我们承认朱莉Heward帮助中的hNSC的准备，Lavaniya Thanabalasundaram她与HeLa细胞培养和转染技术支持。

在三维（3D）文化1. HNSC分化注:隔离并从中获得的hNSC，从伦理认可的组织，在以前的出版物2描述。请遵循道德和制度指引下此过程时。 1.1）三维培养用12孔板<ol><li>使用无菌技术在整个手术过程。在生物安全柜，重新水合物支架通过加入2ml /孔的70％乙醇使用水进行灌溉（WFIr）制备。避免配药的70％乙醇下来每个壁以及接触支架。 </li><li>小心吸出70％的乙醇，并立即用2ml /孔的DMEM洗涤支架:F12，持续1分钟。重复洗涤过程两次。最后一次洗涤后F12:小心吸的DMEM。 </li><li>涂层的支架通过添加层粘连蛋白2毫升/孔（10微克/毫升）的DMEM:F12。在5％CO 2的孵育哼声板在37℃下idified培养箱中培养至少2小时的。洗衣板用温水DMEM:F12。 </li><li>种子每个支架有大约500,000的hNSC在150微升的RMM +政府飞行服务队的卷。 </li><li>孵育板3小时，在37℃，在5％CO 2的加湿培养箱中以允许细胞沉降到支架。 </li><li>添加3.5毫升RMM向每个孔中，注意不要从支架移走细胞。 </li><li>孵育板7天;后，从第一馈送2-3天再次更换RMM介质（3.5毫升/孔）后第1天（第一次喂养）和。 </li><li> 7天后，取出培养基并修复细胞，用4％多聚甲醛（PFA）进行免疫细胞化学（ICC）或者添加500微升/孔裂解试剂总RNA提取（存储板在≤-80℃或进行总RNA提取） 。 </li></ol> 1.2）测量轴突生长过程<ol><li>染色的4％的PFA按照一个标准的ICC协议20使用β固定有区别的hNSC3微管蛋白（TUBB3）与荧光标记的第二抗体检测第一抗体。染液细胞核用Hoechst（1μM）。 </li><li>捕获使用荧光显微镜β3微管蛋白染色的细胞的代表性图像;图像保存为TIFF或JPEG。使用软件的测量工具（ 如图像-Pro Plus中）的轴突生长过程中测量。 </li><li>打开图像，在工具栏中选择测量选项，然后选择跟踪功能。 </li><li>要启动跟踪，通过点击鼠标右键选择对轴突生长的起点。沿着轴突生长的整个长度跟踪;右键点击来完成每个跟踪。重复测量的视场的所有轴突outgrowthswith。 </li><li>快速测量为像素或微米（待定校准设置）。出口长度测量为样本的比较和统计分析的电子表格程序。 </li></ol> 2. miRNA的表达使用干细胞发展途径聚焦的miRNA PCR阵列 2.1）的miRNA总RNA提取<ol><li>在支架和2D成长样品进行miRNA的提取（分化和未分化，hNSC控制）。 </li><li>解冻支架，插在一个12孔板中，如果需要的话。结合的2孔中的裂解试剂内容（500微升）在同一1.5毫升的Eppendorf管中，终体积1毫升裂解试剂。照顾留在井中的脚手架。 </li><li>对于以前收集的干细胞沉淀2D成长样品（分化hNSC 2D文化和未分化的控制），加入1 ml裂解试剂和内容转移到1.5 ml离心管。 </li><li>用1ml注射器和一个短20至25号针头匀浆各试验样品。用1ml注射器多达5次，直到均相裂解物实现通过针通过裂解物。 </li><li>加入200μl的氯仿，以每Eppendorf管和帽牢固。反转和涡流管到混合内容。 </li><li>离心机各自含有匀浆15分钟。≥12,000×g下1.5 ml离心管。 </li><li>传输各样品的上层水相（避免传输任何相间，粉红色液体）到含有750微升的100％乙醇的一个新的1.5 ml离心管。颠倒调匀。 </li><li>吸取高达700微升每种测试样品的，包括任何沉淀，放入2 ml收集管迷你柱。盖上盖子并离心≥8,000XG为在室温下大约30秒。丢弃的流量通过。重复使用的样本的剩余部分这一步。 </li><li>执行的DNA消化15分钟。 </li><li>加入700μlBuffer RWT的小柱和离心约30秒，≥8,000×g的。丢弃的流量通过。 </li><li>通过加入500μl的缓冲液RPE和≥8,000×g下离心约30秒洗涤迷你柱。丢弃的流量通过。重复wash.Centrifuge在≥12,000×g离心1分钟来干燥膜进一步。 </li><li>通过加入40微升不含RNA酶的水至微型柱中，在≥8,000×g下离心1分钟洗脱总RNA在1.5ml Eppendorf管中。测量的总RNA的浓度与适当的分光光度计的助剂。 </li></ol> 2.2）的miRNA基因的制备<ol><li>制备反转录主混合物。每个样本需要4微升5×的miScript的HiSpec缓冲液，2微升10×nucleics组合，2微升的miScript逆转录组合。 </li><li>等分试样8微升主混合物在PCR管中，添加的总RNA（250纳克）的所需体积，并加入无RNase水到20微升的最终体积。 </li><li>孵育60分钟，在37°C.Incubate为5分钟，在95℃下失活的miScript逆转录酶混合。 </li><li>加入200μl无RNase水以各20微升反转录反应，储存在-20℃，或进行真悌我立刻PCR。 </li></ol> 2.3）干细胞与发育途径专注的miRNA PCR阵列<ol><li>通过加入1375微升2×SYBR绿色主混合物，100微升的miRNA的cDNA制备，275微升10倍的miScript通用引物，和1000微升不含RNA酶的水（总体积2750微升）准备在一个15毫升管的PCR成分组合。 </li><li>转移在PCR装载水库PCR组件组合。小心地将其从密封袋中的96孔板PCR阵列。 </li><li>使用8通道移液器，或仅使用8提示一个12通道移液器加入25微升PCR成分混合到PCR阵列的每个孔中。改变下列各移液步骤，以避免井之间的交叉污染枪头。 </li><li>封板与光学粘合剂膜，离心1分钟，在1000×g离心，在室温（15-25℃）以除去气泡。目视检查板从下方以确保无气泡存在于所述孔中。 </li><li>放置在96孔板的PCR阵列中的实时PCR仪。 </li><li>程序的实时PCR仪相应地:1个循环的95℃10分钟，45个循环持续15秒，在95℃，并在60℃下1分钟，设置一个单一的（荧光数据集合）。 </li><li>导出Ct值的所有井空Excel电子表格与PCR阵列数据分析模板Excel和基于网络的软件使用。 注:软件可在<a href="http://www.SABiosciences.com/pcrarraydataanalysis.php">www.SABiosciences.com/pcrarraydataanalysis.php</a> 。 </li></ol> 3.计算分析miRNA靶标预测与靶mRNA的验证记者质粒转染和Ddual萤光素酶检测。 3.1）的miRNA靶标预测计算分析<ol><li>浏览PicTar（ <a href="http://pictar.mdc-berlin.de/">http://pictar.mdc-berlin.de/</a> ）21，在网上提供算法的miR鉴定NA目标预测的mRNA。 </li><li>点击"点击此处查看PicTar脊椎动物预测"。一个新的页面将打开。 </li><li>在新的页面，PicTar Web界面，选择品种在下拉菜单并插入在miRNA的ID中的利益的miRNA。点击搜索了miRNA的目标。 </li><li>观察靶mRNA由aPicTar成绩排名的名单。 注:PicTar计算一个分数反映一个给定的非编码区将由所选的miRNA靶向，并且基于种子互补，热力学和在套共表达的miRNA 22组合预测为共同目标的可能性。 </li><li>同样使用DIANA-LAB（检索靶mRNA的列表<a href="http://diana.cslab.ece.ntua.gr/microT/">http://diana.cslab.ece.ntua.gr/microT/</a> ）23，和TargetScan（ <a href="http://www.targetscan.org/">http://www.targetscan.org/</a> ）24，替代上可用的线算法。 </li><li>编译米对比表iRNAs根据使用不同的软件工具排名上获得。 PicTar位列由得分，DianaLab通过精密（基于信噪比和精度的分数的预测结果的意义的评价）由骨料的PCT和TargetScan（基于种子的互补性，结合热力学自由能，节约了不同的物种）， 见表1。 </li></ol>靶mRNA的3.2），验证通过记者质粒转染和双萤光素酶检测。 注:3&#39;UTR荧光素酶记者的靶mRNA一直是验证前面所述25。 <ol><li>转染<ol><li>种子的HeLa细胞以10 5细胞/孔在24孔板的密度。 </li><li> 24小时后，共转染的HeLa细胞用1.5微升转染试剂，要么100纳克NR4A3 3&#39;UTRor SOX5 3&#39;端非编码区的质粒的，以及为20nM的miRNA模拟物（HSA-的miR-96-5p为SOX5 3217; UTR，和HSA-MIR-7-5p，分别HSA-MIR-17-5p为NR4A3 3&#39;UTR）每口井。 </li><li>联合转染控制井NR4A3 3&#39;UTRor SOX5 3&#39;UTRplasmid和荧光标记阴性对照的siRNA。对照孔与荧光标记阴性对照的siRNA转染允许的转染效率的评估，占模仿miRNA的3&#39;UTR结合特异性。 </li></ol></li><li>双荧光素酶活动的测量<ol><li>测量萤火虫和海肾荧光素酶的活动，24小时后转染。除去细胞生长培养基中。 </li><li>制备工作溶液I通过加入50μl底物I的到10ml溶液I的;调匀。加入300微升工作解决方案，我到每一个24孔的。 </li><li>将每个24的内容早已进入3口井96白色板，100微升每个。等待10分钟，并测量萤火虫荧光素酶为发光收购光度计集。 </li><li>准备工作SOLUT离子II通过加入50μl底物二到10ml溶液II;调匀。加入100微升的工作解决方案，我已经到含有100微升工作方案一，每96以及</li><li>等待10分钟，并测量海肾荧光素酶为发光收购光度计集。从萤火虫荧光素酶的Renilla荧光素酶的发光计算的比率。 </li></ol></li></ol>

