We thank Miriam B&#252;nning for technical support. We acknowledge support from the Deutsche Forschungsgemeinschaft (DFG) (PR1527/5-1 to A.P.), Spark and Berlin Institute of Health (BIH) (BIH Validation Funds to A.P.), the United Mitochondrial Disease Foundation (UMDF) (Leigh Syndrome International Consortium Grant to A.P.), University Hospital Duesseldorf (Forschungskommission UKD to A.P.), and the German Federal Ministry of Education and Research (BMBF) (e:Bio young investigator grant AZ 031L0211 to A.P.).Work in the laboratory of C.R.R. was supported by the DFG (FOR 2795 &#34;Synapses under stress&#34;, Ro 2327/13-1).

NOTE: The use of human iPSCs may require an ethical approval. iPSCs used in this study were derived from healthy control individuals following local ethical approval (#2019-681). All cell culture procedures must be performed under a sterile cell culture hood, carefully disinfecting all reagents and consumables before transferring under the hood. Human iPSCs used for differentiation should have a passage number below 50 to avoid potential genomic aberrations that may occur upon extensive culture. The pluripotent state of the cells should be validated before organoid generation, for example, by monitoring the expression of pluripotency-associated markers such as NANOG or OCT4. Mycoplasma tests should be conducted weekly to ensure mycoplasma-free cultures.
1. Generation of brain organoids
<ol>
	<li>Culture of human iPSCs
	<ol>
		<li>Culture human iPSCs under feeder-free conditions in iPSC medium (see the Table of Materials) on coated 6-well plates and keep them in a humidified tissue culture incubator at 37 &#176;C and 5% CO2. 
		&#8203;NOTE: The carryover of feeder cells may hamper the organoid differentiation. Passage the cells at least once in feeder-free conditions.</li>
		<li>Passage the iPSCs at 80% confluency using enzyme-free detachment medium in ratios ranging from 1:4 to 1:12. To increase cell survival, add 10 &#181;M Rho-associated protein kinase (ROCK) inhibitor (Y27632) after each splitting.</li>
	</ol>
	</li>
	<li>Dissociate the iPSCs (80% confluency)-Day 0.
	<ol>
		<li>Prepare Cortical Differentiation Medium I (CDMI) (Table 1). Prewarm CDMI medium at room temperature (22-25 &#176;C) before adding it to the cells.</li>
		<li>Wash the wells containing the iPSCs with phosphate-buffered saline (PBS) to remove dead cells and debris.</li>
		<li>Add 500 &#181;L of prewarmed Reagent A (Table of Materials) to each well and incubate for 5 min at 37 &#176;C. Check under the microscope to ensure cell detachment.</li>
		<li>Add 1 mL of iPSC medium to dilute reagent A to neutralize its activity.</li>
		<li>Use a 1000 &#181;L pipette to dissociate the cells by pipetting up and down and transfer the cell suspension to a 15 mL centrifuge tube.</li>
		<li>Gently centrifuge the iPSCs at 125 x g for 5 min at room temperature (22-25 &#176;C).</li>
		<li>Carefully aspirate the supernatant to avoid disturbing the cell pellet.</li>
		<li>Resuspend the pellet with 1 mL of CDMI to obtain a single-cell suspension, and count the cell number.</li>
		<li>Prepare the seeding medium with 9,000 iPSCs per 100 &#181;L in CDMI supplemented with 20 &#181;M ROCK inhibitor, 3 &#181;M WNT-catenin inhibitor (IWR1), and 5 &#181;M SB431542.</li>
		<li>Add 100 &#181;L of seeding medium per well to a 96-well v-bottom plate.</li>
		<li>Keep the plate in a humidified tissue culture incubator at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Neurosphere generation
	<ol>
		<li>On day 1, observe that round cell aggregates (neurospheres) with defined smooth borders are forming. Note the dead cells around the aggregates. Continue to culture in the incubator at 37 &#176;C and 5% CO2.</li>
		<li>On day 3, agitate the plate by tapping on the sides three times to detach dead cells.</li>
		<li>Add 100 &#181;L of CDMI supplemented with 20 &#181;M ROCK inhibitor, 3 &#181;M IWR1, and 5 &#181;M SB431542 to each well.</li>
		<li>Return the plate to the incubator at 37 &#176;C and 5% CO2.</li>
		<li>On day 6, carefully remove 80 &#181;L of the supernatant medium from each well. Avoid touching the bottom of the well.</li>
		<li>Add 100 &#181;L of CDMI supplemented with 3 &#181;M IWR1 and 5 &#181;M SB431542 to each well. Return the plate to the incubator at 37 &#176;C and 5% CO2.</li>
		<li>Repeat steps 5 and 6 every 3 days until day 18.</li>
	</ol>
	</li>
	<li>Transfer of neurospheres
	<ol>
		<li>On day 18, prepare Cortical Differentiation Medium II (CDMII) (Table 1) and add 10 mL to a 100 mm ultra-low attachment cell culture plate.</li>
