We thank the Yale Stem Cell Core (YSCC), and the Yale Cancer Center (YCC) for assistance. We thank Dr. Jung Kim for his neuropathology review. This work was supported by Connecticut Regenerative Medicine Research Fund, March of Dimes, and NHLBI R01HL131793 (S.G.K.), the Yale Cancer Center and the Yale Cancer Biology Training Program NCI CA193200 (E.B.) and a generous unrestricted gift from Joseph and Lucille Madri.

1. Stem Cell Maintenance
<ol>
	<li>Maintain H9 hESCs on a layer of growth factor reduced basement membrane matrix (see the Table of Materials, henceforth simply referred to as matrix) according to the manufacturer&#39;s instructions.
	<ol>
		<li>To coat one 6-well plate or one 10 cm dish, combine 100 &#181;L of matrix with 5.9 mL of ice-cold Dulbecco&#39;s modified Eagle medium (DMEM)/F12 media. Wrap plates in paraffin film and store overnight at 4 &#176;C. Use them on the next day for passaging cells after the excess matrix/media is aspirated.</li>
	</ol>
	</li>
	<li>Culture the cells week to week at approximately a 1:12 split ratio every 7 days. Maintain the cells using mTESR-1 media in a 37 &#176;C, low oxygen incubator (5% O2, 5% CO2). Refresh media daily. Weed out differentiating cells from the culture between passages using glass tools.</li>
	<li>H9 cells should be passaged four to six days prior to utilizing them to produce organoids. The cells should be passaged at approximately a 1:8 ratio of cell clusters. To do this, start by rinsing the cells with DMEM F12 media and dissociate the cells with a neutral protease (e.g., dispase, henceforth referred to simply as protease), rinse with DMEM/F-12, and plate as 30-60 cell clusters across 4 plates (6-well or 10 cm) at ~20% confluency. Two days prior to harvest, transition them to a regular incubator (21% O2, 5% CO2). Plates should reach ~80% confluency when starting organoid formation.</li>
</ol>
2. Dissociation of the hESCs for Organoid Culture
<ol>
	<li>Aliquot the protease stock solution (5 U/mL). 
	NOTE: We typically freeze down 1 mL aliquots at -20 &#176;C for use over several months.</li>
	<li>Dilute the protease stock solution to the working concentration by adding 1 mL of the stock solution plus 5 mL of DMEM/F12 for each 6-well or 10 cm plate of hESCs.</li>
	<li>Aspirate and remove cell culture media, then cover the hESCs with the protease solution. Place plates in the incubator for 10-15 min or until the edges of the colonies round up and begin to separate from the matrix.</li>
	<li>Tilt the plate, aspirate the protease solution, and gently wash the cells with DMEM/F12 three times. Use 2 mL/well for each wash when using a 6-well plate and 6 mL when using a 10 cm plate. Make sure colonies stay attached to the matrix when performing this step.</li>
	<li>Add back about 1.5 mL of fresh mTESR media to each well (or 5 mL for a 10 cm plate) and flush the cells off the plate using gentle pipetting.</li>
	<li>Using a 10 mL pipette, gently aspirate and dispense hESC within the plate until they reach approximately 1/30th of their original size. Colony clusters should resemble ~250-350 &#181;m sized squares at the completion of these steps.</li>
</ol>
3. Generation of Organoids
<ol>
	<li>Transfer cells into a single ultra-low attachment T75 flask containing 30 mL of mTESR media without basic fibroblast growth factor (bFGF).</li>
	<li>The next day, tilt the flask(s) such that the live cells pool in the corner (this may take 5-10 min on the first day, but will get quicker as the clusters get larger). 
