此项工作的财政支持ANR（新电国立德拉Recherche），AFM（协会法语反对LES肌病），PACA和IRME FEDER研究所DE RECHERCHE SUR LA Moelle épinière ET L&#39; Encéphale我们衷心感谢玛丽皮埃尔布兰查德（让罗氏研究所），她在时间的推移录制过程中的有效帮助。

1。收集大鼠嗅黏膜<ol><li>首先，准备3张35毫米的Petri菜与F12在一个干净的文化引擎盖DMEM /火腿培养基充满。 </li><li>任何"安乐死"的方法，必须由该机构的动物护理和使用委员会的事先批准，由合格的专业人员进行的。大鼠戊巴比妥钠或其他如氯胺酮/甲苯​​噻嗪注射麻醉形式进入深麻醉后，斩首和消除皮肤。应避免吸入麻醉药。麻醉充足，将评估前脚趾捏斩首。取出，用剪刀下颚和咬骨钳的帮助下，消除双方的面部肌肉。 </li><li>从门牙的背面，用咬骨钳的鼻腔，一次一侧的骨覆盖删除。嗅觉鼻甲进入人们的视线，橙色/棕色机关在位于鼻子后面。 </li><li>娇柔与镊子丢弃的鼻甲。使用26号针头，隔离嗅粘膜，隔躺在沿着三条线切割组织:弧的垂直板，筛板和鼻腔上限。 </li><li>对双方收集的活组织切片检查和转让他们在DMEM /火腿F12 -填充培养皿。这个过程不应该需要更长的时间超过10分钟从一开始安乐死。 </li><li>现在，以去除黏液，转移中充满培养皿两次活组织切片检查。 </li></ol> 2。在人类嗅黏膜收集<ol><li>此过程应进行一个耳鼻喉科（ENT）操刀，根据与当地有关的伦理委员会（S），每门诊应签署知情同意书。 </li><li>使用0 °或30 °刚性内窥镜（直径4 mm），两个鼻腔检查和评估假定存在的息肉或炎性病变。选择最佳的鼻腔，考虑到隔的偏差。 </li><li>使用棉花撒施，申请与肾上腺素，如利多卡因局部麻醉，10分钟。 </li><li>随着throughcut筛窦钳，收集了两个平方毫米活检在根中鼻甲内侧面或隔背内侧区。 </li><li>然后转移的嗅觉活检，是使用消毒针头，进入无菌2毫升1毫升的DMEM / HAM F12满管。提示管倒挂，以确保活检是在培养液中浸泡。 </li><li>插入管和一个冷藏集装箱运输研究实验室。在这个阶段，可用于活检本身比较分子的研究集中在特定的大脑疾病或产生干细胞的处理。 </li></ol> 3。从人类和大鼠胃黏膜嗅觉干细胞的分离<ol><li> F12的DMEM /火腿洗净的活组织切片检查。在充满1毫升dispase II解决方案（2.4 IU / ml）的一个培养皿培养活检1小时，在37 ° C。 </li><li>接下来，解剖显微镜下的衍射倒光，嗅觉上皮被删除从基础层，用微型抹刀固有。 </li><li>嗅上皮更薄，看起来比黑色的背景相比，固有层，这是条纹的半透明橙/棕。上皮细胞在一个白色背景，看起来灰色和固有层，褐色。 </li><li>纯化后，转移到培养皿用DMEM / HAM F12填补了固有层。 </li><li>如果从啮齿类动物的组织，然后切成小块，与两个25表针固有层。然后，充满了1毫升胶原酶IA 15毫升管件。 </li><li>在管内，用无菌塑料吸管，游离于组织。然后，管孵育10分钟，37 ° C。 </li><li>要终止的解离，轻轻摇动试管，加9毫升的无钙和镁无PBS离心5分钟，在200克。 </li><li> DMEM /火腿F12文化与10％小牛血清，抗生素和塑料培养皿板培养基重悬细胞沉淀。 </li><li>现在，如果该组织是人类，然后片成3至4件厚度从200至500微米不等的固有层。 </li><li>插入到它自己的直径2厘米的培养皿每个地带，并覆盖无菌直径1.3厘米的玻璃盖玻片的组织。 </li><li>然后，每个培养皿中添加500μL培养基（DMEM /火腿F12与10％小牛血清和抗生素的补充）。 </li><li>对于任何一个组织的类型，更新培养液中，每2至3天。 </li><li>五到七天后，干细胞就开始侵入了培养皿中，两个星期后，他们应融合。当汇合，通过转移的细胞培养瓶中。 </li></ol> 4。圈的形成和神经Differentiation的嗅觉干细胞<ol><li>要生成干细胞球，两个小时的烧瓶在37 °彗星与聚- L -赖氨酸。 （文:5 g/cm2）。 </li><li>板的细胞密度在每平方厘米1.6万细胞在治疗烧瓶。 </li><li>每隔两天，饲料0.2毫升每平方厘米的培养基（DMEM培养液/火腿F12补充胰岛素，转铁蛋白，硒（ITS - X，​​1％），表皮生长因子（50毫微克/毫升）和FGF2（细胞50毫微克/毫升））。 </li><li> 2到5天之后，收集漂浮的细胞球，要么重制版或游离移植细胞治疗的动物模型前。 </li><li>嗅觉干细胞分化成神经元样细胞，培养21天Neurobasal培养基中含有的B - 27，青霉素，链霉素，谷氨酰胺和谷氨酸。 </li><li>然后进行中的变化，每3天。神经元样细胞后，应该会出现两至三个星期。 </li></ol> 5。代表性的成果: 鼻人类植体outgrowing干细胞（图2A）除以迅速汇合，可一到两周内达成。一个关键的功能，干性，nestin表达，是评估（图2B）。聚- L -赖氨酸时用无血清培养与表皮生长因子（50毫微克/毫升）和FGF2（50毫微克/毫升）的培养基中生长，嗅觉干细胞引起的领域（图2c）。在含血清培养基中生长时，新镀领域引起GFAP的表达（〜50％），微管蛋白表达的细胞（约10-15％）和O4的表达细胞（约2-5％） 9（图2D - F）的。然而，球体源性细胞的命运可以被修改。例如，当一个Neurobasal文化与B27与谷氨酸，最鼻腔嗅觉干细胞向神经元样细胞表达的β- III微管蛋白（图2G）和微管相关蛋白2（图2H）培养基中生长。 <img src="/files/ftp_upload/2762/2762fig1.jpg" alt="图1"/> 图1实验的总体方案。切除大鼠或人体鼻腔嗅粘膜活检。外植体本身可用于比较，目的是查明在脑部疾病的生物标志物的分子生物学研究。隔离嗅觉干细胞，固有层和神经上皮细胞之间的相互作用与dispase二酶被破坏，45分钟后，上皮细胞是用微锅铲删除。啮齿动物的嗅觉干细胞进一步游离胶原酶IA固有层选择。对人体组织，件嗅觉固有层玻璃盖玻片培养干细胞的侵入，直到outgrowing以及整个。扩散后，用适当的培养基，嗅觉干细胞可以生成领域，或分化成神经元样细胞。嗅觉干细胞可用于I）修复脑部疾病或外伤或ii）确定中枢神经系统疾病的分子标记。插图与施维雅医药艺术的帮助。 <img src="/files/ftp_upload/2762/2762fig2.jpg" alt="图2"/> 图2，文化和人类鼻嗅干细胞的分化。越来越多的固有层植（一）人类干细胞迅速分裂，在含血清培养基培养。干细胞表达的干性标记巢（二）。当镀上聚- L -赖氨酸涂层的塑料，在无血清培养与EGF和FGF2培养基栽培，嗅觉干细胞生成领域（三）。球衍生的细胞，在含血清培养基镀时，会引起GFAP的表达细胞（〜50％），微管蛋白表达细胞（10-15％）和O4的表达细胞（约2-5％） 9（DF）的。在Neurobasal文化与B27与谷氨酸培养基生长时，它们分化成神经元样细胞表达的β- III微管蛋白（G）和微管相关蛋白2（H）。

