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 organoids can be cultured in standard tissue culture 6-well plates positioned onto an orbital shaker inside the incubator. This procedure also enables the parallel cultivation of several plates containing different organoids derived from various individual lines, thereby increasing the throughput of the experiments and allowing the monitoring of potential differences emerging in the growth profiles of different organoids. We tested this protocol using different iPSCs derived from healthy controls and individuals affected by mitochondrial diseases, with consistent results.
For mitochondrial disease modeling, it is essential to use different markers to visualize the morphology and organization of the mitochondrial network. This procedure enables the investigation of whether mitochondrial number, morphology, or distribution might be altered in brain organoids derived from patients with mitochondrial diseases. The presence and organization of neural progenitors within the brain organoids could be of crucial importance for modeling mitochondrial disorders. We recently discovered that mutations causing the mitochondrial disease, Leigh syndrome, disrupt the cellular architecture and distribution of neural progenitor cells within patient-derived brain organoids18.
For performing bioenergetic profiling, we have adapted a method that was previously described for assessing the bioenergetics of pluripotent stem cells21. A recent protocol described how to carry out bioenergetic profiling of organoids derived from mouse small intestine, human colon, and colorectal tumors22. However, those organoids are quite small compared to brain organoids, and therefore, a different protocol, such as the one reported here, is needed for brain organoids. We recently employed this protocol for assessing the bioenergetic profile of human brain organoids carrying mutations in the surfeit locus protein 1 gene (SURF1) that causes the severe mitochondrial disease, Leigh syndrome18. We found that the OCR profile is particularly affected in Leigh syndrome organoids, as shown by a significant decrease in the basal OCR level, the ATP production rate, and the maximal respiration rate18.
In conclusion, we present here a detailed protocol for the robust generation of human brain organoids and describe how to perform experiments that would be important for the investigation of the disease mechanisms underlying mitochondrial diseases. Human brain organoids may also be of critical importance for elucidating the mitochondrial diversity in the human brain and its role in human health and diseases23. It is important to clarify that brain organoids generated with currently available protocols, including the one described here, still bear limitations. These include, for example, the lack of vascularization and the absence of microglia population24. These aspects need to be taken into consideration to interpret the results correctly.
For example, the lack of vasculature and microglia could limit compensatory mechanisms that may be in place in vivo. Patient-derived brain organoids might thus exhibit defects that are stronger than the ones observed in patients17,18. Moreover, despite a general reproducibility of this protocol20, line-to-line heterogeneity can be observed. To this end, when performing disease modeling studies, it is always important to systematically quantify the uniformity of control and patient organoids by assessing the patterns of morphology (size, layer) and the distribution of molecular markers across different organoids.
Finally, it is not possible to generate brain organoids from a single iPSC, limiting the feasibility of large-scale genetic screening with CRISPR/Cas9. Given the pace of research, it is likely that some of the current limitations of the protocol described here will soon be overcome. Optimized protocols will become available. These 3D models of mitochondrial diseases will hopefully enable the eventual discovery of implementable therapies for mitochondrial diseases, which are detrimental, and for incurable diseases with highly unmet medical needs.

