We thank Jenny Gringer Richards for her thorough work in validating our control subjects, from which we generated the control iPSCs. This work was supported by NIH T32 MH019113 (to D.A.M. and K.A.K.), the Nellie Ball Trust (to T.H.W. and A.J.W.), NIH R01 MH111578 (to V.A.M. and J.A.W.), NIH KL2 TR002536 (to A.J.W.), and the Roy J. Carver Charitable Trust (to V.A.M., J.A.W., and A.J.W.).&#160;The figures were created with BioRender.com.

The human subjects research was approved under the University of Iowa Institutional Review Board approval number 201805995 and the University of Iowa Human Pluripotent Stem Cell Committee approval number 2017-02. Skin biopsies were obtained from the subjects after obtaining written informed consent. The fibroblasts were cultured in DMEM with 15% fetal bovine serum (FBS) and 1% MEM-non-essential amino acids solution at 37 &#176;C and 5% CO2. Fibroblasts were reprogrammed using an episomal reprogramming kit following the manufacturer&#39;s protocol (see Table of Materials) using a nucleofector for electroporation. All procedures were performed in a Class II Type A2 biological safety cabinet (&#34;hood&#34; for short). All cell culture media were antibiotic-free; therefore, every component that entered the hood was cleaned with 70% ethanol. All cell culture media and components were sterile filtered or opened in the hood to maintain their sterility.
1. Experimental preparation
<ol>
	<li>Prepare basement membrane matrix (BMM)-coated plates. 
	NOTE: BMM solidifies more quickly at warmer temperatures. Plates must be prepared rapidly and immediately placed at 4 &#176;C for storage.
	<ol>
		<li>Thaw the BMM (see Table of Materials) on ice, at 4 &#176;C for at least 2 h, or overnight.</li>
		<li>Mix DMEM/F12 and BMM to a final concentration of 80 &#181;g/mL. Distribute the BMM solution in tissue culture dishes (1 mL for 35 mm and 2 mL for 60 mm dishes) and incubate at 37 &#176;C for at least 1 h or overnight before plating the cells. 
		NOTE: The unused dishes can be stored at 4 &#176;C for 2 weeks.</li>
	</ol>
	</li>
	<li>Prepare poly-L-ornithine/laminin (PLO/laminin)-coated plates.
	<ol>
		<li>Prepare 20 &#181;g/mL PLO (see Table of Materials) in sterile DPBS+/+ and add 1 mL to each well of a 6-well plate. Incubate overnight at 37 &#176;C in the incubator.</li>
		<li>The following day, aspirate the PLO with a vacuum aspirator and wash two times with DPBS+/+. Air dry in the hood. 
		NOTE: Air-dried plates can be stored at 4 &#176;C for up to 2 weeks, wrapped in aluminum foil, for future use.</li>
		<li>Prepare 10 &#181;g/mL laminin (see Table of Materials) in DPBS+/+ and add 1 mL to each well of a 6-well plate. Incubate at least for 3 h or overnight at 37 &#176;C in the incubator.</li>
		<li>Aspirate the laminin with a vacuum aspirator and add either 1 mL of medium or sterile DPBS+/+. 
		NOTE: The laminin coating must not dry out. PBS or an appropriate culture medium should be added immediately to prevent this. Coated plates can be stored at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Prepare PSC passaging solution. 
	NOTE: An osmometer (see Table of Materials) is required to make PSC passaging solution10.
	<ol>
		<li>Dissolve 11.49 g of potassium chloride (KCl) and 0.147 g of sodium citrate dihydrate (HOC(COONa)(CH2COONa)2*2H2O) in 400 mL of sterile cell culture grade water (see Table of Materials).</li>
		<li>Measure the volume of the solution and record it as the initial volume (Vi).</li>
		<li>Measure the osmolarity and adjust it to 570 mOsm by adding sterile cell culture grade water using the formula Vi &#215; Oi = Vf &#215; 570 mOsm.</li>
		<li>Filter-sterilize the solution with a 0.20 &#181;m filter and make 10 mL aliquots. Store the aliquots at room temperature (RT) for up to 6 months.</li>
	</ol>
	</li>
	<li>Prepare pluripotent stem cell (PSC) medium. 
	NOTE: iPSCs are maintained in a medium containing heat-stable FGF2 (see Table of Materials), providing a weekend-free feeding schedule. Other commercially available PSC media have not been tried for this protocol.
	<ol>
		<li>Thaw the PSC medium supplement overnight at 4 &#176;C.</li>
		<li>Add 10 mL of PSC medium supplement to 500 mL of basal PSC medium. Make 25 mL aliquots and store at &#8722;20 &#176;C. 
		NOTE: Frozen aliquots can be stored at &#8722;20 &#176;C for 6 months. Thawed aliquots must be stored at 4 &#176;C and used within 2 weeks. Cells can be frozen for long-term storage in a PSC medium with 10% (v/v) dimethyl sulfoxide (DMSO).</li>
	</ol>
	</li>
	<li>Prepare PSC thawing medium.
	<ol>
		<li>Supplement PSC medium with 50 nM chroman, 1.5 &#181;M emricasan, 1x polyamine supplement, and 0.7 &#181;M trans-ISRIB (CEPT cocktail11) (see Table of Materials).</li>
		<li>Filter-sterilize with a 0.20 &#181;m filter and store at 4 &#176;C for up to 4 weeks.</li>
	</ol>
	</li>
	<li>Prepare pulled glass pipettes.
	<ol>
		<li>Pull 22.9 cm (9 in) glass Pasteur pipettes into two pieces above a Bunsen burner, ~2 cm below the neck, creating two pipettes, with the thinner side being ~4 cm shorter than the other.</li>
		<li>With the help of the flame, bend the tips of the pulled side to create a smooth &#34;r&#34;.</li>
