CRM is a Damon Runyon-Genentech Clinical Investigator. This work was supported, in part, by grants to CRM from the Damon Runyon Cancer Research Foundation (CI-45-09), Department of Defense (W81XWH-09-2-0042), and University of North Carolina University Cancer Research Fund (UCRF). The authors wish to thank Daniel Roth for mouse husbandry assistance. The authors also wish to thank Hannah Chae, Carter McCormick, Demi Canoutas, Stephanie Gillette, and Susannah Krom for tissue culture and immunofluorescence assistance.

所有的动物研究批准了北卡罗莱纳州的机构动物照顾及使用委员会的大学。 1，培养星形胶质细胞从新生小鼠<ol><li>准备<ol><li>揉碎2-3棘手的任务的组织，并放入含70％乙醇的烧瓶的底部。将解剖剪刀，弯钳和2对直微型镊子这个烧瓶中。该组织被用来避免弯曲的微型镊子。 </li><li>加入1 ml HBSS至60毫米组织培养皿。准备一个单独的菜每个动物和菜保持在冰上。 </li><li>麻醉每个机构规定的动物。 </li><li>暖7毫升DMEM中含有10％胎牛血清和1％青霉素 - 链霉素（完全DMEM），用于在37℃的水浴中各动物。注：对于5只小鼠，步骤1.1.1-1.1.4将采取〜15分钟。 </li></ol></li><li>组织收获<ol><li>牺牲新生小鼠（1-3天）每institutional法规。 </li><li>从乙醇中取出解剖剪刀，让他们滴干，并在皮肤上矢状切下来的脊髓鼻子的头盖骨。 </li><li>使在颅骨的切口沿矢状缝，从脊髓开始和过去嗅球延伸。注意保持剪刀头颅骨的内表面附近，以尽量减少脑组织损伤。 </li><li>用弯钳，轻轻剥离颅骨的每个半球横向远离大脑。 </li><li>采用直板微型镊子，轻轻捏每个客场半球皮质的背部分，将其放入含有的HBSS的组织培养皿。小心避免小脑和嗅球。不要把下面的胼胝体的任何组织。 </li><li>用解剖显微镜和2对微型镊子，轻轻地从每个半球皮质去除脑膜。返回的组织培养皿冰，直到开始组织homogenizatioñ。 </li><li>重复步骤1.2.1至1.2.6每增加动物。注：对于5只小鼠，步骤1.2.1-1.2.7将采取〜30分钟。 </li></ol></li><li>组织匀浆<ol><li>用干净的刀片，细切块的半球皮质和板移动到组织文化引擎盖。 </li><li>转移切块组织成一个无菌的15 mL锥形管。用1ml冰冷的HBSS洗涤平板，并转移到含有组织的试管中。 </li><li>等到组织沉降到管底，然后用移液管小心地除去过量的HBSS中。 </li><li>加入2毫升室温胰蛋白酶/ EDTA。吸管〜10倍以1毫升吸管进一步分解细胞。 </li><li>孵育的细胞悬浮液，在37℃下进行15 - 20分钟。通过倒置仔细混合，每隔5分钟。 </li><li>加入3 ml完全DMEM中抑制胰蛋白酶。吸管〜10倍以1毫升吸管混合的解决方案。 </li><li>离心机在200×g离心5分钟，在室温下进行。 </li><li>删除上清液用1毫升移液管。不要吸出培养基，因为颗粒可能松动。 </li><li>悬浮于4毫升37°的小球C完整的DMEM。细胞悬液转移到75厘米螺杆顶端的组织培养烧瓶中。注：对于5只小鼠，步骤1.3.1-1.3.9将采取〜50分钟。 </li><li>大约星形胶质细胞采收后16小时，用4毫升37℃的HBSS洗烧瓶中，再加入4毫升37°C完整的DMEM。保持在37℃，在5％CO 2中的星形胶质细胞。注：该洗涤工序是用于去除未贴壁的细胞和碎片从培养皿重要。 </li><li>当培养物是〜95％汇合时，摇动过夜，于37℃在5％CO 2中 ，删除包含分离的细胞的培养基，然后用4ml的37℃下的HBSS洗烧瓶。加4毫升37℃，5％CO2完全DMEM，并维持细胞在37℃。注意：这一步是非常重要的，以丰富的文化为星形胶质细胞，如污染小胶质细胞和少突胶质细胞祖细胞的分离与此过程，并从培养41被除去。 </li><li>每个动物重复步骤1.3.1-1.3.11。 </li></ol></li><li>诱导基因重组<ol><li> 24小时完成步骤1.3.11后，用2ml的37°更换培养基C完成的DMEM。加入1毫升37℃单片机含1微升Ad5CMVCre或Ad5GFAPCre&gt; 10 10 PFU /毫升。处理时，使用BSL2安全防范措施重组腺病毒：在5％CO 2 .CAUTION孵育6小时，在32℃。 </li><li>删除病毒载媒体及无病毒增加37℃，完全DMEM的星形胶质细胞。注意：处理BSL2使用安全注意事项时，重组腺病毒并放弃每制度规定含有病毒的媒介。注：对于5星形胶质细胞培养，步骤1.4.1 - 1.4.3将采取〜15分钟，不包括6小时的潜伏期。 </li><li>展开皮质星形胶质细胞培养物中体内和体外表征。注：确定突变的下列酶Cre-重组，通过PCR用特异于重组基因或通过免疫印迹对任一特定突变的蛋白质或它的信令后果的引物存在下进行。 Cre的重组后所需的皮质星形细胞培养的表型稳定的时间将取决于所利用的突变，并且必须凭经验确定（ 图2）。 </li></ol></li></ol>从新生小鼠2培养神经干细胞<ol><li>准备<ol><li>在开始解剖前，准备每脑4毫升消化溶液中加入3.1毫克木瓜蛋白酶和1.3毫克半胱氨酸到4ml解离介质- 98毫米的Na 2 SO 4，30mM的K 2 SO 4，5.8毫米MgCl 2，0.25mM的氯化钙 ，1毫米肝素钠（1 M羟乙基pH为7.4股），20毫米的葡萄糖，0.001％酚红，和0.125毫米氢氧化钠。除了木瓜蛋白酶和半胱氨酸的分解介质，有可能使溶液变黄。 </li><li>孵育37℃水浴15分钟。颠倒定期混匀。 </li><li>添加0.1 M氢氧化钠滴，直到消化液返回到亮红色。 </li><li>在组织培养罩，无菌过滤器使用注射器滤器（0.22μm孔径）的消化液。置于冰上的消化液。 </li><li>通过混合44.5毫升NSC基础培养基，加入5ml NSC补充，0.5 ml的青霉素 - 链霉素，50微升0.2％的肝素，10微升100微克/毫升的EGF，和10微升100微克/毫升制备50毫升干细胞培养基（SCM）的bFGF的。 SCM是稳定1个星期保存在4℃下时。 </li><li>完整的HBSS（cHBSS）通过混合50毫升10×HBSS，1.25毫升的1M HEPES（pH7.4）中，制备15毫升的1M D-葡萄糖，加入5ml的100mM的CaCl 2，加入5ml的100mM MgSO 4干燥，和2ml 1 M的NaHCO 3。使总体积至500ml与DDH 2 O。 </li><li>过滤消毒cHBSS机智HA 0.2微米孔径过滤器和储存在4℃。 </li><li>揉碎2-3棘手的任务的组织，并放入含70％乙醇的烧瓶的底部。将解剖剪刀，弯钳和2对直微型镊子这个烧瓶中。该组织被用来避免弯曲的微型镊子。注：对于5只小鼠，步骤2.1.1-2.1.8将采取〜45分钟。 </li></ol></li><li>组织收获<ol><li>牺牲每个机构的规定新生儿（1-3天）小鼠。 </li><li>从70％的乙醇去除解剖剪刀，让他们滴干，并在皮肤上矢状切下来的脊髓鼻子的头盖骨。 </li><li>使在颅骨的切口沿矢状缝，从脊髓开始和过去嗅球延伸。注意保持剪刀头颅骨的内表面附近，以尽量减少脑组织损伤。 </li><li>用弯钳，轻轻剥离颅骨的每个半球横向距离大脑。 </li><li>轻轻地取出整个大脑和发生在冰冷cHBSS。 </li><li>用解剖显微镜下，小心地从大脑解剖细移除工具脑膜。 </li><li>将大脑其底面和去除小脑/后脑和嗅球/额叶皮层与垂直（日冕）削减了大小11手术刀。 </li><li>旋转90°，剩下的大脑将其放置在尾部分，从而产生脑的冠状图。找到侧脑室。 </li><li>切出含有脑室下区（SVZ）一个立方体部分。 </li><li>转让SVZ含组织，以60毫米组织培养皿新鲜冰冷cHBSS切末组织与规模11手术刀。 </li><li>转移组织糜到无菌的15ml试管中。放置在冰上的管中。 </li><li>洗培养皿1-2毫升冰冷cHBSS。传送洗涤溶液含有组织的试管中。 </li><li>重复额外neonata步骤2.2.1-2.2.12升鼠如果必要的。转让全部15ml试管，以组织培养罩的协议的其余部分。注：对于5只小鼠，步骤2.2.1 - 2.2.13将采取〜45分钟。 </li></ol></li><li>组织匀浆<ol><li>将在37℃水浴消解液（步骤2.1.1准备）5分钟。此外，温暖的2毫升单片机每大脑在37℃水浴。 </li><li>小心地用1毫升移液管取出的组织糜的上清液。不要吸。 </li><li>添加每15ml试管2毫升37℃消化解决方案，并通过倒置精心搭配。 </li><li>孵育37℃水浴15分钟。通过倒置混合，每隔5分钟。 </li><li>添加另外2毫升37℃消化解决方案，以每管并重复步骤2.3.4。 </li><li>使细胞沉降到管通过重力的底部，然后用1毫升移液管取出从消化的组织上清。 </li><li>加入2毫升10微米的E-64稀释在cHBSS并通过倒置仔细混匀。 </li><li&gt;孵育3分钟，在室温下进行。 </li><li>使细胞沉降到管通过重力的底部，然后从消化的组织上清。 </li><li>加入1毫升37℃cHBSS，均质1毫升枪头（〜20次）。避免组织均化过程中注入空气泡。 </li><li>通过40微米孔径的细胞过滤器的组织匀浆，然后用清水冲洗过滤用5毫升37℃cHBSS。 </li><li>离心匀浆以125×g离心5分钟。 </li><li>而不干扰细胞沉淀除去95％的上清液用1毫升移液管。仔细重悬细胞沉淀在200微升37℃单片机与200微升枪头。 </li><li>添加1.8毫升37℃SCM与细胞悬浮液转移到一个无菌的6孔板的孔中。注：对于5只小鼠，步骤2.3.1-2.3.14将采取〜75分钟。 </li></ol></li><li>神经干细胞培养<ol><li>通过添加另一个2毫升37°补充后第3天的培养基中Ç单片机。 </li><li> 7天后，传输神经球，以15毫升管，让神经球解决了重力&gt; 5分钟。 </li><li>除去上清液，加入2mL 37℃单片机。 </li><li>转移的神经球到6孔板的一个新井。 </li><li>加入2毫升37℃单片机每隔3-4天，并改变中完全每隔7天。 </li><li>如果神经球是&gt; 100微米直径，仔细游离干细胞分离液和replate国科会在需要的细胞密度。 </li></ol></li><li>诱导基因重组<ol><li>加入1毫升37℃的SCM含有1微升Ad5CMVCre或Ad5GFAPCre&gt; 10 10 PFU / ml的给2毫升单片机目前在细胞上。注意：处理重组腺病毒使用时BSL2的安全防范措施。 </li><li>在5％CO 2温育6小时，在32℃。 </li><li>传送单片机与神经球入15ml试管并让球解决由重力5分钟。 </li><li>用1毫升吸管先取出上清液，然后用200微升吸管。注意：处理BSL2使用安全注意事项时，重组腺病毒并放弃每制度规定含有病毒的媒介。 </li><li>无病毒加入2毫升37℃单片机到NSC，然后转移到一个6孔板。