<ol>
	<li>Feron, F., Perry, C., McGrath, J. J., Mackay-Sim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+techniques+for+biopsy+and+culture+of+human+olfactory+epithelial+neurons.">New techniques for biopsy and culture of human olfactory epithelial neurons.</a> Arch Otolaryngol Head Neck Surg. 124, 861-866 (1998).</li><li>Ronnett, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+biopsies+demonstrate+a+defect+in+neuronal+development+in+Rett's+syndrome.">Olfactory biopsies demonstrate a defect in neuronal development in Rett's syndrome.</a> Ann Neurol. 54, 206-218 (2003).</li><li>Feron, F., Perry, C., Hirning, M. H., McGrath, J., Mackay-Sim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+adhesion,+proliferation+and+death+in+neural+cultures+from+adults+with+schizophrenia.">Altered adhesion, proliferation and death in neural cultures from adults with schizophrenia.</a> Schizophr Res. 40, 211-218 (1999).</li><li>Matigian, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-specific,+neurosphere-derived+cells+as+models+for+brain+disorders.">Disease-specific, neurosphere-derived cells as models for brain disorders.</a> Dis Model Mech. 3, 11-12 (2010).</li><li>Arnold, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+epithelium+amyloid-beta+and+paired+helical+filament-tau+pathology+in+Alzheimer+disease.">Olfactory epithelium amyloid-beta and paired helical filament-tau pathology in Alzheimer disease.</a> Ann Neurol. 67, 462-469 (2010).</li><li>Boone, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+stem+cells,+a+new+cellular+model+for+studying+molecular+mechanisms+underlying+familial+dysautonomia.">Olfactory stem cells, a new cellular model for studying molecular mechanisms underlying familial dysautonomia.</a> PLoS One. , (2010).</li><li>McCurdy, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+alterations+in+biopsied+olfactory+neuroepithelium+in+schizophrenia+and+bipolar+I+disorder+using+cell+culture+and+gene+expression+analyses.">Cell cycle alterations in biopsied olfactory neuroepithelium in schizophrenia and bipolar I disorder using cell culture and gene expression analyses.</a> Schizophr Res. 82, 163-173 (2006).</li><li>Roisen, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+human+olfactory+stem+cells.">Adult human olfactory stem cells.</a> Brain Res. 890, 11-22 (2001).</li><li>Murrell, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+stem+cells+from+adult+olfactory+mucosa.">Multipotent stem cells from adult olfactory mucosa.</a> Dev Dyn. 233, 496-515 (2005).</li><li>Tome, M., Lindsay, S. L., Riddell, J. S., Barnett, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+nonepithelial+multipotent+cells+in+the+embryonic+olfactory+mucosa.">Identification of nonepithelial multipotent cells in the embryonic olfactory mucosa.</a> Stem Cells. 27, 2196-2208 (2009).</li><li>Delorme, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+nose+harbors+a+niche+of+olfactory+ectomesenchymal+stem+cells+displaying+neurogenic+and+osteogenic+properties.">The human nose harbors a niche of olfactory ectomesenchymal stem cells displaying neurogenic and osteogenic properties.</a> Stem Cells Dev. 19, 853-866 (2010).</li><li>Xiao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adult+olfactory+neural+progenitors+rescue+axotomized+rodent+rubrospinal+neurons+and+promote+functional+recovery.">Human adult olfactory neural progenitors rescue axotomized rodent rubrospinal neurons and promote functional recovery.</a> Exp Neurol. 194, 12-30 (2005).</li><li>Xiao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adult+olfactory+neural+progenitors+promote+axotomized+rubrospinal+tract+axonal+reinnervation+and+locomotor+recovery.">Human adult olfactory neural progenitors promote axotomized rubrospinal tract axonal reinnervation and locomotor recovery.</a> Neurobiol Dis. 26, 363-374 (2007).</li><li>Pandit, S. R., Sullivan, J. M., Egger, V., Borecki, A. A., Oleskevich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Effects+of+Adult+Human+Olfactory+Stem+Cells+on+Early-Onset+Sensorineural+Hearing+Loss.">Functional Effects of Adult Human Olfactory Stem Cells on Early-Onset Sensorineural Hearing Loss.</a> Stem Cells. , (2011).</li><li>Murrell, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+mucosa+is+a+potential+source+for+autologous+stem+cell+therapy+for+Parkinson's+disease.">Olfactory mucosa is a potential source for autologous stem cell therapy for Parkinson's disease.</a> Stem Cells. 26, 2183-2192 (2008).</li><li>Nivet, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftment+of+human+nasal+olfactory+stem+cells+restores+neuroplasticity+in+mice.">Engraftment of human nasal olfactory stem cells restores neuroplasticity in mice.</a> The Journal of Clinical Investigation. , (2011).</li><li>Bianco, J. I., Perry, C., Harkin, D. G., Mackay-Sim, A., Feron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin+3+promotes+purification+and+proliferation+of+olfactory+ensheathing+cells+from+human+nose.">Neurotrophin 3 promotes purification and proliferation of olfactory ensheathing cells from human nose.</a> Glia. 45, 111-123 (2004).</li><li>Mackay-Sim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+olfactory+ensheathing+cell+transplantation+in+human+paraplegia:+a+3-year+clinical+trial.">Autologous olfactory ensheathing cell transplantation in human paraplegia: a 3-year clinical trial.</a> Brain. 131, 2376-2386 (2008).</li></ol>

