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 border="0" cellpadding="0" cellspacing="0" width="573">
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			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="20" style="height:20px;">DMEM:F12</td>
			<td>Invitrogen</td>
			<td>21331-020</td>
			<td>For RMM medium preparation</td>
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			<td height="20" style="height:20px;">Human Albumin Solution&#160;</td>
			<td>Baxter health care limited&#160;</td>
			<td>1501052</td>
			<td>For RMM medium used at a final concentration of&#160; 0.03%</td>
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			<td height="20" style="height:20px;">Transferrin, Human recombinant</td>
			<td>Sigma</td>
			<td>T8158</td>
			<td>For RMM medium used at a final concentration of&#160; 5 &#181;g/ml</td>
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			<td height="20" style="height:20px;">Putrescine DiHCl&#160;</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td>For RMM medium used at a final concentration of&#160; 16.2 &#181;g/ml</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Insulin, Human recombinant&#160;&#160;</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td>For RMM medium used at a final concentration of&#160; 5 &#181;g/ml</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Progesterone&#160;</td>
			<td>Sigma</td>
			<td>P6149</td>
			<td>For RMM medium used at a final concentration of&#160; 60 ng/ml</td>
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		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine&#160;</td>
			<td>Invitrogen</td>
			<td>25030-24</td>
			<td>For RMM medium used at a final concentration of&#160; 2 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Selenite&#160;&#160;</td>
			<td>Sigma</td>
			<td>S9133</td>
			<td>For RMM medium used at a final concentration of 40 ng/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">bFGF&#160;&#160;&#160;&#160;&#160;</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 height="20">
			<td height="20" style="height:20px;">EGF&#160;&#160;&#160;&#160;&#160;&#160;&#160;</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 height="20">
			<td height="20" style="height:20px;">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>
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		<tr height="20">
			<td height="20" style="height:20px;">Laminin</td>
			<td>AMS Biotech</td>
			<td>3400-010-10</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">TrypZean/EDTA</td>
			<td>Lonza&#160;</td>
			<td>BE02-034E</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Water for irrigation</td>
			<td>Baxter Healthcare</td>
			<td>UKF7114</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Defined Trypsin Inhibitor (DTI) (store -20&#176;C)</td>
			<td>Invitrogen</td>
			<td>R007-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alvetex 12 well plate&#160;</td>
			<td>Reinnervate Ltd</td>
			<td>AVP002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol&#160;</td>
			<td>Merck&#160;</td>
			<td>1.00986.1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;III tubulin antibody</td>
			<td>Sigma</td>
			<td>T8660</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst</td>
			<td>Sigma</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">miRNeasy mini kit</td>
			<td>Qiagen&#160;</td>
			<td>217004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase free DNase&#160;</td>
			<td>Qiagen&#160;</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform</td>
			<td>Sigma</td>
			<td>C7559-5VL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">miScript II RT Kit&#160;</td>
			<td>Qiagen&#160;</td>
			<td>218160</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SYBR green PCR kit</td>
			<td>Qiagen&#160;</td>
			<td>218073</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Development &#38; Differentiation miScript miRNA PCR Array</td>
			<td>Qiagen/ SABiosciences</td>
			<td>MIHS-103Z</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lightcycler 480 white 96 plate</td>
			<td>Roche</td>
			<td>&#160;4729692001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lightcycler 480&#160; sealing foil</td>
			<td>Roche</td>
			<td>&#160;4729757001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vector NR4A3 miRNA 3&#39; UTR target clones&#160;</td>
			<td>GeneCopeia</td>
			<td>HmiT019538-MT0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vector SOX5 miRNA 3&#39; UTR target clones&#160;</td>
			<td>GeneCopeia</td>
			<td>HmiT017632-MT01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Allstars negative control siRNA AF 488 (Qiagen).</td>
			<td>Qiagen&#160;</td>
			<td>1027284</td>
			<td>MiRNA transfection control</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luc-Pair miR Luciferase Assay</td>
			<td>GeneCopeia</td>
			<td>LPFR-M010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lightcycler 480&#160;</td>
			<td style="width:71px;">Roche</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GloMax 96 microplate luminometer&#160;</td>
			<td style="width:71px;">Promega</td>
			<td style="width:119px;"></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

9.8K 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

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

在SH-SY5Y人神经母细胞瘤分化

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

科研

发育生物学

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

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 ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

人胎脑神经干细胞 Zika 病毒感染的免疫细胞化学分析

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

三维脊髓前庭和听觉感官器官的培养

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...