		<li>Place the pulled pipettes in autoclave sleeves and autoclave for sterilization.</li>
	</ol>
	</li>
	<li>Prepare cerebellar differentiation medium (CDM).
	<ol>
		<li>Mix IMDM and Ham&#39;s F12 nutrient mix in a 1:1 ratio. Supplement the mix with 1x L-alanine-L-glutamine supplement, 1% (v/v) chemically defined lipid concentrate, 0.45 mM 1-thio-glycerol, 15 &#181;L/mL apo-transferrin, 5 mg/mL bovine serum albumin (BSA), and 7 &#181;g/mL insulin (see Table of Materials).</li>
		<li>Filter-sterilize with a 0.20 &#181;m filter, store at 4 &#176;C, and use within 1 month.</li>
	</ol>
	</li>
	<li>Prepare cerebellar maturation medium (CMM).
	<ol>
		<li>Supplement neurobasal medium with 1x L-alanine-L-glutamine supplement and 1x N-2 supplement (see Table of Materials).</li>
		<li>Filter-sterilize with a 0.20 &#181;m filter, store at 4 &#176;C, and use within 2 weeks.</li>
	</ol>
	</li>
</ol>
2. Feeder-free iPSC culture
<ol>
	<li>Thaw the cells following the steps below.
	<ol>
		<li>Place a BMM plate (step 1.1) into the 37 &#176;C incubator to gel for at least 1 h or overnight before thawing the cells.</li>
		<li>Pre-warm 10 mL of PSC thawing medium (step 1.5) at 37 &#176;C.</li>
		<li>Transfer the cryovial with cells from liquid nitrogen to a water bath at 37 &#176;C. 
		NOTE: To avoid generating a source of contamination in the cell culture space, using a standard water bath is not recommended. Instead, fresh water can be heated in a small beaker to 37 &#176;C to generate a one-time-use water bath.</li>
		<li>When there is a small piece of ice left in the tube, remove it from the water bath, dry the tube, and spray with 70% ethanol. Transfer the tube to the hood and use a 2 mL or 5 mL serological pipette (see Table of Materials) to gently transfer the cells to a 15 mL conical tube.</li>
		<li>Add 8 mL of PSC thawing medium to the cells dropwise while swirling the conical tube. Centrifuge at 200 x g for 5 min at RT.</li>
		<li>Carefully aspirate the supernatant with a vacuum aspirator without disturbing the cell pellet. Resuspend the cells in 2 mL of PSC thawing medium.</li>
		<li>Aspirate the BMM from the plate and add the resuspended cells dropwise all over the plate for even distribution.</li>
		<li>Place the plate in the incubator at 37 &#176;C, 5% CO2. Refresh the medium the next day with PSC medium. After that, change the medium daily.</li>
	</ol>
	</li>
	<li>Maintain the cells following the steps below.
	<ol>
		<li>Pre-warm the necessary volume of PSC medium at 37 &#176;C (e.g., 1 mL of medium per 35 mm plate).</li>
		<li>Investigate the plates for differentiated cells or colonies and remove the differentiated cells with a pulled glass pipette (step 1.6). 
		NOTE: iPSC colonies have smooth edges with morphologically identical cells.</li>
		<li>Change the spent medium daily and passage every 3-4 days or when 70% confluency is reached.</li>
	</ol>
	</li>
	<li>Passage the cells.
	<ol>
		<li>Place the necessary amount of BMM plates into the incubator for at least 1 h or overnight before passaging. Pre-warm the necessary amount of PSC medium (step 1.3) at 37 &#176;C.</li>
		<li>Using a pulled glass pipette, clean off any differentiated cells or colonies.</li>
		<li>Aspirate the spent medium with a vacuum aspirator and add PSC dissociation medium, 1 mL per 35 mm dish. Incubate at 37 &#176;C for 1-3 min. 
		NOTE: If colonies are lifting off in the PSC passaging solution before adding PSC medium, the incubation time can be reduced.</li>
		<li>Aspirate the PSC passaging solution and add pre-warmed PSC medium.</li>
		<li>Using a sterile 200 &#181;L pipette tip, scratch lines parallel to each other in one direction, turn the plate 90&#176;, and make a second set of scratch lines perpendicular to the previous ones (making a cross-hatched pattern across the plate).</li>
		<li>Aspirate the BMM solution from the set plates. Collect the colonies using a serological pipette and distribute them to new BMM plates.</li>
		<li>Incubate at 37 &#176;C, 5% CO2. Change the medium daily.</li>
	</ol>
	</li>
	<li>Freeze the cells.
	<ol>
		<li>Prepare cryovials by labeling the content and sterilize under ultraviolet (UV) light in the hood (see Table of Materials) for 30 min.</li>
		<li>Prepare PSC freezing medium by supplementing PSC medium with 10% (v/v) DMSO. Follow steps 2.3.2-2.3.5.</li>
		<li>Transfer the cells to a 15 mL conical tube with a 5 mL serological pipette and centrifuge at 200 x g for 5 min at RT.</li>
		<li>Carefully aspirate the medium and resuspend the cell pellet in PSC freezing medium.</li>
		<li>Distribute into cryovials. Place the vials into a freezing container and place the container in a &#8722;80 &#176;C freezer.</li>
		<li>The following day, transfer the cryovials to liquid nitrogen for long-term storage.</li>
	</ol>
	</li>
</ol>
3. Cerebellar differentiation
NOTE: Before starting the differentiation, iPSCs are passaged to six 35 mm dishes and are ready for the differentiation when they are at 70% confluency. Each 35 mm plate will be transferred to one well of the 6-well plate.
<ol>
	<li>On day 0, lift the healthy iPSC colonies for EB formation.