没有利益冲突的声明。

这里介绍的技术使鼠类和人类嗅粘膜一个有用的模型，为临床研究神经发育和神经退行性疾病以及修复病态或精神创伤的脑的工具的原因。该协议是相对简单的，并可以方便地进行由经验丰富的细胞生物学家。活检和文化技术的成功率是很高的。 关键步骤<ul><li>对于啮齿类动物的嗅黏膜集合，它是建议不超过10分钟之间安乐死和嗅觉组织的最后切除的时间限制。 </li><li>通常10分钟后取得的啮齿类动物嗅觉固有层的分离。孵化胶原酶IA。如果没有，我们建议收集上清固有层浮动的未解离位，其中，离心机，200克和机械分解replating细胞在新井之前，使用巴斯德吸管的浮动层后的一天。第一口井，包含已附着的细胞充满了新鲜培养液。 </li><li>为人类的固有层，这是比啮齿类动物组织更紧凑，我们不建议酶解。组织切片和每个外植体之间插入的塑料盘底部和玻璃盖玻片。成功的文化，包括外植体，其厚度范围从200至500微米。 </li></ul>可能的修改<ul><li>目前的协议，可以略作修改，以产生嗅觉神经元在体外。为此，神经上皮不拆除，整个嗅黏膜切片与McIlwain斩波器（厚度为200微米）。每个外植体，接种在培养皿中，部分干一小时，然后与含有FCS的培养基水化。在第一天电镀，上皮细胞和间质细胞生长出来的外植体。然后，神经祖细胞会迁移这个细胞层的顶部，并分化成神经元。 </li><li>嗅觉干细胞的分离纯化，可以实现用流式细胞仪检测。从表征纸11公布的名单，可检索的特异性表面标志。 </li><li>用于移植的实验，它可以使用老鼠的嗅觉干细胞GFP阳性菌株。这大鼠株（只Dawley，EGFP的PGK启动子驱动）ITERT（法国南特）。在人类嗅觉干细胞的绿色荧光蛋白基因插入，我们使用基于慢病毒感染的方法。 </li><li>本文的重点是嗅觉ecto间质干细胞。然而，另一种兴趣，嗅鞘细胞，细胞类型，可净化来自同一组织。我们描述了一个方法的收集和净化1,17，和我们人类的鼻鞘细胞用于I期/ 第 IIa 18例截瘫患者的临床试验。 </li><li> OE - MSCs的间质干细胞超家族的成员，有能力，适当的培养条件下，分化成脂肪细胞，骨细胞和肌细胞11。 </li></ul>未来的应用<ul><li>我们已经使用了人类嗅觉活检研究自闭症，躁郁症，家族dysautonomia，帕金森氏症，阿尔茨海默氏症和精神分裂症2-7患者的细胞和分子异常。从理论上说，所有的脑部疾病，可使用鼻腔活检或外围的嗅觉干细胞研究。 </li><li>老鼠和人类的鼻嗅干细胞已经嫁接失忆症，帕金森氏症，耳蜗损伤和脊髓损伤 12-16的动物模型。可以想象的是这些细胞移植到阿尔茨海默氏症，多发性硬化症，脑缺血的动物模型。 </li></ul>

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td colspan="3"> 收集大鼠嗅黏膜 </td></tr><tr><td> F12的DMEM /火腿</td><td> Invitrogen公司</td><td> 31331-028 </td></tr><tr><td>戊巴比妥钠</td><td></td><td></td></tr><tr><td>咬骨钳</td><td>福斯特</td><td></td></tr><tr><td> 26号针</td><td> Terumo公司</td><td> NN - 2613R </td></tr><tr><td>镊子</td><td></td><td></td></tr><tr><td colspan="3"> 在人类嗅黏膜收集 </td></tr><tr><td>刚性内窥镜</td><td>卡尔斯托斯或理查德沃尔夫医学</td><td></td></tr><tr><td>利多卡因</td><td></td><td></td></tr><tr><td>肾上腺素</td><td></td><td></td></tr><tr><td> Throughcut筛窦钳</td><td>卡尔斯托斯或理查德沃尔夫医学</td><td></td></tr><tr><td colspan="3"> 嗅觉干细胞的分离</td></tr><tr><td> Dispase二</td><td>罗氏公司</td><td> 10 295 825 001 </td></tr><tr><td>解剖显微镜</td><td></td><td></td></tr><tr><td>微型抹刀</td><td>福斯特</td><td></td></tr><tr><td>胶原酶IA </td><td> Sigma - Aldrich公司</td><td> C9891 </td></tr><tr><td> Ca-free/Mg-free PBS </td><td> Invitrogen公司</td><td> 14190-250 </td></tr><tr><td>胎牛血清</td><td> Invitrogen公司</td><td> 10270098 </td></tr><tr><td>玻璃盖玻片</td><td> Knittel格拉泽</td><td> 001/35 </td></tr><tr><td colspan="3"> 球体的形成和神经元分化 </td></tr><tr><td>聚- L -赖氨酸</td><td> Sigma - Aldrich公司</td><td> P - 1274 </td></tr><tr><td>胰岛素转硒（简称ITS） </td><td> Invitrogen公司</td><td> 51500056 </td></tr><tr><td>表皮生长因子</td><td> R＆D系统</td><td> 236 - EG </td></tr><tr><td> FGF2 </td><td> R＆D系统</td><td> 233 - FB </td></tr><tr><td> Neurobasal培养基</td><td> Invitrogen公司</td><td> 21103-049 </td></tr><tr><td> B - 27无血清的补充</td><td> Invitrogen公司</td><td> 17504-044 </td></tr><tr><td>青霉素/链霉素</td><td> Invitrogen公司</td><td> 15140122 </td></tr><tr><td>谷氨酰胺</td><td> Invitrogen公司</td><td> 25030024 </td></tr><tr><td>谷氨酸</td><td> Sigma - Aldrich公司</td><td></td></tr></tbody></table>

