Name: generation of human brain organoids for mitochondrial disease modeling
Uploaded: 2021-06-21T14:00:00
Description: Watch this Scientific Journal Video about Generation of Human Brain Organoids for Mitochondrial Disease Modeling at JoVE.com

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 ...

<ol>
	<li>Koopman, W. J., Willems, P. H., Smeitink, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monogenic+mitochondrial+disorders.">Monogenic mitochondrial disorders.</a> New England Journal of Medicine. 366 (12), 1132-1141 (2012).</li><li>Gorman, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+diseases.">Mitochondrial diseases.</a> Nature Review Disease Primers. 2, 16080 (2016).</li><li>Vafai, S. B., Mootha, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+disorders+as+windows+into+an+ancient+organelle.">Mitochondrial disorders as windows into an ancient organelle.</a> Nature. 491 (7424), 374-383 (2012).</li><li>Carelli, V., Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNA:+impacting+central+and+peripheral+nervous+systems.">Mitochondrial DNA: impacting central and peripheral nervous systems.</a> Neuron. 84 (6), 1126-1142 (2014).</li><li>Russell, O. M., Gorman, G. S., Lightowlers, R. N., Turnbull, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+diseases:+hope+for+the+future.">Mitochondrial diseases: hope for the future.</a> Cell. 181 (1), 168-188 (2020).</li><li>Weissig, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+development+for+the+therapy+of+mitochondrial+diseases.">Drug development for the therapy of mitochondrial diseases.</a> Trends in Molecular Medicine. 26 (1), 40-57 (2020).</li><li>Tyynismaa, H., Suomalainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+mitochondrial+DNA+defects+and+their+relevance+for+human+disease.">Mouse models of mitochondrial DNA defects and their relevance for human disease.</a> EMBO Reports. 10 (2), 137-143 (2009).</li><li>Ma, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+rescue+in+pluripotent+cells+from+patients+with+mtDNA+disease.">Metabolic rescue in pluripotent cells from patients with mtDNA disease.</a> Nature. 524 (7564), 234-238 (2015).</li><li>Galera-Monge, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+and+calcium+dysregulation+in+Leigh+syndrome+induced+pluripotent+stem+cell+derived+neurons.">Mitochondrial dysfunction and calcium dysregulation in Leigh syndrome induced pluripotent stem cell derived neurons.</a> International Journal of Molecular Science. 21 (9), 3191 (2020).</li><li>Zheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alleviation+of+neuronal+energy+deficiency+by+mTOR+inhibition+as+a+treatment+for+mitochondria-related+neurodegeneration.">Alleviation of neuronal energy deficiency by mTOR inhibition as a treatment for mitochondria-related neurodegeneration.</a> Elife. 5, 13378 (2016).</li><li>Lorenz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPSC-derived+neural+progenitors+are+an+effective+drug+discovery+model+for+neurological+mtDNA+disorders.">Human iPSC-derived neural progenitors are an effective drug discovery model for neurological mtDNA disorders.</a> Cell Stem Cell. 20 (5), 659-674 (2017).</li><li>Inak, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+induced+pluripotent+stem+cell-based+drug+discovery+for+mitochondrial+disease.">Concise review: induced pluripotent stem cell-based drug discovery for mitochondrial disease.</a> Stem Cells. 35 (7), 1655-1662 (2017).</li><li>Chiaradia, I., Lancaster, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+organoids+for+the+study+of+human+neurobiology+at+the+interface+of+in+vitro+and+in+vivo.">Brain organoids for the study of human neurobiology at the interface of in vitro and in vivo.</a> Nature Neuroscience. 23 (12), 1496-1508 (2020).</li><li>Lancaster, M. A., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+cerebral+organoids+from+human+pluripotent+stem+cells.">Generation of cerebral organoids from human pluripotent stem cells.</a> Nature Protocol. 9 (10), 2329-2340 (2014).</li><li>Liput, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+and+approaches+for+analyzing+the+role+of+mitochondria+in+health,+development+and+disease+using+human+cerebral+organoids.">Tools and approaches for analyzing the role of mitochondria in health, development and disease using human cerebral organoids.</a> Developmental Neurobiology. , (2021).</li><li>Winanto, K. Z. J., Soh, B. S., Fan, Y., Ng, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+cultures+of+MELAS+neural+cells+reveal+hyperactive+Notch+signaling+that+impacts+neurodevelopment.">Organoid cultures of MELAS neural cells reveal hyperactive Notch signaling that impacts neurodevelopment.</a> Cell Death and Disease. 11 (3), 182 (2020).</li><li>Romero-Morales, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPSC-derived+cerebral+organoids+model+features+of+Leigh+Syndrome+and+reveal+abnormal+corticogenesis.">Human iPSC-derived cerebral organoids model features of Leigh Syndrome and reveal abnormal corticogenesis.</a> bioRxiv. , (2020).</li><li>Inak, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+metabolic+programming+impairs+early+neuronal+morphogenesis+in+neural+cultures+and+an+organoid+model+of+Leigh+syndrome.">Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome.</a> Nature Communications. 12 (1), 1929 (2021).</li><li>Falk, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodevelopmental+manifestations+of+mitochondrial+disease.">Neurodevelopmental manifestations of mitochondrial disease.</a> Journal of Developmental &amp; Behavioral Pediatrics. 31 (7), 610-621 (2010).</li><li>Velasco, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+brain+organoids+reproducibly+form+cell+diversity+of+the+human+cerebral+cortex.">Individual brain organoids reproducibly form cell diversity of the human cerebral cortex.</a> Nature. 570 (7762), 523-527 (2019).</li><li>Pfiffer, V., 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=Assessing+the+bioenergetic+profile+of+human+pluripotent+stem+cells.">Assessing the bioenergetic profile of human pluripotent stem cells.</a> Methods in Molecular Biology. 1264, 279-288 (2015).</li><li>Ludikhuize, M. 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>

