这项工作得到了新泽西州州长自闭症医学研究和治疗委员会 (CAUT13APS010) 的支持;CAUT14APL031;CAUT15APL041), 南茜. Mindworks 标志着家庭基金会, 慈善领导信任, 以及更大 MetroWest 新泽西州的犹太社区基金会。

作者声明他们没有竞争的财政利益。

这里提出的协议说明了快速和简单的方法来研究基本神经发育过程和测试生长因子和药物使用 hiPSC 衍生神经前体细胞。hiPSC 技术已经革命性的研究神经发育疾病的发病机制, 为我们提供了前所未有的机会, 从受影响的人的活体神经细胞细胞。事实上, 已经有许多 hiPSC 研究的神经发育紊乱, 包括 Rett 综合征, 蒂莫西综合征, 脆性 x 综合征, 精神分裂症, 发现了疾病特异的畸变在树突, 突触和神经元函数4,33,34,35,36。大多数这些研究主要集中在晚期分化的后有丝分裂神经元, 虽然认为相对功能不成熟, 排除了研究早期神经发育过程, 如增殖和迁移。这些后处理已严重牵连神经发育紊乱的发病机制, 并保证进一步研究8,9,10,11,12,35,37,38. 使用 npc 可以让我们研究这些重要的早期事件, 同时也提供机会调查更成熟的过程, 如细胞的能力延长未成熟的轴突/树突 (突起)。此外, 其中一些化验也可以扩展到研究其他参数, 如突起长度, 数量, 分支或最远的距离所移动的细胞。
一些较新的研究使用 organoid 模型系统研究早期发展事件在3维 "微型脑子系统"1,39,40。然而, 即使在这些 organoid 系统中, 增生性前体细胞数量有限, 早期成熟和迁移很难研究15,39。除了限制对早期发展现象的研究外, 使用晚期分化的神经元或 organoids 往往费时, 代价高昂, 并且限制了可以在系统中评估的变量数。这是因为使神经元和 organoids 可能需要病毒感应协议, 特殊的孵化器和设备, 多周的时间, 和大量的媒体。与此相反, 除了 DNA 合成化验 (下文后面所述) 外, 本议定书可以很容易地应用于神经发育紊乱的研究, 不需要广泛的训练、昂贵的工具、资源或软件。在这些化验中添加药物和生长因子的简便性和相对低廉的成本, 使该协议成为一种有用的高通量技术, 用于测试神经发育和精神疾病的各种潜在治疗方法。此外, 由于生长因子作用于通过定义的细胞信号通路, 它们也可以用作测试开发系统中潜在信号缺陷的工具。最后, 由于 npc 是一个增生的自我更新的人口, 大量的细胞可以生产和冷冻保存, 使实验能够有效地进行, 而不必从 iPSCs 的 npc 每次。
要成功地使用这些化验, 重要的是要注意以下关键步骤。该协议将 npc 置于不同的维护和扩展环境中, 而不是实验。具体地说, 虽然 npc 是诱导, 生长和传代中含有神经诱导补充 (NIS), 我们的实验条件减少 70%, 这使细胞在一个限制的环境, 使我们有机会增加回并测试重要生长因子的影响。其次, 必须跟踪 npc 的通过。在我们的研究中, 我们通常限制了 P3-P8 细胞系的通道数。在比 P3 更早的段落中, 有些线条不能有力地表达特征标记。在较高的通道, 虽然一些细胞系有非常一致的生长速率或反应的生长因子, 其他细胞系可能有戏剧性的变化, 细胞生长或反应。虽然没有例行报告, 我们和许多其他人经历了这种戏剧性的变化, 在较高的通道增生率。原因是不确定的, 但这一变化可能反映的是有限的自我更新能力的 npc. 定义扩展通道上的扩散率变化的原因和方式可能会对发展和疾病发病机制提供洞察, 但进一步研究将需要完成。最后, 我们使用的商用神经感应协议有时会产生质量较差的神经干细胞, 特别是如果起始 iPSCs 不是高质量的 (i. e), 则在细胞菌落的边界上有区别细胞,核型异常)。在某些情况下, 细胞形态学被扭曲, 标记的表达不存在。不要使用这些区域性。在其他情况下, npc 生长与平坦的 "污染物" 细胞, 可以删除使用差异细胞分离解决方案治疗, 以确保几乎纯鼻咽癌在使用前的实验。拥有高质量的 npc 对于正确的结果是至关重要的: 请参阅材料和设备部分, 以获得一个连接到神经感应协议的链接, 在那里可以找到高和低质量的 npc 图像。