		<li>Use a 200 &#181;L pipette with the tip cut off to transfer the round neurospheres from the 96-well plate to the 100 mm ultra-low attachment cell culture plate. 
		NOTE: Be gentle to avoid damaging the neurospheres by making sure the opening of the tip is wide enough and that the aggregates are not aspirated too quickly.</li>
		<li>Remove 5 mL of medium from the plate containing the neurospheres and add 5 mL of fresh CDMII. 
		NOTE: This procedure helps to reduce the amount of CDMI medium that may have carried over from the transfer of neurospheres.</li>
		<li>Place the plate on an orbital shaker at 70 rpm inside a humidified tissue culture incubator at 37 &#176;C and 5% CO2. 
		NOTE: Visually inspect the neurospheres the next day. Increase the speed of the orbital shaker if the neurospheres are clumped together or attached to the bottom of the plate.</li>
		<li>Every 3 days, carefully aspirate the supernatant medium and replace it with fresh CDMII. Leave a small amount of the medium to prevent the neurospheres from drying out.</li>
		<li>On day 35, prepare Cortical Differentiation Medium III (CDMIII) (Table 1). 
		NOTE: The matrix component should be dissolved in cold CDMIII.</li>
		<li>Aspirate the medium from the plate and add 10 mL of cold CDMIII. 
		NOTE: It is more effective to use cold medium so that the matrix component can coat the organoids without forming clumps.</li>
		<li>After changing the medium, place the plate back on an orbital shaker at 70 rpm inside a humidified tissue culture incubator at 37 &#176;C and 5% CO2.</li>
		<li>Change the medium every 3-5 days depending on the rate of growth, as indicated by the color of the medium.</li>
		<li>On day 70, prepare Cortical Differentiation Medium IV (CDMIV) (Table 1). Use CDMIV medium until the desired age of organoids is reached. During this period, keep the plate on an orbital shaker set at 70 rpm inside a humidified tissue culture incubator (37 &#176;C and 5% CO2).</li>
		<li>Change the medium every 3-5 days, depending on the growth rate.</li>
	</ol>
	</li>
</ol>
2. Immunostaining of brain organoids
<ol>
	<li>Tissue preparation
	<ol>
		<li>Prepare 4% paraformaldehyde (PFA) solution, and place it under a safety hood. 
		NOTE: Wear personal safety equipment when handling PFA.</li>
		<li>Collect brain organoids and gently transfer them with a blunt-tipped 3 mL plastic Pasteur pipette to a 6-well plate filled with PFA. 
		NOTE: Use organoids older than 40 days to allow the visualization of structures with higher cellular complexity.</li>
		<li>Keep the organoids in the PFA solution for 1 h at room temperature.</li>
		<li>Carefully remove the PFA with a 3 mL plastic Pasteur pipette, and wash the fixed organoids three times using PBS.</li>
		<li>Store the fixed organoids at 4 &#176;C in PBS until further use.</li>
	</ol>
	</li>
	<li>Preparation of brain organoid slices
	<ol>
		<li>Prepare a 3% agar solution and heat slowly until liquefied.</li>
		<li>Place the mold (the cut end of a 10 mL syringe) on a piece of absorbent filter paper (smooth side up). Place a droplet of agar onto it.</li>
		<li>Quickly take a single organoid out of the 6-well plate with a spatula and remove excessive PBS with filter paper. 
		NOTE: Be careful not to touch the organoid directly with filter paper.</li>
		<li>Place the organoid onto the agar droplet.</li>
		<li>Repeat this procedure with up to three organoids. 
		NOTE: Work fast to avoid solidification of the agar during this step.</li>
		<li>Refill the mold with agar until all the organoids are fully covered.</li>
		<li>Wait until the agar begins to solidify, and then gently transfer the entire mold containing the organoids, including the absorbent filter paper, onto a cooling element. 
		NOTE: If a cooling element is unavailable, store the organoids for a few minutes in a refrigerator at 4 &#176;C.</li>
		<li>In the meantime, prepare for the slicing procedure: place a razor blade (cleaned with acetone and washed with double-distilled water) into the holder of the vibratome, mount on the bath, and fill it with PBS.</li>
		<li>Remove the mold from (solidified) agar and use a scalpel to trim it to form a cube.</li>
		<li>Attach the agar cube containing the organoids on the carrier plate of the vibratome with adhesive gel (see the Table of Materials), and place it in the bath containing PBS.</li>
		<li>Adjust the vibratome (see the Table of Materials) to cut slices at a thickness of 150 &#181;m. 
