This work was supported by Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia (FCT), Portugal (UIDB/04565/2020 through Programa Operacional Regional de Lisboa 2020, Project N. 007317, PD/BD/105773/2014 to T.P.S and PD/BD/128376/2017 to D.E.S.N.), projects co-funded by FEDER (POR Lisboa 2020&#8212;Programa Operacional Regional de Lisboa PORTUGAL 2020) and FCT through grant PAC-PRECISE LISBOA-01-0145-FEDER-016394 and CEREBEX Generation of Cerebellar Organoids for Ataxia Research grant LISBOA-01-0145-FEDER-029298. Funding was also received from the European Union&#39;s Horizon 2020 Research and Innovation Programme, under the Grant Agreement number 739572&#8212;The Discoveries Centre for Regenerative and Precision Medicine H2020-WIDESPREAD-01-2016-2017.

1. Passaging and maintenance of human iPSCs in monolayer culture
<ol>
	<li>Preparation of plates
	<ol>
		<li>Thaw the basement membrane matrix (see Table of Materials) stock at 4 &#176;C and prepare 60 &#956;L aliquots. Freeze the aliquots at -20 &#176;C.</li>
		<li>To coat the wells of a 6 well plate, thaw one aliquot of the basement membrane matrix on ice. Once thawed add 60 &#181;L to 6 mL of DMEM-F12. Gently resuspend by pipetting up and down.</li>
		<li>Add 1 mL of diluted basement membrane matrix solution to each well of a 6 well plate and incubate at RT for at least 1 h before passaging or store at 4 &#176;C for up to 1 week.</li>
	</ol>
	</li>
	<li>Passaging of iPSC colonies with EDTA
	<ol>
		<li>Maintain iPSCs in monolayer culture in 6 well plates in the incubator at 37 &#176;C, 95% humidity, and 5% CO2. 
		NOTE: In this protocol, three distinct human iPSC lines were used: F002.1A.1321, human episomal iPSC line (iPSC6.2)22, and commercially obtained iPS-DF6-9-9T.B (see Table of Materials).</li>
		<li>Before passaging, incubate the stored plates (step 1.1) at room temperature (RT) for 15 min and prepare the mTesR1 medium (Table 1).</li>
		<li>Aspirate the solution from the plate using a serological pipette and immediately add 0.5 mL of mTeSR1 medium to each well.</li>
		<li>Aspirate the spent medium from the well containing iPSCs and wash once using 1 mL of 0.5 mM EDTA per well.</li>
		<li>Add 1 mL of 0.5 mM EDTA to each well and incubate at RT for 5 min.</li>
		<li>Aspirate EDTA and remove the cells from the wells by gently adding mTeSR1 medium and pipetting the colonies using a P1000 micropipette. Collect the cells in a conical tube. 
		NOTE: Do not pipette cells up and down more than 3x.</li>
		<li>Add 1 mL of cell suspension (diluted 1:4) to each well so that each well contains 1.5 mL of medium after the cell suspension is added. Return cells to the incubator at 5% CO2, 37 &#176;C.</li>
		<li>Replace the spent medium daily and passage every 3 days when 75%&#8211;80% confluence is achieved.</li>
	</ol>
	</li>
</ol>
2. Seeding of human iPSCs in the bioreactor
<ol>
	<li>Incubate iPSCs grown as monolayers in mTeSR1 supplemented with 10 &#181;M of ROCK inhibitor Y-27632 (ROCKi). Add 1 mL of supplemented medium to each well from a 6 well tissue culture plate and incubate for 1 h at 37 &#176;C, 95% humidity, and 5% CO2. 
	NOTE: ROCKi is used to protect dissociated iPSCs from apoptosis23.</li>
	<li>After 1 h of incubation, aspirate the spent medium from each well and wash 1x with 1 mL of 1&#215; PBS per well.</li>
	<li>Add 1 mL of the cell detachment medium (see Table of Materials) to each well of a 6 well plate and incubate at 37 &#176;C for 7 min until cells detach easily from the wells with gentle shaking.</li>
	<li>Pipette the cell detachment medium up and down with a P1000 micropipette until the cells detach and dissociate into single cells. Add 2 mL of complete cell culture medium to each well to inactivate enzymatic digestion and pipette the cells gently into a sterile conical tube.</li>
	<li>Centrifuge at 210 &#215; g for 3 min and remove the supernatant.</li>
	<li>Resuspend the cell pellet in culture medium (i.e., mTeSR1 supplemented with 10 &#181;M of ROCKi) and count the iPSCs with a hemocytometer using trypan blue dye.</li>
	<li>Seed 15 &#215; 106 single cells in the bioreactor (maximum volume of 100 mL) with 60 mL of mTeSR1 supplemented with 10 &#181;M of ROCKi at a final cell density of 250,000 cells/mL.</li>
	<li>Insert the vessel containing the iPSCs in the universal base unit placed in the incubator at 37 &#176;C, 95% humidity, and 5% CO2. 
	NOTE: The bioreactor stirring is maintained for 24 h by setting the universal base unit control to 27 rpm to promote iPSC aggregation.</li>
</ol>
3. Differentiation and maturation of human iPSC-derived aggregates in cerebellar organoids
<ol>
	<li>Define the day of single cell seeding as day 0.</li>
	<li>On day 1, collect 1 mL of the iPSC aggregates sample using a serological pipette. Maintain the bioreactor under agitation as before by placing the universal base unit with the bioreactor containing the aggregates in a sterile flow prior to collecting the sample. Plate the cell suspension in an ultra-low attachment 24 well plate. Check that iPSC-derived aggregates are formed.</li>