对于所介绍的每种检测方法, 必须注意以下关键步骤、潜在错误和故障排除提示。对于细胞数、DNA 合成和突起的测定, 将细胞作为分离的单细胞而不是团簇, 是很重要的, 因为这可能会扭曲 DNA 合成措施、细胞计数和突起行为。为了确保细胞团块不被镀, 样品小体积的细胞与 P1000, 板在幻灯片上, 并检查是否有细胞团簇是否存在。如果注意到团簇, 在电镀之前, 吸管细胞向上和向下手动分离团块。对于 DNA 合成和细胞数测定, 用酶法提取细胞进行计数和分析。这是关键的视觉确认细胞已完全从培养容器中获得准确计数, 如果需要的话, 可以使用更长的酶潜伏期。在低细胞计数或低 CPMs 的 DNA 合成试验中, 细胞可以在较高的初始密度下被镀, 放射性氚 [3H]-胸苷可以增加双倍的时间 (4 小时而不是 2), 或氚 [3H]-胸苷浓度可以加倍。对于突起化验, 初始电镀密度可以加倍, 而不会增加细胞间接触的风险。注意在盘子上的细胞分布, 并计数1厘米行, 使每一部分的盘子被取样。如果突起百分比过低, 在48小时, 可以延长6天或涂层基质类型或浓度可以改变, 以促进更大比例的突起。然而, 重要的是要注意的是, 我们在协议中提出的涂层时间和方法被选择为最佳突起和细胞健康测试后, 许多不同的基质, 基质浓度, 和涂层时间。对于干细胞测定, 使用较小的 pipettors (P20, P200) 可以导致较大球体的剪切和破损。因此, 吹打是必要的, 用 P1000 或血清学吸管轻轻地做。对于造粒 neurospheres, 还建议降低速度 (100 x g 而不是 300 x g), 以防止干细胞离解。在球形电镀过程中, 确保球体与球体之间的接触能适当地间隔, 从而影响迁移。在迁移速度过快或过慢的情况下, 孵化时间可以分别减少或增加。涂层基体也可以改变, 以更改迁移率。
虽然所使用的技术快速, 简单, 适用于神经发育紊乱的研究, 但也存在一定的局限性。其中的许多分析 (细胞数化验, 突起化验, 迁移化验) 要求调查人员作出主观决定 (例如, 这是突起吗？这个牢房死了吗？可能导致研究者的偏倚和较低的重现性。然而, 分析盲目和制定严格标准的每一个决定, 如方法中所示, 可以改善这些偏见。同样, 这些化验需要人工测量和计数, 这可能是耗时和劳动密集型。但是, 在具有设备和技术资源的实验室中, 可以通过使用自动化的单元格计数器和程序来加速这些检测,41、42将自动进行测量。在 DNA 合成试验中, 这些方法是特定于我们的细胞收割机和闪烁机 (见材料和设备);然而, 还有其他可用的模型和方法, 可以用来获得相同的信息, 如 Omnifilter-95 细胞收割机。对某些机构来说, 使用放射源可能是不可行的。在这种情况下, 使用荧光胸苷模拟的替代方法, 例如, 在荧光微板块阅读器分析, 将允许获得相同的信息, 对 DNA 合成的大量分析 44.
我们的低密度培养系统将 npc 分为单个细胞或小团簇, 这种情况与发育中的神经管中 npc 的密集包装性质不同。但是, npc 是健康的, 表达适当的标记 (图 1, 图 10)。此外, 我们以前对老鼠和大鼠皮层培养的研究显示了体外区域性的并行发现,体内模型表明使用此方法的效用和价值16,17,18,19,24. 此外, 该系统还为了解细胞的成熟和研究细胞亚群提供了有力的途径。例如, 免疫组化可以进行突起化验, 以确定什么特定的神经元细胞类型是延长突起。最终, 尽管有一些限制, 这个独特的协议提供了简单, 强大, 快速的方法来研究神经发育紊乱。

