这项工作是由美国陆军医学研究和装备司令部授予＃05178001 NS041069 - 06由美国国家神经疾病与中风研究所的支持。我们感谢乔尔Vonnahme议定书的有益的意见。

1。 AGS神经干细胞和介质的制备<ol><li>成人海马北极地松鼠（AGS）神经干细胞分离的方式描述9现在可以得到商业冻存瓶。细胞被运往含有干冰，以确保这些细胞在冷冻保存的状态保持在绝缘包过夜。为了保持完整性，解压后立即接收和储存的细胞，在低比-150 ° C，或在气相的液氮杜瓦。 </li><li>准备agsNSC基础培养基+ 5％FBS加入5毫升FBS（LS - 1012）95毫升agsNSC基础培养基</li><li>准备加入500μL的B - 27补编（Invitrogen公司）50毫升ag​​sNSC基础培养基agsNSC扩展介质。分成毫升分装和储存在-20 ° C，直到使用的B27的其余部分。 </li><li>暖25毫升的agsNSC基础培养基+ 5％FBS和20毫升在37℃水浴agsNSC扩展中等。 </li><li>为扩大与聚- L -鸟氨酸的大衣烧瓶<ol><li>解冻聚- L -鸟氨酸（10微克/毫升）的工作解决方案。存储解决方案之间的2-8 ° C为1个月，一旦解冻。 </li><li>加入8毫升聚- L -鸟氨酸工作的解决方案之一发泄的T - 75药剂，并均匀地分布在整个文化的表层的容量瓶的解决方案。 （其他文化的船只，可用于使用0.5调整工作液量-聚- L -鸟氨酸1.0毫升每10平方厘米文化的表层的解决方案。） </li><li>在37 ° C孵育至少24小时瓶。 （孵化器使用不添加二氧化碳 ，是首选的聚- L -鸟氨酸涂层的培养容器的潜伏期。）涂层烧瓶可以保存长达两个星期的孵化器。 </li><li>在使用之前，从烧瓶中，吸聚- L -鸟氨酸的工作解决方案，并用无菌组织培养级水冲洗两次。吸液烧瓶中，直到使用前几乎是干。 </li></ol></li><li>取出一小瓶从杜瓦和检查，安全密封瓶盖。保持，37℃水浴约60-90秒，或留在瓶小瓶的下半部的顶端，直到小瓶几乎完全解冻;少量的冰应该仍然可见。为了避免潜在的污染，保持瓶盖的水。不要过分解冻，因为这样做可能会损坏细胞。 </li><li>喷雾小瓶，用70％乙醇或异丙醇的外观，然后放置在生物安全柜的小瓶。第小心取出，以避免污染或飞溅。 </li><li>轻轻重新暂停使用无菌吸管加入1毫升的agsNSC基底媒体+ 5％FBS的小瓶细胞。重悬细胞转移到25毫升温暖agsNSC基础培养基+ 5％FBS在50 mL离心管中。 </li><li>一次冲洗冷冻小瓶，用稀释的细胞悬液。离心6分钟150 XG细胞。为了获得最佳结果，计算出每一个人的离心机类型的速度。 </li><li>虽然细胞旋转，添加10毫升ag​​sNSC扩展中等的干聚- L -鸟氨酸涂层的T - 75烧瓶中，加入40μLFGF基本RH（40ng/mL）。离心后，吸出上清液，小心不要吸出沉淀。重新悬浮在10毫升ag​​sNSC扩展中等的细胞。在agsNSC扩展介质中传输到媒体的T - 75烧瓶重悬细胞。 </li><li>轻轻摇动培养容器的一侧到另一侧，均匀地分布细胞内的船只。 </li><li>种子放置在37℃，5％的CO 2培养箱培养容器。细胞应重视的容量瓶中，并开始在两个小时播种内形成的过程。如果细胞仍然浮动，并揉成团内介质，再离心5％胎牛血清的细胞和运行步骤1.8-1.10新鲜培养基和烧瓶。 </li></ol> 2。扩展的AgsNSC <ol><li>细胞解冻和播种后两天（48小时），一个完整的介质更换应使用20毫升ag​​sNSC扩展介质加一个基本RH的FGF - 40μL，穗。 </li><li>穗40μLRH翌日接种FGF -基本瓶。这些细胞将准备trypsinize时，他们已达到75-80％汇合，接种后的第三或第四天。培养接种后4天以上的未分化的干细胞是不建议，因为潜伏期较长，将使细胞开始分化为神经元和神经元，可能是由胰蛋白酶在第3步中受伤。 RH FGF基本可以存储长达一个星期在4 ° C，仍然保持活动。 RH FGF基本也可以通过一个额外的冻结/解冻在-20 ° C，如果需要更多的时间。 </li></ol> 3。单元胰蛋白酶的<ol><li> agsNSC应传代接种后3〜4天。聚- L -鸟氨酸涂层烧瓶之间的24小时，一个星期前准备如果agsNSC将再次扩大，传代。 </li><li>吸出从培养容器的介质。加入2.5 mL的0.05％Trypsin/0.02％EDTA（CM - 0017）为每75厘米表面积的2船只。涡流轻轻地，以确保所有细胞都涂有胰蛋白酶/ EDTA。