<ol>
	<li>Falanga, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+tissue+repair+and+regeneration.">Stem cells in tissue repair and regeneration.</a> J Invest Dermatol. 132 (6), 1538-1541 (2012).</li><li>Pollock, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conditionally+immortal+clonal+stem+cell+line+from+human+cortical+neuroepithelium+for+the+treatment+of+ischemic+stroke.">A conditionally immortal clonal stem cell line from human cortical neuroepithelium for the treatment of ischemic stroke.</a> Exp Neurol. 199 (1), 143-155 (2006).</li><li>Hodges, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+stem+cell+lines+suitable+for+transplantation.">Making stem cell lines suitable for transplantation.</a> Cell Transplant. 16 (2), 101-115 (2007).</li><li>Smith, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+site+and+lesion+topology+determine+efficacy+of+a+human+neural+stem+cell+line+in+a+rat+model+of+chronic+stroke.">Implantation site and lesion topology determine efficacy of a human neural stem cell line in a rat model of chronic stroke.</a> Stem Cells. 30 (4), 785-796 (2012).</li><li>Stroemer, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+stem+cell+line+CTX0E03+promotes+behavioral+recovery+and+endogenous+neurogenesis+after+experimental+stroke+in+a+dose-dependent+fashion.">The neural stem cell line CTX0E03 promotes behavioral recovery and endogenous neurogenesis after experimental stroke in a dose-dependent fashion.</a> Neurorehabil Neural Repair. 23 (9), 895-909 (2009).</li><li>Kalladka, D., Muir, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+repair:+cell+therapy+in+stroke.">Brain repair: cell therapy in stroke.</a> Stem Cells Cloning. 7, 31-44 (2014).</li><li>Fire, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391 (6669), 806-811 (1998).</li><li>Anokye-Danso, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+miRNA-mediated+reprogramming+of+mouse+and+human+somatic+cells+to+pluripotency.">Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency.</a> Cell Stem Cell. 8 (4), 376-388 (2011).</li><li>Li, X., Jin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+small+regulatory+RNAs+in+determining+neuronal+identity.">Roles of small regulatory RNAs in determining neuronal identity.</a> Nat Rev Neurosci. 11 (5), 329-338 (2010).</li><li>Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+as+a+scaffold+for+tissue+reconstruction.">The extracellular matrix as a scaffold for tissue reconstruction.</a> Semin Cell Dev Biol. 13 (5), 377-383 (2002).</li><li>Buxboim, A., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+feel+the+difference.">Stem cells feel the difference.</a> Nat Methods. 7 (9), 695-697 (2010).</li><li>Li, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+nanofibrous+scaffold+for+cartilage+tissue+engineering+using+human+mesenchymal+stem+cells.">A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells.</a> Biomaterials. 26 (6), 599-609 (2005).</li><li>Lutolf, M. P., Gilbert, P. M., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+materials+to+direct+stem-cell+fate.">Designing materials to direct stem-cell fate.</a> Nature. 462 (7272), 433-441 (2009).</li><li>Ortinau, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+3D-scaffold+formation+on+differentiation+and+survival+in+human+neural+progenitor+cells.">Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells.</a> Biomed Eng Online. 9 (1), 70 (2010).</li><li>Saha, K., Pollock, J. F., Schaffer, D. V., Healy, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+synthetic+materials+to+control+stem+cell+phenotype.">Designing synthetic materials to control stem cell phenotype.</a> Curr Opin Chem Biol. 11 (4), 381-387 (1016).</li><li>Knight, E., Murray, B., Carnachan, R., Przyborski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alvetex(R):+polystyrene+scaffold+technology+for+routine+three+dimensional+cell+culture.">Alvetex(R): polystyrene scaffold technology for routine three dimensional cell culture.</a> Methods Mol Biol. 695, 323-340 (2011).