注：对于5 NSC文化，步骤2.5.1-2.5.5将采取〜15分钟，不包括6小时的潜伏期。 </li><li>第二天，重复步骤2.5.1-2.5.5，接着神经球传代时必要的。该领域现在可以扩大体外表征和原位注射在小鼠的大脑在体内之前2-3代。注：确定突变的下列酶Cre-重组，通过PCR用特异于重组基因或通过免疫印迹对任一特定突变的蛋白质或它的信令后果的引物存在下进行。酶Cre-重组后需要国科会的文化表型稳定的时间将取决于上使用的突变，并且必须凭经验确定（ 图2和3）。 </li></ol></li></ol> 3，原位注射核重组细胞进​​入收件人Iimmunocompetent小鼠脑<ol><li> 5％甲基纤维素制备方法<ol><li>溶解将5克去离子水中的15厘泊的甲基纤维素于50毫升的100毫升的最终体积螺丝帽瓶中。添加的粉末慢慢地在4℃下搅拌的同时，以防止结块。它最多需要在4℃下搅拌所有的甲基纤维素进入溶液中的24小时。 </li><li>称量瓶中，并记录重量。 </li><li>高压釜中加入5％的甲基纤维素溶液。一旦蒸压，甲基纤维素将成为半固体凝胶。 </li><li>称量瓶中，并计算在高压灭菌过程中丢失的重量。 </li><li>在无菌条件下加入1毫升无菌水为每克重量的丢失。 </li><li>放置于冰上，加入50毫升冰水中冷的2×DMEM。&lt;/ LI&gt; <li>隔夜混合使用磁力搅拌器在4℃。使用无菌技术来划分的5％甲基纤维素溶液20毫升等分试样。店内分装在4℃下长达六个月或直至2倍的DMEM到期。注：在5％的甲基纤维素溶液中的步骤制备3.1.1-3.1.7将采取〜2.5天。 </li></ol></li><li>注射制剂皮质星形胶质细胞<ol><li>嘉实星形胶质细胞时〜90％汇合。 </li><li>收获，抽吸完全DMEM并用37℃的HBSS等体积的洗涤板。 </li><li>加入足够的胰蛋白酶覆盖板。轻轻倾斜和酶标板，直到细胞开始分离。 </li><li>胰蛋白酶失活与37°等体积的C完整的DMEM。 </li><li>转移细胞到50ml锥形管中，用37℃的完全DMEM同一体积中3.2.4用于洗涤平板。 </li><li>洗涤介质转移到含有细胞的试管中。离心机在200×g离心3分钟。 </li><li>吸Šupernatant和悬浮细胞在1毫升37°C完整的DMEM。 </li><li>计数使用血球或其他适当的细胞计数器，以确定细胞/微升数量的细胞悬液。 </li><li>单元的期望数量转移到一个新的15毫升锥形管中。离心机在200×g离心3分钟。 </li><li>悬浮细胞在800微升37℃，完全DMEM。确定细胞沉淀的体积（细胞悬浮液的总体积 - 800微升）。注：重要的是要实现精确的细胞浓度注射。因此，将细胞沉淀的体积必须在此步骤，以便计算的5％甲基纤维素的量，以在步骤3.2.12添加确定。 </li><li>转移细胞到无菌的1.5 ml离心试管中，然后离心分离，在200×g离心3分钟。 </li><li>从细胞沉淀中吸出上清，重悬在冰冷的5％甲基纤维素的适当量的星形胶质细胞（所需的总体积 - 体积将细胞沉淀物），得到细胞/微升的期望数目。 </li><li>将细胞置于冰上，直到注射。 </li><li>将250μL玻璃注射器插入重复抗原饮水机然后把钝18G的针头到注射器中。 </li><li>在皮质星形胶质细胞悬液针拔出柱塞慢;会有气泡以上，必须除去的细胞中，由于气泡将压缩和减少实际注入到大脑的体积。 </li><li>持针刚好高于皮质星形细胞悬浮液的前端和推动柱塞在很快。气泡会移动比细胞悬浮液更快，将大部分排出。 </li><li>重复步骤3.2.16，直到所有气泡从针取出。 </li><li>填充注射器约〜200微升，然后按住针上拉活塞，直到其停止。小气泡可以由保持针尖和迅速推动柱塞在约5被移除0微升然后慢慢退出来满负荷生产。 </li><li>保持尖端直立并重复步骤4-5倍3.2.18。 </li><li>弃18号针头和适应27G的针头到注射器中。 </li><li>按下抗原饮水机的按钮，直到液体从针头排出。注：1星形胶质细胞，步骤3.2.1-3.2.21将采取〜60分钟。 </li></ol></li><li>注射制剂国科会<ol><li>当神经球是&gt; 100微米直径，转移至15ml试管，让神经球解决了重力&gt; 5分钟。 </li><li>除去上清液，并解离用温和解离试剂的神经球，例如干细胞解离溶液，或根据制造商的说明书或％0.05胰蛋白酶-EDTA作为描述42的Accutase。 </li><li>根据不暴露NSC血清制造商的说明抑制解离试剂。离心机在100×g离心5分钟。 </li><li&gt;吸出上清液，再重悬细胞在1毫升37℃cHBSS生成一个单细胞悬液。 </li><li>算使用血细胞计数器或另一合适的细胞计数以确定细胞/微升的数目的细胞。 </li><li> NSC所需数量转移到新的15 mL锥形管。离心机在100×g离心5分钟。 </li><li>重悬在800微升37℃HBSS细胞。确定细胞沉淀的体积（细胞悬浮液的总体积 - 800微升）。注：重要的是要实现精确的细胞浓度注射。因此，将细胞沉淀的体积必须在此步骤，以便计算的5％甲基纤维素的量，以在步骤3.3.9添加确定。 </li><li>将细胞转移到无菌的1.5 ml离心试管中，然后离心分离，在100×g离心5分钟。 </li><li>吸出上清液，并最终制成细胞原位注射在3.2.12-3.2.21。注：1 NSC细胞系，步骤3.3.1-3.3。9将用时约60分钟。 </li></ol></li><li>注射制剂的鼠标<ol><li>通过腹腔注射250毫克/千克阿佛丁（2,2,2三溴乙醇）或100毫克/公斤氯胺酮加10毫克/公斤甲苯噻嗪，每个机构IACUC准则麻醉受体动物。注：本文所用的小鼠在3-6个月的年龄移植主机。小鼠在不同的年龄可被利用来研究发育大脑年龄对肿瘤发生的微环境的影响。 </li><li>剃过的头皮切口部位用指甲刀或剪刀。 </li><li>带3交替的70％乙醇和聚维酮碘的棉签消毒手术部位。 </li><li>应用眼药膏眼睛，防止因眨眼反射损失的任何损害。 </li><li>评估麻醉踏板撤离（趾捏）反射的深度。注：对于5只小鼠，步骤3.4.1-3.4.5将采取〜15分钟。 </li></ol></li><li>原位移植<ol><li>确保动物在立体定位架。 </li><li>麦ê切口在矢状缝耳朵和眼睛之间约为0.5厘米长。 </li><li>找到冠状面和矢状缝（前囟门）的交汇点，以及人字和矢状缝（拉姆达）的交叉点。确保前囟和Lambda是在同一水平面上。 </li><li>附上含有细胞悬浮液，并重复抗原分配器的立体框架的头注射器。把27G的针尖与前囟门接触。这是将用于三维坐标的操作的起点。 </li><li>提高针稍微偏离颅骨表面移动2毫米横向和前囟门1毫米喙。 </li><li>通过头骨的前囟门目的地4毫米腹侧小心地将针头。注意：我们利用这些坐标立体定向到目标基底神经节。不同的立体定向坐标可以被利用来研究不同的微环境的影响大脑区域对肿瘤的发生。 </li><li>注入5μl的细胞悬浮液通过激活的重复抗原分配器一次。离开针就位，持续2分钟，以允许颅内压力达到平衡。 </li><li>拔出针头慢慢地过了一段30秒。用棉签将压力施加到任何可能发生的出血。 </li><li>接近伤口边缘，并使用组织粘合剂关闭切口。辖20μL利多卡因皮下的切口部位。注：对于5只小鼠，步骤3.5.1-3.5.9将采取〜50分钟。联合国军司令部IACUC批准术后仅使用局部麻醉。咨询当地的机构IACUC建议就在此过程后，术后镇痛药的使用要求。 </li></ol></li><li>手术后的护理<ol><li>将动物在一个干净，温暖的笼子收回。动物不应该被直接放到床上用品，因为可能会发生意外的愿望。 </li><li>观察动物恢复正常的行为，如梳洗，进食，排便。注：正常的行为恢复可能需要〜60分钟。 </li></ol></li></ol>

