这项工作是支持由美国国立卫生研究院NINDS授予R01 NS074388 581（CL Limoli），美国加州再生医学研究所（CIRM）批准RS1 - 00413（CLL）的培训资助，CIRM TG2 - 0115丙烯酸甲酯（MMA），和CIRM格兰特的Jove支持视频文件。

我们的实验计划是在图1示意图解。 1。人类神经干细胞（NS​​Cs）的生长和移植准备<ol><li>在这项研究中使用的EnStem细胞系（EMD的微孔）。神经干细胞多能标记nestin和SOX2的表达，为高层次和低层次的多能性标志物Oct - 4的有能力分化为多种神经细胞的表型，并保持正常的核型后，表达的制造商定期验证多个通道。我们的成长条件下，这些神经干细胞显示丰富的表达Sox2和巢，并保留自己的能力来区分，如前面描述 1 。神经干细胞生长聚L -鸟氨酸（20微克/毫升）和层粘连蛋白（5微克/毫升）涂层组织培养处理的烧瓶。细胞神经扩展介质，辅以L -谷氨酰胺（2毫米）（Millipore公司）和碱性成纤维细胞生长生长因子（bFGF的; 20毫微克/毫升）。神经干细胞贴壁单层传代，隔日（1:2）作为分解剂 1 accutase 。用于移植的研究，神经干细胞在通道10以下。 </li><li> 5 - 溴-2&#39; - 脱氧尿苷（BrdU）标记神经干细胞移植前3天与4μm的BrdU增长媒体补充。为了验证BrdU标记指数（ 即 BrdU阳性细胞的百分比），细胞接种在玻片和BrdU使用标准的免疫组织化学方法检测处理。细胞移植利用常规展品标签超过90％2指数。另外，移植的人类干细胞被检测到使用人的具体核抗原标记（HuNu）2 。 </li><li>移植当天，accutase和神经的扩张媒体预加热在37℃水浴。细胞放置在孵化中期（含10微米的神经扩展媒体Y - 27632）为一小时。 Y - 27632（石抑制剂，电解二氧化锰，Calbiochem）用于改善神经干细胞移植后的生存。 </li><li>一个小时后，潜伏期媒体被删除，细胞与accutase治疗5分钟。这个简短的治疗后，加入同等体积的神经扩展媒体中的accutase和应变通过70微米的细胞滤网细胞。 </li><li>血球计数细胞，并准备1.0 × 10 5住神经干细胞在注射液（神经扩充介质含有Y - 27632，40 ng / ml的碱性成纤维细胞生长因子，20 ng / ml的BDNF的10微米），每微升。 </li><li>细胞在神经扩充媒体储存（如1.5所述），直到移植时保持冰，并应6h内使用，以尽量减少长时间冷藏细胞死亡。 </li></ol> 2。放射治疗 - 治疗规划和照射<ol><li>镇静无胸腺裸大鼠（S）被放置在实验室的核磁共振成像扫描仪在容易发生positio<html> n与卓越的最终头骨（第一头）。这3特斯拉的扫描仪是专为小动物扫描。它可以提供精美的软组织对比度高空间分辨率的横截面（轴向）图像。颅骨地区和形象厚度0.8毫米的22 T2加权像E60的集中扫描生成。这些图像提供必要的信息来确定范围内的老鼠的大脑左，右海马。 </li><li>核磁共振成像扫描（24小时），动物镇静剂[麻醉的鸡尾酒（30毫克/千克，甲苯噻嗪，2.5毫克/千克和acepromazine，1毫克/公斤氯胺酮，）]和放置在一个放射肿瘤CT扫描仪另一横断面（轴向）生成图像体积。老鼠被放置在相同或接近相同的位置，用于核磁共振成像扫描和CT的单位设置为扫描颅骨区域。一个形象厚度0.8毫米的研究106 CT图像生成成为CT治疗计划数据。 </li><li>治疗计划房委会"&gt; <li> MRI和CT图像数据被转移到蚀（瓦里安医疗系统，帕洛阿尔托，加利福尼亚）治疗计划软件通过DICOM（医学数字成像和通信）。 Eclipse是一个复杂的和市售的软件，在放射肿瘤学使用新型和外观设计的最佳辐射病人的治疗计划。它集成了器官的密度信息，提取CT图像数据，并用来计算身体内的辐射剂量分布。 </li><li>封面使用一个小的只有头部的手术悬垂性暴露的动物。牢牢插入外耳道耳酒吧内的立体框架（基准数字，徕卡myNeurolab）动物的头。时要格外小心滑入耳道耳栏的一角。门齿栏挂钩老鼠的上切牙和栏的高度调整到标准的参考点，然后再拧紧鼻子钳。 </li><li>位置中央头之间最小的横向运动（± 4毫米），以实现立体零耳酒吧。 </li><li>在这个过程中的下一步是确定要使用辐照技术。 RapidArc形式的弧调强放射治疗（IMRT）和容积调制治疗（VMAT）2高精度照射技术，使用6 MV光子束提供辐射剂量 3-6 。这些技术之间的区别是，调强放射治疗提供了一个剂量使用多个静态轨迹每个对应一个不同的方向，但更适合非常小的目标卷的目标卷接轨。 RapidArc另一方面，通过对重点目标区域（ 图4）的一个或多个弧动态提供了一个剂量。 </li><li>无论交付技术，目标剂量的信息，符合目标和重要器官的剂量约束要在Eclipse的剂量优化窗口进入。 T他的信息，以及与器官卷是用来定制的剂量分布，这是在不断变化的照射光束衰减来实现。 </li><li>一旦计划生成，Eclipse提供计算剂量叠加轴向，冠状面和矢状面图像（ 图5）以及剂量体积直方图（DVH）格式（ 图6）。