我们要感谢他的洞察力和反馈，在使用玻璃纤维细胞德文 - 加利博士。

介绍在这个协议中，我们展示如何提取，选择和板从E13小鼠胎儿的脊髓神经胶质限制易制毒化学（GRP）的细胞。这些细胞内oligodendrocytic和星形细胞系的早期前兆，并通过他们表达A2B5定义。在玻璃纤维介质与FGF-2的补充，A2B5 +的GRPS将开始表达早期oligodendrocytic宗族标记PDGFαR和NG2 1,2的 。这些胶质细胞的细胞系，通过一系列不同表型的阶段，他们每个人特点是形态的变化，以及表达的特定发展阶段的标志。 作为一个基于细胞的治疗方法，在中枢神经系统白质疾病，包括多发性硬化症，leukodystrophies和脑室周围白质软化症（PVL）3,4,5,6的潜在来源，目前正在研究这些前体细胞</s达&gt;。利用人类脑源性胶质细胞谱系前兆的研究报道，在大脑的shiverer鼠标dysmyelination模型以及脱髓鞘脊髓啮齿动物模型6,7,8,9成功的髓鞘。这些胶质前体细胞也被用在其他白质疾病，如脊髓损伤和肌萎缩性侧索硬化症10,11,12,13模型的研究。 我们的实验室已开发的早期产后缺氧缺血小鼠模型，我们正在评估在PVL的，脑瘫14,15,16领导的事业的恢复方法推导出玻璃纤维细胞脊髓的疗效。还有啮齿类动物的脑源性OPC模型已被广泛用于研究脑白质损伤以来GRP细胞有一种倾向，分化成星形细胞通路17。已经进入的OPC型胶质细胞谱系通路，现象​​似乎irreve的rsible。然而，我们感兴趣的是在评估的少突胶质祖细胞，包括GRPS，甚至可能在10.5天所得的非符合资格人士的广泛响应。出于这个原因，我们采取了脊髓，为我们的工作得到玻璃钢模型。 除了 ​​细胞替代使用的GRPS，这些细胞脑白质疾病从野生型和转基因啮齿动物模型的推导允许胶质细胞分化， 在体外设置正常和疾病条件下进一步研究。以前的出版物表明选择性的oligodendrocytic系前体细胞的成熟依赖的谷氨酸，氧化应激，神经发炎18,19牵连的选择因素等各种外部压力的脆弱性的证据。我们的研究都集中在背后的发展，这些白质损伤的细胞和分子机制的了解，评估细胞的敏感性，在胶质细胞谱系谱侮辱以及分析公认的治疗方法。 物料所有涉及动物的程序符合PHS政策和约翰霍普金斯大学IACUC。所有程序应在层流罩，以保持无菌条件下进行。常用解剖是在横流罩和垂直流罩在体外工作。所有媒体都用冷在解剖。在我们的研究使用的小鼠的CD-1株野生型和在（C57/BL6）背景的转基因绿色荧光蛋白。通常一个CD-1小鼠大坝产生10 +的胎儿，这是足够的种​​子1-2的T25瓶。通常情况下，两个水坝牺牲汇集成1的T25每大坝瓶镀的时间和胎儿的脊髓。一旦细胞已被他们可扩展，并在随后的研究体外特点推导。 1。媒体和烧瓶的准备<ul type="disc"><li>玻璃钢股票夹层培养基：DMEM培养基/ F12键1:1（Invitrogen公司）+ B27的（50倍; Invitrogen公司）N2（100倍; Invitrogen公司）的补充和牛血清白蛋白（BSA的0.5％W / V）作为介质。 </li><li>解离介质：夹层介质相同。 </li><li>培养基：GRP股票介质+ FGF-2（10-20毫微克/毫升; Invitrogen公司）和肝素（1微克/毫升;西格玛）。 </li><li> PLL /粘连涂层75厘米或25厘米的T75或T25）2瓶（。 </li><li>组织培养瓶（75厘米2，猎鹰）15-20μg/ml聚-L-赖氨酸（PLL;西格玛）在蒸馏水中H 2 O在37°C孵育1小时，然后吸气，涂上。瓶洗然后涂上15-20微克/毫升层粘连蛋白（Sigma公司）在PBS PBS在37°C，1小时，然后吸气，新鲜的PBS或媒体说，直到细胞准备是镀1X。继涂层，瓶不应该被允许干，但可以在PBS或媒体保持在4°C使用前的短暂。 </li></ul> 2。仪器和另一个M动脉插管<ul type="disc"><li>培养皿：4个水坝：3 Petri网的解剖菜（75毫米），为20毫米收集收获脊髓的菜。 </li><li>大剪刀（43毫米操作剪刀，Roboz）和组织钳开放大坝的腹部。 </li><li>小剪刀，解剖子宫，取出胎儿（15毫米微型解剖剪刀，Roboz）。 </li><li>微弹簧剪刀解剖（6毫米），解剖胎儿的脊髓。 </li><li>微解剖直角钳，锚定在手术过程中的大坝和胎儿身体后取出脊髓一旦暴露。 </li><li> 40或70微米电镀前去除细胞碎片，细胞过滤。 </li><li> 0.05％胰蛋白酶预热至37°C。 </li><li> 10 mg / ml的DNA酶1（Sigma公司），作为100X股票准备（1克/毫升）。 </li><li>生物危害动物尸体袋。 </li><li> 15日和50毫升处理的离心管中提取的脊髓。 </li></ul> 3。确定定时箴egnancy 孕鼠水坝下令从E12的胚胎一天或胎儿发育怀孕或内部确定的区块E13指定交付的商业来源。简单地说，两女都放在一起在傍晚的男性一起离开一夜之间。第二天观察女性的阴道位于粘液栓的存在。粘液栓只是一个迹象表明，交配发生，使动物其后体重每天监测的增重率。被定义为胚胎的第一天（E1）的粘液栓观察一天。 4。组织推导<ol><li>横流罩下工作，以保持无菌条件下。 </li><li>喷用70％乙醇的发动机罩和工作区。 </li><li>反应釜工具或喷雾用70％乙醇宽松。 </li><li>准备使用的工具，媒体和显微镜。 </li><li> 20毫升冷夹层培养基倒入无菌的Petri DISH。 </li><li>麻醉与水合氯醛腹腔注射（IP）的颈椎脱位或斩首（500毫克/千克）的动物。 </li><li>大坝腹部喷洒70-90％的乙醇（消毒） </li><li>打开腹部大剪刀和镊子。 <ol type="a"><li>在CD1和（C57/BL6）菌株：一般8至14胎儿会在子宫内。对胎儿的数量是依赖应变。 </li></ol></li><li>提取包括幼仔和地方在一个干净的培养皿中鲜冷夹层介质洗从胎儿血液和组织碎片小鼠子宫。 </li><li>每个胎儿从子宫角的提取和使用小剪刀，他们从胚胎囊和胎盘分离。新鲜夹层介质转移到一个新的培养皿中提取的胎儿。 <ol type="a"><li>夹层介质应该是在低温条件下，降低代谢活动和维护源性细胞的可行性。 </li><li>与两钳，从现在开始微解剖弹簧剪刀。 </li></ol></li><li>在解剖显微镜下工作，削减皮肤沿微型手术剪，剥离皮肤暴露脊髓与脊髓。 </li><li>关于C1的显微剪刀和尾巴开始在横切的脊髓。 </li><li>钝钳取出脊髓，确保所有骨骼和软骨被删除，并转移到第三夹层介质的Petri菜。 </li><li>为了防止在随后的细胞培养脑膜组织过度生长，尝试从脊髓脑膜尽可能的。由于新生突出的末梢神经，这是比从大脑中提取的脑膜组织更加困难。 </li><li>一旦已被删除脑膜，脊髓转移至20毫米培养皿</li><li>清理彻底喷涂面积70-90％的乙醇。 </li><li>分离以下步骤应迅速完成维护一个派生的脊髓组织的高生存能力。 </li></ol> 5。文化建设<ol><li>胰蛋白酶应该被预温在37°CH 2浴至少30分钟。应去掉一个适当的等分，从股票，股票的风险，以减少温度波动。 </li><li>收获的脊髓转移50 mL离心管中10毫升预热的胰蛋白酶（0.05％;质量生物）和100μLDNA酶（10毫克/毫升），并简要地磨碎。 </li><li>在37℃孵育的CH 2浴10分钟的O型。 </li><li>磨碎和孵化另一个10分钟。 </li><li>加入5毫升玻璃钢中型（定义见上文）和1000 RPM离心5分钟。 </li><li> 10毫升的玻璃钢中型吸上清，重悬沉淀，并加入DNA酶-1（10毫克/毫升;西格玛）。 </li><li>在37℃孵育的CH 2浴10分钟的O型。 </li><li>在1000 rpm离心5分钟，吸出上清液。 </li><li>重悬浮颗粒与频率SH玻璃纤维介质过滤器碎片40 - 70微米的细胞过滤。 </li><li>钢板上涂25厘米2瓶（T25），并于37°C，湿度95％和5％CO 2的PLL /粘连。 </li><li> 100％的媒体在翌日更改。 </li><li>在这一点上，GRPS连接，并开始扩大进程。 </li></ol> 6。 Immunopanning的玻璃钢人口<ol><li> PLL /粘连，直至烧瓶瓶涂层板脊髓组织是85-90％汇合或一个星期左右。 </li><li>收获细胞刮瓶用橡皮警察或0.05％胰蛋白酶，彻底磨碎，但轻轻地，在1000转速为5分钟，彻底重悬沉淀3毫升玻璃钢中型离心。 </li><li>每次1毫升转移的T25 3瓶或3毫升到1的T75烧瓶，加入适当体积中等，完全覆盖细胞，使烧瓶达到80-90％汇合。 </li><li>改变媒体的每一天，迟早如果介质消耗很快。 </li><li>大衣细菌PE三菜（75毫米），4℃过夜，抗小鼠IgM抗体（南方生物技术），10微克/毫升的PBS。 </li><li>吸干多余的缓冲区，并用3倍的PBS的培养皿。 </li><li>添加浓度为5微克/毫升稀释在PBS孵育1小时在室温（RT）A2B5抗体（Millipore公司）。 </li><li>再次用PBS 3倍的盘子洗净，然后加入8毫升玻璃纤维介质，以防止干燥板。 </li><li>转让5毫升玻璃钢中型刮瓶用橡皮警察分离所有细胞从培养皿和收获的GRP细胞</li><li>磨碎细胞悬液简要分手团块和转移5毫升细胞悬液涂的A2B5细菌培养皿。 </li><li> 1小时在室温下孵育无晃动。 </li><li> 1小时后抽吸媒体和洗涤板用PBS 8X。 </li><li>橡胶警察在2毫升玻璃钢媒体轻轻刮去的细胞，然后转移到适当的音量与玻璃钢中型PLL /粘连涂层板或瓶。 </l我&gt; </ol> 7。源性干细胞的冷冻和储存<ol><li>从85-90％汇合的T25或T75瓶2毫升0.05％胰蛋白酶细胞收获。 </li><li>胰蛋白酶稀释和灭活与8毫升GRP股票介质和离心5分钟在1000转。 </li><li>的T25瓶的颗粒悬浮于1毫升玻璃钢冷冻介质（玻璃钢股票的1​​0％（V / V）的DMSO和选择性，20％胎牛血清培养基补充）。较大颗粒的T75瓶稀释2倍。 </li><li> 1.5 - 3×10 10,11,12,13细胞的细胞悬液转移到低温瓶（NALGENE），并储存在低温容器异丙醇-80°C至慢慢冻结隔夜（NALGENE）过夜。 </li><li>继隔夜冷冻细胞被转移到冷冻箱，6个月至1年，并保存在-80°C或较长期的N（L）。解冻的细胞通常是可行的60-80％的PLL /粘连涂层的质量在很大程度上取决于。 </li></ol&gt; 8。代表结果 CO 2用于麻醉的动物，因为可能对下游影响胎儿和最终源性细胞的可行性。此外，这将是非常困难的推导过程中，从脊髓脑膜完全消除，但玻璃钢介质和immunopanning的的结合，将消除这些快速生长的细胞。同样，尽管整个脊髓收获更大的GRP人口，由于其起源于脊髓腹侧和迁移背侧20腹侧浓度。然而，玻璃钢中型选择玻璃钢型，人口由A2B5 immunopanning最大化。电镀脊髓组织后， 在体外培养，培养两个段落或一个星期左右。经过2天的文化，不同形态的细胞，可以观察到，在组织培养瓶，表明异质geneity的人口，但玻璃钢中型玻璃钢型选择。这些细胞是一种组合A2B5 +玻璃钢，电子NCAM的神经禁区前体（NRP）和nestin +，A2B5，神经上皮细胞，并可能纤维连接蛋白+脑膜细胞。此外，更加成熟的表型可能是目前表达的标记，包括Doublecortin的神经谱系Tuj1，星形胶质细胞GFAP和PDGFaR和GALC寡血统，为了最大限度地从开始的异构池（ 图1A）高度的玻璃纤维丰富的人口，细胞双immunopanned的选择和淘汰的E-NCAM的细胞选择为A2B5 +玻璃钢的人口，一旦细胞密度已增至约85-90％汇合的T75瓶。这些玻璃纤维细胞保持他们的能力，尽管在少突胶质细胞的表型选择的媒介，因此后续A2B5 immunopanning可能有必要保持高度丰富的玻璃钢人口成为星形胶质细胞。 immunopanning产生盟友收益率高达95％A2B5 +细胞，但更大的产量，A2B5共轭磁珠（美天旎生物技术公司）可以用来提供高达97％的产量。在常规介质的变化，如维修玻璃钢文化的警惕，将最大限度地减少非玻璃纤维细胞类型的细胞增殖。即使在没有BMP-4星形胶质细胞可能仍然被发现和贫化媒体似乎特别星形细胞表型诱导分化。虽然B27的补充包含三脱碘激素（T3），有没有观察到的趋势分化成少突胶质细胞在GRP人口，只有当更高水平的T3添加到中期，这一现象发生。然而，在T3免费B27的补充，可以取代的，如果有一个成熟的少突胶质细胞（ 图1B）污染的关注。这些玻璃纤维细胞可以很容易地确定其形态与2个或3个短流程的小SOMA但屏幕上的其它类型的细胞可能存在的，IM可以munocytochemistry以前命名的共同发展和细胞类型的特定标志。通常会是一个小A2B5-细胞，神经上皮（NEP）的，能够成为玻璃钢或重建方案的持久性人口。幸运的是，维持较长的细胞在GRP中就越有可能，将选择的GRP型中等。 <img alt="图1。" src="/files/ftp_upload/3462/3462fig1.jpg" /> 图1）异构形态镀脊髓细胞的观察后，2天中文化。 二）Immunopanning最大限度地从开始的异构池高度的玻璃纤维丰富的人口。

