This work is a component of the National Cooperative Reprogrammed Cell Research Groups (NCRCRG) to study mental illness and was supported by NIH grant U19MH107367. Initial work was also supported by NIH grant NS070297. We thank Drs. Carol Marchetto and Fred Gage, The Salk Institute, for providing the WT 126 line of neural progenitor cells, and Drs. Eugene Yeo and Lawrence Goldstein, UC San Diego for providing the CVB WT24 line of neural progenitor cells. We thank Deborah Pre in the laboratory of Dr. Anne Bang, Sanford Burnham Prebys Medical Discovery Institute, for useful discussions.

1. Differentiation Period Prior to Replating
<ol>
	<li>Differentiate neurons on 10 cm dishes, using a protocol of choice7,8 until neurons have formed a thick network with their processes and express not only early neuronal markers such as MAP2 or TuJ1, but also late markers such as NeuN.</li>
	<li>Change half the medium of choice every 4 days during the neuronal differentiation process. 
	NOTE: More extensive or frequent medium changes dilute essential trophic factors, and could disfavor maturation.
	<ol>
		<li>For the iPSC-derived WT126 neurons, use the following post-differentiation culturing media: 5 mL 100x N2 supplement, 10 ng/mL BDNF, 10 ng/mL GDNF, 1 &#181;g/mL laminin, 200 &#181;M ascorbic acid, 1 &#181;M dibutyryl-cAMP and 10 mL SM1 for 500 mL Dulbecco&#8217;s modified Eagle&#8217;s medium/nutrient mixture F-12 (DMEM/F12). Gradually, using half-media changes, replace with neural basal medium (Table of Materials) and the same supplements.</li>
		<li>For the iPSC-derived CVB WT neurons, use the following post-differentiation culturing medium: 500 mL neural basal A medium (Table of Materials) and 500 mL DMEM/F12 medium with 2 &#181;g/mL laminin, 10 mL glutamine supplement (Table of Materials), 0.75 mg/mL sodium bicarbonate, 5 mL minimum essential medium (MEM) nonessential amino acids, 0.2 mM ascorbic acid, 10 ng/mL BDNF, 20 mL 50x B27, and 10 mL 100x N2 supplement.</li>
	</ol>
	</li>
</ol>
2. Coating Multiwells
<ol>
	<li>The day before replating, coat with poly-L-ornithine (PLO). Dissolve PLO in sterile water to make a stock solution (10 mg/mL). Store this stock at -20 &#176;C. Dilute PLO 1:100 in water to yield a concentration of 50 &#181;g/mL when coating glass and 1:1,000 ratio (10 &#181;g/mL) when coating plastic.</li>
	<li>Apply the coating directly to the target plates. Use the volume of coating appropriate to the plate size (i.e., for a 24-well plate apply 500 &#181;L of PLO solution per well).</li>
	<li>Allow plates to sit in the dark overnight at room temperature.</li>
	<li>Retrieve coated plates on the day of replating and transfer to a sterile biosafety cabinet.</li>
	<li>Aspirate the PLO solution and rinse twice with sterile water.</li>
	<li>Dilute laminin (1.15 mg/mL) in phosphate-buffered saline (PBS) at 1:400 dilution. 
	NOTE: Thaw laminin at 4 &#176;C and quickly add to PBS to avoid aggregation of laminin and uneven coating.</li>
	<li>Aspirate sterile water and apply 500 &#181;L of laminin coating to wells previously coated with PLO.</li>
	<li>Place plates in a 37 &#176;C incubator for a minimum of 4-6 h. Use longer incubations, up to 16 h, for glass surfaces. Use consistent incubation times.</li>
</ol>
3. Replating Differentiated Neurons
<ol>
	<li>Rinse plate of differentiated neurons with PBS once gently. Disperse PBS gently down the wall of the plate, and not directly onto the cells, to avoid disrupting them.</li>