细胞通常开始从表面分离，在60秒内，根据细胞的汇合。不要超过trypsinize，因为这样做会损害细胞。轻轻拍打培养容器从几个方面，将鼓励支队。 </li><li>一旦细胞分离，agsNSC基础培养基添加10毫升+ 5％FBS的烧瓶中。轻轻的漩涡，以确保所有的胰蛋白酶溶液中和。使用无菌实验室技术，无菌的离心管中含有10毫升ag​​sNSC基底介质5％胎牛血清的细胞吸管。重复洗涤烧瓶再次用一个额外的10毫升的agsNSC基础培养基+ 5％FBS和结合起来，先洗。 </li><li>如上所述，在6分钟150 XG离心机细胞。离心后，细胞应形成一个清洁的松散颗粒。吸从离心管中的胰蛋白酶和中期瓦解和重新暂停在10毫升的细胞沉淀（每吨75烧瓶）预热agsNSC基础培养基+ 5％FBS轻轻吹打向上和向下与10毫升吸管的2倍。执行如下所述的细胞计数。 </li></ol> 4。电镀细胞的标准计算<ol><li>使用无菌技术操作，漩涡细胞悬液15μL细胞悬液转移到离心管。加入15微升0.4％台盼蓝。轻轻混匀，装入10μL细胞悬液计数商会（＃1490目录：Hausser科学，光明如清洁hemacytometer商会每个<a href="http://www.hausserscientific.com/hausserbrightlinedirect.htm">http://www.hausserscientific.com/hausserbrightlinedirect.htm</a> ）。 </li><li>统计的hemacytometer至少4个象限。蓝台盼蓝阳性的细胞都死了，不应该算。对于准确的细胞计数，每象限的最佳细胞数应该是25-75细胞。 </li><li>细胞计数后，计算平均4个象限。 10,000乘以稀释倍数的细胞计数平均（2如果使用推荐的卷），以确定每毫升细胞数。注意，如果总的百分比生存能力小于92％有可能是一个原因。低生存能力的一个原因可能是随着胰蛋白酶，前中和胰蛋白酶与胎牛血清。另一个原因可能是，血药浓度（FBS），胰蛋白酶太低，继续胰酶消化细胞。第三，细胞可能已经过于融合，使干细胞分化细胞 - 细胞接触，导致受伤期间胰蛋白酶。如果此方法是紧随其后的3个或更多倍增的收益率可能会预期，给人一种&gt; 400万〜50万的初始细胞人口。 </li></ol> 5。另一个扩充瓶接种<ol><li>要确定接种一艘船所需的细胞总数，乘以所需的播种密度（6600细胞每平方厘米2或50万的T - 75烧瓶细胞）接种的船只比表面积（平方厘米）。如果接种超过一艘船只，船只总数乘以每血管细胞的数量。要确定接种每艘船所需的细胞悬液的体积（ml），活菌数/ ml的细胞悬液，接种一艘船所需的细胞分裂的次数。如果接种超过一艘船计算需要乘一艘船需要接种的船只总数的体积总量的细胞悬液。 </li><li>离心机在agsNSC基础培养基中的细胞所需的音量+ 5％FBS，留下一个松散的颗粒。通常情况下，50万细胞带来到总体积为25 ml +5％FBS如前所述，在agsNSC基础培养基。吸液，重新悬浮在10毫升的这种额外的离心步骤是必需的删除FBS接种前agsNSC扩展中等松散颗粒。添加10毫升预热agsNSC扩展中等的T - 75烧瓶。添加10毫升的T - 75烧瓶悬浮细胞悬液，再加入40μLRH FGF基本烧瓶。考虑胰蛋白酶和接种时，应采取不超过1.5小时决定的最大数量的烧瓶工作在任何一个时间。还应该有完整的媒体足够的介质变化，如果使用大量的烧瓶。 </li><li>轻轻摇动烧瓶从一侧到另一侧，均匀地分布在细胞，并将其放置到37℃，5％的CO 2培养箱如前所述，培养容器。 </li></ol> 6。细胞分化孔板接种稀释<ol><li>以及任何多格式可能被使用，但到BD Biocoat聚- L -赖氨酸包被96孔板接种是DEScribed这里。确定为您的实验需要乘以96多孔板接种0.2毫升每口井的井数的细胞悬液的体积和增加约10％，以支付任何损失量在接种。计算稀释到7.5万细胞/ mL的细胞密度。确定要分化培养基中加入终浓度达到2％FBS（见表1）FB的数量。混合FBS和分化培养基。仔细混合细胞的分化与胎牛血清的介质的计算量。 （注：在离心细胞在agsNSC基础培养基+ 5％FBS虽然胎牛血清在离心保护细胞和促进细胞粘附胎牛血清抑制differention稀释FBS的2％，将允许多以及表面的细胞粘附。 2％FBS稀释进一步促进分化。胰岛素转硒（ITS - E）也包括在分化培养基促进分化中。 </li><li>在生物安全柜中，取无菌combitip水库200稀释的细胞悬液，UL，每孔聚- L -赖氨酸包被96多道移液器和大口提示板以及使用。 