This manuscript was supported by the following: 2019 MSCRFF 5119 (AT). K08NS102526 NIH/NINDS (CWH), 2020 Doris Duke Charitable Foundation Clinical Scientist Career Development Award (CWH). 1R01NS117604-01NIH/NINDS (NJM), DOD ALSRP W81XWH2010161 (NJM), 2019 MSCRFD 5122 (NJM). We thank Dr. Raha Dastgheyb and Dr. Norman Haughey for providing the MEA platform and data analysis software we have utilized to validate the described electrophysiological platform.&#160;We would like to thank Khalil Rust for their assistance with protocol demonstration and filming.

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	<li>Ferraiuolo, L., Maragakis, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-review:+Induced+pluripotent+stem+cells+and+the+search+for+new+cell-specific+ALS+therapeutic+targets.">Mini-review: Induced pluripotent stem cells and the search for new cell-specific ALS therapeutic targets.</a> Neuroscience Letters. 755, 135911 (2021).</li><li>Taga, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+human-induced+pluripotent+stem+cell-derived+spinal+cord+astrocytes+in+the+functional+maturation+of+motor+neurons+in+a+multi-electrode+array+system.">Role of human-induced pluripotent stem cell-derived spinal cord astrocytes in the functional maturation of motor neurons in a multi-electrode array system.</a> Stem Cells Translational Medicine. 8 (12), 1272-1285 (2019).</li><li>Klapper, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+lineage+cells+are+essential+for+functional+neuronal+differentiation+and+synapse+maturation+in+human+iPSC-derived+neural+networks.">Astrocyte lineage cells are essential for functional neuronal differentiation and synapse maturation in human iPSC-derived neural networks.</a> Glia. 67 (10), 1893-1909 (2019).</li><li>Zhao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+C9orf72+human+iPSC-derived+astrocytes+cause+non-cell+autonomous+motor+neuron+pathophysiology.">Mutant C9orf72 human iPSC-derived astrocytes cause non-cell autonomous motor neuron pathophysiology.</a> Glia. 68 (5), 1046-1064 (2020).</li><li>Almad, A. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+neuronal+and+glial+subtypes+from+human+pluripotent+stem+cells.">Specification of neuronal and glial subtypes from human pluripotent stem cells.</a> Cellular and Molecular Life Sciences. 68 (24), 3995-4008 (2011).</li><li>Borghese, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+notch+signaling+in+human+embryonic+stem+cell-derived+neural+stem+cells+delays+G1/S+phase+transition+and+accelerates+neuronal+differentiation+in+vitro+and+in+vivo.">Inhibition of notch signaling in human embryonic stem cell-derived neural stem cells delays G1/S phase transition and accelerates neuronal differentiation in vitro and in vivo.</a> Stem Cells. 28 (5), 955-964 (2010).</li><li>Shi, Y., Kirwan, P., Smith, J., Robinson, H. 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The authors have nothing to disclose.

To date, hiPSC- and MEA-based methods for electrophysiological recordings of astrocyte-neuron co-cultures have found limited application in the field of ALS6 and still not in fully human platforms, in contrast to their more widespread use for in vitro modeling of epilepsy9. This platform, however, has the potential to address pathophysiologically relevant questions in ALS research, such as the mechanisms of neuronal hyperexcitability, astrocyte contribution to neur...