使用简单的有机体和老鼠模型阐明了基本的大脑发展和疾病发病机制。尽管有这些进展, 许多精神疾病的病因仍然难以捉摸, 因为并非所有的研究结果都与人类疾病的复杂方面直接相关。此外, 人脑的更大复杂性往往使人的发育和动物疾病的建模变得困难。随着人类诱导多潜能干细胞 (hiPSCs) 技术的发展和发展, 体细胞可以重新编程成干细胞, 然后分化为神经细胞来研究人类疾病。hiPSCs 和 "涨价可能" 技术的进步 (基因组学、转录组学、蛋白质组学、新陈代谢) 有望彻底改变人类大脑发育的认识。这些技术现在可以在个案的基础上对神经精神疾病的特征进行 "精确医学" 处理。
目前在 hiPSC 疾病建模领域的主要内容是将细胞分化成单层的特定神经元亚型, 或者使用一个叫做 organoid 的3D 培养系统来重述大脑发育的各个方面1,2, 3。这些系统在研究和揭露人类发展和疾病的独特方面非常有价值4,5,6,7。然而, 在他们准备学习之前, 神经元文化和 organoids 通常需要从几周到几个月的文化。这些协议的耗时性和维护这些文化系统所需的资源量常常限制可以进行的实验数量和可测试的变量 (如生长因子或药物) 的数量。此外, 许多利用有丝分裂后神经元和 organoids 的研究都集中在后来的发展过程, 如枝晶突起或突触形成。虽然这些过程牵涉到发育紊乱的病理学, 如自闭症和精神分裂症, 早期的发展事件发生之前, 明确的神经元分化也重要的疾病发病机制8 ,9,10,11,12,13。事实上, 最近的基因组研究表明, 由增殖、过程和迁移组成的中胎儿期在自闭症发病机制中尤为重要11,14。因此, 研究神经干细胞和祖细胞群, 以更好地了解这些早期的过程是很重要的。Organoid 系统由于其3D 性质和组织结构而被认为可以更好地概括人脑发育, 但它包含了一个前体池, 用于研究这些早期事件。然而, organoids 的祖群往往稀疏, 更像是径向胶质细胞, 而不是神经干细胞或祖细胞5,15。因此, 有一个高吞吐量的方法来研究神经发育在积极增殖细胞的早期阶段是有益的。
在实验室中, 我们创建了一个使用 hiPSC 神经前体细胞 (npc) 的协议, 一个神经干细胞和祖细胞的混合种群, 高度增生, 以研究神经发育过程, 如增殖、细胞迁移和初始过程 (突起) 扩展。这些化验是从我们实验室使用的技术中开发的, 数十年来成功地研究了老鼠和老鼠皮层培养的神经发育 16, 17, 18, 19, 20, 21,22,23。重要的是, 它还表明, 在鼠和小鼠培养系统中定义的表型和调控信号是高度预测的机制, 是活跃的在体内, 表明这些技术的价值16, 17,18,19,24。在 hiPSCs 与 npc 的初步分化之后, 这些方法使我们能够在几天内研究重要的发育过程。这些方法有许多优点: (1) 它们需要很少的复杂设备, 易于实现, (2) 可以在短时间内进行大量的实验复制, 从而快速确认结果的重现性, (3)培养变量, 如涂层基质, 生长因子的影响, 药物的活性, 可以快速和经济高效地测试。此外, 我们利用细胞外生长因子作为不同发育过程的关键调控者的良好作用。npc 被暴露在选择发展信号, 直接刺激诸如扩散, 突起和细胞迁移等事件, 并发现它们增强了识别在控制条件下不明显的缺陷的能力19,25,26,27,28. 同样, 评估药物的容易性为采用精确的医学技术来测试各种治疗干预措施的功效提供了有力的途径。因此, 本议定书促进了高通量、重现性和直截了当的方法, 研究早期大脑发育、疾病发病机制以及生长因子和药物对神经发育表型的潜在有益影响。

