这项研究得到了日本科学促进会（JSPS）17H05661和18K19903、核心-2核心项目和超越人工智能研究所的支持。

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在该协议的一部分中，一项专利已授权给吉克萨克生物工程公司，该公司是由川田吉罗创立的。

此协议描述了运动神经器官 （MNO） 的形成，该有机体具有从人类 iPS 细胞生成的运动神经元球体中延伸的轴束。形成的轴束厚、灵活，在单向结构中组织良好。通过解剖轴突束，可以充分获得高纯度轴蛋白和RNA进行生化分析。神经元活性可以通过钙成像在轴突束和球形中测量。西方的污点没有检测到轴突利酸盐中核蛋白和树突蛋白的污染，这表明我们的方法有效地将轴突与细胞体和树突分离出来。
此协议的优点之一是 MNO 配备轴成捆绑包的快速分化和生成，其中所有流程均可在 4 周内使用 3D 协议完成，使用 2D 协议可在 5-6 周内完成。与其他通常需要 3-4 周才能从胚胎干细胞和 iPS 细胞17 中分化成 MN 的协议相比，这很短，并且需要额外的 2-4 周才能获得轴突拉伸。与 2D 协议相比，3D 协议通常优先于 2D 协议，因为与 2D 协议相比，没有分离步骤的分化时间更短、步骤更少、技术风险更小。基于微流体的组织培养芯片的设计方式使MNS的轴突可以通过微通道向另一个腔室拉长，从而通过诱导轴突的相互作用和轴突之间的亲和力，促进轴突束的形成。由于简单的实验设置，这里描述的所有方案不仅可以由生物工程师执行，他们熟悉组织培养芯片的操作，而且生物学家和神经科学家也不熟悉微流体和微压造技术。需要注意的是，第 1 步和第 2 步也可以使用外部制造服务执行。
完成协议的关键步骤之一是文化媒介的连续变化。建议在分化过程中每一步彻底改变文化介质，使乏力介质中的因素不干扰MN分化。此协议的另一个关键点是保持未分化的 iPS 细胞质量良好。初始 iPS 细胞培养物的质量显著影响获得运动神经元和 MNO 的效率。另一点是MNS的直径应该小于芯片孔的大小（1.5毫米）。较大的球体无法进入腔室，并有可能在中心部分经历严重的缺氧坏死。MNS 的大小可以通过更改 iPS 细胞的初始播种数量（在 3D 协议中）或运动神经元（在 2D 协议中）来控制。应针对每个 iPS 细胞线优化细胞的播种密度。
带微胶和小孔滤镜的分体微流体设备已被广泛用于将轴突与细胞体和树突分离。这种技术还可以将轴突与细胞体和树突分离出来，在捆绑组织中具有超强的轴突丰度。与其他方法相比，该方法的一个主要局限性是，在组织培养芯片的当前设计中，它不能分离两种不同的培养基，这妨碍了需要两种不同介质的两种不同细胞的共生能力。另一个限制是 PDMS 芯片对组织大小施加了预先确定的限制。大于孔的球体不能进入腔室，轴束不能比微流道的宽度更厚。
这种方法可以应用于其他类型的神经元。我们小组已经显示出使用修改的方法结合脑器官技术18对脑路进行建模的能力。皮质球体被引入舱室，轴突自发地向每个球体相互拉长，随后自发形成轴突束。因此，两个皮质球体可以通过轴束连接，组织可以作为一块获得。这表明，无论神经元细胞类型如何，这种方法都具有高度的多功能性，可以形成轴突束组织。在此协议中，使用了人类iPS细胞，但是，其他干细胞，包括人类ES细胞和人类神经干细胞，可以与修改提出的协议。神经元的3D球形可以通过不同的协议19，20产生。这种用轴束制作组织的方法将来有可能与其他分化方案相结合，用于制造3D MN球形。此外，只需改变组织培养芯片微通道的宽度和高度，即可控制轴束的厚度和长度，以备将来发展之用。
我们相信，这一协议可用于药物测试和筛选，并有助于了解轴感筋膜发育和疾病背后的机制。

