Name: simple generation of a high yield culture of induced neurons from human adult skin fibroblasts
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Description: Watch this Scientific Journal Video about Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts at JoVE.com

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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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><tbody><tr><td>&lt;strong&gt;Cell Lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adult human dermal fibroblasts&amp;nbsp;</td><td></td><td></td><td>[C2 passage #7] Donor was a 67 year old female. Cells obtained from the Parkinson&amp;rsquo;s Disease Research and Huntington&amp;rsquo;s disease clinics at the John van Geest Centre for Brain Repair (Cambridge, UK).</td></tr><tr><td>&lt;strong&gt;Reagents for Fibroblast Culture, Transduction and Conversion&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS) [-CaCl&lt;sub&gt;2&lt;/sub&gt;, -MgCl&lt;sub&gt;2&lt;/sub&gt;]</td><td>Gibco</td><td>14190094</td><td></td></tr><tr><td>Trypsin-EDTA [0.5%]</td><td>Gibco</td><td>15400-054</td><td>Dilute to 0.05% in DPBS.</td></tr><tr><td>Virkon (agent used to neutralize virus)</td><td>Viroderm</td><td>7511&amp;nbsp;</td><td>Dilute to 1% solution with warm water.</td></tr><tr><td>Milli-Q Water</td><td>Millipore</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Basal medium&lt;/strong&gt; - Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM) + GlutaMax</td><td>Gibco</td><td>61965059</td><td></td></tr><tr><td>Penicillin/Streptomycin [10,000 U/mL]</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>CryoMACS&amp;reg;&amp;nbsp;dimethyl sulfoxide (DMSO) 10</td><td>Miltenyi</td><td>170-076-303</td><td></td></tr><tr><td>&lt;strong&gt;Neural differentiation medium&lt;/strong&gt; - NDiff 227</td><td>Takara-Clontech</td><td>Y40002</td><td></td></tr><tr><td>LM-22A4&amp;nbsp;</td><td>Tocris</td><td>4607</td><td>Dilute 10 mg in 1450 &amp;micro;L DMSO. &lt;strong&gt;Stock concentration: 20 mM.&lt;/strong&gt;</td></tr><tr><td>Glial cell line-derived neurotrophic factor (GDNF) [recombinant human]</td><td>R&amp;amp;D systems</td><td>212-GD-010</td><td>Dilute 10 ug in 500 &amp;micro;L 0,1% BSA in DPBS. &lt;strong&gt;Stock concentration: 20 &amp;micro;g/mL.&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>NT3 [recombinant human]</td><td>R&amp;amp;D systems</td><td>267-N3-025</td><td>Dilute 25 &amp;micro;g in 2,5 mL 0,1% BSA in DPBS. &lt;strong&gt;Stock concentration: 10 &amp;micro;g/mL.&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>db-cAMP&amp;nbsp;</td><td>Sigma Aldrich</td><td>D0627</td><td>Dilute 1 g in 40,7 mL Milli-Q water. Filter and make 500 &amp;micro;L aliquots or stock tubes of 10 mL. &lt;strong&gt;Stock concentration: 50 mM.&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>CHIR99021</td><td>Axon</td><td>1386</td><td>Dilute 2 &amp;micro;g in 429,8 &amp;micro;L DMSO. &lt;strong&gt;Stock concentration: 10 mM.&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>SB-431542</td><td>Axon</td><td>1661</td><td>Dilute 5 mg in 595 &amp;micro;L DMSO. &lt;strong&gt;Stock concentration: 20 mM.&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>Noggin [recombinant human]</td><td>Miltenyi</td><td>130-103-456</td><td>Dilute 100 &amp;micro;g in 100 &amp;micro;L of Milli-Q water + 900 &amp;micro;L 0,1% BSA in DPBS. &lt;strong&gt;Stock concentration: 100 &amp;micro;g/mL.&lt;/strong&gt;</td></tr><tr><td>LDN-193189</td><td>Axon</td><td>1509</td><td>Dilute 2 mg in 360 &amp;micro;L DMSO. &lt;strong&gt;Stock concentration: 10 mM.&lt;/strong&gt;</td></tr><tr><td>Valproic acid sodium salt (VPA)</td><td>Merck Millipore</td><td>676380</td><td>Dilute 5 g in Milli-Q water to acheive a &lt;strong&gt;stock concentration of 1 M. CAUTION:&lt;/strong&gt; Avoid ingestion, contact with skin, and breathing dust formation.</td></tr><tr><td>&lt;strong&gt;Reagents for Coatings&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gelatin&amp;nbsp;</td><td>Sigma Aldrich</td><td>G2500</td><td>Dilute to 0.1% in Milli-Q water.</td></tr><tr><td>Poly-L-ornithine</td><td>Sigma Aldrich</td><td>P3655</td><td>Dissolve in Milli-Q water. &lt;strong&gt;Use at 15&amp;micro;g/mL.&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>Fibronectin</td><td>ThermoFisher Scientific</td><td>33010-018</td><td>2 mL of Milli-Q water + 70 &amp;micro;L 0,25 M NaOH. &lt;strong&gt;Use at 5 &amp;micro;g/mL.&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>Laminin</td><td>ThermoFisher Scientific</td><td>23017-015</td><td>Store at -80&amp;deg;C. Thaw on ice, keep cool and aliquot 30 &amp;micro;L. &lt;strong&gt;Use at 5 &amp;micro;g/mL.