这项工作是由（部分）由亥姆霍兹联盟ICEMED - 成像和固化环境代谢性疾病，通过倡议和网络基金亥姆霍兹联合会，来自否则Kroener-费森尤斯基金会的资助，并从德意志研究联合会的资助（ SFB 655:细胞进入组织）。

这项工作坚持Helsinski的宣言和动物ARVO声明。动物根据动物设施在德累斯顿大学的指示用于组织收集和所有相关的方法随访。动物根据实验动物的使用和保养德国联邦指导方针处理和安置，并研究批准Landesdirektion德累斯顿。请与您所在机构的有关适当的安乐死方法的兽医政策（IACUC或其它板）进行咨询。 具体的股票和试剂的工作浓度可在随同此协议的试剂表上找到。 1。文化这道菜准备<ol><li>外套菜肴为75微克足够体积/毫升多聚-L-鸟氨酸（ 即 2毫升/孔的6孔平板）过夜的细胞培养孵化器之前预定夹层2天。 </li><li&gt;吸聚-L-鸟氨酸翌日和5分钟，洗净各孔2×用3ml PBS中。 </li><li>取出最后一次洗涤，再加入纤维连接蛋白（1:250稀释，4微克/毫升的PBS）的板，然后将菜在孵化过夜。 </li><li>吸出纤连蛋白上剥离的日子和洗碗2倍的PBS。返回仍然包含最终PBS洗涤的孵化器，直到分离的组织准备培养板。在那个时候，除去PBS之前电镀组织。 </li><li>涂有聚-L-ornithinein的培养箱中培养达3周存储板。板涂覆有纤连蛋白可以存储多达1周。纤连蛋白涂层可以在少至2小时，如果紧急工作要做。纤维连接蛋白易受变性，不要搅动溶液或让菜干。 </li></ol> 2。分割/电镀<ol><li>该协议适用于使用3成年大鼠（3-6月龄）的。 </li><li> CHIL升大脑前安乐死的大鼠切片上的冰块。 </li><li>地方，包括5毫升加bFGF对冰氮气介质15毫升猎鹰管。 </li><li>根据您所在机构的兽医安乐死政策的影响。 </li><li>通过做一个切口向下头部的中线从颅骨中取出脑。使用镊子剥离骨头允许仔细提取的大脑。 </li><li>将脑入含冰冷的PBS 10cm的培养皿中。这将消除任何残余的头发和血液。 （ 图1A） </li><li>将脑入冷冻切片块。 （ 图1B） </li><li>插入一个新的干净的刀片成块如图示于图1C。 </li><li>插入第二个干净的刀片3毫米尾椎到第一如图1D。 </li><li>小心地从块举起刀片，同时与他们感兴趣的部分。 </li><li>浮动秒通过浸入在PBS TION关闭刀片。 （ 图1E） </li><li>从感兴趣的区域收获组织使用＃5钳子（在这个例子中，我们所用的前连合，（前）ACA， 图1F），并收集到15毫升锥形管中含有1毫升含有bFGF的N2培养基中。 </li><li>将管插入一个层流细胞培养罩，持续的协议的其余部分的无菌条件下进行。 </li><li>使用连接到P1000移液器1毫升移液管尖端机械离解的组织。移液器上下多次（约20倍，以每秒1移液循环的速率）的同时按下移液管尖的对锥形管（ 图1G 1-3）的底部开口。停止研磨时，溶液变得均匀。 </li><li>让溶液静置2分钟，以允许任何较大nondissociated组织沉降至底部。 （ 图1G 4） </li><li>收集均质出口导向单位上清液，并放入锥形管中含有8.5毫升N2培养基中加20毫微克/毫升的bFGF，500纳克/毫升的Dll4，500纳克/毫升的Ang2和200 nMJAK抑制剂。 </li><li>板状成一个6孔板的3个孔用3毫升稀释后的细胞溶液/孔。 </li><li>将板放入湿润的细胞培养箱中，在37℃，5％CO 2，5％O 2。 </li></ol> 3。日常护理<ol><li>更换介质上含有bFGF的，Ang2的，Dll4的，和JAK镀抑制剂后24小时培养皿用N2中。 </li><li>添加bFGF的，Dll4的，Ang2的，并且在第二天JAK抑制剂的大丸剂。 </li><li>继续这种交替的媒介变化和因素增加，共10-14天的过程。 </li></ol> 4。诱导分化<ol><li>撤回的bFGF，Dll4的，Ang2的，和JAK抑制剂含有介质，并将其与不包含任何其它附加因素N2培养基更换。 </li><li>每隔一天改变介质。 </li><li> 10天后固定细胞和免疫细胞化学进行。 </li></ol> 5。免疫细胞化学检测<ol><li>吸出培养基并修复细胞培养板用2毫升4％的多聚甲醛20分钟。 </li><li>洗涤2×3毫升的PBS，每次5分钟。 </li><li>吸出PBS，然后透化，并加入2毫升的PBS中含有5％正常驴血清（NDS）和0.1％的Triton X-100的20分钟，阻断细胞。 </li><li>制备含有5％NDS的第一抗体混合的PBS中。抗巢蛋白抗体可用于识别的SC，而TUJ1（III类β-微管蛋白），GFAP，和CNPase抗体可一起用于分化标记三重染色。 </li><li>吸去封闭溶液并加入1.5毫升该初级抗体混合物的各孔中。在室温下孵育90分钟。 </li><li>除去第一抗体，并用3毫升的PBS洗2倍，1倍和用3ml PBS中含有5％NDS，每次5分钟。 </li><li> PreparË含5％的NDS在PBS的二级抗体。 </li><li>吸出最后一次洗涤并加入1.5毫升二抗溶液，以每孔中。孵育在黑暗中40分钟，在室温下进行。 </li><li>去除二级抗体溶液并加入2毫升新鲜制备的DAPI染色（在PBS中的500纳克/毫升）。孵育3分钟。 </li><li>吸出的DAPI溶液然后洗3次用3ml PBS中，每次5分钟。细胞现在可以可视化用荧光显微镜。 </li></ol>

