This manuscript was supported by R01NS127829 NIH/NINDS (LTD). Figure 1 has been generated using biorender.com.

Recording Network Activity in Brain Assembloids: Multi-Electrode Array Approach

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The authors have no relevant disclosures.

MEA-based methods for electrophysiological recordings of network activity in iPSC-derived assembloids have been used for in vitro modeling of epilepsy22,23. This proposed platform integrating excitatory and inhibitory synaptic connections has the potential to address mechanisms of neuronal hyperexcitability and the role of cortical interneurons in the process of epileptogenesis. Additionally, this platform allows for the stabilization of assembloids and ...

Human pluripotent stem cell (hPSC)-derived brain organoids are spatially self-organized 3D structures mirroring the tissue architecture and developmental trajectories in vivo. They are composed of multiple cell types, including progenitors (neuroepithelial cells, radial glia, neuronal progenitors, glial progenitors), neurons (cortical-like excitatory neurons and inhibitory interneurons), and glial cells (astrocytes and oligodendrocytes)1,2. Assembloids represent the next generation of brain organoids, capable of integrating multiple brain regions and/or cell lineages within a 3D culture. They provide a useful tool for modeling connections between various brain regions resembling the in vivo counterparts, capturing interactions between neurons and astrocytes to better mimic mature and complex neural networks, and to study the assembly of neural circuits. Therefore, assembloids are becoming a widely used tool to recapitulate hallmarks of epilepsy pathophysiology, in which functional measures are necessary to interrogate aberrant neural networks that may underlie the cause of the disease3,4,5,6.
To model interactions between cortical glutamatergic neurons and GABAergic interneurons, several groups developed separate organoids resembling the dorsal and ventral brain and then fused them together into a multi-region assembloid7,8,9,10. Here, a previously described assembloid culture protocol with regionally specific neural subtypes was applied9. However, a significant barrier is the lack of reproducible functional assays to monitor neural network activity during neurodevelopment. Many functional tests of the networks in organoids yield results that have high variability among batches of differentiations and cell lines. Techniques that involve slicing or dissociating organoids alter their inherent networks by severing synaptic connectivity8.
Multi-electrode arrays (MEA) provide a large-scale view of network activity over time with high temporal resolution to characterize electrophysiological properties of organoids without disrupting culture conditions or cell membrane integrity11. Compared to patch clamp electrophysiology, MEA enables high-throughput data acquisition based on large populations of neurons rather than single cells. MEA platforms vary in their electrode density, catering to different needs in brain organoid research12. The widely used systems, as shown in this protocol, record from 8 to 64 electrodes per well13,14,15. High-density MEAs with up to 26,400 electrodes per well enable increased spatial and temporal resolution, quantification of action potential propagation speed, and combination with optogenetic stimulation14,16,17. Therefore, MEA serves as a powerful tool for modeling epilepsy in vitro and a translational paradigm for anti-seizure medication screening.
One major challenge is to stabilize the large-sized assembloids on a hydrophobic metal surface for long-term recordings. This protocol outlines a detailed methodology for plating intact assembloids on MEA plates for long-term longitudinal recording together with pharmacological assays. Unique advantages of the protocol include stable attachment of assembloids to the electrode surface without losing electrical activity, use of commercially available neurophysiological basal media to accelerate functional network maturation after plating, feasibility to conduct downstream functional assays like drug treatment, and wide application to organoids generated with other region-specific protocols.
The goal is to provide a functional assay with high temporal resolution to investigate network activity, examine disease-specific changes, and test drugs with therapeutic potential in epilepsy. Video instructions are provided for the most challenging steps of this protocol, showing the techniques for plating assembloids on MEA plates, as well as representative recordings from these cultures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm Corning Non TC-treated culture dishes</td>
			<td>Corning</td>
			<td>08-772-32</td>
			<td>For suspension culture on the shaker</td>
		</tr>
		<tr>
			<td>100 mL Beaker&#160;</td>
