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>

<ol>
	<li>Lancaster, M. A., 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=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 501, 373-379 (2013).</li><li>Eiraku, M., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+embryonic+stem+cell+culture+for+generation+of+three-dimensional+retinal+and+cortical+tissues.">Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.</a> Nat Protoc. 7, 69-79 (2012).</li><li>Nakano, 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=Self-formation+of+optic+cups+and+storable+stratified+neural+retina+from+human+ESCs.">Self-formation of optic cups and storable stratified neural retina from human ESCs.</a> Cell Stem Cell. 10, 771-785 (2012).</li><li>Gabriel, E., 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=CPAP+promotes+timely+cilium+disassembly+to+maintain+neural+progenitor+pool.">CPAP promotes timely cilium disassembly to maintain neural progenitor pool.</a> EMBO J. 35 (8), 803-819 (2016).</li><li>Al-Dosari, M. S., Shaheen, R., Colak, D., Alkuraya, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+CENPJ+mutation+causes+Seckel+syndrome.">Novel CENPJ mutation causes Seckel syndrome.</a> J Med Genet. 47, 411-414 (2010).</li><li>Wollnik, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+mechanism+for+microcephaly.">A common mechanism for microcephaly.</a> Nat Genet. 42, 923-924 (2010).</li><li>Thornton, G. K., Woods, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+microcephaly:+do+all+roads+lead+to+Rome?.">Primary microcephaly: do all roads lead to Rome?.</a> Trends Genet. 25, 501-510 (2009).</li><li>Barbelanne, M., Tsang, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+basis+of+autosomal+recessive+primary+microcephaly.">Molecular and cellular basis of autosomal recessive primary microcephaly.</a> Biomed Res Int. 2014, 547986 (2014).</li><li>Homem, C. C., Repic, M., 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=Proliferation+control+in+neural+stem+and+progenitor+cells.">Proliferation control in neural stem and progenitor cells.</a> Nat. Rev. Neurosci. 16, 647-659 (2015).</li><li>Gabriel, E., 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=CPAP+promotes+timely+cilium+disassembly+to+maintain+neural+progenitor+pool.">CPAP promotes timely cilium disassembly to maintain neural progenitor pool.</a> EMBO J. 35, 803-819 (2016).</li><li>Mak, S. K., 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=Small+molecules+greatly+improve+conversion+of+human-induced+pluripotent+stem+cells+to+the+neuronal+lineage.">Small molecules greatly improve conversion of human-induced pluripotent stem cells to the neuronal lineage.</a> Stem Cells Int. , (2012).</li><li>Morizane, A., Doi, D., Kikuchi, T., Nishimura, K., Takahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+bone+morphogenic+protein+and+activin/nodal+signals+promote+highly+efficient+neural+induction+from+human+pluripotent+stem+cells.">Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells.</a> J. Neurosci. Res. 89, 117-126 (2011).</li><li>Shi, Y., Kirwan, P., Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+pluripotent+stem+cells+to+cerebral+cortex+neurons+and+neural+networks.">Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks.</a> Nat Protoc. 7, 1836-1846 (2012).</li><li>Zhou, J., 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=High-efficiency+induction+of+neural+conversion+in+human+ESCs+and+human+induced+pluripotent+stem+cells+with+a+single+chemical+inhibitor+of+transforming+growth+factor+beta+superfamily+receptors.">High-efficiency induction of neural conversion in human ESCs and human induced pluripotent stem cells with a single chemical inhibitor of transforming growth factor beta superfamily receptors.</a> Stem Cells. 28, 1741-1750 (2010).</li><li>Takahashi, K., 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Okita, K., 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=A+more+efficient+method+to+generate+integration-free+human+iPS+cells.">A more efficient method to generate integration-free human iPS cells.</a> Nat Methods. 8, 409-412 (2011).</li><li>Yu, J., 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+induced+pluripotent+stem+cells+free+of+vector+and+transgene+sequences.">Human induced pluripotent stem cells free of vector and transgene sequences.</a> Science. 324, 797-801 (2009).</li><li>Lieu, P. T., Fontes, A., Vemuri, M. C., Macarthur, C. C. <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+induced+pluripotent+stem+cells+with+CytoTune,+a+non-integrating+Sendai+virus.">Generation of induced pluripotent stem cells with CytoTune, a non-integrating Sendai virus.</a> Methods Mol. Biol. 997, 45-56 (2013).</li><li>Bai, Q., 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=Temporal+analysis+of+genome+alterations+induced+by+single-cell+passaging+in+human+embryonic+stem+cells.">Temporal analysis of genome alterations induced by single-cell passaging in human embryonic stem cells.</a> Stem Cells Dev. 24, 653-662 (2015).</li><li>Garitaonandia, 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=Increased+risk+of+genetic+and+epigenetic+instability+in+human+embryonic+stem+cells+associated+with+specific+culture+conditions.">Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions.</a> PLoS One. 10, e0118307 (2015).</li><li>Ohnuma, K., 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=Enzyme-free+passage+of+human+pluripotent+stem+cells+by+controlling+divalent+cations.">Enzyme-free passage of human pluripotent stem cells by controlling divalent cations.</a> Sci. Rep. 4, 4646 (2014).</li><li>Currle, D. S., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+freezing+and+cryosectioning+E12.5+mouse+brain.">Flash freezing and cryosectioning E12.5 mouse brain.</a> J Vis Exp. , (2007).</li><li>Yingling, J., 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=Neuroepithelial+stem+cell+proliferation+requires+LIS1+for+precise+spindle+orientation+and+symmetric+division.">Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division.</a> Cell. 132, 474-486 (2008).