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我们什么都没有透露。

许多成功的分离，扩增，并从成年脑的神经干细胞分化的关键步骤是在共同与标准的组织培养技术共享。要记住的目标是，从神经干细胞在成人脑中分离应保持尽可能多的其在体内的特性越好（ 图2J）。往往需要必要的谨慎和适当的速度之间的平衡被击中。护理与从头骨大脑的隔离会使该地区的利益的识别显著更容易。取出组织尽快下列动物安乐死，并在收获和洗涤保持头脑?...

神经生物学的中心法则，由大脑细胞构筑一个多世纪前由拉蒙·卡哈尔华的根本看法放下，认为神经发生是不可能的青春期鉴于在中枢神经系统1中找到的神经网络的复杂性之后。尽管奥特曼在20世纪60年代的工作，后来卡普兰，这表明3 H-胸苷可以在成熟神经元，表明实际上正在在成人大脑的不同区域所产生的神经元被发现，教条继续持有2，3。证据继续与诺特博姆的研究描述了季节的变化出现在鸣禽脑中4神经元的数目安装。但直到1999年，当在与联想学习任务，在大鼠的表现，以及对Kornack和Rakic ​​demonstra观察海马神经元增加的生成古尔德等人发表的作品婷继续在神经发生，成年猕猴是一个刚性较差，更多的塑料大脑的概念，确认5 6。 寻求对这些从头生成的神经元细胞源导致干细胞（SCS）的驻留在大脑区域的离散人口的发现被称为龛7。脑室下区和海马的子颗粒区被认为是两个主要的神经区域8,9。细胞从这些位置中分离显示的胚胎或胎儿衍生的SC经典特性，自我更新和分化潜能。在神经干细胞（NS​​Cs）的情况下，它们可以分化成神经元，星形胶质细胞和少突胶质。另外，这些的SCs染色阳性胎儿NSC的标记，如中间丝蛋白巢蛋白10。更近期的工作着重指出的SCs可能不限于这些吨窝区，并且实际上本地化整个大脑的紧密脉管11相关联的小区的主要静态人口。 该许旺动员响应于损伤的观察暗示了能够利用这些细胞用于再生目的，在从神经退行性疾病和中风12,13的恢复，以帮助的可能性。这是没有什么不同的角色间充质干细胞（MSCs）在结缔组织愈合玩，而被发现的血管周围细胞有潜力成为成骨细胞，软骨细胞，脂肪细胞14。然而，神经干细胞，不能以相同的方式收集来自骨髓的MSC通过常规抽吸和密度梯度离心技术和自体基于细胞的疗法随后利用。其结果是，细胞的其他来源，如使用从EMB来自胎儿神经干细胞或神经元前体的ryonic干细胞已被广泛地探讨了在动物疾病和损伤模型不同程度的成功15。利用体细胞来源的诱导多能干细胞的技术提供了另一种潜在的途径用于生产治疗上有用的基于细胞的疗法的广泛应用，克服了有限的可用的和关于使用胚胎干细胞和胚胎组织16的伦理问题。然而，这些研究结果的临床平移已被证明是一项困难的任务，如在治疗各种神经疾病的SC为基础的治疗的斗争接近17,18，以及一个曲折的路径，以调节间隙。作为一个替代方法，具体介绍药物治疗可以调节NSC数字和促进复苏，帕金森氏症和中风19的模型。无论策略可能，了解如何有效地处理这些细胞需要一个可访问的体外系统。 神经干细胞的培养物可进行无论是作为骨料培养，也称为神经球，或作为单层8,20。这两种方法已被广泛使用，允许建立确定的培养条件下， 即使用表皮生长因子（EGF）或碱性成纤维细胞生长因子（bFGF）作为有丝分裂原来源，即提供多能前体的扩大。而神经球培养物可能更适合用于研究的分离的细胞类型的无性繁殖能力，该系统已被证明扩张21中产生的细胞的混合群。此外，神经球的闭合结构使药物操纵细胞的不切实际，而这些因素可能产生影响的解释可能会由于自身的神经球内建立微环境羞愧。单层培养物，在另一方面，可在小分子文库高通量筛选聘用，提供了强大的工具来探索规范SC的生长和分化，并打开机会发现新的化合物，专门针对这一细胞群的信号转导机制。 作为结果，可再现地产生由大脑中的兴趣区域不同成人神经干细胞培养物的能力，可在研究应用的广谱性使用，从中枢神经系统（CNS）的发育研究来探索新的再生医疗方法。这里介绍的协议演示了如何解剖和评估CNS种姓从成年啮齿类动物脑中分离的分化潜能。