<table border="0" cellpadding="0" cellspacing="0" width="1091">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:323px;">PSC Neural Induction Medium: 
			Protocol Link: https://goo.gl/euub7a&#160;</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td style="width:111px;">A1647801</td>
			<td style="width:472px;">This is a kit that consists of Neurobasal (NB) medium and a 50x Neural Induction Supplement (NIS). The NIS is used to make 1X Neural Induction Medium and 100% Expansion Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Advanced DMEM/F12 Medium</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td style="width:111px;">12634-010</td>
			<td style="width:472px;">Component of 100% Expansion Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Neurobasal Medium</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td style="width:111px;">21103049</td>
			<td style="width:472px;">Component of both NIM and 100% Expansion Medium&#160;</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:323px;">hESC-qualified Matrigel</td>
			<td style="width:185px;">Corning</td>
			<td style="width:111px;">354277</td>
			<td style="width:472px;">hESC-qualified extracellular matrix-mimic gel (ECM-mimic gel)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Y-27632 (2HCl), 1 mg</td>
			<td style="width:185px;">Stem Cell Technologies</td>
			<td style="width:111px;">72302</td>
			<td style="width:472px;">ROCK inhibitor</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:323px;">6 well plates</td>
			<td style="width:185px;">Corning</td>
			<td style="width:111px;">COR-3506</td>
			<td style="width:472px;">Polystyrene plates used for NPC maintenance and for Neurosphere Migration Assay&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">24 well plates</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td style="width:111px;">2021-05</td>
			<td style="width:472px;">Polystyrene plates: Used for NPC DNA Synthesis Assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">35 mm dishes</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td style="width:111px;">2021-01</td>
			<td style="width:472px;">Polystyrene plates: Used for NPC S-Phase Entry and Neurite Assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">Natural Mouse Laminin</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:111px;">23017-015</td>
			<td style="width:472px;">Substrate for coating plates: Used for NPC DNA Synthesis, S-Phase Entry, and Cell Number Assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Fibronectin</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:111px;">F1141</td>
			<td style="width:472px;">Substrate for coating plates: Used for Neurite Assay&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Poly-D-Lysine</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:111px;">P0899</td>
			<td style="width:472px;">Substrate for coating plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">Penicillin/Streptomycin</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td style="width:111px;">15140122</td>
			<td style="width:472px;">Antibiotic, component of NIM, 100% Expansion and 30% Expansion Media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">StemPro Accutase</td>
			<td style="width:185px;">Gibco</td>
			<td style="width:111px;">A11105-01</td>
			<td style="width:472px;">1X Cell Detachment Solution&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2.5% Trypsin (10X)</td>
			<td style="width:185px;">Gibco</td>
			<td style="width:111px;">15090-046</td>
			<td style="width:472px;">10X enzymatic solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">0.5 M EDTA</td>
			<td style="width:185px;">ThermoFischer Scientific</td>
			<td>AM9261</td>
			<td style="width:472px;">used in trypsin solution for lifting cells for DNA synthesis assay</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">tritiated [3H]-thymidine</td>
			<td style="width:185px;">PerkinElmer</td>
			<td style="width:111px;">NET027E001</td>
			<td style="width:472px;">Radioactive tritium, thymidine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Fisherbrand 7 mL HDPE Scintillation Vials</td>
			<td style="width:185px;">Fisherbrand</td>
			<td style="width:111px;">03-337-1</td>
			<td style="width:472px;">Vials for liquid scintillation counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">EcoLite(+)</td>
			<td style="width:185px;">MP Biomedicals</td>
			<td style="width:111px;">0188247501&#160;</td>
			<td style="width:472px;">Liquid scintillation cocktail</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">LS 6500 multi-purpose liquid scintillation counter</td>
			<td style="width:185px;">Beckman Coulter</td>
			<td style="width:111px;">8043-30-1194</td>
			<td style="width:472px;">Liquid Scintillation Counter</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:323px;">Skatron Semi-automactic Cell Harvester Type 11019</td>
			<td style="width:185px;">Molecular Devices &#38; Skatron Instruments, Inc.</td>
			<td style="width:111px;"></td>
			<td style="width:472px;">Semi-automatic cell harvester</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Click-iT EdU Alexa Fluor&#174; 488 Imaging Kit</td>
			<td style="width:185px;">ThermoFisher Scientific</td>
			<td style="width:111px;">C10337</td>
			<td style="width:472px;">EdU and staining kit for S-Phase Entry Assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:185px;">ThermoFisher Scientific</td>
			<td style="width:111px;">15250061</td>
			<td style="width:472px;">Assessing viability of cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">Grade GF/C filter paper</td>
			<td style="width:185px;">GE Healthcare Life Sciences, Whatman</td>
			<td style="width:111px;">1822-849</td>
			<td style="width:472px;">Glass fiber filter paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Human Basic FGF-2</td>
			<td style="width:185px;">Peprotech</td>
			<td style="width:111px;">100-18B</td>
			<td style="width:472px;">growth factor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">Pituitary Adenylate Cyclase Activating Polypeptide (PACAP-38)</td>
			<td>BACHEM</td>
			<td>H-8430</td>
			<td>neuropeptide</td>
		</tr>
	</tbody>
</table>

rapid detection of neurodevelopmental phenotypes in human neural precursor cells (npcs)