	<li>Acquire images with an optical microscope using a total magnification of 40x or 100x to measure aggregate diameter.</li>
	<li>Measure the area of the aggregates in each image using FIJI software.
	<ol>
		<li>Select &#8220;Analyze | Set Measurements&#8221; from the menu bar and click on &#8220;Area&#8221; and &#8220;OK&#8221;.</li>
		<li>Select &#8220;File | Open&#8221; from the menu bar to open a stored image file. Select the line selection tool presented in the tool bar and create a straight line over the scale bar presented in the image. Select &#8220;Analyze | Set scale&#8221; from the menu bar.</li>
		<li>In &#8220;Known distance&#8221; add the expanse of the image&#39;s scale bar in &#181;m. Define the &#8220;Unit of length&#8221; as &#181;m. Click on &#8220;Global&#8221; to maintain the settings and &#8220;OK&#8221;. Select Oval Selection in the tool bar.</li>
		<li>For each aggregate delineate the area with the oval tool. Select &#8220;Analyze | Measure&#8221;. Calculate their diameter based on measured area, considering that aggregates are approximately spherical using 
		<img alt="Equation 1" src="/files/ftp_upload/61143/61143equ01.jpg" /> 
		with A as the area of the aggregate.</li>
	</ol>
	</li>
	<li>When the average diameter of the aggregates is 100 &#181;m, replace 80% of the spent medium with fresh mTeSR1 without ROCKi. When aggregates reach 200&#8211;250 &#956;m in diameter, replace all the spent medium with gfCDM (Table 1), letting the organoids settle at the bottom of the bioreactor. 
	NOTE: If the average aggregate diameter exceeds 350 &#956;m do not start the differentiation protocol. Repeat the seeding of single cells. Generally, it takes around 1 day for the aggregate to reach an average diameter of 100 &#181;m.</li>
	<li>Insert the bioreactor containing the aggregates in the universal base unit placed in the incubator at 37 &#176;C, 95% humidity, and 5% CO2.</li>
	<li>Decrease the bioreactor agitation to 25 rpm.</li>
	<li>On day 2, repeat steps 3.2, 3.3, and 3.4 to evaluate the aggregate diameter. Add 30 &#956;L of FGF2 (final concentration, 50 ng/mL) and 60 &#956;L of SB431542 (final concentration, 10 &#956;M) to 60 mL of gfCDM differentiation medium (Table 1). Replace all spent medium from the bioreactor with the supplemented gfCDM. Repeat step 3.6. 
	NOTE: SB431542 is crucial to inhibit mesendodermal differentiation, inducing neural differentiation24. FGF2 is used to promote the caudalization of the neuroepithelial tissue25.</li>
	<li>On day 5, repeat steps 3.2, 3.3, 3.4, and 3.8. 
	NOTE: Aggregate size should increase during the differentiation protocol. However, the diameter is only critical when the differentiation starts, because this parameter could influence the efficacy of differentiation.</li>
	<li>On day 7, repeat steps 3.2, 3.3, and 3.4. Dilute FGF2 and SB431542 to 2/3: Add 20 &#956;L of FGF2 and 40 &#956;L of SB431542 to 60 mL of gfCDM differentiation medium. Replace all spent medium from the bioreactor with supplemented gfCDM. Repeat step 3.6 and increase bioreactor agitation to 30 rpm.</li>
	<li>On day 14, repeat steps 3.2, 3.3, and 3.4. Add 60 &#956;L of FGF19 (final concentration, 100 ng/mL) to 60 mL of gfCDM differentiation medium. Replace all spent medium from the bioreactor with gfCDM supplemented with FGF19. Repeat step 3.6. 
	NOTE: FGF19 is used to promote polarization of mid-hindbrain structures26.</li>
	<li>On day 18, repeat steps 3.2, 3.3, 3.4, and 3.11.</li>
	<li>On day 21, repeat steps 3.2, 3.3, and 3.4. Replace all spent medium from the bioreactor with complete neurobasal medium (Table 1). Repeat step 3.6. 
	NOTE: Neurobasal medium is a basal medium used to maintain the neuronal cell population within the organoid7.</li>
	<li>On day 28, repeat steps 3.2, 3.3, and 3.4. Add 180 &#956;L of SDF1 (final concentration, 300 ng/mL) to 60 mL of complete neurobasal medium. Replace all spent medium from the bioreactor with complete neurobasal medium supplemented with SDF1. Repeat step 3.6. 
	NOTE: SDF1 is used to facilitate the organization of distinct cell layers27.</li>
	<li>On day 35, repeat steps 3.2, 3.3, and 3.4. Replace all spent medium from the bioreactor with complete BrainPhys medium (Table 1). Repeat step 3.6. 
	NOTE: BrainPhys is a neuronal medium that supports synaptically active neurons28.</li>
	<li>Replace 1/3 of the total volume every 3 days with complete BrainPhys medium until day 90 of differentiation.</li>
</ol>
4. Preparation of organoids for cryosectioning and immunohistochemistry
<ol>
	<li>Collection of organoids for immunostaining
	<ol>
		<li>Collect 1 mL of sample of medium containing organoids with a serological pipette from the bioreactor to a 15 mL conical tube. 
		NOTE: Organoids should be collected at different timepoints to evaluate the efficacy of differentiation, including days 7, 14, 21, 35, 56, 70, 80, and 90.</li>
		<li>Remove the supernatant and wash once with 1 mL of 1&#215; PBS. 
		NOTE: Do not centrifuge the organoids. Let the organoids settle at the bottom of the tube by gravity.</li>
		<li>Remove the supernatant and add 1 mL of 4% paraformaldehyde (PFA). Incubate at 4 &#176;C for 30 min. Remove the spent PFA and add 1 mL of 1&#215; PBS.</li>