	<li>Gently aspirate PBS, being careful to avoid touching the cells directly but to aspirate from the edge of the dish while tipping it towards the researcher.</li>
	<li>Apply at minimum 1 mL of the proteolytic enzyme (Table of Materials) per 10 cm plate and return cells to incubator. Add slightly higher volumes if the tissue culture room exhibits a high evaporation rate due to low humidity.</li>
	<li>Incubate cells with proteolytic enzyme for 40-45 min in order to detach them from the plate and to detach them from other neurons within the neuronal network. 
	NOTE: Timing at this step is critical. Quenching the protease too early can lead to increased cell death after replating. The proteolytic enzyme manufacturer recommends that temperatures much lower than 37 &#176;C be used with longer incubation periods for passaging cell lines (e.g., overnight at 4 &#176;C). However, handling neurons at 4 &#176;C should be avoided, as they often show poor survival after cold exposure. The manufacturer also states that a 60 min incubation with the enzyme at 37 &#176;C leads to its enzymatic inactivation. However, in authors&#39;&#160;experience, a 40-45 min incubation of hiPSC-derived neuronal cultures at 37 &#176;C is sufficient for efficient dissociation and excellent neuronal survival upon replating.</li>
	<li>Check neurons on a phase-contrast microscope during the incubation time and allow protease treatment to continue until the neural network completely detaches from the plate and starts to break apart in smaller sheets upon briefly shaking the plate under the microscope.</li>
	<li>Quench the protease activity using 5 mL of fresh DMEM media per 1 mL of protease in the 10 cm plate to stop the digestion. Gently triturate cells against plate 5-8 times to disrupt network, using serological pipettes. Be careful not to apply too much pressure when triturating, as differentiated neurons are fragile. Do not use a P1000 tip, as the end is too sharp and narrow, and thus can sheer or damage the neurons.</li>
	<li>Apply solution with cells through a cell strainer with 100 &#181;m diameter mesh into a fresh 50 mL conical tube drop by drop.</li>
	<li>Rinse strainer with an additional 5 mL of fresh DMEM media, after cells have filtered through.</li>
	<li>Spin cells in benchtop centrifuge at 1,000 x g for 5 min.</li>
	<li>Return conical tube to the biosafety cabinet and aspirate most of the media, leaving around 250 &#181;L to ensure cells retain moisture.</li>
	<li>Resuspend cells gently in 2 mL of fresh DMEM media. Do not pipette the pellet against the side of the tube. Instead, gently invert conical 2-3 times and pass cells through the end of a 5 mL serological pipet to dislodge them.</li>
	<li>Apply 10 &#181;L of resuspended neurons onto the edge of a hemocytometer.</li>
	<li>Add 8-10 &#181;L of trypan blue to droplet of cells to assess cell viability during this resuspension step. Apply 10 &#181;L of this mixture to the hemocytometer or other automatic cell counters. Assess as viable those cells that are phase bright and exclude the trypan blue dye.</li>
	<li>Determine amount of viable cells/mL, and prepare to dilute the contents of the conical tube according to the desired cell density.</li>
	<li>Plate ~10,000 cells per well for a 384-well plate; plate ~150,000-200,000 cells per well for a 24-well plate.</li>
