通过形成类胚体诱导人羊水多能干细胞向心肌细胞分化

由人羊水中分离羊水多能性干细胞,通过形成类胚体诱导其向心肌细胞分化.取人羊水标本进行体外培养,分离得到人羊水干细胞,已连续传代培养至42代,采用免疫细胞化学、RT-PCR和流式细胞仪技术对羊水干细胞的生物学特性进行检测.取10～15代羊水干细胞,悬浮培养使其形成类胚体,进而向心肌细胞诱导分化.培养的羊水干细胞呈成纤维样,表达部分胚胎干细胞特异标志基因,悬浮培养可形成类胚体.类胚体碱性磷酸酶(AP)检测呈阳性,表达三胚层特异标志基因fgf5、ζ-globin和α-fetoprotein.羊水干细胞形成类胚体后进行诱导,得到α-actin阳性细胞,表达心肌细胞特异标志基因Tbx5、Nkx2.5、GATA4和α-MHC.试验结果表明,从人羊水标本中可分离得到具有胚胎干细胞特性的细胞,经初步检测确定为羊水干细胞,并能通过形成类胚体诱导其向心肌细胞分化.     

心肌样微环境诱导脂肪干细胞分化的研究

微环境在促进干细胞分化过程中起着重要的作用,研究心肌样微环境介导脂肪干细胞向心肌细胞分化有重要意义．将脂肪干细胞与心肌细胞直接或通过细胞培养小室间接共培养,检测脂肪干细胞的分化情况．对于直接共培养体系,采用绿色荧光蛋白CFSE对脂肪干细胞进行标记,然后与心肌细胞以1∶5混合后进行直接共培养,2周后,通过流式细胞仪分选分化的脂肪干细胞,并检测其分化情况．检测方法包括：扫描电镜和透射电镜观察细胞的超微结构;免疫细胞化学检测心肌特异性肌球蛋白重链(MHC)、肌钙蛋白(TnⅠ)和连接蛋白(Cx43);Western blot定量分析;RT-PCR检测心脏特异性转录因子mRNA的表达．结果表明,分化的脂肪干细胞呈现心肌样超微结构,并表达心肌特异性蛋白和转录因子,并且直接共培养体系中分化的脂肪干细胞其表达率明显高于间接共培养体系中的表达率．因此,在心肌样微环境中,除了可溶性细胞因子对分化起作用以外,心肌细胞产生的机械牵拉对脂肪干细胞向心肌细胞分化也起着重要的作用．     

microRNA499 慢病毒载体转染诱导大鼠骨髓间充质干细胞 向心肌样细胞分化*

目的:探讨microRNA 499(miR-499)慢病毒转染对诱导大鼠骨髓来源间充质干细胞(BM-MSCs)向心肌样细胞分化的作用。方法:取第四代Wistar大鼠骨髓来源间充质干细胞进行流式细胞检测,鉴定干细胞表面特异标记物。使用符合干细胞鉴定标准的细胞批次用于后续实验。实验设置miR499慢病毒转染、慢病毒空白转染2个处理组,分别于处理后即日、1d,3d,5d,7d收集细胞进行下列实验:实时荧光定量PCR检测心肌重要转录因子GATA4、NKx2.5和MEF2C的mRNA表达,western-blot检测心肌特异蛋白I(cTnI)的表达。结果:培养第四代Wistar大鼠骨髓来源间充质干细胞表达干细胞表面特异标记物,可用于实验。大鼠骨髓来源间充质干细胞microRNA 499慢病毒载体转染后microRNA 499表达明显升高,且转染后1d,3d,5d,7d,GATA4、NKx2.5和MEF2C的mRNA表达逐渐增强。慢病毒空白转染组未见明显变化。western-blot检测自第3天开始可见cTnI阳性表达条带,慢病毒空白转染组未检测到明显阳性表达条带。结论:microRNA 499可诱导大鼠骨髓来源间充质干细胞向心肌样细胞分化。     