		<li>Store the organoids in 1 mL of 1&#215; PBS at 4 &#176;C until processing for cryosectioning. 
		NOTE: Store the organoids in 1x PBS for no more than 1 week after fixation.</li>
	</ol>
	</li>
	<li>Preparation of organoids for cryosectioning
	<ol>
		<li>Remove the supernatant from the stored organoids. Add 1 mL of 15% sucrose (w/v, diluted in 1&#215; PBS), mix well by gentle swirling, and incubate overnight at 4 &#176;C.</li>
		<li>Prepare a solution of 15% sucrose/7.5% gelatin (Table 2) and maintain at 37 &#176;C during preparation to avoid gelatin to solidify.</li>
		<li>Remove the 15% sucrose solution, add 1 mL of 15% sucrose/7.5% gelatin to the organoids, and quickly mix by gentle swirling. Incubate at 37 &#176;C for 1 h.</li>
		<li>Add 15% sucrose/7.5% gelatin solution to a plastic container up to half of its volume. Wait for solidification at RT.</li>
		<li>After a 1 h incubation, carefully place a sucrose/gelatin drop containing the organoids on the solidified gelatin with a Pasteur pipette. Leave to solidify at RT for about 15 min. Make sure to avoid bubble formation.</li>
		<li>Place 15% sucrose/7.5% gelatin on top of the organoids until the container is filled. Wait for complete solidification at RT.</li>
		<li>After solidification, incubate 20 min at 4 &#176;C.</li>
		<li>Cut the gelatin into a cube containing the organoids in the center and fix the gelatin cube on a piece of cardboard with a drop of O.C.T. compound.</li>
		<li>Place 250 mL of isopentane in a 500 mL cup and fill an appropriate container with liquid nitrogen. Using forceps and thick gloves, carefully place the cup containing isopentane on the surface of liquid nitrogen and cool the isopentane to -80 &#176;C.</li>
		<li>When -80 &#176;C is reached, place the gelatin cube into the cup containing isopentane until it freezes, keeping the temperature at -80 &#176;C. Depending on the size of the cube, it might take 1&#8211;2 min. 
		NOTE: Avoid temperatures below -80 &#176;C or excessive freezing time, because it might cause cracking of the cube.</li>
		<li>When frozen, quickly store the gelatin cube at -80 &#176;C and store until cryosectioning.</li>
	</ol>
	</li>
	<li>Cryosectioning of organoids
	<ol>
		<li>Turn on the cryostat and define both specimen (OT) and cryochamber (CT) temperatures at -25 &#176;C.</li>
		<li>When both temperatures stabilize, fix the gelatin cube containing the organoids on the specimen by using O.C.T. compound.</li>
		<li>Define section thickness at 12 &#181;m.</li>
		<li>Cut the cube and collect 3&#8211;4 slices on adhesion microscope slides (see Table of Materials).</li>
		<li>Store at -20 &#176;C until use.</li>
	</ol>
	</li>
	<li>Immunostaining of organoids slices
	<ol>
		<li>Place the microscope slides containing organoid sections in a copling jar with 50 mL of prewarmed 1x PBS, holding up to 10 slides back-to-back. 
		NOTE: All organoid sections should be submerged with liquid.</li>
		<li>Incubate for 45 min at 37 &#176;C to degelatinize slides.</li>
		<li>Wash 1x with 50 mL of 1&#215; PBS for 5 min at RT: Transfer the slides to a copling jar containing fresh 1&#215; PBS.</li>
		<li>Transfer the slides to a copling jar containing 50 mL of freshly prepared glycine (Table 2) and incubate for 10 min at RT.</li>
		<li>Transfer the slides to a copling jar containing 50 mL of 0.1% triton (Table 2) and permeabilize for 10 min at RT.</li>
		<li>Wash with 1&#215; PBS for 5 min 2x.</li>
		<li>Prepare the immunostaining dish with 3 mm paper soaked in 1&#215; PBS. Dry slides with a tissue all around the slices and place them onto 3 mm paper. With a Pasteur pipette, cover the whole surface of the slides with blocking solution (Table 2) with ~0.5 mL per slide. Incubate for 30 min at RT.</li>
		<li>Remove excess blocking solution and dry the slides with a tissue all around the slices. Place 50 &#181;L of the primary antibody (Table 3) diluted in blocking solution over the sections and cover with the coverslips. Place the slices in a previously prepared immunostaining dish. Incubate overnight at 4 &#176;C.</li>
		<li>Transfer the slides to a copling jar with 50 mL of TBST (Table 2), let the coverslips fall, and wash with TBST for 5 min 3x.</li>
		<li>Place 50 &#181;L of the secondary antibody diluted in blocking solution over the sections and cover with the coverslips. Place the slices in the previously prepared immunostaining dish. Incubate for 30 min at RT, protected from light.</li>
		<li>Transfer the slides to a copling jar again and wash with 50 mL of TBST for 5 min 3x.</li>
		<li>Dry the slides with a tissue all around the slices and place the slices in a previously prepared immunostaining dish. Add 0.5 mL of DAPI solution over the whole surface of the slides with a Pasteur pipette. Incubate for 5 min at RT.</li>
		<li>Repeat step 4.4.9.</li>
		<li>Carefully dry the slides with a tissue. Add 50 &#181;L of mounting medium drop by drop along the slide and then carefully lower a coverslip onto each slide, slightly bending it to avoid bubbles.</li>
	</ol>
	</li>
</ol>