	NOTE: If there are a large number of cells that have adhered to the bottom of the flask at this step or any subsequent steps, transfer the cells to a new flask. It is normal to have a high population of dead cells for the first two days. When performing media changes, be sure to remove as much of the cell debris as possible.</li>
	<li>Once the cells settle, aspirate off the media and dead cells leaving about 10 mL of media containing the live cells.</li>
	<li>Add ~20 mL of low bFGF media (DMEM/F12 supplemented with 1x N2, 1x B27, 1x L-glutamine, 1x NEAA, 0.05% bovine serum albumin (BSA), and 0.1 mM monothioglycerol (MTG) supplemented with 30 ng/mL bFGF).</li>
	<li>Check the cells on day 2. If most of the cells look healthy and bright, there is no need to do anything. However, if more than a third of the cells appear dark, replace the media (using the same tilting technique as in step 3.2) with ~20 mL of low bFGF media supplemented with 20 ng/mL bFGF.</li>
	<li>On day 3, replace half of the media (using the tilting technique in step 3.2) with 20 mL of low bFGF media supplemented with 10 ng/mL bFGF.</li>
	<li>On day 5, replace half of the medium (using the tilting technique in step 3.2) with 20 mL of neural induction media (NIM: DMEM/F12, 1x N2 supplement, 0.1 mM MEM NEAA, 2 &#181;g/mL heparin). 
	NOTE: If there are any large clusters of cells or organoids that are much larger than the others, they should be removed from the culture. Size is estimated by appearance under the microscope; for example, using an eyepiece with reticle. The majority of organoids are similarly sized (roughly 100 &#177; 20 &#181;m). We removed organoids that were approximately 2x smaller or larger than the others.</li>
	<li>Replace half of the medium (~15 mL) (using the tilting technique) with NIM every other day.</li>
	<li>After 3 weeks in culture, add 100x penicillin/streptomycin to the media (NIM: DMEM/F12, 1x N2 supplement, 0.1 mM MEM NEAA, 2 &#181;g/mL heparin) at a final concentration of 1x if desired. Refresh the media every other day. 
	NOTE: In this fashion, we maintained the organoids for up to 6 months in culture.</li>
</ol>
4. RNA Extraction and Preparation
<ol>
	<li>Gently extract approximately 15 organoids (depending upon size) from the flask using a 10 mL pipette and place into a 1.5 mL tube.
	<ol>
		<li>Gently pellet the organoids in the centrifuge (200 x g for 1 min), and rinse with 1x Dulbecco&#39;s PBS (DPBS) three times.</li>
	</ol>
	</li>
	<li>Extract RNA using validated system or protocol (e.g., RNeasy kit).</li>
	<li>Measure the optical density value of each sample at 260 and 280 nm.</li>
	<li>Prepare cDNA using a validated system or protocol (e.g., iScript cDNA synthesis kit).</li>
	<li>Perform qRT-PCR using pre-validated primers (Table 1) including at least one housekeeping gene.</li>
</ol>
5. Immunohistochemistry
<ol>
	<li>Fixation
	<ol>
		<li>Prepare a 4% paraformaldehyde (PFA) solution and place it at 4 &#176;C.</li>
		<li>Using a sterile razor, cut the tip off of a sterile transfer pipette.</li>
		<li>Gently extract organoids using the cut transfer pipette, as they can be easily broken apart, especially when they grow large, and place them into a 6-well plate with additional media or DPBS.</li>
		<li>Tilt the plate, aspirate the media, and replace with 1x DPBS. Rinse the cells with 1x DPBS two additional times.</li>
		<li>Replace the DPBS with 4% PFA solution and place on a shaker at 4 &#176;C. 
		NOTE: While we fixed for 2 days (for small organoids) to 7 days (for organoids &#62;3 months), shorter times (e.g., 16-24 h) may also be possible.</li>
		<li>Prepare 30%, 20% and 10% sucrose solutions in DPBS.</li>
