We thank the Opal lab members for their suggestions. This work was supported by NIH grants 1RO1 NS062051 and 1RO1NS08251 (Opal P)

All animal experiments were performed in compliance with the NIH&#8217;s Guide for the Care and Use of Laboratory Animals (2011) and were approved by the Northwestern University IACUC (Protocol IS00011368).
1. Preparation of Solutions
<ol>
	<li>Prepare tissue dissociation solution made from sterile phenol red-containing Dulbecco&#8217;s phosphate-buffered saline (DPBS) with papain (100 U/ml), cysteine (0.2 mg/ml) and DNase (250 U/ml).</li>
	<li>To prepare DNase solution dilute&#160;100 mg of the lyophilized powder of DNase I (one bottle) in 50 mL of H2O. Mix well and filter the stock solution. Prepare 10 mL stock aliquots. Divide one stock tube into 0.5 mL single use aliquots. Store these single use aliquots at -80 &#176;C.</li>
	<li>Prepare magnetic separation reagents by preparing magnetic column buffer X: 0.5% bovine serum albumin (BSA) and 2 mM EDTA solution.</li>
	<li>To prepare neurosphere media, use neurobasal medium containing penicillin/streptomycin with L-glutamine and supplement with 2% B27, 20 ng/mL human recombinant epidermal growth factor (EGF), and 20 ng/mL human recombinant basic fibroblast growth factor (bFGF).</li>
	<li>To prepare differentiation media, use neurobasal medium and supplement with 10 ng/mL differentiated factor platelet-derived growth factor (PDGF-AA) or 10 ng/mL leukemia inhibitory factor (LIF) and 2% B27.</li>
	<li>Use ultra-low attachment 12 well (3.5 cm2) and 6 well (9.6 cm2) culture plates.</li>
</ol>
2. Dissection of Cerebellum
<ol>
	<li>Anesthetize mouse pups (P3-P7) with isoflurane and decapitate using surgical scissors.</li>
	<li>Spray the separated head of the pups with 70% ethanol.</li>
	<li>Transfer each head into an empty sterile 10 cm culture dish. Separate the skin using the microdissection scissors, then remove the skull by running the scissors sagittal along the midline.</li>
	<li>Using #7 forceps, peel off the skull bones starting caudally from the brainstem. Carefully lift the brain using a spatula, keeping the cerebellum intact, and transfer the brain to a fresh sterile 10.0 cm dish containing 15 mL of ice-cold Hanks&#39; Balanced Salt solution (HBSS) solution.</li>
	<li>Place the dish containing the brain under a dissection microscope. Using fine #5 forceps, remove the meninges and large blood vessels from the cerebellum and separate the cerebellum from the brainstem using the spatula.</li>
	<li>Transfer the cerebellum into a 15 mL centrifuge tube containing 5.0 mL of ice-cold HBSS solution. Wash the cerebellum by rinsing and decanting 3x with 5.0 mL of HBSS. 
	NOTE: Each cerebellum should be placed in its own tube for further processing.</li>
</ol>
3. Cell Suspension Preparation
<ol>
	<li>After the last rinse, add 5 mL of papain-based tissue dissociation solution (based on previous work13,18) that has been pre-warmed to 37 &#176;C. Incubate the tissue for 15 min at 37 &#176;C in a water bath. Slowly mix the content by inverting the tube up and down 3x&#8211;5x every 3 min either using a nutating mixer or by hand.</li>
	<li>Prepare a wide diameter (regular glass pipette) and narrow diameter Pasteur pipette (fire-polished by heating over a Bunsen burner) as described previously19.</li>
	<li>Wash the tissue 3x with 5 mL of HBSS solution, avoiding loss of the tissue while decanting by hand.</li>
	<li>Remove the last HBSS wash and add 5 mL of DPBS solution containing 250 &#956;L of DNase solution to the tissue. Dissociate the tissue by triturating 10x&#8211;15x using the wide diameter Pasteur pipette. Perform this step gently to avoid the formation of bubbles.</li>
	<li>Then incubate the slurry for an additional 10 min at 37 &#176;C in the water bath, mixing by inverting and straightening the tube.</li>
	<li>Use the reduced diameter Pasteur pipette to further triturate the tissue slurry 10x. If large pieces of tissue remain, press the tissue pieces against the bottom of the tube with the tip of the pipette gently and continue pipetting until the cells reach a fine suspension.</li>
	<li>Incubate the tissue at 37 &#176;C for an additional 10 min, repeating the previous mixing steps.</li>
</ol>
4. Immunolabeling of Stem Cells
<ol>
	<li>Place the centrifuge tubes on ice and use ice-cold solutions for the next steps. Strain the dissociated cells through a 40 &#956;m cell strainer into a 50 mL centrifuge tube. Top the filter with 10.0 mL of HBSS solution to ensure that cells pass through the mesh in this additional solution.</li>
	<li>Transfer the filtered cells into a fresh 15 mL centrifuge tube and centrifuge the cell suspension at 300 x g for 10 min at 4 &#176;C. Aspirate and discard the supernatant completely by&#160;carefully using a vacuum aspirator.</li>
	<li>Resuspend the pellet in 160 &#956;L of magnetic column buffer. To obtain the single-cell suspension before magnetic labeling, pass cells through a 30 &#956;m nylon mesh to remove cell clumps, which can otherwise clog the column.</li>
	<li>Add 40 &#956;L of anti-prominin-1 microbeads to each 15 mL tube, mix, and incubate in a refrigerator for 15 min in order for the antibody to bind to prominin-1-expressing cells.</li>
	<li>Wash the cells by adding 1.0&#8211;2.0 mL of column buffer X and centrifuge at 300 x g for 10 min. Aspirate the supernatant completely.</li>
	<li>Resuspend the pellet in 1.0 mL of column&#160;buffer X. 
