Name: Modeling Human Cerebellar Development In Vitro in 2D Structure (Video) | JoVE Journal
Uploaded: 2022-09-16T14:30:00.000+00:00
Duration: 6 min 14 s
Description: Learn to generate 2D cerebellar cell monolayers from iPSCs for studying early human cerebellar development and neuropsychiatric disorder mechanisms. This cost-efficient protocol enables detailed developmental and pathological research.

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5 minutes

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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<li>Fossat, N., Courtois, V., Chatelain, G., Brun, G., Lamonerie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternative+usage+of+Otx2+promoters+during+mouse+development%5BTitle%5D)+AND+%22Developmental+Dynamics%22%5BJournal%5D)">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/pubmed?term=((The+PsychENCODE+project%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Genomics%22%5BJournal%5D)">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/pubmed?term=((Comparative+analysis+of+Six3+and+Six6+distribution+in+the+developing+and+adult+mouse+brain%5BTitle%5D)+AND+%22Developmental+Dynamics%22%5BJournal%5D)">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/pubmed?term=((The+role+of+the+cerebellum+in+schizophrenia%5BTitle%5D)+AND+%22Biological+Psychiatry%22%5BJournal%5D)">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/pubmed?term=((An+MRI+study+of+cerebellar+vermis+morphology+in+patients+with+schizophrenia%3A+Evidence+in+support+of+the+cognitive+dysmetria+concept%5BTitle%5D)+AND+%22Biological+Psychiatry%22%5BJournal%5D)">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>
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<li>Telley, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dual+function+of+NRP1+in+axon+guidance+and+subcellular+target+recognition+in+cerebellum%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Differentiation+of+human+induced+pluripotent+stem+cells+to+mature+functional+Purkinje+neurons%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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/pubmed?term=((Primary+culture+of+neurons+isolated+from+embryonic+mouse+cerebellum%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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

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 c...

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, ...

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

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JoVE Journal

Neuroscience

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...

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

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.

Modeling Neuronal Injury in Mouse Primary Cerebellar Granule Neurons

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.

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

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.

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

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.

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

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.

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

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.

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

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

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.

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

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Learn to generate 2D cerebellar cell monolayers from iPSCs for studying early human cerebellar development and neuropsychiatric disorder mechanisms. This cost-efficient protocol enables detailed developmental and pathological research.

Modeling Human Cerebellar Development In Vitro in 2D Structure

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