</li><li>Bokhari, M., Carnachan, R. J., Przyborski, S. A., Cameron, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emulsion-+templated+porous+polymers+as+scaffolds+for+three+dimensional+cell+culture:+effect+of+synthesis+parameters+on+scaffold+formation+and+homogenity.">Emulsion- templated porous polymers as scaffolds for three dimensional cell culture: effect of synthesis parameters on scaffold formation and homogenity.</a> J. Mater. Chem. 17, 4088-4094 (2007).</li><li>Bokhari, M., Carnachan, R. J., Cameron, N. R., Przyborski, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cell+culture+device+enabling+three-dimensional+cell+growth+and+improved+cell+function.">Novel cell culture device enabling three-dimensional cell growth and improved cell function.</a> Biochem Biophys Res Commun. 354 (4), 1095-1100 (2007).</li><li>Stevanato, L., Sinden, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+microRNAs+on+human+neural+stem+cell+differentiation+in+two-+and+three-dimensional+cultures.">The effects of microRNAs on human neural stem cell differentiation in two- and three-dimensional cultures.</a> Stem Cell Res Ther. 5 (2), 49 (2014).</li><li>Ippolito, D. M., Eroglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+synapses:+an+immunocytochemistry-based+assay+to+quantify+synapse+number.">Quantifying synapses: an immunocytochemistry-based assay to quantify synapse number.</a> J Vis Exp. (45), (2010).</li><li>Chen, K., Rajewsky, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+selection+on+human+microRNA+binding+sites+inferred+from+SNP+data.">Natural selection on human microRNA binding sites inferred from SNP data.</a> Nat Genet. 38 (12), 1452-1456 (2006).</li><li>Krek, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+microRNA+target+predictions.">Combinatorial microRNA target predictions.</a> Nat Genet. 37 (5), 495-500 (2005).</li><li>Maragkakis, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+microRNA+target+prediction+correlates+with+protein+repression+levels.">Accurate microRNA target prediction correlates with protein repression levels.</a> BMC Bioinformatics. 10, 295 (2009).</li><li>Lewis, B. P., Burge, C. B., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+seed+pairing,+often+flanked+by+adenosines,+indicates+that+thousands+of+human+genes+are+microRNA+targets.">Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.</a> Cell. 120 (1), 15-20 (2005).</li><li>Aldred, S. F., Collins, P., Trinklein, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+targets+of+human+micrornas+with+the+LightSwitch+Luciferase+Assay+System+using+3'UTR-reporter+constructs+and+a+microRNA+mimic+in+adherent+cells.">Identifying targets of human micrornas with the LightSwitch Luciferase Assay System using 3'UTR-reporter constructs and a microRNA mimic in adherent cells.</a> J Vis Exp. (55), (2011).</li><li>Ponnio, T., Conneely, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nor-1+regulates+hippocampal+axon+guidance,+pyramidal+cell+survival,+and+seizure+susceptibility.">nor-1 regulates hippocampal axon guidance, pyramidal cell survival, and seizure susceptibility.</a> Mol Cell Biol. 24 (20), 9070-9078 (2004).</li><li>Martinez-Morales, P. L., Quiroga, A. C., Barbas, J. A., Morales, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX5+controls+cell+cycle+progression+in+neural+progenitors+by+interfering+with+the+WNT-beta-catenin+pathway.">SOX5 controls cell cycle progression in neural progenitors by interfering with the WNT-beta-catenin pathway.</a> EMBO Rep. 11 (6), 466-472 (2010).</li><li>Kwan, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX5+postmitotically+regulates+migration,+postmigratory+differentiation,+and+projections+of+subplate+and+deep-layer+neocortical+neurons.">SOX5 postmitotically regulates migration, postmigratory differentiation, and projections of subplate and deep-layer neocortical neurons.</a> Proc Natl Acad Sci U S A. 105 (41), 16021-16026 (2008).</li><li>Lai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX5+controls+the+sequential+generation+of+distinct+corticofugal+neuron+subtypes.">SOX5 controls the sequential generation of distinct corticofugal neuron subtypes.</a> Neuron. 57 (2), 232-247 (2008).</li></ol>