	<ol>
		<li>Add 1 mL of CDM (step 1.7) supplemented with 10 &#181;M Y-27632 and 10 &#181;M SB431542 (see Table of Materials) to each well of a 6-well ultra-low attachment (ULA) plate and place in the incubator until the lifted colonies are ready to be added to the wells.</li>
		<li>Clean the differentiated cells using a pulled glass pipette. Aspirate the medium and add 1 mL of PSC passaging solution for each 35 mm dish. Incubate for 3 min at 37 &#176;C, aspirate, and add 2 mL of CDM supplemented with 10 &#181;M Y-27632 and 10 &#181;M SB431542. 
		NOTE: If colonies are lifting off in the PSC passaging solution before adding CDM, the incubation time can be reduced.</li>
		<li>Under a transmitted-light inverted microscope, gently lift the colonies using the bent edge of the pulled glass pipette using 4x magnification. Once every colony is lifted, gently transfer all to one well of the 6-well ULA plate using a 10 mL serological pipette. Repeat this process for each iPSC plate. Incubate the cells at 37 &#176;C with 5% CO2. 
		NOTE: If the colonies are too large or are merged, they can be sliced with the tip of the pulled glass pipette to create smaller EBs.</li>
	</ol>
	</li>
	<li>On day 2, add FGF2 to each well to a final concentration of 50 ng/mL. 
	NOTE: The FGF2 used for cerebellar differentiation is not heat-stable. This protocol has not been tested with heat-stable FGF2.</li>
	<li>On day 7, do a 1/3 medium change. For 3 mL of total medium in a well, with a 1,000 &#181;L pipettor, gently aspirate 1 mL of spent medium and replace it with 1 mL of fresh CDM. Incubate the EBs for 7 days.</li>
	<li>On day 14, gently aspirate nearly all the spent medium using a 1,000 &#181;L pipettor. To ensure minimal damage to the EBs, swirl the plate to gather all the EBs in the center of the plate, and then tilt the plate and aspirate slowly from the edge.
	<ol>
		<li>As the medium amount decreases, slowly lay the plate flat and continue to aspirate. Leave enough medium to avoid drying the EBs out. Afterward, add 3 mL of fresh CDM supplemented with 10 &#181;M Y-27632. Transfer the EBs using a 10 mL serological pipette to a PLO/laminin-coated dish (step 1.2). 
		NOTE: Depending on the downstream application, the EBs can be transferred to a 6-well PLO/laminin plate or single EBs can be transferred to a single well of a PLO/laminin-coated 24-well plate or coverslip.</li>
	</ol>
	</li>
	<li>On day 15, aspirate the medium and replace it with fresh CDM. 
	NOTE: It is important to add enough medium to the wells to ensure enough medium between feedings as, over time, there will be evaporation (e.g., for a well in a 6-well plate, add 3 mL of medium). If the medium has started to acidify (turn clear and yellow), refresh the medium even if it is not on the feeding schedule until the end of the protocol.</li>
	<li>On day 21, aspirate the spent medium and replace it with CMM. On day 28, change the medium with fresh CMM. On day 35, harvest the cells for further applications.</li>
</ol>
4. Sample preparation for RNA isolation
<ol>
	<li>Aspirate the medium and add 500 &#181;L of cell dissociation reagent (see Table of Materials) to each well of the 6-well plate. Leave it on for a couple of seconds and aspirate.</li>
	<li>Incubate the plate, with the lid on, at room temperature for 2 min. Tap the sides of the plate to detach the cells.</li>
	<li>Add 1 mL of CMM (step 1.8) and collect the cells into a 1.5 mL tube.</li>
	<li>Pellet the cells by centrifugation using a benchtop mini centrifuge (see Table of Materials) for 30 s at RT (max speed 2000 x g), discard the medium, and add 1 mL of 1x PBS pH 7.4 to wash the cell pellet.</li>
	<li>Pellet the cells again using a benchtop mini centrifuge for 30 s at RT and wash with PBS once more.</li>
	<li>Pellet the cells and discard the PBS. Lyse the cells in phenol and guanidine isothiocyanate containing reagent (see Table of Materials). 
	&#8203;NOTE: Cells lysed in phenol and guanidine isothiocyanate&#160;containing&#160;reagent can be stored at &#8722;80 &#176;C for up to 1 year. Cells can be lysed directly in the dish depending on the RNA isolation method and reagents being used. Due to the low number of cells in a dish, isolating RNA using silica spin columns (see Table of Materials) will result in higher yields of RNA.</li>
</ol>
5. Preparing cells for immunofluorescent staining
NOTE: For a 24-well plate, one EB per well is sufficient.
<ol>
	<li>On day 35, aspirate the spent medium and wash the cells by adding 1 mL of DPBS+/+ per well (for one well of a 24-well plate).</li>
	<li>Aspirate the DPBS+/+ and add 500 &#181;L of cold 4% paraformaldehyde (PFA, see Table of Materials) prepared in DPBS+/+ per well. Incubate for 20 min at RT.</li>
	<li>Remove 4% PFA and wash the cells twice with DPBS+/+, as in step 5.1. Add 1 mL of DPBS+/+ and store the cells at 4 &#176;C until staining is done. 
	NOTE: It is advised to do the immunofluorescent staining within 1 week after fixation to avoid cell detachment and the loss of antigens. However, depending on the antibody and the cellular location of the target, staining can be done at later times up to 4 weeks after fixation.</li>
</ol>