	<li>Add fresh DMEM to the conical tube of resuspended cells to achieve appropriate dilution and add additional appropriate supplements such as B27 and/or BDNF, depending on the requirements of the specific cell line.</li>
	<li>Gently tilt conical tube to mix 2-3 times.</li>
	<li>Aspirate laminin coating from the 24-well plate, or from the 384-well multiwell plate using a 16-channel pipet and rinse once with PBS.</li>
	<li>Aspirate PBS using a P1000 for the 24-well plate, or the 16-channel pipette for 384-well plates.</li>
	<li>Apply cell solution to each well in a figure eight motion to avoid clumping. Plating uniformity might also be optimized by using automated liquid handling devices. 
	NOTE: The addition of laminin to the media starting 2-4 days post-replating also helps maintain a homogenous distribution of the cells.</li>
	<li>Repeat steps 3.19 and 3.20 for each well.</li>
	<li>Return the plate to incubator set at 37 &#176;C and 5% CO2.</li>
	<li>After 2 days, start changing half the medium every 4 days using the post-differentiation culturing media described in section 1.2. After the desired maturation time, fix cultures using 3.7% formaldehyde at 37 &#176;C and stain cells according to the experimental requirements.</li>
</ol>
4. Cell Viability Assays Post-replating
<ol>
	<li>Add the early cell death reporter (VivaFix: 0.5 &#181;L of the 50 &#181;L stock solution per 500 &#181;L culture media per well) after briefly vortexing the stock solution.</li>
	<li>Image the live and dead cells cultured on glass-bottom multiwells, after 20 min, without any washing step, since the dye fluoresces only once inside the cells, or fix and co-stain with 4&#39;,6-diamidino-2-phenylindole (DAPI) and/or other antibodies before imaging using a confocal microscope.</li>
</ol>
5. Immunostaining
<ol>
	<li>Fix cultures with 3.7% formaldehyde in PBS plus 120 mM sucrose for 20 min at 37 &#176;C.</li>
	<li>Rinse and permeabilize with 0.2% Triton X-100 for 5 min at room temperature, and then block for 30 min with 2% bovine serum albumin (BSA).</li>
	<li>Aspirate the BSA without rinsing and incubate for 1 h at room temperature with rabbit anti-MAP2 antibody 1:1,000, mouse anti-&#946;3 tubulin (TuJ1) antibody 1:2,000, chicken anti-NeuN antibody (1:100) and rat anti-CTIP2 antibody (1:500), or with mouse anti-PSD95 (1:200), rabbit anti-synapsin 1 (1:200) and chicken anti-MAP2 antibody for synaptic staining.</li>
	<li>Rinse with PBS, and incubate with Alexa Fluor-conjugated secondary antibodies plus DAPI for 45 min at 37 &#176;C.</li>
	<li>Finally, wash twice with PBS before imaging.</li>
</ol>
6. Calcium imaging
<ol>
	<li>Infect cultured cells with AAV8-syn-jGCAMP7f-WPRE (The Salk Institute for Biological Studies, GT3 Core Facility) at 10 days post-replating and image to assess spontaneous calcium transients at 3-4 weeks post-replating.</li>
</ol>
7. Image acquisition and Analysis
NOTE: For details on the acquisition system please refer to Calabrese et al.12.
<ol>
	<li>Use an imaging module for manual or automated image acquisition, and an image analysis software module, such as CellProfiler13, for morphometric measurements.</li>
	<li>Calculate average length of TuJ1 positive neurites and MAP2 positive dendrites by measuring total length per field of view divided by the number of neurons (DAPI + MAP2 or TuJ1 positives cells) within the same field.</li>
</ol>

