我们感谢玛丽. Vejgården 的技术援助。导致这些结果的研究得到了来自纽约干细胞基金会, 欧洲研究理事会根据欧洲联盟的第七框架方案: FP/2007-2013 神经干细胞修复 (602278 号) 和紧急救济会赠款协议的资助。30971, 瑞典研究理事会 (赠款协议 521-2012-5624, 2016-00873 和 70862601/Bagadilico), 瑞典帕金森基金会 (Parkinsonfonden), 和战略研究领域在隆德大学 Multipark (多学科研究帕金森氏症)。珍妮尔 Drouin-乌莱特由加拿大卫生研究院 (研究院) 研究金 (#358492) 提供支持, 而罗杰. 巴克则由 NIHR 生物医学研究中心赠款资助剑桥大学/Addenbrooke 医院。马林 malinparmar 是纽约干细胞基金会罗伯逊调查员。谢尔比 Shrigley 是由欧洲联盟2020地平线方案 (H2020-MSCA-ITN-2015) 资助的玛丽居里夫人-居里创新训练网络和赠款协议 No. 676408。

成人真皮成纤维细胞是从帕金森病研究和亨廷顿疾病诊所在约翰 van Geest 中心的大脑修复 (剑桥, 英国) 和使用在当地的道德批准 (REC 09/H0311/88)。有关皮肤活检取样过程的详细信息, 请参阅参考8。
1. 皮肤成纤维细胞的制备
<ol><li>使用自动细胞解冻系统或37° c 水浴, 解冻成人人真皮成纤维细胞 (aHDFs) 和板材20万每 T75 烧瓶 (计数与一个自动细胞计数) 在10毫升成纤维细胞介质 (参见表 1) 在37° c 在 5% CO2。</li>
	<li>第二天用成纤维细胞培养基进行完整的培养基改变。</li>
	<li>每3-4 天改变成纤维细胞培养基, 直到 95% 70-100。 注: 一个汇合瓶将包含大约100万细胞。aHDFs 可以冻结, 以建立一个细胞线的股票 (第2节) 或直接重新镀准备就绪 (第3节)。</li>
</ol>2. 皮肤成纤维细胞的冷冻
<ol><li>将受控速率的冷冻容器放在有冰或4° c 的冰箱里。</li>
	<li>将细胞与0.05% 胰蛋白酶 (1.5 毫升每 T75 烧瓶) 分离, 在37° c 为3-5 分钟。</li>
	<li>添加成纤维细胞培养基 (含胎牛血清 (FBS)) 中和胰蛋白酶 (每 T75 瓶每冲洗3毫升), 并通过冲洗两次烧瓶中的细胞来收集15毫升试管中的分离细胞。</li>
	<li>使用自动单元计数器计数单元格;(推荐) 每瓶冷冻约 50万 aHDFs。</li>
	<li>向下旋转 400 x g 的细胞5分钟。</li>
	<li>重1毫升的冷冻培养基中的细胞颗粒 (见表 1) 并转移到低温管中。将管子直接放入受控速率的冷冻容器中。</li>
	<li>将受控速率冷冻装置贮存在-80 ° c 过夜。第二天, 把管子转到-140 ° c 的冰箱, 直到需要。</li>
</ol>3. 重新编程的电镀 (日− 1)
注: 建议使用明胶涂层进行短期试验 (最多30天);另外, 对于长期的实验, 建议从多聚 l-鸟氨酸、纤维连接蛋白和层粘连蛋白 (韧带) 涂层开始。
<ol><li>60分钟前电镀 aHDFs 为重新编程, 大衣一个24井板与0.1% 明胶 (250 µL/井) 和孵化在37° c。</li>
	<li>在 aHDFs 上吸取成纤维细胞培养基。用 DPBS 洗一次。将细胞与0.05% 胰蛋白酶 (1.5 毫升每 T75 烧瓶) 分离, 在37° c 为3-5 分钟。</li>
	<li>添加成纤维细胞培养基以中和胰蛋白酶 (每 T75 瓶每冲洗3毫升), 并通过冲洗两次烧瓶中的细胞来收集15毫升试管中的分离细胞。</li>
	<li>在1毫升成纤维细胞培养基上, 旋下 400 x g 的细胞, 5 分钟丢弃上清, 重细胞颗粒。</li>
	<li>使用自动单元计数器计数单元格 (确保良好的质量转换检查, 细胞存活率在90% 以上与台盼蓝染色)。</li>
	<li>对于一个完整的24孔板, 准备在13.2 毫升成纤维细胞培养基中悬浮132万细胞, 以达到悬浮10万细胞/毫升培养基 (或5.5万细胞/井在550µL 成纤维细胞培养基乘以所需的井数)。</li>
	<li>从盘子里抽出明胶, 用 DPBS 洗两次。在 5% CO2中, 在每个井中添加500µL 的细胞悬浮液, 在37° c 的夜间孵育。</li>
</ol>4. 病毒转导 (0 天)
注: 使用慢颗粒需要2类设备和利用代理来中和病毒。同时强烈推荐佩戴双手套。
<ol><li>加热13.2 毫升成纤维细胞培养基到37° c。</li>
	<li>将包含转录因子Ascl1和Brn2的慢向量解冻为两个短发夹 rna (shRNA), 在室温下以静止为目标。 注意: 参考引用8;该结构可在质粒存储库中使用。有关生产慢的程序的详细信息, 请参阅参考9。</li>
	<li>添加必要的慢量, 以感染的 aHDFs 的多样性 (莫伊) 的20到培养基没有任何促转促进剂。 <img alt="Equation" src="/files/ftp_upload/56904/56904eq1v2.jpg">
</li>
	<li>用含有慢载体的成纤维细胞培养基取代24井板中的培养基 (500 µL/井), 在 5% CO2中在夜间孵育37° c。</li>
	<li>第二天, 用新鲜的成纤维细胞培养基取代水井中的培养基, 无慢载体。 注: 培养基被认为具有传染性7天, 因此, 应在病毒转导后的第一周使用适当的保护和处理程序。</li>
</ol>5. 转换单元的维护
注意: 一旦转换开始, 细胞就容易被提升;注意把盘子顶起来, 在移除介质时使用1000µL 吸管, 以避免细胞分离。
<ol><li>3天, 移除成纤维细胞培养基, 加入500µL 早期神经元转化培养基 (参见表 1)。</li>
	<li>每周两到三次, 从井中取出225µL 的旧培养基, 并加入250µL 的新鲜早期神经元转化培养基。</li>
	<li>在18天, 从每个井中取出所有介质并替换为500µL 的晚期神经元转换介质 (请参见表 1)。</li>
	<li>继续改变一半的培养基, 如以上的晚神经元转换培养基每2-3 天, 直到25天或实验终点 (图 1A)。 注: 如果实验中未对电池进行电镀, 请用 PBS 或水填充空井, 以防止介质的明显蒸发, 并尽量减少井间的变化。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>库存集中</td>
			<td>工作浓度</td>
		</tr>
<tr>
<td colspan="3">成纤维细胞培养基</td>
		</tr>
<tr>
<td>基介质</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
<tr>
<td>青霉素/链霉素</td>
			<td>1万 U/毫升</td>
			<td>100毫克/毫升</td>
		</tr>
<tr>
<td>fbs</td>
			<td>N/A</td>
			<td>10%</td>
		</tr>
<tr>
<td colspan="3">冻结介质</td>
		</tr>
<tr>
<td>成纤维细胞培养基</td>
			<td>N/A</td>
			<td>45%</td>
		</tr>
<tr>
<td>fbs</td>
			<td>N/A</td>
			<td>45%</td>
		</tr>
<tr>
<td>砜</td>
			<td>N/A</td>