<table border="0" cellpadding="0" cellspacing="0" width="596">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">6-well tissue culture dishes</td>
			<td style="width:95px;">BD Biosciences</td>
			<td style="width:119px;">353934</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="190" rowspan="1" style="height:190px;width:216px;">Poly-L-ornithine</td>
			<td rowspan="1" style="width:95px;">Sigma-Aldrich</td>
			<td rowspan="1" style="width:119px;">P-3655</td>
			<td style="width:167px;">
			5 mg/ml stock solution prepared in double distilled water (Stable for several months at -20 &#176;C).
			Working concentration 0.5 mg/ml in water (Stable for 1 month at 4 &#176;C).
			</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fibronectin</td>
			<td style="width:95px;">R&#38;D Systems</td>
			<td style="width:119px;">1030-Fn</td>
			<td style="width:167px;">Do not agitate stock solution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Filtration Apparatus</td>
			<td style="width:95px;">Corning Life Sciences</td>
			<td style="width:119px;">430769</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">DMEM/F-12</td>
			<td style="width:95px;">Mediatech</td>
			<td style="width:119px;">10-090-CV</td>
			<td style="width:167px;">See note below for complete N2 media preparation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Apo-transferrin</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">T-2036</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Insulin</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">I-0516</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:216px;">Putrescine</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-5780</td>
			<td style="width:167px;">1 M stock solution in ddH2O (Stable at 20 &#176;C for 6 months)</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:216px;">Sodium Selenite</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">S-5261</td>
			<td style="width:167px;">500 &#181;M stock solution in ddH2O (Stable at -20 &#176;C for 6 months)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Progesterone</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-8783</td>
			<td style="width:167px;">100 &#181;M stock solution in ethanol (Stable at -20&#176;C for 6 months)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:95px;">Invitrogen</td>
			<td style="width:119px;">15140-122</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:95px;">Mediatech</td>
			<td style="width:119px;">21-040-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Basic Fibroblast Growth Factor (bFGF)</td>
			<td style="width:95px;">R&#38;D Systems</td>
			<td style="width:119px;">233-FB</td>
			<td style="width:167px;">Working concentration 20 ng/ml&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Delta4 (Dll4)</td>
			<td style="width:95px;">R&#38;D Systems</td>
			<td style="width:119px;">1389-D4</td>
			<td style="width:167px;">Working concentration&#160; 500 ng/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Angiopoetin 2 (Ang2)</td>
			<td style="width:95px;">R&#38;D Systems</td>
			<td style="width:119px;">623-AN</td>
			<td style="width:167px;">Working concentration&#160; 500 ng/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">JAK Inhibitor</td>
			<td style="width:95px;">Calbiochem</td>
			<td style="width:119px;">420099</td>
			<td style="width:167px;">Working concentration 200 nM&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">A-2058</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">15- and 50-ml Conical tubes</td>
			<td style="width:95px;">Corning Life Sciences</td>
			<td style="width:119px;">430053, 430829</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="243">
