Name: rapid neuronal differentiation of induced pluripotent stem cells for measuring network activity on micro-electrode arrays
Uploaded: 2017-01-08T13:00:00.000+00:00
Duration: 9 min 20 s
Description: Watch this Scientific Journal Video about Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays at JoVE.com

The authors thank Jessica Classen for performing the whole-cell patch-clamp experiments. The hiPSCs used in our experiments were kindly provided by Huiqing Zhou and Willem van den Akker from the Radboud University Nijmegen. The transfer vectors used in this protocol were kindly provided by Oliver Br&#252;stle, Philipp Koch and Julia Ladewig from the University of Bonn Medical Centre.

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The authors have nothing to disclose.

在这里，我们已经实现了由张等人发表了高效的hiPSC分化协议。 （2013年）12用于测量多边环境协定hiPSC衍生神经元网络的网络活动。我们通过创建诱导神经元分化之前rtTA / Ngn2阳性hiPSC线调整原来的协议。此附加的步骤使我们能够控制在MEA的神经元细胞密度。控制了神经元密度是一个重要的先决条件适应协议的MEA和确保一致性。来测量使用的MEA的神经元网络的活性，神经元需要以形成直接在MEA电极17,18的顶端密集网络。这必然需要在神经元的铺板密度严密控制。该rtTA / Ngn2阳性hiPSC线实现了神经元密度，因为这种战术不依赖于人iPS细胞分化前急性慢病毒转导的控制;因此rtTA / Ngn2阳性hiPSC线几乎消除在最终产率的任何变化，由于，例如，慢病毒毒性和可变感染效率。 的实验步骤的另一关键步骤是大鼠星形胶质细胞被共培养与分化人iPS细胞的数目。星形胶质细胞积极推动通过控制突触的形成，维护和消除，所有这些都是神经元功能的重要过程开发的神经回路的细化。本文提出的协议是高度星形胶质细胞依赖性:充分成熟，形成功能性突触，神经元需要从星形胶质细胞的支持。我们经历星形胶质细胞的数量应大致等于hiPSC衍生神经元的数目，以支持神经元的成熟和表现出自发性活动的神经元网络的形成。由于我们的星形胶质细胞的协议收益率原代细胞的小路具有有限寿命Tures的，大鼠星形胶质细胞的分离，必须定期进行。 我们由张等人公布了协议的适配。 （2013年）12与MEA技术的使用可能会显著提高我们的学习hiPSC衍生的网络的网络活动的能力。此前，用于研究hiPSC派生的神经元网络与多边环境协议依靠耗时分化过程13-16。从张某等人的协议。 （2013）提供了一种快速的替代，并且提供了修改删除变异性的来源，这使得它现在更可行使用hiPSC衍生的神经元结合的MEA的技术，尤其是在高通量或药理学研究。另外，由于由Zhang 等人公布的方法。 （2013年）12得到上层皮层神经元的同质人口，我们的改编协议成为可能重点研究进入网络这种特殊神经元的子集ctivity。 尽管如此，这种方法也有一些限制。首先，将培养的均一性也可被认为是一个缺点，因为培养的可能性较小体内网络中，不同类的神经元（ 即抑制性和兴奋性神经元）构成的非均相网络类似于。为了进一步提高使用具有MEA技术hiPSC衍生的神经元，这将是重要的，以开发其他的神经元细胞群快速（基于转基因-）分化协议。如果协议变得可用， 在体外网络将更加紧密地模拟体内网络。第二，目前大鼠星形胶质细胞必须被添加到生长的支持hiPSC衍生的神经元，因此，引起的神经元网络不是人类神经网络狭义。可靠的协议，在未来的溶胶人iPS细胞分化成星形胶质细胞可能已经这个问题26。第三，二维神经元网络，如这里描述的，可用于研究复杂的三维体内神经元网络有限模型。幸运的是，描述的大鼠原代神经元的三维培养物中结合MEA技术协议已经可用27,28。前瞻性，用于获得hiPSC衍生神经元和星形胶质细胞的三维培养技术和MEA技术迅速分化协议的组合应提供新颖的洞察的生物学机制底层神经障碍。