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R., 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=Inhibition+of+Activin/Nodal+signaling+promotes+specification+of+human+embryonic+stem+cells+into+neuroectoderm.">Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm.</a> Developmental Biology. 313 (1), 107-117 (2008).</li><li>Yaguchi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+(FGF)+gene+expression+in+the+developing+cerebellum+suggests+multiple+roles+for+FGF+signaling+during+cerebellar+morphogenesis+and+development.">Fibroblast growth factor (FGF) gene expression in the developing cerebellum suggests multiple roles for FGF signaling during cerebellar morphogenesis and development.</a> Developmental Dynamics. 238 (8), 2058-2072 (2008).</li><li>Fischer, 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=Fgf15-mediated+control+of+neurogenic+and+proneural+gene+expression+regulates+dorsal+midbrain+neurogenesis.">Fgf15-mediated control of neurogenic and proneural gene expression regulates dorsal midbrain neurogenesis.</a> Developmental Biology. 350 (2), 496-510 (2011).</li><li>Bagri, 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=The+chemokine+SDF1+regulates+migration+of+dentate+granule+cells.">The chemokine SDF1 regulates migration of dentate granule cells.</a> Development. 129 (18), 4249-4260 (2002).</li><li>Bardy, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+medium+that+supports+basic+synaptic+functions+and+activity+of+human+neurons+in+vitro.">Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro.</a> Proceedings of the National Academy of Sciences. 112 (20), 2725-2734 (2015).</li><li>Bauwens, C. L., 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=Control+of+Human+Embryonic+Stem+Cell+Colony+and+Aggregate+Size+Heterogeneity+Influences+Differentiation+Trajectories.">Control of Human Embryonic Stem Cell Colony and Aggregate Size Heterogeneity Influences Differentiation Trajectories.</a> Stem Cells. 26 (9), 2300-2310 (2008).</li><li>Bratt-Leal, A. M., Carpenedo, R. L., McDevitt, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+embryoid+body+microenvironment+to+direct+embryonic+stem+cell+differentiation.">Engineering the embryoid body microenvironment to direct embryonic stem cell differentiation.</a> Biotechnology Progress. 25 (1), 43-51 (2009).</li><li>Miranda, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+control+of+cell+aggregation+efficiently+directs+human+pluripotent+stem+cells+towards+neural+commitment.">Spatial and temporal control of cell aggregation efficiently directs human pluripotent stem cells towards neural commitment.</a> Biotechnology Journal. 10 (10), 1612-1624 (2015).</li><li>Miranda, C. C., Fernandes, T. G., Diogo, M. M., Cabral, J. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+up+a+chemically-defined+aggregate-based+suspension+culture+system+for+neural+commitment+of+human+pluripotent+stem+cells.">Scaling up a chemically-defined aggregate-based suspension culture system for neural commitment of human pluripotent stem cells.</a> Biotechnology Journal. 11 (12), 1628-1638 (2016).</li><li>Bardy, 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=Microcarrier+Suspension+Cultures+for+High-Density+Expansion+and+Differentiation+of+Human+Pluripotent+Stem+Cells+to+Neural+Progenitor+Cells.">Microcarrier Suspension Cultures for High-Density Expansion and Differentiation of Human Pluripotent Stem Cells to Neural Progenitor Cells.</a> Tissue Engineering Part C Methods. 19 (2), 166-180 (2013).</li><li>Rigamonti, 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=Large-Scale+Production+of+Mature+Neurons+from+Human+Pluripotent+Stem+Cells+in+a+Three-Dimensional+Suspension+Culture+System.">Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System.</a> Stem Cell Reports. 6 (6), 993-1008 (2016).</li><li>Arora, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+process+engineering+approach+to+increase+organoid+yield.">A process engineering approach to increase organoid yield.</a> Development. 144 (6), 1128-1136 (2017).</li><li>Gimeno, L., Martinez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+chick+Fgf19+and+mouse+Fgf15+orthologs+is+regulated+in+the+developing+brain+by+Fgf8+and+Shh.">Expression of chick Fgf19 and mouse Fgf15 orthologs is regulated in the developing brain by Fgf8 and Shh.</a> Developmental Dynamics. 236 (8), 2285-2297 (2007).</li></ol>