The authors have nothing to disclose.

最关键的步骤，以确保适当的收获和培养星形胶质细胞的有：1）切除皮质而不采取组织下面的胼胝体，2）以除去脑膜，3），彻底解离的细胞，以及4）以富集GFAP +星形胶质细胞。虽然我们使用机械（摇动）和遗传（遗传重组的限制与显性转化的转基因的Ad5GFAPCre载体或利用（TgGZT 121）根据GFAP启动子控制）的方法来富集GFAP +胶质细胞，其他的技术已被用来净化?...

星形细胞瘤是最常见的原发性脑肿瘤和胶质母细胞瘤（GBM），IV级星形细胞瘤，是最常见的和积极的亚型12-15 1,2个月，中位生存期。弥漫性星形细胞瘤，尤其是大紫荆勋贤，入侵排除手术完全切除，限制了辅助治疗的效果，并不可避免地导致治疗后复发3。患者最初存在要么从头（主）GBM或更低级别星形细胞瘤不可避免地发展到（二级）GBM 4。 GBM是基因组异质性和基因支配三个核心信号通路特征是相互排斥的，共同发生突变：在G1 / S 期 （RB）细胞周期检验点，受体酪氨酸激酶（RTK）和TP53途径5-7。 GBM由四个基因与亚型，类似于大脑不同的细胞类型不同的表达模式，表明GBM亚型是influ原产6,8,9其小区enced。更好的星形细胞瘤模型须在星形细胞瘤的发病机制来定义在特定细胞类型的突变的特定组合的作用。利用这些模型进行更有效的临床药物开发将最终有助于改善患者的预后。当前星形细胞瘤模型包括已建立的人细胞系，来自患者的异种移植物（PDX），遗传修饰的正常人星形细胞和神经干细胞（NSC），以及基因工程化小鼠（GEM）10-14。我们开发了一个替代方案，非生殖细胞创业板（nGEM）模型采用15原发性脑细胞-星形胶质细胞和NSC -从创业板窝藏两侧装接loxP致癌等位基因的不同组合收获。我们的目标是生成星形细胞瘤模型与遗传限定的细胞可以被表型特征在于在体外和在体内和潜在地用于临床药物开发第immune能力的小鼠。 建立的人细胞系是最常用的模型星形细胞瘤的发病机制及在体外和体内的药物反应，它们在技术上是简单的，广泛使用的，并且已经在免疫缺陷小鼠10,11,16-18限定在原位异种移植动力学和致瘤性。其缺点包括不能产生从低级别星形细胞瘤的细胞株，限制研究仅向高级别星形细胞瘤;缺乏定义的起源细胞;的复杂基因组的异常，通常与明显不同的原患者样品的基因组谱的存在;和易感性血清11,17,19-22串行 ​​培养过程中的表型和基因型漂移。在建立人类GBM细胞系个人致癌基因突变的表型结果可以通过多种异常，这实际上是目前，这往往妨碍eluc被屏蔽idation直接基因型 - 表型的影响。 通过病人隔离星形细胞瘤细 ​​胞在免疫缺陷小鼠中，或通过它们的培养中所定义的无血清培养基中之前，原位注射非粘附球状体到免疫缺陷小鼠12,23的脑部皮下通道产生PDX。 PDX更准确地保持人体星形细胞瘤的基因组景观，而是类似建立的人细胞系中，个体的致癌基因突变的表型效果可以由于它们的基因组的复杂性19,24所掩盖。要定义特定的致癌基因突变表型的后果，特别是在应对新的治疗方法，建立了人类细胞系或PDX的面板常常用来建立基因型 - 表型的相关性，显示出普遍性，并尽量减少对细胞的特定影响的可能性。虽然PDX准确地概括星形细胞瘤，INC。的病理特点泸定入侵，建立人类细胞系原位移植瘤一般不21,23,25。另外，正常人星形细胞和NSC已被基因工程化以定义的致癌基因突变，以星形细胞瘤肿瘤的体外和体内 13,14,26建模。这些细胞缺乏建立的人细胞系和PDX的基因组的复杂性，准确地概括人星形细胞瘤组织病理学，但需要异种移植在体内的免疫缺陷的啮齿动物。因为所有的人细胞模型需要免疫缺陷啮齿动物宿主以防止免疫介导的异种移植排斥反应，这些模式不能概括同系系统的天然肿瘤 - 基质相互作用和缺少一个完整的免疫系统，从而限制基质靶向和免疫调节的临床前调查治法10,11。 致癌墓塔预定组合的表型后果创业板证考试期间原位肿瘤发生系统蒸发散体内 。而非条件GEM有所有组织中的突变在整个发展，有条件GEM已经两侧装接loxP的致癌等位基因，使突变的，通过使用细胞类型特异性启动子10,11,15,18限制酶Cre介导的重组到特定的细胞类型定位。有条件的星形细胞瘤GEM已利用一个完整的脑11内阐明在不同的细胞类型中的致癌基因突变的功能角色。 