脊髓运动神经元 （MN） 将轴突扩展到骨骼肌肉以控制身体运动。它们的轴突轨迹在发展过程中具有高度的组织性和规范性。尽管对轴承扩展和指导1进行了许多研究，但组织轴束形成机制仍在调查中。运动神经元的轴突经常受到肌萎缩性侧索硬化症（ALS）2等神经退行性疾病的损害，但对轴突筋膜损伤的病理生理机理知之甚少。因此，需要一个生理和病理模型来回顾轴束的形成和回归。
人类干细胞源性运动神经元是了解ALS3等发育和疾病的有希望的平台。人类诱导的多能干细胞（iPS细胞）可用于使用患者衍生细胞模拟疾病。迄今为止，从多能干细胞到MN的各种分化方法已报告4，5，6。然而，二维培养中神经元的轴突是随机定向的，在发育中的神经中，轴突通过密集的轴突-轴位相互作用7进行单向组装，不能在体内微环境中重述。为了克服这个问题，我们开发了一种技术，从人类iPS细胞8中产生类似于运动神经的三维组织，并将组织命名为运动神经器官（MNO）。MNO由位于运动神经元球形（MNS）中的细胞体和从球形延伸出去的轴状筋膜组成。圆柱体中的轴突是单向方向的，类似于发展运动神经的轴突。因此，MNOs独特地提供了一种生理轴突微环境，这是任何其他以前开发的神经元培养方法所没有的。
在此协议中，我们描述了已开发芯片中组织培养芯片制造、快速运动神经元分化和运动神经器官形成的方法。我们的组织培养芯片非常简单，它只包含一个接受球形的隔间，一个用于形成轴束的微通道，以及一个用于安装轴星终端的隔间。该设备不包含复杂的结构，包括微流道或微孔过滤器，通常用于分离轴突和细胞体的大小9，10。因此，如果有光刻设置，则可以通过遵循本协议中描述的步骤轻松制造我们的设备。
通过诱导和模式因子（SB431542、LDN-193189、转录剂酸（RA）和平滑激动剂（SAG）和加速因子（SU5402 和 DAPT）的优化组合，实现了人类 iPS 细胞的快速分化。据报道，SU5402和DAPT的结合加速了外周神经元和神经峰细胞11的分化。在此协议中，我们提供了三种不同的方法来生成 MNO，以便读者可以决定最适合其需求的方法。我们建议在形成球形（3D方法）后对人体iPS细胞进行分化，因为分化的MNS可以直接转移到组织培养芯片中。或者，人类iPS细胞可以在单层（2D）培养中分化成运动神经元，然后创建成三维运动神经元球形，正如我们之前报道的8。我们更新了协议，通过本协议中描述的三维分化方法，可以避免从 2D 到 3D 的过渡，并且无需分离步骤即可以更短的分化时间、更少的步骤和更低的技术风险获得 MNO。商业上可用的神经元也可用于生成 MNS 以减少分化时间。
为了生成 MNO，我们在组织培养芯片中培养了 MNS。轴突从球体拉长，并延伸到轴突聚集和单向对齐的微通道。这有利于斧轴相互作用和在微通道中自发形成紧密组装的轴突单向束组织，这是本协议所独有的，而无论是自发捆绑形成还是引导轴突定向单独可以通过其他协议12、13、14来实现。在一个典型的实验中，很少有细胞从球体迁移到微通道，而且大多数细胞都留在球形附近。这种方法允许轴突自发地从球体中分离出来，而无需使用依赖大小的物理屏障（例如微沟或微孔过滤器）将轴突与细胞体分离。
由此产生的 MNO 可接受各种检查，包括形态学、生化和物理分析。细胞体和扩展轴束可以通过切割进行物理分离，并可单独分析以进行下游实验，例如生化检测。生物材料，包括RNA和蛋白质，可以从几个轴成束中分离，用于常规生化检测，包括RT-PCR和西方印迹。在这里，我们描述了从iPS细胞中生成运动神经器官的协议，它提供了一个有吸引力的生理和病理模型来研究轴心筋膜发育和疾病的机制。

