Name: generation of ipsc-derived human brain organoids to model early neurodevelopmental disorders
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Description: Watch this Scientific Journal Video about Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders at JoVE.com

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,<sup class="xr...

<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. 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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. 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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...

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 ...

The overall goal of this protocol for generating iPSC-derived Human Brain Organoids is to model early human neurodevelopmental disorders. This method can help answer key questions in the human brain development field such as the role of synthesomes and cilia in novel neurogenesis and primary microcephaly. The main advantage of this technique is that it is a robust and quick model to obtain reproducible results from patient's iPSC-derived brain organites.Demonstrating the procedure will be Elke Gabriel, a post-doc from my laboratory at the Center for Molecular Medicine, Cologne. To begin this procedure, collect the neurospheres with a 200 microliter micropipette using a two millimeter tip previously cut with sterile scissors. Next, place the neurospheres approximately five milliliters away from each other on a paraffin film in a 100 milliliter dish.And carefully remove as much of the remaining medium as possible. Then, add a drop of EHS matrix onto each single neurosphere. Incubate the EHS matrix drops with the neurospheres for 15 minutes at 37 degrees celsius.After that, wash the neurospheres carefully from the paraffin film by flushing them with neurosphere medium. Subsequently, incubate the neurospheres for the next four days and add two millimeters of fresh neurosphere medium on day two. In this procedure, add 100 milliliters of brain organoid medium to each spinner flask through its side arms.And prewarm the medium in the incubator at 37 degrees celsius for at least 20 minutes. Then, make sure the neurospheres are all separated. If two or more are connected through EHS matrix, separate them by cutting the connecting matrix with a scalpel.Set up a stirring program at 25 RPM. Carefully transfer the EHS matrix embedded neurospheres into the spinner flasks containing 100 milliliters of organoid medium. Following that, space the spinner flasks on a magnetic stirring platform in the incubator for 14 days at 37 degrees celsius.This is day zero of the organoid culture. Change the medium once a week by removing half of the medium and adding the same amount of the fresh medium. In this step, collect the organoids from the spinner flask culture on day 14 with a pre-cut one milliliter micro pipette.Put all of them in a 60 millimeter dish and wash them once with five milliliters of warm DMEM F12 for three minutes. Afterward, prepare a 1.5 milliliter tube with 500 microliters of warm, 4%PFA. Place each organoid separately in each tube and fix them for 30-60 minutes at room temperature.Afterward, use an inoculation loop to removed the organoids. Then, remove PFA and wash the fixed organoids twice for 10 minutes each time with one milliliter of PBS. Subsequently, store the organoids in one milliliter of PBS at four degrees celsius for up to 7 days for further use.To embed the organoids for cryosectioning, remove PBS and add 1 milliliter of 30%sucrose solution in each 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 four degrees celsius.By the next day, the organoids should've sunk down to the bottom of the tube. Following that, fill a vinyl specimen mold with 400 milliliters of 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. Then, freeze the organoid-containing mold at negative 80 degrees celsius until cryosectioning. Subsequently, coat the glass cryoslides with 0.1%PLL and PBS for five minutes at room temperature.Then, let the slides dry for three hours. Store the slides at four degrees celsius and warm them up to room temperature before use. Following that, section the cryo-frozen organoids into 20 to 50 micrometer thick slices on the PLL-coated glass cryoslides.Let the sections dry from one hour at room temperature and store the sections at negative 80 degrees celsius until further processing. Immunofluorescent imaging of cryosectioned organoids displaying a typical VZ and the primitive cortical plate. The VZ spans from the apical side to the basil side.Note that the cells within the VZ display paliside-like nucleide suggesting that they are radial glial cells. The right panel shows the immunofluorescent staining of nested positive NPCs within the VZ and TUJ1 positive neurons in the primitive cortical plate. Shown here are two examples of VZs stained with PVIM and ARL 13B.The regions marked by white squares are magnified. Apical radial glial cells dividing at the apical side of the VZ are PVIM positive. The division plane of an anaphase cell is horizontal to the lumen surface line which is marked by ARL 13B staining.Once mastered, this technique can be done in 23 days if it is performed properly. While attempting this procedure, it's important to remember to perform periodic quality check and microplasma check on cultures. After its development, this technique paved the way for researchers in the field of development in neurobiology to explore the mechanisms of neurodevelopment in human brain organoids.After watching this video, you should have a good understanding of how to generate human brain organoids from various iPSC cell lines including patient cell lines with compromised cellular functions.

generation-ipsc-derived-human-brain-organoids-to-model-early

Research

JoVE Journal

Developmental Biology

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 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 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 ...

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

Differentiation and Mechanical Stimulation of hiPSC-Derived Endothelial Cells

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

Author Spotlight: Studying hiPSC-Derived Endothelial Cells Cultured Under Fluidic-Mediated Mechanical Stimulation 

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.