1.5-2小时板块在37℃，5％的CO 2。附着在细胞，约90至120分钟后，小心地取出50％（96孔板100μL）的介质从每口井采用大孔枪头。小心地更换用温水agsNSC分化培养基中删除的卷，再次使用大孔枪头。 （标准孔板的提示使用可能会损害细胞）。 </li><li>接种后2天（48小时），执行50％的中型企业使用agsNSC分化培养基和大孔枪头的变化。 </li></ol> 7。神经元的维修<ol><li>准备NeuraLife，谷氨酰胺和B27的神经元维持液，按照指示使用。神经维持液应每周准备。 </li><li>两天后最后agsNSC分化培养基的变化，一般4天接种后，执行50％的中型企业使用神经维持液的更换。 </li><li>继续执行50％的中型企业的更新换代产品，每2至3天使用神经维持液。应该出现改变神经元的维持液几天内的神经元突起。应采用细胞被转移到神经元的维持液在3个星期。神经元可使用市售的神经元的标记数目确定。星形胶质细胞也将始终遵循。 </li><li>文化的人群通常表达的神经元细胞总数的比例大于40％。使用的神经元比NeuraLife维护媒体其他可能提供劣质细胞不看形态成熟，可能无法生存，只要在文化的。 </li></ol> 8。代表性的成果： 北极地松鼠成体神经干细胞会继续增加一倍时，至少有两个通道，3至5天，每24小时在500,000-600,000 cells/75mm容量瓶种子。这些细胞就会很容易区分basicFGF和B27的拆除和存在1-2％胎牛血清和1％的胰岛素/转/硒，将成为TUJ1（Covance公司）阳性神经元在14-21天（见图2）。神经元细胞总数的比例将介于40-60％，依赖于文化的年龄（图3）。神经元可能被用于从播种后12天的试验，通过至少21天播种后。 <img src="/files/ftp_upload/2199/2199fig1.jpg" alt="图1" /> 图1。 AGS成年海马神经干细胞的显微照片。 AGS成年海马神经干细胞在聚- L -鸟氨酸涂层烧瓶agsNSC基底媒体3天的增长期的显微照片。加倍发生，大约每24小时。照片在100X的放大倍率。 <img src="/files/ftp_upload/2199/2199fig2.jpg" alt="图2" /> 图2。荧光显微镜的区别的AGS成年海马神经干细胞 AGS神经干细胞分化媒体为4天，然后在Neuralife保持17天前固定。固定细胞，用4％多聚甲醛和确定使用自Invitrogen TUJ1初级抗体（Covance公司）的Alexa Fluor 568二级抗体（绿色）的神经元。 Hoechst染料33343染色所有的细胞核（蓝色）。细胞数量，神经元数量和神经元的比例确定使用突起生长的软件和Cellomics Arrayscan仪器。这张照片代表约57％的神经元。 10倍的放大倍率<img src="/files/ftp_upload/2199/2199fig3.jpg" alt="图3" /> 图3。 AGS分化的神经干细胞的比例在三个神经元数量和神经元文化年龄 受到AGS神经干细胞分化媒体4天，然后在Neuralife保持为12，15和17天前，以固定。固定细胞和神经元TUJ1初级抗体（Covance公司）确定。 Hoechst染料染色所有核细胞总数识别。细胞数量，神经元数目％的神经元的比例分别确定使用突起生长的软件和Cellomics Arrayscan仪器。值代表平均每15井条件+ SD。 表1样品用于计算传代和播种<ol><li>使用70万细胞/ ml的细胞悬液</li><li>使用的75000细胞/ ml或15,000细胞的细胞密度每孔，每孔0.2 mL体积。 </li><li>以及= 19.2毫升或理想的最终容积21毫升每0.2毫升接种于96孔。 </li><li> （最终浓度X最终成交量）÷初始浓度=初始音量 （21毫升x 75000细胞/ mL）÷ 700000 = 2.25毫升</li><li> FB的细胞悬液量。 2.25毫升= 2.25毫升× 0.05 = 0.1125毫升</li><li> FB的卷需要21毫升= 21毫升x 0.02 = 0.42毫升</li><li> FB的卷添加到分化培养基= 0.42毫升 - 0.1125毫升= 0.3075毫升</li><li>分化培养基量= 21毫升 - 2.25 mL细胞悬液 - 0.3075毫升5％FBS = 18.44毫升。 </li><li>结合0.308毫升FBS的分化培养基和18.44毫升，加入2.25 ml的细胞悬液。 </li></ol>

