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><tbody><tr><td>&lt;strong&gt;Post Replating Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>L-Ascorbic Acid</td><td>Sigma</td><td>A4403</td><td>Add 1 mL of 200 mM stock to 1 L of N2B27 media</td></tr><tr><td>Dibutyryl-cAMP</td><td>Sigma</td><td>D0627</td><td>Add 1 &amp;micro;M</td></tr><tr><td>Human BDNF</td><td>Peprotech</td><td>450-02</td><td>10 ng/ml final concentration</td></tr><tr><td>B27 (50x)</td><td>Thermofisher Scientific</td><td>17504044</td><td>Add 20 mL to 1 L N2B27 media</td></tr><tr><td>DMEM/F12 with Glutamax</td><td>Thermofisher Scientific</td><td>31331093</td><td>Add N2 and distribute in 50 mL conicals; parafilm wrap lids</td></tr><tr><td>Human GDNF</td><td>Peprotech</td><td>450-10</td><td>10 ng/mL final concentration</td></tr><tr><td>Glutamax</td><td>Thermofisher Scientific</td><td>35050038</td><td>Add 10 ml to 1 L N2B27 media; glutamine supplement</td></tr><tr><td>Mouse Laminin</td><td>Sigma</td><td>P3655-10mg</td><td>Add 100 &amp;micro;L to 50 mL N2B27</td></tr><tr><td>MEM Nonessential Amino Acids</td><td>Thermofisher Scientific</td><td>11140035</td><td>Add 5 mL to 1 L N2B27 media</td></tr><tr><td>N2 (100x) Supplement</td><td>Life Technologies</td><td>17502048</td><td>Add 5 mL to 500 mL media</td></tr><tr><td>Neurobasal A Media</td><td>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><td>Neurobasal Media</td><td>Thermofisher Scientific</td><td>21103049</td><td>for WT126 cells; neural basal media</td></tr><tr><td>SM1 Supplement</td><td>StemCell Technologies</td><td>5711</td><td>Add 1:50 to media</td></tr><tr><td>Sodium bicarbonate</td><td>Thermofisher Scientific</td><td>25080-094</td><td>Add 10 mL to 1 L N2B27 media</td></tr><tr><td>&lt;strong&gt;Plate Preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 cm Tissue Culture Dishes</td><td>Fisher Scientific</td><td>08772-E</td><td>Plastic TC-treated dishes</td></tr><tr><td>6-well Tissue Culture Dishes</td><td>Thomas Scientific</td><td>1194Y80</td><td>NEST plates</td></tr><tr><td>Mouse Laminin</td><td>Life Technologies</td><td>23017-015</td><td>Add 1:400 on plastic</td></tr><tr><td>Poly-Ornithine</td><td>Sigma</td><td>P3655-10mg</td><td>Add 1:1,000 on plastic</td></tr><tr><td>UltraPure Distilled Water</td><td>Life Technologies</td><td>10977-015</td><td>To dilute Poly-L-Ornithine</td></tr><tr><td>&lt;strong&gt;Replating Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>100 mM Cell Strainer</td><td>Corning</td><td>431752</td><td>Sterile, individually wrapped</td></tr><tr><td>384-well plate, uncoated</td><td>PerkinElmer</td><td>6007550</td><td>Coat with PLO and Laminin</td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190144</td><td>Dulbecco&#039;s phosphate-buffered saline</td></tr><tr><td>Poly-D-Lysine-Precoated 384-well Plates</td><td>PerkinElmer</td><td>6057500</td><td>Rinse before coating with laminin</td></tr><tr><td>StemPro Accutase</td><td>Life Technologies</td><td>A1110501</td><td>Apply 1 mL/10 cm plate for 30-45 min; proteolytic enzyme</td></tr><tr><td>&lt;strong&gt;Fixation Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>37% Formaldehyde</td><td>Fisher Scientific</td><td>F79-1</td><td>Dissolved in PBS</td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-12</td><td>0.8 g per 10 mL of fixative</td></tr><tr><td>&lt;strong&gt;Immunostaining Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 488 Goat anti-mouse</td><td>Invitrogen</td><td>A-11001</td><td>secondary antibody</td></tr><tr><td>Alexa Fluor 568 Goat anti-chicken</td><td>Invitrogen</td><td>A-11041</td><td>secondary antibody</td></tr><tr><td>Alexa Fluor 647 Goat anti-chicken</td><td>Invitrogen</td><td>A-21449</td><td>secondary antibody</td></tr><tr><td>Alexa Fluor 561 Goat anti-rat</td><td>Invitrogen</td><td>A-11077</td><td>secondary antibody</td></tr><tr><td>DAPI</td><td>Biotium</td><td>40043</td><td>visualizes DNA</td></tr><tr><td>Mouse antibody against b3-tubulin (TuJ-1)</td><td>Neuromics</td><td>MO15013</td><td>early stage neuronal marker</td></tr><tr><td>Rat antibody against CTIP2</td><td>Abcam</td><td>ab18465</td><td>layer 5/6 cortical neurons</td></tr><tr><td>Chicken antibody against MAP2</td><td>LifeSpan Biosciences</td><td>LS-B290</td><td>early stage neuronal marker</td></tr><tr><td>Chicken antibody against NeuN</td><td>Millipore</td><td>ABN91</td><td>late stage neuronal marker</td></tr><tr><td>Rabbit antibody against MAP2</td><td>Shelley Halpain</td><td>N/A</td><td>early stage neuronal marker</td></tr><tr><td>Mouse antibody against PSD-95</td><td>Sigma</td><td>P-246</td><td>post-synaptic marker</td></tr><tr><td>Rabbit antibody against Synapsin 1</td><td>Millipore</td><td>AB1543</td><td>pre-synaptic marker</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>GE Healthcare Life Sciences</td><td>SH30574.02</td><td>10% in PBS for blocking</td></tr><tr><td>Titon X-100</td><td>Sigma</td><td>9002931</td><td>Dilute to 0.2% on PBS for permeabilization</td></tr><tr><td>&lt;strong&gt;Viability Markers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Vivafix 649/660</td><td>Biorad</td><td>135-1118</td><td>cell death marker</td></tr><tr><td>&lt;strong&gt;Calcium Imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Name of Reagent/ Equipment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>AAV8-syn-jGCAMP7f-WPRE</td><td>THE SALK INSTITUTE, GT3 Core Facility</td><td>N/A</td><td>calcium reporter in a viral delivery system</td></tr><tr><td>&lt;strong&gt;hiPSC-derived NPCs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>WT 126 (Y2610)</td><td>Gage lab</td><td>N/A</td><td>Marchetto et al., 2010</td></tr><tr><td>CVB WT24</td><td>Yeo and Goldstein labs</td><td>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 ...

