Name: isolation of neural stem/progenitor cells from the periventricular region of the adult rat and human spinal cord
Uploaded: 2015-05-14T13:00:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord at JoVE.com

The authors would like to acknowledge support from Spinal Cord Injury Ontario, the Ontario-China Research and Innovation Fund, the Toronto General and Western Hospital Foundation, and Physicians&#8217; Services Incorporated Foundation. The authors thank Dr. Tasneem Zahir for expert advice in human spinal cord NSPC culture, and Drs. Cindi Morshead and Iris Kulbatski for their expert advice in rat spinal cord NSPC isolation.

所有动物的程序都是由大学健康网络的动物保健委员会，多伦多加拿大批准，按照既定的指南编制的加拿大委员会关于动物保护实验动物的护理和使用的政策。对于人类脊髓组织的收获，从大学健康网络研究伦理委员会，并从延龄-礼品生活基金会负责监督器官捐赠在加拿大安大略省获得批准。 1.准备解剖缓冲区和文化传媒<ol><li>对于大鼠脊髓的隔离，制备100ml夹层缓冲液（1×PBS + 0.6％葡萄糖+ 2％青霉素 - 链霉素）和冷藏。对于人类脊髓制备100ml清扫缓冲液（1×HBSS + 0.6％葡萄糖+ 2％青霉素 - 链霉素），并冷藏。 </li><li>制备100ml无血清培养基（SFM）和温暖的在37℃。为了制备SFM，加入2毫摩尔L-谷氨酰胺，100微克/立方米升青霉素 - 链霉素，2％B27，和10％的激素组合，以的Neurobasal-A培养基。为了制备激素混合，得到1:含0.6％葡萄糖1 DMEM / F12培养基，3毫碳酸氢钠 ，5mM的HEPES，25微克/毫升胰岛素，100微克/毫升脱辅基转铁蛋白，10μM腐胺，为30nM硒，和20nM的孕酮。 </li><li>制备EFH生长因子富含电镀媒体（至SFM加入20毫微克/毫升的EGF，20纳克/ ml的bFGF，和2微克/毫升肝素）和温暖的在37℃。保证人体EFH包含人类重组生长因子（见表材料）。 </li></ol> 2.收获和成年大鼠脑室周围脊髓组织的解剖<ol><li>消毒解剖仪器在乙醇和冲洗在无菌盐水中，或高压釜文书提前。给老鼠-根据自己的机构（6〜8周龄）一个致命注射戊巴比妥钠（1毫升65毫克/毫升）或麻醉剂（ 即 4％异氟醚）的过量批准的动物协议。 D乌斯的大鼠的背部，用70％的乙醇，拆下上背表面的皮肤具有大夹层剪刀。 </li><li>持垂直于背水面的剪刀，横向切割上面的后肢脊柱。使用更小剪刀纵向切割的背部肌肉覆盖脊柱的喙方向露出棘突。 </li><li>开始在暴露的尾端，插入骨钳或小钝骨切割器械extradurally成在脊髓和脊柱之间的空间内的椎管的外侧面。使小切入薄片（左骨弓和棘突的上椎骨的每一侧右），并仔细剥离薄片以暴露脊髓（ 图1A-D）。确保叶片的角度是浅而平行于线这样的底层脊髓没有损坏。 </li><li>继续在全椎板切除喙方向博览会Ë胸椎和脊髓型颈椎病。 </li><li>轻轻地用生硬的组织钳和使用microscissors脊柱切除脊髓仔细切断根系释放线。将切除脊髓中含有4℃无菌鼠清扫缓冲区（1.1如上所述）培养皿。冲洗在新鲜制备的4℃解剖缓冲器组织并用剪刀横向切割脊髓1厘米的段（ 图1E）。 </li><li>对于组织的每个部分，用一只手将与细镊子组织。另一只手，用microscissors小心取出覆脑膜，白质，和大多数的灰质与解剖范围的援助。或者，使用细镊子（杜蒙＃4），以剥离大部分的灰质和白质，而使脊髓它包括室管膜和少量周围灰质（ 图1F-H）的仅脑室区域。</li><li>池解剖脑室周围组织到含有冷解剖老鼠缓冲10cm的无菌培养皿。 </li></ol> 3.采收和成人脑室周围人的脊髓组织的解剖注意:人的脊髓组织从成年器官移植供体收获（供体年龄从2至60岁的年龄范围）的其他器官已被删除为器官移植后。生活计划的延龄，礼品获得同意切除组织以研究为目的，从病人家属，并通知我们收获的团队及时，使得我们已经能够在2小时以内主动脉阻断的收获线。男，女成年捐赠者的负面血清学和感染没有接受。 <ol><li>使用相同的前曝光收获组织在手术室无菌方式通过前路已解剖器官移除由器官运输兰特收割队。 </li><li>暴露通过回缩脊髓前柱与大肋撒播和剩余的组织和器官，包括大血管的大桨拉钩。 </li><li>使用安装有一个长叶片穿过椎间盘的脊柱的所需长度的喙和尾部端削减要切除胸骨锯。角锯约45°向内从椎体停止只是短暂椎管外侧部切开一个三角槽。 </li><li>整块除去具有弯曲的骨凿和槌注意避免通过硬脑膜切割的帮助下的所需的段的椎体的内侧方面。然后轻轻抬起硬脑膜覆盖脊髓用有齿镊，尖锐地切开线的喙和尾椎末端。用长剪刀和镊子进行双边横切个人根源。 </li><li>将脊髓的切除段在4°的温度不育缓冲液（1×HBSS + 0.6％葡萄糖+ 2％青霉素 - 链霉素）在大试管运输到组织培养室。 </li> <li> 注意:通常，3 -脊髓从上部胸椎6厘米段和/或中到低胸椎水平在无菌条件下在手术室除去尽快停止流通后，置于冷的无菌缓冲。循环停止并除去脊髓之间所经过的时间随收获靶器官捐赠所需要的时间，并已介于45分钟至2小时。如果心脏和肺部也被拆除，夹层可以采取远吻侧收获下颈椎脊髓的一部分也是如此。 </li><li>在3小时以内尽快解剖人类脊髓组织收获后，一般。取出硬脑膜和其他脑膜钳。在冲洗新鲜的4℃解剖缓冲脊髓组织横向切成1厘米的段。 </li><li>随着解剖范围的帮助下，用组织钳，细镊子和microscissors每个组织切除段的覆脑膜，白质，和大多数的灰质。凝聚解剖脑室周围组织，其包括室管膜和少量的周围灰质，到含有冷人类解剖缓冲器10cm的无菌培养皿。 </li></ol> 4.培养分离成年大鼠和人类脊髓NSPCs和<ol><li>执行以下步骤在无菌条件下在层流罩。 <ol><li>剁碎的解剖老鼠或人类脑室周围组织成1mm的3个与microscissors。 </li><li>酶分解使用的木瓜蛋白酶解离套件，在材料/试剂表中所示的蛋白水解酶的​​组织糜。注:试剂成分包括4瓶;瓶1:Earle氏平衡盐溶液（EBSS），小瓶2:木瓜蛋白酶containin克L-半胱氨酸和EDTA，小瓶3:脱氧核糖核酸酶I（DNA酶），小瓶4:卵类粘蛋白的蛋白酶抑制剂与牛血清白蛋白（BSA）。 注意:注意 :木瓜蛋白酶是一种巯基蛋白酶具有广泛的特异性蛋白底物。木瓜蛋白酶本文从番木瓜植物的胶乳衍生并降低最蛋白质底物相比更广泛胰蛋白酶。在离解过程中存在一些细胞损伤引起DNA释放到解离介质，这将增加粘度和使移液困难。因此，DNA酶被包括在细胞分离过程中消化的DNA，而不会损坏完整细胞。 Ovomucoids被糖蛋白用于抑制解离步骤后，木瓜蛋白酶活性的蛋白酶抑制剂。 </li></ol></li><li>在第一次使用，重组白蛋白卵类粘蛋白的抑制剂混合物（瓶4）32毫升EBSS（瓶1），然后储存在​​4℃和用于后续隔离。注意:这产生以10mg卵类粘蛋白抑制剂和10mg每ml白蛋白的有效浓度的溶液。 </li><li>添加EBSS（瓶1）5毫升，以木瓜蛋白酶小瓶（小瓶2），得到在20个单位的木瓜蛋白酶/ ml的溶液在1毫L-半胱氨酸用0.5mM EDTA中。检查时完全溶解的木瓜蛋白酶溶液出现清楚，否则将小瓶在37℃水浴中十分钟，直到木瓜蛋白酶完全溶解，以确保酶的全部活性。 </li><li>添加EBSS（1瓶）的500微升到DNA酶小瓶（瓶3），轻轻混匀DNA酶敏感剪切变性。加入250微升DNA酶溶液到含有木瓜蛋白酶小瓶，导致大约20单位/ ml的木瓜蛋白酶和0.005％DNA酶的终浓度。保存DNA酶小瓶的平衡以后使用。 </li><li>放置在木瓜蛋白酶溶液中的切碎大鼠或人的组织中制备的上面的步骤4.4。对于人体组织分离，分裂组织糜同样两者之间木瓜蛋白酶小瓶（约为0.2克/小瓶）。 </li><li>孵育在37℃下与上摇杆平台恒定搅拌以解离组织中活化的木瓜蛋​​白酶溶液。孵育大鼠组织45分钟到1小时，和人体组织为取决于组织糜，约0.2的量为1〜2小时 - 分别0.4克。 </li><li>用10毫升吸管研磨该混合物以分解任何残留的组织块以产生混浊的细胞悬浮液。转移的细胞悬浮液（不包括任何片未解离的组织）到无菌的15毫升锥形管中并离心，在300×g离心5分钟，在RT。 </li><li>重悬沉淀的细胞在含有介质卵类粘蛋白，木瓜蛋白酶抑制剂。 <ol><li>通过混合2.7毫升EBSS（瓶1）用300微升重组白蛋白的卵类粘蛋白的抑制剂溶液（瓶4）的在15毫升的锥形试管制备的卵类粘蛋白溶液。加入150微升的DNA酶溶液（瓶3）保存在上面的步骤4.4。 </li><li>丢弃上清该沉淀的细胞，并立即重新悬浮在稀释的DNA酶白蛋白抑制剂混合物的细胞沉淀。 </li></ol></li><li>分离完整细胞通过离心从细胞膜通过单一步骤不连续密度梯度。 <ol><li>通过加入5毫升白蛋白抑制剂溶液（瓶4）至15ml管中，并用5毫升吸管轻轻制备不连续密度梯度，缓慢层细胞悬浮液（其制备如步骤4.8所述）上的顶白蛋白 - 抑制剂溶液。 </li><li>离心，在70×g离心6分钟，在室温。 </li><li>注意:渐变的两个层之间的界面应清晰可见，尽管在此边界最小混合不会影响结果。膜碎片留在接口和脱落的细胞沉淀在试管底部。 </li></ol></li><li>对大鼠细胞，弃去上清，重悬在1ml大鼠EFH介质的细胞沉淀（预加热至37℃）。计数与血球活细胞密度和板的细胞进入一个T25培养烧瓶中，在10个细胞/微升EFH的密度。从大鼠组织细胞的典型产率为约2×10 6个细胞与约80％viability.If克隆培养物所需的，种子细胞在低于10个细胞/微升的密度。孵育烧瓶在37℃在5％CO 2，并允许培养物生长不受干扰1周，以避免球体的聚集。 </li><li>对于人细胞，弃去上清，重悬细胞沉淀在10ml人类EFH培养基（预加热至37℃）。通过一个40微米尼龙细胞过滤器过滤该细胞悬浮液，接着用30ml EFH以除去髓鞘和细胞膜碎片。离心机在300×g离心5分钟，重悬细胞沉淀在1ml EFH培养基。 </li><li>计数活细胞密度用血球计和在总体积板的人类细胞于6孔培养板，在10 5个细胞/孔的密度5毫升EFH /孔。从人体组织细胞的典型产量是约1×10 6个细胞用约70％的生存能力。预涂井粘附基材，如基质胶（参见下面的注释）。稀释在40微升基质胶的比率1毫升SFM。 </li><li> 注意:可替换地，也可以使用其他粘合基材如聚-D-赖氨酸/层粘连蛋白，纤连蛋白或胶原。 </li><li>孵育所述板在37℃在5％CO 2，并允许培养物生长静置1周。 </li></ol> 5.