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

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

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

generation of human brain organoids for mitochondrial disease modeling

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

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

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

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

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

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

This protocol aims to use brain organoids to study mitochondrial diseases. It was developed for generating reproducible brain organoids in the assessment of their mitochondrial properties. This method does not require expensive bioreactors and time-consuming embedding procedures.Therefore, it is easy to implement and upscale. This protocol makes it possible to generate reproducible brain organoids and to test their mitochondrial properties. This can lead to identification of interventional targets for untreatable mitochondria diseases.To begin, culture human iPSCs under feeder-free conditions in iPSC medium on coated six-well plates and keep them in a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide. Passage the iPSCs at 80%confluency using enzyme-free detachment medium in ratios ranging from one by four to one by 12. To increase cell survival, add 10 micromolar rho-associated protein kinase or ROCK inhibitor after each splitting.Dissociate the 80%iPSCs at day zero. Prepare cortical differentiation medium one or CDM1 and pre-warm it at room temperature before adding it to the cells. Wash the wells containing the iPSCs with PBS to remove dead cells and debris.Add 500 microliters of pre-warmed reagent A to each well and incubate for five minutes at 37 degrees Celsius. Check under the microscope to ensure cell detachment. Add one milliliter of iPSC medium to dilute reagent A to neutralize its activity.Use a 1, 000 microliter pipette to dissociate the cells by piping up and down and transfer the cell suspension to a 15 milliliter centrifuge tube. Gently centrifuge the iPSCs at 125 times G for five minutes at room temperature, then carefully aspirate the supernatant to avoid disturbing the cell pellet. Resuspend the pellet with one milliliter of CDM1 to obtain a single cell suspension and count the cell number.Prepare the seeding medium with 9, 000 iPSCs per 100 microliters in CDM1 supplemented with 20 micromolar ROCK inhibitor, three micromolar WNT catenin inhibitor or IWR-1, and five micromolar SB-431542. Add 100 microliters of seeding medium per well to a 96-well V bottom plate. Keep the plate in a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide.To generate neurospheres, on day one, observe that round cell aggregates with defined smooth borders are forming. Note the dead cells around the aggregates. Continue to culture in the incubator at 37 degrees Celsius and 5%carbon dioxide.On day three, agitate the plate by tapping on the sides three times to detach dead cells, then add 100 microliters of CDM1 supplemented with 20 micromolar ROCK inhibitor, three micromolar IWR-1, and five micromolar SB-431542 to each well. Keep the plate to the incubator at 37 degrees Celsius and 5%carbon dioxide. On day six, carefully remove 80 microliters of the supernatant medium from each well.Avoid touching the bottom of the well. Add 100 microliters of CDM1 supplemented with three micromolar IWR-1 and five micromolar SB-431542 to each well and incubate the plate. Repeat the previous step after every three days until day 18.On day 18, to transfer neurospheres, prepare CDM2 and add 10 milliliters to a 100 millimeter ultra low attachment cell culture plate. Use a 200 microliter pipette with the tip cut off to transfer the round neurospheres from the 96-well plate to the 100 millimeter ultra low attachment cell culture plate. Remove five milliliters of medium from the plate containing the neurospheres and add five milliliters of fresh CDM2.Place the plate on an orbital shaker at 70 rotations per minute inside a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide. Every three days, carefully aspirate the supernatant medium and replace it with fresh CDM2. Leave a small amount of the medium to prevent neurospheres from drying out.On day 35, prepare CDM3. Aspirate the medium from the plate and add 10 milliliters of cold CDM3. After changing the medium, place the plate back on an orbital shaker at 70 rotations per minute inside a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide.Change the medium every three to five days depending on the rate of growth as indicated by the color of the medium. On day 70, prepare CDM4. Use CDM4 medium until the desired age of organoids is reached.During this period, keep the plate on an orbital shaker set at 70 rotations per minute inside a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide. Change the medium every three to five days depending on the growth rate. Prepare 4%PFA solution and place it under a safety hood.Collect brain organoids and gently transfer them with a blunt tipped three milliliter plastic Pasteur pipette to a six-well plate filled with PFA. Keep the organoids in the PFA solution for one hour at room temperature. Carefully remove the PFA with a three milliliter plastic Pasteur pipette and wash the fixed organoids three times using PBS.Store the fixed organoids at four degrees Celsius in PBS until further use. The organoids generated using this protocol contain mature neurons that can be visualized using protein markers specific for axons and dendrites. Mature organoids contain not only neuronal cells, but also glial cells.Using sliced brain organoids analysis, it is possible to monitor SMI 312 positive axons and MAP2 positive dendrites or the S100 beta glial cells. Further, confocal images help to investigate the detailed distribution and organization of SOX2 positive neural progenitors with respect to beta-3 tubulin positive neurons. Brain organoids were stained for mitochondria-specific markers, such as translocase of outer membrane or TOM20.Bioenergetic profiling of brain organoids was performed by measuring both mitochondrial metabolisms using the oxygen consumption rate, or OCR, and the glycolytic metabolism using the extracellular acidification rate, or ECAR. Oligomycin causes a drop in the OCR profile and therefore identifies the OCR needed for ATP production. Upon oligomycin treatment, there may also be a compensatory increase in ECAR suggesting that the cells can upregulate glycolysis to prevent the metabolic stress.When transferring the neurospheres from the 96-well plate to a culture plate or during the fixing procedure, it is crucial to handle the organoids gently to avoid damaging them. Following the generation of brain organoids, multi-electrode array or calcium imaging would be ideal methods to test the functionality of the organoids.