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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=Spatial+and+cell+type+transcriptional+landscape+of+human+cerebellar+development.">Spatial and cell type transcriptional landscape of human cerebellar development.</a> Nature Neuroscience. 24 (8), 1163-1175 (2021).</li><li>Fossat, N., Courtois, V., Chatelain, G., Brun, G., Lamonerie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+usage+of+Otx2+promoters+during+mouse+development.">Alternative usage of Otx2 promoters during mouse development.</a> Developmental Dynamics. 233 (1), 154-160 (2005).</li><li>Akbarian, 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=The+PsychENCODE+project.">The PsychENCODE project.</a> Nature Neuroscience. 18 (12), 1707-1712 (2015).</li><li>Aijaz, 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=Expression+analysis+of+SIX3+and+SIX6+in+human+tissues+reveals+differences+in+expression+and+a+novel+correlation+between+the+expression+of+SIX3+and+the+genes+encoding+isocitrate+dehyhrogenase+and+cadherin+18.">Expression analysis of SIX3 and SIX6 in human tissues reveals differences in expression and a novel correlation between the expression of SIX3 and the genes encoding isocitrate dehyhrogenase and cadherin 18.</a> Genomics. 86 (1), 86-99 (2005).</li><li>Conte, I., Morcillo, J., Bovolenta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+Six3+and+Six6+distribution+in+the+developing+and+adult+mouse+brain.">Comparative analysis of Six3 and Six6 distribution in the developing and adult mouse brain.</a> Developmental Dynamics. 234 (3), 718-725 (2005).</li><li>Andreasen, N. C., Pierson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+cerebellum+in+schizophrenia.">The role of the cerebellum in schizophrenia.</a> Biological Psychiatry. 64 (2), 81-88 (2008).</li><li>Nopoulos, P. C., Ceilley, J. W., Gailis, E. A., Andreasen, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+MRI+study+of+cerebellar+vermis+morphology+in+patients+with+schizophrenia:+Evidence+in+support+of+the+cognitive+dysmetria+concept.">An MRI study of cerebellar vermis morphology in patients with schizophrenia: Evidence in support of the cognitive dysmetria concept.</a> Biological Psychiatry. 46 (5), 703-711 (1999).</li><li>Jacobsen, L. 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=Quantitative+morphology+of+the+cerebellum+and+fourth+ventricle+in+childhood-onset+schizophrenia.">Quantitative morphology of the cerebellum and fourth ventricle in childhood-onset schizophrenia.</a> American Journal of Psychiatry. 154 (12), 1663-1669 (1997).</li><li>Saywell, V., Cioni, J. -. M., Ango, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+gene+expression+profile+of+axon+guidance+cues+in+Purkinje+cells+during+cerebellar+circuit+formation.">Developmental gene expression profile of axon guidance cues in Purkinje cells during cerebellar circuit formation.</a> The Cerebellum. 13 (3), 307-317 (2014).</li><li>Kim, D., Ackerman, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+UNC5C+netrin+receptor+regulates+dorsal+guidance+of+mouse+hindbrain+axons.">The UNC5C netrin receptor regulates dorsal guidance of mouse hindbrain axons.</a> Journal of Neuroscience. 31 (6), 2167-2179 (2011).</li><li>Maier, V., 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=Semaphorin+4C+and+4G+are+ligands+of+Plexin-B2+required+in+cerebellar+development.">Semaphorin 4C and 4G are ligands of Plexin-B2 required in cerebellar development.</a> Molecular and Cellular Neuroscience. 46 (2), 419-431 (2011).</li><li>Telley, 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=Dual+function+of+NRP1+in+axon+guidance+and+subcellular+target+recognition+in+cerebellum.">Dual function of NRP1 in axon guidance and subcellular target recognition in cerebellum.</a> Neuron. 91 (6), 1276-1291 (2016).</li><li>Wang, 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=Differentiation+of+human+induced+pluripotent+stem+cells+to+mature+functional+Purkinje+neurons.">Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons.</a> Scientific Reports. 5, 9232 (2015).</li><li>Shabanipour, 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=Primary+culture+of+neurons+isolated+from+embryonic+mouse+cerebellum.">Primary culture of neurons isolated from embryonic mouse cerebellum.</a> Journal of Visualized Experiments. (152), e60168 (2019).</li></ol>