<table><tbody><tr><td>(Tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane</td><td>Sigma</td><td>440302</td><td></td></tr><tr><td>16% Paraformaldehyde (formaldehyde) aqueous solution</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>200&amp;micro;l Wide Bore Pipet Tips</td><td>BMBio</td><td>BMT-200WRS</td><td></td></tr><tr><td>6-well plates</td><td>Violamo</td><td>2-8588-01</td><td></td></tr><tr><td>Accutase</td><td>ICT</td><td>AT104</td><td></td></tr><tr><td>B-27 Supplement (50X)</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A6003</td><td></td></tr><tr><td>Brain-derived neurotrophic factor (BDNF)</td><td>Wako</td><td>020-12913</td><td></td></tr><tr><td>CO2 incubator</td><td>Panasonic</td><td>MCO-18AIC</td><td></td></tr><tr><td>Cryostor CS10</td><td>Stem Cell Technologies</td><td>07959</td><td></td></tr><tr><td>DAPT</td><td>Sigma</td><td>D5942</td><td></td></tr><tr><td>DMEM/F12</td><td>Sigma</td><td>D8437</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Dojindo Laboratories</td><td>CS22</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>Growth factor reduced Matrigel (basement membrane matrix)</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>HB9 Antibody</td><td>Santa Cruz</td><td>sc-22542</td><td></td></tr><tr><td>HBSS</td><td>Wako</td><td>085-09355</td><td></td></tr><tr><td>Hoechst 33342</td><td>Sigma</td><td>14533</td><td></td></tr><tr><td>iCell motor neuron (commercially available human iPS cell-derived motor neurons)</td><td>Cellular Dynamics</td><td>R1051</td><td></td></tr><tr><td>Isopropyl alcohol (IPA)</td><td>Wako</td><td>166-04836</td><td></td></tr><tr><td>Knock Out Serum Replacement</td><td>Gibco</td><td>10828028</td><td></td></tr><tr><td>LDN193189</td><td>Sigma</td><td>SML0559</td><td></td></tr><tr><td>MEA probe</td><td>Alpha MED Scientific inc</td><td>MED-P5004A</td><td></td></tr><tr><td>MEM Non-essential Amino Acid Solution (100x) (NEAA)</td><td>Sigma</td><td>M7145</td><td></td></tr><tr><td>Microscope Glass</td><td>Matsunami</td><td>S9111</td><td></td></tr><tr><td>mTeSR Plus</td><td>Stem Cell Technologies</td><td>05825</td><td></td></tr><tr><td>N2 supplement</td><td>Wako</td><td>141-08941</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Photoresist SU-8 2100</td><td>Microchem</td><td>#SU-8 2100</td><td></td></tr><tr><td>Prime surface 96U</td><td>Sumitomo Bakelite</td><td>MS-9096U</td><td></td></tr><tr><td>ReLeSR (passaging reagent)</td><td>Stem Cell Technologies</td><td>05872</td><td></td></tr><tr><td>Retinoic acid</td><td>Wako</td><td>186-01114</td><td></td></tr><tr><td>SAG</td><td>Sigma</td><td>SML1314</td><td></td></tr><tr><td>SB431542</td><td>Wako</td><td>192-16541</td><td></td></tr><tr><td>Silicon wafer</td><td>SUMCO</td><td>PW-100-100</td><td></td></tr><tr><td>Silpot 184 w/c kit</td><td>Dow Toray</td><td>Silpot 184 w/c kit</td><td></td></tr><tr><td>Smi32 Antibody</td><td>Biolegend</td><td>801701</td><td></td></tr><tr><td>SU5402</td><td>Sigma</td><td>SML0443</td><td></td></tr><tr><td>SU-8 Developer</td><td>Microchem</td><td>Y020100</td><td></td></tr><tr><td>Synapsin I Antibody</td><td>Millipore</td><td>Ab1543</td><td></td></tr><tr><td>TrypLE Express liquid without phenol red (dissociation solution)</td><td>Gibco</td><td>12604-021</td><td></td></tr><tr><td>Tuj1 Antibody</td><td>Biolegend</td><td>801202</td><td></td></tr><tr><td>Y-27632</td><td>Wako</td><td>030-24021</td><td></td></tr></tbody></table>