		<li>After fixation in PFA, replace with the 10% sucrose solution and place on a shaker at 4 &#176;C for 24 h.</li>
		<li>Replace the 10% sucrose with 20% sucrose and place on a shaker at 4 &#176;C for 24 h.</li>
		<li>Replace the 20% sucrose with 30% sucrose and place on a shaker at 4 &#176;C for 24 h.</li>
	</ol>
	</li>
	<li>Frozen sections
	<ol>
		<li>Prepare a flat layer of dry ice and place a labeled plastic mold on top of it.</li>
		<li>Pour a thin layer of optimal cutting temperature medium (OCT) into the mold and let it start to harden (within a few seconds).</li>
		<li>Place a few organoids on the top of the OCT in the mold using a transfer pipette with the tip cut off and pay close attention to the location of the organoids.</li>
		<li>Slowly add in OCT until the mold is full and the organoids are covered. Let it harden completely for an additional 10 min. 
		NOTE: While freezing over 10 min helps ensure ideal relative placement of multiple organoids for sectioning, it is possible to use an ethanol-dry ice mix or liquid nitrogen to freeze more quickly.</li>
		<li>Mark the relative location of the organoids with a marker to make it easier to find them when cutting.</li>
		<li>Place the molds in a bag or box and store at -80 &#176;C until ready to cut the sections.</li>
		<li>Using a cryostat, slice 10 &#181;m sections and place the tissue onto labeled, positively charged slides.</li>
	</ol>
	</li>
	<li>Staining
	<ol>
		<li>Prepare blocking solution (0.3% Triton X-100, 4% normal donkey serum in PBS).</li>
		<li>Use a hydrophobic pap pen to draw around the perimeter of the tissue.</li>
		<li>Rinse the slides with PBS 3 times for 5 min each.</li>
		<li>Replace PBS with blocking solution for 1 h at room temperature.</li>
		<li>Replace the blocking solution with antibody solution (antibody at appropriate concentration, 0.1% Triton X-100, 4% normal donkey serum in PBS) at 4 &#176;C overnight.</li>
		<li>On the following day, wash the slide 3 times with PBS for 10 min each.</li>
		<li>Replace PBS with the appropriate secondary antibody (at the appropriate concentration) diluted in antibody solution for 1 h at room temperature.</li>
		<li>Rinse 3 times for 10 min each time with 1x PBS.</li>
		<li>Apply the 4&#39;,6-diamidino-2-phenylindole (DAPI) stain and rinse three times for 10 min each with 1x PBS.</li>
		<li>Affix coverslips to the front of the slides with mounting solution, let dry at room temperature in the dark, and store in the dark at 4 &#176;C.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Northcutt, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+vertebrate+brain+evolution.">Understanding vertebrate brain evolution.</a> Integrative and Comparative Biology. 42 (4), 743-756 (2002).</li><li>Roth, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Long Evolution of Brains and Minds. , (2013).</li><li>Herculano-Houzel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+brain+in+numbers:+a+linearly+scaled-up+primate+brain.">The human brain in numbers: a linearly scaled-up primate brain.</a> Frontiers in Human Neuroscience. 3, 31 (2009).</li><li>Roth, G., Dicke, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+brain+and+intelligence.">Evolution of the brain and intelligence.</a> Trends in Cognitive Sciences. 9 (5), 250-257 (2005).</li><li>Eiraku, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organized+formation+of+polarized+cortical+tissues+from+ESCs+and+its+active+manipulation+by+extrinsic+signals.">Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals.</a> Cell Stem Cell. 3 (5), 519-532 (2008).</li><li>Watanabe, 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=Directed+differentiation+of+telencephalic+precursors+from+embryonic+stem+cells.">Directed differentiation of telencephalic precursors from embryonic stem cells.</a> Nature Neuroscience. 8 (3), 288-296 (2005).</li><li>Kadoshima, 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-organization+of+axial+polarity,+inside-out+layer+pattern,+and+species-specific+progenitor+dynamics+in+human+ES+cell-derived+neocortex.">Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (50), 20284-20289 (2013).</li><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 (7467), 373-379 (2013).</li><li>Li, X. 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=Specification+of+motoneurons+from+human+embryonic+stem+cells.">Specification of motoneurons from human embryonic stem cells.</a> Nature Biotechnology. 23 (2), 215-221 (2005).</li><li>Zhang, S. C., Wernig, M., Duncan, I. D., Brustle, O., Thomson, 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=In+vitro+differentiation+of+transplantable+neural+precursors+from+human+embryonic+stem+cells.">In vitro differentiation of transplantable neural precursors from human embryonic stem cells.</a> Nature Biotechnology. 19 (12), 1129-1133 (2001).</li><li>Pasca, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cortical+neurons+and+astrocytes+from+human+pluripotent+stem+cells+in+3D+culture.">Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture.</a> Nat Methods. 12 (7), 671-678 (2015).</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</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 (5), 1238-1254 (2016).</li><li>Sloan, S. A., Andersen, J., Pasca, A. M., Birey, F., Pasca, S. P. <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+assembly+of+human+brain+region-specific+three-dimensional+cultures.">Generation and assembly of human brain region-specific three-dimensional cultures.</a> Nature Protocols. 13 (9), 2062-2085 (2018).</li><li>Kelava, I., Lancaster, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cell+Models+of+Human+Brain+Development.">Stem Cell Models of Human Brain Development.</a> Cell Stem Cell. 18 (6), 736-748 (2016).</li><li>Boisvert, E. M., Means, R. E., Michaud, M., Madri, J. A., Katz, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minocycline+mitigates+the+effect+of+neonatal+hypoxic+insult+on+human+brain+organoids.">Minocycline mitigates the effect of neonatal hypoxic insult on human brain organoids.</a> Cell Death and Disease. 10 (4), 325 (2019).</li><li>Bergmann, 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=Blood-brain-barrier+organoids+for+investigating+the+permeability+of+CNS+therapeutics.">Blood-brain-barrier organoids for investigating the permeability of CNS therapeutics.</a> Nature Protocols. 13 (12), 2827-2843 (2018).</li><li>Mansour, A. 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=An+in+vivo+model+of+functional+and+vascularized+human+brain+organoids.">An in vivo model of functional and vascularized human brain organoids.</a> Nature Biotechnology. 36 (5), 432-441 (2018).</li><li>Pham, M. 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=Generation+of+human+vascularized+brain+organoids.">Generation of human vascularized brain organoids.</a> Neuroreport. 29 (7), 588-593 (2018).</li><li>Boisvert, E. M., Denton, K., Lei, L., Li, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specification+of+telencephalic+glutamatergic+neurons+from+human+pluripotent+stem+cells.">The specification of telencephalic glutamatergic neurons from human pluripotent stem cells.</a> Journal of Visualized Experiments. (74), (2013).</li></ol>

