The authors would like to thank all colleagues at Ksilink for their valuable help and discussions that lead to the design of the presented protocol.

1. Preparation of medium and plates for neuron seeding (Day 1)
<ol>
	<li>To prepare the plates for neuron seeding on Day-1, warm Laminin to room temperature (RT) just before use. Prepare the Laminin solution by diluting the Laminin stock solution (0.1 mg/mL) 1/10 in cold PBS+/+ (with Ca2+ and Mg2+). 
	NOTE: All reagents are listed in the Table of Materials. The compositions of solutions and buffers are described in Tables 1-4.</li>
	<li>Then, add 25 &#181;L of the Laminin solution to each well of a Poly-D-Lysine (PDL) precoated 384-well plate, and incubate overnight at 4 &#176;C. The coated plates can be stored at 4 &#176;C for up to one week. 
	NOTE: The protocol can be paused here for up to one week. Seal plates using plastic film.</li>
	<li>Prepare Complete Maintenance Media and store at 4 &#176;C for up to one month (Table 1).</li>
</ol>
2. Thawing of neurons (Day 0)
<ol>
	<li>To thaw the neurons on Day 0, prewarm a water bath to 37 &#176;C, and equilibrate the Complete Maintenance Media to RT, protected from light.</li>
	<li>Take the vial containing the commercially obtained frozen neurons (see Table of Materials) out of the liquid nitrogen tank and place it on dry ice. Then place the vial in the water bath for 2 min. Once the liquid is completely thawed, disinfect the vial with 70% ethanol.</li>
	<li>Aspirate the thawed neurons with a P1000 pipette (around 370 &#181;L) and transfer them in a 50 mL centrifuge tube without up-and-down pipetting. Next, rinse the vial with 630 &#181;L of Complete Maintenance Medium, dispense drop by drop (45&#176; angle) in the 50 mL tube. Agitate slowly while dispensing. 
	NOTE: It is critical to dispense the medium slowly, drop by drop, to avoid osmotic rupture of the cells. A 45&#176; angle and light agitation minimizes high local osmotic pressure during pipetting.</li>
	<li>Similarly, add 1 mL of Complete Maintenance Medium in the 50 mL centrifuge tube with a P1000 pipette. Then, slowly add 2 mL of Complete Maintenance Medium. Agitate carefully while dispensing.</li>
	<li>Count the cells. Prepare a microtube with 10 &#181;L Trypan Blue and 10 &#181;L cell suspension and add to a counting chamber slide (10 &#181;L). Perform counting using an automated cell counter (see Table of Materials) or manually.</li>
	<li>After counting, centrifuge the 50 mL tube containing the neurons at 400 x g for 5 min at RT, and remove the supernatant. Carefully resuspend the pellet using a P1000 pipette and 1 mL of Complete Maintenance Medium. Then add the necessary volume to reach the desired concentration (300,000 cells/mL, see also next step).</li>
</ol>
3. Seeding of neurons on prepared plates (Day 0)
<ol>
	<li>To seed neurons on the prepared plates on Day 0, take the coated plates out of the refrigerator, place them under the cell culture hood and let them equilibrate to RT for about 30 min.</li>
	<li>Just before seeding, aspirate 15 &#181;L coating solution with an automated liquid handler or a 16-channel pipette. Leave approximately 10 &#181;L per well to prevent damage to the coating.</li>
	<li>Next, dispense 50 &#181;L of the cell solution containing 300,000 cells/mL (prepared in step 2.6) per well with a 16-channel pipette, resulting in 15,000 seeded neurons per well and a final volume per well of 60 &#181;L.</li>
	<li>In a 384-well plate, avoid using columns 1, 2, 23, 24, and rows A, B, O, and P to minimize possible edge effects which can impact the phenotypic profiles. Fill the unused empty wells with 80 &#181;L of PBS. Incubate the plates at 37 &#176;C and 5% CO2.</li>
</ol>
4. Medium change or compound treatment (Day 3)
<ol>
	<li>Depending on the number wells, pre-warm an appropriate volume of Complete Maintenance Medium at RT. Protect from light.</li>
	<li>If a medium change is required, proceed to step 4.4. If compound treatment is desired, verify the compound stock concentration and the used solvent (water, DMSO, methanol, etc.).</li>