Authors YH and SJ are employees of PBS Biotech. The author BL is CEO and co-founder of PBS Biotech, Inc. These collaborating authors participated in the development of the bioreactors used in the manuscript. This does not alter the authors&#8217; adherence to all the policies of the journal on sharing data and materials. All other authors declare no conflict of interest.

The need for large cell numbers as well as defined culture conditions to generate specific cell types for drug screening and regenerative medicine applications has been driving the development of scalable culture systems. In recent years, several groups have reported the scalable generation of neural progenitors and functional neurons32,33,34, providing significant advances in the development of new models for neurodegenerative ...

The emergence of human pluripotent stem cells (PSCs) represents a excellent tool for regenerative medicine and disease modeling, because these cells can be differentiated into most cell lineages of the human body1,2. Since their discovery, PSC differentiation using diverse approaches has been reported to model different diseases, including neurodegenerative disorders3,4,5,6.
Recently, there have been reports of 3D cultures derived from PSCs resembling human cerebral structures; these are called brain organoids3,7,8. The generation of these structures from both healthy and patient-specific PSCs provides a valuable opportunity to model human development and neurodevelopmental disorders. However, the methods used to generate these well-organized cerebral structures are difficult to apply for their large-scale production. To produce structures that are large enough to recapitulate tissue morphogenesis without necrosis inside the organoids, protocols rely on the initial neural commitment in static conditions, followed by encapsulation in hydrogels and subsequent culture in dynamic systems3. However, such approaches may limit the potential scale-up of organoid production. Even though efforts have been made to direct PSC differentiation to specific regions of the central nervous system, including cortical, striatal, midbrain, and spinal cord neurons9,10,11,12, the generation of specific brain regions in dynamic conditions is still a challenge. In particular, the generation of mature cerebellar neurons in 3D structures has yet to be described. Muguruma et al. pioneered the generation of culture conditions that recapitulate early cerebellar development13 and recently reported a protocol that allows for human embryonic stem cells to generate a polarized structure reminiscent of the first trimester cerebellum7. However, the maturation of cerebellar neurons in the reported studies requires the dissociation of the organoids, sorting of cerebellar progenitors, and coculture with feeder cells in a monolayer culture system7,14,15,16. Therefore, the reproducible generation of the desired cerebellar organoids for disease modeling under defined conditions is still a challenge associated with culture and feeder source variability.
This protocol presents optimal culture conditions for 3D expansion and efficient differentiation of human PSCs into cerebellar neurons using single-use vertical wheel bioreactors (see Table of Materials for specifications), hereafter called bioreactors. Bioreactors are equipped with a large vertical impeller, which in combination with a U-shaped bottom, provide a more homogeneous shear distribution inside the vessel, allowing gentle, uniform mixing and particle suspension with reduced agitation speeds17. With this system, shape and size-controlled cell aggregates can be obtained, which is important for a more homogeneous and efficient differentiation. Moreover, a larger number of iPSC-derived organoids can be generated in a less laborious manner.
The main feature of the organoids, which are 3D multicellular structures usually formed from stem cells, is the self-organization of different cell types that forms specific shapes like those seen in human morphogenesis18,19,20. Therefore, organoid morphology is an important criterion to be evaluated during the differentiation process. Cryosectioning of organoids and further immunostaining of organoid slices with a specific set of antibodies allow for the spatial visualization of molecular markers to analyze cell proliferation, differentiation, cell population identity, and apoptosis. With this protocol, by immunostaining organoid cryosections, an initial efficient neural commitment is observed by the 7th day of differentiation. During differentiation, several cell populations with cerebellar identity are observed. After 35 days in this dynamic system, the cerebellar neuroepithelium organizes along an apicobasal axis, with an apical layer of proliferating progenitors and basally located postmitotic neurons. During the maturation process, from days 35&#8211;90 of differentiation, distinct types of cerebellar neurons can be seen, including Purkinje cells (Calbindin+), granule cells (PAX6+/MAP2+), Golgi cells (Neurogranin+), unipolar brush cells (TBR2+), and deep cerebellar nuclei projection neurons (TBR1+). Also, a nonsignificant amount of cell death is observed in the generated cerebellar organoids after 90 days in culture.
In this system, human iPSC-derived organoids mature into different cerebellar neurons and survive for up to 3 months without the need for dissociation and feeder coculture, providing a source of human cerebellar neurons for disease modeling.