</li><li>Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+step+for+the+cell,+a+giant+leap+for+mankind:+a+hypothesis+of+neocortical+expansion+during+evolution.">A small step for the cell, a giant leap for mankind: a hypothesis of neocortical expansion during evolution.</a> Trends Neurosci. 18, 383-388 (1995).</li><li>Hu, B. Y., 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=Neural+differentiation+of+human+induced+pluripotent+stem+cells+follows+developmental+principles+but+with+variable+potency.">Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 4335-4340 (2010).</li><li>Wilson, P. G., Stice, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+differentiation+of+neural+rosettes+derived+from+human+embryonic+stem+cells.">Development and differentiation of neural rosettes derived from human embryonic stem cells.</a> Stem Cell Reviews. 2, 67-77 (2006).</li><li>Dhara, S. K., Stice, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+differentiation+of+human+embryonic+stem+cells.">Neural differentiation of human embryonic stem cells.</a> J. Cell Biochem. 105, 633-640 (2008).</li><li>Christie, V. B., 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=Synthesis+and+evaluation+of+synthetic+retinoid+derivatives+as+inducers+of+stem+cell+differentiation.">Synthesis and evaluation of synthetic retinoid derivatives as inducers of stem cell differentiation.</a> Org Biomol Chem. 6, 3497-3507 (2008).</li><li>Kim, D. 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=Robust+enhancement+of+neural+differentiation+from+human+ES+and+iPS+cells+regardless+of+their+innate+difference+in+differentiation+propensity.">Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity.</a> Stem Cell Reviews. 6, 270-281 (2010).</li><li>Gabriel, E., 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=Recent+Zika+Virus+Isolates+Induce+Premature+Differentiation+of+Neural+Progenitors+in+Human+Brain+Organoids.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+Characterization+of+a+MYF5+Reporter+Human+iPS+Cell+Line+Using+CRISPR/Cas9+Mediated+Homologous+Recombination.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+genomic+correction+methods+in+human+iPS+cells+using+CRISPR-Cas9+system.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Isogenic+Human+iPS+Cell+Line+Precisely+Corrected+by+Genome+Editing+Using+the+CRISPR/Cas9+System.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cerebral+organoids+recapitulate+gene+expression+programs+of+fetal+neocortex+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-Region-Specific+Organoids+Using+Mini-bioreactors+for+Modeling+ZIKV+Exposure.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midbrain-like+Organoids+from+Human+Pluripotent+Stem+Cells+Contain+Functional+Dopaminergic+and+Neuromelanin-Producing+Neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FOXG1-Dependent+Dysregulation+of+GABA/Glutamate+Neuron+Differentiation+in+Autism+Spectrum+Disorders.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+in+3D+culture:+challenges+and+perspectives.">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 border="0" cellpadding="0" cellspacing="0" width="670">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-mouse 488</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">A-11001</td>
			<td style="width:212px;">Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-rabbit 647</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">A-21245</td>
			<td style="width:212px;">Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Arl13b</td>
			<td style="width:123px;">proteintech</td>
			<td style="width:119px;">17711-1-AP</td>
			<td style="width:212px;">ARL13B rabbit polyclonal antibody&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CELLSPIN system</td>
			<td style="width:123px;">IBS Integra Bioscience</td>
			<td style="width:119px;">183001</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DAPI</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">32670</td>
			<td style="width:212px;">4&#8242;,6-Diamidino-2-phenylindole dihydrochloride; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM/F-12</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">31331093</td>
			<td style="width:212px;">Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dorsomorphin</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">P5499</td>
			<td style="width:212px;">Compound C; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Embedding medium</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A9011, 0100</td>
			<td style="width:212px;">Mowiol; embedding medium; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Engelbreth-Holm-Swarm (EHS) matrix</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">354277</td>
			<td style="width:212px;">Matrigel hESC-qualified matrix; important: hESC qualified</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fish gelatin&#160;</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">G7765-250ML</td>
			<td style="width:212px;">Gelatin from cold water fish skin; multiple suppliers; autoclave after adding to PBS to dissolve and sterilize, store at 4&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glycine</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A1067,1000</td>
			<td style="width:212px;">Glycine for molecular biology; multiple suppliers&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inoculation loop with needle, disposable (1 &#181;l)</td>
			<td style="width:123px;">Sigma Aldrich, US</td>
			<td style="width:119px;">BR452201-1000EA</td>
			<td style="width:212px;">multiple suppliers&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insulin</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">I3536-100MG</td>
			<td style="width:212px;">multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-glutamine</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">25030081</td>
			<td style="width:212px;">L-glutamine (200 mM)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Medium A</td>
			<td style="width:123px;">Stem cell technologies</td>
			<td style="width:119px;">#05850</td>