	<li>Prepare a 1.5x concentrated compound solution for all desired concentrations to be tested. Prepare the compound dilutions using Complete Maintenance Medium. 
	NOTE: As a neutral control, use the respective solvent at the same concentration as the tested compound. If multiple or unknown compounds at different concentrations are used, it is advisable to perform a separate dose-response experiment to measure solvent effects on the phenotypic profile.</li>
	<li>If an automatic pipetting system is used, add 60 &#181;L of the 1.5x compound solution to a 384-well storage plate. Alternatively, use a 16-channel pipette. If a medium change is required, add 60 &#181;L of Complete Maintenance Medium instead.</li>
	<li>Using the automatic pipetting system, aspirate and discard 40 &#181;L of medium per well from the neuron-containing plate to keep 20 &#181;L/well. Then add 40 &#181;L/well of 1.5x compound solution from the 384-well storage plate to each well to obtain the desired final concentration. 
	NOTE: It is critical not to perform full medium changes but to always leave residual medium in the well to prevent damage to the neuronal carpet or the coating.</li>
	<li>In case neuronal culture is performed for more than the 6 days described in this protocol, change the medium every 2-3 days.</li>
</ol>
5. Neuron fixation and staining (Days 6 to 7)
<ol>
	<li>To fix and stain the neurons on Days 6 and 7, make use of an automated liquid handler for all dispensing and washing steps. Alternatively, use a 16-channel pipette.</li>
	<li>Prepare a 10% Triton X-100 solution by diluting Triton X-100 in 1x PBS. Vortex until the solution is homogeneous. Store at 4 &#176;C.</li>
	<li>To fix the neurons, dispense 20 &#181;L/well of 16% PFA resulting in a 4% final concentration. Incubate the plate for 30 min at RT, and wash it three times with 1x PBS. Leave 20 &#181;L/well of PBS after the last wash. 
	CAUTION: PFA is recognized as a hazardous substance known to cause oral, dermal, and respiratory toxicity. It also poses a threat to the eyes and may lead to genetic mutations and cancer. Proper handling of PFA requires the use of suitable personal protective equipment, such as eye and face protection, in addition to ensuring proper ventilation. It is important to prevent the release of PFA into the environment.</li>
	<li>For permeabilization and blocking, prepare a 2x blocking solution (Table 2).</li>
	<li>Add 20 &#181;L/well of 2x blocking solution (1x final concentration), incubate for 1 h at RT, and wash once with PBS. Keep 20 &#181;L/well of PBS after washing.</li>
	<li>For primary antibody (see Table of Materials) staining, prepare a 2x primary staining buffer (Table 3).</li>
	<li>Add 20 &#181;L/well of 2x primary staining buffer (1x final concentration) and incubate overnight at 4 &#176;C. The next morning on Day 7, wash three times with PBS. Leave 20 &#181;L/well of PBS after washing.</li>
	<li>For secondary antibody (see Table of Materials) staining, prepare a 2x secondary staining buffer (Table 4).</li>
	<li>Add 20 &#181;L/well of 2x secondary staining buffer (1x final concentration), incubate for 2 h at RT away from light, and wash three times with PBS. Leave 100 &#181;L PBS/well after the last wash.
	<ol>
		<li>Add aluminum sealing on the plate to minimize evaporation. Alternatively, cover the plate using plastic film and aluminum foil. Proceed to image acquisition, or store the plate at 4 &#176;C. 
		&#8203;NOTE: The protocol can be paused here for up to one week. If background fluorescence is observed, consider increasing the blocking time. If the staining is insufficient, try increasing the primary antibody concentration or incubation time.</li>
	</ol>
	</li>
</ol>
6. Imaging of fluorescently stained neurons (Day 7)
<ol>
	<li>Acquire images of the plated, cultured, and stained neurons on Day 7. Ideally, use an automated confocal fluorescence microscope (see Table of Materials). Alternatively, acquire images manually.</li>
	<li>Acquire the Hoechst, TH, &#945;-synuclein, and MAP2 channels using 405 nm, 488 nm, 561 nm, and 647 nm lasers, respectively (Figure 2). 