S.G.K. is a SAB member for Dansar IT and Gene-in-Cell.

Similar to other organoid models, this is an artificial system that comes with several caveats. Although there was little batch to batch variation in terms of overall expression levels, individual organoids did exhibit differences. For example, the location of Sox-2 positive areas were not identical in every organoid (Figure 3). While qPCR is suitable to look for overall changes in batches of cells, additional techniques such as single cell RNAseq will be utilized in future studies to gather more informa...

Due to myriad practical and ethical limitations, there has been a great deal of difficulty in studying the human brain. While studies utilizing rodents have been critical to our understanding of the human brain, the mouse brain has many dissimilarities1,2. Interestingly, mice have a neuronal density that is at least 7 times less than the primate brain3,4. Although primates are closer to humans than rodents from an evolutionary standpoint, it is not practical for most researchers to work with them. The purpose of this protocol was to recapitulate many important features of the human brain using a simplified and less expensive method without the need for a heterologous basement membrane matrix or exogenous growth factors while maintaining brain cell diversity and cellular organization.
Formative work from the Sasai lab used the serum free culture of embryoid bodies (SFEBq) method to generate two- and three- dimensional neuronal cell types from signalized embryonic stem cells (ESCs)5,6. Many human brain organoid methods have followed a relatively similar path from signalized ESCs7,8. In contrast, this protocol starts with clusters of detached human ESCs (hESCs), similar to the initial steps of seminal work of the Thomson and Zhang laboratories prior to the plating steps9,10 as well as the initial step of the brain organoid protocol of the Pasca laboratory before the addition of exogenous growth factors11. Basement membrane matrices (e.g., matrigel) have been utilized in many brain organoid protocols and it has been shown to be an effective scaffold8. However, most commonly used basement membrane matrices do not come without complications as they co-purify with unknown quantities of growth factors with batch to batch variability during production12. In addition, these matrices can complicate imaging, and increase the risk of contamination and cost.
While human brain organoids can be used to answer many questions, there are certain limitations to bear in mind. For one, starting from embryonic stem cells, organoids more closely resemble immature brains than aged brains and as such may not be ideal models for diseases that occur in old age, like Alzheimer&#39;s disease. Second, while our protocol found markers of forebrain, midbrain and hindbrain development which are useful to study the effect of a treatment or disease on cells from multiple brain regions in concert, other protocols can be followed to concentrate on specific brain regions13,14. Finally, another limitation of organoid models is that of size, while the average length of a human brain approximately 167 mm, brain organoids made with the use of agitation grow up to 4 mm8 and the organoids formed by this protocol grow to 1-2 mm by 10 weeks. Nonetheless, this protocol provides an important tool to study human brain tissue and the interaction of multiple cell types.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 goat anti-rabbit</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>A11035</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Gibco, Waltham, MA, USA</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Life Technologies, Carlsbad, CA, USA</td>
			<td>PHG0263</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr>
			<td>BX43 microscope</td>
			<td>Olympus, Shinjuku, Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI stain</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>STEMCELL Technologies, Vancouver, Canada</td>
			<td>07913</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco, Waltham, MA, USA</td>
			<td><a href="https://www.thermofisher.com/order/catalog/product/10010023" target="_blank">10010023</a></td>
			<td></td>
		</tr>
		<tr>
			<td>FluroSave</td>
			<td>MilliporeSigma, Burlington, MA</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>NeuroMab, Davis, CA</td>
			<td>N206A/8</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth Factor Reduced Matrigel (Matrix)</td>
			<td>Corning, Corning, NY, USA</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>H9 hESCs</td>
			<td>WiCell, Madison, WI, USA</td>
			<td>WA09</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>9041-08-1</td>
			<td></td>
		</tr>
		<tr>
			<td>iQ SYBR Green Supermix</td>
			<td>Bio-Rad, Hercules, CA, USA</td>
			<td>1708880</td>
			<td></td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad, Hercules, CA, USA</td>
			<td>1708891</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco, Waltham, MA, USA</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Monothioglycerol</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>mTESR media</td>
			<td>STEMCELL Technologies, Vancouver, Canada</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 NeuroPlex</td>
			<td>Gemini Bio Products, West Sacramento, CA, USA</td>
			<td>400-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Gibco, Waltham, MA, USA</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum (NDS)</td>
			<td>ImmunoResearch Laboratories Inc., West Grove, PA, USA</td>
			<td><a href="https://www.jacksonimmuno.com/catalog/products/017-000-121" target="_blank">017-000-121</a></td>
			<td></td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Sakura Finetek, Torrance, CA, USA</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Electron Microscopy Sciences, Hatfield, PA</td>
			<td>RT15710</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR machine</td>
			<td>Bio-Rad, CFX96, Hercules, CA, USA</td>
			<td>1855196</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Sox2</td>
			<td>MilliporeSigma, Burlington, MA</td>
			<td>AB5603</td>
			<td></td>
		</tr>
		<tr>
			<td>TMS-F microscope</td>
			<td>Nikon, Melville, NY, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>T8787-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low attachment T75 flasks</td>
			<td>Corning, Corning, NY, USA</td>
			<td>3814</td>
			<td></td>
		</tr>
	</tbody>
</table>

a static self-directed method for generating brain organoids from human embryonic stem cells

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

Figure 1 shows representative brightfield images of several time points to demonstrate what the cells/organoids look like throughout the different stages of the protocol. The hESCs were removed from the tissue culture plate, broken into small pieces, and placed in a T75 ultra-low attachment flask where they formed spheres. It is important to note that the cells look bright and similar in size, without dark, dying cells in the centers of these clusters. The cells were gradually weaned off bFG...

Watch this Scientific Journal Video about A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells at JoVE.com

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

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

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

a-static-self-directed-method-for-generating-brain-organoids-from

Research

JoVE Journal

Developmental Biology

8.7K Views.  Yale University School of Medicine. This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

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

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

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. ...

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...