The authors have no conflicts of interest to declare.

The ability to model human cerebellar development in vitro is important for disease modeling as well as furthering the understanding of normal brain development. Less complicated and cost-effective protocols create more opportunities for replicable data generation and broad implementation across multiple scientific labs. A cerebellar differentiation protocol is described here using a modified method of generating EBs that does not require enzymes or dissociation agents, using growth factors reported by Muguruma ...

Understanding human cerebellar development and the critical time windows of this process is important not only for decoding the possible causes of neurodevelopmental disorders but also for identifying new targets for therapeutic intervention. Modeling human cerebellar development in vitro has been challenging, yet over time, many protocols differentiating human embryonic stem cells (hESCs) or iPSCs with cerebellar lineage fates have emerged1,2,3,4,5,6,7,8. Furthermore, it is important to develop protocols that generate reproducible results, are relatively simple (to reduce error), and are not heavy on monetary costs.
The first protocols for cerebellar differentiation were generated from 2D cultures from plated embryoid bodies (EBs), inducing cerebellar fate with various growth factors similar to in vivo development, including WNT, BMPs, and FGFs1,9. More recent published protocols induced differentiation primarily in 3D organoid culture with FGF2 and insulin, followed by FGF19 and SDF1 for rhombic lip-like structures3,4, or used a combination of FGF2, FGF4, and FGF85. Both cerebellar organoid induction methods resulted in similar 3D cerebellar organoids as both protocols reported similar cerebellar marker expression at identical time points. Holmes and Heine extended their 3D protocol5 to show that 2D cerebellar cells can be generated from hESCs and iPSCs, which start as 3D aggregates. In addition, Silva et al.7 demonstrated that cells representing mature cerebellar neurons in 2D can be generated with a similar approach to Holmes and Heine, using a different time point for switching from 3D to 2D and extending the time of growth and maturation.
The current protocol induces cerebellar fate in feeder-free iPSCs by generating free-floating embryoid bodies (EBs) using insulin and FGF2 and then plating the EBs on PLO/laminin-coated dishes on day 14 for 2D growth and differentiation. By day 35, cells with cerebellar identity are obtained. The ability to recapitulate the early stages of cerebellar development, especially in a 2D environment, allows researchers to answer specific questions requiring experiments with a monolayer structure. This protocol is also amenable to further modifications such as micropatterned surfaces, axonal outgrowth assays, and cell sorting to enrich the desired cell populations.

<table><tbody><tr><td>10 mL Serological pipette</td><td>Fisher Scientific</td><td>13-678-26D</td><td></td></tr><tr><td>1-thio-glycerol</td><td>Sigma</td><td>M6145</td><td></td></tr><tr><td>2 mL Serological pipette</td><td>Fisher Scientific</td><td>13-678-26B</td><td></td></tr><tr><td>250 mL Filter Unit, 0.2 &amp;micro;m aPES, 50 mm Dia</td><td>Fisher Scientific</td><td>FB12566502</td><td></td></tr><tr><td>35 mm Easy Grip Tissue Cluture Dish</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>4D Nucleofector core unit</td><td>Lonza</td><td>276885</td><td>Nucleofector</td></tr><tr><td>5 mL Serological pipette</td><td>Fisher Scientific</td><td>13-678-25D</td><td></td></tr><tr><td>60 mm Easy Grip Tissue Culture Dish</td><td>Falcon</td><td>353004</td><td></td></tr><tr><td>6-well ultra-low attachment plates</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>9&quot; Disposable Pasteur Pipets</td><td>Fisher Scientific</td><td>13-678-20D</td><td></td></tr><tr><td>Apo-transferrin</td><td>Sigma</td><td>T1147</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A9418</td><td></td></tr><tr><td>Cell culture grade water</td><td>Cytiva</td><td>SH30529.02</td><td></td></tr><tr><td>Chemically defined lipid concentrate</td><td>Gibco</td><td>11905031</td><td></td></tr><tr><td>Chroman 1</td><td>Cayman</td><td>34681</td><td></td></tr><tr><td>Class II, Type A2, Biological safety Cabinet</td><td>NuAire, Inc.</td><td>NU-540-600</td><td>Hood, UV light</td></tr><tr><td>Costar 24-well plate, TC treated</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>Costar 6-well plate, TC treated</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>DAPI solution</td><td>Thermo Scientific</td><td>62248</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965092</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11320033</td><td></td></tr><tr><td>DMSO (Dimethly sulfoxide)</td><td>Sigma</td><td>D2438</td><td></td></tr><tr><td>DPBS+/+</td><td>Gibco</td><td>14040133</td><td></td></tr><tr><td>Emricasan</td><td>Cayman</td><td>22204</td><td></td></tr><tr><td>Epi5 episomal iPSC reprogramming kit</td><td>Life Technologies</td><td>A15960</td><td></td></tr><tr><td>Essential 8-Flex</td><td>Gibco</td><td>A2858501</td><td>PSC medium with heat-stable FGF2</td></tr><tr><td>EVOS XL Core Imaging system</td><td>Life Technologies</td><td>AMEX1000</td><td></td></tr><tr><td>Fetal bovine serum - Premium Select</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>FGF2</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>GlutaMAX supplement</td><td>Gibco</td><td>35050061</td><td>L-alanine-L-glutamine supplement</td></tr><tr><td>Ham&#039;s F12 Nutrient Mix</td><td>Gibco</td><td>11765054</td><td></td></tr><tr><td>HERAcell VIOS 160i CO2 incubator</td><td>Thermo Scientific</td><td>50144906</td><td></td></tr><tr><td>Human Anti-EN2, mouse</td><td>Santa Cruz Biotechnology</td><td>sc-293311</td><td></td></tr><tr><td>Human anti-Ki67/MKI67, rabbit</td><td>R&amp;amp;D Systems</td><td>MAB7617</td><td></td></tr><tr><td>Human anti-PTF1a, rabbit</td><td>Novus Biologicals</td><td>NBP2-98726</td><td></td></tr><tr><td>Human anti-TUBB3, mouse</td><td>Biolegend</td><td>801213</td><td></td></tr><tr><td>IMDM</td><td>Gibco</td><td>12440053</td><td></td></tr><tr><td>Insulin</td><td>Gibco</td><td>12585</td><td></td></tr><tr><td>Laminin Mouse Protein</td><td>Gibco</td><td>23017015</td><td></td></tr><tr><td>Matrigel Matrix</td><td>Corning</td><td>354234</td><td>Basement membrane matrix</td></tr><tr><td>MEM-NEAA</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>Mini Centrifuge</td><td>Labnet International</td><td>C1310</td><td>Benchtop mini centrifuge</td></tr><tr><td>Monarch RNA Cleanup Kit (50 &amp;micro;g)</td><td>New England BioLabs</td><td>T2040</td><td>Silica spin columns</td></tr><tr><td>Monarch Total RNA Miniprep Kit</td><td>New England BioLabs</td><td>T2010</td><td>Silica spin columns</td></tr><tr><td>N-2 supplement</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>PFA 16%</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Polyamine supplement</td><td>Sigma</td><td>P8483</td><td></td></tr><tr><td>Poly-L-Ornithine (PLO)</td><td>Sigma</td><td>3655</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>746436</td><td></td></tr><tr><td>SB431542</td><td>Sigma</td><td>54317</td><td></td></tr><tr><td>See through self-sealable pouches</td><td>Steriking</td><td>SS-T2 (90x250)</td><td>Autoclave pouches</td></tr><tr><td>Sodium citrate dihydrate&amp;nbsp;</td><td>Fisher Scientific</td><td>S279-500</td><td></td></tr><tr><td>Syringe filters, sterile, PES 0.22 &amp;micro;m, 30 mm Dia</td><td>Research Products International</td><td>256131</td><td></td></tr><tr><td>Trans-ISRIB</td><td>Cayman</td><td>16258</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Invitrogen</td><td>15596018</td><td>Phenol and guanidine isothiocyanate</td></tr><tr><td>TrypLE Express Enzyme (1x)</td><td>Gibco</td><td>12604039</td><td>Cell dissociation reagent&amp;nbsp;</td></tr><tr><td>Vapor pressure osmometer</td><td>Wescor, Inc.</td><td>Model 5520</td><td>Osmometer</td></tr><tr><td>Y-27632</td><td>Biogems</td><td>1293823</td><td></td></tr></tbody></table>

