作者感谢梁美芬 l. 杨和 Burri 哈佛医学院的帮助与初步传播和量化的 ZIKV。我们还要感谢纳撒尼尔 d 帕特里克的成像支持。K.E. 得到了斯坦利精神病研究中心和哈佛干细胞研究所的支持。

1. 从 iPSC/干细胞2D 区域性创建脑 Organoids (天 0) 注意: 此协议假定在委员会介质中使用受精或 Geltrex 进行干细胞 (SC) 维护基.如果使用一种替代性的高蛋白干细胞培养培养基, 在启动化形成之前, 宜将 SC 培养物转变为委员会至少两个通道。该协议尚未通过 feeder-based SC 培养方法进行测试. 
<ol> <li> 使用常规 SC 维护方法 25 , 26 , 将区域性带到 50%-70% 汇合处。这通常是3-4 天后传代, 虽然这可能是依赖于文化方法和细胞线. 
	 注意: 大约2口井从一个6井板材是需要的生产一个充分的96井板材 organoids. </li>
	<li> 通过明显微镜检查培养基的 10 x-20 x 放大倍数, 以确保健康的菌落形态, 无可察觉的分化. </li>
	<li> 将委员会带到37和 #176; 在热水浴中进行加热, 并解冻50和 #181; 50 毫米 Y-27632 (岩石抑制剂) 和2毫升酶分离试剂到室温. </li>
	<li> 准备一个超低附着 (ULA) U 底96井板, 多通道 P200 吸管和25毫升试剂库. </li>
	<li> 分45毫升的委员会在50毫升锥形管, 并添加45和 #181; L 50 毫米 Y-27632。由研制彻底混合. </li>
	<li> 真空抽吸两个含 SCs 的井, 并使用 P1000 吸管将1毫升无酶的分离试剂迅速添加到每个油井。在37和 #176 中孵育盘; C 孵化4分钟. </li>
	<li> 真空抽吸处理好的井, 并使用 P1000 吸管将1毫升的酶分离试剂添加到每个井中。
	<ol> <li> 在37和 #176 中孵化板; C 孵化器为额外的5分钟 </li>
		<li> 卸下板并检查处理过的井。轻轻拍打盘子以分解聚集的细胞。如果大的细胞簇仍然可见, 在37和 #176 孵育板2额外的分钟; c. 重复此步骤, 直到肉眼看不到簇. </li>
	</ol> </li> <li> 一旦适当分离细胞, 将1毫升的委员会添加到每个井中, 以解除离解酶。池4毫升的细胞悬浮在一个50毫升锥管, 并采取小分细胞计数。
	<ol> <li> 将维修板中的任何未处理的井返回到37和 #176; C 恒温箱. </li>
	</ol> </li> <li> 在计数时, 将单元格悬浮在 300 x g 处离心5分钟 </li>
	<li> 确定单元格的总数, 并计算实现6万单元/mL 悬浮液所需的委员会 + Y-27632 解决方案的数量. </li>
	<li> 小心吸真空吸取上清, 并增加委员会 + Y-27632 溶液的计算量。混合5-10 次5毫升吸管, 确保一个统一的细胞悬浮. </li>
	<li> 立即将电池悬浮液转移到试剂库, 并将吸管150和 #181; L 插入 ULA U 底96孔板的每一个井中, 使用多通道 P200 吸管。这将产生96个单独的 organoids, 每个最初由9000单元组成. </li>
	<li> 离心板在 150 x g 为 1 min. </li>
	<li> 将板放入37和 #176; C 恒温箱, 请勿干扰板48小时. </li>
</ol> 2。脑化维护 (2 天) <ol> <li> 准备17毫升委员会与17和 #181; L 50 毫米 Y-27643 在50毫升锥形管。将委员会 + Y-27632 溶液加热到37和 #176; C 在热水浴中. </li>
	<li> 从孵化箱中取化板, 并使用多通道 P200 吸管设置为75和 #181; L, 从第一排的井边慢慢画出介质。一旦培养基被抽出, 迅速将培养基排出回井中, 以释放出松散的细胞和 organoids。对每行重复此操作. 
	 注意: 这项技术, 以后称为 #34;d ispersion, #34; 将细胞碎片和化吊起。化迅速下沉到井的底部, 而较小的残骸仍然悬浮, 允许它被去除与媒介变动。这有助于防止化在文化中的细胞残骸中休息。
	<ol> <li> 在所有井都被分散后, 等待十五年代, 以确保 organoids 已下沉到每口井的底部. </li>
	</ol> </li> <li> 缓慢而小心地吸入75和 #181; 从每个井中的培养基中, 使用多通道 P200 吸管并分配到一个废物库. 
	 注意: 大多数用户在常规的 feed 中偶尔会抽化。这可能发生大约1% 的时间。请相应地计划, 以确保足够的 organoids 生存到实验时间点. </li>
