Name: synaptic microcircuit modeling with 3d cocultures of astrocytes and neurons from human pluripotent stem cells
Uploaded: 2018-08-16T14:00:00
Description: Watch this Scientific Journal Video about Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells at JoVE.com

我们要感谢埃里克 Ullian 博士 (UCSF) 在设计这些程序方面的知识投入, 迈克尔. 沃德博士 (NIH) 就 iNeuron 分化和 Barlas 进行初步图像分析提供技术咨询。

1. 细胞培养和试剂制备
注: 本节中的协议按照它们在差异化协议中的显示顺序编写 (第2节)。请参阅材料和目录编号的材料表。
<ol><li>
为细胞培养制备涂层板材。
	<ol>
<li>稀释细胞外基质 (ECM) 涂层溶液与 DMEM/F12 介质, 以准备1毫克/毫升的库存解决方案。整除稀释的 ECM 库存溶液成30个3毫升的锥形管, 并立即存储在-20 °c。对于一个工作的解决方案, 并用?...

在本协议中, 我们描述了一个系统的方法来生产3D 球体的神经 cocultures。球体由星形胶质细胞和神经元组成, 独立于 hPSCs。虽然不是本协议的重点, 但从 hPSCs28中生成的纯星形胶质细胞是一个关键步骤, 如果没有事先的经验, 它在技术上可能具有挑战性。这些突触集成电路的第一步应该是细致的定时和注意细节。hPSC 星形胶质细胞的使用限制是长的分化过程;然而, 生产大量的细胞, 可?...

星形胶质细胞是中枢神经系统 (CNS) 内一种高度丰富的神经细胞类型, 其功能职责超出结构支持范围。通过可溶性 synaptogenic 因子和细胞外基质 (ECM) 组分的分泌, 星形胶质细胞在发育过程中帮助成熟突触的建立和聚集1。通过胞外信号2、3、4、5, 它们在保持突触的健康和可塑性方面发挥着关键作用, 并有助于维持稳定的长期稳定性。环境通过调节胞外钾和谷氨酸, 以及能量基质的分泌和 ATP6,7,8。最后, 它们可以通过影响 extrasynaptic 电流9来促进神经传递, 并且可以通过其他细胞类型 (如促进髓鞘形成10) 间接地影响活动。重要的是, 由于星形胶质细胞的异常或功能障碍会导致许多神经发育综合征和成人神经病理学, 显然需要将星形胶质细胞与神经网络中的神经元包括在一起, 以改善内源性脑环境模型。星形胶质细胞的一个整体特征是它们能够形成与神经元突触1,11,12的动态相互作用。在没有胶质细胞的情况下, 神经元形成数量有限的突触, 一般也缺乏功能成熟度13。
人星形胶质细胞显示形态学, 转录和功能特征-如增加的大小和复杂性的分支, 以及物种特异基因-不概括在啮齿目动物12,14, 15。因此, 利用人类多潜能干细胞 (hPSC) 衍生的神经细胞的研究已被广泛接受为一种检测中枢神经系统相关疾病的方法, 同时开发新的疗法, 伤害模型和文化范例16 ,17。此外, hPSCs 允许研究人类突触的形成和功能, 而不需要初级组织18,19。
我们理解各种细胞类型和信号对突触电路功能的贡献的障碍是缺乏相关的人脑模型。需要一个适当的平台来重述其突触网络的高保真度和重现性。最近, 3D 文化系统 (广为人知的 "organoids"、"球体" 或 "迷你大脑") 的生产中出现了兴趣,在细胞和宏观层面上对复杂的三维 (3D) 结构建模。3D. 在典型的2D 共培养范式21、22中, 文化系统保留 ECM 和细胞间的相互作用, 通常不存在或受到限制。为培养3D 神经球体23、24、25, 存在大量的技术;然而, 许多人需要长时间的文化周期 (几个月到几年) 来进行自发的开发和层的保存, 用户对输出的控制很少。
在这里, 我们说明了一个系统的方法, 迅速和一贯生物工程的神经相互作用之间的多种细胞类型 (预分化神经元和星形胶质细胞) 从 hPSCs 的组装细胞成球形 cocultures (' 小行星 ')26在3D 中概括了人类特有的形态学复杂性。这种高密度的神经系统产生均匀分散的神经亚型, 随着时间的推移而逐渐成熟, 可以用高通量的方式进行筛选或测定。我们首次证明, 人类星形胶质细胞诱发突触网络爆裂活动在这些 3D cocultures。此外, 该协议很容易适应产生不同大小的球体, 利用指定给中枢神经系统不同区域特性的细胞, 并根据需要研究多种其他细胞类型的相互作用。