原位 gliomagenesis使用条件GEM的临床前程序是由若干因素，包括1）缺乏体外互相关联，2）难以产生小鼠的大样本与复基因型的原位肿瘤的发展，3）长潜伏期不限，4）和随机肿瘤进展。因为原位肿瘤缺乏对应的体外模型中， 在体外药物测试无法进行瓦特第i个传统的条件创业板模型。在对比其它癌症，脑星形细胞瘤的条件GEM模型由单个致癌基因的突变11很少被诱导。因此，复杂的育种计划必须产生具有多个致癌基因突变的条件GEM。此外，星形细胞瘤起始时具有可变的外显率在这些模型中很长的潜伏期后，而进展到高级别星形细胞瘤一般发生在一个非均匀，随机的方式，最终会引起肿瘤的复杂基因组的风景和快速生长动力学27 28。可变外显率和恶性进展的随机性在有条件的创业板模型要求，个别小鼠经X线影像学筛查的临床前药物试验入组前检测的高级别星形细胞瘤的存在和位置。综合起来，这些限制阻碍了需要公关的产生和创业板条件的大型同伙测试电子临床药物试验。 将RCAS-TVA GEM系统，它利用禽逆转录病毒（RCAS）载体来感染GEM改造以表达病毒受体（TVA）上的特定神经细胞类型中，已被广泛用于模拟星形细胞肿瘤11。相反，创业板的条件，该模型系统能够引进特定细胞类型的多种致癌突变没有复杂的育种计划的要求。然而，它是由可变外显率，为积极分裂的细胞来实现病毒整合的要求，并在随机插入 ​​转基因到宿主基因组29限定。 非种系GEM（nGEM）模型，其中利用细胞从GEM收获，正变得越来越重要，因为它们克服了许多其他模型系统15的限制。发起于星形细胞瘤发病机制的细胞类型和共同发生突变的作用都难以阻止矿用建立的人GBM细胞系或PDX因为它们是从恶性进展的过程中，已经积累了大量的基因突变中未定义的细胞类型，终末期肿瘤衍生的。相比之下，星形细胞瘤各年级可以使用nGEM通过诱导特异性纯化脑细胞类型11,30中定义的基因突变进行建模。因此，特定的基因突变和在细胞和分子表型细胞类型的影响，可以在体外和体内测定。类似建立的人GBM细胞系中，使用nGEM初始体外药物测试可用于优先级药物的体内试验采用相同的细胞。肿瘤在体内然后可通过同种异体移植nGEM细胞原位成的免疫能力的同基因同窝30的大脑来确定。因此，这些原位移植模型允许在体内试验，不仅传统的细胞毒性á次有针对性的治疗，但免疫调节和基质靶向治疗为好。最后，对肿瘤的发生和发展的微环境的作用，可通过使用相同的突变在相同的细胞类型进行比较nGEM和常规GEM模型之间的结果来确定。 我们和其他人已经开发出星形细胞瘤nGEM使用原代细胞-星形胶质细胞，国科会，还是少突胶质前体细胞（OPC） -由创业板30-34收获。后面的星形细胞瘤nGEM发展的理论基础是建立一个模型，以确定在特定的细胞类型的致癌基因突变，可能在免疫感受态的动物可用于体外临床药物试验和体内表型的影响。我们收获的野生表型星形胶质细胞和NSC非Cre表达，维持在&gt; 94％的C57 / BL6背景，两侧装接loxP Rb途径有条件的创业板-皂苷Rb1 的loxP / loxP位 ，或TgGZT121 -和两侧装接loxP RTK / RAS / PI3K通路- NF1 的loxP / loxP位 ， 嘉仕G12D和PTEN 的loxP / loxP位 -基因的不同组合35-39。我们采用编码Cre重组酶的腺病毒载体诱导的基因重组体外 。因为皮质星形细胞收获含有细胞类型的混合物中，我们使用Ad5GFAPCre载体或显性致癌基因的转基因，如TgGZT 121从人GFAP启动子驱动的，以富 ​​集在这些培养物GFAP +星形胶质细胞。我们定义G 1 / S的表型结果（RB），MAPK和PI3K途径的突变在星形胶质细胞和NSC 的体外和体内 。 MAPK和PI3K途径活化的G1 / S期缺陷型星形胶质细胞的分子拟仿人体前神经GBM，一经原位注射，在一个预先定义的位置的均匀生长动力学，短的等待时间形成的肿瘤，和组织病理学人标志人类GBM 30。 体内纵向监测肿瘤的生长有助于治疗反应40通过治疗组群和肿瘤生长的定量分析的归一化的临床前药物测试。我们通过注射荧光素酶表达星形胶质细胞的小鼠的纵向生物发光成像确定肿瘤的生长动力学。因此，星形胶质细胞和NSC从创业板有条件衍生为的星形细胞瘤相关的基因突变功能的后果和临床前药物开发的潜在模型系统定义中的易处理的模型系统。