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The authors have nothing to disclose.

We have demonstrated a straight-forward procedure for the resuspension and replating of human neuronal cultures that optimizes viability, differentiation success, and subcellular imaging in a manner that is compatible with high content screening platforms, and other assays relevant to drug discovery. Figure 6 illustrates the overall workflow and examples of such applications.
Although here we focused on hiPSC-derived neurons that are directed toward a cortical neu...

Human pluripotent stem cell (hiPSC)-derived neurons are increasingly relevant in the areas of basic research, drug development, and regenerative medicine. Workflows and procedures to optimize their culture and maintenance, and improve the efficiency of differentiation into specific neuronal subtypes, are evolving rapidly1,2. To improve the utility and cost-effectiveness of human stem cell-derived neurons as model systems amenable to high-content analyses in drug discovery and target validation, it is useful to decrease the culturing time required to generate mature, functional neurons, while retaining maximum robustness, reproducibility, and phenotype relevance. Although 3-dimensional organoid cultures are driving breakthroughs in neurodevelopment research3, 2-dimensional monolayer cultures are especially compatible with automated imaging-based applications due to their minimal tissue thickness.
However, the adaptation of imaging-based screening methods to models of human neurological and neurodevelopmental disease faces a major challenge. The protracted timeframe over which the human nervous system matures in vivo necessitates extended time in culture to accommodate natural programs of gene expression and achieve neuronal maturation.
One practical consequence of the lengthy neuronal differentiation program is that the maintenance of hiPSC-derived monolayer cultures must be sustained for many weeks to achieve adequate synapse maturity. During this time, neural progenitors that remain undifferentiated continue to divide. These can quickly overgrow the culture and usurp the nutrient content required to maintain viable postmitotic neurons. Vigorously dividing neural progenitor cells (NPCs) can also compete with neurons for the growth substrate. This can render such cultures subject to problems of poor adhesion, a condition unsuitable for imaging-based assays. Moreover, many investigators find that the smaller the culture volume, the greater the difficulty in maintaining healthy populations of differentiated neurons long enough to observe the late stages of neuronal differentiation. In other words, assays of synapse maturation using high content screening (HCS) approaches can be very challenging for human-derived neurons.
To circumvent some of these problems, a procedure of resuspending and replating previously differentiated hiPSC-derived neurons has been used. Firstly, it allows the study of neurite outgrowth (or, more accurately, neurite regeneration) in a population of fully committed neurons. Secondly, the replating of previously differentiated neurons from a large volume format (like 10 cm plates or larger), down to small volume formats (like HCS-compatible 96- or 384-well microtiter plates) enables a significant reduction in total culturing time in the small volume condition. This facilitates the study of synapse assembly and maturation over subsequent weeks in vitro.
However, the replating of mature neurons that have already established long neurites and a complex connectivity network presents several challenges, one of which is the sometimes high and variable rate of cell death. Here, we describe a replating procedure that results in excellent cell survival and reproducibility. Commonly, neurons are exposed to proteolytic enzymes for short incubation periods (typically ~3-10 min) in order to detach cells from the substrate prior to trituration. This brief proteolysis time is customarily used for resuspending and passaging many types of dividing cells, including non-neuronal cells and undifferentiated progenitors4,5,6. However, for differentiated neurons bearing long, interconnected neurites, it is essential not only to detach cells from the substrate but also to disrupt the dendritic and axonal network in order to isolate individual cells while minimizing damage. Indeed, a thick meshwork of neurons usually tends to detach from the substrate as a single sheet, rather than as individual cells. If care is not taken to loosen the thick network of neurites, neurons not only become irreversibly damaged during trituration, but many of them fail to pass through the filter used to remove clumps, resulting in poor cell yield. Below we describe a simple modification to a widely-used protease incubation procedure to counter these difficulties.
In the protocol described below, neurons are incubated for 40-45 min with a mild protease, such as the proteolytic enzyme (e.g., Accutase). During the first 5-10 min after adding the enzyme, the neuronal network lifts off from the substrate as a sheet. Incubation with the protease proceeds for an additional 30-40 min before proceeding with gentle trituration and filtering. This extra incubation time helps ensure that the digestion of the material relaxes the intercellular network, thereby ensuring that subsequent trituration produces a suspension of individual cells. This procedure maximizes the uniformity of cell distribution upon replating while minimizing cell death. We have successfully applied this replating method to hiPSC-derived neuronal cultures generated by various differentiation protocols7,8 and from various lines of hiPSCs. The procedure is nominally suitable for use with most or all lines of stem cell-derived neurons. We have observed that an extended protease incubation time is not absolutely essential for replating cultures from small format plates (e.g., 35 mm diameter); however, as we show here, it provides a significant benefit when replating from large diameter plates (e.g., 10 cm diameter or larger), probably because neurites in such plates can extend very long processes and form a densely interconnected array.
Here we demonstrate this method and briefly illustrate its application in assays for early neuritogenesis and for synapse maturation, which involves clustering of pre- and postsynaptic proteins along the dendrites and axons, followed by their later colocalization at synaptic sites. The examples highlight the advantages this protocol offers in preserving cell viability and reproducibility. First, it permits investigators to study early steps in human neuritogenesis. The experimental setting is similar to the commonly used primary cultures of rodent cortical or hippocampal neurons, where cells are extracted from late fetal or early postnatal brain, dissociated by trituration after gentle protease treatment, and allowed to initiate neurites or to regenerate neurites that were severed in the procedure9,10. Similar to such rodent primary neurons, hiPSC-derived neurons begin to form or regenerate their neurites within hours after replating, allowing imaging of growth cones and neurite morphology in an environment optimal for high spatiotemporal imaging with fewer surrounding undifferentiated cells. We have observed that neurite outgrowth is more synchronized compared to the variable delays and different outgrowth rates seen when neurons first begin to differentiate from a progenitor population. In addition, replating enables assays of neurons expressing neuronal subtype markers that typically appear later in neural development, such as the cortical layer 5/6 transcription factor CTIP2 (Chicken ovalbumin upstream promoter transcription factor-interacting protein 2), or the pan-neuronal marker NeuN11. An especially useful feature of the replating approach is that synaptogenesis proceeds within a time frame compatible with HCS.