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

Research

JoVE Journal

Neuroscience

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Three-Dimensional Shape Modeling and Analysis of Brain Structures

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

An In Situ Immunostaining Protocol for Parkinson&#039;s Disease Study

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

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

Author Spotlight: Establishing a New Fluorescence-Based Protocol for In Vivo Mitochondrial Morphology Analysis in Parkinson&#039;s Disease

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

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

Modeling the Functional Network for Spatial Navigation in the Human Brain

Spatial navigation is a complex function involving the integration and manipulation of multisensory information. Using different navigation tasks, many promising results have been achieved on the specific functions of various brain regions (e.g., hippocampus, entorhinal cortex, and parahippocampal place area). Recently, it has been suggested that a non-aggregate network process involving multiple interacting brain regions may better characterize the neural basis of this complex function. This paper presents an integrative approach for constructing and analyzing the functionally-specific network for spatial navigation in the human brain. Briefly, this integrative approach consists of three major steps: 1) to identify brain regions important for spatial navigation (nodes definition); 2) to estimate functional connectivity between each pair of these regions and construct the connectivity matrix (network construction); 3) to investigate the topological properties (e.g., modularity and small worldness) of the resulting network (network analysis). The presented approach, from a network perspective, could help us better understand how our brain supports flexible navigation in complex and dynamic environments, and the revealed topological properties of the network can also provide important biomarkers for guiding early identification and diagnosis of Alzheimer's disease in clinical practice.

This paper presents an integrative approach to investigating the functional network for spatial navigation in the human brain. This approach incorporates a large-scale neuroimaging meta-analytic database, resting-state functional magnetic resonance imaging, and network modeling and graph-theoretical techniques.

Spatial navigation is a complex function involving the integration and manipulation of multisensory information. Using different navigation tasks, many ...