three-dimensional motor nerve organoid generation

轴突的筋膜是神经系统中观察到的主要结构图案之一。轴感筋膜的破坏可能导致发育和神经退行性疾病。虽然已经对轴突进行了大量研究，但由于缺乏坚固的体外模型，我们对轴突筋膜的形成和功能障碍的理解仍然有限。在这里，我们描述了一个循步骤的协议，从微流体为基础的组织培养芯片中从人类诱导的多能干细胞（iPS）细胞中快速生成运动神经器官（MNO）。首先，描述了用于该方法的芯片的制造。从人类iPS细胞中，形成运动神经元球形（MNS）。接下来，区分的 MNS 将传输到芯片中。此后，轴突自发地从球形中长出来，在芯片中装备的微通道内组装成一个圆柱体，从而产生一个MNO组织，携带一束从球形延伸的轴突。对于下游分析，MNO 可以从芯片中取出，用于形态分析或剖析以进行生化分析，以及钙成像和多极阵列记录。通过该协议产生的多国机构可以促进药物测试和筛选，并有助于了解轴位筋膜发育和疾病的基本机制。

在3D分化程序中，运动神经元在12-14天内被分化（图4和图5）。重要的是，超过60%的细胞在分化过程中表示运动神经元标记HB9。免疫化学显示，MNS中大约80%的细胞是SMI32阳性运动神经元。HB9和SMI32是已建立的早期运动神经元标记15，16。HB9 和 SMI32 的表达是需要确认以确保运动神经元细胞识别的关键参数。在将 MNS 引入培养芯片后，轴突扩展到通道和轴突捆绑形式。由于微通道充当物理导引，轴突从 MNS 拉长，并通过轴突交互（图 6A）形成捆绑。必须通过微观观察确认轴束的形成，以确认MNO的生成。成功的 MNO 承载一个宽于 50μm 的轴突捆绑包，通道中的捆绑包中几乎没有分离的轴突。在引入球体24小时后，可以观察到轴突的初始拉长。在接下来的3-4天内，轴突到达微通道的中心，然后在10天内到达另一端（图6A）。因此，轴突在芯片的2-3周内组装并形成了一个直线和单向束，之后观察到神经元活动。
运动神经器官可以通过将 PDMS 从显微镜玻璃中分离以进行生物分析（图 6B）从芯片中收集。在显微镜下使用手术刀或钳子切割，可以解剖和分离轴束和细胞体（图6B）。这些生物材料，包括RNA和蛋白质，可用于常规生化检测，如RT-PCR和西方印迹。在多核子的轴突束中，在西方印迹（图6C）中未检测到核蛋白或对气质制造蛋白。
结合钙指标（Fluo-4 AM），神经元活性可以在组织培养芯片中捕获。在MNO中观察到球形和轴束中运动神经元的自发活动。此外，还使用多极阵列系统观察到神经活动。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61544/61544fig01.jpg"> 图1:PDMS组织培养芯片的尺寸。 （A） 组织培养芯片的光面具。（B） 组织培养芯片中微通道的尺寸。支撑运动神经元球形的基室直径为2毫米，腔体上方的PDMS孔为1.5毫米。微通道桥接两个腔室的宽度和高度均为 150 μm。 <a href="https://www.jove.com/files/ftp_upload/61544/61544fig01large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61544/61544fig02.jpg"> 图2:运动神经元分化的示意图。 （A） 分化步骤涉及神经诱导、运动神经元血统的模式以及运动神经元的成熟。（B） 从iPS细胞中创建运动神经元球形（MNS）的两种选择:3D协议和具有运动神经元分离步骤的二维协议。运动神经器官 （MNO） 可以通过这两种协议获得。<a href="https://www.jove.com/files/ftp_upload/61544/61544fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61544/61544fig03.jpg"> 图3:地下室膜基质涂层和运动神经元球形引入的分步协议。 （A） 组织培养芯片通道中的地下室膜基质涂层。（B） MNS 引入芯片的孔。（C） 文化媒介因耗尽媒介的愿望而改变。 <a href="https://www.jove.com/files/ftp_upload/61544/61544fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61544/61544fig04.jpg"> 图4:2D和3D运动神经元分化。 （A） 具有代表性的 3D MNS 分化（3D 协议）的时间过程。随着时间的推移，MNS 的大小逐渐增加。比例:500μm。（B） -D2、D0、D1、D6、D12（2D协议）的2D分化时间过程。比例:500μm。<a href="https://www.jove.com/files/ftp_upload/61544/61544fig04large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61544/61544fig05.jpg"> 图5:运动神经元的特征。 （A） （左） 沾有SMI-32抗体和DAPI的MNS的低温剖析。（中）在地下室膜矩阵涂层表面上重镀 MNS 的相位对比图像。观察到轴拉伸。（右）重镀 Mns 的轴染色为 Synapsin I 和 Tuj1 抗体。刻度条:500μm（左、中）和50μm（右）。（B） 二维运动神经元免疫与 Tuj 和 HB9 抗体的代表性图像。比例:200μm。 <a href="https://www.jove.com/files/ftp_upload/61544/61544fig05large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61544/61544fig06.jpg"> 图6:培养芯片中产生的运动神经器官（MNO）的特征。 （A） D32 上轴伸长和厚轴束形成的代表性图像。比例:500μm。（B） SMI-32和DAPI对运动神经器官（MNO）进行免疫。轴突和细胞体可以通过物理切割进行分离。刻度条:1毫米（C）来自轴突和细胞体的蛋白质纯度，由西方印迹量化。MAP2，一种树突标记物，没有在轴蛋白中检测到，而轴蛋白Tau1富含轴蛋白。 <a href="https://www.jove.com/files/ftp_upload/61544/61544fig06large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>

Watch this Scientific Journal Video about Three-Dimensional Motor Nerve Organoid Generation at JoVE.com

Three-Dimensional Motor Nerve Organoid Generation

该协议提供了一个全面的程序，通过自发组装从组织培养芯片中的球形延伸的强健的轴突束来制造人类iPS细胞衍生的运动神经器官。

三维运动神经器官生成

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...

three-dimensional-motor-nerve-organoid-generation

Research

JoVE Journal

Neuroscience

9.2K 次观看.  The University of Tokyo. 该协议提供了一个全面的程序，通过自发组装从组织培养芯片中的球形延伸的强健的轴突束来制造人类iPS细胞衍生的运动神经器官。