传代成年大鼠和人类脊髓NSPCs的<ol><li>鼠NSPCs会形成小神经球约70微米直径1日当周首次接种的范围内;通道大鼠NSPCs每周收集细胞悬浮液，在300×g离心离心5分钟，并机械捣碎细胞沉淀以解离的神经球。 </li><li>种子细胞分离到含有新鲜的大鼠中EFH T25烧瓶。或者，用50％的大鼠条件培养基传代。鼠NSPCs的典型产量在传代为约5×10 6个细胞，其与通道数呈指数地增加。 </li><li>为人类文化，到初始电镀后一周，更换一半的新鲜EFH培养基每周两次，以允许细胞粘附到基质上。一般地，1 - 2周后，一旦细胞已经附着到衬底，每周用新鲜EFH替换培养基的整个体积的两倍。 </li><li> 8周 - 4之间达到汇合前传代细胞。 <ol><li>传代培养的细胞通过从基板酶促分离细胞（见表材料）用2ml酶每孔在37℃下温育约10分钟。收集细胞悬浮液，离心分离，在300×g离心5分钟，并轻轻重悬细胞沉淀在1ml新鲜制备EFH培养基。 </li><li>计数活细胞用血细胞计数器和板将细胞于6孔培养板（预共如上在步骤4.12），在10 5个细胞的密度/孔在5毫升EFH的总体积/孔ated。人类NSPCs的典型产率在传代为约2×10 6个细胞，通常这将增加一倍与通路。通常，维持培养物用50％的空调EFH介质2 - 每周3次传代之间。 </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+expansion+of+the+adult+mouse+neural+stem+cells+using+the+neurosphere+assay.">Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay.</a> J Vis Exp. , (2010).</li><li>Reynolds, B. A., Rietze, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+and+neurospheres--re-evaluating+the+relationship.">Neural stem cells and neurospheres--re-evaluating the relationship.</a> Nat Methods. 2, 333-336 (2005).</li><li>Singec, I., 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=Defining+the+actual+sensitivity+and+specificity+of+the+neurosphere+assay+in+stem+cell+biology.">Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology.</a> Nat Methods. 3, 801-806 (2006).</li><li>Louis, S. A., Azari, H., Sharififar, S., Vedam-Mai, V., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural-colony+forming+cell+assay:+an+assay+to+discriminate+bona+fide+neural+stem+cells+from+neural+progenitor+cells.">Neural-colony forming cell assay: an assay to discriminate bona fide neural stem cells from neural progenitor cells.</a> J Vis Exp. , (2011).</li><li>Louis, S. 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=Enumeration+of+neural+stem+and+progenitor+cells+in+the+neural+colony-forming+cell+assay.">Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.</a> Stem Cells. 26, 988-996 (2008).</li><li>Conti, L., 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=Niche-independent+symmetrical+self-renewal+of+a+mammalian+tissue+stem+cell.">Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.</a> PLoS Biol. 3, e10 (2005).</li><li>Pollard, S. M., Conti, L., Sun, Y., Goffredo, D., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherent+neural+stem+(NS)+cells+from+fetal+and+adult+forebrain.">Adherent neural stem (NS) cells from fetal and adult forebrain.</a> Cereb Cortex. 16, i112-i120 (2006).</li></ol>