	<li> 将委员会 + Y-27632 溶液转移到试剂库中, 并将150和 #181; L 使用多通道 P200 吸管进入每个化井. </li>
	<li> 将板材放回 37 #176; C 恒温箱. </li>
</ol> 3。脑化维护 (3 天) <ol> <li> 温暖17毫升的委员会到37和 #176; C, 此时 没有 Y-27643. </li>
	<li> 用多通道 P200 吸管设置为100和 #181; L, 分散化板的所有井 (见步骤 2.2)。等待十五年代, 以确保 organoids 已沉没到他们的井底. </li>
	<li> 准备一个废弃的水库, 并将温热的委员会转移到一个源油藏中. </li>
	<li> 小心地抽吸125和 #181; 从每个井中吸出培养基 l, 用150和 #181 取代; 用多通道 P200 吸管代替新鲜委员会. </li>
	<li> 将板材放回 37 #176; C 恒温箱. </li>
</ol> 4。脑化维护 (4 天 +) 注意: 每隔一天进行一次定期维护, 直到感染为止. 
<ol> <li> 在开始4天的提要之前, 准备神经感应 (NI) 培养基。
	<ol> <li> 解冻250和 #181; 分2毫克/毫升肝素, 以及一个5毫升的 100X N-2 补充剂。添加250和 #181; 5 毫升的 100X N-2, 5 毫升的100X 记忆 NEAA 到500毫升的 DMEM/F12+GlutaMAX。无菌过滤器的混合解决方案在一个500毫升过滤器. 
		 注意: 不使用时, 将 NI 保持在4和 #176;在新培养基准备好之前, NI 可持续两周. </li>
	</ol> </li> <li> 热17毫升 NI 培养基到37和 #176; C. </li>
	<li> 用多通道 P200 吸管设置为100和 #181; L, 分散化板的所有井 (见步骤 2.2)。等待十五年代, 以确保 organoids 已沉没到他们的井底. </li>
	<li> 准备一个废弃的水库, 并将温热的 NI 转移到一个源油藏中. </li>
	<li> 小心地抽吸125和 #181; 从每个井中抽出培养基, 用125和 #181 替换; 用多通道 P200 吸管代替新鲜镍. </li>
	<li> 将板材放回 37 #176; C 恒温箱. </li>
</ol> 5。化感染与 ZIKV (~ 天 24) 注意: 在约3周的差异, 有 wi我将是大型上皮结构和花环可见的化, 将高度敏感 ZIKV 感染. 
<ol> <li> 将厄尔和 #39 的1X 平衡盐溶液 (EBSS)、1X PBS 和 NI 到37和 #176; C 在热水浴中. </li>
	<li> 稀释 ZIKV 的已知浓度在 1X EBSS 到目标感染多重感染 (莫伊). 
	 注意: 建议滴定不同的 MOIs, 以达到病毒剂量反应。这可能因病毒株而异;对于 ATCC VR-1838 和 ATCC VR-1843, 典型的莫伊值范围介于0.1 和10之间). </li>
	<li> 使用单通道 P200 吸管小心地从化中取出所有介质。快速添加200和 #181; L 1X PBS 每井使用 P200 多通道吸管清洗 organoids. </li>
	<li> 使用单通道 P200 吸管小心地从化中取出所有介质。快速添加50和 #181; L 1X EBSS 只 (模拟感染) 或 ZIKV 病毒解决方案, 并确保化完全淹没。重复每一个化, 将被感染的研究. </li>
	<li> 将板材放回37和 #176; C 型恒温箱2小时. 
	 注意: 与未受干扰的 organoids 相比, 在这段时间内孵化模拟感染的 organoids 不应显著影响其生长. </li>
	<li> 在病毒孵化完成前约30分钟, 分25毫升的 NI 成50毫升的锥形管, 并带到37和 #176; C 在热水澡. </li>
	<li> 在病毒暴露完成后, 用200和 #181 清洗 organoids; 每个井的 1X pbs, 允许 organoids 定居十五年代, 然后使用单一通道 P200 吸管删除 1X pbs. </li>
	<li> 添加200和 #181; 每个井都有新鲜的 NI, 然后放回孵化器。
	<ol> <li> 可选: 一天0时间点, organoids 可能会在感染后一小时被成像. </li>
	</ol> </li> <li> 通过替换125和 #181 继续进行区域性操作; 我每隔一天使用色散方法 (请参见步骤 4)。一定要正确处理废弃的介质和提示, 因为这包含有传染性的 ZIKV 粒子. </li>
</ol>