		NOTE: Vibratome settings (proper angle, amplitude, frequency, and velocity of the blade) may be similar to those used for slicing fixed brain tissue derived from early postnatal animals. However, the ideal settings depend strongly on the type of the vibratome and must be determined in a first step to prevent distortion or even ripping of the tissue while cutting.</li>
		<li>Start the cutting procedure. Use a glass pipette or a spatula to gently transfer each freshly cut slice into a 24-well plate filled with PBS.</li>
		<li>Store the plate containing slices at 4 &#176;C (for up to a few days) until further processing.</li>
		<li>Transfer the slices out of the plate with a glass pipette or a spatula onto microscope slides. Use a minimum of 2 slices per slide.</li>
		<li>Carefully remove the agar and excess PBS with a syringe.</li>
		<li>Allow the slices to dry until they adhere to the slides. 
		NOTE: While microscope slides can be stored in plastic chambers filled with PBS at 4 &#176;C, they should be stained as soon as possible after the slicing procedure.</li>
	</ol>
	</li>
	<li>Immunohistochemical staining
	<ol>
		<li>Prepare the blocking solution (Table 1).</li>
		<li>Use a PAP pen to draw a hydrophobic border around the slices on the slide to help keep all the solutions on the slides.</li>
		<li>Carefully add the blocking solution on the slide, and incubate for 1 h at room temperature (22-25 &#176;C). To avoid destroying the tissue, do not add the solution directly on top of the slices.</li>
		<li>Aspirate the blocking solution and apply the desired primary antibody diluted in the blocking solution.</li>
		<li>Incubate the slide overnight in a humidified chamber at 4 &#176;C.</li>
		<li>Rinse the slide three times with 1x PBS for 10 min each.</li>
		<li>Incubate the slices with the specific secondary antibody diluted in the blocking solution and perform Hoechst staining (1:2,500) for 1 h at room temperature in the dark. 
		NOTE: Remember to perform negative controls to confirm there are no non-specific binding or auto-fluorescence.</li>
		<li>Rinse three times with 1x PBS for 10 min each in the dark.</li>
		<li>Add one drop of mounting medium to the slice, place a coverslip on the edge of the drop, and slowly lay the coverslip down toward the slice to avoid air bubbles.</li>
		<li>Allow the slide to rest overnight at room temperature. Apply nail polish on the border of the coverslip to further seal the slide. For long-term storage, store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Documentation of staining
	<ol>
		<li>To scan large images for stitching, utilize a motorized upright wide-field microscope equipped with (see the Table of Materials for details) a high-quality objective; DAPI filter set (e.g., excitation (EX): 340-380 nm, dichroic mirror (DM): 400 nm, barrier filter (BA): 435-485 nm); fluorescein isothiocyanate filter set (e.g., EX: 465-495 nm, DM: 505 nm, BA: 515-555 nm); Alexa594 filter set (e.g., ET 575/40; T 600 LPXR; HC 623/24); digital camera; high-performance acquisition software allowing for automated stitches and stack operations.</li>
		<li>For image handling, use an image processing program capable of generating 8-bit tif-files, cropping stitches, adjusting contrast and brightness, merging the channels (e.g., blue, green, and red), and adding scale bars.</li>
		<li>To scan details, use a motorized confocal laser scanning microscope equipped with a high-quality objective, a UV laser (EX: 408 nm), an Argon laser (EX: 488 nm), a Helium-Neon laser (EX: 543), imaging software for a confocal microscope.</li>
		<li>For image handling of details, use an image processing program capable of generating maximum-intensity projections of confocal z-stacks (e.g., optical sections of 0.6 &#181;m each), generating 8-bit tif-files, adjusting contrast and brightness, merging the channels (e.g., blue, green, and red), adding scale bars.</li>
		<li>Use a graphic editor to arrange the figures.</li>
	</ol>
	</li>
</ol>
3. Bioenergetic profiling of brain organoids
<ol>
	<li>Preparation of organoids for bioenergetic profiling
	<ol>
		<li>Prepare the papain and DNase solution following the manufacturer&#39;s protocol.</li>
		<li>Transfer 3-5 organoids into a 6-well plate. Wash them two times with prewarmed PBS.</li>
		<li>Add 2 mL of prewarmed activated papain solution containing DNase. Using a blade, cut the organoids into small pieces.</li>
		<li>Place the plate onto an orbital shaker set at 27 rpm inside a cell culture incubator (at 37 &#176;C, 5% CO2), and incubate for 15-20 min. 