The authors have nothing to disclose.

在大鼠脊髓组织护理的解剖应采取在执行椎板切除不要损坏脊髓。这是比较容易分离脑室周围组织时的脊髓节段都完好无损。的组织片段应全部浸入夹层缓冲器和上覆脑膜和白质可以被切掉为的纵向条带microscissors。另外，精细组织钳可以用来剥白质离开。 为隔离程序NSPCs，我们使用了木瓜蛋白酶EDTA解离法结合机械研磨以解离的大鼠和人的脊髓组织。相比于其他蛋白酶，木瓜?...

NSPCs致力于能够自我更新和体外易于扩展神经系多能细胞。我们将这些细胞作为神经干/祖细胞的混合群，因为它们显示自我更新的多能干细胞和更限制性祖细胞的性质。 NSPCs被发现在胎儿和成人脑和脊髓1,2-两者。在成人中，NSPCs通常静态和驻留特定龛包括脑室下区衬侧脑室2-4，以及围绕脊髓5,6的中央管室周区域内。 通常情况下，NSPCs如在无血清培养基中自由浮动的神经球或以贴壁单层补充EGF和bFGF，这对于干细胞/祖细胞群选择分裂素中培养。最初由雷诺和Weiss 2开发的神经球测定中，是最常用的培养和扩大的神经干细胞。 NSPCs显示多能的时候都接种于生长因子 - 自由含培养基的血清，分化成神经元，星形胶质细胞和少突胶质。多能，自我更新NSPCs可以分离和培养从成年啮齿动物脊髓当培养组织包括中央管6,7的区域。使用从相对于其他区域产生的细胞在成人脊髓生成NSPCs的潜在的优点是，这些组织特异性细胞最相似的细胞在脊髓被丢失或损坏的以下的损伤或疾病。 以前的工作表明，从成人脊髓衍生神经球可能不会传播长期或传代来产生足够数量的细胞8,9。但是，在培养条件的修改中，我们报道了成人脊髓源性NSPCs膨胀和移植，这表明自我更新和多能NSPCs可以从器官移植供体10的成人脊髓中分离。首先，大多数的白质解剖和这些培养细胞中除去生长因子富含介质粘附基板上选择用于增殖成人脊髓NSPCs。在这个协议中，我们描述了从成年大鼠和人类器官移植的供体，脑室周围组织的解剖，并NSPCs的分离，培养和扩大了脊髓的收获。