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作者声明他们没有竞争的金融利益。

在使用人脑 organoids 研究 ZIKV 感染时, 有几个注意事项需要考虑。一个重要的考虑是, 化的形成和结构是高度依赖于干细胞线, 从它们形成。在比较细胞系时要小心, 包括基因的工程线;最好的结论是使用多个 subclones, 理想的是多条干细胞系。此外, 由于这种差异的细胞线, 试点实验, 以确保适当的差异是发生在 organoids 的建议。虽然上皮结构是可见的明显微镜, 也建议进行 qRT PCR 或 cryosectioning 实验寻找神经分化标记, 如 PAX6 或磷-波形。
在同一细胞系内, 也应预测 organoids 之间的相当大的变异性。由于协议的 unpatterned 性质, 上皮结构的数量和大小在个体 organoids 之间会有所不同。通常, 你可以期望至少两个大 (&gt; 200 µm 直径由 D24) 神经花环每化。因此, 纵向研究, 如观察化大小的变化对感染, 尤其有启发意义。批次之间的可变性也可能发生, 其中最可能的原因是不准确的细胞计数或播种。建议进行多次计数, 并确保在 ULA U 底96孔板上播种前, 细胞悬浮液混合良好。
当培养 organoids 在 ULA U 底 96-井板, 计划看到可观的蒸发作用在板材的边缘附近。这是理想的填充这些井与 PBS 和只工作与中心60井。由于蒸发, organoids 在外部井将暴露在不同的条件比那些在中心, 将可能影响他们的成长和分化。在计划需要进行实验的 organoids 的数量时, 请考虑这一点。另外, organoids 将开始满96井格式在大约30天以后, 将是可看见的由于文化媒介的变色。如果计划采取 organoids 过去的30天, 建议将 organoids 转移到一个 ULA 24-井板。要进行转移, 使用剪刀削减 P1000 小费, 使开幕式比 organoids 本身大得多, 并转移 organoids 的移。
人脑 organoids 在促进该领域对神经和疾病的认识方面具有巨大的潜力。鼓励研究人员在必要时修改该协议。例如, 你可以实施模式的因素, 以提高分化效率, 以特定的细胞类型, 使他们能够探索病毒影响的某些解剖区域。ZIKV 的神经发育效应仍未得到很好的理解, 但这种新的方法将使研究人员有一个可访问、快速和高通量的方法来调查感染机制。

