人羊膜间充质细胞具有向心肌样细胞分化的特性

摘 要 探讨人羊膜间充质细胞（human amniotic mesenchymal cells,hAMCs）向心肌细胞分化的能力。采用胶原酶消化法分离hAMCs,用流式细胞仪进行表型鉴定;用5-氮杂胞苷和碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）诱导hAMCs向心肌细胞分化,免疫荧光染色法检测诱导后细胞中特异蛋白结蛋白和α-辅肌动蛋白的表达,RT-PCR检测心肌特异性转录因子Nkx2.5 、GATA-4和心肌特异性收缩蛋白α-肌球蛋白重链（α-myosin heavy chain,α-MHC）mRNA的表达。结果显示：①hAMCs原代培养至第6 d,贴壁细胞汇合度可达80%,呈漩涡状生长。传代后hAMCs增殖迅速,3~4 d细胞汇合度可达100%,细胞呈梭形或多角形。②hAMCs表达CD44和波形蛋白,不表达CK19。③hAMCs经诱导分化8~10 d后细胞排列紧密,多为长梭形。③hAMCs诱导2 w和4 w表达α-辅肌动蛋白和心肌特异性转录因子Nkx2.5。④诱导前后的hAMCs均表达结蛋白和GATA-4,但均未见α-MHC表达。说明hAMCs具有向心肌样细胞分化的能力,可望成为细胞心肌成形术（cellular cardiomyoplasty,CCM）的候选细胞。     

体外化学诱导人骨髓间充质干细胞分化为心肌样细胞

To investigate the potential of adult mesenchymal stem cells (hMSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure of 5-azacytidine in vitro. A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073 g/mL Percoll and cultured in the right cell culturing medium as previously described. The phonotypes of hMSCs were identified by flow cytometry. The stem cells were cultured in cell culture medium (as control) and medium mixed with 5-azacytidine (5-aza, 3, 5, 10 micromol/L) (n=5, respectively) for cellular differentiation. We examined respectively with immunohischemistry at 21 days of inducement on desmin, cardiac-specific cardiac troponin I (cTnI), GATA4 & connexin43. The ultrastructures of induced cells were examined by transmission electron microscope. The results indicated that the hMSCs showed a fibroblast-like morphology with vortex distribution in their peak propagation, and express high level of CD44 but negative for CD34 and CD45. 20%-30% cells grown after 5, 10 microl/L 5-aza treatment connected with adjoining cells and coalesced into myotube structures after 14 days. After 21 days of culturing, immunohistochemistry revealed expression of desmin, GATA4, cTnI and connexin43 in 5, 10 micromol/L showed positive, but no cardiac specific protein were found in neither 3 micromol/L nor in control group. The ratio of cTnI positive stained cells in 10 micromol/L group were higher than that in 5 micromol/L group (65.3+/-4.7% vs 48.2+/-5.4%, p<0.05). Electron microscopy revealed myofilaments were formed. The results indicated that purified hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration.     

枸杞多糖诱导人脐血间充质干细胞向神经元样细胞分化的实验研究

目的:探讨枸杞多糖诱导人脐血间充质干细胞(MSCs)向神经元样细胞分化的可行性及其机制。方法:无菌条件下收集正常足月儿的脐带血,经肝素抗凝,用相对密度1.077的淋巴细胞分离液分离脐血的单个核细胞,用低糖DMEM培养基进行培养和纯化扩增。选取第3代细胞进行诱导实验,当传代细胞长满瓶底的80%以上时,先用含15?S和10ng/ml bFGF的DMEM完全培养基预诱导24小时,然后用不含血清含1g/L枸杞多糖的DMEM培养基诱导,光镜下观察细胞形态,用免疫组化技术检测细胞Nestin和NF的表达。结果:预诱导后MSCs没有变化,而经枸杞多糖诱导4h后细胞即出现形态学上的改变,细胞变成不规则形,立体感增强,从胞体伸出突起。免疫组化检测显示,细胞Nestin、NF呈阳性。结论:人脐血间充质干细胞经枸杞多糖诱导可转化为神经元样细胞,其诱导机制可能与枸杞多糖的抗氧化作用有关。     