			<td colspan="4" height="243" style="height:243px;width:596px;">Other necessary items include: General dissection instruments, including razor blade and forceps, Adult rat (3-6 months old; Sprague-Dawley or Long Evans), CO2 intoxication chamber, Laminar flow hood for cell culture and incubator Incubator (humidified, 37 &#176;C, 5% CO2, 5% O2). Note: For complete N2 media preparation, to one bottle of DMEM/F-12 (500 ml) add 0.05 g of apotransferin, 0.0125 g of insulin (freshly predissolved in 1 ml of 10 mM NaOH), 50 &#956;l of putrescine, 30 &#956;l of sodium selenite, 100 &#956;l of progesterone stocks, and 5 ml of penicillin/streptomycin solution.&#160; Adjust pH to 7.2, if needed. Filter-sterilize and store at 4 &#176;C for up to 3 weeks and protect from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td>[header]</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td>Immunofluorescence Reagents Table</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Paraformaldehyde</td>
			<td style="width:95px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15719</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Normal Donkey Serum (NDS)</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">D-9663</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Triton X-100</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">T-8787</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">4,6-Diamidino-2-phenylindole (DAPI)</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">D-8417</td>
			<td style="width:167px;">5 mg/ml stock solution in methanol</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Primary Antibody Table</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">Nestin</td>
			<td rowspan="1" style="width:95px;">Chemicon</td>
			<td rowspan="1" style="width:119px;">MAB353</td>
			<td style="width:167px;">
			Dilution Factor: 1:400
			Species: Mouse IgG1
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">Hes3</td>
			<td rowspan="1" style="width:95px;">Santa Cruz</td>
			<td rowspan="1" style="width:119px;">sc-25393</td>
			<td style="width:167px;">
			Dilution Factor: 1:100
			Species: Rabbit IgG
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">Sox2</td>
			<td rowspan="1" style="width:95px;">R&#38;D Systems</td>
			<td rowspan="1" style="width:119px;">MAB2018&#160;</td>
			<td style="width:167px;">
			Dilution Factor: 1:100
			Species: Mouse IgG2a
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">CNPase</td>
			<td rowspan="1" style="width:95px;">Chemicon</td>
			<td rowspan="1" style="width:119px;">MAB326</td>
			<td style="width:167px;">
			Dilution Factor: 1:200
			Species: Mouse IgG1
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">&#946;-tubulin III (TUJ1)</td>
			<td rowspan="1" style="width:95px;">R&#38;D Systems</td>
			<td rowspan="1" style="width:119px;">MAB1195</td>
			<td style="width:167px;">
			Dilution Factor: 1:500
			Species: Mouse IgG2a
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">Glial Fibrillary Acidic Protein (GFAP)</td>
			<td rowspan="1" style="width:95px;">Dako North America</td>
			<td rowspan="1" style="width:119px;">Z0334</td>
			<td style="width:167px;">
			Dilution Factor 1:500
			Species: Rabbit
			</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Secondary Antibody Table</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="1" style="height:64px;width:216px;">Alexa&#160;&#160; 568</td>
			<td rowspan="1" style="width:95px;">Invitrogen</td>
			<td rowspan="1" style="width:119px;">A-21124</td>
			<td style="width:167px;">
			Dilution Factor: 1:200
			Species: Goat anti Mouse IgG1
			</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="1" style="height:64px;width:216px;">Alexa 488</td>
			<td rowspan="1" style="width:95px;">Invitrogen</td>
			<td rowspan="1" style="width:119px;">A-21131</td>
			<td style="width:167px;">
			Dilution Factor: 1:200
			Species: Goat anti Mouse IgG2a
			</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="1" style="height:64px;width:216px;">Cy5</td>
			<td rowspan="1" style="width:95px;">Jackson ImmunoResearch</td>
			<td rowspan="1" style="width:119px;">59883</td>
			<td style="width:167px;">
			Dilution Factor: 1:200
			Species: Goat anti Rabbit
			</td>
		</tr>
	</tbody>
</table>