		NOTE: The time of incubation depends on the organoid stage. Early-stage organoids can be used as they are. For organoids older than 3 months, it is recommended to cut the organoids into 2-3 pieces before dissociation and incubate the pieces at a rocking speed set at 27 rpm for 15-20 min at 37 &#176;C. This procedure can help remove necrotic tissue that may be present in the later-stage organoids.</li>
		<li>Collect the digested tissues into a 15 mL tube and add 5 mL of organoid culture medium CDMIV (Table 1).</li>
		<li>Triturate the tissue with a 10 mL plastic pipette by pipetting up and down 10-15 times. Let the undissociated tissue settle down to the bottom of the tube.</li>
		<li>Carefully transfer the cell suspension to a 15 mL tube, avoiding any pieces of undissociated tissue. Filter the solution through a 40 &#181;m cell strainer (e.g., polystyrene round-bottom tubes with cell strainer caps).</li>
		<li>Pellet the cells by centrifuging at 300 x g for 5 min at room temperature.</li>
		<li>Assess the cell number and quality using trypan blue.</li>
		<li>Plate the desired number (~20,000/well) of cells onto coated 96-well microplates. Change the medium 6-8 h after plating to Neuronal Medium (Table 1).</li>
		<li>Incubate the coated 96-well microplate in a CO2 incubator (37 &#176;C, 5% CO2) for 4 days.</li>
	</ol>
	</li>
	<li>Bioenergetic profiling
	<ol>
		<li>On day 3 after replating the dissociated cells, add 200 &#181;L of calibration solution into each well of the bottom part of the 96-well microplate, and place the top green sensor cartridge onto the hydrated microplate. 
		NOTE: Place the sensor cartridge on top of the microplate in the correct orientation, and ensure that the calibrant solution covers all the sensors.</li>
		<li>Incubate the hydrated 96-well microplate in a non-CO2 incubator at 37 &#176;C overnight.</li>
		<li>Turn on the analyzer to allow the instrument to stabilize at 37 &#176;C overnight.</li>
		<li>On day 4 after replating, inspect the disassociated organoid culture on the 96-well microplate under the microscope to ensure that the cells appear as a confluent monolayer.</li>
		<li>Prepare Assay Medium (Table 1).</li>
		<li>Remove Neuronal Medium from all wells with a pipette without touching the bottom of the well to prevent cell damage. Alternatively, carefully invert the whole plate and then dry it on clean paper. Work quickly to avoid cell death.</li>
		<li>Wash the cells twice with prewarmed 200 &#181;L of Assay Medium. Add Assay Medium to a final volume of 180 &#181;L per well. Incubate the 96-well microplate in a non-CO2 incubator at 37 &#176;C for 1 h.</li>
		<li>Prepare 10 &#181;M solutions of mitochondrial inhibitors in Assay medium. Note that the final concentration after injection is 1 &#181;M.</li>
		<li>Load the sensor cartridge placed in the hydrated microplate with 10x solutions of the mitochondrial inhibitors.
		<ol>
			<li>Add 18 &#181;L of mitochondrial inhibitor 1 into port A.</li>
			<li>Add 19.8 &#181;L of mitochondrial inhibitor 2 into port B.</li>
			<li>Add 21.6 &#181;L of mitochondrial inhibitor 2 into port C.</li>
			<li>Add 23.4 &#181;L of mitochondrial inhibitor 3 into port D.</li>
		</ol>
		</li>
		<li>Place the loaded cartridge in the hydrated microplate in a non-CO2 incubator at 37 &#176;C until the start of the assay.</li>
		<li>Set up a running protocol in the instrument&#39;s software (Table 2).</li>
		<li>Press START. Take the loaded cartridge from the non-CO2 incubator and place it into the analyzer for calibration. 
		NOTE: Make sure the plate is inserted in the correct orientation and without the lid.</li>
		<li>Once the calibration step ends, remove the calibration plate. Take the 96-well microplate from the non-CO2 incubator and place it into the analyzer. Click on CONTINUE to start the measurements.</li>
		<li>When the run is finished, remove the 96-well cell culture microplate from the analyzer and collect the medium from all the wells without disturbing the cells. 
		NOTE: The medium can be stored at -20 &#176;C and used later for measuring the amount of lactate released by the cells in the medium using an appropriate lactate assay kit.</li>
		<li>Wash the cells with 200 &#181;L of 1x PBS in each well.</li>
		<li>After removing the PBS, freeze the plate at -20 &#176;C. 
		NOTE: The frozen plate can be used to quantify cells, proteins, or DNA in each well of the microplate. This quantification will be needed for normalizing the obtained bioenergetic rates. Follow the manufacturer&#180;s instructions for cell, protein, or DNA quantification assays.</li>
	</ol>
	</li>
</ol>