体外化学诱导人骨髓间充质干细胞分化为心肌样细胞

为了探讨人骨髓间充质干细胞(MSCs)的体外培养及化学诱导向心肌细胞分化的过程及条件,我们用1.073g/mL密度梯度离心法分离健康人骨髓单个核细胞,经骨髓间充质干细胞培养基传代培养后用流式细胞仪检测细胞表面抗原,在完全培养基中分别加入3、5、10μmol/L的5氮胞苷(每组n=5)进行化学诱导分化,阴性对照组采用完全培养基培养,诱导后21天细胞爬片免疫荧光法鉴定,透射电镜观察细胞超微结构。结果显示人MSCs为形态均一的梭形细胞,生长旺盛时呈旋涡样分布,流式细胞仪检测细胞表面CD44阳性,CD34、CD45阴性;5、10μmol/L的5氮胞苷进行化学诱导后细胞形态变长,诱导后14天时20%-30%细胞融合形成多核肌管样结构,3μmol/L组MSCs未出现肌管结构,诱导后21天5、10μmol/L组MSCs中desmin、心肌早期转录因子GATA4、心肌特异性cTnI及闰盘蛋白connexin43的表达阳性,10μmol/L组cTnI阳性染色细胞数目(65.3±4.7%)高于5μmol/L诱导组(48.2±5.4%)(p<0.05);3μmol/L组及阴性对照组无心肌特异性蛋白的表达。细胞诱导后28天透射电镜下可见肌丝形成。本实验说明,人MSCs在体外经化学诱导可分化为心肌样细胞,而且5-氮胞苷对于心肌相关蛋白的表达呈浓度依赖性正相关。     

诱导体外培养的骨髓间充质干细胞向心肌样细胞的分化

选用Wistar大鼠分离骨髓间充质干细胞作体外培养及鉴定其表达抗原CD44、CDw90;采用10μmol／L 5-氮胞苷诱导第1代的骨髓间充质干细胞,于诱导后2、4周进行免疫细胞化学反应检测α-横纹肌肌动蛋白、肌钙蛋白T。证实体外培养的第1代骨髓间充质干细胞经5-氮胞苷诱导可分化为心肌样细胞,为指导体外诱导的心肌细胞应用于。临床提供一定的理论依据和技术手段。     

体外诱导骨髓间充质干细胞向肝细胞样细胞方向分化

探讨成纤维生长因子-2(FGF-2)在体外定向诱导大鼠骨髓间充质干细胞(BM-MSCs)向肝细胞样细胞分化的作用及量化关系。体外分离培养大鼠BM-MSCs,将第3代BM-MSCs采用不同剂量的FGF-2诱导。诱导后,在显微镜下观察细胞形态学的改变;用免疫细胞化学法检测白蛋白和CK19的分泌;Shiff染色法检测糖原的分泌。诱导后BM-MSCs由梭形向多角形、卵圆形方向变化,白蛋白、CK19和糖原12 d即有阳性表达,以后随着诱导时间的延长阳性率逐渐升高。20 ng/mL FGF-2诱导比10 ng/mL FGF-2诱导细胞白蛋白、CK19和糖原的表达量均多。20 ng/mL FGF-2具有较强的诱导BM-MSCs向肝细胞样细胞分化的能力。     

