Name: modeling human cerebellar development in vitro in 2d structure
Uploaded: 2022-09-16T14:30:00
Description: Watch this Scientific Journal Video about Modeling Human Cerebellar Development In Vitro in 2D Structure at JoVE.com

我们感谢Jenny Gringer Richards在验证对照受试者方面的全面工作，我们从中生成了对照iPSC。这项工作得到了NIH T32 MH019113（致D.A.M.和K.A.K.），Nellie Ball Trust（致T.H.W.和A.J.W.），NIH R01 MH111578（致V.A.M.和J.A.W.），NIH KL2 TR002536（致A.J.W.）和Roy J. Carver慈善信托基金（致V.A.M.，J.A.W.和A.J.W.）的支持。这些数字是用 BioRender.com 创建的。

人类受试者研究获得了爱荷华大学机构审查委员会批准号201805995和爱荷华大学人类多能干细胞委员会批准号2017-02的批准。在获得书面知情同意后，从受试者那里获得皮肤活检。成纤维细胞在DMEM中用15%胎牛血清（FBS）和1%MEM-非必需氨基酸溶液在37 °C和5%CO2下培养。按照制造商的方案（参见 材料表）使用用于电穿孔的核苷载体，使用游离体重编程试剂盒对成纤维细胞进行重编程?...

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在 体外模拟 人类小脑发育的能力对于疾病建模以及进一步了解正常大脑发育非常重要。不太复杂且具有成本效益的协议为可复制的数据生成和跨多个科学实验室的广泛实施创造了更多机会。这里使用Muguruma等人报告的生长因子使用不需要酶或解离剂的改进方法来描述小脑分化方案，该方法不需要酶或解离剂。图4 和改进的2D单层细胞生长方案类似于Holmes等人的方法<e...

了解人类小脑发育和这一过程的关键时间窗口不仅对于解码神经发育障碍的可能原因很重要，而且对于确定治疗干预的新靶点也很重要。在体外模拟人类小脑发育一直具有挑战性，但随着时间的推移，已经出现了许多区分人类胚胎干细胞（hESC）或具有小脑谱系命运的iPSCs的方案1，2，3，4，5，6，7，8.此外，重要的是开发产生可重复结果的协议，相对简单（以减少错误），并且不承担沉重的货币成本。
小脑分化的第一个方案是从板状胚体（EB）的2D培养物中产生的，用类似于体内发育的各种生长因子诱导小脑命运，包括WNT，BMP和FGFs1，9。最近发表的方案主要在用FGF2和胰岛素诱导3D类器官培养中诱导分化，然后是FGF19和SDF1用于菱形唇样结构3，4，或使用FGF2，FGF4和FGF8的组合5。两种小脑类器官诱导方法都产生了相似的3D小脑类器官，因为两种方案都报告了在相同时间点相似的小脑标志物表达。Holmes和Heine扩展了他们的3D协议5，以表明2D小脑细胞可以从hESC和iPSCs产生，它们以3D聚集体的形式开始。此外，Silva等人7证明，代表2D成熟小脑神经元的细胞可以用与Holmes和Heine类似的方法生成，使用不同的时间点从3D切换到2D并延长生长和成熟时间。
目前的协议通过使用胰岛素和FGF2产生自由漂浮的拟胚体（EB），然后在第14天将EB接种在PLO /层粘连蛋白包被的培养皿上以进行2D生长和分化，从而诱导无饲养层iPSC中的小脑命运。到第35天，获得具有小脑身份的细胞。概括小脑发育早期阶段的能力，特别是在2D环境中，使研究人员能够回答需要单层结构实验的特定问题。该协议也适用于进一步的修改，例如微图案表面，轴突生长测定和细胞分选，以富集所需的细胞群。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-26D</td>
			<td></td>
		</tr>
		<tr>
			<td>1-thio-glycerol</td>
			<td>Sigma</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-26B</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Filter Unit, 0.2 &#181;m aPES, 50 mm Dia</td>
			<td>Fisher Scientific</td>
			<td>FB12566502</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Easy Grip Tissue Cluture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>4D Nucleofector core unit</td>
			<td>Lonza</td>
			<td>276885</td>
			<td>Nucleofector</td>
		</tr>
		<tr>
			<td>5 mL Serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-25D</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Easy Grip Tissue Culture Dish</td>
			<td>Falcon</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well ultra-low attachment plates</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>9&#34; Disposable Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture grade water</td>
			<td>Cytiva</td>
			<td>SH30529.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemically defined lipid concentrate</td>
			<td>Gibco</td>
			<td>11905031</td>
			<td></td>
		</tr>
		<tr>
			<td>Chroman 1</td>
			<td>Cayman</td>
			<td>34681</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II, Type A2, Biological safety Cabinet</td>
			<td>NuAire, Inc.</td>
			<td>NU-540-600</td>
			<td>Hood, UV light</td>
		</tr>
		<tr>
			<td>Costar 24-well plate, TC treated</td>
			<td>Corning</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 6-well plate, TC treated</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td>Thermo Scientific</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO (Dimethly sulfoxide)</td>
			<td>Sigma</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS+/+</td>
			<td>Gibco</td>
			<td>14040133</td>
			<td></td>
		</tr>
		<tr>
			<td>Emricasan</td>
			<td>Cayman</td>
			<td>22204</td>
			<td></td>
		</tr>
		<tr>
			<td>Epi5 episomal iPSC reprogramming kit</td>
			<td>Life Technologies</td>
			<td>A15960</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential 8-Flex</td>
			<td>Gibco</td>
			<td>A2858501</td>
			<td>PSC medium with heat-stable FGF2</td>
		</tr>
		<tr>
			<td>EVOS XL Core Imaging system</td>
			<td>Life Technologies</td>
			<td>AMEX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum - Premium Select</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>L-alanine-L-glutamine supplement</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12 Nutrient Mix</td>
			<td>Gibco</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>HERAcell VIOS 160i CO2 incubator</td>
			<td>Thermo Scientific</td>
			<td>50144906</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Anti-EN2, mouse</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-293311</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-Ki67/MKI67, rabbit</td>
			<td>R&#38;D Systems</td>
			<td>MAB7617</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-PTF1a, rabbit</td>
			<td>Novus Biologicals</td>
			<td>NBP2-98726</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-TUBB3, mouse</td>
			<td>Biolegend</td>
			<td>801213</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440053</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Gibco</td>
			<td>12585</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein</td>
			<td>Gibco</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>MEM-NEAA</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Centrifuge</td>
			<td>Labnet International</td>
			<td>C1310</td>
			<td>Benchtop mini centrifuge</td>
		</tr>
		<tr>
			<td>Monarch RNA Cleanup Kit (50 &#181;g)</td>
			<td>New England BioLabs</td>
			<td>T2040</td>
			<td>Silica spin columns</td>
		</tr>
		<tr>
			<td>Monarch Total RNA Miniprep Kit</td>
			<td>New England BioLabs</td>
			<td>T2010</td>
			<td>Silica spin columns</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA 16%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyamine supplement</td>
			<td>Sigma</td>
			<td>P8483</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine (PLO)</td>
			<td>Sigma</td>
			<td>3655</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>746436</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Sigma</td>
			<td>54317</td>
			<td></td>
		</tr>
		<tr>
			<td>See through self-sealable pouches</td>
			<td>Steriking</td>
			<td>SS-T2 (90x250)</td>
			<td>Autoclave pouches</td>
		</tr>
		<tr>
			<td>Sodium citrate dihydrate&#160;</td>
			<td>Fisher Scientific</td>
			<td>S279-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filters, sterile, PES 0.22 &#181;m, 30 mm Dia</td>
			<td>Research Products International</td>
			<td>256131</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-ISRIB</td>
			<td>Cayman</td>
			<td>16258</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td>Phenol and guanidine isothiocyanate</td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1x)</td>
			<td>Gibco</td>
			<td>12604039</td>
			<td>Cell dissociation reagent&#160;</td>
		</tr>
		<tr>
			<td>Vapor pressure osmometer</td>
			<td>Wescor, Inc.</td>
			<td>Model 5520</td>
			<td>Osmometer</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Biogems</td>
			<td>1293823</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling human cerebellar development in vitro in 2d structure