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) are powerful tools for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro and provide a translational paradigm for drug discovery strategies1. Researchers have demonstrated that the co-culture of hiPSC-A with hiPSC-N enhances the morphological, molecular, electrophysiological, and pharmacological maturation of both cell types, generating complex neuronal networks and astrocyte-neuron interactions that resemble their in vivo counterparts2,3. Similar co-culture experiments can recapitulate hallmarks of ALS pathobiology such as astrocyte-mediated neurotoxicity4,5 and neuronal hyper-excitability6. Additionally, with advancements in differentiation protocols, human induced pluripotent stem cells (hiPSC) can be differentiated into regionally-specific neural subtypes, including cortical and spinal cord hiPSC-A and hiPSC-N7,8. These strategies provide the potential for modeling cortical and spinal motor neuron pathology in ALS as well as the astrocytic influence on both. However, this requires that there is a reproducible functional assay to determine these effects.
Recently it was shown that multi-electrode array (MEA) recording is particularly suitable for the electrophysiological characterization of neuron-astrocyte co-cultures2. As opposed to single-cell electrophysiological analyses, these high-density electrode arrays passively record extracellular field potentials from large populations of neurons without disrupting culture conditions and preserving cell membranes' integrity. These platforms are particularly useful for recording the cellular and network activity of cultures over time and in response to pharmacologic manipulation. Finally, when the presence of astrocytes is a culture variable, MEA recordings can provide functional insights into astrocyte-neuron bidirectional interactions2,9.
Presented here is an optimized protocol for the differentiation of hiPSC into spinal cord hiPSC-A and hiPSC-motor neurons (MN) that has been previously validated2. The spinal cord hiPSC-A differentiation protocol consistently results in astrocyte cultures, which are positive for S100 calcium-binding protein B (S100&#946;), glial fibrillary acidic protein (GFAP), and Homeobox B4 (HOXB4) in up to 80%, 50%, and 90% of cells, respectively, indicating a maturing glial and spinal cord specification2,10. The hiPSC-MN differentiation protocol generates neurons that are &gt;90% positive for choline acetyltransferase (ChAT), suggestive of a mature alpha-motor neuron identity2. Additionally, the protocol describes techniques for the generation of hiPSC-A/MN co-cultures previously demonstrated to result in neurons with enhanced morphological complexity by Scholl analysis and immunofluorescent microscopy when compared to neuronal cultures without astrocytes or with rodent astrocytes2. While these descriptions are specific to spinal cord hiPSC-A and hiPSC-N, a unique advantage is that the initial independent culture of astrocytes and neurons followed by steps to co-culture at later time points can be translated to study the effects of neuron-astrocyte interactions from other specific regions as well as disease-specific cells7,8. Finally, the protocol describes how to grow these cultures on MEA plates so that functional activity as a factor of co-culture composition can be studied over time with the ability to manipulate cellular composition as well as culture conditions.
The goal of these protocols is to provide a functional assay to investigate astrocyte-neuron interactions, examine disease-specific changes, and test drugs with therapeutic potential in the field of ALS. Video instructions are provided for the most challenging steps of this protocol.