<table>
	<tbody>
		<tr>
			<td>6 well plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Olympus Plastics</td>
			<td>28-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma</td>
			<td>A6964-100ML</td>
			<td>Detachment solution</td>
		</tr>
		<tr>
			<td>AggreWell plate</td>
			<td>Stemcell Technologies</td>
			<td>34850</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution</td>
			<td>Stemcell Technologies</td>
			<td>7010</td>
			<td>Prevent cell adhesion to microwell plates</td>
		</tr>
		<tr>
			<td>Anti/anti</td>
			<td>Thermofisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermofisher</td>
			<td>17504044</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>BrainPhys neuronal medium</td>
			<td>Stemcell Technologies</td>
			<td>5790</td>
			<td>Neurophysiological basal medium alternative</td>
		</tr>
		<tr>
			<td>Circular glass coverslips</td>
			<td>Neuvitro</td>
			<td>GG-12-oz</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostor CS10</td>
			<td>Stemcell Technologies</td>
			<td>7930</td>
			<td>Cryopreservation medium with 10% DMSO</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermofisher</td>
			<td>10565-042</td>
			<td>With GlutaMAX supplement</td>
		</tr>
		<tr>
			<td>DMH-1</td>
			<td>Stemcell Technologies</td>
			<td>73634</td>
			<td>HAZARD: Toxic if swallowed. Working concentration: 2 uM</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Lampire Biological Laboratories</td>
			<td>7332100</td>
			<td>Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer</td>
		</tr>
		<tr>
			<td>Doxycycline Hydrochloride (Dox)</td>
			<td>Sigma</td>
			<td>D3072-1ml</td>
			<td>HAZARD: Toxic for pregnant women. Working concentration: 2 ug/mL</td>
		</tr>
		<tr>
			<td>Epidermal growth factor (EGF)</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor-2 (FGF)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Fluoromount-G mounting solution</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisherbrand</td>
			<td>22-037-246</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Lampire Biological Laboratories</td>
			<td>7332500</td>
			<td>Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer</td>
		</tr>
		<tr>
			<td>Hemacytometer or automatic cell counter</td>
			<td>Life Technologies</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149-50KU</td>
			<td>Working concentration: 2 mg/mL</td>
		</tr>
		<tr>
			<td>Magnetic plate</td>
			<td>DLAB</td>
			<td>8030170200</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel membrane matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td>ECM coating solution. Working concentration: 80 ug/ml. Prepare on ice and ensure that pipettes, tubes, and media are pre-chilled.</td>
		</tr>
		<tr>
			<td>MEA 2100 System</td>
			<td>Multichannel Systems</td>
			<td>MEA2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Thermofisher</td>
			<td>17502048</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>Tissue embedding solution for cryosectioning</td>
		</tr>
		<tr>
			<td>Pap Pen (Aqua Hold)</td>
			<td>Scientific Device Laboratory</td>
			<td>9804-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Acros Organics</td>
			<td>169650025</td>
			<td>HAZARD: Toxic if inhaled. Working concentration: 4% in PBS</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Stemcell Technologies</td>
			<td>CA008-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-l-ornithine (PLO)</td>
			<td>Sigma</td>
			<td>P3655-100MG</td>
			<td>Working concentration: 0.5 mg/mL</td>
		</tr>
		<tr>
			<td>Rectangular glass cover slips</td>
			<td>Fisherfinest Premium Superslip</td>
			<td>12-545-88</td>
			<td></td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stemcell Technologies</td>
			<td>5872</td>
			<td>Detachment and passaging reagent</td>
		</tr>
		<tr>
			<td>Rho-Kinase Inhibitor Y27632- (Y)</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Working concentration: 10 uM</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemcell Technologies</td>
			<td>72234</td>
			<td>Working concentration: 2 uM</td>
		</tr>
		<tr>
			<td>Spinner flasks</td>
			<td>Fisher Scientific</td>
			<td>4500-125</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Chemical</td>
			<td>S5-3</td>
			<td>Working concentration: 20% or 30% in PBS</td>
		</tr>
		<tr>
			<td>T25 Culture Flask</td>
			<td>Olympus Plastics</td>
			<td>25-207</td>
			<td>Vented caps</td>
		</tr>
		<tr>
			<td>T75 Culture Flask</td>
			<td>Olympus Plastics</td>
			<td>25-209</td>
			<td>Vented caps</td>
		</tr>
		<tr>
			<td>Terg-A-zyme</td>
			<td>Sigma</td>
			<td>Z273287-1EA</td>
			<td>Detergent. Working concentration: 1%</td>
		</tr>
		<tr>
			<td>TeSR-E8 basal medium</td>
			<td>Stemcell Technologies</td>
			<td>5940</td>
			<td>Human pluripotent stem cell (hPSC) medium</td>
		</tr>
		<tr>
			<td>TeSR-E8 supplements</td>
			<td>Stemcell Technologies</td>
			<td>5940</td>
			<td>Supplements for human pluripotent stem cell medium</td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
			<td>Detergent for cell permeabilization. Working concentration: 0.25% in blocking buffer</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 488</td>
			<td>Thermofisher</td>
			<td>A-11029</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>AlexaFluor 594</td>
			<td>Thermofisher</td>
			<td>A-11037</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Ezrin</td>
			<td>Thermofisher</td>
			<td>MA5-13862</td>
			<td>Primary antibody; astrocytes perisynaptic</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Chemicon</td>
			<td>MAB360</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>Aves</td>
			<td>GFP-1020</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>Glt1</td>
			<td>Gift from Dr. Jeffrey Rothstein</td>
			<td>n/a</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>Homer</td>
			<td>Synaptic Systems</td>
			<td>160 011</td>
			<td>Primary antibody; neurons, post-synaptic</td>
		</tr>
		<tr>
			<td>MAP2</td>
			<td>Synaptic Systems</td>
			<td>188 004</td>
			<td>Primary antibody; neurons</td>
		</tr>
		<tr>
			<td>PSD95</td>
			<td>Abcam</td>
			<td>ab2723</td>
			<td>Primary antibody; neurons, post-synaptic</td>
		</tr>
		<tr>
			<td>S100</td>
			<td>Abcam</td>
			<td>ab868</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>Synapsin 1</td>
			<td>Synaptic Systems</td>
			<td>106 103</td>
			<td>Primary antibody; neurons, pre-synaptic</td>
		</tr>
		<tr>
			<td>TuJ1/&#946;3-tubulin (TUBB3)</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>Primary antibody; neurons</td>
		</tr>
	</tbody>
</table>