再生胰腺提取物诱导人羊膜间充质干细胞定向分化为胰岛素分泌细胞

利用天然生物诱导剂大鼠再生胰腺提取物(Rgenerating pancreatic extract,RPE)定向诱导人羊膜间充质干细胞(Human amniotic mesenchymal stem cells,hAMSCs)向胰岛素分泌细胞分化。切除大鼠60%胰腺刺激胰腺再生,而后制备RPE,以终浓度为20 mg/L的RPE诱导hAMSCs。实验通过形态学鉴定、双硫腙染色、免疫荧光分析、RT-PCR基因检测和高糖刺激胰岛素分泌等实验鉴定细胞诱导结果。实验结果显示P3代hAMSCs经RPE诱导后形态变化明显,诱导15 d后细胞呈簇状生长,经双硫腙染色可见棕红色细胞团;免疫荧光染色结果显示诱导细胞呈胰岛素阳性表达;RT-PCR实验证明诱导细胞阳性表达人胰岛相关基因Pdx1和insulin;高糖刺激实验证明培养液中有胰岛素成分产生,且分泌量随刺激时间的延长先增加而后趋于稳定。实验结果表明hAMSCs在体外经RPE诱导可以分化为胰岛素分泌细胞。     

中药诱导骨髓间充质干细胞多向分化研究进展

干细胞（stem cell）是指具有自我更新、高度增殖及多分化潜能的未分化细胞,它能产生出表型与基因型完全相同的子细胞,是机体其他细胞的起源。根据其发展阶段和来源不同,干细胞可分为胚胎干细胞和成体干细胞。     

鼠骨髓间充质干细胞的分离培养及定向诱导分化为内皮样细胞

目的：探讨体外大鼠骨髓间充质干细胞（rBMMSCs）的分离培养和血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）对其定向诱导为内皮样细胞（ELCs）的可行性。方法：采用Percoll（1.073g/ml）分离液分离骨髓单个核细胞,用含10%胎牛血清（FBS）的LG-DMEM培养基贴壁纯化培养,倒置显微镜、免疫细胞化学法、流式细胞仪、MTT法、透射电镜（TEM）联合对rBMMSCs形态、表型、生长曲线、细胞周期以及超微结构进行鉴定;诱导后的细胞,采用倒置显微镜观察细胞形态,免疫细胞化学法检测CD31、CD144（VE-cadherin）和CD34表达以及摄取Dil-ac-LDL、结合FITC-UEA-1的功能特点。结果：rBMMSCs呈长梭形,漩涡状排列。细胞生长曲线显示潜伏期、对数生长期和平台期,符合干细胞的生长规律。透射电镜结果表明：rBMMSCs有两种不同的形态结构,其中体积较小、核质比大、胞质内细胞器稀少者为处于未分化或分化较低状态的幼稚型rBMMSCs。细胞周期分析显示：第4代细胞G0/G1期为95.67%,表明绝大部分细胞处于非增殖状态;诱导后的部分细胞形态可见类似ELCs改变,表达血管内皮细胞（ECs）特异性表面标志CD31、CD34和CD144,具有摄取Dil-ac-LDL以及结合FITC-UEA-1的功能特点。结论：采用Percoll密度梯度离心与贴壁培养相结合的方法所培养的rBMMSCs在体外具有定向诱导分化为ELCs的潜能,可能成为血管组织工程理想的种子细胞来源。     

大鼠脂肪组织来源的间充质干细胞向心肌样细胞分化的细胞特征

目的探索大鼠脂肪组织来源的间充质干细胞(ADMSCs)向心肌细胞定向诱导分化的细胞特征。方法胰酶消化法分离出ADMSCs,对培养第3代细胞分别进行HE染色和CD44免疫细胞化学染色,利用光镜和激光共聚焦扫描显微镜观察其细胞特征和CD44的表达;5-氮杂胞苷(5-aza)诱导,应用肌球蛋白重链(MHC)抗体进行免疫细胞化学染色,鉴定分化后的心肌样细胞;利用免疫组织化学观察脂肪组织中CD44阳性细胞的表达分布。结果大鼠脂肪组织分离的细胞生长迅速,培养后分为两类:一类为大型细胞,其数量较多、体积较大;另一类为小型细胞,其数量较少、体积较小。免疫细胞化学和激光共聚焦显微镜观察显示大细胞呈CD44弱阳性反应,小细胞呈强阳性反应。5-aza诱导后,发现7处细胞团明显分化为心肌样细胞,肌球蛋白呈强阳性反应。HE染色显示呈典型的心肌细胞的特征。脂肪组织中CD44阳性细胞位于脂肪小叶之间,数量较少,形态较小。结论培养的大鼠脂肪组织来源的ADMSCs分为大、小两类,小型细胞可能具有较强的分化为心肌样细胞的能力。     