			<td>10%</td>
		</tr>
<tr>
<td colspan="3">早期神经元转化培养基 (国立)</td>
		</tr>
<tr>
<td>神经分化培养基</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
<tr>
<td>青霉素/链霉素</td>
			<td>1万 U/毫升</td>
			<td>100毫克/毫升</td>
		</tr>
<tr>
<td>CHIR99021</td>
			<td>10毫米</td>
			<td>2µM</td>
		</tr>
<tr>
<td>SB-431542</td>
			<td>20毫米</td>
			<td>10µM</td>
		</tr>
<tr>
<td>脑袋</td>
			<td>100µg/毫升</td>
			<td>0.5 µg/毫升</td>
		</tr>
<tr>
<td>LDN-1931189</td>
			<td>10毫米</td>
			<td>0.5 µM</td>
		</tr>
<tr>
<td>vpa</td>
			<td>1 M</td>
			<td>1毫米</td>
		</tr>
<tr>
<td>LM-22A4</td>
			<td>20毫米</td>
			<td>2µM</td>
		</tr>
<tr>
<td>gdnf</td>
			<td>20µg/毫升</td>
			<td>2 ng/毫升</td>
		</tr>
<tr>
<td>NT3</td>
			<td>10µg/毫升</td>
			<td>10 ng/µL</td>
		</tr>
<tr>
<td>db 阵营</td>
			<td>50毫米</td>
			<td>0.5 毫米</td>
		</tr>
<tr>
<td colspan="3">晚期神经元转化培养基 (LNM)</td>
		</tr>
<tr>
<td>神经分化培养基</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
<tr>
<td>青霉素/链霉素</td>
			<td>1万 U/毫升</td>
			<td>100毫克/毫升</td>
		</tr>
<tr>
<td>LM-22A4</td>
			<td>20毫米</td>
			<td>2µM</td>
		</tr>
<tr>
<td>gdnf</td>
			<td>20µg/毫升</td>
			<td>2 ng/毫升</td>
		</tr>
<tr>
<td>NT3</td>
			<td>10µg/毫升</td>
			<td>10 ng/µL</td>
		</tr>
<tr>
<td>db 阵营</td>
			<td>50毫米</td>
			<td>0.5 毫米</td>
		</tr>
<tr>
<td colspan="3">外地资产缓冲区</td>
		</tr>
<tr>
<td>HBSS 1x [-钙-镁-酚红]</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
<tr>
<td>波塞军</td>
			<td>N/A</td>
			<td>1%</td>
		</tr>
<tr>
<td>DNAase</td>
			<td>N/A</td>
			<td>0.05%</td>
		</tr>
</tbody></table>表 1: 使用的不同媒体的组成.完整描述该协议所需的所有介质的组成, 包括成纤维细胞培养基、冷冻培养基、早期神经元转化培养基、晚期神经元转化培养基、和外地资产缓冲区。
6. 电生理记录用玻璃片
注: 建议穿上实验室大衣、护目镜、双层手套, 并在油烟罩中完成所有工作。此协议是从10改编的。
<ol><li>将玻璃片在玻璃盘底部, 而不重叠。</li>
	<li>添加一般实验室清洁洗涤剂, 以淹没所有的片, 而不会冒着在晃动时溢出的危险 (约30毫升)。以慢速2小时放置在轨道振动筛上。</li>
	<li>用蒸压去离子水洗涤六次 (30 分钟)。</li>
	<li>2小时加95% 乙醇</li>
	<li>除去乙醇, 等到片干燥。</li>
	<li>干燥后, 将片到玻璃烧杯中, 加入70% 的硝酸, 直到片沉入水中。</li>
	<li>将玻璃烧杯放入 sonicator 浴缸60分钟。</li>
	<li>除去硝酸, 用蒸压去离子水洗涤三次。 注意: 总是在水中加酸, 而不是在其他方面, 因为这会导致剧烈的反应。</li>
	<li>从烧杯中取出尽可能多的水, 并加入浓盐酸 (HCI), 直到片被淹没;旋转烧杯, 用石蜡薄膜覆盖。</li>
	<li>几种为60分钟 (47-63 赫兹, 30 W)。</li>
	<li>尽可能地从片中取出尽可能多的 HCI, 并放入适当的垃圾桶中。用蒸压的去离子水冲洗两次。</li>
	<li>把片从引擎盖上取出, 用蒸气去离子水冲洗20次 (或更多), 直到所有的 HCI 都被移除。</li>
	<li>干燥后, 将片放入无菌24井板中。</li>
	<li>把盘子放在紫外线 (UV) 灯下过夜。第二天, 片准备使用。 注: 如果使用玻璃片, 建议使用韧带涂层。简单地把一个片到一个24井板的每个井 (使用无菌钳), 并遵循下面的协议。</li>
</ol>7. 长期养殖用韧带涂料
<ol><li>用多聚 l-鸟氨酸 (500 µL/井) 将24井板涂上, 在37° c 下过夜, 5% CO2。</li>
	<li>吸入聚 l-鸟氨酸, 并等待, 直到它是干的, 足以形成滴在上面。</li>
	<li>在每个井的中心做一滴 (大约60µL) 的层粘连蛋白, 并覆盖整个表面的片。在 5% CO2中, 在37° c 下离开2ĥ45分钟。</li>
	<li>用 DPBS 洗三次。</li>
	<li>添加纤维连接蛋白 (500 µL/井), 并在37° c 5% CO2过夜。</li>
	<li>在添加单元格之前用 DPBS 清洗一次。 注: 韧带涂层可用于 iNs 的长期文化 (超过100天), 虽然渐进细胞死亡预计从30天。</li>
</ol>8. 外地资产管制
注: 在进行分类后, 重新对细胞进行重组, 预先准备好一个韧带涂层的48小时板。细胞可以被分类使用神经细胞黏附分子 (分子) 抗体从天20起跟随转导。
<ol><li>用1000µL 吸管取出介质 (不要清洗以避免细胞分离)。</li>
	<li>添加细胞游离剂 (250 µL/井), 离开10-20 分钟直到细胞提升, 并作为单细胞漂浮。</li>
	<li>在此期间准备资产管制的缓冲区。</li>
	<li>用1000µL 吸管轻轻研制。如果有些结块, 再孵育一点。</li>
	<li>一旦一个单细胞悬浮得到, 从井和地方去除它入1.5 毫升管子。</li>
	<li>冲洗井两次与晚神经元转换介质和地方到相同的1.5 毫升管。</li>
	<li>在 400 x g 处旋转5分钟, 丢弃上清液。</li>
	<li>重200µL 的细胞团缓冲。</li>
	<li>在 400 x g 处旋转5分钟的细胞并丢弃上清液。</li>
	<li>重复步骤8.8 和8.9 两次。</li>
	<li>重50µL 中的细胞颗粒, 含有人类 CD56 (分子) 抗体, 浓度为 1:50, 在冰上15分钟。免受光。</li>
	<li>向下旋转 400 x g 的细胞5分钟。</li>
	<li>重在200µL 的缓冲区, 以洗涤和旋转的细胞在 400 x g 5 分钟。</li>