Zika 病毒 (ZIKV) 迅速蔓延在密克罗尼西亚, 法属波利尼西亚和美洲, 最近被证明跨越胎盘屏障1,2感染发育中的胎儿大脑, 导致神经发育疾病, 如作为小头3,4,5,6 。长期影响这些患者的生活, 目前缺乏治疗, 导致研究人员和临床医生, 以争夺更好地了解的机制背后的 ZIKV 感染和复制。以往的研究已经检查了 ZIKV 感染的体外系统在各种生理相关的细胞类型7,8,9, 以及在体内10与免疫和免疫缺陷小鼠11,12,13和非人类灵长类动物141516。除了这些更传统的技术, 几个小组已经实施干细胞衍生脑 organoids, 以了解更多关于 ZIKV 感染的发展中的人脑。这些组已利用人脑 organoids 确认小头表型17,18, 调查与病毒条目相关的受体19, 检查对感染的生理反应20,21, 潜在的筛选药物候选者22。这里描述了一种快速产生和感染干细胞衍生脑 organoids 的技术, 如前所示19, 以提高对发展中人脑的 ZIKV 感染的认识。
由于 organoids 是由标准多能干细胞 (PSC) 培养而成的, 因此这种技术可以对发展中的大脑的病毒感染问题进行各种科学的解答。例如, CRISPR 工程可用于在化感染之前以更快的速度修改这些 PSC 线, 而不是大多数在体内遗传研究19。另外, 与标准二维 (2D) 分化培养不同, organoids 展示了复杂的细胞结构, 这对 corticogenesis 是至关重要的, 最近的研究表明, 这种体系结构可能在感染时被破坏。21。最后, 相对于体内模型而言, 生成 organoids 的成本较低, 可以进行更高的吞吐量试验和筛选。另一方面, organoids 在 ZIKV 研究中的运用也存在一些弊端。虽然 organoids 比2D 文化更具生物学意义, 但由于其三维 (3D) 的性质, 在 organoids 的评估中还有其他的挑战。organoids 的成像和离解往往涉及更多的设备和比标准2D 文化更大的资源投资。此外, organoids 缺乏的血管和免疫成分存在于体内模型, 因此, 研究人员对这些方面的病毒感染建议寻求一个替代协议。
在文化中有许多技术来形成人类的 organoids 和球, 它们通常属于图案或unpatterned类别。图案化方法实现了调节 Wnt、BMP、TGFβ和其他信号通路的因素, 以将分化推向特定的血统23。Unpatterned 方法, 如这里描述的, 利用诱导的多能干细胞 (干细胞) 和人类胚胎干细胞 (干细胞) 的倾向, 以区别于神经血统的默认24。经过大约三周的分化, 由此产生的 organoids 包括大型, 仿生上皮结构, 包含几个细胞类型, 在早期发育的大脑中观察到。
提出了从标准、无饲养的 PSC 文化中生产 organoids 的技术, 并将这些 organoids 与 ZIKV 进行了充分的研究。关于无馈线公司所需的培养方法的指导, 请参阅以前的方法出版物25,26。此外, 为了在多个实验中应用一致的病毒数量, 重要的是要提前计算语言教学。这是通过进行感染的维罗细胞, 其次是治疗的叠加培养基, 孵化, 和免疫。此技术的描述和方法以前已被描述为19,27。
一旦 PSC 文化达到 50%-70% 的目标汇流, 细胞就会被离解并聚集到超低附着的 96-井板中。细胞被维护3天在无异的干细胞维护媒介 (委员会), 然后转换成神经归纳媒介为文化的剩下的人。一旦 organoids 有分化3周, 感染可以进行。通过在感染后一周内例行拍摄图像, 研究人员将观察化细胞的渐进性死亡和破坏。研究人员也可能在这个时候分离 organoids 进行转录或蛋白质组分析。Cryosectioning 和 lightsheet 方法是建议的成像, 研究人员可以期望看到高水平的感染和病毒复制, 特别是在神经祖细胞 (NPC) 的人口在 organoids。最终, 这项技术使研究人员能够迅速检测到低成本和有限设备的人脑病毒感染的机制。

<table style="border-collapse:collapse;width:1358pt" width="1811">
	<colgroup>
		<col style="width:317pt" width="423" />
		<col style="width:158pt" width="211" />
		<col style="width:134pt" width="178" />
		<col style="width:749pt" width="999" />
	</colgroup>
	<tbody>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="width:317pt;height:26.25pt" width="423">Xeno-Free Stem Cell Maintenance Media (SCMM), TeSR-E8</td>
			<td class="xl19" style="width:158pt" width="211">StemCell Technologies</td>
			<td class="xl19" style="width:134pt" width="178">5940</td>
			<td class="xl19" style="width:749pt" width="999">Use for stem cell maintenance and early stages of organoid formaiton.&#160; Store at 4 &#7506;C for up to two weeks after prepared from individual components</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Enzyme-Free Detachment Reagent, ReLeSR</td>
			<td class="xl19">StemCell Technologies</td>
			<td class="xl19">5873</td>
			<td class="xl19"></td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Enzymatic Detachment Reagent, Accutase</td>
			<td class="xl19">Gibco</td>
			<td class="xl19">A11105-01</td>
			<td class="xl19">Store aliquoted at -20 &#7506;C, do not keep thawed for longer than necessary</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Y-27632</td>
			<td class="xl19">Selleck Chem</td>
			<td class="xl19">S1049</td>
			<td class="xl19">Reconstitute to 50mM in DMSO, and keep at -20 &#7506;C in 50&#181;L aliquots.</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Dimethyl Sulfoxide (DMSO)</td>
			<td class="xl19">Sigma-Aldrich</td>
			<td class="xl19">D2650-100ML</td>
			<td class="xl19"></td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Glutamate-supplemented DMEM/F12 (1X)</td>
			<td class="xl19">Gibco</td>
			<td class="xl19">10565-018</td>
			<td class="xl19">(+)2.438 g/L sodium bicarbonate, (+)sodium pyruvate</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">N-2 Supplement (100X)</td>
			<td class="xl19">Gibco</td>
			<td class="xl19">17502-048</td>
			<td class="xl19"></td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">MEM Non-Essential Amino Acids (MEM-NEAA)</td>
			<td class="xl19">Gibco</td>
			<td class="xl19">11140-050</td>
			<td class="xl19"></td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Heparin</td>
			<td class="xl19">Sigma-Adrich</td>
			<td class="xl19">H4784-1G</td>
			<td class="xl19">Reconstitute to 2 mg/mL in sterile DI water and store at -20 &#7506;C in 250&#181;L aliquots</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Earle&#39;s Balanced Salt Solution (EBSS)</td>
			<td class="xl19">GE Healthcare Life Sciences</td>
			<td class="xl19">SH30029.02</td>
			<td class="xl19"></td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Stericup Filter (500mL)</td>
			<td class="xl19">Millipore</td>
			<td class="xl19">220009-140</td>
			<td class="xl19"></td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Costar Ultra-Low Attachment Plate, 96-Well, Round Bottom</td>
			<td class="xl19">Corning</td>
			<td class="xl19">7007</td>
			<td class="xl19"></td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Low Residual Volume Reagent Reservoir (25mL)</td>
			<td class="xl19">Integra</td>
			<td class="xl19">4312</td>
			<td class="xl19"></td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Pipet-Lite Multi Pipette L12-200XLS+</td>
			<td class="xl19">Rainin</td>
			<td class="xl19">17013810</td>
			<td class="xl19"></td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Large Centrifuge, Culture Plate-Compatible</td>
			<td class="xl19">Thermo Scientific</td>
			<td class="xl19">75007210</td>
			<td class="xl19"></td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Culture Plate Centrifuge Rotor</td>
			<td class="xl19">Thermo Scientific</td>
			<td class="xl19">75005706</td>
			<td class="xl19"></td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Vero Cells</td>
			<td class="xl19">ATCC</td>
			<td class="xl19">CCL-81</td>
			<td class="xl19">For virus expansion, if necessary</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Zika Virus (Uganda Strain)</td>
			<td class="xl19">ATCC</td>
			<td class="xl19">VR-1838</td>
			<td class="xl19">Other strains may be used</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">Zika Virus (Puerto Rico Strain)</td>
			<td class="xl19">ATCC</td>
			<td class="xl19">VR-1843</td>
			<td class="xl19">Other strains may be used</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">phospho-vimentin</td>
			<td class="xl19">MBL</td>
			<td class="xl19">D076-3</td>
			<td class="xl19">mouse polyclonal,&#160; 1:250 dilution</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">TBR2</td>
			<td class="xl19">Abcam</td>
			<td class="xl19">ab23345</td>
			<td class="xl19">rabbit polyclonal, 1:300 dilution</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">MAP2</td>
			<td class="xl19">Novus</td>
			<td class="xl19">NB300-213</td>
			<td class="xl19">chicken polyclonal, 1:1500 dilution</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">GFAP</td>
			<td class="xl19">Cell Signaling Technologies</td>
			<td class="xl19">CST12389S</td>
			<td class="xl19">rabbit polyclonal, 1:200 dilution</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">D1-4G2 flavivirus envelope protein</td>
			<td class="xl19">EMD Millipore</td>
			<td class="xl19">MAB10216</td>
			<td class="xl19">mouse monoclonal, 1:500 dilution</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">cleaved caspase-3</td>
			<td class="xl19">Cell Signaling Technologies</td>
			<td class="xl19">9661</td>
			<td class="xl19">rabbit polyclonal, 1:200 dilution</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">488 anti-mouse IgG</td>
			<td class="xl19">Thermo Fisher</td>
			<td class="xl19">A-11001</td>
			<td class="xl19">goat polyclonal, 1:1000</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">594 anti-rabbit IgG</td>
			<td class="xl19">Thermo Fisher</td>
			<td class="xl19">A-11037</td>
			<td class="xl19">goat polyclonal, 1:1000 dilution</td>
		</tr>
		<tr height="35" style="height:26.25pt">
			<td class="xl19" height="35" style="height:26.25pt">647 anti-chicken IgY</td>
			<td class="xl19">Thermo Fisher</td>
			<td class="xl19">A-21449</td>
			<td class="xl19">goat polyclonal, 1:1000 dilution</td>
		</tr>
	</tbody>
</table>

modelling zika virus infection of the developing human brain in vitro using stem cell derived cerebral organoids

最近 Zika 病毒 (ZIKV) 在易感人群中的出现导致了新生儿小头和其他神经发育状况的突然增加。虽然蚊子是病毒传播的主要途径, 但它也被证明通过性接触和垂直 mother-to-fetus 传输传播。在后一种情况下的传输, 由于独特的病毒取向的 ZIKV, 该病毒被认为是主要目标的神经祖细胞 (npc) 发展的大脑。
这里描述了 ZIKV 感染的建模方法, 以及当人类大脑 organoids 暴露于活 ZIKV 时产生的小头。organoids 在其神经祖群中显示高水平的病毒, 并随着时间的推移表现出严重的细胞死亡和小头。这个三维脑化模型允许研究人员进行物种配对实验, 观察和潜在干预与发展中的人脑的 ZIKV 感染。该模型提供了改进的相关性比标准 two-dimensional 方法, 并包含了人类特有的细胞结构和蛋白表达, 是不可能的动物模型。

在化形成的那一天, SC 文化应该在日志生长阶段, 在其中他们是 50%-70% 汇合, 如图 1(A-c)所示。区域性应形成具有不同边缘的圆形菌落, 并且井应明确区分图 1(D-E)。如果发生差异, 建议在创建 organoids 前至少一天内进行 pick-to-remove, 以清理板块内部受影响的区域。如果文化的大部分被区分, 可能有必要进行额外的通道, 解冻新的小瓶, 或改变干细胞培养技术, 以确保 organoids 是由纯干细胞培养形成。
由无酶和酶分离试剂的结合处理的离解应该导致多数单细胞, 虽然可能仍然有小聚集细胞观察在计数期间。小团聚体的存在不会破坏化的形成, 尽管它可能会影响计数精度。建议每个样本进行多次计数, 如果计数变化超过 20%, 则可能需要进一步增加离 singularize 细胞的离解时间。
一旦细胞被播种并在一个 ULA 的 U 底96井板 (图 2a) 中旋转下来, 它们就会在每个井的底部显示为扁平的、致密的细胞板。细胞在接下来的两天内会慢慢聚集。最好不要在这48小时内扰乱板块, 因为机械力会扰乱松散形成的骨料。在前10天, 化将保持主要球形, 并将增长从〜300µm 到600-800 µm (图 2B)。在10天和20天 (图 2C-E)之间的化中, 更可观察到的结构开始出现。在24天, 研究人员应该能够通过明显微镜观察玫瑰样的结构 (图 2F)。在继续进行区域性的同时, 这些上皮结构可能会在数天内增加数量和大小 (图 2G)。在喂食过程中, 研究者会注意到在每一个井的底部收集大量的碎片, 特别是在最初的2周的文化中。步骤2.2 中描述的色散技术去除了大部分的细胞碎片, 以防止凋亡的碎片堆积在多个源上 (图 3A)。这些碎片可以干扰成像, 它可以使它更具挑战性的观察和量化感染诱导细胞死亡, 它可能提供不对称信号的邻近化, 可能会影响分化。如果对色散技术进行了正确的处理, 则应减少此方法 (图 3B-C, 3 d-E)。
建议进行 cryosections 或 lightsheet 成像, 以检查在 organoids 内形成的皮层结构。在25天 organoids, 莲座丛结构和心室区在 DAPI (图 4A) 和磷-波形 (图 4B) 染色中清晰可见。TBR2 (图 4C) 的存在表明中间的祖细胞正在形成, 其形态学表明向外迁移。MAP2+ (图 4D) 神经元填充每个莲座丛的外部区域。从这些蛋白质, 你可以可视化的心室区 (VZ), 脑室区 (SVZ), 中间区 (伊兹), 皮质板 (CP) 在每个花环 (图 4E,4E ")。你能继续培养这些 organoids 为了观察发展的以后阶段。虽然皮质分层在这种类型的化不突出, 自然开关到 gliogenesis 确实发生了很像它在体内。这可以在108天的 organoids 中观察到, 其中很大一部分细胞表示 MAP2 或胶质蛋白 (图 4F-I)。
用户可能希望在 ZIKV 感染后的大约3天内看到化大小的差异, 具体取决于应用于 organoids (图 5A-b) 的病毒语言。在这3天之后, 与模拟井相比, 受感染水井中的细胞碎片也会增加。化大小的这种差异将在接下来的一周内增加, 直到感染的 organoids 开始分离 (图 5C-d)。在这一点上可以使用多种化验方法来调查感染机制, 包括病毒 RNA 提取和 cryosectioning (在材料表中报告的推荐抗体)。在 cryosectioning, 用户将观察到在上皮结构的顶端区域有大量的病毒存在, 这表明神经祖细胞 (npc) 对 ZIKV 感染的敏感性 (图 6)。免疫荧光切片 organoids 也将显示在被感染的 organoids 的 caspase-3 表达增加 (图 7)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56404/56404fig1.jpg"> 
图 1: 在化形成之前, 干细胞菌落形态. 殖民地应该是 50%-70% 汇合在分离时产生大量的一致 organoids。(A)稀疏、最近播种的区域性尚未达到日志增长阶段, 不会产生许多 organoids。(B)一旦细胞到达适当的汇合处, 它们就可以分离。同样重要的是, 这些殖民地的检查, 以确保它们是明确的分化。(C)太远的殖民地将达到 over-confluence, 不建议化形成。这种状态下的区域性更可能包含区分单元格。(D)菌落边缘应是不同的, 中心的圆角单元格具有一致的细胞形态学, 边缘上有稍拉长的细胞。(E)在区域性中应存在最小差异。如果在菌落的中心或边缘 (白色箭头) 观察到更大的扁平细胞, 并构成超过大约1% 的培养物, 建议 pick-to-remove 或其他通道进行, 以形成一个均匀的干细胞种群。<a href="//ecsourc href=""></a href="//ecsourc>文件/ftp_upload/56404/56404fig1large. jpg "目标 =" _blank ">> 请点击这里查看这个数字的一个更大的版本。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56404/56404fig2.jpg"> 