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.

Overview of the 3D to 2D cerebellar differentiation 
Cerebellar cells are generated starting from iPSCs. Figure 1A shows the overall workflow and the addition of major components for differentiation. On day 0, EBs are made by gently lifting the iPSC colonies (Figure 1B) using a pulled glass pipette in CDM containing SB431542 and Y-27632 and placed into ultra-low-attachment plates. FGF2 is added on day 2. On day 7, one-third of the medium is...

Watch this Scientific Journal Video about Modeling Human Cerebellar Development In Vitro in 2D Structure at JoVE.com

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.

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, ...

modeling-human-cerebellar-development-in-vitro-in-2d-structure

Research

JoVE Journal

Neuroscience

1.6K Views.  University of Iowa. Deze methode kan helpen bij het bestuderen van de vroege ontwikkeling van cerebellen bij de mens en hoe deze kan worden veranderd bij neuropsychiatrische aandoeningen. Het belangrijkste voordeel van deze methode is het genereren van ontwikkelingsmatig vroege menselijke cerebellaire cellen in een 2D-laagstructuur zonder ingewikkelde stappen uit te voeren. Het is ook kostenefficiënt.Het maken van pull pipetten kan in het begin lastig zijn, maar met oefening en tijd wordt het gemakkelijk...

Video: Modeling Human Cerebellar Development In Vitro in 2D Structure

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Developmental Biology

Understanding Cerebellar Pattern Formation

I am Kathy Millan. I'm an Assistant professor at the University of Chicago in the Department of Human Genetics and also in the Department of Neurology. And I work on brain development, and specifically I work on Cere Butler development, the cerebellum, the part of the brain back here at the back of the skull.And it's most famous for its role in motor development, but it turns out it's involved in a lot of things. And it's a really interesting part of the brain Pattern Formation is the development of pattern in a biological system. And it's one of these words that developmental biologists use kind of in a squishy way to mean almost anything.And it's not really clear what it means. I think every developmental biologist would give you a different version of what it means to me. What it means is how does the brain, or in particular the cerebellum, know from top and bottom, how do, how does you define left and right?How do you decide which part of the structure is going to be, which part? And once you've made those critical, fundamental early embryonic decisions, how do you bring all of the cellular structure together to form the exquisitely patterned part of the brain that we call the Cerebellum? So we're really Interested in cerebellar development and pattern information in the developing cerebellum.Because the cerebellum is an exquisitely patterned tissue. It's got not that many cell layers and not that many cell types. So relative to other parts of the brain, it's especially, well, it's a relatively simple structure.And also because the cerebellum has a very specific function, or one of many functions is motor coordination. When mice or humans have cerebellar abnormalities, one of the resulting phenotypes is motor uncoordination. And so over the a hundred years or so of mouse genetics, there have been multiple spontaneous mouse mutants that had been discovered and characterized to some extent, but also just isolated and sitting on the shelf at Jackson Labs where they're mice with motor abnormalities.And a large number of those mice, I think there's over about 60 spontaneous strains have cerebellar developmental abnormalities. And that resource is a really valuable rich resource to look at what are the developmental mechanisms that drive cerebellar pattern formation. So one thing my lab is focused on is to take all the tools that we have from the Human Genome Project and the Mouse Genome Project, and use those molecular reagents to go and find the genes that are mutate mutant in all of those spontaneous mutants.And by finding those mutants, we take a phenotype centric approach to finding genes. So we're not randomly taking genes and knocking them out. We're finding phenotypes and finding the genes because by using that approach, we're specifically finding genes that are particularly relevant to the system of interest.So our lab and many other labs have cloned a number of these spontaneous mouse mutants. And those spontaneous mouse mutants have led to really interesting new biology. Because we're taking this phenotype driven approach.We're not having any preconceptions about what kind of genes we're going to find. We're not specifically looking for dal ho analogs or channel mutations. We are getting to the genes that the phenotypes dictate.And because of that, we're learning a lot of new biology. So for example, one of the genes or one of the phenotypes that my lab has worked on and together when I was a postdoc with another colleague, Jim Linig, we worked on a mouse mutant called dre, which is German for spinner or spinning like a top. And these mouse mutants first arose in 1929 in Germany and have been accumulating at Jackson Labs.There are multiple alleles of this particular locus, and it was known that the cerebellum was incorrectly formed. So all the cerebellar cell types were there, but the pattern of the cerebellum was abnormal. The middle part was missing the middle part's called the verus.And we discovered that there was an early embryonic phenotype in these mice that led to that adult phenotype. And the phenotype was a result of mutation in a gene called LMX one A, which stands for limb homeo domain containing box one. So L LMX one A gene is actually turned into a really interesting and important gene in developmental biology because is one of the most critical genes in defining what is up in the central nervous system or what is dorsal.And without having this rare mutation, nobody actually knew what this gene did. In fact, it had been found for other reasons in insulin producing cells in the pancreas. And other people had been studying it for potential role in diabetes.But in fact, it turns out that the LMX one aging really by itself has no role in pancreatic development or eyelet cell development. And in fact, its main role in development is driving dorsal patterning in the central Nervous system. When we first cloned the DR Gene, the LMX one A gene, we realized that it's only expressed in the dorsal part of the central nervous system.And in fact, it's not only expressed in the dorsal part of the central nervous system or the the roof plate that it's called just around the cerebellum. It's actually expressed in the dorsal part of the central nervous along the whole axial length of the mouse embryo. And in fact, it's expressed along the whole axial length of every vertebrate that we've looked at so far.And because it's expressed dorsally everywhere, we reasoned that it must have a role dorsally in other parts of the, in the central nervous