	<li>重复步骤8.13。</li>
	<li>重200µL 中含有碘碘化物 (PI; 10 µg/毫升) 的外地资产管制缓冲区。根据无分子抗体或 pi 染色的对照样品的荧光强度水平, 采用门控法对分子阳性 (iNs) 和 pi 负 (活) 细胞进行分类。</li>
	<li>在含有晚期神经元转化介质的管内收集分子阳性细胞。</li>
	<li>使用自动单元计数器计数单元格, 在韧带包覆的盘中, 在晚期神经元转换介质中, 在高密度 (5万细胞/cm2) 中重新板细胞。</li>
	<li>在实验终点 (图 1A) 的情况下, 继续改变一周中2-3 次的一半, 并以晚期神经元转换介质。</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+age+in+human+neurons+generated+by+microRNA-based+neuronal+conversion+of+fibroblasts.">Maintenance of age in human neurons generated by microRNA-based neuronal conversion of fibroblasts.</a> Elife. 5, (2016).</li><li>Drouin-Ouellet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REST+suppression+mediates+neural+conversion+of+adult+human+fibroblasts+via+microRNA-dependent+and+-independent+pathways.">REST suppression mediates neural conversion of adult human fibroblasts via microRNA-dependent and -independent pathways.</a> EMBO Mol Med. , (2017).</li><li>Tiscornia, G., Singer, O., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+purification+of+lentiviral+vectors.">Production and purification of lentiviral vectors.</a> Nat Protoc. 1, 241-245 (2006).</li><li>Richner, M., Victor, M. B., Liu, Y., Abernathy, D., Yoo, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-based+conversion+of+human+fibroblasts+into+striatal+medium+spiny+neurons.">MicroRNA-based conversion of human fibroblasts into striatal medium spiny neurons.</a> Nat Protoc. 10, 1543-1555 (2015).</li><li>Lau, S., Rylander Ottosson, D., Jakobsson, J., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+neural+conversion+from+human+fibroblasts+using+self-regulating+and+nonintegrating+viral+vectors.">Direct neural conversion from human fibroblasts using self-regulating and nonintegrating viral vectors.</a> Cell Rep. 9, 1673-1680 (2014).</li><li>Pfisterer, U., Wood, J., Nihlberg, K., Hallgren, O., Bjermer, L., Westergren -Thorsson, G., Lindvall, O., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+induction+of+functional+neurons+from+adult+human+fibroblasts.">Efficient induction of functional neurons from adult human fibroblasts.</a> Cell Cycle. 10, 3311-3316 (2011).</li><li>Caiazzo, M., Dell'Anno, M. T., Dvoretskova, E., Lazarevic, D., Taverna, S., Leo, D., Sotnikova, T. D., Menegon, A., Roncaglia, P., Colciago, G., Russo, G., Carninci, P., Pezzoli, G., Gainetdinov, R. R., Gustincich, S., Dityatev, A., Broccoli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+generation+of+functional+dopaminergic+neurons+from+mouse+and+human+fibroblasts.">Direct generation of functional dopaminergic neurons from mouse and human fibroblasts.</a> Nature. 476, 224-227 (2011).</li><li>Masserdotti, G., Gasc&#243;n, S., G&#246;tz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+neuronal+reprogramming:+learning+from+and+for+development.">Direct neuronal reprogramming: learning from and for development.</a> Development. 143, 2494-2510 (2016).</li><li>Pereira, M., Pfisterer, U., Rylander, D., Torper, O., Lau, S., Lundblad, M., Grealish, S., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+generation+of+induced+neurons+from+human+fibroblasts+that+transplantation+into+the+adult+rat+brain.">Highly efficient generation of induced neurons from human fibroblasts that transplantation into the adult rat brain.</a> Sci Rep. 4, 6330 (2014).</li></ol>