人类大脑发育通过一系列精确编排的过程进行, 早期的阶段以增殖、迁移和突起为特色;后阶段的特征是轴突/枝晶生长和突触形成。在神经发育紊乱中, 通常有一个或多个过程被打乱, 导致大脑形成和功能异常。随着人类诱导的多潜能干细胞 (hiPSC) 技术的出现, 研究人员现在有大量的人类细胞供应, 可以被区分成几乎任何细胞类型, 包括神经元。这些细胞可以用来研究正常的大脑发育和疾病发病机制。一些使用 hiPSCs 模型神经精神疾病的协议使用晚期分化的神经元或使用3D 的文化系统称为 organoids。这些方法在研究人类疾病发病机制方面已证明是宝贵的, 但也存在一些弊端。hiPSCs 分化为神经元和 organoids 的产生是冗长而昂贵的过程, 可能会影响可以评估的实验和变量的数量。此外, 在有丝分裂后的神经元和 organoids 允许研究与疾病有关的过程, 包括枝晶生长和 synaptogenesis, 它们排除了对早期的过程, 如扩散和迁移的研究。在神经发育紊乱, 如自闭症, 丰富的遗传和验尸证据表明早期发育过程中的缺陷。神经前体细胞 (npc), 一个高度增生的细胞群体, 可能是一个合适的模型, 其中提出问题的发育过程和疾病的启动。我们现在扩展了从研究老鼠和大鼠皮质文化的发展到人类 npc 的方法学。使用 npc 可以让我们调查与疾病有关的表型, 并定义不同的变量 (例如, 生长因子, 药物) 如何影响发展过程, 包括增殖, 迁移, 并在短短几天的分化。最终, 此工具集可用于重现性和高通量的方式, 以确定神经发育紊乱疾病的特定机制和表型。