脐血间充质干细胞诱导向神经元样细胞分化的实验研究

目的:研究脐血间充质干细胞生物学特性及向神经元样细胞分化的潜能。方法:采用密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,观察细胞生长情况,描绘生长曲线,流式细胞仪检测细胞表面标志物,分别向成骨细胞、脂肪细胞、神经元样细胞进行诱导分化,通过茜素红染色、油红O染色检测脐血间充质干细胞成骨、成脂肪细胞诱导分化能力,而以免疫组织化学检测诱导后细胞表面神经标志物的表达。结果:纯化的脐血间充质干细胞贴壁生长,呈均一梭形,生长曲线呈S型,并以P3代增殖能力最强,细胞表面不表达或弱表达CD34、CD35、CD106,高表达CD29、CD44、CD105。成骨诱导2周后,可检测到钙化基质的形成,成脂肪诱导3周后,可检测到脂滴的形成。向神经元样细胞诱导分化后,可观察到典型的神经元样形态改变,且NSE、NF、GFAP阳性表达。结论:分离纯化的脐血间充质干细胞具有较强的增殖能力与分化潜能,并在体外诱导条件下可以向神经元样细胞定向分化。     

猪脂肪间充质干细胞的分离培养及其成脂分化

脂肪间充质干细胞(Adipose mesenchymal stemcell,AMSCs)是一类来源于脂肪组织并具有多向分化潜能的干细胞。近年来的研究证明,脂肪组织具有取材方便和干细胞含量高的优势,有望在研究与应用领域成为骨髓干细胞的替代物。猪是一种比啮齿类更接近人类的模式动物,具有较强的脂肪沉积能力。本研究探讨了猪脂肪间充质干细胞的体外分离纯化、培养扩增和向脂肪细胞诱导分化的条件。采用Ⅰ型胶原酶消化分离脂肪微管基质成分,传代培养扩增,流式细胞仪检测细胞表面标记。取第3-7代AMSCs,采用不同方法诱导AMSCs向脂肪细胞分化,光学显微镜下可观察到诱导后的细胞内有高折光性的小脂滴出现,油红O染色成阳性,不同诱导方法诱导率不同。被诱导细胞用RT-PCR可检测到脂肪细胞分化标志基因LPL和PPARγ的表达。结果表明可以从脂肪组织中分离培养出AMSCs,经传代后可提高其纯度。CD44、CD105表达呈阳性,CD14、CD34、S-100、HLA-DR呈阴性,在合适的诱导条件下,可向脂肪细胞分化。     

经HIF-1α与EGFP双基因重组腺病毒转染大鼠心肌干细胞后HIF-1α基因的表达

常规经基因修饰腺病毒转染细胞后,即进行细胞的移植,并不对病毒转染效果进行检验。本研究采用HIF-1α与EGFP双基因重组腺病毒转染大鼠心肌干细胞,察看基因重组腺病毒是否能够转染心肌干细胞内;研究经HIF-1α与EGFP(enhanced green fluorescence protein,EGFP)双基因重组腺病毒转染心肌干细胞后HIF-1α基因的表达情况。在荧光显微镜下,去察看病毒转染细胞的情况,确定病毒最适MOI值;采用实时定量PCR(real-time quantitative PCR,RT-qPCR)评价HIF-1α基因表达变化;应用Western blotting方法检测HIF-1α蛋白表达变化,进而获得良好的实验结果。     

脐带华通胶间充质干细胞向Flk1 阳性细胞分化的研究*

目的:诱导脐带华通胶间充质干细胞向Flk1阳性细胞分化。方法:胶原酶法分离培养脐带华通胶间充质干细胞,第3代细胞以含2-巯基乙醇的分化培养基培养,应用RT-PCR和流式细胞仪从mRNA和蛋白水平检测Flk1阳性细胞分化水平。结果:脐带华通胶间充质干细胞Flk1mRNA及蛋白表达极低,分化培养基培养后表达上调,48h达高峰(P<0.05),之后表达降低。结论:2-巯基乙醇可诱导脐带华通胶间充质干细胞向Flk1阳性细胞分化,为从中分选Flk1阳性细胞进行进一步研究提供了依据。     