作者没有什么可透露的。

这一步/单矢量重编程方法为获得成人成纤维细胞的 iNs 提供了一种有效的途径。成年成纤维细胞通常比胎儿成纤维细胞更难隐蔽, 而先前报告的效率有限, 大约 5-10%12,13。然而, 有了这个新的协议, 就有可能实现神经元的产量 (测量为 MAP2+ 细胞) 约 50%8。此外, 我们的协议可用于真皮成纤维细胞已多次传代, 而不失去效率的转换。到目前为止, 我们已经使用的细胞传代高达14倍, 而没有检测到任何降低转换效率。此外, 在52和87岁之间, 在我们手里的成纤维细胞的重新编程效率没有区别。有关使用此构造测试的其他单元格线的年龄和疾病的详细信息, 请参阅参考8。其他研究也报告了在0和89年4之间使用基于慢和小分子增强的协议在转换效率上没有差异。此外, 所有年龄的成纤维细胞都报告了与 miRNA 的神经元重新编程的一致适用性, 在0和86年之间的捐赠者为7。通过这一途径, 成熟的神经元可以获得大约12-15 周体外或大约8周后移植体内8。这是有利的, 因为它可以接触到疾病和病人特定的人从组织, 是容易接近的。虽然这个协议是有效的, 它不会产生一个100% 的神经元产量, 并作为这样一个净化步骤, 使用外地资产管制所需要的实例。
该协议中最关键的步骤是病毒转导 (协议4节)。这是至关重要的病毒滴度是精确的, 除了镀了准确数量的 aHDFs 转换。推荐使用此协议的效价介于 4 x 108和 4 x 109之间。不建议使用低于 1 x 108的任何一种效价, 因为添加大量病毒将对细胞有害。此外, 由于成纤维细胞开始向 iNs 隐蔽, 它们将变得更加脆弱, 容易被提升。在改变媒体的时候, 必须要温柔, 不要太多地打扰细胞。这可以通过去除液体缓慢与1000µL 吸管。最后, 当电镀为重编程 (协议部分 3) 在开始实验之前保证一个健康成纤维细胞的人口是重要的;这是由90% 以上的细胞生存能力与台盼蓝染色。在到达 95% 70-100 之前, aHDFs 应该总是传代的。
如果在病毒转导前有明显的细胞死亡, 不要开始转换: 双检查细胞的生存能力高于 90%, 并且没有问题与涂层的板材。它预计将有少量细胞死亡在病毒转导之后, 然而, 这不应该是重要的。在这种情况下, 确认准确播种 5万 aHDFs/井, 并检查病毒滴度。如果在转换过程中油井之间有明显的不一致, 首先检查每口井中是否含有等量的介质, 并在边缘处不发生显性蒸发 (如果需要额外的介质可以添加到边缘井中)。或者, 检查步骤 4.4, 确保在换时进行均匀悬浮的适当混合。在将慢加入到井中之前, 先将其添加到介质中是至关重要的。直接在油井中加入慢, 将会增加井的变异性, 而且对细胞也有毒性。最后, 在添加到细胞之前, 始终检查培养基是否预热到37° c。
该协议包括可选的部分, 用于长期的文化 iNs 和外地资产管制局分类, 以提高神经元纯度的涂层条件。对于希望研究 iNs 功能特性的实验, 还包括了用于制备电生理学的玻璃片的协议。此处的转换协议设置为使用24井板;如果需要, 可以修改为 6, 12, 48, 96 井板或烧瓶。在这种情况下, 请将所有的音量调整到所使用的板或烧瓶的表面积。
此协议使用Ascl1和Brn2的强制表达式, 并将所有打包在一个单矢量8中的 REST 击键组合在一起, 以生成泛神经元表型的 iNs。然而, 这种方法产生的任何神经胶质亚型都没有被评估。因此, 此方法需要修改, 以便与其他重编程因子一起使用, 以获得亚型特定神经元。直接重新编程已经显示了产生运动神经元, 感觉神经元, 光, 纹中刺神经元和多巴胺能神经元10,14的可能性。这将是有益的, 当研究神经疾病的特定神经元亚型优先受影响, 例如帕金森病和多巴胺能神经元。
直到最近, 直接神经重新编程技术不允许以标准化和高效的方式生产 iNs-到一个大规模的毒理学和药物筛选化验所需要的水平。这种新的方法是非常有效的, 可以用于成纤维细胞已被传代多次, 这样, 它现在消除这些限制, 并开辟了大量的研究, 不仅在人类神经系统, 但也在一个系统, 可以有耐心的具体。这种方法的简单性使得任何想要在内部进行类似研究的群体都能获得技术, 而且可以很容易地用于大规模的生物医学应用, 如药物筛选和毒理学检测, 同时也支持从动物模型和人类宰后组织样本获得的数据。