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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPSC-derived+cerebral+organoids+model+features+of+Leigh+Syndrome+and+reveal+abnormal+corticogenesis.">Human iPSC-derived cerebral organoids model features of Leigh Syndrome and reveal abnormal corticogenesis.</a> bioRxiv. , (2020).</li><li>Inak, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+metabolic+programming+impairs+early+neuronal+morphogenesis+in+neural+cultures+and+an+organoid+model+of+Leigh+syndrome.">Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome.</a> Nature Communications. 12 (1), 1929 (2021).</li><li>Falk, M. 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C., Meerlo, M., Burgering, B. M. T., Colman, R. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+to+profile+the+bioenergetics+of+organoids+using+Seahorse.">Protocol to profile the bioenergetics of organoids using Seahorse.</a> STAR Protocols. 2 (1), 100386 (2021).</li><li>Menacho, C., Prigione, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tackling+mitochondrial+diversity+in+brain+function:+from+animal+models+to+human+brain+organoids.">Tackling mitochondrial diversity in brain function: from animal models to human brain organoids.</a> International Journal of Biochemestry &amp; Cell Biology. 123, 105760 (2020).</li><li>Del Dosso, A., Urenda, J. P., Nguyen, T., Quadrato, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upgrading+the+physiological+relevance+of+human+brain+organoids.">Upgrading the physiological relevance of human brain organoids.</a> Neuron. 107 (6), 1014-1028 (2020).</li></ol>