人类诱导多能干细胞（iPS细胞）分化的协议的发展， 在体外产生提供了用于研究神经系统疾病一个强大的新工具的人类神经元。直到最近，这些疾病的研究受到严重缺乏使用人类神经元模型系统的阻碍。虽然啮齿类动物可用于研究神经系统疾病，这些研究的结果不能容易地转换为人类1。鉴于这些限制，hiPSC衍生的神经元是可以用来阐明分子机制神经障碍和用于体外药物筛选一个有前途的替代模型。 在过去的十年中，多个协议转换人iPS细胞向神经元已经开发2-8。但是，这些协议在许多方面仍然受到限制。首先，协议通常费时:有足够的成熟产生的神经元（ 即 synap本身形成）和功能活动需要培养程序，这使得很难9大规模研究个月。此外，hiPSC到神经元的转换效率是低的。协议通常产生神经元的异质群体，并因此不允许神经元细胞的特定亚群的研究。此外，该协议是不可复制的，产生不同的结果，不同的iPSC系10,11。最后，成熟阶段，将所得神经元的功能性质也是可变的10。 为了解决这些问题，Zhang 等。 （2013）12开发的，通过过表达转录因子神经元素-2快速，可重复产生的iPS细胞的神经元人类的协议。所报告的作者，分化发生相对快速地（仅2 - 诱导神经元素-2的表达后3周），该协议是可重复的（神经元特性独立于的Starting hiPSC线），以及hiPSC到神经元的转换效率高（几乎100％）。与他们的协议产生的神经元的人口均匀（类似上层皮层神经元），允许神经元疾病的细胞类型的具体贡献进行调查。此外，它们的hiPSC衍生神经元仅20天之后显示出成熟的属性（ 例如 ，能力，以形成突触和健壮的功能活性）。 在网络层面表征hiPSC衍生神经元的电生理特性是hiPSC技术面前的一个重要的先决条件可以为人类疾病的研究中被利用。出于这个原因，许多研究小组最近开始探讨采用微电极阵列（MEA）的设备（多声道系统，Reutlingen，德国）13-16在网络级干细胞衍生的神经元。一个MEA的电极嵌在其上的神经元细胞可以培养的基板。的MEA可以用来探索神经网络和它们的活性的体外发育的电生理特性。目前，多边环境协定仅用于与需要几个月的时间来产生成熟的网络协议的分化组合。因此，具有快速分化方案相结合的多边环境协定应有助于神经系统疾病的大规模研究中使用这种技术。 这里，我们提出的Zhang 等人的修改。 （2013年）12协议，使其适用于多边环境协定中使用。特别是，而不是依赖于急性慢病毒转导，我们转而创建hiPSC线诱导分化前稳定表达rtTA / Ngn2。我们这样做主要是为了具有在神经元细胞密度重现的控制中，由于神经元细胞密度为神经网络形成的关键，并且为神经元和MEA 17,18的电极之间的良好接触。 Althoug小时张某等人。协议是非常有效的相对于转导人iPS细胞的转化，这是相对于神经元从人iPS细胞的数目的最终产率镀最初（见图2E在Zhang 等人 ）12固有的变量。凭借稳定的线路，我们消除了许多问题引起的变异，如慢病毒毒性和感染效率。然后，我们优化的可靠地生产多边环境协定hiPSC派生的神经元网络的参数，获取网络的成熟（ 如 ，涉及到广大渠道同步事件）的第三周。这种快速和可靠的协议应该使来自不同（ 即患者特异性）hiPSC线以及用于药理学研究提供健壮一致性来源的神经元之间的直接比较。

