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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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 border="0" cellpadding="0" cellspacing="0">
	<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&#160;</td>
			<td>Life Technologies</td>
			<td>#10888-022</td>
			<td>Culture medium</td>
		</tr>
		<tr>
			<td>L-glutamine, 200mM</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>NaHCO3</td>
			<td>Sigma</td>
			<td># S-5761</td>
			<td>Prepare 7.5% NaHCO3 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 &#956;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 &#956;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 &#956;g/ml stocks in B27 and aliquot; EFH medium&#160;</td>
		</tr>
		<tr>
			<td>Heparin, 10000U</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 &#8211; 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&#160;</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 &#38; Lomb</td>
			<td>N1430</td>
			<td></td>
		</tr>
		<tr>
			<td>10mm Petri dishes&#160;</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 &#956;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 &#956;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

12.1K 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 Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

科研

发育生物学

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

分离和成人犬海马神经前体扩张

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

脐带血诱导的多能干细胞的软骨形成颗粒形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

人脐血内皮祖细胞的分离

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

人脑神经前体细胞神经发育表型的快速检测 (npc)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

人成体和胎心标本中原发性心外膜细胞的分离培养

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

早产儿和足月婴儿脐带源性间充质干细胞的分离与鉴定

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.