<table><tbody><tr><td>3MM paper</td><td>WHA3030861</td><td>Merck</td><td></td></tr><tr><td>Accutase</td><td>A6964 - 500mL</td><td>Sigma</td><td>cell detachment medium</td></tr><tr><td>Anti-BARHL1 Antibody</td><td>HPA004809</td><td>Atlas Antibodies</td><td></td></tr><tr><td>Anti-Calbindin D-28k Antibody</td><td>CB28</td><td>Millipore</td><td></td></tr><tr><td>Anti-MAP2 Antibody</td><td>M4403</td><td>Sigma</td><td></td></tr><tr><td>Anti-N-Cadherin Antibody</td><td>610921</td><td>BD Transduction</td><td></td></tr><tr><td>Anti-NESTIN Antibody</td><td>MAB1259-SP</td><td>R&amp;amp;D</td><td></td></tr><tr><td>Anti-OLIG2 Antibody</td><td>MABN50</td><td>Millipore</td><td></td></tr><tr><td>Anti-PAX6 Antibody</td><td>PRB-278P</td><td>Covance</td><td></td></tr><tr><td>Anti-SOX2 Antibody</td><td>MAB2018</td><td>R&amp;amp;D</td><td></td></tr><tr><td>Anti-TBR1 Antibody</td><td>AB2261</td><td>Millipore</td><td></td></tr><tr><td>Anti-TBR2 Antibody</td><td>ab183991</td><td>Abcam</td><td></td></tr><tr><td>Anti-TUJ1 Antibody</td><td>801213</td><td>Biolegend</td><td></td></tr><tr><td>Apo-transferrin</td><td>T1147</td><td>Sigma</td><td></td></tr><tr><td>BrainPhys Neuronal Medium N2-A &amp;amp; SM1 Kit</td><td>5793 - 500mL</td><td>Stem cell tecnhnologies</td><td></td></tr><tr><td>Chemically defined lipid concentrate</td><td>11905031</td><td>ThermoFisher</td><td></td></tr><tr><td>Coverslips 24x60mm</td><td>631-1575</td><td>VWR</td><td></td></tr><tr><td>Crystallization-purified BSA</td><td>5470</td><td>Sigma</td><td></td></tr><tr><td>DAPI</td><td>10236276001</td><td>Sigma</td><td></td></tr><tr><td>Dibutyryl cAMP</td><td>SC- 201567B -500mg</td><td>Frilabo</td><td></td></tr><tr><td>DMEM-F12</td><td>32500-035</td><td>ThermoFisher</td><td></td></tr><tr><td>Fetal bovine serum</td><td>A3840001</td><td>ThermoFisher</td><td></td></tr><tr><td>Gelatin from bovine skin</td><td>G9391</td><td>Sigma</td><td></td></tr><tr><td>Glass Copling Jar</td><td>E94</td><td>ThermoFisher</td><td></td></tr><tr><td>Glutamax I</td><td>10566-016</td><td>ThermoFisher</td><td></td></tr><tr><td>Glycine</td><td>MB014001</td><td>NZYtech</td><td></td></tr><tr><td>Ham&amp;rsquo;s F12</td><td>21765029</td><td>ThermoFisher</td><td></td></tr><tr><td>Human Episomal iPSC Line</td><td>A18945</td><td>ThermoFisher</td><td>iPSC6.2</td></tr><tr><td>IMDM</td><td>12440046</td><td>ThermoFisher</td><td></td></tr><tr><td>Insulin</td><td>91077C</td><td>Sigma</td><td></td></tr><tr><td>iPS DF6-9-9T.B</td><td></td><td>WiCell</td><td></td></tr><tr><td>Iso-pentane</td><td>PHR1661-2ML</td><td>Sigma</td><td></td></tr><tr><td>L-Ascorbic acid</td><td>A-92902</td><td>Sigma</td><td></td></tr><tr><td>Matrigel</td><td>354230</td><td>Corning</td><td>basement membrane matrix</td></tr><tr><td>Monothioglycerol</td><td>M6154</td><td>Sigma</td><td></td></tr><tr><td>Mowiol</td><td>475904</td><td>Millipore</td><td>mounting medium</td></tr><tr><td>mTeSR1</td><td>85850 -500ml</td><td>Stem cell technologies</td><td></td></tr><tr><td>N2 supplement</td><td>17502048</td><td>ThermoFisher</td><td></td></tr><tr><td>Neurobasal</td><td>12348017</td><td>ThermoFisher</td><td></td></tr><tr><td>Paraformaldehyde</td><td>158127</td><td>Sigma</td><td></td></tr><tr><td>PBS-0.1 Single-Use Vessel</td><td>SKU: IA-0.1-D-001</td><td>PBS Biotech</td><td></td></tr><tr><td>PBS-MINI MagDrive Base Unit</td><td>SKU: IA-UNI-B-501</td><td>PBS Biotech</td><td></td></tr><tr><td>Recombinant human BDNF</td><td>450-02</td><td>Peprotech</td><td></td></tr><tr><td>Recombinant human bFGF/FGF2</td><td>100-18B</td><td>Peprotech</td><td></td></tr><tr><td>Recombinant human FGF19</td><td>100-32</td><td>Peprotech</td><td></td></tr><tr><td>Recombinant human GDNF</td><td>450-10</td><td>Peprotech</td><td></td></tr><tr><td>Recombinant human SDF1</td><td>300-28A</td><td>Peprotech</td><td></td></tr><tr><td>ROCK inhibitor Y-27632</td><td>72302</td><td>Stem cell technologies</td><td></td></tr><tr><td>SB431542</td><td>S4317</td><td>Sigma</td><td></td></tr><tr><td>Sucrose</td><td>S7903</td><td>Sigma</td><td></td></tr><tr><td>SuperFrost Microscope slides</td><td>12372098</td><td>ThermoFisher</td><td>adhesion microscope slides</td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>25608-930</td><td>VWR</td><td></td></tr><tr><td>Tris-HCL 1M</td><td>T3038-1L</td><td>Sigma</td><td></td></tr><tr><td>Triton X-100</td><td>9002-93-1</td><td>Sigma</td><td></td></tr><tr><td>Tween-20</td><td>P1379</td><td>Sigma</td><td></td></tr><tr><td>UltraPure 0.5M EDTA, pH 8.0</td><td>15575020</td><td>ThermoFisher</td><td></td></tr></tbody></table>