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 S100 calcium-binding protein B (S100&#223;)) (Figure 1B).
By analyzing sliced brain organoids using confocal microscopy, it is possible to identify and monitor the detailed distribution and organization of different cell types and cellular structures. This could provide new insight into how mitochondrial diseases might affect nervous system development. For example, it is possible to monitor neuronal axons (SMI312-positive) and dendrites (MAP2-positive) (Figure 2A) or the mutual occurrence of neuronal cells (MAP2-positive) and glial cells (S100&#223;-positive) (Figure 2A). Confocal images may also help to investigate in more detail the distribution and organization of neural progenitors ((sex determining region Y) box-2 (SOX2)-positive) with respect to neurons (beta-III tubulin (TUJ1)-positive) (Figure 2B). Finally, brain organoids can be stained for mitochondria-specific markers (such as the outer mitochondrial membrane protein, translocase of outer membrane 20 kDa subunit (TOM20)) (Figure 2C).
The described protocol enables researchers to perform bioenergetic profiling of brain organoids. Using this procedure, it is possible to measure both mitochondrial metabolism using the oxygen consumption rate (OCR) (Figure 2D) and the glycolytic metabolism using the extracellular acidification rate (ECAR) (Figure 2E). Bioenergetic profiling allows monitoring how cells may modify their OCR and ECAR profiles in response to a sequential administration of mitochondrial inhibitors.
First, the ATP synthase inhibitor, oligomycin, can be applied. Oligomycin causes a drop in the OCR profile (Figure 2D), and therefore, identifies the OCR needed for ATP production. Upon oligomycin treatment, there may also be a compensatory increase in ECAR (Figure 2E), suggesting that the cells can upregulate glycolysis to prevent the metabolic stress caused by the reduction in mitochondrial metabolism. The subsequent double application of the proton ionophore, carbonyl cyanide-p-trifluoromethoxyphenylhydrazon (FCCP), causes the loss of the mitochondrial membrane potential. As the oxygen molecules are now free to move, this causes a rapid increase in OCR (Figure 2D).
These changes in the OCR profile identify the maximal respiration capacity of the cells. The final administration of rotenone plus antimycin A causes a block of the electron transport, and therefore, a steep decrease in OCR (Figure 2D).&#160;ECAR may show fluctuation after treatment with FCCP and rotenone plus antimycin A (Figure 2E), depending on the residual glycolytic capacity of the cells. The OCR and ECAR profiles may be dramatically altered in brain organoids derived from mitochondrial patients.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62756/62756fig01.jpg" /> 
Figure 1: Generation of brain organoids from human iPSCs. (A)&#160;Schematic representation of the protocol used to produce brain organoids with corresponding transmission images. Day 0 corresponds to the dissociation of iPSCs and seeding in a 96-well plate with V-bottom using CDMI supplemented with a ROCK inhibitor, a WNT inhibitor, and SB431542. At day 18, neurospheres are transferred from the 96-well plates to 100 mm cell culture dishes with CDMII supplemented with N2. From this point onwards, the cultures are positioned on an orbital shaker. At day 35, the medium is switched from CDMII to CDMIII, which also contains a dissolved matrix component (Table 1). From day 70 onwards, CDMIII is switched to CDMIV supplemented with B27. A representative neurosphere image was taken at day 12 using a microscope camera with 10x magnification. An early organoid image was taken at day 22 using a microscope camera at 4x magnification. A mature organoid image was taken at day 40 using a microscope camera with 4x magnification. (B)&#160;The overall structure and cellular organization of brain organoids can be visualized using wide-field microscopy. Representative stitched wide-field images are shown to visualize the relationships between dendrites (MAP2-positive) and axons (SMI312-positive), and between neuronal cells (MAP2-positive) and presumed astrocytes (S100&#223;-positive). The cells were counterstained with Hoechst to reveal the nuclei. All images were taken using 78 day-old brain organoids. The right column shows the overlay of three channels (merge). Scale bars = 500 &#181;m. Abbreviations: iPSC = induced pluripotent stem cell; CDM = Cortical Differentiation Medium; ROCK = Rho kinase; MAP2 = microtubule-associated protein 2; S100&#223;= S100 calcium-binding protein B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62756/62756fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62756/62756fig02.jpg" /> 