<table border="0" cellpadding="0" cellspacing="0" style="width:1076px;" width="1076">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Post Replating Media</td>
			<td style="width:276px;"></td>
			<td style="width:119px;"></td>
			<td style="width:377px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">L-Ascorbic Acid</td>
			<td style="width:276px;">Sigma</td>
			<td>A4403</td>
			<td>Add 1ml of 200mM stock to 1L of N2B27 media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dibutyryl-cAMP</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>Add 1 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Human BDNF</td>
			<td style="width:276px;">Peprotech</td>
			<td style="width:119px;">450-02</td>
			<td>10 ng/ml final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">B27 (50X)</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td style="width:119px;">17504044</td>
			<td>Add 20 ml to 1L N2B27 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">DMEM/F12 with Glutamax</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td>31331093</td>
			<td>Add N2 and distribute in 50 mL conicals; parafilm wrap lids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Human GDNF</td>
			<td style="width:276px;">Peprotech</td>
			<td>450-10</td>
			<td>10 ng/ml final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Glutamax</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td>35050038</td>
			<td>Add 10 ml to 1L N2B27 media; glutamine supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Mouse Laminin</td>
			<td style="width:276px;">Sigma</td>
			<td>P3655-10mg</td>
			<td>Add 100 &#181;l to 50 mL N2B27</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">MEM Nonessential Amino Acids</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td style="width:119px;">11140035</td>
			<td>Add 5ml to 1L N2B27 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">N2 (100X) Supplement</td>
			<td style="width:276px;">Life Technologies</td>
			<td>17502048</td>
			<td>Add 5ml to 500mL media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Neurobasal A Media</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td>10888022</td>
			<td>Combine with DMEM/F12 to generate N2B27 media for CVB wt cells; neural basal A media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Neurobasal Media</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td>21103049</td>
			<td>for WT126 cells; neural basal media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">SM1 Supplement</td>
			<td style="width:276px;">StemCell Technologies</td>
			<td>5711</td>
			<td>Add 1:50 to media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium bicarbonate</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td>25080-094</td>
			<td>Add 10ml to 1L N2B27 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Plate Preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">10cm Tissue Culture Dishes</td>
			<td style="width:276px;">Fisher Scientific</td>
			<td>08772-E</td>
			<td>Plastic TC-treated dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">6-well Tissue Culture Dishes</td>
			<td style="width:276px;">Thomas Scientific</td>
			<td>1194Y80</td>
			<td>NEST plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Mouse Laminin</td>
			<td style="width:276px;">Life Technologies</td>
			<td>23017-015</td>
			<td>Add 1:400 on plastic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Poly-Ornithine</td>
			<td style="width:276px;">Sigma</td>
			<td>P3655-10mg</td>
			<td>Add 1:1000 on plastic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">UltraPure Distilled Water</td>
			<td style="width:276px;">Life Technologies</td>
			<td style="width:119px;">10977-015</td>
			<td>To dilute Poly-L-Ornithine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Replating Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">100mM Cell Strainer</td>
			<td style="width:276px;">Corning</td>
			<td style="width:119px;">431752</td>
			<td>Sterile, individually wrapped</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">384-well plate, uncoated</td>
			<td style="width:276px;">PerkinElmer</td>
			<td>6007550</td>
			<td>Coat with PLO and Laminin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">DPBS</td>
			<td style="width:276px;">Life Technologies</td>
			<td style="width:119px;">14190144</td>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Poly-D-Lysine-Precoated 384-well Plates</td>
			<td style="width:276px;">PerkinElmer</td>
			<td>6057500</td>
			<td>Rinse before coating with laminin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">StemPro Accutase</td>
			<td style="width:276px;">Life Technologies</td>
			<td>A1110501</td>
			<td>Apply 1mL/10cm plate for 30-45 minutes; proteolytic enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Fixation Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">37% Formaldehyde</td>
			<td style="width:276px;">Fisher Scientific</td>
			<td style="width:119px;">F79-1</td>
			<td>Dissolved in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Sucrose</td>
			<td style="width:276px;">Fisher Scientific</td>
			<td style="width:119px;">S5-12</td>