&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents for Fluorescence Activated Cell Sorting&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell dissociation agent - Accutase (Stem Pro)</td><td>ThermoFisher Scientific</td><td>A1110501</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS) 1X [-calcium, -magnesium, - phenol red]</td><td>ThermoFisher Scientific</td><td>14175-046</td><td></td></tr><tr><td>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><td>DNAase</td><td>Sigma Aldrich</td><td>DN-25</td><td>Use at 0.05% concentration.</td></tr><tr><td>Anti-Human CD56 (NCAM) Antibody [Mouse]</td><td>BD Pharmingen</td><td>555518</td><td>Use at 1 : 50.</td></tr><tr><td>Propidium iodide</td><td>Sigma Aldrich</td><td>P4170</td><td>Dilute to 1mg/mL in PBS and keep sterile. &lt;strong&gt;Use at 1:1000 to achieve a concentration of 10&amp;micro;g/mL.&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents for Glass Coverslips&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Autoclaved deionized water</td><td></td><td></td><td></td></tr><tr><td>Alconox detergent</td><td>Sigma Aldrich</td><td>Z273228</td><td></td></tr><tr><td>Ethanol 95%</td><td></td><td></td><td></td></tr><tr><td>Nitric Acid</td><td>Sigma Aldrich</td><td>438073-M</td><td>&lt;strong&gt;CAUTION:&lt;/strong&gt; Concentrated nitric acid is highly corrosive, and its vapours are potentially harmful.</td></tr><tr><td>Concentrated hydrochloric acid (HCI)</td><td></td><td></td><td>&lt;strong&gt;CAUTION:&lt;/strong&gt; Concentrated hydrochloric acid is highly corrosive, and its vapours are potentially harmful.</td></tr><tr><td>&lt;strong&gt;Reagents for Immunocytochemistry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Merck Millipore</td><td>1040051000</td><td>Use at a concentration of 4%. &lt;strong&gt;CAUTION:&lt;/strong&gt; PFA is a potent fixative. Avoid ingestion and contact with skin</td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>10254640</td><td>Use at a concentration of 0.1%.</td></tr><tr><td>Serum [Donkey]</td><td>Merck Millipore</td><td>S30-100ML</td><td></td></tr><tr><td>Anti-MAP2 Antibody [Chicken]</td><td>Abcam</td><td>ab5392</td><td>Use at a concentration of 1 : 5,000.</td></tr><tr><td>Tau HT7 Monoclonal Antibody [Mouse]</td><td>ThermoFisher Scientific</td><td>MN1000</td><td>Use at a concentration of 1 : 500.</td></tr><tr><td>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><td>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><td>4&amp;prime;,6-Diamidine-2&amp;prime;-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&amp;deg;C, &lt;strong&gt;light sensitive&lt;/strong&gt;. Use at a concentration of 1 : 500.</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>T75 flask [Nunclon Delta Surface]</td><td>ThermoFisher Scientific</td><td>156499</td><td></td></tr><tr><td>24-well plate [Nunc]</td><td>ThermoFisher Scientific</td><td>142485</td><td></td></tr><tr><td>1.5 mL polypropylene tube</td><td>Sigma Aldrich</td><td>Z336769</td><td></td></tr><tr><td>15 mL falcon tube&amp;nbsp;</td><td>Sarstedt</td><td>62.554.502</td><td></td></tr><tr><td>50 mL falcon tube&amp;nbsp;</td><td>Sarstedt</td><td>62.547.254</td><td></td></tr><tr><td>CryoPure tube 1.6 mL</td><td>Sarstedt</td><td>72.380</td><td></td></tr><tr><td>Pippette controller</td><td></td><td></td><td>For pipetting volumes 1-25 mL.</td></tr><tr><td>Sterile serological pipettes: 5, 10 and 25 mL&amp;nbsp;</td><td>Sarstedt</td><td>86.1253.001, 86.1254.001, 86.1685.001</td><td></td></tr><tr><td>Adjustable volume pipettors: 5, 20, 200, and 1,000 &amp;micro;L&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Sterile pipette tips</td><td></td><td></td><td>For pipetting volumes of 0.5 - 1,000 &amp;micro;L.</td></tr><tr><td>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><td>Glass dish</td><td></td><td></td><td>Approximately 150mm diameter.</td></tr><tr><td>Glass beaker</td><td></td><td></td><td>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><td>Parafilm M</td><td>VWR</td><td></td><td></td></tr><tr><td>ThawSTAR Automated Cell Thawing System</td><td>BioCision</td><td>BCS-601</td><td></td></tr><tr><td>Countess II Automated Cell Counter</td><td>ThermoFisher Scientific</td><td>AMQAX1000</td><td></td></tr><tr><td>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><td>CoolCell Cell LX Controlled-rate Freezing Container</td><td>Biocision</td><td>BCS-405</td><td></td></tr><tr><td>Laminar flow hood</td><td></td><td></td><td></td></tr><tr><td>Humidified 5% CO&lt;sub&gt;2&lt;/sub&gt; 37 &amp;deg;C incubator</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td>Suitable for 1,5, 15 and 50 mL tubes.</td></tr><tr><td>Orbital shaker</td><td></td><td></td><td></td></tr><tr><td>Sonicator - Bransonic Model B200 cleaner</td><td>Sigma Aldrich</td><td>Z305359</td><td>Frequency = 50-60Hz, Amplitude = 30 Watts</td></tr><tr><td>FACS Aria III cell sorter</td><td>BD Pharmingen</td><td></td><td></td></tr><tr><td>Phase contrast microscope</td><td>Olympus</td><td>CKX31</td><td></td></tr><tr><td>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