The authors declare no competing financial or non-financial interests.

This paper describes the reproducible generation of human iPSC-derived brain organoids and their use for mitochondrial disease modeling. The protocol described here is modified based on a previously published work20. One major advantage of the present protocol is that it does not require the manual embedding of each organoid into a scaffolding matrix. In fact, the matrix solution is simply dissolved into the cell culture medium. Moreover, there is no need to employ expensive bioreactors, as organo...

Mitochondrial diseases represent the largest class of inborn errors of metabolism1. They are caused by genetic mutations disrupting different mitochondrial processes, including oxidative phosphorylation (OXPHOS)2, respiratory chain assembly, mitochondrial dynamics, and mitochondrial DNA transcription or replication3. Tissues with energy requirements are particularly affected by mitochondrial dysfunction4. Accordingly, patients with mitochondrial diseases typically develop early-onset neurological manifestations.
There are currently no treatments available for children affected with mitochondrial diseases5. A major hindrance for drug development of mitochondrial diseases is the lack of effective models recapitulating the human disease course6. Several of the currently studied animal models do not exhibit the neurological defects present in the patients7. Hence, the mechanisms underlying the neuronal pathology of mitochondrial diseases are still not fully understood.
Recent studies generated iPSCs from patients affected by mitochondrial diseases and used these cells to obtain patient-specific neuronal cells. For example, genetic defects associated with the mitochondrial disease, Leigh syndrome, have been found to cause aberrations in cellular bioenergetics8,9, protein synthesis10, and calcium homeostasis9,11. These reports provided important mechanistic clues on the neuronal impairment occurring in mitochondrial diseases, paving the way for drug discovery for these incurable diseases12.
Two-dimensional (2D) cultures, however, do not enable the investigation of the architectural complexity and regional organization of 3D organs13. To this end, the use of 3D brain organoids derived from patient-specific iPSCs14 may allow researchers to gain additional important information and thereby help to dissect how mitochondrial diseases impact the development and function of the nervous system15. Studies employing iPSC-derived brain organoids to investigate mitochondrial diseases are beginning to uncover the neurodevelopmental components of mitochondrial diseases. 
Spinal cord organoids carrying mutations associated with the mitochondrial disease, mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes syndrome (MELAS), showed defective neurogenesis and delayed motor neuron differentiation16. Cortical organoids derived from patients with the mitochondrial disease, Leigh syndrome, showed reduced size, defects in neural epithelial bud generation, and loss of cortical architecture17. Brain organoids from Leigh syndrome patients showed that the disease defects initiate at the level of neural progenitor cells, which cannot commit to mitochondrial metabolism, causing aberrant neuronal branching and morphogenesis18. Thus, neural progenitors may represent a cellular therapeutic target for mitochondrial diseases, and strategies promoting their mitochondrial function may support the functional development of the nervous system.
The use of brain organoids might help uncover the neurodevelopmental components of mitochondrial diseases. Mitochondrial diseases are mainly considered as early-onset neurodegeneration5. However, neurodevelopmental defects are also present in patients affected by mitochondrial diseases, including developmental delay and cognitive impairment19. Patient-specific brain organoids may help address these aspects and elucidate how mitochondrial diseases may impact human brain development. Mitochondrial dysfunction could also play a pathogenetic role in other more common neurological diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease4. Hence, elucidating the impact of mitochondrial defects in neurodevelopment using brain organoids might also be instrumental for the study of those diseases. This paper describes a detailed protocol for generating reproducible brain organoids that can be used for conducting disease modeling of mitochondrial diseases.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Gibco</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Affinity Designer</td>
			<td>Serif (Europe) Ltd</td>
			<td></td>
			<td>Layout software; Vector graphics editor</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-guinea pig</td>
			<td>Sigma Aldrich</td>
			<td>SAB4600033-250UL</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-mouse</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-31571</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma Aldrich</td>
			<td>1397-94-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin III (TUJ-1)</td>
			<td>Sigma Aldrich</td>
			<td>T8578</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>Argon Laser</td>
			<td>Melles Griot</td>
			<td></td>
			<td>Any other Laser, e.g., diode lasers emitting 488 is fine, too</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A92902</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 with Vitamin A</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>3% in PBS, store solution at -20 &#176;C</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-0811</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Star cell culture 6 well plate</td>
			<td>Greiner-Bio-One</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemically Defined Lipid Concentrate</td>
			<td>Gibco</td>
			<td>11905031</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope C1</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td>Modular confocal microscope system</td>
		</tr>
		<tr>
			<td>Corning Matrigel Growth Factor Reduced (GFR) Basement membrane matrix, Phenol Red-free, LDEV-free</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Matrix component</td>
		</tr>
		<tr>
			<td>CyQUANT Cell Proliferation Assay Kit</td>
			<td>Thermo Fisher</td>
			<td>C7026</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>ThermoFisher</td>
			<td>31330038</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2660-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-goat Cy3</td>
			<td>Merck Millipore</td>
			<td>AP180C</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse Cy3</td>
			<td>Merck Millipore</td>
			<td>AP192C</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit Cy3</td>
			<td>Merck Millipore</td>
			<td>AP182C</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>DS-Q1Mc camera</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse 90i upright widefield microscope</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse E 600FN upright microscope</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse Ts2 Inverted Microscope</td>