在这一点上，它是计划时，必须进行评估，以确定它是否适合于交付或需要加以改进。 </li></ol></li><li>辐射过程<ol><li>一旦计划被批准交付，从CT在Eclipse治疗计划数据生成的数字重建的X光片（DRR）图像。这些骨质密度加权突出的头骨和其他骨性标志性建筑的正交图像。此后，治疗计划和DRRS发送到提供治疗通过DICOM计算机系统。 </li><li>输送系统控制在Varian三部曲直线加速器ñormally用于人类放疗。加速器配备120计算机控制的薄multileaf准直器（MLC）和诊断质量X射线成像系统，包括在主板上的成像系统（OBI）。这种提供系统上传Eclipse的具体信息，并重新配置提供计划剂量的加速器准直仪参数。 </li><li>此时老鼠准备进行辐照，镇静剂和一张纸毯覆盖，以保持它的温暖。几分钟后，大鼠，其毯子，但与颅骨暴露，被放置在治疗表用来产生CT扫描同一位置。这是一个关键的一步，因为定位精度直接相关剂量交货的准确性。 </li><li>一旦老鼠被定位正确的治疗表上，是一组正交的X射线图像使用的三部曲的欧倍德系统。这些高分辨率图像，随后融合到Eclipse生成ð DRRS使用欧倍德图像融合软件。 </li><li> X射线图像和DRRS数字匹配使用软件提供上表中的位置必须作出转变，实现图像的合作（ 图 7）注册的信息。审查信息和验证，不小心带入加速器和治疗表之间的任何冲突所造成的转变后，计算机应用的转变和治疗表自动移动到所需位置。 </li><li>在这一点上，开始照射。为了与6场调强放射治疗计划提供了10 Gy的剂量，束时间约10分钟，而两个弧RapidArc〜3分钟。治疗后，即完成交货，老鼠是从治疗室，并允许恢复保存在一个加热垫的控股笼。 </li></ol></li></ol> 3。海马内移植的人类干细胞立体定向手术<ol><li>一nimals畜牧业和手术的准备<ol class="lalpha"><li>在这项研究中，我们采用了2个月的ATN大鼠从国立癌症研究所（株0N01训练班:NIH - RNU）收购。动物被关在无菌笼，并保持在温度和光控屏障 - 12-h/12-h光/暗周期的环境。大鼠蒸压食物和水随意和照顾是每星期清洗眼睛Vetropolycin眼膏（西医供应，阿卡迪亚，CA），以防止眼部感染。 </li><li>对于立体定向手术麻醉，大鼠腹腔注射麻醉鸡尾酒（氯胺酮，30毫克/千克，甲苯噻嗪，2.5毫克/千克和acepromazine，1毫克/公斤）。完成麻醉诱导后10-15分钟，镇静监测使用扪反射（趾捏）。如果在手术过程中需要的，可给予15-20％的原剂量，以维持足够的麻醉。 </li><li>从使用电动快船头皮毛。约为手术区的200％应剃光头发，以防止感染。清洁剃光部分使用providone /碘（3倍），其次是70％的酒精前切口（3次）。 </li></ol></li><li>立体定向手术<ol class="lalpha"><li>立体定向仪器（数字显示器和帧），微电机钻，干涸的河床灭菌器和控制器应保持在层流罩，以防止在手术过程中在ATN的大鼠过程可能感染。可以使用一个嵌入式电热垫的立体舞台，在手术过程中保持温暖的动物。我们使用手术thoughout的"金佰利Safeskin紫色丁腈无菌检查手套"。这些手套是单独包装在无菌袋（货号55093）。此外，外科医生在手术过程中使用70％酒精喷雾消毒双手。 </li><li>封面使用一个小的只有头部的手术悬垂性暴露的动物。动物头插入耳BA牢牢内的立体框架（基准数字，徕卡myNeurolab）RS进入外耳道。时要格外小心滑入耳道耳栏的一角。门齿栏挂钩老鼠的上切牙和栏的高度调整到标准的参考点，然后再拧紧鼻子钳。 </li><li>中心的头部位置之间最小的横向运动（± 4毫米），以实现立体零耳酒吧。 </li><li>这些定位步骤，适用于润滑眼膏，以防止干燥和保护他们免受可能的碘酒或酒精泄漏。在手术过程中，使用这药膏至少2次。 </li><li>使用无菌手术刀沿头皮中线切开皮肤（2厘米），并清洁使用无菌棉放倒撒施。在尾鳍和颈部肌肉地区，避免损坏。 </li><li>通过解剖拉钩，保持骨膜开放和明确的软组织，用棉倾斜涂药蘸1％的过氧化氢（的PBS）。止血，至少有1英里保持坚定的压力N使用棉头涂药或纱布。直到颅骨缝线，前囟和lambda是可见的，申请公司刮干净的头骨和涂抹对皮肤的议案的议案。 </li><li>精确的立体坐标，参照前囟门，确定使用大鼠脑图谱 7 。干细胞移植是在四个不同地点使用下面的立体坐标为每个半球: <ol class="lroman"><li> Anterio后（AP）3.0毫米，从囟门，德尔梅迪奥外侧（ML）1.8毫米从中线，背腹（DV）3.2毫米从大脑表面。 </li><li> AP，3.6毫米;毫升，2.5毫米的DV，3.2毫米</li><li> AP，4.2毫米，3.2毫米毫升; DV，3.2毫米</li><li> AP，4.2毫米，3.2毫米毫升; DV，3.2毫米</li></ol></li><li>前囟门已被确定后，所有三个坐标（AP，ML和DV）应归零数字显示/控制器（数字立体框架）。然后进行标记的精确移植网站（使用上述坐标）与细点记号笔在框架上的一个小探头支架。 </li><li>通过使用脚踏板控制微电机钻（莱卡myNeurolab）的头骨上钻一个0.35毫米孔（1 / 4号牙科毛刺，碳化钒钻头）。注意避免损坏硬脑膜膜。如果出血发生在钻井过程中，适用于公司的压力，用棉倾斜涂药。请勿使用酒精或碘在演练现场，因为这将导致刺激的动物。 </li><li>大鼠双侧海马内注射，暂停注射神经干细胞在1μL，5μL汉密尔顿microsyringe（规格30）的最大使用量。根据实验的细节，本卷内的细胞的确切数量会有所不同。而要做到这一点，microsyringe被连接到一个小探头支架上的立体框架。直至到达大脑的表面（ 即脑膜），小心地插入针在颅骨的一角。此时，DV的配TE上的数字显示/控制器应归零，然后轻轻地插入到所需的深度（DV，3.2毫米）的针。等待发起的任何细胞注射前1分钟。 </li><li>注入0.25μL的体积/分钟（缓慢释放）使用一个计时器。共1μL注入后，等待从移植部位回缩针前8分钟。在此时间之后，慢慢地缩回从移植部位（0.5毫米/分钟）针针轨道，以防止通过毛细血管回流注射内容。 </li><li>按照其余的移植网站（4）每半球步骤3.2（JK）。 </li><li>从头骨取下的皮肤拉钩，用生硬的镊子轻轻地拉回来缩回的皮肤，并使用Autoclips手术缝合应用方（莱卡myNeurolab），适用于4-5无菌不锈钢剪辑。 </li><li>从框架中取出的动物后，注入镇痛（Buprenorphin，0.1毫克/千克，SC，每12h）和乳酸林格氏液（5 mL/250克成年大鼠，SC）。 </li><html><li>应该保持在一个加热垫，将它在笼中的动物回。监测动物，直到它变成自觉，然后返回其控股房间。放置一些沾在一个单独的每个动物笼中的Petri菜颗粒（动物州城）和transgel。另外，DietGel恢复（ClearH 2 O波特兰，ME）可以作为水分和养分的来源。 </li><li>监视动物在他们的恢复时间和应用镇痛（丁丙诺啡0.02毫克/千克（每2天12小时）。检查疼痛，窘迫，发红或手术部位感染的迹象。应用providone /碘或neosporin软膏，每天一次（至2日-3日），以防止可能的感染。，如果感染，疼痛或痛苦的任何迹象，坚持在12个小时的手术内的镇痛药或抗生素治疗后，获得进一步的帮助或照顾动物的工作人员咨询兽医。 </li></ol></li></ol> 4。代表性的成果: <html> iles/ftp_upload/3107/3107fig1.jpg"/&gt; 图1:我们的实验计划的示意图。 <img alt="图2" src="/files/ftp_upload/3107/3107fig2.jpg" /> 图2 CT（骨解剖）和MRI（软组织）在Eclipse软件的图像融合。匹配轴向，冠状和矢状面的部分，允许每个成像方式所产生的大鼠脑的关键解剖特点的合作注册表。 <img alt="图3" src="/files/ftp_upload/3107/3107fig3.jpg" /> 图3。轮廓的大脑海马区域。图像融合后，不包括海马的海马和大脑识别和轮廓轴向定义这些机关的具体体积地区。这些地区提供必要的解剖和卷的信息，为调整剂量分布的预期目标（S）。 图4"SRC ="/ files/ftp_upload/3107/3107fig4.jpg"/&gt; 图4。高精度照射强度调控放射治疗（IMRT）或容积调控弧形治疗（VMAT）选项使用RapidArc形式。这些技术提供了6 MV光子束为多个静态的轨迹，可以衔接在非常小的目标卷（IMRT）作为重点目标区域（RapidArc）一个或多个圆弧或动态。 <img alt="图5" src="/files/ftp_upload/3107/3107fig5.jpg" /> 图5。ECLIPSE的计算叠加轴位图像的剂量。剂量是单或双海马内照射治疗计划。 <img alt="图6" src="/files/ftp_upload/3107/3107fig6.jpg" /> 图6。日食计算剂量体积直方图。数据对比照射与非照射下单hippoc的海马体积百分比ampus的治疗方案如图。 5。 <img alt="图7" src="/files/ftp_upload/3107/3107fig7.jpg" /> 图7。图像引导放射治疗大鼠定位。正交数字重建的X光片（DRR）从CT在Eclipse治疗计划数据生成图像融合三部曲板上成像（欧倍德）系统所采取的治疗表大鼠正交X射线图像。 DRRS加权骨质密度，突出的头骨和其他骨性标志性建筑，促进合作与欧倍德图像登记。这些集的匹配提供治疗必须实现的CT和X射线图像的合作登记表中的位置的变化。 <img alt="图8" src="/files/ftp_upload/3107/3107fig8.jpg" /> 图8。立体定向手术后移植的神经干细胞的位置。在移植后1个月，动物灌注，大脑被sectioneD和用BrdU（检测移植的神经干细胞）和计数器染色与苏木染。针道（ 新台币 ，红色线），表明注射轨迹，存放在神经干细胞移植发布网站（TR），略低于胼胝体（CC）和CA1区以上。移植的神经干细胞显示广泛的整个主机海马齿状回，危险品;齿状回门区，卫生署; CA1和CA3子域;放大倍率X4 入门迁移。比例尺，200微米。