Figure 2: Visualization and bioenergetic profiling of brain organoids for mitochondrial disease modeling. The detailed organization and architecture of organoids can be analyzed using confocal microscopy. All images were taken using 78 day-old brain organoids and counterstained with Hoechst to reveal the nuclei. The right column shows the overlay of three channels (merge). Scale bars = 50 &#181;m.&#160;(A) Representative extended-focus projections (44-48 optical planes, 0.6 &#181;m each) addressing the interplay between dendrites (MAP2-positive, arrowheads) and axons (SMI312-positive, arrows), and between neuronal cells (MAP2-positive, arrowheads) and presumed astrocytes (S100&#223;-positive, arrows). (B) Representative extended-focus projections (14-31 optical planes, 0.6 &#181;m each) showing the distribution of neurons (TUJ1-positive, arrowheads) with respect to neural progenitors (SOX2-positive, arrows). (C)&#160;Representative extended-focus projections (20 optical planes, 0.6 &#181;m each) showing the distribution within neurons (TUJ1-positive, arrowheads) of mitochondria (visualized using antibodies against the outer mitochondrial membrane protein TOM20, arrows). (D) Mitochondrial respiration of brain organoids can be monitored based on the profile of the OCR after sequential administration of different mitochondrial inhibitors (see text for details). (E) Glycolytic activity of brain organoids can be monitored based on the ECAR upon sequential administration of mitochondrial inhibitors (see text for details). For bioenergetic profiling, approximately 10-15 brain organoids were dissociated to obtain enough cells for replating onto the 96-well microplate. The bars indicate the SEMs based on the results obtained in two independent experiments. Abbreviations: MAP2 = microtubule-associated protein 2; S100&#223; = S100 calcium-binding protein B; TUJ1 = beta-III tubulin; SOX2 = (sex determining region Y) box-2; TOM20 = translocase of outer membrane 20 kDa subunit; OCR = oxygen consumption rate; ECAR = extracellular acidification rate; Oligom. = oligomycin; FCCP = carbonyl cyanide-p-trifluoromethoxyphenylhydrazon; R = rotenone; AntA = Antimycin A; SEMs = standard error of means. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62756/62756fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media Composition</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CDMI (Day 0-18)</td>
			<td></td>
			<td></td>
			<td>Final conc.</td>
		</tr>
		<tr>
			<td>Glasgow-MEM</td>
			<td>Gibco</td>
			<td>11710-035</td>
			<td>[1:1]</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement (KSR)</td>
			<td>Gibco</td>
			<td>10828010</td>
			<td>20%</td>
		</tr>
		<tr>
			<td>MEM-NEAA (MEM non-essential amino acid solution)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>0.1 mM</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>2-mercaptethanol</td>
			<td>Gibco</td>
			<td>31350-010</td>
			<td>0.1 mM</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100 U/mL and 100 &#956;g/mL</td>
		</tr>
		<tr>
			<td>CDMII (Day 18-35)</td>
			<td></td>
			<td></td>
			<td>Final conc.</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>31330038</td>
			<td>[1:1]</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Chemically Defined Lipid Concentrate</td>
			<td>Gibco</td>
			<td>11905031</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100 U/mL and 100 &#956;g/mL</td>
		</tr>
		<tr>
			<td>CDMIII (Day 35-70)</td>
			<td></td>
			<td></td>
			<td>Final conc.</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>31330038</td>
			<td>[1:1]</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Chemically Defined Lipid Concentrate</td>
			<td>Gibco</td>
			<td>11905031</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100 U/mL and 100 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Merck</td>
			<td>H3149-25KU</td>
			<td>5 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>CDMIV(Day 70+)</td>