视频: Three-Dimensional Motor Nerve Organoid Generation

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

<meta charset="utf8"></head><body>类器官的三维成像以研究离体器官发生过程中的原发性纤毛发生

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

类器官的三维成像以研究 离体 器官发生过程中的原发性纤毛发生

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

科研

遗传学

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

在自制迷你生物反应器中从诱导多能干细胞中生成脑类器官

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

发育生物学

Innervation of Human Intestinal Organoids

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques for generating human intestinal organoids (HIOs) resembling human small intestine have been previously reported. HIOs contain epithelium and mesenchyme but lack other critical components of functional intestine such as the enteric nervous system (ENS), immune cells, vasculature, and microbiome. Two independent research groups have published distinct methods to innervate HIOs with an ENS. Here we discuss a unique method of incorporating the ENS into an HIO-derived bioengineered small intestine, which utilizes components of these prior reports to optimize progenitor cell identity as well as developmental timing.
Human pluripotent stem cells (hPSCs) are differentiated to independently generate HIOs and enteric neural crest cells (ENCCs) by temporal regulation of differentiation markers over a period of several days per published protocols. Once HIOs reach the mid-hindgut spheroid stage (approximately day 8), day 15-21 ENCC spheroids are dissociated, co-cultured with HIOs, and suspended within clear three-dimensional (3D) basement membrane matrix droplets. HIO + ENCC co-cultures are maintained in vitro for 28-40 days before transplantation into &#62;9-week-old immunodeficient mice for further development and maturation. Transplanted HIOs (tHIOs) with ENS can be harvested 4-20 weeks later. This method integrates elements from two previously published techniques by utilizing ENCCs generated from hPSCs and co-culturing them with HIOs at an early stage of development to maximize exposure to early developmental cues that likely contribute to the formation of a more mature intestinal morphology.

Human intestinal organoids must be innervated to better recapitulate the structure and function of the native human intestine. Here, we present one method for incorporating an enteric nervous system into these constructs.

人肠道类器官的神经支配

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques ...

Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate.
The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain&#39;s inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain.
The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson&#39;s disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.

This protocol describes a novel method for creating 3D midbrain organoids from human induced pluripotent stem cells, guiding their formation to mimic native midbrain tissue, thereby aiding in the study of development and disorders.

从人诱导多能干细胞生成 3D 中脑类器官

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

生物工程

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker &#945;-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.

We describe a method to generate human motor units in commercially available microfluidic devices by co-culturing human induced pluripotent stem cell-derived motor neurons with human primary mesoangioblast-derived myotubes resulting in the formation of functionally active neuromuscular junctions.

在微流体装置中生成具有功能性神经肌肉接头的人类运动单元

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle ...

神经科学

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 ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

用于类器官培养和 3D 成像的高通量平台

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...

生物学

Generating Neuromuscular Junctions Using a Microfluidic Device

Source: Stoklund Dittlau, K. et al., <a href="https://app.jove.com/v/62959">Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices.</a>&#160;J. Vis. Exp. (2021)
The video demonstrates the formation of neuromuscular junctions using a microfluidic device. Neural precursor cells and muscle progenitor cells are seeded into different wells within the device and left to mature into motor neurons and myotubes, respectively. The creation of a volume and chemical gradient leads to the development of active neuromuscular junctions.

使用微流体设备生成神经肌肉接头

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
All reagents and equipment used in ...

三维运动神经器官生成

摘要

探索更多视频

类器官的三维成像以研究离体器官发生过程中的原发性纤毛发生

类器官的三维成像以研究离体器官发生过程中的原发性纤毛发生

在自制迷你生物反应器中从诱导多能干细胞中生成脑类器官

人肠道类器官的神经支配

从人诱导多能干细胞生成 3D 中脑类器官

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

在微流体装置中生成具有功能性神经肌肉接头的人类运动单元

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

用于类器官培养和 3D 成像的高通量平台

使用微流体设备生成神经肌肉接头

三维运动神经器官生成

摘要

探索更多视频

类器官的三维成像以研究 离体 器官发生过程中的原发性纤毛发生

类器官的三维成像以研究 离体 器官发生过程中的原发性纤毛发生

在自制迷你生物反应器中从诱导多能干细胞中生成脑类器官

人肠道类器官的神经支配

从人诱导多能干细胞生成 3D 中脑类器官

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

在微流体装置中生成具有功能性神经肌肉接头的人类运动单元

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

用于类器官培养和 3D 成像的高通量平台

使用微流体设备生成神经肌肉接头

类器官的三维成像以研究离体器官发生过程中的原发性纤毛发生

类器官的三维成像以研究离体器官发生过程中的原发性纤毛发生