	NOTE: If small clumps are seen after the resuspension of pellet with 1.0 mL of column buffer X, they&#160;should be removed carefully using a tip of the Pasteur pipette,&#160;otherwise&#160;these clumps can block the magnetic column during cell sorting.</li>
</ol>
5. Magnetic Column Preparation, Cell Sorting, and Plating
NOTE: The magnetic separation from different genotype conditions (disease vs. control) must be performed at same time, since any delay may affect the neurosphere morphology.
<ol>
	<li>Prepare the magnetic columns by placing them on the magnetic stand exposed to the magnetic field. Rinse the column once with 500 &#956;L of buffer X by applying buffer that drips into a centrifuge tube to be discarded. 
	NOTE: Prepare fresh magnetic column buffer X for each experiment; if not, then stem cell yield will be low.</li>
	<li>Apply the labeled cell suspension onto the column. Collect the flowthrough containing unlabeled cells that mainly consist of cerebellar neuronal/glial mixed cells into fresh 15 mL tubes (Figure 1A).</li>
	<li>Wash the column 3x with 500 &#956;L of buffer X (each wash takes around 2&#8211;4 min). 
	NOTE: Cerebellar neuronal/glial mixed culture enriched with cerebellar granular neurons can serve as a useful byproduct of this purification step.</li>
	<li>Remove the column from the magnetic field and place in a 1.5 mL tube. Add 1.0 mL of culture medium (neurosphere medium) to the column and push the plunger into the column to flush out the cells tagged with prominin-1 beads into a fresh 1.5 mL falcon tube.</li>
	<li>To enhance the purity of prominin-1 labeled cells, pass the eluted cells over a second column following steps 5.1&#8211;5.4.</li>
	<li>Count the cells with a hemocytometer. The typical yield is 107 cells per cerebellum. Plate the cells onto ultra-low attachment plates (6 or 12 well, based on the density required for downstream experiments).</li>
</ol>
6. Passaging of Neurospheres and Differentiation
<ol>
	<li>Plate the prominin-1-labeled stem cells on ultra-low attachment 12 well plates in neurosphere medium (5,000 cells/well).</li>
	<li>After 7&#8211;10 days, the cells divide to yield ball shaped floating neurospheres (primary neurospheres). 
	NOTE: In this experiment, secondary neurospheres that expand in numbers are generated for further use. Primary neurosphere populations are not used for these experiments, since they may contain contaminating cells that do not have stem cell properties and can clump together with neurospheres.</li>
	<li>For passaging, transfer the primary neurospheres along with the culture media using 1.0 mL pipette tips to a 15 mL sterile centrifuge tube. Pellet the neurospheres by centrifugation at 300 x g for 5 min and discard the supernatant.</li>
	<li>Resuspend the pellet in 5 mL of tissue dissociation media that contains papain or 0.05% trypsin solution. Incubate at 37 &#176;C for 10 min.</li>
	<li>Centrifuge the cell suspension at 300 x g for 5 min. Resuspend the cells in 5 mL of neurosphere medium and dissociate the cells mechanically using a plastic Pasteur pipette and slowly&#160;pipetting up and down 10x.</li>
	<li>Plate the cells (again) in neurosphere media as described earlier. After 7&#8211;10 days in culture, the plate should be enriched with secondary neurospheres. 
	NOTE: These cultures can be passaged up to 8x with good efficiency, after which neurosphere size tends to be smaller and suggestive of a decrease in proliferation.</li>
	<li>Count the cells on the hemocytometer and plate the optimal number for future experiments on poly-D-lysine coated plates (6 or 12 wells).</li>
	<li>To differentiate the stem cells, collect neurospheres from the second to eighth passage by centrifugation as described earlier, except add differentiation medium to the pellet. 