大鼠脂肪干细胞向雪旺细胞诱导分化能力的研究

目的：研究脂肪干细胞（ADSCs）向雪旺细胞的诱导分化,为神经组织工程提供新的种子细胞。方法：取SD大鼠项背处的皮下脂肪,分离出脂肪干细胞并培养传代,流式细胞仪检测细胞表面特异标记CD29,CD34,CD44,CD45,CD90,以评价干细胞的生物学特性;采用b-FGF和forskolin等诱导脂肪干细胞向雪旺细胞分化,光镜观察诱导后细胞形态的变化;免疫荧光染色鉴定雪旺细胞特异性标记物S100、P75和GFAP的表达;PCR检测诱导前后雪旺细胞特异性标记物S100、P75的表达。结果：分离培养的鼠脂肪干细胞CD29、CB90表达呈阳性,而CD34、CD44和CD45表达呈阴性,具有脂肪干细胞的生物学特性;脂肪干细胞经过胶质细胞生长因子的作用,光镜下发现诱导的细胞形态与雪旺细胞相似;免疫荧光染色S100、P75和GFAP阳性;RT-PCR结果显示诱导的雪旺细胞标记物S100和P75表达上调。结论：脂肪干细胞可诱导分化成雪旺细胞,其表型和分子特征与雪旺细胞相似,诱导分化的脂肪干细胞是一种理想的神经组织工程的种子细胞。     

脐血间充质干细胞诱导向神经元样细胞分化的实验研究

目的：研究脐血间充质干细胞生物学特性及向神经元样细胞分化的潜能。方法：采用密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,观察细胞生长情况,描绘生长曲线,流式细胞仪检测细胞表面标志物,分别向成骨细胞、脂肪细胞、神经元样细胞进行诱导分化,通过茜素红染色、油红O染色检测脐血间充质干细胞成骨、成脂肪细胞诱导分化能力,而以免疫组织化学检测诱导后细胞表面神经标志物的表达。结果：纯化的脐血间充质干细胞贴壁生长,呈均一梭形,生长曲线呈S型,并以P3代增殖能力最强,细胞表面不表达或弱表达CD34、CD35、CD106,高表达CD29、CD44、CD105。成骨诱导2周后,可检测到钙化基质的形成,成脂肪诱导3周后,可检测到脂滴的形成。向神经元样细胞诱导分化后,可观察到典型的神经元样形态改变,且NSE、NF、GFAP阳性表达。结论：分离纯化的脐血间充质干细胞具有较强的增殖能力与分化潜能,并在体外诱导条件下可以向神经元样细胞定向分化。     

Nurr-1基因转染促进脂肪干细胞的神经分化

目的:研究孤儿核受体相关基因1(Nurr-1)对脂肪干细胞(adipose tissue-derived stem cells,ADSC)向神经元方向分化的潜在作用。方法:流式细胞术与成骨、成脂诱导技术鉴定脂肪干细胞;Nurrr-1基因转染脂肪干细胞后,应用神经特异性标志物MAP-2,β-tubulin的免疫荧光染色评估其向神经方向分化的能力。结果:流式细胞术结果表明培养的细胞CD29,CD44表达90%以上,CD45,CD90表达均低于1.5%,经过诱导后,油红O、茜素红S染色均呈阳性,表明所培养的细胞为脂肪干细胞;慢病毒转染Nurr-1基因后,免疫荧光染色检测MAP-2,β-tubulin的免疫荧光强度显著增加;RT-PCR结果显示Nurr-1转染的脂肪干细胞的MAP-2、β-tubulin、NF200的表达量显著提高。结论:Nurr-1基因转染能促进脂肪干细胞向神经方向分化,为神经损伤和神经退行性病变的治疗提供了新途径。     

Gata4, Tbx5 and Baf60c induce differentiation of adipose tissue-derived mesenchymal stem cells into beating cardiomyocytes

The adipose tissue-derived mesenchymal stem cells (ADMSCs) are extensively utilized in tissue engineering, regenerative medicine and cell therapy. ADMSCs can differentiate into cardiomyocytes, and it has been shown that over-expression of a cocktail of factors can induce ectopic heart formation and program cardiogenesis in ESCs. However, which genes are responsible for differentiation of ADMSCs into beating cardiomyocyte-like cells remains unknown. In this study we have shown that the combination of Gata4, Tbx5 and Baf60c is sufficient for inducing ADMSCs to form cardiomyocytes. It also appears that, while Gata4 and Baf60c are key inducers of myocardial differentiation, Tbx5 is essential for the ability of cardiac cells to contract. These findings provide additional experimental references for myocardial tissue engineering in the emerging field of cell-based therapy of heart diseases.     

重组腺病毒介导HGF修饰ADSCs对大鼠肝损伤模型的治疗作用

目的：建立重组腺病毒介导肝细胞生长因子HGF促ADSCs 定向分化肝细胞的方法,并对其参与肝损伤修复能力进行验证,为作为治疗肝损伤细胞来源提供参考。方法：采用消化培养的方法,分离SD 大鼠腹股沟脂肪组织ADSCs 细胞,连续传代3 次对其进行纯化培养,利用形态学鉴定、流式细胞术检测ADSCs 表面标志物方法对其间充质干细胞样特征进行鉴定,加入成脂肪细胞诱导液观察其分化成脂肪细胞的能力;构建腺病毒表达HGF载体Adeno-HGF-EGFP,并转染ADSCs 细胞,利用免疫细胞化学染色方法检测肝细胞标志分子表达水平;最后建立大鼠肝损伤动物模型,观察Adeno-HGF-EGFP 转染的ADSCs 细胞参与肝损伤修复能力情况。结果：分离的ADSCs 细胞形态较为一致,绝大多数呈梭形,排列不规则。流式细胞术结果显示,该细胞表达CD29、CD90、CD106 等间充质干细胞细胞表面标记物,低表达造血干细胞细胞表面标记物CD34、CD45,同时,分离的ADSCs 细胞具有诱导分化成脂肪细胞能力;Adeno-HGF-EGFP 转染ADSCs后,AFP、ALB、CK18 等肝细胞特异性分子表达水平升高;经尾静脉注射ADSCs 细胞后,肝损伤大鼠的AST、ALT、TBIL 等分子表达水平恢复正常。结论：建立了重组腺病毒介导肝细胞生长因子HGF促ADSCs定向分化肝细胞的方法,并且表达HGF的ADSCs 细胞具有修复大鼠肝损伤模型能力,这为通过细胞治疗肝损伤提供了新的细胞来源。     

Isolation, identification and multipotential differentiation of mouse adipose tissue-derived stem cells

Bone marrow and adipose tissue have provided two suitable sources of mesenchymal stem cells. Although previous studies have confirmed close similarities between bone marrow-derived stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs), the molecular phenotype of ADSCs is still poorly identified. In the present study, mouse ADSCs were isolated from the inguinal fat pad of 12-14 weeks old mice. Freshly isolated and three passaged ADSCs were analyzed for the expression of OCT4, Sca-1, c-kit and CD34 by RT-PCR. Three passaged ADSCs were analyzed by flow cytometry for the presence of CD11b, CD45, CD31, CD29 and CD44. Moreover, cardiogenic, adipogenic and neurogenic differentiation of ADSCs were induced in vitro. Freshly isolated ADSCs showed the expression of OCT4, Sca-1, c-kit and CD34, and two days cultured ADSCs were positively immunostained with anti-OCT4 monoclonal antibody. After three passages, the expression of OCT4, c-kit and CD34 eliminated, while the expression of Sca-1 showed a striking enhancement. These cells were identified positive for CD29 and CD44 markers, and they showed the lack of CD45 and CD31 expression. Three passaged ADSCs were differentiated to adipocyte-, cardiomyocyte- and neuron-like cells that were identified based on the positive staining with Sudan black, anti-cardiac troponin I antibody and anti-map-2 antibody, respectively. In conclusion, adipose tissue contains a stem cell population that seems to be a good multipotential cell candidate for the future cell replacement therapy.     