<table><tbody><tr><td>Lebovitz&#039;s L-15 medium</td><td>Gibco</td><td>11415-064</td><td></td></tr><tr><td>B-27 supplement</td><td>Gibco</td><td>0080085SA</td><td></td></tr><tr><td>Poly-D-Lysine (PDL)</td><td>Sigma-Aldrich</td><td>P6407</td><td></td></tr><tr><td>Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;-free HBSS&amp;nbsp;</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>0.05% Trypsin-EDTA&amp;nbsp;</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>2.5% Trypsin</td><td>Gibco</td><td>15090-046</td><td></td></tr><tr><td>High-glucose DMEM</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Sigma-Aldrich</td><td>F2442-500ML</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>70 &amp;micro;m cell strainer</td><td>BD Falcon</td><td>352350</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>psPAX2 lentiviral packaging vector</td><td>Addgene</td><td>Plasmid #12260</td><td></td></tr><tr><td>pMD2.G lentiviral packaging vector</td><td>Addgene</td><td>Plasmid #12259</td><td></td></tr><tr><td>Basement membrane matrix</td><td>Gibco</td><td>A1413201</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11320-074</td><td></td></tr><tr><td>Cell detachment solution</td><td>Sigma-Aldrich</td><td>A6964</td><td></td></tr><tr><td>E8 medium</td><td>Gibco</td><td>A1517001</td><td></td></tr><tr><td>ROCK inhibitor</td><td>Gibco</td><td>A2644501&amp;nbsp;</td><td>Alternatively, ROCK inhibitors like thiazovivin can be used.</td></tr><tr><td>Polybrene</td><td>Sigma-Aldrich</td><td>H9268-5G</td><td></td></tr><tr><td>G418</td><td>Sigma-Aldrich</td><td>G8168-10ML</td><td></td></tr><tr><td>Puromycin</td><td>Sigma-Aldrich</td><td>P9620-10ML</td><td></td></tr><tr><td>Vitronectin</td><td>Gibco</td><td>A14700</td><td></td></tr><tr><td>6-well MEAs</td><td>Multi Channel Systems</td><td>60-6wellMEA200/30iR-Ti-tcr</td><td></td></tr><tr><td>Glass coverslips</td><td>VWR</td><td>631-0899</td><td></td></tr><tr><td>Poly-L-Ornithine (PLO)</td><td>Sigma-Aldrich</td><td>P3655-10MG</td><td></td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020-1MG</td><td></td></tr><tr><td>Doxycyclin</td><td>Sigma-Aldrich</td><td>D9891-5G</td><td></td></tr><tr><td>N-2 supplement</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Sigma-Aldrich</td><td>M7145</td><td></td></tr><tr><td>NT-3, human recombinant</td><td>Promokine</td><td>C66425</td><td></td></tr><tr><td>BDNF, human recombinant</td><td>Promokine</td><td>C66212</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>L-alanyl-L-glutamine</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>Cytosine &amp;beta;-D-arabinofuranoside&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C1768-100MG</td><td></td></tr><tr><td>Straight fine-tipped forceps</td><td>Fine Science Tools</td><td>11251</td><td></td></tr><tr><td>Fine-tipped spring scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>91500-09</td><td></td></tr></tbody></table>

rapid neuronal differentiation of induced pluripotent stem cells for measuring network activity on micro-electrode arrays

从人类诱导多能干细胞来源的神经元（iPS细胞）为研究神经系统疾病有前途的新工具。在过去的十年中，用于区分人iPS细胞向神经元的许多协议已被开发出来。然而，这些协议通常具有较高的变异性，重复性低，效率低下缓慢。此外，这些协议中得到的神经元通常未成熟和缺乏无论是在单细胞和网络电平足够功能活性除非神经元是几个月中培养。部分由于这些限制，hiPSC衍生神经元网络的功能性质仍然没有得到很好的表征。在这里，我们适应最近公布的协议，说明产量从iPS细胞人的神经元的转录因子神经元素- 2 月12日被迫表达。这个协议是快速（产生3周内成熟神经元）和高效，具有transduc近100％的转换效率编细胞（&gt; DAPI阳性细胞95％是MAP2阳性）。此外，该协议产生兴奋性神经元，将允许对神经障碍的细胞类型的具体贡献的调查的均质群体。我们通过产生稳定转导hiPSC细胞，让我们在神经元的总数显式控制修改的原始协议。然后将这些细胞用于生成微电极阵列hiPSC衍生的神经元网络。以这种方式，hiPSC衍生的神经元网络的自发电生理学活性可以测量和表征，而在细胞密度方面保持interexperimental一致性。所提出的协议是广泛适用的，尤其是对人类神经元网络的机械和药理研究。