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	<li>Barnes, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze+avoidance+in+a+mammal:+body+temperatures+below+0+degree+C+in+an+Arctic+hibernator.">Freeze avoidance in a mammal: body temperatures below 0 degree C in an Arctic hibernator.</a> Science. 244, 1593-1595 (1989).</li><li>Dave, K. R., Prado, R., Raval, A. P., Drew, K. L., Perez-Pinzon, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arctic+ground+squirrel+brain+is+resistant+to+injury+from+cardiac+arrest+during+euthermia.">The Arctic ground squirrel brain is resistant to injury from cardiac arrest during euthermia.</a> Stroke. 37, 1261-1265 (2006).</li><li>Dave, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+epsilon+activation+delays+neuronal+depolarization+during+cardiac+arrest+in+the+euthermic+arctic+ground+squirrel.">Protein kinase C epsilon activation delays neuronal depolarization during cardiac arrest in the euthermic arctic ground squirrel.</a> J Neurochem. 110, 1170-1179 (2009).</li><li>Ross, A. P., Christian, S. L., Zhao, H. W., Drew, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+tolerance+to+oxygen+and+nutrient+deprivation+and+N-methyl-D-aspartate+in+cultured+hippocampal+slices+from+hibernating+Arctic+ground+squirrel.">Persistent tolerance to oxygen and nutrient deprivation and N-methyl-D-aspartate in cultured hippocampal slices from hibernating Arctic ground squirrel.</a> J Cereb Blood Flow Metab. 26, 1148-1156 (2006).</li><li>Christian, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arctic+ground+squirrel+(Spermophilus+parryii)+hippocampal+neurons+tolerate+prolonged+oxygen-glucose+deprivation+and+maintain+baseline+ERK1/2+and+JNK+activation+despite+drastic+ATP+loss.">Arctic ground squirrel (Spermophilus parryii) hippocampal neurons tolerate prolonged oxygen-glucose deprivation and maintain baseline ERK1/2 and JNK activation despite drastic ATP loss.</a> J Cereb Blood Flow Metab. 28, 1307-1319 (2008).</li><li>Jin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+stroke-induced+neurogenesis+in+the+human+brain.">Evidence for stroke-induced neurogenesis in the human brain.</a> Proc Natl Acad Sci U S A. 103, 13198-13202 (2006).</li><li>Maysami, S., Lan, J. Q., Minami, M., Simon, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferating+progenitor+cells:+a+required+cellular+element+for+induction+of+ischemic+tolerance+in+the+brain.">Proliferating progenitor cells: a required cellular element for induction of ischemic tolerance in the brain.</a> J Cereb Blood Flow Metab. 28, 1104-1113 (2008).</li><li>Sung, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia/reoxygenation+stimulates+proliferation+through+PKC-dependent+activation+of+ERK+and+Akt+in+mouse+neural+progenitor+cells.">Hypoxia/reoxygenation stimulates proliferation through PKC-dependent activation of ERK and Akt in mouse neural progenitor cells.</a> Neurochem Res. 32, 1932-1939 (2007).</li><li>Brewer, G. J. <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+culture+of+adult+rat+hippocampal+neurons.">Isolation and culture of adult rat hippocampal neurons.</a> J Neurosci Methods. 71, 143-155 (1997).</li><li>Palmer, T. D., Takahashi, J., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+rat+hippocampus+contains+primordial+neural+stem+cells.">The adult rat hippocampus contains primordial neural stem cells.</a> Mol Cell Neurosci. 8, 389-404 (1997).</li></ol>