			<td>Nikon Microsope Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-C1 Silver Version 3.91</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td>Imaging software for confocal microscope</td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma Aldrich</td>
			<td>370-86-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-291</td>
			<td></td>
		</tr>
		<tr>
			<td>Glasgow MEM</td>
			<td>Gibco</td>
			<td>11710-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Brand</td>
			<td>747715</td>
			<td>Inverted</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Helium-Neon Laser</td>
			<td>Melles Griot</td>
			<td></td>
			<td>Every other Laser, e.g., diode lasers emitting 594 is fine, too</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Merck</td>
			<td>H3149-25KU</td>
			<td></td>
		</tr>
		<tr>
			<td>HERACell 240i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026331</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>1:2500</td>
		</tr>
		<tr>
			<td>Image J 1.53c</td>
			<td>Wayne Rasband National Institute of Health</td>
			<td></td>
			<td>Image processing Software</td>
		</tr>
		<tr>
			<td>Injekt Solo 10 mL/ Luer</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Gibco</td>
			<td>10828010</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser (407 nm)</td>
			<td>Coherent</td>
			<td></td>
			<td>Any other Laser, e.g., diode lasers emitting 407 is fine, too</td>
		</tr>
		<tr>
			<td>Map2</td>
			<td>Synaptic Systems</td>
			<td>No. 188004</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Maxisafe 2030i</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAA</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR Plus</td>
			<td>Stemcell Technology</td>
			<td>85850</td>
			<td>iPSC medium</td>
		</tr>
		<tr>
			<td>Multifuge X3R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>10325804</td>
			<td></td>
		</tr>
		<tr>
			<td>MycoAlert Mycoplasma Detection Kit</td>
			<td>Lonza</td>
			<td># LT07-218</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle for single usage (23G x 1&#8221; TW)</td>
			<td>Neoject</td>
			<td>10016</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Aadvanced Research 3.2</td>
			<td>Nikon</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>Oligomycin A</td>
			<td>Sigma Aldrich</td>
			<td>75351</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker Heidolph Unimax 1010</td>
			<td>Heidolph</td>
			<td>543-12310-00</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP Pen</td>
			<td>Sigma</td>
			<td>Z377821-1EA</td>
			<td>To draw hydrophobic barrier on slides.</td>
		</tr>
		<tr>
			<td>Papain Dissociation System kit</td>
			<td>Worthington</td>
			<td>LK003150</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>818715</td>
			<td>4% in PBS, store solution at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Pasteur pipette 7mL</td>
			<td>VWR</td>
			<td>612-1681</td>
			<td>Graduated up to 3 mL</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plan Apo VC 20x / 0.75 air DIC N2&#160; &#8734;/0.17 WD 1.0</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td>Dry Microscope Objective</td>
		</tr>
		<tr>
			<td>Plan Apo VC 60x / 1.40 oil DIC N2 &#8734;/0.17 WD 0.13</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td>Oil Immersion Microscope Objective</td>
		</tr>
		<tr>
			<td>Polystyrene Petri dish (100 mm)</td>
			<td>Greiner Bio-One</td>
			<td>664161</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene round-bottom tube with cell-strainer cap (5 mL)</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Roth</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Glass Antifade Moutant</td>
			<td>Invitrogen</td>
			<td>P36980</td>
			<td></td>
		</tr>
		<tr>
			<td>Qualitative filter paper</td>
			<td>VWR</td>
			<td>516-0813</td>
			<td></td>
		</tr>
		<tr>
			<td>Rock Inhibitior</td>
			<td>Merck</td>
			<td>SCM075</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma</td>
			<td>83-794</td>
			<td></td>
		</tr>
		<tr>
			<td>S100&#946;</td>
			<td>Abcam</td>
			<td>Ab11178</td>
			<td>1:600</td>
		</tr>
		<tr>
			<td>SB-431542</td>
			<td>Cayman Chemical Company</td>
			<td>13031</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Heinz Herenz Hamburq</td>
			<td>1110918</td>
			<td></td>
		</tr>
		<tr>
			<td>SMI312</td>
			<td>Biolegend</td>
			<td>837904</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Merck/Sigma</td>
			<td>31437-1kg-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Roth</td>
			<td>3957</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate</td>
			<td>Applichem</td>
			<td>131965</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>SOX2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Sc-17320</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Gibco/StemPro</td>
			<td>A1110501</td>
			<td>Reagent A</td>
		</tr>
		<tr>
			<td>Super Glue Gel</td>
			<td>UHU</td>
			<td>63261</td>
			<td>adhesive gel</td>
		</tr>
		<tr>
			<td>SuperFrost Plus</td>
			<td>VWR</td>
			<td>631-0108</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe for single usage (1 mL)</td>
			<td>BD Plastipak</td>
			<td>300015</td>
			<td></td>
		</tr>
		<tr>
			<td>TB2 Thermoblock</td>
			<td>Biometra</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC Plate 24 Well</td>
			<td>Sarstedt</td>
			<td>83.3922</td>
			<td></td>
		</tr>
		<tr>
			<td>TC Plate 6 Well</td>
			<td>Sarstedt</td>
			<td>83.392</td>
			<td></td>
		</tr>
		<tr>
			<td>TGFbeta3</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-007</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>ThermoFisher</td>
			<td>51032711</td>
			<td></td>
		</tr>
		<tr>
			<td>TOM20</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-11415</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Merck</td>
			<td>X100-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5M EDTA</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome Microm HM 650 V</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Production terminated, any other adjustable microtome is fine, too.</td>
		</tr>
		<tr>
			<td>Vibratome Wilkinson Classic Razor Blade</td>
			<td>Wilkinson Sword</td>
			<td>70517470</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Benchkote</td>
			<td>Merck/Sigma</td>
			<td>28418852</td>
			<td></td>
		</tr>
		<tr>
			<td>Wnt Antagonist I</td>
			<td>EMD Millipore Corp</td>
			<td>3378738</td>
			<td></td>
		</tr>
		<tr>
			<td>XF 96 extracellular flux analyser</td>
			<td>Seahorse Bioscience</td>
			<td>100737-101</td>
			<td></td>
		</tr>
		<tr>
			<td>XF Assay DMEM Medium</td>
			<td>Seahorse Bioscience</td>
			<td>103680-100</td>
			<td></td>
		</tr>
		<tr>
			<td>XF Calibrant Solution</td>
			<td>Seahorse Bioscience</td>
			<td>100840-000</td>
			<td></td>
		</tr>
		<tr>
			<td>XFe96 FluxPak (96-well microplate)</td>
			<td>Seahorse Bioscience</td>
			<td>102416-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of human brain organoids for mitochondrial disease modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