	NOTE: To generate sufficient amount of detailed data for phenotypic profiling, use a 40x objective and acquire 16 fields/well using Z-stacks consisting of 3 Z-slices separated by 2 &#181;m. In case there are pipetting-related damages in the neuronal carpet, try to avoid imaging these areas.</li>
	<li>Depending on the microscope and camera, adjust the exposure times and excitation intensities for each of the four fluorescent channels separately to obtain an optimal dynamic range of the fluorescent intensities. 
	&#8203;NOTE: Imaging software often provides a histogram to determine the ideal exposure time. If the histogram is shifted too much to the left in the low signal range, then the exposure time is too short or the excitation intensity is too low. If there is a sharp cliff at the maximum signal level on the right, then the signal value is saturated. In this case, reduce the excitation intensity or shorten the exposure time.</li>
	<li>Store images in a loss-free and open format such as .tif.</li>
</ol>
7. Image processing (Day 8)
<ol>
	<li>Image segmentation and phenotypic feature extraction are required for the creation of quantitative phenotypic profiles. Use the PhenoLink software for image segmentation and feature extraction (Table of Materials). Instructions for installing PhenoLink can be found in the GitHub repository (https://github.com/Ksilink/PhenoLink). 
	NOTE: Image segmentation is used to identify and separate different objects or regions in an image, while phenotypic feature extraction is used to analyze and extract relevant information from those regions. Several alternative software solutions, such as CellProfiler10, ImageJ/FIJI11, Napari12, or the Knime Analytics Platform13 are available to extract quantitative information from multichannel fluorescence images.</li>
	<li>Perform image segmentation on illumination-corrected raw images. Determine the respective fluorescent channel intensity thresholds empirically per plate so that the background signal is minimal, and the desired segmented signal corresponds to the signal in the raw image. Plates processed and stained on the same day typically require comparable channel intensity thresholds for segmentation.</li>
	<li>Define the nucleus size and intensity to separate living from dead cells. When using 40x images, keep all other default parameters and run the software. One hundred twenty-six quantitative image features will be calculated per well (Supplementary Table 1).</li>
	<li>Use the resulting tabular quantitative data to construct phenotypic profiles and compare phenotypic profiles from different cell lines or treatment conditions. Each row corresponds to a biological condition (well) and each column corresponds to a determined phenotypic feature. 
	&#8203;NOTE: We provide an example output file together with the data analysis pipeline to illustrate its use (see Table of Materials). Additionally, Figure 3 shows the composition of a phenotypic profile.</li>
</ol>
8. Phenotypic profile generation and visualization (Day 8)
<ol>
	<li>If you do not have Python and Jupyter installed on your computer, install the Anaconda Distribution and open the Jupyter software. Download the provided Jupyter notebook and all other provided files and save them in the same directory (see Table of Materials). Open the Jupyter notebook file using the Jupyter software. 
	NOTE: Anaconda is a free and open-source platform for programming languages such as Python. This platform comes with the Python interpreter Jupyter which can execute the provided Jupyter notebook to create and analyze phenotypic profiles (https://github.com/Ksilink/Notebooks/tree/main/Neuro/DopaNeuronProfiling).</li>
	<li>Execute the Jupyter notebook cell by cell using the Jupyter software. The provided sample data .fth and requirements .txt files need to be located in the same directory as the Jupyter notebook. Each Jupyter notebook cell is annotated to explain its functionality. 
	NOTE: It is crucial to use the Jupyter notebook in the correct order starting with data loading and scaling and to proceed cell by cell to the end. All data and graphics output will be stored in a newly created folder in the Jupyter notebook source directory. Figure 4 depicts the workflow and the workflow output.</li>
</ol>