<ol>
	<li>Acharya, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+ionizing+radiation-induced+damage+in+human+neural+stem+cells.">Consequences of ionizing radiation-induced damage in human neural stem cells.</a> Free. Radic. Biol. Med. , (2010).</li><li>Acharya, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+radiation-induced+cognitive+impairment+through+cranial+transplantation+of+human+embryonic+stem+cells.">Rescue of radiation-induced cognitive impairment through cranial transplantation of human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 19150-19155 (2009).</li><li>Boyer, A., Khan, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+11.">Ch. 11.</a> Treatment planning in Radiation Oncology. , (2007).</li><li>Lawson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+application+of+IMRT+based+radiosurgery+to+treat+multiple+or+large+irregular+lesions+and+verification+of+infra-red+frameless+localization+system.">Intracranial application of IMRT based radiosurgery to treat multiple or large irregular lesions and verification of infra-red frameless localization system.</a> J. Neurooncol. 97, 59-66 (2010).</li><li>Ling, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commissioning+and+quality+assurance+of+RapidArc+radiotherapy+delivery+system.">Commissioning and quality assurance of RapidArc radiotherapy delivery system.</a> Int. J. Radiat. Oncol. Biol. Phys. 72, 575-581 (2008).</li><li>Otto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volumetric+modulated+arc+therapy:+IMRT+in+a+single+gantry+arc.">Volumetric modulated arc therapy: IMRT in a single gantry arc.</a> Med. Phys. 35, 310-317 (2008).</li><li>Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in stereotaxic coordinates. , (2007).</li><li>Hess, D. C., Borlongan, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+therapy+in+ischemic+stroke.">Cell-based therapy in ischemic stroke.</a> Expert. Rev. Neurother. 8, 1193-1201 (2008).</li><li>Hefferan, M. P., Johe, K., Feldman, E. L., Lunn, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+immunosuppressive+therapy+for+spinal+grafting+of+human+spinal+stem+cells+in+a+rat+model+of+ALS.">Optimization of immunosuppressive therapy for spinal grafting of human spinal stem cells in a rat model of ALS.</a> Cell. Transplant. , (2011).</li></ol>