	NOTE:&#160;After&#160;7 days in vitro, the stem cells differentiate into neurons, astrocytes, and oligodendrocytes, as demonstrated by staining with the neuronal marker &#946;-III tubulin, astrocytic marker GFAP, and oligodendrocyte maker O4.</li>
</ol>

<ol>
	<li>Glickstein, M., Strata, P., Voogd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellum:+history.">Cerebellum: history.</a> Neuroscience. 162, 549-559 (2009).</li><li>Carta, I., Chen, C. H., Schott, A. L., Dorizan, S., Khodakhah, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+modulation+of+the+reward+circuitry+and+social+behavior.">Cerebellar modulation of the reward circuitry and social behavior.</a> Science. 363, (2019).</li><li>Sathyanesan, 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=Emerging+connections+between+cerebellar+development,+behaviour+and+complex+brain+disorders.">Emerging connections between cerebellar development, behaviour and complex brain disorders.</a> Nature Reviews Neuroscience. 20, 298-313 (2019).</li><li>Wagner, M. J., Kim, T. H., Savall, J., Schnitzer, M. J., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+granule+cells+encode+the+expectation+of+reward.">Cerebellar granule cells encode the expectation of reward.</a> Nature. 544, 96-100 (2017).</li><li>Araujo, A. P. B., Carpi-Santos, R., Gomes, F. C. A. <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+Astrocytes+in+the+Development+of+the+Cerebellum.">The Role of Astrocytes in the Development of the Cerebellum.</a> Cerebellum. , (2019).</li><li>Seto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+identity+transition+from+Purkinje+cell+progenitors+to+GABAergic+interneuron+progenitors+in+the+cerebellum.">Temporal identity transition from Purkinje cell progenitors to GABAergic interneuron progenitors in the cerebellum.</a> Nature Communication. 5, 3337 (2014).</li><li>Marzban, H., 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=Cellular+commitment+in+the+developing+cerebellum.">Cellular commitment in the developing cerebellum.</a> Frontiers in Cell Neurosciences. 8, 450 (2014).</li><li>Koziol, L. F., 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=Consensus+paper:+the+cerebellum's+role+in+movement+and+cognition.">Consensus paper: the cerebellum's role in movement and cognition.</a> Cerebellum. 13, 151-177 (2014).</li><li>Ben-Arie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+is+essential+for+genesis+of+cerebellar+granule+neurons.">Math1 is essential for genesis of cerebellar granule neurons.</a> Nature. 390, 169-172 (1997).</li><li>Machold, R., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+is+expressed+in+temporally+discrete+pools+of+cerebellar+rhombic-lip+neural+progenitors.">Math1 is expressed in temporally discrete pools of cerebellar rhombic-lip neural progenitors.</a> Neuron. 48, 17-24 (2005).</li><li>Wang, V. Y., Rose, M. F., Zoghbi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+expression+redefines+the+rhombic+lip+derivatives+and+reveals+novel+lineages+within+the+brainstem+and+cerebellum.">Math1 expression redefines the rhombic lip derivatives and reveals novel lineages within the brainstem and cerebellum.</a> Neuron. 48, 31-43 (2005).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+population+of+Nestin-expressing+progenitors+in+the+cerebellum+exhibits+increased+tumorigenicity.">A population of Nestin-expressing progenitors in the cerebellum exhibits increased tumorigenicity.</a> Nature Neurosciences. 16, 1737-1744 (2013).</li><li>Lee, 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=Isolation+of+neural+stem+cells+from+the+postnatal+cerebellum.">Isolation of neural stem cells from the postnatal cerebellum.</a> Nature Neurosciences. 8, 723-729 (2005).</li><li>Toren, 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=CD133-positive+hematopoietic+stem+cell+"stemness"+genes+contain+many+genes+mutated+or+abnormally+expressed+in+leukemia.">CD133-positive hematopoietic stem cell "stemness" genes contain many genes mutated or abnormally expressed in leukemia.</a> Stem Cells. 23, 1142-1153 (2005).</li><li>Zhu, 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=Prominin+1+marks+intestinal+stem+cells+that+are+susceptible+to+neoplastic+transformation.">Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation.</a> Nature. 457, 603-607 (2009).</li><li>Man, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+Role+for+the+DNA+Sensor+AIM2+in+Stem+Cell+Proliferation+and+Cancer.">Critical Role for the DNA Sensor AIM2 in Stem Cell Proliferation and Cancer.</a> Cell. 162, 45-58 (2015).</li><li>Fleming, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Purkinje+neuron+acts+as+a+central+regulator+of+spatially+and+functionally+distinct+cerebellar+precursors.">The Purkinje neuron acts as a central regulator of spatially and functionally distinct cerebellar precursors.