小脑的精确和及时发育不仅对准确的运动协调和平衡至关重要，而且对认知也至关重要。此外，小脑发育的中断与许多神经发育障碍有关，包括自闭症、注意力缺陷多动障碍 （ADHD） 和精神分裂症。人类小脑发育的研究以前只能通过尸检或神经成像进行，但这些方法不足以了解早期 发育过程中体内 发生的分子和细胞变化，这是许多神经发育障碍的起源。从体细胞产生人类诱导多能干细胞（iPSC）的技术的出现以及进一步将iPSC重新分化为神经元的能力为早期大脑发育的 体外 建模铺平了道路。本研究为需要二维（2D）单层结构的应用提供了生成小脑细胞的简化步骤。代表早期发育阶段的小脑细胞通过以下步骤 来源 于人iPSC：首先，在三维（3D）培养中制造胚状体，然后用FGF2和胰岛素处理以促进小脑命运规范，最后，在聚l-鸟氨酸（PLO）/层粘连蛋白包被的底物上最终分化为单层。在分化 35 天时，iPSC 来源的小脑细胞培养物表达小脑标志物，包括 ATOH1、PTF1α、PAX6 和 KIRREL2，表明该方案产生谷氨酸能和 GABA 能小脑神经元前体，以及浦肯野细胞祖细胞。此外，分化的细胞显示出独特的神经元形态，并且对神经元身份的免疫荧光标志物（如TUBB3）呈阳性。这些细胞表达轴突引导分子，包括信号蛋白-4C、丛蛋白-B2和神经皮林-1，可以作为研究神经突生长和突触连接的分子机制的模型。该方法生成可用于下游应用的人类小脑神经元，包括需要 2D 单层格式的基因表达、生理和形态学研究。

3D 至 2D 小脑分化概述 小脑细胞从iPSC开始产生。 图 1A 显示了整个工作流程以及为差异化添加的主要组件。在第0天，使用含有SB431542和Y-27632的CDM中的拉式玻璃移液管轻轻提起iPSC菌落（图1B）并放入超低附着板中，从而制备EB。在第 2 天添加 FGF2。在第7天，改变三分之一的培养基，并观察到EB形成（图1C）。在第14天，...