system. And so we actually spent a lot of time characterizing the phenotype of the DR mice in the spinal cord because the spinal cord is actually much better model system for understanding CNS development than the cerebellum because it's much less three dimensionally complicated. It's got a lot less three dimensional structure.Switching to the chick, we were actually able to take advantage of a large number of reagents to figure out what the role of LMX one A was in the spinal cord. And from the spinal cord, we switched back to the cerebellum. And frankly, I don't think without the technology of chick ation, we really would've had a great understanding of what the LMX one a gene does, a postdoc in my laboratory, Victor Chiko, was the person who really took these experiments and ran with them.And he was the person who actually figured out what the LMX one a gene does, and its primary role in central nervous system by using the Chick electroporation system. And I honestly don't think that we would've been able to make as much advance as we did without chick electroporation. So Chick electroporation is a really valuable technology because you can over express genes rapidly in the developing chick nervous system, get a result after two or three days of incubation and get analyzable phenotypes.If you were to actually try and do gene manipulations in mice for transgenic animals that would take several months and for knockouts, et cetera, that would take at least a year and a half. And so the chick ation system is a really valuable system because you can manipulate gene expression and you can find out phenotypes rapidly. So the LMX one aging when it's over expressed in the developing chick nervous system, in particular, the developing spinal cord, caused the whole dorsal part of the central nervous system or the spinal cord to turn into a dorsal part of the central nervous system or the roof plate.And so what that told us in combination with a bunch of other experiments that Victor did was that LMX one A by itself is responsible for driving the whole differentiation program of the roof plate. And that's important because the roof plate's a critical signaling center in the developing nervous system. If you don't have a roof plate, you don't secrete morphogen to tell adjacent neural tube cells to turn into dorsal sensory neurons.And without those signals, those cells go into a default state of an intermediate cell type. And you lose most of the dorsal neurons in the, in the, in the spinal cord, but also actually in the developing cerebellum. And I don't think we would've easily been able to come to that conclusion using any other system except the chicken Neural tube.We're really interested In cerebellar development, not only because of the basic biology in in in mouse systems and also in the chick, but also humans have cerebellar malformations. And those human cerebellar malformations are really poorly understood. And so in collaboration with a colleague, Dr.William Dobbins, one of the world's experts in human brain malformations, together, we've initiated a project to find human cerebellar malformation genes.And we're taking several approaches. One of the biggest things that we've done is we've built a DNA database and a clinical database where we've recruited over 500 patients with defined human cerebellar malformations and had collected images of MRIs and also correlated those with DNA samples so that we can use that as a resource for gene hunting because it turns out that human cerebellar malformations are not well categorized at all. And I would honestly say that out of every 10 scans we receive for through our studies, at least one of those scans is some malformation that's never been described and has not really been recognized before.The most common human cerebellar malformation is called Dandy Walker malformation, and it's the one that's most recognized by clinicians. But it turns out it's sort of a garbage diagnosis of the human cerebellar world. Any physician or many physicians, when they see an abnormal cerebellum classified as dandy walker.And so we recruit patients into our Dandy Walker malformation study, and we find all kinds of different interesting cerebellar malformations. So we're using this resource to find new cerebellar developmental genes, because as a developmental biologist, what I think is that humans are one great big mutagenesis experiment. They're one great source for finding new developmental genes.So we're taking several approaches to find those genes. One of those approaches is to take genes that we've figured out in mouse that have roles in cerebellar development and sequencing those genes in humans. And in fact, we have just recently identified the very first human LMX one A mutation.So so far there's Been really limited applications of our basic science research into clinical research. But that's really rapidly changing. As we're finding new and new genes, we can provide new genetic counseling information to families of affected children.And frankly, that's where I see the biggest impact Of our research. So my Vision for the field of five year in five years is that a lot more gene discovery will have happened both by characterization of mouse models and also by work in human cerebellar malformations. We're gonna know more about the set of genes that are involved in cerebellar development.And then the goal is gonna have to be how do those genes interact with each other, and how do those genes drive the actual biology? How do they drive all the different cellular events that take place to make a cerebellum? And I think the cerebellum's really an ideal model for the whole central nervous system because frankly, there are only five to seven principle neuronal cell types.They're all arranged in a various stereotypical manner. We're learning more and more about the molecules that drive the specification and the early differentiation of those progenitors. We're understanding more and more about how those cells proliferate.So I think more than five years from now, but ultimately I see the field being able to go tube development all the way through cerebellar development to achieve the mature form of the cerebellum. And I think because the cerebellum's a relatively simple system, we have a hope of actually figuring out the molecules and the developmental events that drive the formation of the entire central of the entire cerebellum. And I think that's really important, not only for the field of genetic diagnosis in humans, and it's important for the field of cerebellar development, but I think we're also gonna have applications of those mechanisms to other parts of the brain.The cerebellum's a relatively simple structure, but many of the developmental events aren't going to be exclusive to the cerebellum. We can take what we learned from the cerebellum to other more complicated regions of the central nervous system like the cerebral cortex, and take the lessons and the pathways that we learned and figure out how those apply to the rest Of the brain.