JA凯莱赫，安德森是Neuronascent创始人和北极黄鼠专利申请的发明。 Neuronascent接收AGS媒体和干细胞的销售特许权使用费。 RC麦吉是受雇于生命线，公司分配媒体和AGS神经干细胞。

AGS成人神经干细胞是强大的，可扩大许多段落，使他们基本的神经干细胞特性的研究神经干细胞的极好来源。扩张的速度大约是一增加一倍，每二十四小时，但这些细胞必须达到汇合前传，因为细胞之间的接触将有利于星形胶质细胞分化。联系介导的分化将因此导致的神经元细胞总数的比例急剧下降，如果分化开始之前，胰蛋白酶和通过。图1说明了一个AGS神经干细胞培养，应在24小时内传。 <...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>agsNSC</td>
<td>&nbsp;</td>
<td>Lifeline</td>
<td>FC-0004</td>
<td>&nbsp;</td>
<tr>
<td>agsNSC Expansion kit</td>
<td>&nbsp;</td>
<td>Lifeline</td>
<td>LL-0008</td>
<td>Alternative source for DMEM/F12, B27 and rhFGFb (<a href="http://www.invitrogen.com">www.invitrogen.com</a>)</td>
</tr>
<tr>
<td>agsNSC differentiation kit</td>
<td>&nbsp;</td>
<td>Lifeline</td>
<td>LL-0009</td>
<td>Alternative source for DMEM/F12 and ITS-x (<a href="http://www.invitrogen.com">www.invitrogen.com</a>)</td>
</tr>
<tr>
<td>NeuraLife maintenance media kit</td>
<td>&nbsp;</td>
<td>Lifeline</td>
<td>LL-0012</td>
<td>Alternative sources are NeuraBasal (LifeLine) or Neurobasal (in vitrogen), glutamine and B27 (<a href="http://www.invitrogen.com">www.invitrogen.com</a>)Differentiated cells are maintained for up to 3 weeks in NeuroLife, but no more than 2 weeks in Neurobasal (<a href="http://www.invitrogen.com">www.invitrogen.com</a>)</td>
</tr>
<tr>
<td>Micrometer (such as a Bright Line Counting Chamber)</td>
<td>&nbsp;</td>
<td>Hausser Scientific</td>
<td>1490</td>
<td><a href="http://www.hausserscientific.com/hausserbrightlinedirect.htm">http://www.hausserscientific.com/hausserbrightlinedirect.htm</a></td>
</tr>
<tr>
<td>Biocoat Poly-L-Lysine coated 96 well plates</td>
<td>&nbsp;</td>
<td>BDBioscience</td>
<td>356516</td>
<td><a href="http://www.bdbiosciences.com">www.bdbiosciences.com</a></td>
</tr>
<tr>
<td>Large orifice pipette tips</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>02-707-141</td>
<td>Avoids neuronal cell damage when pippetting. <a href="http://www.fishersci.com">http://www.fishersci.com</a></td>
</tr>		</tbody>
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growth and differentiation of adult hippocampal arctic ground squirrel neural stem cells