Cyclosporin-A potently induces highly cardiogenic progenitors from embryonic stem cells

Though cardiac progenitor cells should be a suitable material for cardiac regeneration, efficient ways to induce cardiac progenitors from embryonic stem (ES) cells have not been established. Extending our systematic cardiovascular differentiation method of ES cells, here we show efficient and specific expansion of cardiomyocytes and highly cardiogenic progenitors from ES cells. An immunosuppressant, cyclosporin-A (CSA), showed a novel effect specifically acting on mesoderm cells to drastically increase cardiac progenitors as well as cardiomyocytes by 10-20 times. Approximately 200 cardiomyocytes could be induced from one mouse ES cell using this method. Expanded progenitors successfully integrated into scar tissue of infracted heart as cardiomyocytes after cell transplantation to rat myocardial infarction model. CSA elicited specific induction of cardiac lineage from mesoderm in a novel mesoderm-specific, NFAT independent fashion. This simple but efficient differentiation technology would be extended to induce pluripotent stem (iPS) cells and broadly contribute to cardiac regeneration.     

Ex vivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells

Bone marrow mesenchymal stem cells have been shown to transdifferentiate into cardiomyocytes after 5-azacytidine treatment or co-culturing with rodent cardiomyocytes. We investigate if adult human bone marrow stem cells can be differentiated ex vivo into cardiomyocyte-like cells (CLCs) independent of cytotoxic agents or co-culturing technique. Sternal bone marrow was collected from 16 patients undergoing coronary artery bypass surgery. Mesenchymal stem cells were differentiated in a cardiomyogenic differentiation medium containing insulin, dexamethasone, and ascorbic acid. Differentiation towards CLCs was determined by induced expression of cardiomyocyte-specific proteins. Differentiated CLCs expressed multiple structural and contractile proteins that are associated with cardiomyocytes. Thin filament associated myofibrillar proteins were detected early in the cells, with cardiac troponin I, sarcomeric tropomyosin, and cardiac titin among the first expressed. Some CLCs were found to develop into a nascent cardiomyocyte phenotype with cross-striated myofibrils characterized by alpha-actinin-positive Z bands after 4-5 passages in differentiated culture. These lineage-defined CLCs may be potentially useful for repairing damaged myocardium.     

Ghrelin promotes the differentiation of human embryonic stem cells in infarcted cardiac microenvironment

Ghrelin is broadly expressed in myocardial tissues, where it exerts different functions. It also has been found to have a wide variety of biological functions on cell differentiation and tissue development. The aim of this study was to investigate the effect of ghrelin on human embryonic stem cell (hESC) differentiation in infarcted cardiac microenvironment. The hESCs grown on feeder layers expressed several pluripotential markers including alkaline phosphatase (AKP). Four weeks after transplantation into rat infarcted hearts, the hESCs and their progeny cells survived and formed intracardiac grafts were 54.7% and 19.6% respectively in ghrelin- and phosphate-buffered saline (PBS)-treated groups. Double immunostaining with anti-human Sox9 and anti-HNA or anti-human fetal liver kinase-1 (Flk1) and anti β-tubulin showed that the human grafts were in development. However, double positive stains were only found in the ghrelin-treated group. In addition, the hESC injection protocol was insufficient to restore heart function of the acute myocardial infarction model. Our study, therefore, provides a new insight of ghrelin on promoting hESC survival and differentiation in rat infarcted cardiac microenvironment. This may give a clue for therapy for myocardial infarction by hESCs or progeny cells.     

