1。制备<ol><li>准备甲苯噻嗪，氯氨酮溶液（氯胺酮是一种受控物质。应保持详细的记录和存储在一个安全，锁定位置的解决方案）。 </li><li>清洁和消毒设备。 </li><li>准备擦拭无菌剂，然后用无菌纸毛巾覆盖手术区，并成立了显微。 </li></ol> 2。供移植的细胞的制备<ol><li>应重悬到10万个细胞/ E L浓度的细胞，虽然只有25万细胞移植是每鼠，过量的细胞所需的注射器加载的目的（准备至少30万个接收细胞的小鼠细胞）。 </li><li>洗细胞移植的HBSS 3倍，在50 ml锥形瓶。细胞计数前的最后旋转。 </li><li>经过最后的自旋向下，倒出的HBSS，并留在小瓶倒挂位置，以防止水滴达到颗粒。 </li><li>用紫外线照射，无菌Kimwipe，干燥的内壁。不要让干的颗粒。 </li><li>管直立，慢慢的，非常微妙的细胞重悬在一半的最终所需体积的HBSS。 </li><li>测量成枪头吹打多数暂停，然后调整pipetter拨号，直到整​​个悬挂在枪头总量。这会告诉你是多少更多的HBSS所需浓度的需要。 </li><li>使加入的HBSS体积所需的金额。 </li><li>冰回原位。检查细胞的活力，如果他们必须在冰时间超过2小时。 </li></ol> 3。手术和移植的小鼠的制备<ol><li>通过腹腔注射氯胺酮（100mg/kg）和甲苯噻嗪（10mg/kg的）〜100μL剂量或等效的麻醉剂麻醉鼠标。从手术预习缝合，整个过程将需要30-40分钟。 </li><li> （可选：申请编号，彩色胶带，每个鼠标的尾部，以确保鉴定） </li><li>从腰背颈部剃须鼠标背部的面积，延长双侧从中线2厘米，与电动快船。头发应削减（它可能有必要对该地区去几次）尽可能接近。 </li><li>要除去剩余的头发，申请用纱布一层薄薄的脱毛膏（奈尔）放倒撒施。 </li><li> 1-2分钟后，轻轻用肥皂水浸湿的纱布擦拭奈尔。准备区应清洁裸露的皮肤没有任何杂散件，可以在随后的外科手术伤口的头发。 碘化钾溶液消毒准备区。 </li></ol> 4。椎板切除术<ol><li>经常变化和/或整个过程中消毒手套。位置鼠标背侧头指向左侧（如果你是右撇子）。悬垂性动物，以确保不育和，只露出剃光的面积是。请laminecomy网站的有关胸椎T8跨越到T12的垂直切口（〜1.3厘米）。 </li><li>左手举行的格雷夫钳，牢固地固定在T9（ 图1A）脊柱，解除鼠标夸大脊柱弯曲。 </li><li>使用手术刀将比分T10和T11之间的交界处，两者之间的刺状突起的空间。进一步暴露，小心杀而去揭露骨（ 见图1B，C），肌肉层的交界处。 </li><li>使用剪刀，以进一步明确的肌肉离椎板和小剪蒂周围。这将开辟一个椎骨之间的小空间。缓慢和微妙的这种差距插入一个刀片和剪刀剪断椎弓根。确保剪刀的弧度总是横向位置，远离电源线。在另一边重复上述步骤。 （参见图1D，E） </li><li>抬起椎板，显露脊髓和小心地剪断它关闭。确保不留下任何免费或锯齿状的碎骨落后。 （ 见图1楼 ） </li><li>注射前，任何血液，用无菌棉签清洁。 </li></ol> 5。注射细胞<ol><li>附加汉密尔顿注射器的针头，针螺母和干净，用清水冲洗，然后70％的乙醇几次，终于HBSS中。每个水，70％的乙醇，或HBSS中填写后插入柱塞。 </li><li>汉密尔顿注射器准备装载细胞取出柱塞和针螺母松动，拔针头从注射器中，以防止背压。确保无菌手套，小心处理针和螺母。 </li><li>装入枪头细胞15μl和尖紧紧压入注射器的后端装入注射器细胞。 </li><li>插入柱塞约5mm，然后拧紧针螺母。 </li><li>压下柱塞联合国TIL细胞悬液退出针。 </li><li>确保有注射器中无气泡和引力的梯度，以防止细胞的水平位置奠定注射器。 </li><li> laminectomized鼠标抓住spinalis背阔肌肌肉连接T8和T9（ 图2B，C），刺。 </li><li>左（垂直）的显微手臂夹紧止血钳，使老鼠的前爪在空气中，其后方的爪子轻轻抚摸无菌纸毛巾的平台，在图 2a和2b。 </li><li>将注射器的权利显微臂（70 °角）和滑动至最低位置有可能前夹紧注射器。 </li><li>稳定的鼠标，它的尾巴，寄托对纸巾和慢慢放下注射器（ 图 2B ）。 </li><li>降低对线针和针1毫米插入对面半球的背中线（ 图2C）。针尖应在靠近中央管灰质。 </li><li>慢慢注入细胞2.5μl。在加入1μl/ 5秒的速度注入。注射细胞后，等待10秒，一次收回针一转的十分之一，每10秒，直到针的线。注意细胞悬液，可能流出。 </li><li>快速缩回的注射器和脱离显微手臂。打下的注射器水平。 </li><li>释放鼠标和转移缝合表。 </li><li>重复步骤为每个鼠标5.7-5.14，直到注射器空。工具消毒（灭菌）和动物之间的针（用乙醇擦拭）。放弃细胞和重装，如果是可见的聚集。 </li><li> 5.1之间的负载步清洁的注射器。 </li></ol> 6。缝合和术后护理<ol><li>缝合切口。缝合针插入切口两侧的浅筋膜。线程串通过，拉动浅筋膜（ 图3A），从而掩盖了脊髓暴露在拆除椎板现场。不要缝合皮肤肌附着在皮肤上或背面的骨骼肌。拉动整个穿过，留下约1 / 2“。使用持针器，三节的形成和线程修剪尽可能接近的结。 </li><li>关闭切口，用两到三个订书钉（切口大小而定），皮肤（ 图 3B ）。小心地拉出皮肤远离鼠标，以避免装订底层肌肉。 </li><li>使用26G3 / 8针注入0.5毫升乳酸林格分cutaneously腰背远离切口。 </li><li>它在笼中放置鼠标。为了确保它能够舒适地呼吸，而在麻醉状态下，鼠标应该放在其副作用在笼子里，避免手术的部位和笼床上用品接触。网箱应放在加热垫。 </li><li>小鼠监测麻醉后消失，以确保出血消退，缝合线仍然封闭，老鼠恢复到手术前的流动性。 </li><li>治疗小鼠的镇痛丁丙诺啡（0.05-0.1毫克/公斤），手术后一次。 </li><li>确保受损的老鼠有足够的食物和水，矿泉水瓶都配有扩展3.5英寸的嘴和老鼠，无法走路都是手工美联储水和/或高热量的膳食补充剂（Cal的营养，Tomlyn）。 </li></ol> 7。代表性的成果： 预期的结果将缺乏在注射细胞悬液的外排和脊髓完整的外观，下面的过程识别。为此，在椎板切除术，并在注射明亮，直接鼠标的脊椎上的照明是至关重要的。最佳照明条件是促进光纤照明（ 图2A）。 <img src="/files/ftp_upload/2834/2834fig1.jpg" alt="图1"/> 图1 -椎板切除术 （a）一旦椎T9是扎扎实实地举行格雷夫钳，（B，C）得分与手术刀的T10和T11之间的脊椎，以方便入境的微型剪刀。 （四）小心地将通过空间之间的微型剪刀T10和T11（箭头和插图，E）和削减每侧蒂（破折号，E），免费背椎板。 （F）的翻转椎板rostrally，就把它砍下来。 <img src="/files/ftp_upload/2834/2834fig2.jpg" alt="图2"/> 图2。 （一） 神经干细胞注射。右臂，左手臂和汉密尔顿注射器在70角连接鼠标的止血显微常规设置。 （二）止血举行的spinalis肌连接T8和T9的刺。 （三）的针是降低通过T他中线到对面半球的灰质，中央管近端。 <img src="/files/ftp_upload/2834/2834fig3.jpg" alt="图3"/> 图3。 （一） 缝线和伤口缝合。的缝线适用于切口两侧的浅筋膜。 （二）关闭切口需要2-3订书钉（一主食上的伤口需​​要3所示）。