图 2: 脑化随时间增长. 
(a)从2D 公司中维护的 organoids 的生产图, 无异、无馈线介质。(B)在最初的8天内, organoids 将仍然主要是球形, 很少有可检测的特征。化的直径将范围从大约300µm 到500µm. (C-E)在10天, 更清晰的区域将开始被探测周围的 organoids, 他们将失去他们的球形形状。它们的大小将继续增加到直径约1毫米, 这主要取决于用于生产 organoids 的细胞线。(F)在大约20天的差异化后, 用户将观察上皮结构 (黑色箭头) 在大多数 organoids 的清晰外围区域的形成。这些有环形的形态学, 顶端和基底结构模仿那些发展中的皮层。(G)这些上皮结构将在大约20-30 天的时间内继续增长并增加大小。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56404/56404fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56404/56404fig3.jpg"> 
图 3: 通过分散饲料技术清除细胞碎片. 在化生长期间, 大量的细胞死亡是预期的, 并可能导致堆积的细胞碎片周围的基地的化。(A)所描述的色散技术允许研究人员在每次进食时移除大部分碎片。要做到这一点, 使用多通道 P200 吸管, 慢慢地绘制使用的培养基, 并迅速排出, 以解除化和细胞碎片悬浮。化将迅速下落到井的底部, 在这个时候可以进行常规饲料。在(B)和(C)后的6天化的例子中, 分散喂养显示了大量的碎片减少。这将持续数周, 如在(D)之前的18天化和(E)分散喂养之后显示。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56404/56404fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56404/56404fig4.jpg"> 
图 4: 通过免疫组织化学表征脑 organoids. Lightsheet 脑 organoids 的图像显示, 提供一个细胞结构的例子, 形成。在25天, DAPI (A)和磷光-波形(B)分别表示单独的花环和心室区。(C) TBR2 标签正向外迁移的中间祖和 MAP2 (D)标签在皮质板中形成的神经元。(e, e ')这些蛋白质的结合清楚地显示了脑化模型中皮质发育的重述。(F-I)在108天, gliogenesis 已经发生, 与混合的胶质蛋白 + 和 MAP2+ 细胞填充大部分的化。VZ = 心室区, SVZ = 脑室区, 伊兹 = 中间区, CP = 皮质板。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56404/56404fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56404/56404fig5.jpg"> 
图 5: 感染的 organoids 的降解. 
(A)在莫伊 = 0.1 和10中模拟和 ZIKV 感染的脑 organoids 的明亮场图像。图像被采取后立即感染 (天 0), 以及3和6天感染。(B-c)明周围的细胞碎片的图像(B)模拟和(C) ZIKV 感染在莫伊 = 10 脑 organoids 3 天感染。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56404/56404fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56404/56404fig6.jpg"> 
图 6: 感染 organoids 的 Cryosection 和免疫荧光. 24天 organoids 被暴露在 ZIKV 在莫伊 = 0.1, 和 cryosectioned 6 天后。在这一阶段, 有明显的病毒存在于心室, 脑室, 和中间区的化, 其中神经祖细胞和径向胶质的驻留。(A)通过核染色, 这些上皮区域显然是可见的, 因为 (白色箭头) 到核的高密度, 形成环形结构的顶端表面周围。(B)通过对病毒包络进行染色, 可以观察到这些莲座丛结构 (白色箭头) 中病毒的相对较高的存在。注意, 在各种不明的外围细胞中存在少量的病毒。(C-D)MAP2 或其他神经元特异的污渍表明, 有神经元存在的文化, 似乎没有感染的病毒时, 覆盖的病毒染色。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56404/56404fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56404/56404fig7.jpg"> 
图 7: caspase-3 感染化的免疫荧光. 感染后, 人们可以使用凋亡指标来研究 organoids 内发生的细胞死亡的机制。在这个例子中, 24 天 organoids 被暴露在 ZIKV 在莫伊 = 0.1, 和 cryosectioned 6 天后。(A)未感染的 organoids 显示没有病毒包膜 (4G2 蛋白), 以及在组织中发生的自我平衡的细胞凋亡的少量 caspase-3。(B)受感染的花环开始显示细胞凋亡水平的升高。(C)通过4G2 染色显示严重感染的花环也倾向于在这些区域表现出高水平的劈开 caspase-3。感染的严重性与被感染的花环内的 caspase-3 表达有直接关系。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56404/56404fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids at JoVE.com