发展有效的治疗神经紊乱的障碍是由于有限的获得活体人脑细胞进行机械研究和功能性化验。大约十年前, 随着诱导多能干细胞 (干细胞) 技术的发展1,2, 这种情况发生了根本性的变化。这一点, 再加上对正常人类发育过程中发生的神经分化机制的更好的理解, 允许从病人和疾病的特定材料中产生定义的和多样的神经元亚型。有了这些材料, 现在可以研究神经系统疾病的细胞内机制和不同化合物的潜能, 以缓解这些病理特征3。
虽然干细胞是革命性的神经科学领域, 这些细胞的一个主要缺点是, 他们的老化签名在重新编程过程中被删除, 这样的方式, 恢复活力的神经元不保留与脆弱性相关老化4,5,6。产生的神经元的这一特殊特征可能最终对综述细胞内致病性叶栅的许多方面至关重要, 特别是在老年是一个重要的危险因素的疾病的情况下。
直接神经重新编程是一种技术, 其中体细胞是直接转化成一个在没有通过多能中间阶段。这允许快速生成的人的神经元在体外, 可以是病人和疾病的具体。直接重新编程的一个显著特点是, 供体细胞的起始年龄保持不变, 而且, 它易受衰老过程的影响, 如氧化应激的增加4,7。因此, 与老年痴呆症和帕金森氏症相关的神经系统疾病患者的 iNs 很适合广泛的生物医学应用, 包括疾病建模、药物筛选化验和毒理学研究。.
防止神经退行性疾病患者的 iNs 被广泛使用的主要告诫是, 他们不容易重新编程, 这变得更加困难的成纤维细胞的扩张。因此, 直到最近8才实现了这些类型的应用程序所需的数量的单元格生成。我们现在已经开发了一个简单的方法, 以非常有效的方式重新编程从任何年龄的捐助者的成纤维细胞。该方法将神经元转录因子Ascl1和Brn2的强迫表达式与阻遏蛋白 RE1-silencing 转录因子 (REST) 的击倒结合使用单个向量。在这里, 我们描述的不同步骤导致的 iNs 转换从皮肤成纤维细胞活检从老年捐赠者。