<ol>
	<li>Bergmann, C. C., Lane, T. E., Stohlman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronavirus+infection+of+the+central+nervous+system:+host-virus+stand-off.">Coronavirus infection of the central nervous system: host-virus stand-off.</a> Nat Rev Microbiol. 4, 121-132 (2006).</li><li>Weiner, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenge+of+multiple+sclerosis:+how+do+we+cure+a+chronic+heterogeneous+disease.">The challenge of multiple sclerosis: how do we cure a chronic heterogeneous disease.</a> Ann Neurol. 65, 239-248 (2009).</li><li>Fleming, J. O., Trousdale, M. D., Bradbury, J., Stohlman, S. A., Weiner, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+demyelination+induced+by+coronavirus+JHM+(MHV-4):+molecular+identification+of+a+viral+determinant+of+paralytic+disease.">Experimental demyelination induced by coronavirus JHM (MHV-4): molecular identification of a viral determinant of paralytic disease.</a> Microb Pathog. 3, 9-20 (1987).</li><li>Totoiu, M. O., Nistor, G. I., Lane, T. E., Keirstead, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remyelination,+axonal+sparing,+and+locomotor+recovery+following+transplantation+of+glial-committed+progenitor+cells+into+the+MHV+model+of+multiple+sclerosis.">Remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the MHV model of multiple sclerosis.</a> Exp Neurol. 187, 254-265 (2004).</li><li>Carbajal, K. S., Schaumburg, C., Strieter, R., Kane, J., Lane, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+engrafted+neural+stem+cells+is+mediated+by+CXCL12+signaling+through+CXCR4+in+a+viral+model+of+multiple+sclerosis.">Migration of engrafted neural stem cells is mediated by CXCL12 signaling through CXCR4 in a viral model of multiple sclerosis.</a> Proc Natl Acad Sci U S A. 107, 11068-11073 (2010).</li><li>Hardison, J. L., Nistor, G., Gonzalez, R., Keirstead, H. S., Lane, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+glial-committed+progenitor+cells+into+a+viral+model+of+multiple+sclerosis+induces+remyelination+in+the+absence+of+an+attenuated+inflammatory+response.">Transplantation of glial-committed progenitor cells into a viral model of multiple sclerosis induces remyelination in the absence of an attenuated inflammatory response.</a> Exp Neurol. 197, 420-429 (2006).</li><li>Blakemore, W. F., Crang, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transplantation of glial cells into areas of demyelination in the adult rat spinal cord. , (1992).</li><li>Liu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+differentiate+into+oligodendrocytes+and+myelinate+in+culture+and+after+spinal+cord+transplantation.">Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation.</a> Proc Natl Acad Sci U S A. 97, 6126-6131 (2000).</li><li>Keirstead, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+oligodendrocyte+progenitor+cell+transplants+remyelinate+and+restore+locomotion+after+spinal+cord+injury.">Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury.</a> J Neurosci. 25, 4694-4705 (2005).</li><li>Mayor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+patient+enters+trial+to+test+safety+of+stem+cells+in+spinal+injury.">First patient enters trial to test safety of stem cells in spinal injury.</a> BMJ. 341, c5724-c5724 (2010).</li></ol>