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

科研

发育生物学

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

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

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

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

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

神经科学

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

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

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

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

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

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

使用层粘连蛋白521矩阵整合人诱导多能干细胞的自由衍生

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

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 ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

生物工程

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

生物学

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

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

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

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

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

Isolating Induced Neural Progenitor Cell Colonies Derived From Adult Human Fibroblasts

Source: Meyer, S. et al., <a href="https://app.jove.com/v/52831">Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells.</a>&#160;J. Vis. Exp. (2015).
This video demonstrates a technique for isolating induced neural progenitor cells (iNPCs) derived from adult human fibroblasts. It outlines the steps involved in isolating the iNPC colonies and culturing them in laminin-coated multi-well plate wells in the presence of suitable culture media that facilitate the continued proliferation and expansion of the iNPC population.

分离源自成人成纤维细胞的诱导神经祖细胞集落

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Putative Induced ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Generating Neurons by Reprogramming Human Dermal Fibroblast Cells

Source: Shrigley, S., et al. <a href="https://app.jove.com/v/56904">Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts.</a> J. Vis. Exp. (2018)
The video demonstrates the generation of neurons from adult human dermal fibroblasts (aHDFs). The aHDFs are reprogrammed using a lentiviral vector carrying genes for neuronal transcription factors and short hairpin RNAs (shRNAs). The shRNAs inhibit the expression of a neuronal transcriptional repressor, and the neuronal transcription factors activate genes to differentiate aHDFs into neurons. These reprogrammed aHDFs are cultured in an early and a late neuronal conversion medium to generate mature neurons.

通过重编程人真皮成纤维细胞生成神经元

1. Preparation of Skin Fibroblasts for Reprogramming

	Using an automated cell thawing system or a 37 &#176;C water bath, thaw the adult human dermal ...