<table border="0" cellpadding="0" cellspacing="0" width="1603">
	<tbody>
		<tr height="40">
			<td colspan="4" height="40" style="height:40px;width:1603px;">Cell Lines</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Adult human dermal fibroblasts&#160;</td>
			<td style="width:219px;"></td>
			<td style="width:159px;"></td>
			<td style="width:756px;">[C2 passage #7] Donor was a 67 year old male. Cells obtained from the Parkinson&#8217;s Disease Research and Huntington&#8217;s disease clinics at the John van Geest Centre for Brain Repair (Cambridge, UK).</td>
		</tr>
		<tr height="40">
			<td colspan="4" height="40" style="height:40px;">Reagents for Fibroblast Culture, Transduction and Conversion</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#39;s phosphate-buffered saline (DPBS) [-CaCl2, -MgCl2]</td>
			<td>Gibco</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypsin-EDTA [0.5%]</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td>Dilute to 0.05% in DPBS.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Virkon (agent used to neutralize virus)</td>
			<td>Viroderm</td>
			<td>7511&#160;</td>
			<td>Dilute to 1% solution with warm water.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Milli-Q Water</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Basal medium - Dulbecco&#8217;s Modified Eagle Medium (DMEM) + GlutaMax</td>
			<td>Gibco</td>
			<td>61965059</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin/Streptomycin [10,000 U/mL]</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CryoMACS&#174;&#160;dimethyl sulfoxide (DMSO) 10</td>
			<td>Miltenyi</td>
			<td>170-076-303</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neural differentiation medium - NDiff 227</td>
			<td>Takara-Clontech</td>
			<td>Y40002</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LM-22A4&#160;</td>
			<td>Tocris</td>
			<td>4607</td>
			<td style="width:756px;">Dilute 10 mg in 1450 &#181;L DMSO. Stock concentration: 20 mM.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glial cell line-derived neurotrophic factor (GDNF) [recombinant human]</td>
			<td>R&#38;D systems</td>
			<td>212-GD-010</td>
			<td style="width:756px;">Dilute 10 ug in 500 &#181;L 0,1% BSA in DPBS. Stock concentration: 20 &#181;g/mL.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NT3 [recombinant human]</td>
			<td>R&#38;D systems</td>
			<td>267-N3-025</td>
			<td>Dilute 25 &#181;g in 2,5 mL 0,1% BSA in DPBS. Stock concentration: 10 &#181;g/mL.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">db-cAMP&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D0627</td>
			<td>Dilute 1 g in 40,7 mL Milli-Q water. Filter and make 500 &#181;L aliquots or stock tubes of 10 mL. Stock concentration: 50 mM.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CHIR99021</td>
			<td>Axon</td>
			<td>1386</td>
			<td>Dilute 2 &#181;g in 429,8 &#181;L DMSO. Stock concentration: 10 mM.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SB-431542</td>
			<td>Axon</td>
			<td>1661</td>
			<td>Dilute 5 mg in 595 &#181;L DMSO. Stock concentration: 20 mM.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Noggin [recombinant human]</td>
			<td>Miltenyi</td>
			<td>130-103-456</td>
			<td>Dilute 100 &#181;g in 100 &#181;L of Milli-Q water + 900 &#181;L 0,1% BSA in DPBS. Stock concentration: 100 &#181;g/mL.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LDN-193189</td>
			<td>Axon</td>
			<td>1509</td>
			<td>Dilute 2 mg in 360 &#181;L DMSO. Stock concentration: 10 mM.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Valproic acid sodium salt (VPA)</td>
			<td>Merck Millipore</td>
			<td>676380</td>
			<td style="width:756px;">Dilute 5 g in Milli-Q water to acheive a stock concentration of 1 M. CAUTION: Avoid ingestion, contact with skin, and breathing dust formation.</td>
		</tr>
		<tr height="40">
			<td colspan="4" height="40" style="height:40px;">Reagents for Coatings</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gelatin&#160;</td>
			<td>Sigma Aldrich</td>
			<td>G2500</td>
			<td>Dilute to 0.1% in Milli-Q water.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Poly-L-ornithine</td>
			<td>Sigma Aldrich</td>
			<td>P3655</td>
			<td>Dissolve in Milli-Q water. Use at 15&#181;g/mL.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fibronectin</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:159px;">33010-018</td>
			<td style="width:756px;">2 mL of Milli-Q water + 70 &#181;L 0,25 M NaOH. Use at 5 &#181;g/mL.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laminin</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:159px;">23017-015</td>
			<td style="width:756px;">Store at -80&#176;C. Thaw on ice, keep cool and aliquot 30 &#181;L. Use at 5 &#181;g/mL.&#160;</td>
		</tr>
		<tr height="40">
			<td colspan="4" height="40" style="height:40px;">Reagents for Fluorescence Activated Cell Sorting</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell dissociation agent - Accutase (Stem Pro)</td>
			<td>ThermoFisher Scientific</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hanks&#39; Balanced Salt Solution (HBSS) 1X [-calcium, -magnesium, - phenol red]</td>
			<td>ThermoFisher Scientific</td>
			<td>14175-046</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A2153</td>
			<td>Use at 1% concentration for Anti-Human CD56 (NCAM).</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNAase</td>
			<td>Sigma Aldrich</td>
			<td>DN-25</td>
			<td>Use at 0.05% concentration.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-Human CD56 (NCAM) Antibody [Mouse]</td>
			<td>BD Pharmingen</td>
			<td>555518</td>
			<td>Use at 1 : 50.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Propidium iodide</td>
			<td>Sigma Aldrich</td>
			<td>P4170</td>
			<td>Dilute to 1mg/mL in PBS and keep sterile. Use at 1:1000 to achieve a concentration of 10&#181;g/mL.</td>
		</tr>
		<tr height="40">
			<td colspan="4" height="40" style="height:40px;">Reagents for Glass Coverslips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Autoclaved deionized water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alconox detergent</td>