执行良好的移植，将取决于主要仔细椎板切除术和细胞注射。主要的缺陷，以避免在一个椎板切除脊髓损害。这期间可能出现的程序本身或锋利的骨碎片左下面的过程背后所造成的损害。要避免这些问题，确保弯曲的微型剪刀点总是面临着远离电源线，并仔细检查laminectomized脊椎，以确保所有的碎骨清除和其余椎体结构不公开突起或锯齿状边缘。 如前所述，外排的检测将成为可能，如果光线明亮，并直接在直射暴露脊髓注射。流出是最有可能发生30表针（与33计），如果注射过快。虽然这个协议给了我们很好的效果，有的报道不再收回针注射后8,9前的等待时间（最多5分钟） 。此外，较小的表针是可取的，但我们观察到，有些细胞是太容易溶解在通过通过33表针。 为了最大限度地提高效率，移植小组的人手四个不同的站（鼠标预习，椎板，注射和缝合）是可取的。此外，每道工序的时间应进行优化，以最大限度地减少细胞在冰上等待的时间。例如，我们四个（剂量注射器在每个负载）组移植小鼠，注射细胞的人开始加载后的第三个鼠标已经laminectomized和注射器的人准备的小鼠麻醉以下前一组的第二个鼠标后的集团一直laminectomized。在这种方式下，我们可以移植到小鼠在约3小时40的细胞（或控制媒体），即使每个鼠标将需要约30-40分钟。 目前在10个临床试验的一些中枢神经系统疾病的治疗的细胞替代疗法。有没有替代NSC移植和我们的协议为便于使用的MS的重要模式与病毒引起的脱髓鞘到小鼠脊髓神经干细胞植入体内模型，也可以很容易地适应其他车型。