Culturing Human Midbrain Dopaminergic Neurons for Phenotypic Profiling

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All authors are employed by Ksilink.

Phenotypic profiling is a technique to measure a large number of phenotypes in cells by applying fluorescent stainings, microscopy, and image analysis3. Phenotypic profiles can be obtained and compared across cell lines or other experimental conditions to understand complex changes in cellular biology that might go unnoticed when using a single readout. Here we describe the application of phenotypic profiling to human iPSC-derived mDA neurons, a cell type frequently used to model PD cellular biolo...

A variety of cell biological processes are disturbed in Parkinson&#39;s disease (PD). For example, mitochondrial dysfunction, oxidative stress, protein degradation defects, disruption of vesicular trafficking and endolysosomal function have been associated with midbrain dopaminergic (mDA) neuron loss, are commonly observed in PD1. Therefore, PD appears to involve multiple disease mechanisms that can interact with and worsen each other. One useful way to investigate this mechanistic interplay is the creation of a comprehensive phenotypic fingerprint or profile of midbrain dopaminergic (mDA) neurons.
Phenotypic profiling is an approach that involves creating a profile of a sample based on a collection of measurable characteristics, and second, it involves making predictions about a sample based on this profile2,3. The goal of profiling is to capture a diverse range of features, some of which may not have been previously associated with a disease or treatment3. As a result, profiling can reveal unexpected biological processes. Phenotypic profiling typically relies on fluorescently stained cells, and standardized assays, such as Cell Painting, have been developed to create phenotypic profiles4. Recently, phenotypic profiling has, for example, been applied for the characterization of small molecules or the accurate prediction of PD-subtypes solely based on patient-derived fibroblasts5,6. Despite these advances, phenotypic profiling has rarely been applied to post-mitotic differentiated cells, such as human induced pluripotent stem cell (iPSC)-derived mDA neurons that express PD-linked mutations such as LRRK2 G2019S. Significant challenges of iPSC-derived models include the presence of subtle or variable pathological features across differentiation batches or genotypes, and the fact that isolated PD phenotypes do not capture the full complexity of the disease. Furthermore, while iPSC neuronal models are physiologically relevant, they are rarely used in PD drug discovery processes due to concerns about technical complexity7,8.
We previously developed a robust methodology to measure multiple PD-related pathophysiological phenotypes in human mDA neurons that are both sensitive to genetic and chemical compound-induced phenotypic changes9. This article describes in detail a further optimized version of this methodology to create phenotypic profiles from mDA neurons (Figure 1). This protocol has several advantages over the previously described phenotypic profiling approaches, such as the use of high-quality mDA neurons and technical reproducibility. For the first time, this protocol enables phenotypic profiling in physiologically relevant post-mitotic mDA neurons after chemical perturbations in a highly scalable fashion. Fully differentiated and cryopreserved mDA neurons are commercially available, significantly decreasing batch-to-batch differentiation variability. Secondly, technical variability can be further reduced by using a well-defined experimental design (i.e., culture duration or avoiding edge wells), automated liquid handling and automated microscopy. Additionally, the initial steps of phenotypic profile analysis using unsupervised clustering or supervised classification approaches are outlined here, indicating how phenotypic profiling data can be analyzed. This protocol will be of use for researchers interested in phenotypic changes of mDA neurons induced by genetic or chemical perturbations, specifically when a highly scalable study setup is required, for example, during screening campaigns or when the effects of a smaller number of compounds are to be studied, for example, to determine toxic effects. In summary, it is anticipated that the application of phenotypic profiling of human neurons is a valuable technique to study complex disease-related phenotypes and characterize the cellular effects of drug candidates.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65570/65570fig01.jpg" /> 