			<td>Sigma Aldrich</td>
			<td>Z273228</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethanol 95%</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nitric Acid</td>
			<td>Sigma Aldrich</td>
			<td>438073-M</td>
			<td>CAUTION: Concentrated nitric acid is highly corrosive, and its vapours are potentially harmful.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Concentrated hydrochloric acid (HCI)</td>
			<td></td>
			<td></td>
			<td>CAUTION: Concentrated hydrochloric acid is highly corrosive, and its vapours are potentially harmful.</td>
		</tr>
		<tr height="40">
			<td colspan="4" height="40" style="height:40px;">Reagents for Immunocytochemistry</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde (PFA)</td>
			<td>Merck Millipore</td>
			<td>1040051000</td>
			<td>Use at a concentration of 4%. CAUTION: PFA is a potent fixative. Avoid ingestion and contact with skin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>10254640</td>
			<td>Use at a concentration of 0.1%.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Serum [Donkey]</td>
			<td>Merck Millipore</td>
			<td>S30-100ML</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-MAP2 Antibody [Chicken]</td>
			<td>Abcam</td>
			<td>ab5392</td>
			<td>Use at a concentration of 1 : 5,000.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:469px;">Tau HT7 Monoclonal Antibody [Mouse]</td>
			<td>ThermoFisher Scientific</td>
			<td>MN1000</td>
			<td>Use at a concentration of 1 : 500.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cy3 Anti-Chicken Antibody [Donkey]</td>
			<td>Jackson ImmunoResearch</td>
			<td>703-165-155</td>
			<td>Use at a concentration of 1 : 400.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alexa Fluor488 Anti-Mouse Antibody [Donkey]</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-545-150</td>
			<td>Use at a concentration of 1 : 400.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:469px;">4&#8242;,6-Diamidine-2&#8242;-phenylindole dihydrochloride (DAPI)</td>
			<td>Sigma Aldrich</td>
			<td>D9542</td>
			<td>Reconstitute the powder in Milli-Q water to 1 mg/mL. Aliquot and store at -20&#176;C, light sensitive. Use at a concentration of 1 : 500.</td>
		</tr>
		<tr height="40">
			<td colspan="4" height="40" style="height:40px;">Equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">T75 flask [Nunclon Delta Surface]</td>
			<td>ThermoFisher Scientific</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">24-well plate [Nunc]</td>
			<td>ThermoFisher Scientific</td>
			<td>142485</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">1.5 mL polypropylene tube</td>
			<td>Sigma Aldrich</td>
			<td>Z336769</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">15 mL falcon tube&#160;</td>
			<td>Sarstedt</td>
			<td>62.554.502</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">50 mL falcon tube&#160;</td>
			<td>Sarstedt</td>
			<td>62.547.254</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CryoPure tube 1.6 mL</td>
			<td>Sarstedt</td>
			<td>72.380</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pippette controller</td>
			<td></td>
			<td></td>
			<td>For pipetting volumes 1-25 mL.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sterile serological pipettes: 5, 10 and 25 mL&#160;</td>
			<td>Sarstedt</td>
			<td style="width:159px;">86.1253.001, 86.1254.001, 86.1685.001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Adjustable volume pipettors: 5, 20, 200, and 1,000 &#181;L&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sterile pipette tips</td>
			<td></td>
			<td></td>
			<td>For pipetting volumes of 0.5 - 1,000 &#181;L.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass coverslips</td>
			<td>NeuVitro</td>
			<td>GG-12-1.5-oz</td>
			<td>#1.5 thickness, 12mm diameter, 0.5oz, CE certified, fit 24 well plates.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass dish</td>
			<td></td>
			<td></td>
			<td>Approximately 150mm diameter.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass beaker</td>
			<td></td>
			<td></td>
			<td style="width:756px;">Make sure to have an appropriate size beaker for the sonicator bath available. Water from the sonicator bath should not overflow into the glass beaker.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Parafilm M</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ThawSTAR Automated Cell Thawing System</td>
			<td>BioCision</td>
			<td>BCS-601</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Countess II Automated Cell Counter</td>
			<td>ThermoFisher Scientific</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:469px;">Cell counting chambers [50 slides] and trypan blue [0.4%]</td>
			<td>ThermoFisher Scientific</td>
			<td>C10228</td>
			<td>For use with Countess II Automated Cell Counter.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CoolCell Cell LX Controlled-rate Freezing Container</td>
			<td>Biocision</td>
			<td>BCS-405</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laminar flow hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Humidified 5% CO2 37 &#176;C incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Centrifuge</td>
			<td></td>
			<td></td>
			<td>Suitable for 1,5, 15 and 50 mL tubes.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Orbital shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sonicator - Bransonic Model B200 cleaner</td>
			<td>Sigma Aldrich</td>
			<td>Z305359</td>
			<td>Frequency = 50-60Hz, Amplitude = 30 Watts</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FACS Aria III cell sorter</td>
			<td>BD Pharmingen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Phase contrast microscope</td>
			<td>Olympus</td>
			<td>CKX31</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Inverted fluorescence microscope</td>
			<td>Leica</td>
			<td>DMI6000 B</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple generation of a high yield culture of induced neurons from human adult skin fibroblasts