Heterogeneic nature of adult cardiac side population cells

Side population cells have been found in various types of adult tissue including heart and are presumed to be tissue-specific stem/progenitor cells. In the present study, we confirmed the presence of cardiac side population (cSP) cells, which showed both the Hoechst 33342 efflux ability and ABCG2 expression, in adult murine heart. Flow cytometric analysis showed that more than half of cSP cells expressed the endothelial marker VE-cadherin or the smooth muscle markers, α-smooth muscle actin and desmin. In addition, immunohistochemical analysis demonstrated that ABCG2⁺ cells were mainly localized within vascular walls. Quantitative RT-PCR analysis demonstrated that VE-cadherin⁻ cSP cells progressively expressed Nkx2.5 and cardiac troponin T with time in culture. VE-cadherin⁻ cSP cells also expressed mesodermal-mesenchymal-associated markers and differentiated into osteocytes and adipocytes. These results highlight the heterogeneic nature of cSP cells, consisting of vascular endothelial cells, smooth muscle cells, and mesenchymal stem/progenitor cells including potential cardiomyogenic cells.     

RhoA promotes differentiation of WB-F344 cells into the biliary lineage

When cultured on Matrigel, liver precursor epithelium WB-F344 cells could be induced to differentiate into biliary cells in which RhoA expression was upregulated. To further investigate the role of RhoA in WB cell differentiation initiated by Matrigel treatment, we constructed constitutively active RhoA-expressing vectors and stably transfected them into WB-F344 cells. Accompanying upregulation of biliary lineage markers and morphological changes, cells with ectopically active RhoA expression were found to form bile-duct-like structures even without Matrigel treatment. Besides, ROCK inhibitor Y27632 treatment eliminated luminal morphogenesis. F-actin cytoplasmic staining further verified that the RhoA–ROCK signal pathway was involved in differentiation of WB cells into the biliary lineage. In conclusion, our results suggested that the RhoA–ROCK–stress fibre system plays an obligatory role in Matrigel-induced WB-F344 cell luminal morphogenesis and further differentiation.     

Evaluation of the effect of boron derivatives on cardiac differentiation of mouse pluripotent stem cells

BackgroundThe heart is one of the first organs to form during embryonic development and has a very important place. So much that the formation of a functional heart is completed on the 55th day of human development and the 15th day of mouse development. Myocardial, endocardial and epicardial cells, which are derived from the mesoderm layer, are the cells that form the basis of the heart. Cardiac development, like other embryonic developments, is tightly controlled and regulated by various signaling pathways. The WNT signaling pathway is the most studied of these signaling pathways and the one with the clearest relationship with heart development. It is known that boron compounds and the Wnt/β-catenin pathway are highly correlated. Therefore, this study aimed to investigate the role of boron compounds in heart development as well as its effect on pluripotency of mouse embryonic stem cells for the first time in the literature.MethodsToxicity of boron compounds was evaluated by using MTS analysis and obtained results were supported by morphological pictures, Trypan Blue staining and Annexin V staining. Additionally, the possible boron-related change in pluripotency of embryonic stem cells were analyzed with alkaline phosphatase activity and immunocytochemical staining of Oct4 protein as well as gene expression levels of pluripotency related OCT4, SOX2 and KLF4 genes. The alterations in the embryonic body formation capacity of mouse embryonic stem cells due to the application boron derivatives were also evaluated. Three linage differentiation was conducted to clarify the real impact of boron compounds on embryonic development. Lastly, cardiac differentiation of mESCs was investigated by using morphological pictures, cytosolic calcium measurement, gene expression and immunocytochemical analysis of cardiac differentiation related genes and in the presence of boron compounds.ResultsObtained results show that boron treatment maintains the pluripotency of embryonic stem cells at non-toxic concentrations. Additionally, endodermal, and mesodermal fate was found to be triggered after boron treatment. Also, initiation of cardiomyocyte differentiation by boron derivative treatments caused an increased gene expression levels of cardiac differentiation related TNNT2, Nkx2.5 and ISL-1 gene expression levels.ConclusionThis study indicates that boron application, which is responsible for maintaining pluripotency of mESCs, can be used for increased cardiomyocyte differentiation of mESCs.