Figure 1: Schematic depiction of the experimental protocol to generate image-based phenotypic profiles from human iPSC-derived mDA neurons. <a href="https://www.jove.com/files/ftp_upload/65570/65570fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Anti- chicken &amp;ndash; Alexa 647</td><td>Jackson ImmunoRearch</td><td>703-605-155</td><td>Immunofluorescence</td></tr><tr><td>Anaconda</td><td></td><td>https://www.anaconda.com/download</td><td></td></tr><tr><td>Anti-Map2</td><td>Novus</td><td>NB300-213</td><td>Immunofluorescence</td></tr><tr><td>Anti-mouse - Alexa 488</td><td>Thermo Fisher</td><td>A11001</td><td>Immunofluorescence</td></tr><tr><td>Anti-rabbit - Alexa 555</td><td>Thermo Fisher</td><td>A21429</td><td>Immunofluorescence</td></tr><tr><td>Anti-Tyrosine Hydroxylase</td><td>Merck</td><td>T2928</td><td>Immunofluorescence</td></tr><tr><td>Anti-&amp;alpha;-synuclein</td><td>Abcam</td><td>138501</td><td>Immunofluorescence</td></tr><tr><td>Bravo Automated Liquid Handling Platform with 384ST head</td><td>Agilent</td><td></td><td>If no liquid handler is available, the use of an electronic multichannel pipette is recommended.</td></tr><tr><td>Confocal microscope&amp;nbsp;</td><td>Yokogawa</td><td>CV7000</td><td>The use of an automated confocal fluorescence microscope is recommended to ensure image quality consistency.</td></tr><tr><td>Countess Automated cell counter</td><td>Invitrogen</td><td></td><td>Cell counting before seeding. Can also be done using a manual counting chamber.</td></tr><tr><td>DPBS +/+</td><td>Gibco</td><td>14040-133</td><td>Buffer for washing</td></tr><tr><td>EL406 Washer Dispenser&amp;nbsp;</td><td>BioTek (Agilent)&amp;nbsp;</td><td></td><td>If no liquid handler is available, the use of an electronic multichannel pipette is recommended.</td></tr><tr><td>Formaldehyde Solution (PFA 16 %)</td><td>Euromedex</td><td>EM-15710-S</td><td>Fixation before staining</td></tr><tr><td>Hoechst 33342</td><td>Invitrogen</td><td>H3570</td><td>Nuclear staining</td></tr><tr><td>iCell Base Medium 1</td><td>Fujifilm</td><td>M1010</td><td>Base medium for neurons</td></tr><tr><td>iCell DPN, Donor#01279, Phenotype AHN, lot#106339, 1M</td><td>Fujifilm</td><td>C1087</td><td>Apparently healthy donor</td></tr><tr><td>iCell DPN, Donor#11299, Phenotype LRRK2 G2019S, phenotype PD lot#106139</td><td>Fujifilm</td><td>C1149</td><td>Donor carrying LRRK2 G2019S mutation&amp;nbsp;</td></tr><tr><td>iCell Nervous System Supplement</td><td>Fujifilm</td><td>M1031</td><td>Supplement for base medium</td></tr><tr><td>iCell Neural Supplement B</td><td>Fujifilm</td><td>M1029</td><td>Supplement for base medium</td></tr><tr><td>Jupyter Python Notebook</td><td>In-house development</td><td>https://github.com/Ksilink/Notebooks/tree/main/Neuro/DopaNeuronProfiling</td><td>Notebook to perform phenotypic profile visualization and classification from raw data.</td></tr><tr><td>Laminin</td><td>Biolamina</td><td>LN521</td><td>Plate coating</td></tr><tr><td>PFE-360</td><td>MedChemExpress</td><td>HY-120085</td><td>LRRK2 kinase inhibitor</td></tr><tr><td>PhenoLink</td><td>In-house development</td><td>https://github.com/Ksilink/PhenoLink</td><td>Software for image analysis</td></tr><tr><td>PhenoPlate 384w, PDL coated</td><td>Perkin Elmer</td><td>6057500</td><td>Pre-coated plate for cell culture and imaging. This plate allows imaging of all wells using all objectives of the Yokogawa CV7000 microscope.</td></tr><tr><td>Storage plates Abgene 120 &amp;micro;L</td><td>Thermo Scientific</td><td>AB-0781</td><td>Necessary for compound dispensing using the Vprep pipetting system. If not available, the use of an electronic multichannel pipette is recommended.</td></tr><tr><td>Triton</td><td>Sigma</td><td>T9284</td><td>Permeabilization before lysis</td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154-20ML</td><td>Determination of living cells</td></tr><tr><td>Vprep Pipetting System&amp;nbsp;</td><td>Agilent</td><td></td><td>Medium change and compound dispensing. Alternatively, an electronic multichannel pipette can be used.</td></tr></tbody></table>

