogenous regeneration by stem cells are the main aims of liver-dir…     

间充质干细胞胰岛样细胞分化研究进展

间充质干细胞是存在于成体多种器官的多能干细胞,具有多胚层分化能力.近年研究发现在体内体外特定微环境作用下,它可以分化为胰岛样细胞,分泌一定量的胰岛素,并且具有降血糖的功能.这一技术可以同时解决胰岛移植中供体紧缺及免疫排斥反应两大难题,为临床糖尿病细胞治疗开辟了一条新的途径.     

成人骨髓间充质干细胞体外诱导分化为神经元样细胞的研究

目的 研究成人骨髓间充质干细胞(hMSC)体外定向诱导可否分化为神经元样细胞。方法 Percoll分离液离心分离hMSC,体外扩增,采用两种方法：①含碱性成纤维细胞生长因子(bFGF)预诱导24小时,甲氧酚(BHA)和二甲亚枫(DMSO)联合诱导6小时;②2-巯基乙醇(2-ME)预诱导24小时,BHA和DMSO诱导6小时。免疫组化检测神经元样细胞表达神经元烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)。结果 hMSC在体外扩增传至5代后,流式细胞仪显示hMSC表面抗原CD29、CD44、CD90表达阳性率分别为99．5％,97．8％,98．8％。采用两种方法诱导hMSC后,胞体收缩,突起伸出,较密的部分神经元拉成网状;免疫组化显示诱导的神经元样细胞能特异性表达NSE、NF,不表达GFAP。结论 成人骨髓hMSC在体外可以分化为神经元样细胞。     

Hepatic differentiation capability of rat bone marrow-derived mesenchymal stem cells and hematopoietic stem cells

AIM: To investigate the different effects of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) on hepatic differentiation. METHODS: MSCs from rat bone marrow were isolated and cultured by standard methods. HSCs from rat bone marrow were isolated and purified by magnetic activated cell sorting. Both cell subsets were induced. Morphology, RT-PCR and immunocytochemistry were used to identify the hepatic differentiation grade. RESULTS: MSCs exhibited round in shape after differentiation, instead of fibroblast-like morphology before differentiation. Albumin mRNA and protein were expressed positively in MSCs, without detection of alpha-fetoprotein (AFP). HSCs were polygonal in shape after differentiation. The expression of albumin signal decreased and AFP signal increased. The expression of CK18 was continuous in MSCs and HSCs both before and after induction. CONCLUSION: Both MSCs and HSCs have hepatic differentiation capabilities. However, their capabilities are not the same. MSCs can differentiate into mature hepatocyte-like cells, never expressing early hepatic specific genes, while Thy-1.1(+) cells are inclined to differentiate into hepatic stem cell-like cells, with an increasing AFP expression and a decreasing albumin signal. CK18 mRNA is positive in Thy-1.1(+) cells and MSCs, negative in Thy-1.1(-) cells. It seems that CK18 has some relationship with Thy-1.1 antigen, and CK18 may be a predictive marker of hepatic differentiation capability.     

PI3K／Akt信号通路在骨髓间充质干细胞增殖及成骨分化调控中的作用

目的：研究PI3K/Akt信号通路抑制剂LY294002对骨髓间充质干细胞（mesenchymal stem cells,MSCs）增殖及分化的影响。方法采用贴壁法体外分离人骨髓间充质干细胞（hMSCs）,加入PI3K抑制剂LY294002（1、10μmol/L）,应用MTT法测定细胞增殖,常规成骨诱导分化培养3或7d,采用碱性磷酸酶（ALP）染色观察成骨分化水平,化学比色法测定ALP活性,茜素红染色后观察矿化钙结节数量并定量分析,Westernblot检测磷酸化Akt蛋白表达,应用Realtime-PCR检测各组细胞BMP2、Runx2、OPN及Osterix等成骨分化标记物的基因表达水平。结果从24至72h,LY294002对hMSCs增殖均产生显著抑制,随时间推延,可见抑制增殖效果增强（P＜0.05）。ALP染色和定量测定提示10μmol/L的ALP活性最强,在不同时间显著高于对照组和1μmol/L组（P＜0.05）。成骨诱导培养3和7d,1、10μmol/L组矿化量都显著高于对照组（P＜0.05）。10μmol/L组矿化量在成骨诱导7d也显著高于1μmol/L组（P＜0.05）。Westernblot检测结果证实成骨诱导可激活Akt磷酸化蛋白表达,但LY294002可抑制该蛋白磷酸化。成骨诱导分化7d,1、10μmol/L均明显促进BMP2、Runx2、OPN、Osterix4种基因mRNA表达（均P＜0.05）。结论PI3K/Akt信号通路参与hMSCs增殖和分化过程。成骨分化伴随下游Akt蛋白表达。PI3K抑制剂可抑制hMSCs增殖,但同时促进其向成骨分化和矿化。     

Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells

Mesenchymal stem cells (MSCs), in addition to their multilineage differentiation, have a direct immunosuppressive effect on T-cell proliferation in vitro. However, it is unclear whether they also modulate the immune system by acting on the very first step. In this investigation, we addressed the effects of human MSCs on the differentiation, maturation, and function of dendritic cells (DCs) derived from CD14+ monocytes in vitro. Upon induction with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4), MSC coculture could strongly inhibit the initial differentiation of monocytes to DCs, but this effect is reversible. In particular, such suppression could be recapitulated with no intercellular contact at a higher MSC/monocyte ratio (1:10). Furthermore, mature DCs treated with MSCs were significantly reduced in the expression of CD83, suggesting their skew to immature status. Meanwhile, decreased expression of presentation molecules (HLA-DR and CD1a) and costimulatory molecules (CD80 and CD86) and down-regulated IL-12 secretion were also observed. In consistence, the allostimulatory ability of MSC-treated mature DCs on allogeneic T cells was impaired. In conclusion, our data suggested for the first time that human MSCs could suppress monocyte differentiation into DCs, the most potent antigen-presenting cells (APCs), thus indicating the versatile regulation of MSCs on the ultimate specific immune response.     

Cellular mechanical properties reflect the differentiation potential of adipose-derived mesenchymal stem cells

The mechanical properties of adipose-derived stem cell (ASC) clones correlate with their ability to produce tissue-specific metabolites, a finding that has dramatic implications for cell-based regenerative therapies. Autologous ASCs are an attractive cell source due to their immunogenicity and multipotent characteristics. However, for practical applications ASCs must first be purified from other cell types, a critical step which has proven difficult using surface-marker approaches. Alternative enrichment strategies identifying broad categories of tissue-specific cells are necessary for translational applications. One possibility developed in our lab uses single-cell mechanical properties as predictive biomarkers of ASC clonal differentiation capability. Elastic and viscoelastic properties of undifferentiated ASCs were tested via atomic force microscopy and correlated with lineage-specific metabolite production. Cell sorting simulations based on these "mechanical biomarkers" indicated they were predictive of differentiation capability and could be used to enrich for tissue-specific cells, which if implemented could dramatically improve the quality of regenerated tissues.