所有的作家都员工，股票和/或ReNeuron公司或其母公司的股票期权的持有人。 这项研究是由ReNeuron（RENE.L）的支持。

新的体外实验的发展，评估和提高干细胞的分化一直提出了挑战，它们的功能和治疗能力的调查。长期，稳定附着的hNSC的，所需的强大的3D 体外分化测定法的发展，通过用在材料和结构术语不同特性测试几个三维基材处理。由于试验发展的一部分，我们还评估最佳细胞接种浓度，并确定了支架被涂层粘连蛋白的要求。虽然我们的hNSC可以应自发分化的4-OHT和生长因子的去除，三维培养系统的实施表明由轴突生长过程时相比，标准的2D分化的hNSC中测量其分化能力的显著改善。 探讨3D各色诱导的作用ntiation上hNSC miRNA表达我们使用了PCR阵列。与标准的芯片芯片相比PCR阵列提供的利益的miRNA的直接定量和验证的优势。虽然在PCR阵列中的miRNA的总数每阵列，例如术语在这里比96少的限制，它可以根据具体包括感兴趣的miRNA，在我们的情况下，预先在干细胞发育的报道。的途径集中的miRNA表达异形3D和2D分化的hNSC未分化的hNSC相比。最显著下调的miRNA被选择和分析，以评估它们的推定靶mRNA与确定它们的功能和生物解释使用在线可用算法（DIANA实验室，PicTar和TargetScans）的意图。 单个的miRNA能够识别数百个目标，并基于这个原因，我们验证与3&#39;UTR mRNAsthat miRNA的相互作用可能是合适的人选了xonal调节。 NR4A3，所述HSA-的miR-7和miR-17家族的一个目标，是NR4A家族核受体的，这取决于表达的水平的成员，参与了海马轴突的分化，存活，细胞凋亡和调节指导26。同样SOX5，HSA-的miR-96的目标，据报道，在控制神经祖细胞轴突27 28的长度，迁移，后迁徙分化，神经元29的预测细胞周期进程。既SOX5和NR4A3被确定为HSA-的miR-96的直接靶mRNA和hsa-的miR-7和17分别由双荧光素酶报告测定。双荧光素酶（萤火虫和海肾）依赖于在相同的质粒两种不同的报告基因，并提供一种改进的系统，允许正常化引起的不理想的转染和用单个荧光素酶质粒系统相比的细胞毒作用的效果。由于我们的实验开发的一部分，我们还优化了转conditi附加组件，以获得较高的效率。 所述protocolsdescribed此处可以被利用来进一步优化和表征体外 hNSC分化可能Python实现。python_cpp_mix.jpg intransplantation协议用 ​​于临床前研究和未来的临床应用。

干细胞研究起着再生医学，这也涵盖组织工程，发育细胞生物学，细胞疗法，基因疗法的学科中心作用，和生物材料（支架和矩阵）。干细胞是在两种器官发展和组织修复重要;理解干细胞如何工作的关键是开发新的干细胞基础的治疗1。 CTX0E03是一个克隆，条件永生化人神经干细胞（hNSC）2号线从孕早期胎儿大脑皮层中分离。 CTX0E03定义是根据良好生产规范（GMP）3临床细胞银行至关重要的品质特征。的hNSC可以自发地分化为神经元，神经胶质细胞和少突胶质和已被证明，当在局灶性缺血2,4,5啮齿动物模型移植到改善神经功能缺损。该CTX0E03的hNSC目前在脑卒中disabil临床试验性（NCT01151124 6和NCT02117635，Clinicaltrials.gov）。 miRNA的具有平均22个核苷酸长度并且被证明调节分子7，它们不编码蛋 ​​白质，而是结合mRNA的3首要翻译区（3&#39;UTR），调节它们的稳定性和翻译成蛋白质。 miRNA是据报道参与多种细胞过程，包括发育，增殖和分化8的调节。几个miRNA的有牵连的调 ​​节神经干细胞9的命运和规范。 在二维（2D）标准培养环境中的体内环境的复杂性不匹配，因此hNSC分化的诱导和调节不是最佳的。 在体内 ，细胞通过一个三维（3D）微环境由其他构成包围细胞及细胞外配体，其中包括许多类型胶原，层粘连蛋白和其他基质蛋白（细胞外基质，ECM）的。以前的研究已经报道，模仿ECM和其几何形状影响细胞表型和命运10-15的材料的建筑地形。可许多商用三维支架;我们使用了高多孔聚苯乙烯支架设计具有良好定义的和均匀的架构成200微米厚的膜，它提供了一个三维空间到其中的细胞可以侵入并区分16-18。 此处的二维和三维分化测定法用于评估axonprocess产物。此外，我们描述的方法来分析一个临床级hNSC线的miRNA表达，以进一步探讨其分化的miRNA的效果。示于此处的方法是基于最初在19所描述的那些。