growing neural stem cells from conventional and nonconventional regions of the adult rodent brain

最近的工作表明，中枢神经系统（CNS）的再生和肿瘤发生涉及干细胞群（SCS）的成年大脑内的居民。然而，这些机制通常静止期细胞采用，以确保神经网络的正常运作，以及他们在受伤的神经变性过程和缓解恢复的作用是一知半解。这些细胞存在于称为"死角"，提供了一个涉及从两个血管系统和免疫系统的调节信号，维持环境的区域。隔离，维护和分化而排除未知因素确定的培养条件下的CNS的SCs的，使得它们通过药理学或遗传手段访问处理，从而提供了洞察其在体内的行为。在这里，我们提供从成人大脑的不同区域产生中枢神经系统的种姓文化上的方法的详细信息和方法来评估其Ðifferentiation潜力成神经元，星形胶质细胞，并在体外少突胶质细胞。这种技术产生一个均匀的细胞群体作为可以被可视化研究个别SCs和它们的后代的单层培养。此外，它可以在不同的动物模型系统和临床样品被应用，以前被用来预测在受损成年神经系统的再生反应。

识别感兴趣区域，从中收获神经干细胞是关键的第一步和将确定它需要得到细胞的汇合板所需的时间量。例如，SVZ是一个经典的神经性区域，因此具有神经干细胞有较高的相对比例。然而，这里提出的技术可以被用来与不经常确认为成年期间具有鲁棒神经原性潜力的其他地区。对于本协议的目的，我们使用了前联合（前部）。而一些细胞和碎片会沉淀在电镀中，介质通常将保持浑浊，蜂窝材料的?...

Watch this Scientific Journal Video about Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain at JoVE.com

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

从成人的大脑收获的神经干细胞被利用，应用范围从神经系统发育的基础研究，以探讨在再生医学潜在的临床应用越来越多。这使得严格控制用于种植这些细胞对于合理的实验结果的分离和培养条件。

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 ...

growing-neural-stem-cells-from-conventional-nonconventional-regions

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JoVE Journal

Neuroscience

12.1K 次观看.  University of Dresden. 从成人的大脑收获的神经干细胞被利用，应用范围从神经系统发育的基础研究，以探讨在再生医学潜在的临床应用越来越多。这使得严格控制用于种植这些细胞对于合理的实验结果的分离和培养条件。 

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

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

成人北极黄鼠海马神经干细胞的生长和分化

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

科研

神经科学

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 ...

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 ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

一个鼠标，两种文化:隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

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 ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

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 ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

利用突触锌组织化学在发育和成人大脑中揭示不同的区域和叶片

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

用于下游分析的冷冻啮齿动物大脑区域集合

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.