这些研究的目的之一是确定 npc 的增殖活动, 即细胞数量的增加。这是通过评估 DNA 合成的总细胞总数, 一个高通量的方法, 测量将放射性示踪剂氚胸苷纳入细胞提取物, 并反映所有从事 s-阶段的细胞, 无论是合成5分钟或整整两个小时。此外, 这些化验可以确定进入 S 相和总细胞数的细胞比例, 一种更劳动密集型的单细胞检测。细胞在 s-阶段合成 DNA, 这是有丝分裂和细胞分裂之前的一步, 必须发生以增加细胞数。由于这些过程需要一段时间, DNA 合成的变化估计在48小时可能不会与细胞数量的变化在这个时间点。尽管如此, 发现 DNA 合成在48小时内的变化可靠地预测4和6天内细胞数量的增加。
在评估 DNA 合成时, 细胞的密度为10万细胞 (~ 50% 汇合) 在24井板, 并允许生长48小时前进行测量。使用这种密度可以确保细胞类似于它们的单层环境, 但也不会在48小时内迅速生长, 以至于培养基变得太酸性。酸性太强的培养基会显著影响细胞的新陈代谢, 从而改变增殖结果。如果特定细胞系高度增生, 研究者应考虑改变细胞密度, 媒体体积, 或媒体交换频率, 以防止高酸性的条件。如果条件发生变化, 在比较不同的单元格线时必须保持一致, 因为细胞对细胞的接触依赖变化肯定会影响生长速率。这些化验的直接设计使我们能够测试不同的生长因子。如图 7所示, 增加成纤维细胞生长因子 (10 ng/毫升) 48 小时将 DNA 合成提高了40%。此外, dna 合成检测是可重复的, 因为所有克隆人和个体显示在基因改造后的 dna 合成增加。在表 1中, 在不同的未受影响的个体中, 基线和后代的变异可能刺激 DNA 合成, 以及同一个体中克隆变异的可能性。由于这种变化, 必须对每个个体的多个 iPSC 克隆进行测试, 并评估从每个不同 iPSC 克隆获得的至少3到5个 NPC 线。每条 NPC 线至少进行3项实验。
dna 合成方法使我们能够快速评估大量的实验组在高通量的方式, 这项措施反映了 DNA 合成的总和, 无论 S 阶段的持续时间 (5 分钟到2小时)。为确定细胞在 s 相中的比例, 采用 s-相进入法。对于这种检测, 细胞生长在相同的密度, 如前所述, 以允许单层样的动力学发生, 但随后它们被离解后2天, 并允许简单地坚持板进行单细胞分析。单层细胞的计数可能是困难的, 因为高细胞间的接触和区域变异的板块。这个范式允许我们将细胞建模为单层, 然后将它们作为单个细胞进行分析。它还作为一个方法学独立确认的数据获得的 DNA 合成化验。如图 8所示, 48 小时 (10 ng/毫升) 刺激会使进入 S 期的细胞比例增加25%。
在评估细胞数时, 使用的细胞密度低于上述化验, 5万个细胞被镀24个井板的每一个井。再次, 这个密度被选择, 以确保更快的细胞线不会增长如此迅速超过6天的时间, 培养基 pH 值变得太酸性和变黄。在图 9中, 虽然在2天内, 控件和 10 ng/mL 组之间的单元格数可能没有显著差异, 但 DNA 合成在 48 h (图 7) 中的变化预测了在4和6天内细胞数的变化。
虽然 npc 通常是在高密度培养, 突起化验是在密度5万细胞镀成一个35毫米的菜, 以评估单细胞。即使在这种低密度, npc 文化表达骨架蛋白和转录因子特征的 npc, 如巢, SOX2 和 PAX6 (图 10 A D)。这表明低密度培养并没有显著改变细胞的命运在这个时间框架。此外, 在老鼠和老鼠培养系统中使用类似的低密度条件来检测最终可重现的在体内16,17,18,19, 20,21,22,23。在48小时的孵化后, 一小部分 npc 开始扩展突起, 如图 11A和B,中所示, 并在图 11C中的图表中进行量化。可以测量扩展突起的细胞比例、突起长度和突起/细胞数量, 以评估发育参数。为了准确地评估突起生长细胞的百分比, 重要的是细胞被镀成单细胞或 < 5 细胞团, 而不是大团聚体。如图 10 E F所示, 承载突起 (白色箭头) 的单元格也表示未成熟的神经元标记β III 蛋白 (TUJ1)。如所述方法, 可以获得 TUJ1 染色的 npc 的荧光图像, 然后这些图像可以计算为 TUJ1+ 突起的比例, 以确保神经元的过程的起源。在我们的实验室中, 任何一种方法的分析都产生了统计学上相似的结果。
简单的设计和快速的性质的突起化验也可以让我们测试的影响, 发育相关的生长因子, 细胞因子和肽。例如,图 11显示了神经肽垂体腺苷酸酶活化多肽 (PACAP, 3 nM) 增加了突起在 npc 中的生长. PACAP 是一种重要的发育因子, 在中枢神经系统中具有广泛的表达, 并显示在大脑发育中是重要的。我们实验室和其他实验室的啮齿动物研究发现, PACAP 在后脑和前脑16、22中都具有与阶段相关的发展效应, 如调节突起的生长、迁移和增殖, 29,30,31。最近的研究由 Ataman et 。(2016) 利用培养的人胎儿皮层细胞表明, 神经元活动诱发了 PACAP 基因表达的9倍增加, 表明肽在人类神经发育中的重要性32。实际上, Table 2显示了从未受影响的个人派生的许多行之间的控制突起和 PACAP (3 nM) 条件的百分比。如表 2所示, 在来自同一个人的不同克隆和来自不同个体的 npc 的细胞系中表达的突起的百分比有一定的变异性。然而, 这些未受影响的个体在 PACAP 的反应中增加了突起的生长, 表明了化验的重现性。