在这里，我们已经成功地修改了其中iPS细胞是通过过表达转录因子神经元素-2 12直接分化成皮层神经元的协议，我们已经适应它的使用多边环境协定。这种方法是快速和有效的使我们能够分化诱导后在第三周获得官能神经元和网络活动已经。 在该分化方案的过程中，细胞形态开始类似于神经元:形成小的流程和神经元开始彼此连接（ 图1A）。我们建立了从健康控制hiPSC源性神经元的神经生理学配置文件，通过在开发过程中测量它们的神经元形态和突触的属性。 hiPSC衍生神经元开始后不同天染色MAP2和突触-1/2分化（ 图2A）的。衍生的神经元分化的诱导3周后显示出成熟神经元形态已经。基于突触-1/2免疫组织化学染色突触-1/2泪点（为突触的数量的度量）的数量进行定量。突触-1/2泪点的数量随时间而增加，这表明神经元的连接的电平也增加（ 图2B）。分化诱导23天后突触-1/2泪点的数目是在两个独立的IPS线（ 图2C）类似。在23 DIV最synapsin1 / 2泪点分别并列PSD-95泪点，其表示为功能性突触（ 图2D）。 由Zhang 等人所描述的结果一致，我们产生了人口兴奋上层皮层神经元的，由特定的亚型泛神经元和皮质米确认<html> arkers如BRN2和SATB2（层II / III）。我们没有观察到阳性的深层神经元CTIP2（第五层）或FOXP2（第六层）（ 图2E和F）的神经元表征hiPSC衍生神经元的电生理学活性，我们使用全细胞电流和电压钳记录， 即内在特性和兴奋性输入到这些神经元发育过程中进行测定。神经元能够分化和扣球细胞的百分比的随时间（ 图2G和H）增加后，以产生动作电位已经一周。此外，神经元一周接收已经兴奋性突触输入分化诱导后:兴奋性突触输入的频率和振幅在开发过程中增加（ 图2I - K）。 NT"FO:保together.within页="1"&gt;为了更好地理解单细胞的活性结合，形成网络级的功能，来研究神经元的演唱会是如何工作是必不可少的多边环境协定体外神经元网络培养。构成了一个宝贵的实验模型，为研究神经元的动态。我们的分化诱导后记录从健康控制hiPSC线培养来源的神经元的电网络活动的20分钟的6孔多边环境协定（ 图2M）。几周之后，神经元从健康控制iPS细胞衍生形成功能活跃神经元网络，显示自发事件（0.62±0.05穗/秒; 图2N）。（ 即后16天分化的开始），在这个发展阶段涉及的所有通道没有同步事件。的被检测的MEA（ 图20）的网络活动的水平的发展过程中增加了:在t后第四周<html>他诱导分化的神经元网络显示自发性活动高电平;在该装置的所有孔中（2.5±0.1穗/秒图2N）。该网络还展出了同步网络暴（4.1±0.1爆裂/分钟， 图20），持续时间长（2 100±500毫秒）。 <img alt="图1" src="/files/ftp_upload/54900/54900fig1.jpg" /> 图1:hiPSC 分化为神经元。 iPS细胞分化A.三个时间点变成盖玻片上的神经元。 iPS细胞的多边环境协议B.电镀。 C.星形胶质细胞在T75烧瓶中100％汇合（注意该细胞形成一个棋盘格状的单层）。比例尺:150微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54900/54900fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> e_content"FO: - together.within页保留="1"&gt; <img alt="图2" src="/files/ftp_upload/54900/54900fig2.jpg" /> 图2. hiPSC派生的神经元表征。 答 hiPSC衍生神经元分化开始后染色MAP2（绿色）和在不同的日子突触-1/2（红色）。比例尺:10微米。 B.在两个独立实验突触泪点的量化。在每个实验中至少10个细胞进行分析。 C.在DIV23在来自两个独立的IPS线来源的神经元突触泪点的量化。 D. hiPSC衍生神经元染色的PSD-95（绿色）和分化开始后突触-1/2（红色）23天。突触泪点并列到PSD-95泪点。 E. hiPSC衍生神经元染色MAP2（绿色）和BRN2（红色）或SATB2（红色）分化开始后23 D。 F. MAP2阳性细胞的百分比为阳性表示标记。 G.代表当前钳记录表示动作电位可以早分化开始后第7天产生。在分化诱导后不同日期，显示一个或多个动作电位细胞的百分比H.。 一，分化后不同天hiPSC派生的神经元兴奋性接受性突触后电流（EPSCS）的代表痕迹。在开发过程中兴奋性突触后电流的频率杂志 。在开发过程中兴奋性突触后电流的幅值K.。 EPSC录音L.代表迹线无（对照），并与CNQX（CNQX）。从一个hiPSC系衍生分枝神经元分别在6孔的MEA培养和网络活动示出了用于hiPSC衍生神经元网络16和分化诱导后23 D。从记录的活动（记录的20分钟的5分钟中示出），每个孔（10千赫的采样率）表示用不同的颜色。 N.射击速度16和分化诱导后23 D。 O.破裂率16和分化诱导后23 D。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54900/54900fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 给出的结果，所得到的hiPSC衍生的神经元的质量可以通过使细胞的神经生理信息进行评估。即，分化，形态，突触-1/2的表达和神经元的电生理学的开始可以评估后三至四个星期。在该时间点，所述hiPSC衍生的神经元预计将显示一个神经元样形态，是MAP2，突触/ PSD-95阳性执行时免疫细胞化学，并且exhibi吨自发电生理活动（无论是在单细胞和网络电平）。