Watch this Scientific Journal Video about Modeling Human Cerebellar Development In Vitro in 2D Structure at JoVE.com

Modeling Human Cerebellar Development In Vitro in 2D Structure

本协议解释了从诱导多能干细胞生成2D单层小脑细胞，用于研究小脑发育的早期阶段。

在2D结构中模拟人类小脑体 外 发育

The precise and timely development of the cerebellum is crucial not only for accurate motor coordination and balance but also for cognition. In addition, ...

这种方法可以帮助研究早期人类小脑发育以及它在神经精神疾病中如何改变。该方法的主要优点是在2D层结构中生成发育的早期人小脑细胞，而无需执行复杂的步骤。它也具有成本效益。制作拉移液器在开始时可能很棘手，但随着时间的推移，以更容易使用的方式拉移移液器会变得更容易。开始实验准备，将一个22.9厘米的巴斯德移液器拉到颈部下方约两厘米处，在本生燃烧器上方，以创建两个玻璃移液器。较薄的一面将比另一面短约四厘米。将拉动侧的尖端弯曲在火焰上以形成光滑的R，然后将拉动的移液器放入高压灭菌器套筒中，并在高压灭菌器中对其进行消毒。在第零天，向六孔超低附着或ULA板的每个孔中加入一毫升补充有10微摩尔Y-27632和SB-431542的小脑分化培养基，并将其放入培养箱中，直到提升的菌落准备好添加到孔中。使用拉动玻璃移液管清洁分化的细胞。吸出培养基，每 35 毫米培养皿加入 1 毫米的 iPSC 传代溶液。在37摄氏度下孵育三分钟后，吸出培养基并加入补充有10微摩尔Y-27632和SB-431542的两毫米小脑分化培养基在透射光倒置显微镜的4倍放大倍数下，使用拉动玻璃移液器的弯曲边缘提起菌落。提起每个菌落后，使用 10 毫升血清移液管将所有菌落轻轻转移到六孔 ULA 板的一个孔中。对每个iPSC板重复此过程，并将细胞在37摄氏度下用5%二氧化碳孵育。在第二天，向每个孔中加入FGF2，以调整每毫升50纳克的最终浓度。在第七天，通过吸出一毫升用过的培养基并用一毫升新鲜的小脑分化培养基代替来更换三分之一的培养基。孵育胚状体七天。在第14天，旋转板以将所有胚状体聚集在板的中心。然后倾斜板并使用 1000 微升移液器从边缘吸出所有用过的培养基。随着培养基量的减少，慢慢将板放平并继续吸出，留下足够的培养基以避免将拟胚体拉出。接下来，加入三毫升补充有10微摩尔Y-27632的新鲜小脑分化培养基。然后使用10毫升血清移液管将胚状体转移到聚-L-鸟氨酸层粘连蛋白包被的培养皿中。在第15天，吸出培养基并用新鲜的小脑分化培养基代替。在第0天，将iPSC菌落清洁并提升到含有SB-431542和Y-27632的小脑分化培养基中，并放入超低附着板中以产生胚状体。在第2天添加FGF2，在第7天改变1/3培养基，导致胚胎体在第14天显示增大。细胞在第15天开始沿着涂层表面从拟胚体向外迁移。从第21天开始，培养基完全转变为小脑成熟培养基，到第35天时，细胞具有神经元样形态和复杂性的单层细胞。第35天的基因表达分析显示，这些细胞表达早期小脑祖细胞标志物，如谷氨酸能祖细胞、ATOH1、GABA能祖细胞、PTF1α和浦肯野祖细胞标志物KIRREL2和SKOR2。此外，还观察到菱形唇标志物OTX2和大部分前神经元特异性SIX3的表达。第35天收获的细胞的免疫荧光标记显示小脑标志物EN2和PTF1 α，神经元标志物β微管蛋白III和增殖标志物KI67染色阳性。用 4'6-二脒基-2-苯基吲哚或 DAPI 进行核染色显示细胞核。为了成功分化，从健康和未分化的iPSC菌落开始，并使用尽可能新鲜复溶的试剂至关重要。

Research

JoVE Journal

Neuroscience

Modeling Human Cerebellar Development In Vitro in 2D Structure

Operant Sensation Seeking in the Mouse

在鼠标的操作性感觉寻求

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

科研

神经科学

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

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 ...

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

使用扩频萧条的情节和慢性偏头痛的神经免疫信号建模<em在体外</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Laser Nanosurgery of Cerebellar Axons In Vivo

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

在癌神经侵袭的体外模型：背根神经节型号

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

小鼠原代多元体的体外模拟？

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

在2D结构中模拟人类小脑体 外 发育

在2D结构中模拟人类小脑体外发育