<table><tbody><tr><td>DMEM:F12</td><td>Invitrogen</td><td>21331-020</td><td>For RMM medium preparation</td></tr><tr><td>Human Albumin Solution&amp;nbsp;</td><td>Baxter health care limited&amp;nbsp;</td><td>1501052</td><td>For RMM medium used at a final concentration of&amp;nbsp; 0.03%</td></tr><tr><td>Transferrin, Human recombinant</td><td>Sigma</td><td>T8158</td><td>For RMM medium used at a final concentration of&amp;nbsp; 5 &amp;micro;g/ml</td></tr><tr><td>Putrescine DiHCl&amp;nbsp;</td><td>Sigma</td><td>P5780</td><td>For RMM medium used at a final concentration of&amp;nbsp; 16.2 &amp;micro;g/ml</td></tr><tr><td>Insulin, Human recombinant&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>I9278</td><td>For RMM medium used at a final concentration of&amp;nbsp; 5 &amp;micro;g/ml</td></tr><tr><td>Progesterone&amp;nbsp;</td><td>Sigma</td><td>P6149</td><td>For RMM medium used at a final concentration of&amp;nbsp; 60 ng/ml</td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>Invitrogen</td><td>25030-24</td><td>For RMM medium used at a final concentration of&amp;nbsp; 2 mM</td></tr><tr><td>Sodium Selenite&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>S9133</td><td>For RMM medium used at a final concentration of 40 ng/ml</td></tr><tr><td>bFGF&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Peprotech</td><td>AF-100-15</td><td>To be added to RMM medium, used at a final concentration of 10 ng/ml</td></tr><tr><td>EGF&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Peprotech</td><td>AF-100-188</td><td>To be added to RMM medium, used at a final concentration of 20 ng/ml</td></tr><tr><td>4-OHT</td><td>Sigma</td><td>H7904</td><td>To be added to RMM medium, used at a final concentration of 100 nM</td></tr><tr><td>Laminin</td><td>AMS Biotech</td><td>3400-010-10</td><td></td></tr><tr><td>TrypZean/EDTA</td><td>Lonza&amp;nbsp;</td><td>BE02-034E</td><td></td></tr><tr><td>Water for irrigation</td><td>Baxter Healthcare</td><td>UKF7114</td><td></td></tr><tr><td>Defined Trypsin Inhibitor (DTI) (store -20&amp;deg;C)</td><td>Invitrogen</td><td>R007-100</td><td></td></tr><tr><td>Alvetex 12 well plate&amp;nbsp;</td><td>Reinnervate Ltd</td><td>AVP002</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Merck&amp;nbsp;</td><td>1.00986.1000</td><td></td></tr><tr><td>&amp;beta;III tubulin antibody</td><td>Sigma</td><td>T8660</td><td></td></tr><tr><td>Hoechst</td><td>Sigma</td><td>B2261</td><td></td></tr><tr><td>miRNeasy mini kit</td><td>Qiagen&amp;nbsp;</td><td>217004</td><td></td></tr><tr><td>RNase free DNase&amp;nbsp;</td><td>Qiagen&amp;nbsp;</td><td>79254</td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>C7559-5VL</td><td></td></tr><tr><td>miScript II RT Kit&amp;nbsp;</td><td>Qiagen&amp;nbsp;</td><td>218160</td><td></td></tr><tr><td>SYBR green PCR kit</td><td>Qiagen&amp;nbsp;</td><td>218073</td><td></td></tr><tr><td>Cell Development &amp;amp; Differentiation miScript miRNA PCR Array</td><td>Qiagen/ SABiosciences</td><td>MIHS-103Z</td><td></td></tr><tr><td>Lightcycler 480 white 96 plate</td><td>Roche</td><td>&amp;nbsp;4729692001</td><td></td></tr><tr><td>Lightcycler 480&amp;nbsp; sealing foil</td><td>Roche</td><td>&amp;nbsp;4729757001</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668-027</td><td></td></tr><tr><td>Vector NR4A3 miRNA 3&#039; UTR target clones&amp;nbsp;</td><td>GeneCopeia</td><td>HmiT019538-MT0</td><td></td></tr><tr><td>Vector SOX5 miRNA 3&#039; UTR target clones&amp;nbsp;</td><td>GeneCopeia</td><td>HmiT017632-MT01</td><td></td></tr><tr><td>Allstars negative control siRNA AF 488 (Qiagen).</td><td>Qiagen&amp;nbsp;</td><td>1027284</td><td>MiRNA transfection control</td></tr><tr><td>Luc-Pair miR Luciferase Assay</td><td>GeneCopeia</td><td>LPFR-M010</td><td></td></tr><tr><td>Lightcycler 480&amp;nbsp;</td><td>Roche</td><td></td><td></td></tr><tr><td>GloMax 96 microplate luminometer&amp;nbsp;</td><td>Promega</td><td></td><td></td></tr></tbody></table>

differentiation of a human neural stem cell line on three dimensional cultures, analysis of microrna and putative target genes