没有利益冲突的声明。

相当多的研究正在进行中探索了无数的方法，干细胞可用于临床，以恢复正常功能，损坏，老化和病变组织 8 。这些努力的最终实现将需要一个详细的了解，在完好的正常组织不同的独特的微环境，嫁接细胞的行为。我们的工作已经证明，内照射的组织床，颅嫁接的神经干细胞功能恢复的认知，他们生存，迁移和分化沿神经和神经胶质谱系2。正是这些细胞是如何调解认知的恢复是目前的不确定性，但不依赖于进行了一系列精心控制的实验程序，在重现的方式。我们有详细的这些关键程序，在这里的努力，以加快缓解与相关的不良认知影响的干细胞疗法的翻译的潜力临床管理的大脑和其他形式的癌症。有可能有一个数据质量产生重大影响的其他注意事项强调如下。 的关键试剂，取决于干细胞移植为基础的疗法，而相应的，必须小心以正确的文化特征，保持无菌，并使用通过数字匹配结果的可靠性。移植的人类干细胞用BrdU标记在手术前，为客户提供跟踪它们在体内的手段之一。我们的实验条件下，移植的神经干细胞没有经过广泛的扩散，使稀释BrdU标签是没有问题的。另外，移植的人类干细胞可分辨从宿主细胞的核基质蛋白（H -国统会或hNUMA）9或人类特有的核抗原（HuNu）2，如人类的特异性标志物免疫。人类干细胞也可以标记各种荧光标记，以方便他们的身份，主机内的大脑。 注意照射参数定义精确的剂量交付技术，这里描述的模式，目前在放射肿瘤学临床实践。大脑的干细胞重现移植也很关键，是通过使用数字立体定位仪。正是micromanipulate干细胞移植在一个微量注射器像小海马脑结构的能力，大大减少了人为错误。有了这个仪器，神经干细胞移植后大鼠海马地区跨越前4个不同地点。背腹（DV）坐标测定的基础上无胸腺裸鼠（ATN）大鼠，手术过程不会造成损害海马2的经验。冠状切面显示一个通过大鼠脑海马形成包括关键结构uding齿状回（DG），齿状回门区（DH）和CA1区和CA3子域（ 图8）。移植的神经干细胞，背部，从有形的针道（新台币，红线）介绍重水复疑无路染色（褐色）细胞移植发布网站（TR）略低于胼胝体（CC），随后在整个的se​​pto迁移存放的可视化海马的时空轴。