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Research

JoVE Journal

Neuroscience

9.3K Views.  University of California, San Diego. Dit protocol vergemakkelijkt het gebruik van menselijke pluripotente stamcel-afgeleide neuronen voor hoge inhoud screening toepassingen. Het is bijzonder geschikt voor het testen van synapsen, die in menselijke neuronen vereisen lange cultuurperiodes. We tonen de replating van neuronen van groot formaat gerechten in HCS-compatibele multi putten op een manier die hun levensvatbaarheid behoudt.Contra-intuïtief, tonen we aan dat de uitbreiding van protease incubatie voorafgaand aan resus...

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

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Developmental Biology

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, ...

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 ...

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

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

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

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

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Biology

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...

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 ...

Differentiating Human Induced Pluripotent Stem Cells into Neurons on Micro-Electrode Arrays

Source: Frega, M., et al., <a href="https://app.jove.com/v/54900">Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.</a> J. Vis. Exp. (2017)
The video demonstrates a method for generating excitatory cortical neurons from human-induced pluripotent stem cells (hiPSCs). Upon doxycycline induction, the hiPSCs overexpress neurogenin-2, a neural-specific transcription factor. By using specific media and co-culturing with astrocytes, this process facilitates the hiPSCs&#39; differentiation into neurons with synaptic connections.

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

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