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:241px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:233px;">Comments/Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">Dulbecco&#39;s Modified Eagle Medium (DMEM) (1X)</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">11995065</td>
			<td style="width:233px;">DMEM can also be purchased from other suppliers including GIBCOSigma and Cellgro</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Fetal Bovine Serum, Regular</td>
			<td style="width:191px;">Cellgro</td>
			<td style="width:119px;">35-010-CV</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Penicillin-Streptomycin</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">15140122</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Sharp-Pointed Dissecting Scissors</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">08-395</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Cartilage Thumb Forceps, Curved</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">1631</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Miltex 17-301 Style 1 Jeweler Style Forceps, Fine, 4</td>
			<td style="width:191px;">Miltex</td>
			<td style="width:119px;">17-301</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Ethanol (200 proof)</td>
			<td style="width:191px;">Decon Labs</td>
			<td style="width:119px;">2710</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Razor Blades</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">55411-050</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">14175-095</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">TrypLE Express (1X), Phenol Red</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">12605-010</td>
			<td style="width:233px;">Other Trypsin solutions are also suitable for cortical astrocyte havest and culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">CULTURE DISH, 60 x 15 mm</td>
			<td style="width:191px;">Thomas Scientific</td>
			<td style="width:119px;">9380H77</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">15 ml tubes</td>
			<td style="width:191px;">BD Biosciences</td>
			<td style="width:119px;">352096</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">50 ml tubes</td>
			<td style="width:191px;">BD Biosciences</td>
			<td style="width:119px;">352070</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">Adenovirus stock</td>
			<td style="width:191px;">Gene Transfer Vector Core, U. Iowa</td>
			<td style="width:119px;">Ad5CMVCre</td>
			<td style="width:233px;">Store in 5 &#181;l aliquots at -80 C. Hazardous.&#160; Use Bsl2 safetyy&#160; precautions</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Sodium sulfate (Na2SO4)&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">238597</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Potassium sulfate (K2SO4)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">221325</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Magnesium chloride (MgCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Calcium chloride (CaCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">746495</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">HEPES potassium salt</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">H0527</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">D-(+)- Glucose</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Phenol Red</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">P3532</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Sodium hydroxide (NaOH)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5881</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Papain</td>
			<td style="width:191px;">Worthington</td>
			<td style="width:119px;">LS003127</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">L-Cysteine-HCl</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1276</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Syringe filter (0.22 &#181;m pore size)</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">SLGP033NS</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:241px;">Neurocult proliferation kit, mouse</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">5702</td>
			<td style="width:233px;">This kit contains the NeuroCult NSC Basal Medium and NeuroCult NSC supplement needed for NSC culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">0.2% Heparin solution</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">7980</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">EGF&#160;</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">PMG8041</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">bFGF&#160;</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">PHG0261</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hanks&#39; Balanced Salt Solution (HBSS) (10X)</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">14185-052</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Magnesium sulfate (MgSO4)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Sodium bicarbonate (NaHCO3)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">E-64</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">E3132</td>
			<td style="width:233px;">Make 10 mM stock in DMSO, store at -20 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">6-well plates</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-83</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Cell strainer (40 &#181;m pore size)</td>
			<td style="width:191px;">Corning</td>
			<td style="width:119px;">352340</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Stem cell dissociation solution</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">5707</td>
			<td style="width:233px;">Alternatively, use gentle enzyme solutions such as Accutase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Methyl cellulose 15 cP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7027</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Dulbecco&#39;s Modified Eagle&#39;s Media 2X</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">SLM-202-B</td>
			<td style="width:233px;">For making 5% methyl cellulose solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">1.7mL Snap Cap Microcentrifuge Tube</td>
			<td style="width:191px;">Corning</td>
			<td align="right" style="width:119px;">3620</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hamilton syringe, 250 &#181;l LT no needle</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">14-815-92</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">PB600-1 Antigen Dispenser</td>
			<td style="width:191px;">Hamilton</td>
			<td style="width:119px;">&#160;83700</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Disposable 18 ga needles&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9015638&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">27 ga 1/2&#34; luer tip needle</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">14-826-48</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">2,2,2-Tribromoethanol (Avertin)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">T48402</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Betadine</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9386574</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Puralube Opthalmic Ointment</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9689910</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">&#160;Model 900 Stereotaxic frame</td>
			<td style="width:191px;">Kopf Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">VETBOND</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9259532&#160;</td>
			<td style="width:233px;">Tissue adhesive&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Lidocaine&#160;</td>
			<td style="width:191px;">ShopMedVet</td>
			<td style="width:119px;">RXLIDO-EPI</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)</td>
			<td style="width:191px;">Promega</td>
			<td style="width:119px;">G3580</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Anti-Sox2</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">AB5603</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Polyclonal Rabbit Anti-Glial Fibrillary Acidic Protein (GFAP)</td>
			<td style="width:191px;">Dako</td>
			<td style="width:119px;">Z0334&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">IVIS Kinetic</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td style="width:233px;">For in vivo imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">D-Luciferin - K+ Salt Bioluminescent Substrate</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;">122796</td>
			<td style="width:233px;">For in vivo bioluminescence imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">EdU Imaging Kit</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">C10340</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">MSCV Luciferase PGK-hygro</td>
			<td style="width:191px;">Addgene</td>
			<td style="width:119px;">18782</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling astrocytoma pathogenesis in vitro and in vivo using cortical astrocytes or neural stem cells from conditional, genetically engineered mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

我们开发了一种nGEM模型系统与星形胶质细胞和NSC从新生儿GEM窝藏收获的两侧装接loxP的条件致癌等位基因可表型特征在于在体内和体外 （ 图1）。为了研究的致癌基因突变特异性的体外大鼠星形胶质细胞的影响，它给第一个丰富 ​​的星形胶质细胞是至关重要的。皮质星形胶质细胞的收获包含小胶质细胞，星形胶质细胞，少突胶质细胞，OPC和神经细胞的混合物，?...

Watch this Scientific Journal Video about Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice at JoVE.co

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

modeling-astrocytoma-pathogenesis-vitro-vivo-using-cortical

Research

JoVE Journal

Neuroscience

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

13.3K 次观看.  University of North Carolina School of Medicine. Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

视频: Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

科研

神经科学

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

使用扩频萧条的情节和慢性偏头痛的神经免疫信号建模<em在体外</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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 ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

成像细胞内Ca<sup&gt; 2+</sup&gt;从成年小鼠使用基因编码的钙指标纹状体星形胶质细胞信号

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

小鼠神经干细胞和脑中5-羟基甲基细胞氨酸的检测

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

使用从纤维细胞直接转换为神经元祖先细胞和分化为星形细胞的神经系统疾病体外建模

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...