<table><tbody><tr><td>10 cm sterile culture plates</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; sterile culture flasks</td><td>Falcon</td><td>353136</td><td></td></tr><tr><td>2-Mercaptoethanol (&amp;beta;-ME)</td><td>Thermofisher</td><td>21985023</td><td>Working concentration 110 &amp;micro;M</td></tr><tr><td>500 mL 0.2 &amp;micro;m CA Filter System</td><td>Corning</td><td>430769</td><td></td></tr><tr><td>5 mL pipette</td><td>Falcon</td><td>357543</td><td></td></tr><tr><td>6 well sterile culture plates</td><td>Falcon</td><td>3046</td><td></td></tr><tr><td>Amphotericine B</td><td>Gibco</td><td>15290018</td><td>Working concentration 2.0 &amp;mu;g/mL</td></tr><tr><td>Anionic detergent with protease enzyme - Terg-A-Zyme</td><td>Sigma-Aldrich</td><td>Z273287</td><td>Working concentration 1% m/v</td></tr><tr><td>Ascorbic acid (ASAC)</td><td>Sigma</td><td>A4403</td><td>Dissolve 100 mg into 250 mL of dH&lt;sub&gt;2&lt;/sub&gt;O to get 0.4mg/ml stock. Sterile filter through a 0.22 &amp;micro;m filter, aliquot and freeze at -80 &amp;ordm;C. Dilute at 1:1000 for use. (working concentration 0.4 &amp;micro;g/mL).</td></tr><tr><td>Axion CytoView MEA 24 plates (M384-tMEA-24W)</td><td>Axion</td><td>OPT-24</td><td></td></tr><tr><td>Axion Edge MEA platform</td><td>Axion</td><td>Maestro Edge</td><td></td></tr><tr><td>Basement Membrane matrix - Matrigel</td><td>Corning</td><td>354277</td><td>Details in the protocol</td></tr><tr><td>Benchtop microscope (sterile under cell culture hood)</td><td>Zeiss</td><td>415510-1100-000</td><td>Primo Vert</td></tr><tr><td>Bicuculline</td><td>Sigma Aldrich</td><td>14340</td><td>Working concentration10 &amp;mu;M</td></tr><tr><td>Ciliary neurotrophic factor (CNTF)</td><td>Peprotech</td><td>450-13</td><td>Dissolve 100 &amp;micro;g in 1mL of sterile PBS, and then add 9 mL of sterile 0.1% BSA-PBS to 10 &amp;micro;g/mL. Aliquot and freeze at -80 &amp;ordm;C. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; tanks and regulator for Axion Edge</td><td>AirGas/Harris</td><td>9296NC</td><td></td></tr><tr><td>Compound E</td><td>Abcam</td><td>ab142164</td><td>Dissolve 250 &amp;micro;g in 2.0387 mL of DMSO to get a 250 &amp;micro;M stock. Aliquot and freeze at -80 &amp;ordm;C. Dilute 1:2000 for use. (working concentration 125 nM).</td></tr><tr><td>Cyanquixaline&amp;nbsp; (CNQX)</td><td>Sigma Aldrich</td><td>C239</td><td>Working concentration 50 &amp;mu;M</td></tr><tr><td>Dihydrokainic acid (DHK)</td><td>Tocris</td><td>111</td><td>Working concentration 50 &amp;mu;M and 300 &amp;mu;M</td></tr><tr><td>DMEM/F12</td><td>Thermofisher</td><td>113300</td><td>Working concentration 1x</td></tr><tr><td>Essential 8 Medium + Essential 8 Supplement</td><td>Thermofisher</td><td>A1517001</td><td>Combine 10 mL of Essential 8 (10x)&amp;nbsp; supplement with 500 mL of Essential 8 Growth Medium (1x)</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Thermofisher</td><td>16140071</td><td>Working concentration 1x</td></tr><tr><td>Glial cell line-derived neurotrophic (GDNF)</td><td>Peprotech</td><td>450-10</td><td>Dissolve 100 &amp;micro;g in 1mL of PBS to 100 &amp;micro;g/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 &amp;micro;g/mL. Aliquot and freeze at -80 &amp;ordm;C. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).</td></tr><tr><td>Hemocytometer</td><td>Election Microscopy Sciences</td><td>63510-20</td><td></td></tr><tr><td>Heparin</td><td>Millipore-sigma</td><td>H3149-100KU</td><td>Dissolve 200 mg in 100 mL of PBS to get 2 mg/mL stock solution. Aliquot the stock in 15 mL and 500 &amp;micro;L tubes. Dilute 1:1000 for use. (working concentration 2 &amp;micro;g/mL).</td></tr><tr><td>Humidity controlled Cell culture incubator</td><td>ThermoFisher</td><td>370</td><td>set to 37 &amp;ordm;C, 5 % CO2</td></tr><tr><td>Insulin-like growth factor 1 (IGF-1)</td><td>R&amp;amp;D systems</td><td>291-G1-200</td><td>Dissolve 200 &amp;micro;g in 2 mL of PBS to 100 &amp;micro;g/mL, then add 18 mL of 0.1%BSA-PBS to make 10 &amp;micro;g/mL stock and store at -80 &amp;ordm;C. Aliquot and freeze at -80 &amp;ordm;C. Dilute 10 &amp;micro;g/mL stock at 1: 1000 for use. (working concentration 10 ng/mL).</td></tr><tr><td>Kainc acid</td><td>Abcam</td><td>ab144490</td><td>Working concentration 5 &amp;mu;M</td></tr><tr><td>Knockout Serum Replacement (KSR)</td><td>Thermofisher</td><td>10828</td><td>Working concentration 1x</td></tr><tr><td>Laminin</td><td>Thermofisher</td><td>23017-015</td><td>Stock solution 1 mg/mL, working concentration 10 &amp;micro;g/mL (coating), 1 &amp;micro;g/mL (cell media)</td></tr><tr><td>LDN193189</td><td>Stemgent</td><td>04-0074</td><td>Dissolve 2 mg of LDN into 500 &amp;micro;L of Chloroform to get 10 mM stock. Aliquot this and freeze at -80 &amp;ordm;C. For using, dilute the stock 1 to 10 into DMSO [to 1 mM] first, then dilute 1:5000 of 1 mM into the desired media to get 0.2 &amp;micro;M working solution</td></tr><tr><td>L-Glutamine</td><td>Thermofisher</td><td>25030</td><td>Working concentration 100x</td></tr><tr><td>MEA glass plates</td><td>MultiChannel Systems</td><td>60MEA200/30iR-Ti-gr</td><td></td></tr><tr><td>Multichannel Pipet P200</td><td>Gilson</td><td>PJ22224</td><td></td></tr><tr><td>Neurobasal</td><td>Thermofisher</td><td>21103049</td><td>Working concentration 1x</td></tr><tr><td>Non-Essential Amino Acids (NEAA)</td><td>Thermofisher</td><td>11140050</td><td>Working concentration 100x</td></tr><tr><td>Pencillin/Streptomycin</td><td>Thermofisher</td><td>15140122</td><td>Working concentration 100x</td></tr><tr><td>Polyornithine (PLO)</td><td>Sigma-Aldrich</td><td>P3655</td><td>Dissolve 100 mg in 1 mL of ddW to get 100 mg/mL stock solution. Aliquot the stock in 100 &amp;micro;L tubes. (working concentration 100 &amp;micro;g/mL)</td></tr><tr><td>Potassium chloride (KCl)</td><td>NA</td><td>NA</td><td>Working concentration100 mM</td></tr><tr><td>Purmorphamine (PMN)</td><td>Millipore-Sigma</td><td>540223</td><td>Dissolve 5 mg in 9.6 mL of DMSO to get 1 mM solution. Aliquot and freeze at -80 &amp;ordm;C. Dilute 1:1000 for use. (working concentration 1 &amp;micro;M).</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 &amp;micro;g in 1 mL of PBS to 100 &amp;micro;g/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 &amp;micro;g/mL. Aliquot and freeze at -80 &amp;ordm;C. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).</td></tr><tr><td>Retinoic acid (RA)</td><td>Sigma</td><td>R2625</td><td>Dissolve 100 mg into 3.3 mL of DMSO to get 100 mM stock solution. Aliquot the stock 100 &amp;micro;L/tube and freeze at -80 &amp;ordm;C. Take 200 &amp;micro;L of 100 mM stock and dilute 10x (add 1.8 mL of DMSO) to make 10 mM stock. Aliquot 50 &amp;micro;L/tube&amp;nbsp;and store at -80 &amp;ordm;C. Dilute at 1:10000 for use. (working concentration 2 &amp;micro;M).</td></tr><tr><td>ROCK-I nhibitor</td><td>Peprotech</td><td>1293823</td><td>Dissolve 5 mg in 1480 &amp;micro;L of dH&lt;sub&gt;2&lt;/sub&gt;O to get 10 mM stock, aliquot and freeze at -80 &amp;ordm;C. Dilute at 1: 500 for use. (working concentration 20 &amp;micro;M).</td></tr><tr><td>SB431542</td><td>Sigma</td><td>S4317</td><td>Dissolve 5mg into 1.3 mL of DMSO to get 10 mM stock solution. Aliquot and freeze at -80 &amp;ordm;C. Dilute at 1:1000 for use. (working con 10 &amp;micro;M)</td></tr><tr><td>Sterile cell culture hoods</td><td>Baker Company</td><td>SG-600</td><td></td></tr><tr><td>Supplement B - B27 Supplement</td><td>Thermofisher</td><td>21985023</td><td>Working concentration 50x</td></tr><tr><td>Supplement N - N2 Supplement</td><td>Thermofisher</td><td>17502048</td><td>Working concentration 100x</td></tr><tr><td>Table top cell culture centrifuge</td><td>ThermoFisher</td><td>75004261</td><td>Sorvall Legend X1R</td></tr><tr><td>Thermoplastic&amp;nbsp;film - Parafilm</td><td>PARAFILM</td><td>P7793</td><td></td></tr><tr><td>Tissue dissociation protease - Dispase</td><td>StemCell Technologies</td><td>7923</td><td>Working concentration 1x</td></tr><tr><td>Trypsin Inhibitor</td><td>Sigma</td><td>T6522-1G</td><td>Dissolve 1g in 100mL ddH&lt;sub&gt;2&lt;/sub&gt;O to get 10 mg/mL stock. Aliquot and store at 4 &amp;ordm;C. Dilute 1:10 of trypsin volume for use. (working concentration 1 mg/mL).</td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>Thermofisher</td><td>2530054</td><td>Working concentration 1x</td></tr><tr><td>Waterbath</td><td>ThermoFisher</td><td>2332</td><td>Isotemp</td></tr></tbody></table>