<table border="1"><tbody><tr><td> 材料名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>数字立体定位仪w/45 °和18 ° Earbars </td><td>头颅移植手术的设置</td><td>徕卡显微系统（以前:MyNeuroLab） </td><td> 39463501 </td><td>有用的配件:与加热器和可变的电流源阶段（在手术过程中保持体温） </td></tr></tbody></table>

stem cell transplantation strategies for the restoration of cognitive dysfunction caused by cranial radiotherapy

放射治疗往往提供的只有那些患有原发性或转移性脑肿瘤临床追索。同时有利于脑照射可诱发在认知的渐进性和破坏性的下降，部分原因，可能是引起神经干细胞的枯竭。鉴于诊断患有脑癌，认知健康生活质量的患者的存活率增加，已成为越来越多的关注，尤其是在没有任何令人满意的长期治疗。 为了解决这一严重的健康问题，我们使用干细胞替代作为一项战略，以打击辐射引起的认知功能下降。我们的模型采用无胸腺裸大鼠颅照射。电离辐射是要么全脑或作为一个高度集中的光束， 通过林耳朵ccelerator（加速器）的立体定向放射外科海马交付。两天后的照射下，胡stereotaxically人的神经干细胞（hNSCs）移植到海马。大鼠评估认知，嫁接细胞存活和分化特异性标志物1和照射后4个月的表达变化。我们的认知测试范例表明，与hNSCs嫁接的动物表现出显著改善认知功能。无偏体视显示1和4个月的嫁接细胞移植后，移植细胞的数量和类型依赖的显著生存（10-40％）。广泛栽种的细胞迁移，分化沿胶质细胞和神经细胞的谱系，并表示未成熟和成熟的表型标记的范围。 我们的数据表明，从嫁接的人类干细胞衍生的直接认知利益，表明此过程中，可能有一天买得起一个很有前途的战略长期功能恢复个人受到颅radiot认知herapy。为了促进传播必要的关键程序，复制和扩展我们的研究，我们已提供书面和视​​觉在我们的实验计划的几个关键步骤的文件，强调立体radiosurgey和移植。

Watch this Scientific Journal Video about Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy at JoVE.com

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

脑肿瘤患者定期接受颅放疗，而有益的，这种治疗方法往往衰弱的认知功能障碍的结果。这种严重的悬而未决的问题，目前，临床上无追索权，并推动我们努力制定基于干细胞疗法的辐射诱发的认知递减复苏。

颅放疗引起的认知功能障碍恢复干细胞移植策略

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation ...

stem-cell-transplantation-strategies-for-restoration-cognitive

Nanyang Technological University

Research

JoVE Journal

Medicine

14.4K 次观看.  University of California Irvine. 脑肿瘤患者定期接受颅放疗，而有益的，这种治疗方法往往衰弱的认知功能障碍的结果。这种严重的悬而未决的问题，目前，临床上无追索权，并推动我们努力制定基于干细胞疗法的辐射诱发的认知递减复苏。

视频: Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

主动脉原位移植大鼠模型，研究慢性血管病变的发展

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

科研

医学

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

量化颅放疗引起的认知递减

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

鼠肾移植:移植排斥反应模型

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

对专为递送治疗因素的神经保护策略成人干细胞高通量表征

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

常温<em&gt;离体</em&gt;肾脏的灌注肾的移植保护移植前

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...