<table><tbody><tr><td>1x PBS</td><td>Life Technologies</td><td>#10010023</td><td>Dissection buffer</td></tr><tr><td>1x HBSS</td><td>Life Technologies</td><td>#14175095</td><td>Dissection buffer</td></tr><tr><td>D-glucose</td><td>Sigma</td><td># G-6152</td><td>Prepare 30% glucose stock solution for dissection buffer and hormone mix</td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>#15140-148</td><td>Dissection buffer and culture medium</td></tr><tr><td>Neurobasal-A&amp;nbsp;</td><td>Life Technologies</td><td>#10888-022</td><td>Culture medium</td></tr><tr><td>L-glutamine, 200 mM</td><td>Life Technologies</td><td>#25030-081</td><td>Culture medium</td></tr><tr><td>B27</td><td>Life Technologies</td><td>#12587010</td><td>Culture medium</td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>#11885084</td><td>Hormone mix</td></tr><tr><td>F12</td><td>Life Technologies</td><td>#21700-075</td><td>Hormone mix</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td># S-5761</td><td>Prepare 7.5% NaHCO&lt;sub&gt;3&lt;/sub&gt; stock solution for hormone mix</td></tr><tr><td>HEPES</td><td>Sigma</td><td>#H9136</td><td>Prepare 1M HEPES stock solution for hormone mix</td></tr><tr><td>Insulin</td><td>Sigma</td><td>#I-5500</td><td>Hormone mix</td></tr><tr><td>Apo-transferrin</td><td>Sigma</td><td>#T-2252</td><td>Hormone mix</td></tr><tr><td>Putrescine</td><td>Sigma</td><td># P7505</td><td>Hormone mix</td></tr><tr><td>Selenium</td><td>Sigma</td><td>#S-9133</td><td>Hormone mix</td></tr><tr><td>Progesterone</td><td>Sigma</td><td>#P-6149</td><td>Hormone mix</td></tr><tr><td>EGF, mouse</td><td>Sigma</td><td>#E4127</td><td>Prepare 100 &amp;mu;g/ml stocks in B27 and aliquot; EFH medium</td></tr><tr><td>EGF, human recombinant</td><td>Sigma</td><td>#E9644</td><td>Prepare 100 &amp;mu;g/ml stocks in B27 and aliquot; EFH medium</td></tr><tr><td>bFGF, human recombinant</td><td>Sigma</td><td>#F0291</td><td>Prepare 100 &amp;mu;g/ml stocks in B27 and aliquot; EFH medium&amp;nbsp;</td></tr><tr><td>Heparin, 10,000 U</td><td>Sigma</td><td>#H3149</td><td>Prepare 27.3 mg/ml stocks in hormone mix and aliquot; EFH medium</td></tr><tr><td>Papain dissociation kit</td><td>Worthington Biochemicals</td><td>#LK003150</td><td>Contains EBSS, papain, DNase, ovomucoid protease inhibitor with BSA</td></tr><tr><td>Sodium Pentobarbital</td><td>Bimeda &amp;ndash; MTC Animal Health Inc</td><td>DIN 00141704</td><td></td></tr><tr><td>Tissue Forceps: Addisons</td><td>Fine Science Tools</td><td>#11006-12&amp;nbsp;</td><td>Serrated standard tip; micro-tip also available</td></tr><tr><td>Fine Forceps: Dumont #4</td><td>Fine Science Tools</td><td>#11241-30</td><td></td></tr><tr><td>Microscissors</td><td>Fine Science Tools</td><td>#15024-10</td><td>Round-handled Vannas</td></tr><tr><td>Rongeurs</td><td>Bausch &amp;amp; Lomb</td><td>N1430</td><td></td></tr><tr><td>10 mm Petri dishes&amp;nbsp;</td><td>Nunc</td><td>1501</td><td></td></tr><tr><td>T25 Culture flasks</td><td>Nunc</td><td>156367</td><td></td></tr><tr><td>40 &amp;mu;m nylon cell strainer</td><td>VWR</td><td>CA21008-949</td><td></td></tr><tr><td>6 well plates</td><td>Nunc</td><td>CA73520-906</td><td></td></tr><tr><td>Matrigel, growth factor reduced (100x)</td><td>Corning</td><td>354230</td><td>Thaw according to directions and freeze aliquots; use diluted at a ratio of 40 &amp;mu;l Matrigel: 1 ml SFM</td></tr></tbody></table>