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.

The spinal cord patterning protocol for the generation of hiPSC-MN and spinal cord hiPSC-A is outlined in Figure 1. In this protocol, hiPSCs are maintained and passaged as non-confluent colonies (Figure 2A). Neurogenesis is initiated (neural induction) through dual SMAD inhibition by the addition of LDN193189 and SB431542, inactivating the bone morphogenetic protein (BMP) and the transforming growth factor-beta (TGF-&#946;) pathways, respectively. A monolayer-ba...

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

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) ...

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2.2K Views.  Johns Hopkins University. De techniek die we vandaag presenteren, maakt de analyse van de activiteit van ruggenmergneuronen mogelijk als gevolg van intrinsieke neuronale factor en de invloed van omringende astrocyten. Deze techniek genereert op betrouwbare wijze zowel menselijke neuronen als menselijke astrocyten die specifiek zijn voor het ruggenmerg en maakt gebruik van een functionele outputmaat die kan worden geschaald voor reproduceerbaarheid. De techniek die we presenteren maakt het mogelijk om spinale neuronen ...

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

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

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Developmental Biology

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

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

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

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

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 ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

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

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.

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.

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

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

Biology

Differentiation of Spinal Cord Neural Progenitor Cells into Motor Neurons

Source: Taga, A. et al., <a href="https://app.jove.com/v/62726">Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons.</a> J. Vis. Exp. (2021).
This video showcases a method for differentiating spinal cord neural progenitor cells (NPCs) into motor neurons. It details the culturing process of the NPCs on a coated culture plate using specialized media enriched with specific inhibitors aimed at facilitating NPC differentiation into mature motor neurons.

1. Thawing Neural Progenitor Cells (NPC) cultures

	Coat 10 cm plates with polyornithine (PLO) and laminin the day prior to thawing the cells.
	
		Add 5 ...

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

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