isolating nasal olfactory stem cells from rodents or humans

嗅粘膜，位于鼻腔中，负责检测气味。它也是唯一的神经组织，暴露在外部环境和方便在每一个生命的个体。因此，这个组织是唯一的，目的是查明病理大脑中的分子异常或隔离成体干细胞，细胞疗法的人。 在脑部疾病的分子异常往往是研究用神经组织样本收集的验尸报告。然而，这种材料具有很多局限性。相比之下，嗅粘膜易于访问和安全没有任何意义上的气味 1损失可以活检。因此，嗅黏膜提供一个"开放的窗口"，在成年的人，通过它可以研究发展（如自闭症， 精神分裂症 ）2-4或神经退行性疾病（如帕金森，老年痴呆症 ） 4,5。嗅粘膜，可用于分子要么比较研究4,6或 3,7对神经元的在体外实验。 嗅上皮也是一种神经组织，产生新的神经元每天，以取代那些污染损害的，细菌病毒感染。这个永久性的神经发生持续的祖细胞，但也干细胞在黏膜，即神经上皮和基础固有层8-10两个车厢居住。我们最近开发出一种方法来净化位于固有层中的成体干细胞，后证明，他们是密切相关的骨髓基质干细胞（BM - MSC），我们将它们命名为嗅觉ecto间质干细胞（OE海安）11。 有趣的是，当相比，BM - MSCs的，OE - MSCs的显示高增殖率，高架clonogenicity和分化成神经细胞的倾向。我们注意到这些特点优势，以执行专门推出新的候选基因，在精神分裂症和帕金森氏病4的研究。我们和其他OE - MSCs的12,13脊髓外伤，耳蜗损伤14后或在帕金森氏症的15或16失忆的动物模型，细胞疗法是有希望的候选人，也表明。 在这项研究中，我们目前在老鼠和人类的嗅黏膜活检的方法。上皮细胞和干细胞采集后，固有层酶分离纯化使用酶或一个非酶法。纯净嗅觉干细胞，然后可以要么增加大量库存在液态氮或诱导形成球体或成神经细胞分化。这些干细胞也可以用于比较组学（基因组，转录组，表观基因，蛋白质）的研究。

Watch this Scientific Journal Video about Isolating Nasal Olfactory Stem Cells from Rodents or Humans at JoVE.com

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

我们在这里描述的方法biopsying从大鼠和人鼻腔嗅粘膜。这些活组织切片检查或者确定脑部疾病的分子异常或隔离多能成可利用细胞移植的干细胞在脑外伤/疾病的动物模型，可用于

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

isolating-nasal-olfactory-stem-cells-from-rodents-or-humans

Research

JoVE Journal

Neuroscience

30.6K 次观看.  Aix Marseille University. 我们在这里描述的方法biopsying从大鼠和人鼻腔嗅粘膜。这些活组织切片检查或者确定脑部疾病的分子异常或隔离多能成可利用细胞移植的干细胞在脑外伤/疾病的动物模型，可用于

视频: Isolating Nasal Olfactory Stem Cells from Rodents or Humans

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

从蛾的嗅觉系统的电生理测量

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

科研

神经科学

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

扩展胚胎及成体神经干细胞<em&gt;在子宫内</em电穿孔或病毒立体定向注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

人类神经的聚集体cGMP的适用展开法干细胞和祖细胞来源从多能干细胞或胎儿脑组织

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

从人多能干细胞定向多巴胺能神经元分化

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

通过鼻腔活检获得联合激光捕获显微切割的嗅觉神经元:一种可能的方法来研究治疗反应的精神障碍

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...