			<td style="width:212px;">mTeSR1 (hiPSC medium)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Medium B</td>
			<td style="width:123px;">Stem cell technologies</td>
			<td style="width:119px;">#05835</td>
			<td style="width:212px;">Neural induction medium (NIM); neural differentiation medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Medium C</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">21103049</td>
			<td style="width:212px;">Neural Basal Medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MEM</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">11140035</td>
			<td style="width:212px;">MEM non-essential amino acids solution (100x)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MycoAlert Mycoplasma Detection Kit</td>
			<td style="width:123px;">Lonza, Switzerland</td>
			<td style="width:119px;">#LT07-218</td>
			<td style="width:212px;">Mycoplasma detection kit; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nestin</td>
			<td style="width:123px;">Novus biologicals</td>
			<td style="width:119px;">NBP1-92717</td>
			<td style="width:212px;">Nestin mouse monoclonal antibody (4D11)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Paraformaldehyde (PFA)</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A3813, 0500</td>
			<td style="width:212px;">4% in PBS, store solution at -20&#176;C; caution: wear skin and eye protection and work under hood&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PBS tablets</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">18912014</td>
			<td style="width:212px;">See manufacturer&#180;s instructions; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin-Streptomycin (10.000 U/ml)</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">15140122</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly-L-lysine solution (PLL)</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">P8920-100ML</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pVim</td>
			<td style="width:123px;">MBL</td>
			<td style="width:119px;">D076-3S</td>
			<td style="width:212px;">Phospho-Vimentin (Ser55) mAb</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Reagent A&#160;</td>
			<td style="width:123px;">Stem cell technologies</td>
			<td style="width:119px;"># 05872</td>
			<td style="width:212px;">Note to Protocol 1.1.1.2; ReLSR (Enzyme-free human ES and iPS cell selection and passaging reagent); please follow manufactorer&#180;s protocol; alternative products from muliple suppliers available</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Reagent B&#160;</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">A6964-100ML</td>
			<td style="width:212px;">Accutase solution is an enzymatic solution for single cell dissociation; multiple suppliers; protocol 1.1.2 &#34;enzymatic cell dissociation solution&#8221;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Research Cryostat Leica CM3050 S</td>
			<td style="width:123px;">Leica biosystems</td>
			<td style="width:119px;">CM3050 S</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SB431542</td>
			<td style="width:123px;">Selleckchem.com</td>
			<td style="width:119px;">S1067</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spinner flask 250 ml</td>
			<td style="width:123px;">IBS Integra Bioscience</td>
			<td style="width:119px;">182026</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A4734, 1000</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Superfrost ultra plus microscope slides</td>
			<td style="width:123px;">Thermo scientific, US</td>
			<td style="width:119px;">J3800AMNZ</td>
			<td style="width:212px;">Slides should be labeled with a &#34;+&#34; and positively charged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Supplement 1</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">17502048</td>
			<td style="width:212px;">N-2 supplement (100x)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Supplement 2 w/o Vitamin A</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">12587010</td>
			<td style="width:212px;">B-27 supplement (50x), minus vitamin A; multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue-Tek Cryomold</td>
			<td style="width:123px;">Sakura, NL</td>
			<td style="width:119px;">4565</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:123px;">Sakura, NL</td>
			<td style="width:119px;">4583</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A1388,0500</td>
			<td style="width:212px;">Multiple suppliers multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TUJ1</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">T2200</td>
			<td style="width:212px;">&#946;-Tubulin III (rabbit polyclonal)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TUNEL assay</td>
			<td style="width:123px;">Promega, US</td>
			<td style="width:119px;">G3250</td>
			<td style="width:212px;">DeadEnd Fluorometric TUNEL system; multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween 20 for molecular biology</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A4974,0500</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">waterproof sheet</td>
			<td style="width:123px;">BEMIS company, inc.</td>
			<td style="width:119px;">PM996</td>
			<td style="width:212px;">Parafilm &#8220;M&#8221;; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Y-27632&#160;</td>
			<td style="width:123px;">Selleckchem.com</td>
			<td style="width:119px;">S1049</td>
			<td style="width:212px;">ROCK-inhibitor (Y-27632 2HCL); multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">&#946;-mercaptoethanol</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">31350010</td>
			<td style="width:212px;">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...

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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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19.6K 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

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

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. ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...