			<td></td>
			<td></td>
			<td>Final conc.</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>31330038</td>
			<td>[1:1]</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Chemically Defined Lipid Concentrate</td>
			<td>Gibco</td>
			<td>11905031</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100 U/mL and 100 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Merck</td>
			<td>H3149-25KU</td>
			<td>5 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>2%</td>
		</tr>
		<tr>
			<td>B-27 supplement with Vitamin A 50x</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>2%</td>
		</tr>
		<tr>
			<td>Neuronal Medium</td>
			<td></td>
			<td></td>
			<td>Final conc.</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>31330038</td>
			<td>[1:1]</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>[1x]</td>
		</tr>
		<tr>
			<td>B-27 supplement with vitamin A 50x</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>[1x]</td>
		</tr>
		<tr>
			<td>L-Ascorbic acid</td>
			<td>Sigma Aldrich</td>
			<td>A92902</td>
			<td>[200&#8201;&#181;M]</td>
		</tr>
		<tr>
			<td>db-cAMP (dibutyryl cyclic adenosine monophosphate)</td>
			<td>Sigma Aldrich</td>
			<td>D0627</td>
			<td>500&#8201;&#181;M</td>
		</tr>
		<tr>
			<td>BDNF (brain-derived neutrotrophic factor)</td>
			<td>MACS Miltenyi</td>
			<td>130-093-811</td>
			<td>[10&#8201;ng/mL]</td>
		</tr>
		<tr>
			<td>GDNF (glial cell line-derived neurotrophic factor)</td>
			<td>MACS Miltenyi</td>
			<td>130-096-290</td>
			<td>[10&#8201;ng/mL]</td>
		</tr>
		<tr>
			<td>Human TGF-&#946;3 (transforming growth factor-beta3)</td>
			<td>MACS Miltenyi</td>
			<td>130-094-007</td>
			<td>[1&#8201;ng/mL]</td>
		</tr>
		<tr>
			<td>Assay Medium</td>
			<td></td>
			<td></td>
			<td>Final conc.</td>
		</tr>
		<tr>
			<td>Seahorse XF DMEM medium</td>
			<td>Seahorse Bioscience</td>
			<td>103680-100</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BEBP17-605E</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>50-99-7</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Blocking Solution</td>
			<td></td>
			<td></td>
			<td>Final conc.</td>
		</tr>
		<tr>
			<td>PBS-Tween</td>
			<td></td>
			<td></td>
			<td>[1:1] 0.1% Tween</td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>Sigma Aldrich</td>
			<td>D9663</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Merck</td>
			<td>X100-5ML</td>
			<td>1%</td>
		</tr>
	</tbody>
</table>
Table 1: Details of media and solutions used for organoid generation.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Initialization</td>
			<td>Baseline (X3)</td>
			<td>Oligomycin Injection (X3)</td>
			<td>FCCP Injection (X3)</td>
			<td>FCCP Injection (X3)</td>
			<td>Anti A + Rot Injection (X3)</td>
		</tr>
		<tr>
			<td rowspan="2">Calibrate</td>
			<td>Mix</td>
			<td>Mix</td>
			<td>Mix</td>
			<td>Mix</td>
			<td>Mix</td>
		</tr>
		<tr>
			<td>(04:00)</td>
			<td>(04:00)</td>
			<td>(04:00)</td>
			<td>(04:00)</td>
			<td>(04:00)</td>
		</tr>
		<tr>
			<td rowspan="2">Equilibrate (12:00)</td>
			<td>Wait</td>
			<td>Wait</td>
			<td>Wait</td>
			<td>Wait</td>
			<td>Wait</td>
		</tr>
		<tr>
			<td>(02:00)</td>
			<td>(02:00)</td>
			<td>(02:00)</td>
			<td>(02:00)</td>
			<td>(02:00)</td>
		</tr>
		<tr>
			<td rowspan="2"></td>
			<td rowspan="2">Measure (03:00)</td>
			<td rowspan="2">Measure (03:00)</td>
			<td rowspan="2">Measure (03:00)</td>
			<td>Measure</td>
			<td rowspan="2">Measure (03:00)</td>
		</tr>
		<tr>
			<td>(03:00)</td>
		</tr>
	</tbody>
</table>
Table 2: Protocol setup for bioenergetic profiling. Description of the steps and their length in minutes using the Seahorse Wave Desktop software. Abbreviations: FCCP = carbonyl cyanide-p-trifluoromethoxyphenylhydrazon; Rot = rotenone; Anti A = Antimycin A.

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

Nanyang Technological University

Research

JoVE Journal

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

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

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 ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

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. ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

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 ...