神经干细胞（NS​​Cs）是具有自我更新及分化为神经元，星形胶质细胞和少突胶质细胞在特定的地方微环境。在这里，我们提出了一套用于三维（3D）的分化和克隆人类神经干细胞（hNSC）线的miRNA的分析，目前在中风残疾（NCT01151124和NCT02117635，Clinicaltrials.gov）临床试验的方法。的hNSC来源于道德批准前三个月胎儿大脑皮层和使用的C-mycER TAM的单拷贝逆转录病毒整合建设条件永生化。我们描述如何衡量的hNSC分化三维支架，以及如何使用定量PCR数组中的miRNA表达相关变化的过程中轴突生长。此外，我们举例说明计算分析与选择的miRNA的推定靶的目的。 SOX5和NR4A3被确定为选择的显著合适的miRNA的推定靶下调ð的miRNA在分化hNSC。 miRNA靶标验证是由双报告质粒转染和双荧光素酶检测对SOX5和NR4A3 3&#39;UTRs执行。

在图1中被可视化hNSC分化的三维支架的调查和定量与传统的平坦面培养物（2D）进行比较，并expressedas轴突生长过程的测量。在图2中是representedthe工作流程和利用干细胞的结果为所述miRNA量化和发育途径集中的miRNA阵列的PCR来调查的miRNA在2D和3D分化的hNSC与未分化的对照相比的差异表达。差异化和未分化的hNSC和计算分析目标的预测，SOX5和NR4A3（NOR1）之间以下miRNA的对比分析，确定为假定的靶mRNA。研究miRNA及其上的3&#39;UTR的mRNA，我们使用含有2或者SOX5或NR4A3 3&#39;UTR序列市售的质粒插入到萤火虫荧光素酶报告基因的下游，推定站点之间的直接交互的<html>次包含在同一质粒海肾萤光素酶基因进行标准化。的3&#39;端非编码区的荧光素酶的活性下调的代表性结果示于图3中 。 <img alt="图1" src="/files/ftp_upload/52410/52410fig1.jpg" /> 图1:在三维支架的hNSC的分化，和轴突过程ES生长测定 （A） 的以下标准的ICC染色β3微管蛋白（绿色）和核用Hoechst（蓝色）代表显微照片被收购和轴突过程的测量是用图像分析软件的帮助下进行的。 （B）方框图报告的轴突生长过程中的2D和3D分化的hNSC 1（1W）或3周（3W）进行测量。COM /文件/ ftp_upload / 52410 / 52410fig1highres.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/52410/52410fig2.jpg" /> 图2:miRNA表达谱在分化的hNSC的概略流程的总RNA，包括微RNA，没有从任一对的三维支架和标准的2D培养物，未分化的或分化的控制的hNSC萃取。 250ng的总RNA是复古转录与便于选择性转化成熟miRNA的成cDNA适于miRNA的定量与人类细胞分化和发育的miScript mi​​RNA的PCR阵列的方法。地缘平均SNORD61，SNORD68，SNORD72，SNORD95，SNORD96A和RNU6-2的用于基于所述2-ΔΔCT方法数据分析。显著的变化被定义为+/- 1.5折上下调节到一个站统计学上显著的程度。使用基于网络的软件可在结果表示为一clustergram数据进行分析。从图3月 19日修改。 <a href="https://www.jove.com/files/ftp_upload/52410/52410fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52410/52410fig3.jpg" /> 图3:miRNA靶的验证预测基因使用双荧光素酶报告分析。 （A）中作为一种工具，以验证3&#39;UTR序列与miRNA的直接靶的双荧光素酶报告质粒的代表性示意图。 （B）的代表性的显微镜照片示出转染效率。 （C） &#160;信号，表示为相对荧光素酶活性，从人3＆＃39; UTR记者ofSOX5和NR4A3基因的HSA-MIR-96的存在是显著撞倒，并-HSA的miR-7，分别和17 miRNA的模仿。从图5月 19日修改。 <a href="https://www.jove.com/files/ftp_upload/52410/52410fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td></td><td colspan="2"> DIANALAB </td><td> PicTar </td><td> TargetScan </td></tr><tr><td> miRNA的 </td><td> 靶基因 </td><td> 靶位点/发现基因 </td><td> 精确 </td><td> 得分 </td><td> 总P CT </td></tr><tr><td> HSA-MIR-96 </td><td> SOX5 </td><td>七百六十三分之一千二百一十四</td><td> 1 </td><td> 6.74 </td> &lt;TD&gt; 0.94 </td></tr><tr><td> HSA-MIR-183 </td><td> DUSP10 </td><td>一百九十○分之三百〇二</td><td> 0.72 </td><td> 4.75 </td><td> 0.85 </td></tr><tr><td> HSA-MIR-302A </td><td> NR4A3 </td><td> 473分之863 </td><td> 0.84 </td><td> 3.05 </td><td> NF </td></tr><tr><td> HSA-MIR-182 </td><td> FOXN3 </td><td> 794分之1358 </td><td> 0.94 </td><td> NF </td><td> 0.96 </td></tr><tr><td> HSA-MIR-7 </td><td> NR4A3 </td><td> NF </td><td> NF </td><td> 1.61 </td><td> 0.17 </td></tr><tr><td> HSA-MIR-7 </td><td> FOXN3 </td><td> 136分之399 </td><td> 0.17 </td><td> NF </td><td> 0.88 </td></tr><tr><td> HSA-MIR-20A </td><td> NR4A3 </td><td>八百四十一分之一千六百五十</td><td> 0.85 </td><td> 5.87 </td><td> 0.63 </td></tr><tr><td><strong&gt;的HSA-MIR-20B </td><td> NR4A3 </td><td> 973分之1955 </td><td> 0.9 </td><td> 5.87 </td><td> 0.63 </td></tr><tr><td> HSA-MIR-17 </td><td> NR4A3 </td><td> 961分之1928 </td><td> 0.9 </td><td> 5.38 </td><td> 0.63 + 0.37 </td></tr><tr><td> HSA-MIR-20A </td><td> EIF4G3 </td><td>八百四十一分之一千六百五十</td><td> 0.97 </td><td> NF </td><td> NF </td></tr><tr><td> HSA-MIR-20B </td><td> EIF4G3 </td><td> 973分之1955 </td><td> 0.94 </td><td> NF </td><td> NF </td></tr><tr><td> HSA-MIR-17 </td><td> EIF4G3 </td><td> 961分之1928 </td><td> 0.94 </td><td> NF </td><td> NF </td></tr></tbody></table> 表1:miRNA靶标预测mRNA的列表 。 miRNA的代表性表，目标预测的基因，和算法分析（预测离子采用戴安娜实验室，PicTar和TargetScan分别设有/得分/总）。从表3中 19修改。