isolation of neural stem/progenitor cells from the periventricular region of the adult rat and human spinal cord

成年鼠和人的脊髓神经干/祖在生长因子富集培养基中培养细胞（NSPCs）允许，自我更新的和具有发泡性的神经干细胞的多能增殖。在血清条件下，这些多能NSPCs将分化，产生神经元，星形胶质细胞和少突胶质。所收获的组织被酶促解离以木瓜蛋白酶-EDTA溶液，然后机械地分离，并通过一个不连续密度梯度分离，得到是镀在补充有表皮生长因子（EGF）的Neurobasal培养基，碱性成纤维细胞生长因子的单个细胞悬浮液（bFGF的），和肝素。成年大鼠脊髓NSPCs是生长的贴壁培养的自由浮动的神经球和成人脊髓NSPCs培养。在这些条件下，成人脊髓NSPCs增殖，前体细胞的明确标记，并在通道可连续展开。这些细胞可以bê研究在体外响应于各种刺激，而外部因素可以被用来促进谱系限制检查神经干细胞的分化。多能NSPCs或其后代也可以移植到各种动物模型来评估再生修复。

在EFH培养基中生长在悬浮培养成年大鼠脊髓细胞将配成1日当周首次电镀内小神经球（未分化细胞的克隆）。在原代培养物，最镀细胞就会死亡和生长因子响应的干细胞会增殖，并选择用于EFH介质。由通道3，将有大量的自由浮动的神经球大约100微米的直径（ 图2A）。神经球是圆的和相高亮，并且在高的放大倍率，纤毛状microspikes被看见从细胞上球体它们是神经球不像细胞碎片（ ?...

Watch this Scientific Journal Video about Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord at JoVE.com

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

isolation-neural-stemprogenitor-cells-from-periventricular-region

Research

JoVE Journal

Developmental Biology

12.2K 次观看.  Toronto Western Research Institute and Krembil Neuroscience Center. The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors. 

视频: Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

科研

神经科学

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

骨髓衍生祖细胞的低氧预处理作为成熟雪旺细胞产生的来源

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

发育生物学

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

促进存活神经干细胞与纤维蛋白和生长因子鸡尾酒和分化的严重脊髓损伤后

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

从成年鼠脑的常规和非常规地区日益增长的神经干细胞

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

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 ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

小鼠脊髓损伤模型中内源性神经干细胞活化的干细胞检测

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

胚胎小鼠神经干细胞的分离与培养

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

用于神经干细胞和少突胶质细胞祖细胞分离的大鼠"脑挤奶"

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.
Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail &#34;brain milking&#34;, a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are &#34;released&#34; from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a &#34;release cocktail&#34;. The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor-2. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.
Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well&#160; by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

作者聚焦:使用新型脑挤奶方案从活体动物中收集神经干细胞和祖细胞 

A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

从活大鼠中分离神经干细胞和少突胶质细胞祖细胞的"脑挤奶"方法

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

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此视频中的章节

成年鼠神经干细胞的分离和扩大使用神经干含量

骨髓衍生祖细胞的低氧预处理作为成熟雪旺细胞产生的来源

促进存活神经干细胞与纤维蛋白和生长因子鸡尾酒和分化的严重脊髓损伤后

从成年鼠脑的常规和非常规地区日益增长的神经干细胞

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

分离和成人神经干细胞的培养从鼠标Subcallosal区

小鼠脊髓损伤模型中内源性神经干细胞活化的干细胞检测

胚胎小鼠神经干细胞的分离与培养

从活大鼠中分离神经干细胞和少突胶质细胞祖细胞的"脑挤奶"方法

从活大鼠中分离神经干细胞和少突胶质细胞祖细胞的"脑挤奶"方法