			<td>Fisher Scientific</td>
			<td>FB100100</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP28184-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (&#946;-ME)</td>
			<td>Thermo Fisher</td>
			<td>21985023</td>
			<td>Working concentration 100 &#956;M</td>
		</tr>
		<tr>
			<td>48-well cell culture plate</td>
			<td>Fisher Scientific</td>
			<td>50-202-140</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plate</td>
			<td>Fisher Scientific</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Aggrewell 800</td>
			<td>Fisher Scientific</td>
			<td>501974754</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-14R Refrigerated Centrifuge</td>
			<td>Beckman Coulter&#160;</td>
			<td>BE-AX14R</td>
			<td></td>
		</tr>
		<tr>
			<td>Allopregnanolone</td>
			<td>Cayman</td>
			<td>16930</td>
			<td>Suspended 5mg into DMSO to get 1 mM stock solution. Aliquot and freeze at &#8722;80 &#176;C. Dilute at 1:10,000 for use. Working concentration 100 nM.</td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>Thermo Fisher</td>
			<td>AMQAX2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Axion CytoView MEA 6-well plates&#160;</td>
			<td>Axion Biosystems</td>
			<td>M384-tMEA-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Axion Maestro MEA platform</td>
			<td>Axion Biosystems</td>
			<td>Maestro</td>
			<td>With temp environmental control</td>
		</tr>
		<tr>
			<td>B-27 supplement (regular, with Vitamin A)</td>
			<td>Thermo Fisher</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 supplement (without Vitamin A)</td>
			<td>Thermo Fisher</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Basement membrane matrix- Geltrex</td>
			<td>Thermo Fisher</td>
			<td>A1569601</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead bath</td>
			<td>Fisher Scientific</td>
			<td>10-876-001</td>
			<td>Isotemp</td>
		</tr>
		<tr>
			<td>Benchtop inverted microscope</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td>Kept in laminar flow clean bench</td>
		</tr>
		<tr>
			<td>Bicuculline</td>
			<td>Sigma-Aldrich</td>
			<td>14340</td>
			<td>Working concentration 10 &#956;M</td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>CLOROX</td>
			<td>67619-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Borate buffer 20x</td>
			<td>Thermo Fisher</td>
			<td>28341</td>
			<td>Working concentration at 1x</td>
		</tr>
		<tr>
			<td>BrainPhys media</td>
			<td>StemCell Technologies</td>
			<td>5790</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell dissociation reagent (StemPro Accutase)</td>
			<td>Thermo Fisher</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>Celltron orbital shaker</td>
			<td>HT-Infors</td>
			<td>&#160;I69222&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Detergent/enzyme (Terg-A-Zyme)</td>
			<td>Sigma-Aldrich</td>
			<td>Z273287</td>
			<td>Working concentration 1% m/v</td>
		</tr>
		<tr>
			<td>DMEM/F12 + HEPES/L-Glutamine</td>
			<td>Thermo Fisher</td>
			<td>113300</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>67685</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Sigma-Aldrich</td>
			<td>P5499</td>
			<td>Dissolve 5mg into DMSO to get 10 mM stock solution. Aliquot and freeze at &#8722;20 &#176;C. Dilute at 1:2000 for use. (working concentration 5 &#956;M)</td>
		</tr>
		<tr>
			<td>D-PBS w/o calcium or magnesium</td>
			<td>Thermo Fisher</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Glial cell line-derived neurotrophic (GDNF)</td>
			<td>Peprotech</td>
			<td>450-10</td>
			<td>Dissolve 100 &#956;g in 1mL of PBS to 100 &#956;g/mL. Aliquot and freeze at &#8722;20 &#176;C. Dilute at 1:5000 for use. (working concentration 20 ng/mL).</td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Thermo Fisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Election Microscopy Sciences</td>
			<td>63510-20</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Heraguard ECO Clean Bench</td>
			<td>Thermo Fisher</td>
			<td>51029692</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity controlled cell culture incubator</td>
			<td>Thermo Fisher</td>
			<td>370</td>
			<td>set to 37 &#176;C, 5% CO2</td>
		</tr>
		<tr>
			<td>IWP-2</td>
			<td>Selleckchem</td>
			<td>S7085</td>
			<td>Aliquot and freeze at &#8722;80 &#176;C. It will precipitate if thawed at room temp. Frozen aliquots should be placed directly into 37 &#176;C before use.</td>
		</tr>
		<tr>
			<td>Knockout serum replacement (KOSR)</td>
			<td>Thermo Fisher</td>
			<td>10828010</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSRplus (medium + supplements)</td>
			<td>StemCell Technologies</td>
			<td>100-0276</td>
			<td>cGMP, stabilized feeder-free medium for human iPSC cells</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermo Fisher</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal A</td>