与突起一样, 细胞迁移是大脑正确连接、组织和布线必不可少的重要发展过程。Neurospheres 允许我们研究 npc 迁移的典型高密度条件, 保持细胞之间的联系, 在 npc (图 12)。发育相关因素也可以在 neurospheres 上进行测试, 以评估它们对迁移的影响。例如,图 12显示 PACAP (10 nM) 增加了 npc 的迁移。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56628/56628fig1.jpg"> 图 1: 在段落3中的 npc.npc 在 P3 表达多个阶段特定标记, 包括(A)多能转录因子, SOX2 (B)转录因子特异的前脑 npc, PAX6 和 NPC 骨架蛋白巢。<a href="//cloudflare.jove.com/files/ftp_upload/56628/56628fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56628/56628fig2.jpg"> 图 2: s-相进入检测示意图.S 相进入试验的时间线。<a href="//cloudflare.jove.com/files/ftp_upload/56628/56628fig2large.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/56628/56628fig3.jpg"> 图 3: 量化 s-阶段条目.(A)相位图像, 显示相暗的活细胞 (白色箭头)、相明亮的死细胞 (白星) 和相明亮的活细胞 (绿色箭头)。(B)荧光 DAPI 染色, 显示死细胞 (白星) 中的凝聚核和活细胞中的大核 (白色和绿色箭头)。(C)荧光图像显示明亮的教育局阳性核 (红色箭头) 和斑点的教育局阳性核 (红星)。(D)相位和图像的荧光合并 3-3 c。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56628/56628fig4.jpg"> 图 4: 标识突起.npc 的相对比图像. (A)具有进程 > 2 个单元格的单元格长度, 从而满足突起的标准。(B)具有进程 < 2 个单元格长度的单元格, 因此不考虑突起。(C)表示一个具有2个进程的单元格-为突起标准评估较长的进程。<a href="//cloudflare.jove.com/files/ftp_upload/56628/56628fig4large.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/56628/56628fig5.jpg"> 图 5: 为迁移检测选择 neurospheres.(A)在 24 h 球体上具有代表性的 neurospheres 的相位对比图像不到100微米, 因此不为迁移试验收集。(B) neurospheres 代表字段的相位对比图像, 在72小时。所有球体都在100微米的20µm 范围内, 表明它们已经准备好为迁移试验收集。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56628/56628fig6.jpg"> 图 6: 量化迁移.(A)代表干细胞的相对比图像。(B)蓝色轮廓显示要测量总干细胞区域的跟踪。红色显示用于测量内部细胞质量区域的轮廓。迁移被定义为总干细胞区域-内部细胞质量区域。注意, 白色圆圈显示的单元格不在相邻的地毯中, 因为这些单元格被排除在迁移轮廓之外。<a href="//cloudflare.jove.com/files/ftp_upload/56628/56628fig6large.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/56628/56628fig7.jpg"> 图 7: 评估 DNA 合成.有代表性的对照结果与经基因治疗的 npc (10 ng/毫升) 增加 48 h 的 DNA 合成 (p ≤ 1 x 10-3)。(n = 2-4 井或小组或实验; 3 实验)。误差线代表 SEM。
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56628/56628fig8.jpg"> 图 8: 进入 s-阶段的单元格比例.(A)在对照中孵化的 NPC 细胞的相对比图像 (10 ng/毫升) 处理过的培养基为 48 h. 镶嵌物代表了在控制的荧光和 10 ng/ml 培养基上染色的细胞的更高放大图像。红色箭头表示的单元格是 "正" 和 "白色箭头" 表示的是教育局阴性的细胞。(B)具有代表性的对照结果图 (10 ng/毫升) 处理的 npc. 在 48 h (p ≤1 x 10-3) 中, 其增加 S 相位输入。(n = 2-4 菜/小组/实验; 3 实验)。误差线代表 SEM。
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56628/56628fig9.jpg"> 图 9: 在2、4和6天枚举单元格.(A)控件中的单元格的相位对比图像 (10 ng/毫升) 在2、4和6天处理介质。(B)代表结果图;请注意, 在2天内, ≤0.05 (10 ng/毫升) 并不总是增加细胞数, 但在4和6天增加是明显的 (p)。(n = 2-4 井或小组或实验; 3 实验)。误差线代表 SEM。
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56628/56628fig10.jpg"> 图 10: 低密度条件下的 npc 特性.(A、C、E)低密度条件下的 npc 相位对比图像。(B) npc 快速茎/祖细胞标记巢蛋白 (绿色)、SOX2 (红色) 和核标记 DAPI (蓝色)。(D) PAX6 (红色), DAPI (蓝色)。(F)在低密度下, 扩展突起 (白色箭头) 的细胞也表达未成熟的神经元标记 TUJ1 (绿色)。<a href="//cloudflare.jove.com/files/ftp_upload/56628/56628fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/56628/56628fig11.jpg"> 图 11: 突起的产物。(A) npc 的相位对比图像。黑色箭头指向带有突起的单元格。(B)添加神经肽 PACAP (3 nM) 增加了突起的单元格百分比 (p ≤1 x 10-2)。(C)对控制中的突起的量化以及包含介质的 3 nM PACAP。(n = 2-4 菜/小组/实验; 3 实验)。误差线代表 SEM。
