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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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><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-&amp;beta;-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 &amp;deg;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 CO&lt;sub&gt;2&lt;/sub&gt; 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&amp;rdquo; 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 &amp;deg;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&amp;nbsp; &amp;infin;/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 &amp;infin;/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&amp;beta;</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

6.0K 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

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Developmental Biology

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

Genetic Modification of Primate Cerebral Organoids

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.
As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Author Spotlight: Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification  

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

Bioengineering

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 ...

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 ...

Application of Flow Cytometric Analysis for Measuring Multiple Mitochondrial Parameters in 3D Brain Organoids

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, mitochondrial respiratory chain (MRC) complexes, and mitochondrial DNA (mtDNA) copy number often feature in these processes. Human brain organoids derived from human induced pluripotent stem cells (iPSCs) recapitulate the brain&#39;s three-dimensional (3D) cytoarchitectural arrangement and offer the possibility to study disease mechanisms and screen new therapeutics in a complex human system. Here, we report a unique flow cytometry-based approach to measure multiple mitochondrial parameters in iPSC-derived cortical organoids. This report details a protocol for generating cortical brain organoids from iPSCs, single-cell dissociation of generated organoids, fixation, staining, and subsequent flow cytometric analysis to assess multiple mitochondrial parameters. Double staining with antibodies against the MRC complex subunit NADH: Ubiquinone Oxidoreductase Subunit B10 (NDUFB10) or mitochondrial transcription factor A (TFAM) together with voltage-dependent anion-selective channel 1 (VDAC 1) permits assessment of the amount of these proteins per mitochondrion. Since the quantity of TFAM corresponds to the amount of mtDNA, it provides an indirect estimation of the number of mtDNA copies per mitochondrial content. This entire procedure can be completed within a span of 2-3 h. Crucially, it allows for the concurrent quantification of multiple mitochondrial parameters, including both total and specific levels relative to the mitochondrial mass.

This protocol reports a unique method of using a streaming cytometer and multiple antibodies for simultaneous assessment of multiple mitochondrial functional parameters, including changes in mitochondrial volume, amounts of the mitochondrial respiratory chain (MRC) complex subunits, and mitochondrial DNA (mtDNA) replication.

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, ...

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

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal brain development. The fusion of dorsal with ventral regionally specified brain organoids in vitro generates assembloids, which have functionally integrated microcircuits with excitatory and inhibitory neurons. Due to their structural complexity and diverse population of neurons, assembloids have become a useful in vitro tool for studying aberrant network activity. Multi-electrode array (MEA) recordings serve as a method for capturing electrical field potentials, spikes, and longitudinal network dynamics from a population of neurons without compromising cell membrane integrity. However, adhering assembloids onto the electrodes for long-term recordings can be challenging due to their large size and limited contact surface area with the electrodes. Here, we demonstrate a method to plate assembloids onto MEA plates for recording electrophysiological activity over a 2-month span. Although the current protocol utilizes human cortical organoids, it can be broadly adapted to organoids differentiated to model other brain regions. This protocol establishes a robust, longitudinal, electrophysiological assay for studying the development of a neuronal network, and this platform has the potential to be used in drug screening for therapeutic development in epilepsy.

Author Spotlight: Advancing Genetic Epilepsy Studies with Multi-Electrode Array-Based Long-Term Electrophysiological Monitoring of Human Brain Assembloids

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

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

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

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Chapters in this video

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

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

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

Application of Flow Cytometric Analysis for Measuring Multiple Mitochondrial Parameters in 3D Brain Organoids

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids