This work was supported by the Fritz Thyssen Foundation (Az.10.14.2.152). We are grateful to the tissue embedding facility and the microscope core facility of CMMC. We are grateful for the discussions and technical support provided by the members of the Laboratory for Centrosome and Cytoskeleton Biology. We thank Li Ming Gooi for proofreading the manuscript.

1. Generation of Brain Organoids (23 days)
<ol>
	<li>Initiation of neuroectoderm (5 days) 
	NOTE: The following points should be considered before the start of differentiation. The reprogramming method (lentiviral-, sendai-virus-, episomal-, or microRNA-based etc.) to obtain human iPSCs should ideally be the same for all patient and control iPSC lines. Various reprogramming kits and instructions based on published protocols are available15,16,17,18. The quality of the human iPSC lines is the key to accomplishing an optimal differentiation. Monitor colony and cell morphology with a microscope and validate pluripotency by testing the expression of markers such as Oct3/4, Nanog, or TRA-1-60.
	<ol>
		<li>Culture human iPSCs under feeder-free conditions in medium A on a dish coated with Engelbreth-Holm-Swarm (EHS) matrix. 
		NOTE: Grow human iPSCs feeder-free and serum-free to maintain a defined culture condition and to avoid an additional step for the removal of mouse embryonic feeder cells (MEFs) before the start of differentiation. The optimal passage number for human iPSCs to start differentiation ranges from passage 15 upon reprogramming to passage 70 in total. When passaging, detach hiPSC colonies as cells aggregate using an appropriate cell detachment solution with low mechanical stress and a 2-mL serological pipette to transfer aggregates to a new dish. Avoid dissociation to single cells, as this might induce differentiation and apoptosis in most iPSC lines. It was reported that long-term, single-cell passaging could increase genomic alterations in human iPSCs19,20. Avoid cell detachment procedures, which require a centrifugation step to remove detachment solution, as this reduces the overall viability of iPSCs. Test all cultures for microbial contaminations, particularly mycoplasma, on a regular basis, because this might alter the quality of the iPSCs and their differentiation capacity.
		<ol>
			<li>Coat a 60 mm tissue culture dish with EHS matrix as per the manufacturer&#39;s instructions.</li>
			<li>Thaw an aliquot of 1 x 106 human iPSCs. Seed the iPSCs in a 60-mm tissue culture dish coated in EHS matrix and containing 5 mL of medium A (see the Materials Table). Change the medium daily and passage after 5 to 7 days when the cells reach ~80% confluency. 
			NOTE: Passage the thawed iPSCs at 80% confluency using standard methods, such as the enzyme-free detachment of colonies21, at least once before starting the differentiation. In brief, remove the iPSC medium, wash the cells once with pre-warmed 37 &#176;C Dulbecco&#39;s Modified Eagle&#39;s Medium: Nutrient Mixture F-12 (DMEM/F12), and incubate the iPSCs following the manufacturer&#39;s instructions using reagent A (see the materials table). Don&#39;t exceed the recommended incubation time in order to avoid dissociation to single cells. Human iPSCs should be detached and floating as aggregates, not as single cells. They can then be transferred to new EHS matrix-coated dishes; for example, iPSC aggregates from one 60-mm dish can be distributed to 4 new 60-mm dishes.</li>
			<li>Check for mycoplasma with a mycoplasma detection kit according to the manufacturer&#39;s instructions. Use only mycoplasma-free iPSCs, as mycoplasma can alter the differentiation capability of iPSCs.</li>
		</ol>
		</li>
		<li>Dissociate iPSCs (80% confluent) and prepare a single-cell suspension using reagent B.
		<ol>
			<li>Wash the iPSCs once with pre-warmed (37 &#176;C) DMEM/F12.</li>
			<li>Add pre-warmed reagent B (e.g., 1 mL in a 60 mm dish) and incubate the iPSCs for 5 min at 37 &#176;C and 5% CO2.</li>
			<li>Flick 20 times with a fingertip on the side and bottom of the dish to detach the iPSCs. Check for the detachment of cells under the microscope.</li>
			<li>Pipette the cell suspension up and down in the dish 5 times with a 1-mL micropipette.</li>
			<li>Add 3 mL of medium A to dilute 1 mL of reagent B and collect the cell suspension in a 15-mL centrifuge tube.</li>
			<li>Gently spin down the iPSCs (500 x g) for 4 min at room temperature.</li>
			<li>Resuspend the cell pellet in 1 mL of medium B and count the cell number with a hemocytometer. 
			NOTE: Be aware of using only medium B for resuspension. Avoid using medium A, as it contains a too-high concentration of bFGF, which might inhibit the differentiation.</li>
		</ol>
		</li>
		<li>Dilute the cell suspension to 4.5 x 105 cells per mL in medium B supplemented with 10 &#181;M rho-associated protein kinase inhibitor (Y-27632).</li>
		<li>Add 100 &#181;L per well in a non-adherent, v-bottom, 96-well plate. 
		NOTE: Make sure the cells are equally distributed in the suspension by shaking the tube each time before taking out 100 &#181;L portions. It is important that each well should contain an equivalent cell number in order to obtain neurospheres homogenous in size and shape (round, defined surfaces).</li>
		<li>Gently spin down the plate with the cells at 500 x g and room temperature for 3 min and incubate at 37 &#176;C and 5% CO2.</li>
		<li>Change the medium daily by removing 50 &#181;L and adding 50 &#181;L of fresh medium B into each well for the next 5 days.</li>
	</ol>
	</li>
</ol>
2. Embedding Neurospheres in EHS Matrix (4 days)
<ol>
	<li>Prepare neurosphere medium by mixing the following: 1:1 mixture of DMEM/F12 and medium C (v/v), 1:200 (v/v) supplement 1, 1:100 (v/v) supplement 2 without (w/o) Vitamin A, 1:100 L-glutamine, 0.05 mM non-essential amino acids (MEM), 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 1.6 g/L insulin, and 0.05 mM &#946;-mercaptoethanol.</li>
	<li>Collect the neurospheres with a 200 &#181;L micropipette using a ~2 mm tip previously cut with sterile scissors.</li>
	<li>Place the neurospheres approximately 5 mm away from each other on paraffin film (3 x 3 cm2) in an empty 100 mm dish and carefully remove as much of the remaining medium as possible.</li>
	<li>Add a drop (7 &#181;L) of EHS matrix onto each single neurosphere.</li>
	<li>Incubate the EHS matrix drops with the neurospheres for 15 min in an incubator.</li>
	<li>Wash the neurospheres carefully from the paraffin film by flushing them with neurosphere medium. To flush, use a 1 mL micropipette and a new 100 mm Petri dish containing 10 mL of neurosphere medium.</li>
	<li>Incubate the neurospheres for the next 4 days and add 2 mL of fresh neurosphere medium on day 2. 
	NOTE: Make sure that the shelves in the incubator are flat so that the EHS matrix-embedded neurospheres will not clump together on one side of the dish.</li>
</ol>
3. Organoids in a Rotary Suspension Culture (14 Days)
<ol>
	<li>Prepare brain organoid medium by mixing the following: 1:1 mixture of DMEM/F12 and medium C (v/v), 1:200 (v/v) supplement 1, 1:100 (v/v) supplement 2 w/o Vitamin A, 1:100 L-glutamine, 0.05 mM MEM, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 1.6 g/L insulin, 0.5 &#181;M dorsomorphin, 5 &#181;M SB431542, and 0.05 mM &#946;-mercaptoethanol.</li>
	<li>Add 100 mL of brain organoid medium to each spinner flask through its side arms and place them in an incubator for pre-warming for at least 20 min.</li>
	<li>Set up a stirring program at 25 rpm, according to the manufacturer&#39;s instructions. 
	NOTE: Before transferring the EHS matrix-embedded neurospheres into spinner flasks, make sure that they are all separated. If two or more are connected through EHS matrix, separate them by cutting the connecting matrix with a scalpel.</li>
	<li>Carefully transfer the EHS matrix-embedded neurospheres into spinner flasks containing 100 mL of organoid medium using a 2 mL serological pipette. Use the side arms of the spinner flask to transfer the neurospheres into the flask.</li>
	<li>Place the spinner flasks on a magnetic stirring platform in an incubator at 37 &#176;C and 5% CO2; this is day 0 of the organoid culture.</li>
	<li>Change the medium once per week (or more often when there is a color change) by removing half of the medium and adding the same amount of fresh medium. 
	NOTE: When taking the spinner flasks out of the incubator, wait 3-5 min to let the organoids sink down to the bottom of the flask. Remove the medium by placing the glass pipette tip (connected to a pump) on the surface of the liquid; aspirate the medium carefully through one side opening/arm of the flask. These manipulations must be done under the laminar hood.</li>
</ol>
4. Analysis of Brain Organoids
<ol>
	<li>Fixation of organoids
	<ol>
		<li>Collect the organoids on day 14 of the spinner flask culture with a cut 1 mL micropipette (cut ~5 mm). Put all of them in a 60 mm dish and wash them once with 5 mL of warm DMEM/F12 for 3 min.</li>
		<li>Prepare a 1.5 mL tube with 500 &#181;L of warm 4% paraformaldehyde (PFA). 
		Caution: Wear skin and eye protection and work under a safety hood when handling PFA fixative.</li>
		<li>Place each organoid separately in each tube and fix them for at least 30 min at room temperature. Do not fix the organoids for longer than 60 min. To move the organoids, use an inoculation loop or any other devise that is convenient.</li>
		<li>Remove the PFA and wash the fixed organoids twice for 10 min each with 1 mL of PBS.</li>
		<li>Store the organoids in 1 mL of PBS at 4 &#176;C for up to 7 days, until further use.</li>
	</ol>
	</li>
	<li>Embedding the organoids for cryosectioning
	<ol>
		<li>Remove the PBS and add 1 mL of 30% sucrose in distilled water solution per tube to dehydrate the organoids before cryofreezing them; after adding sucrose solution, the organoids should be floating at the surface. Store the organoids overnight in sucrose solution at 4 &#176;C; by the next day, the organoids should have sunk down to the bottom of the tube. 
		NOTE: Organoids can be stored for up to 3-5 days at 4 &#176;C in sucrose solution, if necessary.</li>
		<li>Fill a vinyl specimen mold with 400 &#181;L of optimum cutting temperature (OCT) compound and use an inoculation loop to place an organoid at the center of the mold. Label the rim of the mold with the sample name.</li>
		<li>Freeze the organoid-containing mold at -80 &#176;C until cryosectioning.</li>
		<li>Coat glass cryoslides with 0.1% poly-l-lysine solution (PLL) in PBS for 5 min at room temperature and let the slides dry for 3 h. Store the slides at 4 &#176;C and warm them up to room temperate before use. 
		NOTE: PLL-coating is an important step, as it will prevent the organoid slices from floating away. Collect and store PLL solution at 4 &#176;C for up to 3 months. Before reuse, filter it with a 0.22-&#181;m syringe and let it warm to room temperature.</li>
		<li>Section cryofrozen organoids into 20-50 &#181;m-thick slices on PLL-coated glass cryoslides22. Let the slides with the sections dry for 1 h at room temperature. Store the sections at -80 &#176;C until further processing.</li>
	</ol>
	</li>
</ol>
5. Immunofluorescent Staining of Organoid Sections
NOTE: For the general characterization of organoids, staining with nestin, a neural progenitor marker, and TUJ1, a pan-neuronal marker, is recommended. As additional examples, immunofluorescent staining with phospho-Vimentin (p-Vim), which labels mitotic apical radial glial cells, and Arl13b, for cilium, are described. To test apoptosis, use the Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. Place the slides in a plastic box during the incubations to protect them from dust, light, and drying out.
<ol>
	<li>Thaw the slides for 30 min at room temperature.</li>
	<li>Wash the slides twice with 200 &#181;L of PBS-glycine (0.225 g of glycine in PBS) for 3 min to quench the PFA-induced autofluorescence.</li>
	<li>Permeabilize the sections with 200 &#181;L of 0.5% Triton X100/0.1% tween in PBS solution for 10 min at room temperature.</li>
	<li>Wash them twice with 200 &#181;L of PBS-glycine solution for 3 min.</li>
	<li>Incubate them with 200 &#181;L of 0.5% fish gelatin/0.1% Triton X100 in PBS for 1 h at room temperature or overnight at 4 &#176;C to block unspecific antigen binding. 
	NOTE: If a TUNEL assay is required, perform the assay as per the manufacturer&#39;s protocol. Start with the TUNEL assay before performing the immunostaining, as it might interfere with secondary antibodies and quench fluorophores when used afterwards.</li>
	<li>Dilute the antibodies in blocking solution at the following concentrations: nestin, 1:200; p-Vim, 1:500; TUJ1, 1:200; Arl13b, 1:20; and secondary antibodies, 1:1,000.</li>
	<li>Incubate with 200 &#181;L of the first primary antibody (e.g., nestin) for 1-2 h at room temperature or overnight at 4 &#176;C.</li>
	<li>Wash 3 x 3 min with 200 &#181;L of blocking solution.</li>
	<li>Dilute the first (anti-mouse 488) secondary antibody 1:1,000 in blocking solution and incubate the slide in 200 &#181;L for 1-2 h at room temperature. From now on, always protect the slides from light.</li>
	<li>Wash 3 x 3 min with 200 &#181;L of blocking solution.</li>
	<li>Incubate with 200 &#181;L of the next primary antibody (e.g., TUJ1) for 1-2 h at room temperature or overnight at 4 &#176;C.</li>
	<li>Wash 3 x 3 min with 200 &#181;L of blocking solution.</li>
	<li>Add 200 &#181;L of the next secondary antibody (anti-rabbit 647) for 1-2 h at room temperature.</li>
	<li>Wash 3 x 3 min. with 200 &#181;L of blocking solution.</li>
	<li>Add 200 &#181;L of 4&#39;,6-diamidino-2-phenylindole (DAPI) at a 30 nM concentration in PBS for 15 min at room temperature for nuclear staining.</li>
	<li>Wash 2 x 3 min with 200 &#181;L of blocking solution.</li>
	<li>Wash 1 x 1 min with 200 &#181;L of distilled water and let the sections dry for 10-20 min, until no obvious water drop is visible any more.</li>
	<li>Mount the sections with embedding medium. Store them protected from light at 4 &#176;C for up to several weeks.</li>
	<li>Proceed with microscopic analysis to image an overview of the organoid, ventricular zone, primitive cortical plate, and other areas of interest.</li>
	<li>Use a confocal microscope with a 63X oil objective and fluorescent filters, chosen according to the fluorescent dye-tagged secondary antibodies used1,10.</li>
</ol>

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<li>Gabriel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+Zika+Virus+Isolates+Induce+Premature+Differentiation+of+Neural+Progenitors+in+Human+Brain+Organoids%5BTitle%5D)+AND+%22Cell+stem+cell%22%5BJournal%5D)">Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids.</a> Cell stem cell. , (2017).</li>
<li>Wu, J., Hunt, S. D., Xue, H., Liu, Y., Darabi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+and+Characterization+of+a+MYF5+Reporter+Human+iPS+Cell+Line+Using+CRISPR%2FCas9+Mediated+Homologous+Recombination%5BTitle%5D)+AND+%22Sci.+Rep%22%5BJournal%5D)">Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination.</a> Sci. Rep. 6, 18759(2016).</li>
<li>Li, H. L., Gee, P., Ishida, K., Hotta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+genomic+correction+methods+in+human+iPS+cells+using+CRISPR-Cas9+system%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.</a> Methods. 101, 27-35 (2016).</li>
<li>Grobarczyk, B., Franco, B., Hanon, K., Malgrange, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+Isogenic+Human+iPS+Cell+Line+Precisely+Corrected+by+Genome+Editing+Using+the+CRISPR%2FCas9+System%5BTitle%5D)+AND+%22Stem+Cell+Reviews%22%5BJournal%5D)">Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System.</a> Stem Cell Reviews. 11, 774-787 (2015).</li>
<li>Camp, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+cerebral+organoids+recapitulate+gene+expression+programs+of+fetal+neocortex+development%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.</a> Proc. Natl. Acad. Sci. U. S. A. 112, 15672-15677 (2015).</li>
<li>Qian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain-Region-Specific+Organoids+Using+Mini-bioreactors+for+Modeling+ZIKV+Exposure%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure.</a> Cell. 165, 1238-1254 (2016).</li>
<li>Jo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Midbrain-like+Organoids+from+Human+Pluripotent+Stem+Cells+Contain+Functional+Dopaminergic+and+Neuromelanin-Producing+Neurons%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons.</a> Cell Stem Cell. 19, 248-257 (2016).</li>
<li>Mariani, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FOXG1-Dependent+Dysregulation+of+GABA%2FGlutamate+Neuron+Differentiation+in+Autism+Spectrum+Disorders%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">FOXG1-Dependent Dysregulation of GABA/Glutamate Neuron Differentiation in Autism Spectrum Disorders.</a> Cell. 162, 375-390 (2015).</li>
<li>D'Avanzo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alzheimer%27s+in+3D+culture%3A+challenges+and+perspectives%5BTitle%5D)+AND+%22Bioessays%22%5BJournal%5D)">Alzheimer's in 3D culture: challenges and perspectives.</a> Bioessays. 37, 1139-1148 (2015).</li>
</ol>

The authors declare that they have no competing financial interests.

MCPH is a complex human neurodevelopmental disorder that cannot be recapitulated in animal models in vivo or in simple human cell culture approaches in vitro. The clinical manifestation of MCPH begins to appear during the first trimester, when early neurogenesis begins. Thus, 3D brain organoids represent a reliable experimental system to model MCPH development. In addition, 3D human brain organoids are an ideal approach since i) they allow for the adaptation of a spectrum of patient samples with various...

Human neurodevelopmental disorders, such as microcephaly, can only be poorly studied in animal models due to the fact that human brains have an extended cortical surface, a unique feature differing from non-human animals.
This aspect makes human brain development a complex process that cannot be sufficiently studied in a 2D, in vitro cell culture system. Emerging 3D culture techniques allow the generation of tissue-like organoids from induced pluripotent stem cells (iPSCs). The in vitro differentiation of pluripotent stem cells in a 3D suspension culture allows the formation of various cell types in a timely and region-specific manner, giving rise to an organized, stratified tissue1,2,3. Thanks to laboratories that pioneered 3D culture technologies and demystified the complexity of organ formation, starting from stem cells, we developed a robust method of generating brain organoids to delineate early events of human brain development and to model microcephaly in vitro1,2,3. It is noteworthy that we adapted the original method developed by Lancaster et al. to generate cerebral organoids1. This method was modified according to our experimental requirements.
The aim of a study from Gabriel et al. was to analyze the cellular and molecular mechanisms of neural stem cell maintenance during brain development. In order to do this, a mechanistic study was performed by analyzing neural progenitor cells (NPCs) in 3D brain organoids derived from a microcephaly patient4. This patient carried a mutation in CPAP, a conserved centrosomal protein required for centrosome biogenesis5. A widely accepted hypothesis is that microcephaly is the result of a depletion of the NPC pool, and this might be due either to cell death or to premature differentiation1,6,7,8,9.
By analyzing the ventricular zones (VZs) of microcephaly brain organoids, it was shown that a significant number of NPCs undergo asymmetrical cell division, unlike brain organoids derived from a healthy donor4. Extensive microscopic and biochemical analyses of microcephalic brain organoids revealed an unexpected role for CPAP in timely cilia disassembly4. Specifically, mutated CPAP is associated with retarded cilium disassembly and delayed cell cycle re-entry, leading to the premature differentiation of NPCs4. These results suggest a role for cilia in microcephaly and their involvement during neurogenesis and brain size control10.
The first part of this protocol is a description of a three-step method to generate homogenous brain organoids. As mentioned before, the original Lancaster protocol was adapted and modified to suit our purpose1. First, human iPSCs are cultured in a defined feeder-free condition on Engelbreth-Holm-Swarm (EHS) matrix. This step avoids the variations of feeder-dependent pluripotent stem cell cultures. In this protocol, the induction of neural differentiation to form neural epithelium starts directly from iPSCs. By skipping the embryoid body (EB) formation step, the neural differentiation proceeds in a more controlled and directed manner. This approach limits the spontaneous and undirected formation of other germ cell layers, such as mesoderm and endoderm. By applying this protocol, neurospheres containing neural rosettes can be harvested on day 5 for EHS matrix embedding and stationary suspension culture. The organoid medium used for the third step of our protocol is supplemented with dorsomorphin and SB431542. Dorsomorphin is a small-molecule inhibitor of bone morphogenic protein (BMP), and SB431542 inhibits the TGF&#946;/activin/nodal signaling pathway. The combination of these factors could promote neural differentiation more efficiently than retinoic acid alone11,12,13,14.
Altogether, these modifications enable the reproducible generation of brain organoids, with minimal variations across organoids. Importantly, this method was applied to robustly generate microcephalic brain organoids from patient iPSCs, which carry mutations in genes that affect centrosomes and cell-cycle dynamics.
The second part of this protocol gives instructions to prepare brain organoids for the analysis and interpretation of cellular defects in microcephaly. This includes fixation, cryosectioning, immunofluorescent staining, and confocal microscopic analysis. This protocol will provide the reader with a detailed description of expected results and with guidance for interpretation.

<table><tbody><tr><td>Anti-mouse 488</td><td>Invitrogen</td><td>A-11001</td><td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488</td></tr><tr><td>Anti-rabbit 647</td><td>Invitrogen</td><td>A-21245</td><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647</td></tr><tr><td>Arl13b</td><td>proteintech</td><td>17711-1-AP</td><td>ARL13B rabbit polyclonal antibody&amp;nbsp;</td></tr><tr><td>CELLSPIN system</td><td>IBS Integra Bioscience</td><td>183001</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich, US</td><td>32670</td><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride; multiple suppliers</td></tr><tr><td>DMEM/F-12</td><td>Gibco, US</td><td>31331093</td><td>Dulbecco&#039;s Modified Eagle Medium: Nutrient Mixture F-12&amp;nbsp;</td></tr><tr><td>Dorsomorphin</td><td>Sigma-Aldrich, US</td><td>P5499</td><td>Compound C; multiple suppliers</td></tr><tr><td>Embedding medium</td><td>AppliChem</td><td>A9011, 0100</td><td>Mowiol; embedding medium; multiple suppliers</td></tr><tr><td>Engelbreth-Holm-Swarm (EHS) matrix</td><td>Corning</td><td>354277</td><td>Matrigel hESC-qualified matrix; important: hESC qualified</td></tr><tr><td>Fish gelatin&amp;nbsp;</td><td>Sigma-Aldrich, US</td><td>G7765-250ML</td><td>Gelatin from cold water fish skin; multiple suppliers; autoclave after adding to PBS to dissolve and sterilize, store at 4 &amp;deg;C</td></tr><tr><td>Glycine</td><td>AppliChem</td><td>A1067,1000</td><td>Glycine for molecular biology; multiple suppliers&amp;nbsp;</td></tr><tr><td>Inoculation loop with needle, disposable (1 &amp;micro;L)</td><td>Sigma Aldrich, US</td><td>BR452201-1000EA</td><td>multiple suppliers&amp;nbsp;</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich, US</td><td>I3536-100MG</td><td>multiple suppliers</td></tr><tr><td>L-glutamine</td><td>Gibco, US</td><td>25030081</td><td>L-glutamine (200 mM)</td></tr><tr><td>Medium A</td><td>Stem cell technologies</td><td>#05850</td><td>mTeSR1 (hiPSC medium)</td></tr><tr><td>Medium B</td><td>Stem cell technologies</td><td>#05835</td><td>Neural induction medium (NIM); neural differentiation medium</td></tr><tr><td>Medium C</td><td>Gibco, US</td><td>21103049</td><td>Neural Basal Medium</td></tr><tr><td>MEM</td><td>Gibco, US</td><td>11140035</td><td>MEM non-essential amino acids solution (100x)</td></tr><tr><td>MycoAlert Mycoplasma Detection Kit</td><td>Lonza, Switzerland</td><td>#LT07-218</td><td>Mycoplasma detection kit; multiple suppliers</td></tr><tr><td>Nestin</td><td>Novus biologicals</td><td>NBP1-92717</td><td>Nestin mouse monoclonal antibody (4D11)</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>AppliChem</td><td>A3813, 0500</td><td>4% in PBS, store solution at -20 &amp;deg;C; caution: wear skin and eye protection and work under hood&amp;nbsp;</td></tr><tr><td>PBS tablets</td><td>Gibco, US</td><td>18912014</td><td>See manufacturer&amp;acute;s instructions; multiple suppliers</td></tr><tr><td>Penicillin-Streptomycin (10.000 U/mL)</td><td>Gibco, US</td><td>15140122</td><td>Multiple suppliers</td></tr><tr><td>Poly-L-lysine solution (PLL)</td><td>Sigma-Aldrich, US</td><td>P8920-100ML</td><td>Multiple suppliers</td></tr><tr><td>pVim</td><td>MBL</td><td>D076-3S</td><td>Phospho-Vimentin (Ser55) mAb</td></tr><tr><td>Reagent A&amp;nbsp;</td><td>Stem cell technologies</td><td># 05872</td><td>Note to Protocol 1.1.1.2; ReLSR (Enzyme-free human ES and iPS cell selection and passaging reagent); please follow manufactorer&amp;acute;s protocol; alternative products from muliple suppliers available</td></tr><tr><td>Reagent B&amp;nbsp;</td><td>Sigma-Aldrich, US</td><td>A6964-100ML</td><td>Accutase solution is an enzymatic solution for single cell dissociation; multiple suppliers; protocol 1.1.2 &quot;enzymatic cell dissociation solution&amp;rdquo;&amp;nbsp;</td></tr><tr><td>Research Cryostat Leica CM3050 S</td><td>Leica biosystems</td><td>CM3050 S</td><td>Multiple suppliers</td></tr><tr><td>SB431542</td><td>Selleckchem.com</td><td>S1067</td><td>Multiple suppliers</td></tr><tr><td>Spinner flask 250 mL</td><td>IBS Integra Bioscience</td><td>182026</td><td></td></tr><tr><td>Sucrose</td><td>AppliChem</td><td>A4734, 1000</td><td>Multiple suppliers</td></tr><tr><td>Superfrost ultra plus microscope slides</td><td>Thermo scientific, US</td><td>J3800AMNZ</td><td>Slides should be labeled with a &quot;+&quot; and positively charged</td></tr><tr><td>Supplement 1</td><td>Gibco, US</td><td>17502048</td><td>N-2 supplement (100x)</td></tr><tr><td>Supplement 2 w/o Vitamin A</td><td>Gibco, US</td><td>12587010</td><td>B-27 supplement (50x), minus vitamin A; multiple suppliers</td></tr><tr><td>Tissue-Tek Cryomold</td><td>Sakura, NL</td><td>4565</td><td>Multiple suppliers</td></tr><tr><td>Tissue-Tek O.C.T. compound</td><td>Sakura, NL</td><td>4583</td><td>Multiple suppliers</td></tr><tr><td>Triton X-100</td><td>AppliChem</td><td>A1388,0500</td><td>Multiple suppliers</td></tr><tr><td>TUJ1</td><td>Sigma-Aldrich, US</td><td>T2200</td><td>&amp;beta;-Tubulin III (rabbit polyclonal)</td></tr><tr><td>TUNEL assay</td><td>Promega, US</td><td>G3250</td><td>DeadEnd Fluorometric TUNEL system; multiple suppliers</td></tr><tr><td>Tween 20 for molecular biology</td><td>AppliChem</td><td>A4974,0500</td><td>Multiple suppliers</td></tr><tr><td>waterproof sheet</td><td>BEMIS company, inc.</td><td>PM996</td><td>Parafilm &amp;ldquo;M&amp;rdquo;; multiple suppliers</td></tr><tr><td>Y-27632&amp;nbsp;</td><td>Selleckchem.com</td><td>S1049</td><td>ROCK-inhibitor (Y-27632 2HCL); multiple suppliers</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Gibco, US</td><td>31350010</td><td>2-mercaptoethanol (50 mM); multiple suppliers</td></tr></tbody></table>

generation of ipsc-derived human brain organoids to model early neurodevelopmental disorders

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

The generation of brain organoids requires at least three weeks of continuous culturing (Figure 1A). To accomplish reproducible results, we recommend that the researcher documents every step and, importantly, avoids any alterations regarding medium components, time points, and cell handling. Here, we give a summary of how to evaluate if critical milestones are reached in order to obtain organoids of sufficient quality at the end of the experiment. The formation of neurosp...

Watch this Scientific Journal Video about Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders at JoVE.com

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

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

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

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20.6K Views.  University of Cologne. Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described. 

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

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

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

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

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

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

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

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

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

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

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

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

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

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

Genetic Modification of Primate Cerebral Organoids

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

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

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

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

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

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

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

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

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

Generating Human Brain Organoids with Resident Microglia Using iPSC-Derived HPCs

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated&#160;into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

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.

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 ...

Neuroscience

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 Vitro Method to Generate Human Brain Organoids

Source: Gabriel, E., et al., <a href="https://app.jove.com/v/55372">Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders.</a> J. Vis. Exp. (2017)
The video demonstrates a method for generating human brain organoids from induced pluripotent stem cells. It shows how the neurosphere forms a neuroectodermal layer, which suppresses the formation of other germ layers. The neurospheres are embedded in a 3D gel matrix and form neuroepithelial buds. When incubated with organoid media, these neuroepithelial buds differentiate into specific brain regions, resulting in the formation of mature brain organoids.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Generation of Brain Organoids (23 ...

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

Summary

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An In Vitro Method to Generate Human Brain Organoids