<table border="1"><tbody><tr><th>试剂/设备名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> Ketaject </td><td>凤凰旗医药</td><td> NDC 57319-542-02 </td><td></td></tr><tr><td>甲苯噻嗪盐酸盐</td><td> MP生物医学</td><td> 158307 </td><td></td></tr><tr><td>奈尔</td><td>教会和德怀特有限公司</td><td></td><td></td></tr><tr><td> 10μL汉密尔顿注射器W /可拆卸针</td><td>汉密尔顿公司</td><td> 7635-01 </td><td></td></tr><tr><td>汉密尔顿针，30G或33 G，½英寸，30 °斜角</td><td>汉密尔顿公司</td><td> 7803-077803-05 </td><td>测试你的细胞的可行性，通过针头通过后，以确定最佳的压力表使用</td></tr><tr><td>微型剪刀</td><td>世界精密仪器</td><td> 555500S </td><td></td></tr><tr><td>小格雷夫钳</td><td>福斯特</td><td> 11053-10 </td><td></td></tr><tr><td>立体定向</td><td> KOPF仪器</td><td> 1772型号通用持有</td><td></td></tr><tr><td>立体定向</td><td> KOPF仪器</td><td> 1773型电极支架</td><td></td></tr><tr><td>立体定向</td><td> KOPF仪器</td><td>型号902的小动物立体定位</td><td></td></tr><tr><td>立体定向</td><td> KOPF仪器</td><td> 960型的左电极载体</td><td></td></tr><tr><td>缝合线</td><td>爱惜康</td><td> 95057-064 </td><td></td></tr><tr><td>乳酸钠林格</td><td> Hospira公司</td><td> NDC 0409-7953-03 </td><td></td></tr><tr><td>斯台普斯</td><td>精细科学</td><td> 12032-07 </td><td></td></tr><tr><td>止血</td><td>福斯特</td><td> 13010-12 </td><td></td></tr><tr><td>解剖刀，尺寸10,11 </td><td>费舍尔</td><td> 268878，268879 </td><td></td></tr><tr><td>缝合线</td><td>爱惜康</td><td> 1676G </td><td> 5-0大小，3 / 8“圆，19毫米针，45CM编织线程</td></tr><tr><td>反射7伤口剪辑撒施</td><td>福斯特</td><td> 12031-07 </td><td></td></tr><tr><td> 7毫米反射伤口剪辑</td><td>福斯特</td><td> 12032-07 </td><td></td></tr><tr><td>奥尔森的Hegar持针器</td><td>福斯特</td><td> 12502-12 </td><td></td></tr></tbody></table>

surgical transplantation of mouse neural stem cells into the spinal cords of mice infected with neurotropic mouse hepatitis virus

与鼠肝炎病毒的嗜神经JHM病毒（MHV）感染的小鼠类似脱髓鞘疾病多发性硬化症（MS）患者的病理和临床结果。我们已经表明，神经干细胞移植到患病老鼠在两个髓鞘和临床结果的显着改善的结果的脊髓。治疗慢性神经系统疾病的细胞替代疗法现在已经成为现实， 并在体内模型了解嫁接的细胞和主机组织的微环境之间的相互作用是至关重要的。此演示文稿提供了一个适应JHMV感染小鼠的脊髓细胞移植到的方法。简而言之，我们提供了一个对我的程序）神经干细胞移植前准备，二）小鼠前术后护理，三）通过椎板切除脊髓暴露，IV）立体定向神经干细胞注射，和IV）术后护理。

Watch this Scientific Journal Video about Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus at JoVE.com

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

小鼠神经干细胞分化到小鼠建立脱髓鞘脊髓移植是详细的。神经干细胞的准备工作，椎板切除胸椎9（T9），神经干细胞移植概述随着小鼠前和术后护理。

小鼠神经干细胞移植手术与器官的诱向性鼠肝炎病毒感染小鼠的脊髓

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

surgical-transplantation-mouse-neural-stem-cells-into-spinal-cords

Research

JoVE Journal

Neuroscience

23.2K 次观看.  University of California, Irvine. 小鼠神经干细胞分化到小鼠建立脱髓鞘脊髓移植是详细的。神经干细胞的准备工作，椎板切除胸椎9（T9），神经干细胞移植概述随着小鼠前和术后护理。

视频: Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

科研

神经科学

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

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

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

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

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

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

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

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

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

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

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

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

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

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

小鼠胚胎干细胞向皮质 Interneuron 前体细胞的分化

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

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

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

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

Interneuron 前体 Homochronic 移植早期小鼠脑的研究

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

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

胚胎小鼠神经干细胞的分离与培养

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

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 ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

神经干细胞向大鼠和小鼠大脑的动脉内传递：对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...