synaptic microcircuit modeling with 3d cocultures of astrocytes and neurons from human pluripotent stem cells

我们理解各种细胞类型和信号对突触电路功能的作用的障碍是缺乏研究人脑的相关模型。解决这个问题的一项新兴技术是使用三维 (3D) 神经细胞培养, 称为 ' organoids ' 或 ' 球体 ', 长期保存细胞间相互作用, 包括细胞外黏附分子。然而, 这些文化系统是费时的, 没有系统地产生。在这里, 我们详细的方法, 以迅速和一贯地产生 3D cocultures 神经元和星形胶质细胞从人类多潜能干细胞。首先, 对预分化的星形胶质细胞和神经元祖先进行离解和计数。接下来, 细胞被结合在球形形成的菜肴与一个蛋白激酶抑制剂, 并在特定的比率, 以产生可再生的大小球体。经过几个星期的文化作为漂浮球体, cocultures (' 小行星 ') 最终被切片的染色或电镀后, 多电极阵列, 以测量突触密度和强度。一般情况下, 该协议将产生3D 神经球体, 显示成熟的细胞类型限制标记, 形成功能性突触, 并表现自发突触网络突发活动。与单层培养相比, 该系统允许对疾病机制进行药物筛选和调查。

当正确执行时, 此协议将产生由 hPSCs (图 1A-1C) 生成的星形胶质细胞28、33、34和神经元35的功能 cocultures 的定义种群, 如详细的前26和这里描述的步骤2.1–2.2。这个逐步的过程, 使用 microwell 板, 预计产生3D 的神经球体的大小和形状一致 ...

Watch this Scientific Journal Video about Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells at JoVE.com

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

在本协议中, 我们的目的是描述一种可重现的方法, 将分离的人类多能干细胞衍生神经元和星形胶质细胞组合成3D 球体 cocultures, 在自由漂浮条件下维持这些球体, 随后用 immunoanalysis 和多电极阵列记录测量球体的突触电路活动。

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Research

JoVE Journal

Developmental Biology

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

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

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

科研

发育生物学

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

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

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

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RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

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

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

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用DMSO对人多能干细胞的暂时性治疗促进分化

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...