北极地松鼠（Urocitellus parryii，AGS）是独特的能力与核心体温接近或低于冻结1休眠。这些动物也抵制在体内 2,3和氧糖剥夺在体外 4,5脑缺血损伤。这些独特的品质提供了动力隔离AGS的神经元的研究神经元固有的特点，可占为AGS神经元的能力，以抵御缺血引起的损伤和细胞死亡和极冷温度。识别蛋白或靶基因，使这些细胞的独特属性，能够在发现有效的治疗缺血性标志和耐冷性研究的援助。成人AGS海马包含神经干细胞继续增殖，从而在培养这些干细胞易于扩展。我们在这里描述的方法，研究人员可以利用这些干细胞和分化的神经元为任意数量的目的。密切下列步骤AGS神经干细胞可以扩大通过两个或以上的通道，然后分化文化TUJ1阳性神经元（〜50％），无需使用有毒化学品，以尽量减少细胞分裂的数量高。缺血诱导神经元的6和神经通过MEK / ERK和PI3K/Akt生存信号通路，有助于缺血抵抗 体内 7 和 8（凯莱赫-安德森， 德鲁等，在准备中）在体外进行。这些独特的神经细胞的进一步鉴定，可以提前在许多方面，使用部分或所有这些方法。

Watch this Scientific Journal Video about Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells at JoVE.com

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

准备从成人非冬眠周岁北极地松鼠（AGS）海马神经干细胞。这些神经干细胞可以通过许多段落扩大，分化和保持了近50:50的神经元的神经胶质文化。

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 ...

growth-differentiation-adult-hippocampal-arctic-ground-squirrel

Research

JoVE Journal

Neuroscience

9.5K 次观看.  University of Alaska at Fairbanks. 准备从成人非冬眠周岁北极地松鼠（AGS）海马神经干细胞。这些神经干细胞可以通过许多段落扩大，分化和保持了近50:50的神经元的神经胶质文化。

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

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

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

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

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

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

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

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

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

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

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

从小鼠胚胎神经管的神经嵴细胞的分离和培养

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

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 ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

使用克隆测定神经干细胞的计数。

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

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 ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

成人希马神经前体细胞的乳化电路特定调节

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

胎儿和成体神经干细胞衍生少突胶质细胞前体细胞培养物的高内涵筛选分化与成熟分析

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...

Methods Article

成人北极黄鼠海马神经干细胞的生长和分化