phenotypic profiling of human stem cell-derived midbrain dopaminergic neurons

Parkinson&#39;s disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro PD cellular models lack complexity and do not take multiple phenotypes into account. Phenotypic profiling in human induced pluripotent stem cell (iPSC)-derived mDA neurons can address these shortcomings by simultaneously measuring a range of neuronal phenotypes in a PD-relevant cell type in parallel. Here, we describe a protocol to obtain and analyze phenotypic profiles from commercially available human mDA neurons. A neuron-specific fluorescent staining panel is used to visualize the nuclear, &#945;-synuclein, Tyrosine hydroxylase (TH), and Microtubule-associated protein 2 (MAP2) related phenotypes. The described phenotypic profiling protocol is scalable as it uses 384-well plates, automatic liquid handling and high-throughput microscopy. The utility of the protocol is exemplified using healthy donor mDA neurons and mDA neurons carrying the PD-linked G2019S mutation in the Leucine-rich repeat kinase 2 (LRRK2) gene. Both cell lines were treated with the LRRK2 kinase inhibitor PFE-360 and phenotypic changes were measured. Additionally, we demonstrate how multidimensional phenotypic profiles can be analyzed using clustering or machine learning-driven supervised classification methods. The described protocol will particularly interest researchers working on neuronal disease modeling or studying chemical compound effects in human neurons.

Phenotypic profiling in mDA neurons is an efficient way to quantify multiple aspects of cellular biology and their changes during the experimental modulation. To exemplify this methodology, this study made use of cryopreserved LRRK2 G2019S and healthy donor mDA neurons. These neurons have been differentiated for approximately 37 days, are post-mitotic and express neuronal markers (TUBB3 and MAP2) and dopaminergic neuron markers, including tyrosine hydroxylase (TH) in combination with FOXA2, while the glial marker Glial F...

Watch this Scientific Journal Video about Generating Neuronal Phenotypic Profiles - A Protocol to Culture and Image Human Midbrain Dopaminergic Neurons at JoVE.com

Author Spotlight: Generating Neuronal Phenotypic Profiles - A Protocol to Culture and Image Human Midbrain Dopaminergic Neurons 

This protocol describes the cell culturing of human midbrain dopaminergic neurons, followed by immunological staining and the generation of neuronal phenotypic profiles from acquired microscopic high-content images allowing the identification of phenotypic variations due to genetic or chemical modulations.

Phenotypic Profiling of Human Stem Cell-Derived Midbrain Dopaminergic Neurons

Parkinson&#39;s disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro ...

phenotypic-profiling-human-stem-cell-derived-midbrain-dopaminergic

Research

JoVE Journal

Neuroscience

1.4K Views.  Ksilink. We construeren fenotypische profielen van gedifferentieerde menselijke cellen zoals neuronen. Ons doel is om de algehele effecten van behandelingen met chemische verbindingen op de cellen beter te begrijpen. Fenotypische profielen kunnen minder bevooroordeelde ingangspunten suggereren voor verdere mechanistische studies.De kwaliteit van protocollen voor het differentiëren van menselijke dopaminerge neuronen is aanzienlijk verbeterd, waardoor de productie van grote batches homogene neu...

Video: Auteur Spotlight: Het genereren van neuronale fenotypische profielen - een protocol voor het kweken en afbeelden van dopaminerge neuronen van de menselijke middenhersenen 

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 ...

Developmental Biology

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

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

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

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

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

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, ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate.
The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain&#39;s inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain.
The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson&#39;s disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.

This protocol describes a novel method for creating 3D midbrain organoids from human induced pluripotent stem cells, guiding their formation to mimic native midbrain tissue, thereby aiding in the study of development and disorders.

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain ...

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

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

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

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

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 ...

Phenotypic Profiling of Human Stem Cell-Derived Midbrain Dopaminergic Neurons

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