scalable generation of mature cerebellar organoids from human pluripotent stem cells and characterization by immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

The protocol was initiated by promoting cell aggregation using the 0.1 L bioreactors (Figure 1A). Single cell inoculation of the iPSCs was performed, with 250,000 cells/mL seeded in 60 mL of medium with an agitation speed of 27 rpm. This was defined as day 0. After 24 h, the cells efficiently formed spheroid-shaped aggregates (day 1, Figure 1B), and the morphology was well-maintained until day 5, with a gradual increase in size, demonstrating a high degree of ho...

Watch this Scientific Journal Video about Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining at JoVE.com

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

scalable-generation-mature-cerebellar-organoids-from-human

Research

JoVE Journal

Bioengineering

10.5K Views.  Universidade de Lisboa. We presenteren voor het eerst een nieuwe aanpak voor reproduceerbare en schaalbare generatie, van IPS-cellen afgeleid bij Cerebellar Organoids, en een chemische gedefinieerde omstandigheden, met behulp van bioreactoren voor eenmalig gebruik. Om cellen te verkrijgen voor het zaaien van de bio-reactor, voeg een milliliter van celloslating medium, aan elke put van een zes put plaat van menselijke iPSC's. Incubeer de plaat op 37 graden Celsius gedurende zeven minuten, tot zacht schudden gemakkeli...

Video: Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

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. ...

Developmental Biology

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

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

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

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

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

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

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

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

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

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

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

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

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

Neuroscience

Modeling Human Cerebellar Development In Vitro in 2D Structure

The precise and timely development of the cerebellum is crucial not only for accurate motor coordination and balance but also for cognition. In addition, disruption in cerebellar development has been implicated in many neurodevelopmental disorders, including autism, attention deficit-hyperactivity disorder (ADHD), and schizophrenia. Investigations of cerebellar development in humans have previously only been possible through post-mortem studies or neuroimaging, yet these methods are not sufficient for understanding the molecular and cellular changes occurring in vivo during early development, which is when many neurodevelopmental disorders originate. The emergence of techniques to generate human-induced pluripotent stem cells (iPSCs) from somatic cells and the ability to further re-differentiate iPSCs into neurons have paved the way for in vitro modeling of early brain development. The present study provides simplified steps toward generating cerebellar cells for applications that require a 2-dimensional (2D) monolayer structure. Cerebellar cells representing early developmental stages are derived from human iPSCs via the following steps: first, embryoid bodies are made in 3-dimensional (3D) culture, then they are treated with FGF2 and insulin to promote cerebellar fate specification, and finally, they are terminally differentiated as a monolayer on poly-l-ornithine (PLO)/laminin-coated substrates. At 35 days of differentiation, iPSC-derived cerebellar cell cultures express cerebellar markers including ATOH1, PTF1&#945;, PAX6, and KIRREL2, suggesting that this protocol generates glutamatergic and GABAergic cerebellar neuronal precursors, as well as Purkinje cell progenitors. Moreover, the differentiated cells show distinct neuronal morphology and are positive for immunofluorescence markers of neuronal identity such as TUBB3. These cells express axonal guidance molecules, including semaphorin-4C, plexin-B2, and neuropilin-1, and could serve as a model for investigating the molecular mechanisms of neurite outgrowth and synaptic connectivity. This method generates human cerebellar neurons useful for downstream applications, including gene expression, physiological, and morphological studies requiring 2D monolayer formats.