			<td>Thermo Fisher</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids (NEAA)</td>
			<td>Thermo Fisher</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>NT3</td>
			<td>Peprotech</td>
			<td>450-03</td>
			<td>Dissolve 100 &#956;g in 1mL of PBS to 100 &#956;g/mL. Aliquot and freeze at &#8722;20 &#176;C. Dilute at 1:5000 for use. (working concentration 20 ng/mL).</td>
		</tr>
		<tr>
			<td>NuFF Human neonatal foreskin fibroblasts</td>
			<td>MTI-GlobalStem</td>
			<td>GSC-3002</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>PARAFILM</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (P10, P200, P1000)</td>
			<td>Eppendorf</td>
			<td>EP4926000034</td>
			<td>Autoclaved cut P1000 tips for organoid collection</td>
		</tr>
		<tr>
			<td>Poly (Ethyleneimine) (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>P3143</td>
			<td>Dilute stock in sterile borate buffer. Working concentration 0.07%. See details in manuscript.</td>
		</tr>
		<tr>
			<td>Recombinant human epidermal growth factor (EGF)</td>
			<td>R&#38;D Systems</td>
			<td>236-EG-200</td>
			<td>Suspended in PBS. Aliquot and freeze at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Recombinant Human fibroblast growth factor (FGF)-basic&#160;</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Suspended in PBS. Aliquot and freeze at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Recombinant human-brain-derived neurotrophic factor (BDNF)</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td>Centrifuge briefly before reconstitution. Dissolve 100 &#956;g in 1 mL of PBS to 100 &#956;g/mL. Aliquot and freeze at &#8722;20 &#176;C. Dilute at 1:5000 for use. (working concentration 20 ng/mL).</td>
		</tr>
		<tr>
			<td>Retinoic acid (RA)</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td>Dissolve 100 mg into 3.3 mL of DMSO to get 100 mM stock solution. Aliquot the stock 100 &#956;L/tube and freeze at &#8722;80 &#176;C. Take 200 &#956;L of 100 mM stock and dilute 10x (add 1.8 mL of DMSO) to make 10 mM stock. Aliquot 50 &#956;L/tube and store at &#8722;80 &#176;C. Dilute at 1:100,000 for use. (working concentration 100 nM).</td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>1:200 from 10 mM stock</td>
		</tr>
		<tr>
			<td>SAG (smoothened agonist)</td>
			<td>Selleckchem</td>
			<td>S7779</td>
			<td>Aliquot and freeze at &#8722;80 &#176;C. Stock concentration 1mM. Use at 1:10,000 dilution (working concentration 100 nM).</td>
		</tr>
		<tr>
			<td>SB-431542</td>
			<td>Tocris</td>
			<td>1614</td>
			<td>Dissolve 5mg into 1.3 mL of DMSO to get 10 mM stock solution. Aliquot and freeze at &#8722;80 &#176;C. Dilute at 1:1000 for use. (working concentration 10 &#956;M)</td>
		</tr>
		<tr>
			<td>Serological pipette filler</td>
			<td>Fisher Scientific</td>
			<td>14-387-166</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip vacuum tube top filter</td>
			<td>Sigma-Aldrich</td>
			<td>SE1M179M6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell culture hoods</td>
			<td>Baker Company</td>
			<td>SG-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue solution (0.4%)</td>
			<td>Thermo Fisher</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermo Fisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Zoom stereomicroscope</td>
			<td>Olympus</td>
			<td>SZ61/SZ51</td>
			<td>Kept in laminar flow clean bench</td>
		</tr>
	</tbody>
</table>

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.

Human dorsal-ventral assembloids were plated on a 6-well MEA plate (n = 6 per differentiation, 3 differentiations), with each well containing 64 electrodes (Figure 2A). Nine out of ten assembloids planned for longitudinal recording were firmly attached to the electrodes for over 50 days in vitro (Figure 2D). 6 out of 8 assembloids designated for pharmacological assays were successfully settled through the wash-out phase ...

Author Spotlight: Advancing Genetic Epilepsy Studies with Multi-Electrode Array-Based Long-Term Electrophysiological Monitoring of Human Brain Assembloids

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

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 ...

a-multi-electrode-array-platform-for-modeling-epilepsy-using-human

Research

JoVE Journal

Neuroscience

970 Views.  Michigan Medicine, Ann Arbor. This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

Video: Author Spotlight: Advancing Genetic Epilepsy Studies with Multi-Electrode Array-Based Long-Term Electrophysiological Monitoring of Human Brain Assembloids

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

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.

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

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

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.

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.

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