诱导神经元 (iNs) 是直接转化为神经元的体细胞的产物, 是一种从组织中获得患者源性神经元的方法, 易于获取。通过这条路线, 成熟的神经元可以在几个星期内得到。在这里, 我们描述了一个简单和快速的一步协议, 以获得从真皮成纤维细胞通过活检样本从成人捐赠人。我们解释的每一个步骤的过程, 包括皮肤成纤维细胞的维护, 冻结程序建立一股细胞线, 种子细胞的重新编程, 以及文化条件在转换过程中。此外, 我们还介绍了用于电生理记录的玻璃片的制备, 长期涂层条件, 以及荧光活化细胞分类。我们还举例说明了预期的结果。这里描述的协议很容易执行, 可以应用于人皮肤成纤维细胞从不同的诊断和年龄的患者皮下活检。该协议产生了足够数量的 iNs, 可用于广泛的生物医学应用, 包括疾病建模, 药物筛选, 和目标验证。

从5天起 (图 1B), 细胞形态学的明显变化应该是可见的。一些细胞死亡是在病毒转导后, 虽然不是公开的。从每个井在一个24井板的总细胞产量2000万细胞应该是预期的25天, 其中大约一半应该已经成为神经元。重要的是要注意, 产量和纯度可以在不同的细胞线, 以及与疾病的状态和病毒批次。
细胞将表达最标准的神经元标记, 包括 MAP2 和 TAU (图 1C) 在25天, 除了展示一个成熟的神经元形态学。通过基于标记分子 (参见8,11) 进行的资产管制系统, 可以获得一个纯净的人口。此后, 细胞可以重新镀在韧带三重涂层上 (参见参考10) 或直接冷冻进行生物分子分析。
如果未对细胞进行分类, 则应进行 MAP2 或 TAU 的免疫荧光标记, 以识别成功转换的细胞并与感兴趣的蛋白共同标记。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56904/56904fig1.jpg"> 图 1: 随时间推移的转换过程.(a) 用于对成人真皮成纤维细胞进行重新编程的慢的实验和结构图的时间轴。每个黑色箭头代表一个中等的变化。(B) 具有代表性的相位对比度图像, 描述在0天到22天之间转换过程中细胞形态学的变化 (如每个面板的右上角所示)。利用10X 目标在相衬显微镜上拍摄图像。(C) 头和 MAP2 双染色的免疫荧光图像在35天后转导。细胞被固定在4% 甲醛和化与0.1% 海卫一在 DPBS 10 min. 细胞被阻拦了 30 min 在5% 血清解答在 DPBS。抗体被稀释在阻断溶液中, 并在4° c 夜间应用。荧光结合二次抗体被稀释在阻断液中, 并应用于 2 h 细胞1-4 与 DAPI 15 分钟, 其次是3洗 DPBS。图像采用倒置荧光显微镜使用20X 目标。缩放条形图 = 100 µm (B、C)。缩写: 国立: 早期神经元介质;LNM: 晚期神经元介质。<a href="//cloudflare.jove.com/files/ftp_upload/56904/56904fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts at JoVE.com

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

直接的神经元重编程产生的神经元保持起始体细胞的年龄。在这里, 我们描述了一个单一的矢量的方法, 产生诱导神经元从真皮成纤维细胞获得从成人捐赠人。

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

simple-generation-high-yield-culture-induced-neurons-from-human-adult

Research

JoVE Journal

Neuroscience

10.2K 次观看.  Lund University. 直接的神经元重编程产生的神经元保持起始体细胞的年龄。在这里, 我们描述了一个单一的矢量的方法, 产生诱导神经元从真皮成纤维细胞获得从成人捐赠人。

视频: Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Organotypic Culture of Full-thickness Adult Porcine Retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

器官型培养的全厚成年猪视网膜

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

谱系重编程的成人大脑的周细胞源性细胞诱导成神经细胞

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

96井格式人诱导多潜能干细胞源神经元高速拉伸损伤的方法

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment. Despite vast differences in the physiology between mammals and fish, primary cell culture protocols from fish are often based on mammalian culture conditions, often with only minor modifications. The environmental differences affect not only body temperature, but also blood serum parameters such as osmolality, pH, and pH buffer capacity. As cell culture media and similar working solutions are meant to mimic characteristics of the extracellular fluid and/or blood serum to which a cell is adapted, it is crucial that these parameters are adjusted specifically to the animal in question.
The current protocol describes optimized primary culture conditions for medaka (Oryzias latipes). The protocol provides detailed steps on how to isolate and maintain healthy dissociated pituitary cells for more than one week and includes the following steps: 1. the adjustment of the osmolality to the values found in medaka blood plasma, 2. the adjustment of the incubation temperature to normal medaka temperature (here in the aquarium facility), and 3. the adjustment of the pH and bicarbonate buffer to values comparable to other fish species living at similar temperatures. The results presented using the described protocol promote physiologically meaningful results for medaka and can be used as a reference guide by scientists making primary cell cultures from other non-mammalian species.

Here we describe a protocol to prepare and maintain primary pituitary cell cultures from medaka (Oryzias latipes). The optimized conditions in this protocol take important parameters such as temperature, osmolality, and pH into consideration by mimicking the physiological conditions of the fish, thereby enabling physiologically more meaningful results.

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

摘要

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此视频中的章节

从胚胎脊髓连合神经的解剖和文化

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

器官型培养的全厚成年猪视网膜

从产前小鼠海马神经元的分离和培养

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

谱系重编程的成人大脑的周细胞源性细胞诱导成神经细胞

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

一个简单的一步解剖协议成人全安装准备

96井格式人诱导多潜能干细胞源神经元高速拉伸损伤的方法

从鱼类垂体制备优质原代细胞培养

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

摘要

探索更多视频

此视频中的章节

从胚胎脊髓​​连合神经的解剖和文化

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

器官型培养的全厚成年猪视网膜

从产前小鼠海马神经元的分离和培养

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

谱系重编程的成人大脑的周细胞源性细胞诱导成神经细胞

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

一个简单的一步解剖协议成人全安装准备

96井格式人诱导多潜能干细胞源神经元高速拉伸损伤的方法

从鱼类垂体制备优质原代细胞培养

从胚胎脊髓连合神经的解剖和文化