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

本协议描述了一种用于开发人脑的 Zika 病毒感染模型的技术。利用野生或工程化的干细胞系, 研究人员可以利用这种技术来发现可能影响早期脑部感染的各种机制或治疗方法, 并在 Zika 病毒感染的胚胎中产生小头。

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

modelling-zika-virus-infection-developing-human-brain-vitro-using

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

10.4K 次观看.  Novartis Institutes for BioMedical Research. 本协议描述了一种用于开发人脑的 Zika 病毒感染模型的技术。利用野生或工程化的干细胞系, 研究人员可以利用这种技术来发现可能影响早期脑部感染的各种机制或治疗方法, 并在 Zika 病毒感染的胚胎中产生小头。

视频: Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

科研

神经科学

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

神经干细胞和祖细胞在小鼠脑开发基因毒性应激后的评估细胞周期进程

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system.
Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.

Here we report a protocol to develop a three-dimensional (3-D) system from human induced-pluripotent stem cells (hiPSCs) called the serum free embryoid body (SFEB). This 3-D model can be used like an organotypic slice culture to model human cortical development and for the physiological interrogation of developing neural circuits.

开发HiPSC衍生的无血清胚胎体用于使用生理相关测定的3-D干细胞培养物的询问

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

使用多体分析在发育中的鼠标大脑中进行翻译分析

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

通过非侵入性经鼻途径在免疫功能正常的小鼠大脑中移植人诱导的多能干细胞衍生小胶质细胞

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

强大且高度可重复的皮质脑类器官生成，用于在体外模拟大脑神经元衰老

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

成人皮层的器官型培养作为人类干细胞移植和验证的 离体 模型

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...