			<td>0.8 g per 10 ml of fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Immunostaining Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa Fluor 488 Goat anti-mouse</td>
			<td style="width:276px;">Invitrogen</td>
			<td>A-11001</td>
			<td>secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa Fluor 568 Goat anti-chicken</td>
			<td style="width:276px;">Invitrogen</td>
			<td>A-11041</td>
			<td>secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa Fluor 647 Goat anti-chicken</td>
			<td style="width:276px;">Invitrogen</td>
			<td>A-21449</td>
			<td>secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa Fluor 561 Goat anti-rat</td>
			<td style="width:276px;">Invitrogen</td>
			<td>A-11077</td>
			<td>secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">DAPI</td>
			<td style="width:276px;">Biotium</td>
			<td>40043</td>
			<td>visualizes DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">mouse antibody against b3-tubulin (TuJ-1)</td>
			<td style="width:276px;">Neuromics</td>
			<td>MO15013</td>
			<td>early stage neuronal marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">rat antibody against CTIP2</td>
			<td style="width:276px;">Abcam</td>
			<td>ab18465</td>
			<td>layer 5/6 cortical neurons</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">chicken antibody against MAP2</td>
			<td style="width:276px;">LifeSpan Biosciences</td>
			<td>LS-B290</td>
			<td>early stage neuronal marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">chicken antibody against NeuN</td>
			<td style="width:276px;">Millipore</td>
			<td>ABN91</td>
			<td>late stage neuronal marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">rabbit antibody against MAP2</td>
			<td style="width:276px;">Shelley Halpain</td>
			<td>N/A</td>
			<td>early stage neuronal marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">mouse antibody against PSD-95</td>
			<td style="width:276px;">Sigma</td>
			<td>P-246</td>
			<td>post-synaptic marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">rabbit antibody against Synapsin 1</td>
			<td style="width:276px;">Millipore</td>
			<td>AB1543</td>
			<td>pre-synaptic marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Bovine serum albumin (BSA)</td>
			<td style="width:276px;">GE Healthcare Life Sciences</td>
			<td>SH30574.02</td>
			<td>10% in PBS for blocking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Titon X-100</td>
			<td style="width:276px;">Sigma</td>
			<td style="width:119px;">9002931</td>
			<td>Dilute to 0.2% on PBS for permeabilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Viability Markers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Vivafix 649/660</td>
			<td style="width:276px;">Biorad</td>
			<td style="width:119px;">135-1118</td>
			<td>cell death marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Calcium Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Name of Reagent/ Equipment</td>
			<td style="width:276px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAV8-syn-jGCAMP7f-WPRE</td>
			<td>THE SALK INSTITUTE, GT3 Core Facility</td>
			<td style="width:119px;">N/A</td>
			<td>calcium reporter in a viral delivery system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">hiPSC-derived NPCs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">WT 126 (Y2610)</td>
			<td style="width:276px;">Gage lab</td>
			<td style="width:119px;">N/A</td>
			<td>Marchetto et al., 2010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">CVB WT24</td>
			<td style="width:276px;">Yeo and Goldstein labs</td>
			<td style="width:119px;">N/A</td>
			<td>unpublished</td>
		</tr>
	</tbody>
</table>

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.

The replating of hiPSCs-derived neurons that have been differentiated for multiple weeks offers several advantages. However, detaching and replating differentiated neurons that have long, interconnected dendrites and axons (Figure 1A) can result in a high fraction of irreversibly damaged neurons.
As described in the protocol section, we used incubation with a proteolytic enzyme to detach the neurons from the substrate. Typically, due to their thic...

Watch this Scientific Journal Video about Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation at JoVE.com

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

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 ...

post-differentiation-replating-human-pluripotent-stem-cell-derived

Research

JoVE Journal

Neuroscience

9.2K Views.  University of California, San Diego. 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.

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

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.

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

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. 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 ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

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 ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

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 ...

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 ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

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 ...