The protocol described here facilitates the robust generation of round organoids (Figure 1A). The generated organoids&#160;contain mature neurons that can be visualized using protein markers specific for axons (SMI312) and dendrites (microtubule-associated protein 2 (MAP2)) (Figure 1B). Mature organoids contain not only neuronal cells (MAP2-positive) but also glial cells (e.g., positive for the astrocyte marker ...

Watch this Scientific Journal Video about Generation of Human Brain Organoids for Mitochondrial Disease Modeling at JoVE.com

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

generation-of-human-brain-organoids-for-mitochondrial-disease-modeling

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Neuroscience

5.9K Views.  Heinrich Heine University. We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Video: Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca2+ buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca2+ concentration. Ca2+ retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca2+ leads to the permeability of the inner mitochondrial membrane, termed the opening of mitochondrial permeability transition pore (mPTP), which leads to the leakage of molecules with a molecular weight less than 1.5 kDa. Ca2+-triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca2+ retention capacity and Ca2+-triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca2+ are added, all steps can be completed in one day and recorded by a microplate reader. Thus, these two simple and effective assays can be adopted to assess the Ca2+-related mitochondrial functions.

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in ...

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

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

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

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

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

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

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

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

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

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

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker &#945;-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.

We describe a method to generate human motor units in commercially available microfluidic devices by co-culturing human induced pluripotent stem cell-derived motor neurons with human primary mesoangioblast-derived myotubes resulting in the formation of functionally active neuromuscular junctions.

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle ...

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 ...

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...

An In Situ Immunostaining Protocol for Parkinson's Disease Study

Histological Examination of Mitochondrial Morphology in a Parkinson's Disease Model

Mitochondria play a central role in the energy metabolism of cells, and their function is especially important for neurons due to their high energy demand. Therefore, mitochondrial dysfunction is a pathological hallmark of various neurological disorders, including Parkinson's disease. The shape and organization of the mitochondrial network is highly plastic, which allows the cell to respond to environmental cues and needs, and the structure of mitochondria is also tightly linked to their health. Here, we present a protocol to study mitochondrial morphology in situ based on immunostaining of the mitochondrial protein VDAC1 and subsequent image analysis. This tool could be particularly useful for the study of neurodegenerative disorders because it can detect subtle differences in mitochondrial counts and shape induced by aggregates of &#945;-synuclein, an aggregation-prone protein heavily involved in the pathology of Parkinson's disease. This method allows one to report that substantia nigra pars compacta dopaminergic neurons harboring pS129 lesions show mitochondrial fragmentation (as suggested by their reduced Aspect Ratio, AR) compared to their healthy neighboring neurons in a pre-formed fibril intracranial injection Parkinson model.

Author Spotlight: Establishing a New Fluorescence-Based Protocol for In Vivo Mitochondrial Morphology Analysis in Parkinson's Disease

This study presents a method to analyze the morphology of mitochondria based on immunostaining and image analysis in mouse brain tissue in situ. It also describes how this allows one to detect changes in mitochondrial morphology induced by protein aggregation in Parkinson's disease models.

Mitochondria play a central role in the energy metabolism of cells, and their function is especially important for neurons due to their high energy ...