<ol>
	<li>Kalyani, A., Hobson, K., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroepithelial+stem+cells+from+the+embryonic+spinal+cord:+isolation,+characterization,+and+clonal+analysis.">Neuroepithelial stem cells from the embryonic spinal cord: isolation, characterization, and clonal analysis.</a> Developmental biology. 186, 202-202 (1997).</li><li>Rao, M. S., Mayer-Proschel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial-restricted+precursors+are+derived+from+multipotent+neuroepithelial+stem+cells.">Glial-restricted precursors are derived from multipotent neuroepithelial stem cells.</a> Developmental biology. 188, 48-48 (1997).</li><li>Goldman, S. A., Lang, J., Roy, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor+cell-based+myelination+as+a+model+for+cell-based+therapy+of+the+central+nervous+system.">Progenitor cell-based myelination as a model for cell-based therapy of the central nervous system.</a> Ernst Schering Research Foundation workshop. (60), 195-195 (2006).</li><li>Goldman, S. A., Schanz, S., Windrem, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+strategies+for+treating+pediatric+disorders+of+myelin.">Stem cell-based strategies for treating pediatric disorders of myelin.</a> Human molecular genetics. 17, 76-76 (2008).</li><li>Goldman, S. A., Windrem, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+replacement+therapy+in+neurological+disease.">Cell replacement therapy in neurological disease.</a> Philosophical transactions of the Royal Society of London. 361, 1463-1463 (2006).</li><li>Walczak, P., All, A. H., Rumpal, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+glial-restricted+progenitors+survive,+proliferate,+and+preserve+electrophysiological+function+in+rats+with+focal+inflammatory+spinal+cord+demyelination.">Human glial-restricted progenitors survive, proliferate, and preserve electrophysiological function in rats with focal inflammatory spinal cord demyelination.</a> Glia. 59, 499-499 (2011).</li><li>Liu, S., Qu, Y., Stewart, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+differentiate+into+oligodendrocytes+and+myelinate+in+culture+and+after+spinal+cord+transplantation.">Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 6126-6126 (2000).</li><li>Windrem, M. S., Schanz, S. J., Guo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+chimerization+with+human+glial+progenitor+cells+can+both+remyelinate+and+rescue+the+otherwise+lethally+hypomyelinated+shiverer+mouse.">Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse.</a> Cell stem cell. 2, 553-553 (2008).</li><li>Groves, A. K., Barnett, S. C., Franklin, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+demyelinated+lesions+by+transplantation+of+purified+O-2A+progenitor+cells.">Repair of demyelinated lesions by transplantation of purified O-2A progenitor cells.</a> Nature. 362, 453-453 (1993).</li><li>Back, S. A., Han, B. H., Luo, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+vulnerability+of+late+oligodendrocyte+progenitors+to+hypoxia-ischemia.">Selective vulnerability of late oligodendrocyte progenitors to hypoxia-ischemia.</a> J. Neurosci. 22, 455-45 (2002).</li><li>Johnston, M. V., Trescher, W. H., Ishida, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+hypoxic-ischemic+injury+in+the+developing+brain.">Neurobiology of hypoxic-ischemic injury in the developing brain.</a> Pediatric research. 49, 735-735 (2001).</li><li>Lepore, A. C., Han, S. S., Tyler-Polsz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+fate+of+multipotent+and+lineage-restricted+neural+precursors+following+transplantation+into+the+adult+CNS.">Differential fate of multipotent and lineage-restricted neural precursors following transplantation into the adult CNS.</a> Neuron glia biology. 1, 113-113 (2004).</li><li>Rothstein, J. D., Dykes-Hoberg, M., Pardo, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockout+of+glutamate+transporters+reveals+a+major+role+for+astroglial+transport+in+excitotoxicity+and+clearance+of+glutamate.">Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.</a> Neuron. 16, 675-675 (1996).</li><li>Johnston, M. V., Ferriero, D. M., Vannucci, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+cerebral+palsy:+which+ones+are+best.">Models of cerebral palsy: which ones are best.</a> Journal of child neurology. 20, 984-98 (2005).</li><li>Comi, A. M., Johnston, M. V., Wilson, 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=Strain+variability,+injury+distribution,+and+seizure+onset+in+a+mouse+model+of+stroke+in+the+immature+brain.">Strain variability, injury distribution, and seizure onset in a mouse model of stroke in the immature brain.</a> Developmental neuroscience. 27, 127-127 (2005).</li><li>Comi, A. M., Weisz, C. J., Highet, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+stroke+and+ischemic+seizures+in+the+immature+mouse.">A new model of stroke and ischemic seizures in the immature mouse.</a> Pediatric neurology. 31, 254-254 (2004).