Watch this Scientific Journal Video about Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays at JoVE.com

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

我们修改和实施描述人类诱导多能干细胞（iPS细胞）的快速，可重复，高效分化成兴奋皮层神经元12先前公布的协议。具体地讲，我们的修改允许对神经元细胞密度和使用的控制上的微电极阵列以测量在网络级电生理特性。

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, ...

rapid-neuronal-differentiation-induced-pluripotent-stem-cells-for

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

26.8K 次观看.  Radboudumc. 我们修改和实施描述人类诱导多能干细胞（iPS细胞）的快速，可重复，高效分化成兴奋皮层神经元12先前公布的协议。具体地讲，我们的修改允许对神经元细胞密度和使用的控制上的微电极阵列以测量在网络级电生理特性。 

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

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

科研

神经科学

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

发育生物学

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

用于循环作用电位记录的多井微电极阵列上的人类 iPSC 衍生心肌细胞网络

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

生物工程

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

分化后再蛋白的人类多能干细胞衍生神经元，用于高含量的内尿成生和突合性成熟筛查

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

建立区域特异性人多能干细胞衍生星形胶质细胞和神经元模拟ALS的电生理平台

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

人诱导多能干细胞及其神经和神经胶质衍生物中多种线粒体参数的流式细胞术分析

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

<meta charset="utf8"></head><body>记录大脑组件中的网络活动：多电极阵列方法

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal brain development. The fusion of dorsal with ventral regionally specified brain organoids in vitro generates assembloids, which have functionally integrated microcircuits with excitatory and inhibitory neurons. Due to their structural complexity and diverse population of neurons, assembloids have become a useful in vitro tool for studying aberrant network activity. Multi-electrode array (MEA) recordings serve as a method for capturing electrical field potentials, spikes, and longitudinal network dynamics from a population of neurons without compromising cell membrane integrity. However, adhering assembloids onto the electrodes for long-term recordings can be challenging due to their large size and limited contact surface area with the electrodes. Here, we demonstrate a method to plate assembloids onto MEA plates for recording electrophysiological activity over a 2-month span. Although the current protocol utilizes human cortical organoids, it can be broadly adapted to organoids differentiated to model other brain regions. This protocol establishes a robust, longitudinal, electrophysiological assay for studying the development of a neuronal network, and this platform has the potential to be used in drug screening for therapeutic development in epilepsy.

<meta charset="utf8"></head><body>作者聚焦：通过基于多电极阵列的人脑组件长期电生理监测推进遗传性癫痫研究

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

使用人类多能干细胞来源的脑组件对癫痫进行建模的多电极阵列平台

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal ...

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

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人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

用于循环作用电位记录的多井微电极阵列上的人类 iPSC 衍生心肌细胞网络

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

分化后再蛋白的人类多能干细胞衍生神经元，用于高含量的内尿成生和突合性成熟筛查

建立区域特异性人多能干细胞衍生星形胶质细胞和神经元模拟ALS的电生理平台

人诱导多能干细胞及其神经和神经胶质衍生物中多种线粒体参数的流式细胞术分析

使用人类多能干细胞来源的脑组件对癫痫进行建模的多电极阵列平台

使用人类多能干细胞来源的脑组件对癫痫进行建模的多电极阵列平台

使用人类多能干细胞来源的脑组件对癫痫进行建模的多电极阵列平台