Watch this Scientific Journal Video about Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes at JoVE.com

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

differentiation-human-neural-stem-cell-line-on-three-dimensional

Research

JoVE Journal

Developmental Biology

9.9K 次观看.  ReNeuron. With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

视频: Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

科研

生物工程

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

发育生物学

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

研究脑癌和神经退行性疾病的人类神经器官

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia ...

神经科学

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

人诱导多能干细胞及其神经和神经胶质衍生物中多种线粒体参数的流式细胞术分析

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

Differentiating Human Neuronal Stem Cells into 3D Culture Using a Porous Scaffold

Source: Stevanato, L. et al., <a href="https://app.jove.com/v/52410">Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes.</a> J. Vis. Exp. (2015)
This video showcases the development and differentiation of human neural stem cells within a three-dimensional scaffold. A laminin coating on the scaffold promotes cell attachment, while a growth factor-free neural medium induces cell differentiation into neural progenitor cells, which extend their processes within a porous architecture in multiple directions, forming a three-dimensional culture

使用多孔支架将人神经元干细胞分化为 3D 培养物

1. HNSC differentiation on Three-Dimensional (3D) Culture
NOTE: Isolate and derive human neural stem cells (hNSCs) from an ethically approved tissue ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Differentiating Non-Transgenic and Transgenic Human Pluripotent Stem Cells into Neural Progenitor Cells

Source: Cvetkovic, C., et al. <a href="https://app.jove.com/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates the generation of neural progenitor cells from non-transgenic and transgenic human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural progenitor cells using a neuronal differentiation medium. While differentiation is induced in non-transgenic cells using small molecules, the transgenic hPSCs are differentiated by adding an antibiotic that induces the promoter for a neuronal differentiation-specific transcription factor.

将非转基因和转基因人多能干细胞分化为神经祖细胞

Ethic approvals were obtained when performing the experiments.
1. Cell Culture and Reagent Preparation

	Prepare coated plates for cell culture.
	
		...

Differentiating Human Embryonic Stem Cells into Neural Progenitors

Plate the cells on a basement membrane matrix coated 6-well plate at a density of 2 x 105 cells per well in 2 milliliters of mTeSR1 with 10 micromolar ROCK inhibitor. After 24 hours, replace the culture medium with neural induction medium supplemented with 1 micromolar dorsomorphin and 5 micromolar SB431542. Change the medium every other day during the first four days of neural induction, then every day, until confluence is reached at day seven.