</li><li>Dietrich, J., Noble, M., Mayer-Proschel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+A2B5++glial+precursor+cells+from+cryopreserved+human+fetal+brain+progenitor+cells.">Characterization of A2B5+ glial precursor cells from cryopreserved human fetal brain progenitor cells.</a> Glia. 40, (2002).</li><li>Alberdi, E., Sanchez-Gomez, M. V., Marino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca(2+)+influx+through+AMPA+or+kainate+receptors+alone+is+sufficient+to+initiate+excitotoxicity+in+cultured+oligodendrocytes.">Ca(2+) influx through AMPA or kainate receptors alone is sufficient to initiate excitotoxicity in cultured oligodendrocytes.</a> Neurobiology of disease. 9, 234-234 (2002).</li><li>Volpe, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+injury+in+premature+infants:+a+complex+amalgam+of+destructive+and+developmental+disturbances.">Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances.</a> Lancet neurology. 8, 110-110 (2009).</li><li>Warf, B. C., Fok-Seang, J., Miller, R. H. <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+the+ventral+origin+of+oligodendrocyte+precursors+in+the+rat+spinal+cord.">Evidence for the ventral origin of oligodendrocyte precursors in the rat spinal cord.</a> J. Neurosci. 11, 2477-2477 (1991).</li><li>Deng, W., Poretz, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendroglia+in+developmental+neurotoxicity.">Oligodendroglia in developmental neurotoxicity.</a> Neurotoxicology. 24, 161-161 (2003).</li><li>Deng, W., Rosenberg, P. A., Volpe, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-permeable+AMPA/kainate+receptors+mediate+toxicity+and+preconditioning+by+oxygen-glucose+deprivation+in+oligodendrocyte+precursors.">Calcium-permeable AMPA/kainate receptors mediate toxicity and preconditioning by oxygen-glucose deprivation in oligodendrocyte precursors.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 6801-6801 (2003).</li><li>Karadottir, R., Cavelier, P., Bergersen, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptors+are+expressed+in+oligodendrocytes+and+activated+in+ischaemia.">NMDA receptors are expressed in oligodendrocytes and activated in ischaemia.</a> Nature. 438, 1162-1162 (2005).</li><li>Pleasure, D., Soulika, A., Singh, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+white+matter:+clinical+and+pathophysiological+aspects.">Inflammation in white matter: clinical and pathophysiological aspects.</a> Mental retardation and developmental disabilities research reviews. 12, 141-141 (2006).</li><li>Gregori, N., Proschel, C., Noble, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tripotential+glial-restricted+precursor+(GRP)+cell+and+glial+development+in+the+spinal+cord:+generation+of+bipotential+oligodendrocyte-type-2+astrocyte+progenitor+cells+and+dorsal-ventral+differences+in+GRP+cell+function.">The tripotential glial-restricted precursor (GRP) cell and glial development in the spinal cord: generation of bipotential oligodendrocyte-type-2 astrocyte progenitor cells and dorsal-ventral differences in GRP cell function.</a> J. Neurosci. 22, 248-248 (2002).</li><li>Rao, M. S., Noble, M., Mayer-Proschel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tripotential+glial+precursor+cell+is+present+in+the+developing+spinal+cord.">A tripotential glial precursor cell is present in the developing spinal cord.</a> Proceedings of the National Academy of Sciences of the United States of America. 95, 3996-3996 (1998).</li><li>Raff, M. C., Miller, R. H., Noble, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+glial+progenitor+cell+that+develops+in+vitro+into+an+astrocyte+or+an+oligodendrocyte+depending+on+culture+medium.">A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium.</a> Nature. 303, 390-390 (1983).</li><li>Strathmann, F. G., Wang, X., Mayer-Proschel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+novel+glial-restricted+cell+populations+in+the+embryonic+telencephalon+arising+from+unique+origins.">Identification of two novel glial-restricted cell populations in the embryonic telencephalon arising from unique origins.</a> BMC developmental biology. 7, 33-33 (2007).</li><li>Nunes, M. C., Roy, N. S., Keyoung, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+isolation+of+multipotential+neural+progenitor+cells+from+the+subcortical+white+matter+of+the+adult+human+brain.">Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain.</a> Nature. 9, 439-439 (2003).</li><li>Roy, N. S., Wang, S., Harrison-Restelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification,+isolation,+and+promoter-defined+separation+of+mitotic+oligodendrocyte+progenitor+cells+from+the+adult+human+subcortical+white+matter.">Identification, isolation, and promoter-defined separation of mitotic oligodendrocyte progenitor cells from the adult human subcortical white matter.</a> J. Neurosci. 19, 9986-9986 (1999).</li><li>Chen, Y., Balasubramaniyan, V., Peng, 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+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nature protocols. 2, 1044-1044 (2007).</li></ol>