Modeling Human Cerebellar Development In Vitro in 2D Structure

The present protocol explains the generation of a 2D monolayer of cerebellar cells from induced pluripotent stem cells for investigating the early stages of cerebellar development.

The precise and timely development of the cerebellum is crucial not only for accurate motor coordination and balance but also for cognition. In addition, ...

Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research

The currently available animal and cellular models do not fully recapitulate the complexity of changes that take place in the aging human brain. A recent development of procedures describing the generation of human cerebral organoids, derived from human induced pluripotent stem cells (iPSCs), has the potential to fundamentally transform the ability to model and understand the aging of the human brain and related pathogenic processes. Here, an optimized protocol for generating, maintaining, aging, and characterizing human iPSC-derived cerebral organoids is presented. This protocol can be implemented to generate brain organoids in a reproducible manner and serves as a step-by-step guide, incorporating the latest techniques that result in improved organoid maturation and aging in culture. Specific issues related to organoid maturation, necrosis, variability, and batch effects are being addressed. Taken together, these technological advances will allow the modeling of brain aging in organoids derived from a variety of young and aged human donors, as well as individuals afflicted with age-related brain disorders, allowing the identification of physiologic and pathogenic mechanisms of human brain aging.

The present protocol provides a step-by-step procedure for the reproducible generation, maintenance, and aging of cerebral organoids derived from human-induced pluripotent stem cells (iPSCs). This method enables culturing and maturing cerebral organoids for extended periods, which facilitates the modeling of processes involved in brain aging and age-related pathogenesis.

The currently available animal and cellular models do not fully recapitulate the complexity of changes that take place in the aging human brain. A recent ...

Fluorescence Immunostaining of Cerebellar Organoid Slices

Source: Silva, T. P., et al., <a href="https://app.jove.com/v/61143">Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining.</a> J. Vis. Exp. (2020)
This video demonstrates the immunofluorescence staining of cerebellar organoid slices to identify specific target antigens expressed in various cerebellar neurons, which are indicative of organoid maturation.

1. Immunostaining of organoid slices

	Place the microscope slides containing organoid sections in a copling jar with 50 mL of prewarmed 1x PBS, holding ...

JoVE Encyclopedia of Experiments

Neuropathology

Developmental and Pediatric Disorders

Generating Induced Pluripotent Stem Cell-Derived Cerebral Organoids

Source: Koch, L. S., et al. <a href="https://app.jove.com/v/64586">Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research.</a> J. Vis. Exp. (2023).
This video demonstrates an assay to generate cerebral organoids from induced pluripotent stem cells (iPSCs). Human iPSCs are induced to form embryoid bodies (EBs). The EBs are cultured over a polymer scaffold using an induction medium, promoting differentiation into neural stem cells, progenitor cells, and immature neurons. Finally, the EBs are embedded in a matrix and transferred to a maintenance medium for cerebral organoid formation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Figure 1 illustrates a schematic ...

Neuronal Culture Techniques

Advanced Neuronal Models

Generating Mature Cerebellar Organoids From Induced Pluripotent Stem Cells Using Single-Use Bioreactors

Source: Silva, T.P., et al. <a href="https://app.jove.com/v/61143">Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining.</a> J. Vis. Exp. (2020)
This video demonstrates a technique for generating mature cerebellar organoids from induced pluripotent stem cells (iPSCs) using a single-use 3D bioreactor. The bioreactor facilitates iPSC aggregate formation, which then undergoes treatment with a series of culture media enriched in growth factors and chemokines that induce the differentiation of the iPSC aggregates into mature cerebellar organoids. Subsequently, the organoids are harvested and cryopreserved for further analysis.

1. Seeding of human iPSCs in the bioreactor

	Incubate iPSCs grown as monolayers in mTeSR1 supplemented with 10 &#181;M of ROCK inhibitor Y-27632 (ROCKi). ...

A Technique to Generate a 2D Monolayer of Cerebellar Cells from Induced Pluripotent Stem Cells

Source: Madencioglu, D. A., et al. <a href="https://app.jove.com/v/64462">Modeling Human Cerebellar Development In Vitro in 2D Structure.</a>&#160;J. Vis. Exp. (2022).
This video demonstrates a protocol for differentiating human induced pluripotent stem cells into cerebellar progenitors. It involves transferring stem cell colonies to an ultra-low attachment plate to form embryoid bodies, promoting differentiation with growth factors, and then transferring the embryoid bodies onto a matrix-coated plate for adhesion. Subsequent steps involve inducing cell migration and adding a maturation medium to guide the development of glutamatergic and GABAergic cerebellar neuronal precursors and Purkinje cell progenitors.

1. Experimental preparation 

	Prepare basement membrane matrix (BMM)-coated plates.
	NOTE: BMM solidifies more quickly at warmer temperatures. Plates ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

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