Expression of cardiac function genes in adult stem cells is increased by treatment with nitric oxide agents

Mesenchymal stem cells (MSCs) have received special attention for cardiomyoplasty because several studies have shown that they differentiate into cardiomyocytes both in vitro and in vivo. Nitric oxide (NO) is a free radical signaling molecule that regulates several differentiation processes including cardiomyogenesis. Here, we report an investigation of the effects of two NO agents (SNAP and DEA/NO), able to activate both cGMP-dependent and -independent pathways, on the cardiomyogenic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs). The cells were isolated, cultured and treated with NO agents. Cardiac- and muscle-specific gene expression was analyzed by indirect immunofluorescence, flow cytometry, RT-PCR and real-time PCR. We found that untreated (control) ADSCs and BM-MSCs expressed some muscle markers and NO-derived intermediates induce an increased expression of some cardiac function genes in BM-MSCs and ADSCs. Moreover, NO agents considerably increased the pro-angiogenic potential mostly of BM-MSCs as determined by VEGF mRNA levels.     

一种体外快速分离脂肪来源干细胞的方法

目的:以小鼠为模型,建立一种基于流式细胞仪为检测手段的快速分离脂肪来源干细胞的方法,解决间充质干细胞进入实际应用过程中遇到的难题。方法:取BALB/c小鼠腹股沟内侧的皮下脂肪组织,采用Ⅰ型胶原酶消化等系列措施,获取脂肪干细胞(adipose-derived stem cells,ADSCs)。所得细胞分离培养4代后,流式细胞仪分选出CD73+CD45-ADSCs后成骨诱导分化。碱性磷酸酶染色和实时荧光定量PCR检测其分化情况。结果:刚分离培养的ADSCs细胞普遍呈圆形或椭圆形,传至第三代的细胞,非MSCs细胞逐渐被淘汰,剩余的细胞形态逐渐变得一致,细胞形态呈梭形。流式细胞仪检测发现ADSCs细胞表面抗原标记CD73+CD45-为20.7%。所得的ADSCs成骨诱导分化后碱性磷酸酶(alkaline phosphatase,ALP)染色呈阳性,实时荧光定量PCR检测成骨标志基因发现它们表达上调,其中ALP的表达高达22倍。结论:本方法可以获得纯度较高的ADSCs,且耗时少成本低;且提示可采用该方法来获得大量的人源ADSCs用于组织工程修复。     

Co-culture with TM4 cells enhances the proliferation and migration of rat adipose-derived mesenchymal stem cells with high stemness

The proliferation and migration of mesenchymal stem cells (MSCs) are the efficiency determinants in MSCs transplant therapy. Sertoli cells considered as “nurse cell” possesses the ability to enhance the proliferation and migration of umbilical cord mesenchymal stem cells (UCMSCs). However, no reports about TM4 cells' effect on the proliferation and migration of adipose tissue-derived mesenchymal stem cells (ADSCs) have been found until at present research work. Therefore, this study investigates the effect of TM4 cells on the proliferation and migration of ADSCs. We found that the performance of proliferation and migration of ADSCs were improved significantly while maintaining their stemness and reducing their apoptosis rate. After co-culturing with TM4 cells, the co-cultured ADSCs demonstrated higher proportion of synthetic phase (S) cells and colony-forming units-fibroblastic (CFU-F) number, lower proportion of sub-G1 phase cells and enhanced osteogenic and adipogenic differentiation ability. Moreover, results confirmed the higher multiple proteins involved in cell proliferation and migration including expression of the phospho-Akt, mdm2, pho-CDC2, cyclin D1 CXCR4, MMP-2, as well as phospho-p44 MAPK and phospho-p38 MAPK in co-cultured ADSCs. Furthermore, the process of TM4 cells promoting the proliferation of ADSCs was significantly inhibited by the administration of the PI3K/AKT inhibitor LY294002. Obtained results indicated that TM4 cells through MAPK/ERK1/2, MAPK/p-38 and PI3K/Akt pathways influence the proliferation and migration of ADSCs. These findings indicated that TM4 cells were found effective in promoting stemness and migration of ADSCs, that proves adopted co-culturing technique as an efficient approach to obtain ADSCs in transplantation therapy.     