这项研究是由美国国立卫生研究所[NICHD的P30HD024061（AWP的），NINDS的K08NS063956（AF）和R01NS028208（MVJ）]和儿童神经基金会。本报告的内容纯属作者的责任，并不代表官方意见的国家卫生研究院，DHHS。作者有没有本研究相关的利益冲突。

<p class="jove_content"&gt;中枢神经系统白质的障碍，包括大量的病因包括遗传，炎症，缺血性和有毒的原因，<sup&gt; 19,21,22,23,24</sup&gt;。虽然多发性硬化症和vasculopathies成人脑白质病的主要病因，与早产儿脑室周围白质软化是在儿童人口的脑白质损伤的最常见的原因。为了进一步揭示疾病发病机制，细胞ofoligodendrocytic血统，这是很大的由围产期侮辱影响的进一步研究，是必要的。的GRPS从啮齿动物胚胎脊髓中分离和证明是性质tripotential，从而产生少突胶质细胞和两种截然不同的星形胶质细胞的人口，但不分化成神经元<sup&gt; 2,25,26</sup&gt;。同样，来自大鼠视神经和胚胎后期或产后早期大鼠脑少突胶质前体细胞（OPC），已被证明在适当的介质条件下运作的少突胶质细胞成熟<sup&gt; 27,28</sup&gt;。此外，胎儿或成人的派生人类的OPC已进一步进入更成熟的少突胶质细胞的表型操纵<sup&gt; 29,30</sup&gt;。</p&gt;<p class="jove_content"E13.5大鼠和小鼠脊髓E12中，13.5&gt;玻璃纤维细胞已被隔离。在大鼠的差异已在背侧和腹侧派生的GRP细胞之间的反应条件，促进与腹侧派生GRPS表现出更大的倾向分化成更成熟的表型，少突胶质细胞或星形胶质细胞的生成<sup&gt; 25,26,28</sup&gt;。这可能是由于新生玻璃纤维细胞，这可能是不太容易接受的分化线索背迁移腹侧。 immunopanning允许亚群，表达A2B5被孤立和维护文化收集。</p&gt;<p class="jove_content"&gt;我们的鼠标玻璃钢文化源自总脊髓组织产生一个异类，汇集人口玻璃纤维细胞的细胞化学（中）和抗体为基础的技术选择。虽然我们的研究利用衍生脊髓鼠标玻璃纤维细胞，它最近报道，也能够发展成为成熟的少突胶质细胞脑源性小鼠少突胶质祖细胞的推导<sup&gt; 31</sup&gt;。这里源性细胞的初步培养，使用适当的培养基丰富PDGFαR+ OPC的人口操纵oligospheres<sup&gt; 31</sup&gt;。我们的研究都集中在少突胶质细胞谱系从A2B5 +PDGFαR-GRP前体（MBP的+）成熟的少突胶质细胞髓鞘碱性蛋白表达谱对缺氧缺血模型对细胞的影响。事实上，我们<em&gt;在体外</em&gt;表征表明，这些不成熟的玻璃纤维细胞成MBP的表达无论在单一或联合培养皮层神经元细胞的成熟。可以操纵这些细胞培养成特定的分化途径参与围产期脑白质损伤的机制研究。有几个小鼠模型脑白质疾病涉及细胞少突胶质细胞系，这部分人口的可用性允许进入机制，造成损伤，以及探索可能的细胞治疗方案的有效性进行彻底调查。</p