Biology

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended effects that extrinsic patterning factors might introduce into human pluripotent stem cell (hPSC) models of brain development or pathophysiology, such as masking disease phenotype. Here, we present two cerebellar differentiation protocols for hPSCs, designed with simpler startup method, fewer patterning factors, and less material requirements than previous protocols. Recently, we developed culture procedures, which generate free-floating 3-dimensional (3D) products consistent with other brain &#34;organoid&#34; protocols, including morphologies relevant to modeling brain development such as sub/ventricular zone- and rhombic lip-like structures. The second uses an adherent, 2D monolayer procedure to complete differentiation, which is shown capable of generating functional cerebellar neurons, as products are positive for cerebellar-associated markers, and exhibit neuron-like calcium influxes. Together, these protocols offer scientists a choice of options suited to different research purposes, as well as a basic model for testing other types of streamlined neural differentiations.

We describe a simplified 3D differentiation protocol for hPSCs, using defined medium and reduced growth factors, capable of generating cell aggregates with early neuroepithelial structures and positive for cerebellar-associated markers, as well as an optional 2D modification for differentiating cells as a monolayer to generate functional neurons.

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended ...

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 ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

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

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

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

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

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.

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 ...

Bioengineering

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in vitro, although scalable,&#160;does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo&#160;mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe&#160;an&#160;ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0&#8211;2 mice, after which they myelinate&#160;ex vivo&#160;for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.

Presented is a protocol for an ex vivo quantitative model of demyelination and remyelination using mouse cerebellar slice cultures. This method closely recapitulates an in vivo model with its full complement of CNS cell types in intact tissue, while maintaining the chemical, genetic, and environmental amenability of an in vitro system.

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in ...

Modeling Neuronal Injury in Mouse Primary Cerebellar Granule Neurons

Source: Laaper, M. et al., <a href="https://app.jove.com/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a>&#160;J. Vis. Exp. (2017).
This video demonstrates the generation of a neuronal injury model in mouse primary cerebellar granule neurons. It outlines the steps involved in inducing neuronal injury using NMDA, an excitatory neurotransmitter receptor agonist, and glycine, a co-agonist. NMDA and glycine bind to specific neuronal receptors, causing their overstimulation and resulting in a massive intracellular calcium influx, which leads to neuronal cell damage and death.

1. Experimental Preparation
NOTE: The following stock solutions can be prepared and maintained until use.

	Dissection Solution
	
		Dissolve 3.62 g of ...

Modeling Human Cerebellar Development In Vitro in 2D Structure

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