Neural Differentiation of Rat Adipose-Derived Stem Cells in Vitro

It is reported that adipose-derived stem cells (ADSCs) had multilineage differentiation potential, and could differentiate into neuron-like cells induced by special induction media, which may provide a new idea for restoration of erectile dysfunction (ED) after cavernous nerve injury. The aim of this research was to explore the neuronal differentiation potential of ADSCs in vitro. ADSCs isolated from inguinal adipose tissue of rat were characterized by flow cytometry, and results showed that ADSCs were positive for mesenchymal stem cell markers CD90 and CD44, but negative for hematopoietic stem cell markers. ADSCs maintained self-renewing capacity and could differentiate into adipocytes and neurocytes under special culture condition. In this research, two methods were used to induce ADSCs. In method 1, ADSCs were treated with the preinduction medium including epithelium growth factor, basic fibroblast growth factor, and brain derived neurotrophic factor (BDNF) for 3?days, then with the neurogenic induction medium containing isobutylmethylxanthine, indomethacin, and insulin. While in method 2, BDNF was not used to treat ADSCs. After induction, neuronal differentiation of ADSCs was evaluated. Neuronal markers, glial fibrillary acidic protein (GFAP), and ??-tubulin III (Tuj-1) were detected by immunofluorescence and Western Blot analyses. The expressions of GFAP and Tuj-1 in method 1 were obviously higher then those in method 2. In addition, the positive rate of the neuron-like cells was higher in method 1. It suggested that ADSCs are able to differentiate into neural-like cells in vitro, and the administration of BDNF in the preinduction medium may provide a new way to modify the culture method for getting more neuron-like cells in vitro.     

Priming with oxytocin and relaxin improves cardiac differentiation of adipose tissue–derived stem cells

Previous studies have identified the heart as a source and a target tissue for oxytocin and relaxin hormones. These hormones play important roles in the regulation of cardiovascular function and repair of ischemic heart injury. In the current study, we examined the impact of oxytocin and relaxin on the development of cardiomyocytes from mesenchymal stem cells. For this purpose, mouse adipose tissue–derived stem cells (ADSCs) were treated with different concentrations of oxytocin or relaxin for 4 days. Three weeks after initiation of cardiac induction, differentiated ADSCs expressed cardiac-specific genes, Gata4, Mef2c, Nkx2.5, Tbx5, α- and β-Mhc, Mlc2v, Mlc2a and Anp, and cardiac proteins including connexin 43, desmin and α-actinin. 10 ⁻⁷ M oxytocin and 50 ng/mL relaxin induced the maximum upregulation in the expression of cardiac markers. A combination of oxytocin and relaxin induced cardiomyocyte differentiation more potently than the individual factors. In our experiment, oxytocin-relaxin combination increased the population of cardiac troponin I-expressing cells to 6.84% as compared with 2.36% for the untreated ADSCs, 3.7% for oxytocin treatment and 3.41% for relaxin treatment groups. In summary, the results of this study indicated that oxytocin and relaxin hormones individually and in combination can improve cardiac differentiation of ADSCs, and treatment of the ADSCs and possibly other mesenchymal stem cells with these hormones may enhance their cardiogenic differentiation and survival after transplantation into the ischemic heart tissue.