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> DMEM培养基/ F12键1:1 </td><td> Invitrogen公司</td><td> 11320033 </td></tr><tr><td> B27的</td><td> Invitrogen公司</td><td> 17504044 </td></tr><tr><td>氮气</td><td> Invitrogen公司</td><td> 17502048 </td></tr><tr><td> RH-FGF-基本</td><td> Invitrogen公司</td><td> PHG0026 </td></tr><tr><td>胰蛋白酶（0.05％），EDTA的</td><td>生物质量，公司</td><td> 118-087-721 </td></tr><tr><td>聚-L-赖氨酸氢溴酸</td><td>西格玛</td><td> P1274-25毫克</td></tr><tr><td>层粘连蛋白</td><td>西格玛</td><td> L2020-1MG </td></tr><tr><td> DNA酶1 </td><td>西格玛</td><td> DN25的</td></tr></tbody></table>

derivation of glial restricted precursors from e13 mice

这是一个胶质禁区前体（GRP）从E13小鼠胎儿的脊髓细胞衍生的协议。这些细胞是内的oligodendrocytic的细胞系的早期前兆。最近，这些细胞进行了研究脑白质疾病的恢复治疗的潜在来源。脑室周围白质软化症（PVL）是在童年的非遗传性脑白质疾病的首要原因，影响极早产儿的50％。这些数据表明了发育中的大脑缺血缺氧，氧化应激和兴奋，有选择性地针对新生的白质的提高的敏感性。胶质禁区前体（GRP），少突胶质祖细胞（OPC）和未成熟的少突胶质细胞（preOL）似乎是在PVL的发展的关键球员，并有继续研究的主题。此外，以往的研究已经确定了中枢神经系统组织的一个子集，易感性增加谷氨酸作为一个发展模式，这种易感性。我们的实验室目前正在调查在玻璃钢的发展阶段，在PVL的和使用的细胞少突胶质祖细胞的作用。我们利用这些派生的玻璃纤维细胞中的几个实验范式，以测试他们的反应，选择一致的应力和PVL。玻璃纤维细胞可以操纵OPCs的和preOL将在体外用鼠标PVL的模型和PVL的类似的侮辱，包括缺血缺氧的体外模型移植实验。利用培养的细胞， 在体外研究中有实验，有利于对数据的解释之间的变异性将减少。培养的细胞，也允许GRP的人口富集，同时最大限度地减少污染的非玻璃钢型细胞的影响。

Watch this Scientific Journal Video about Derivation of Glial Restricted Precursors from E13 mice at JoVE.com

Derivation of Glial Restricted Precursors from E13 mice

该协议概述从胎儿的脊髓神经胶质细胞的限制性前体推导，并保持在体外移植或oligodendrocytic谱系研究。

从E13小鼠胶质限制前体的推导

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

derivation-of-glial-restricted-precursors-from-e13-mice

Research

JoVE Journal

Neuroscience

11.2K 次观看.  Johns Hopkins University. 该协议概述从胎儿的脊髓神经胶质细胞的限制性前体推导，并保持在体外移植或oligodendrocytic谱系研究。

视频: Derivation of Glial Restricted Precursors from E13 mice

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

科研

神经科学

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

丰富的少突胶质细胞培养和Myelinating产后小鼠组织联合培养的少突胶质细胞/神经元的推导

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

从尸检人脑软脑膜捐助外植体培养的推导

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

小鼠胚胎干细胞向皮质 Interneuron 前体细胞的分化

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

将套管植入啮齿动物的麦格纳池中

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

行为小鼠海马空间受限振荡的记录

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Interneuron 前体 Homochronic 移植早期小鼠脑的研究

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...