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/56628/56628fig12.jpg"> 图 12: 干细胞迁移。(A) neurospheres 的相对比图像。(B)添加 PACAP (10 nM) 增加干细胞细胞迁移 (p ≤10-2) (C)量化细胞迁移。(n = 20 个球形或小组或实验, 3 个实验)。误差线代表 SEM。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td></td>
			<td colspan="2">媒体</td>
		</tr>
<tr>
<td rowspan="2">病人</td>
			<td rowspan="2">克隆#</td>
			<td rowspan="2">ctrl</td>
			<td>fgf</td>
		</tr>
<tr>
<td>(10 ng/毫升)</td>
		</tr>
<tr>
<td rowspan="2">病人1</td>
			<td>1</td>
			<td>21853</td>
			<td>47538</td>
		</tr>
<tr>
<td>2</td>
			<td>20336</td>
			<td>38070</td>
		</tr>
<tr>
<td rowspan="2">病人2</td>
			<td>1</td>
			<td>7664</td>
			<td>14060</td>
		</tr>
<tr>
<td>2</td>
			<td>16573</td>
			<td>30087</td>
		</tr>
</tbody></table>表 1:DNA 合成.从两个 iPSC 克隆中提取的两个不受影响个体的 DNA 合成值 (CPMs) 的摘要。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td></td>
			<td colspan="2">媒体</td>
		</tr>
<tr>
<td rowspan="2">病人</td>
			<td rowspan="2">克隆#</td>
			<td rowspan="2">ctrl</td>
			<td>pacap</td>
		</tr>
<tr>
<td>(3 毫微米)</td>
		</tr>
<tr>
<td rowspan="2">病人1</td>
			<td>1</td>
			<td>13.60%</td>
			<td>18.10%</td>
		</tr>
<tr>
<td>2</td>
			<td>16.50%</td>
			<td>21.10%</td>
		</tr>
<tr>
<td rowspan="2">病人2</td>
			<td>1</td>
			<td>8.90%</td>
			<td>14.10%</td>
		</tr>
<tr>
<td>2</td>
			<td>14.20%</td>
			<td>21.10%</td>
		</tr>
</tbody></table>表 2:具有突起的单元格百分比。每两个未受影响的个体从两个 iPSC 克隆中获得的突起细胞百分比的摘要。

Watch this Scientific Journal Video about Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs at JoVE.com

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

神经发育过程, 如增殖, 迁移和突起的生长, 往往是不安的精神疾病。因此, 我们提出的协议, 快速和重现性评估这些神经发育过程中的人类 iPSC 派生的 npc。这些协议还允许评估相关生长因子和疗法对 NPC 发展的影响。

人脑神经前体细胞神经发育表型的快速检测 (npc)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

rapid-detection-neurodevelopmental-phenotypes-human-neural-precursor

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

9.9K 次观看.  Rutgers Robert Wood Johnson Medical School. 神经发育过程, 如增殖, 迁移和突起的生长, 往往是不安的精神疾病。因此, 我们提出的协议, 快速和重现性评估这些神经发育过程中的人类 iPSC 派生的 npc。这些协议还允许评估相关生长因子和疗法对 NPC 发展的影响。

视频: Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

科研

发育生物学

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

从人类心脏组织血管周围多能前体细胞群的分离

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

源自iPSC的人脑组织体的一代新型早期神经发育障碍

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

人胎脑神经干细胞 Zika 病毒感染的免疫细胞化学分析

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

人类诱导多能干细胞衍生神经细胞治疗移植小鼠脑损伤控制皮质影响模型

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...