</a> Developmental Cell. 27, 278-292 (2013).</li><li>Panchision, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+flow+cytometric+analysis+of+central+nervous+system+tissue+reveals+novel+functional+relationships+among+cells+expressing+CD133,+CD15,+and+CD24.">Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24.</a> Stem Cells. 25, 1560-1570 (2007).</li><li>Beaudoin, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+pyramidal+neurons+from+the+early+postnatal+mouse+hippocampus+and+cortex.">Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex.</a> Nature Protocols. 7, 1741-1754 (2012).</li><li>Edamakanti, C. R., Do, J., Didonna, A., Martina, M., Opal, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+ataxin1+disrupts+cerebellar+development+in+spinocerebellar+ataxia+type+1.">Mutant ataxin1 disrupts cerebellar development in spinocerebellar ataxia type 1.</a> Journal of Clinical Investigation. 128, 2252-2265 (2018).</li><li>Erlandsson, A., Enarsson, M., Forsberg-Nilsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immature+neurons+from+CNS+stem+cells+proliferate+in+response+to+platelet-derived+growth+factor.">Immature neurons from CNS stem cells proliferate in response to platelet-derived growth factor.</a> Journal of Neurosciences. 21, 3483-3491 (2001).</li><li>Galli, R., Pagano, S. F., Gritti, A., Vescovi, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neuronal+differentiation+in+human+CNS+stem+cell+progeny+by+leukemia+inhibitory+factor.">Regulation of neuronal differentiation in human CNS stem cell progeny by leukemia inhibitory factor.</a> Developmental Neurosciences. 22, 86-95 (2000).</li><li>Silbereis, J., Cheng, E., Ganat, Y. M., Ment, L. R., Vaccarino, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precursors+with+Glial+Fibrillary+Acidic+Protein+Promoter+Activity+Transiently+Generate+GABA+Interneurons+in+the+Postnatal+Cerebellum.">Precursors with Glial Fibrillary Acidic Protein Promoter Activity Transiently Generate GABA Interneurons in the Postnatal Cerebellum.</a> Stem Cells. 27, 1152-1163 (2009).</li><li>Parmigiani, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+and+Bipotency+of+Astroglial-Like+Cerebellar+Progenitors+along+the+Interneuron+and+Glial+Lineages.">Heterogeneity and Bipotency of Astroglial-Like Cerebellar Progenitors along the Interneuron and Glial Lineages.</a> Journal of Neurosciences. 35, 7388-7402 (2015).</li><li>Wojcinski, 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=Cerebellar+granule+cell+replenishment+postinjury+by+adaptive+reprogramming+of+Nestin(+)+progenitors.">Cerebellar granule cell replenishment postinjury by adaptive reprogramming of Nestin(+) progenitors.</a> Nature Neurosciences. 20, 1361-1370 (2017).</li><li>Yang, Z., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YAP1+is+involved+in+replenishment+of+granule+cell+precursors+following+injury+to+the+neonatal+cerebellum.">YAP1 is involved in replenishment of granule cell precursors following injury to the neonatal cerebellum.</a> Developmental Biology. 1606 (19), 30207 (2019).</li><li>Wang, S. S., Kloth, A. D., Badura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cerebellum,+sensitive+periods,+and+autism.">The cerebellum, sensitive periods, and autism.</a> Neuron. 83, 518-532 (2014).</li><li>Eberhart, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+down+and+one+to+go:+modeling+medulloblastoma+subgroups.">Three down and one to go: modeling medulloblastoma subgroups.</a> Cancer Cell. 21, 137-138 (2012).</li><li>Takahashi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133+is+a+positive+marker+for+a+distinct+class+of+primitive+human+cord+blood-derived+CD34-negative+hematopoietic+stem+cells.">CD133 is a positive marker for a distinct class of primitive human cord blood-derived CD34-negative hematopoietic stem cells.</a> Leukemia. 28, 1308-1315 (2014).</li></ol>

No conflicts of interest are declared.

Prominin-1-expressing cerebellar stem cells reside in the prospective white matter during the first 3 weeks of postnatal life. Their proliferation is tightly controlled by the sonic hedgehog pathway supported by Purkinje cells17. These stem cells/progenitors contribute to later-born GABAergic interneurons called basket cells and stellate cells. These interneurons reside in the molecular layer, where they synapse onto Purkinje cells and sculpt PC topography and function via GABAergic inhibition<sup...

The cerebellum has been long recognized as a major neuronal circuit coordinating voluntary movement1. It receives input from the wide swathes of the neuroaxis, which includes proprioceptive information from the periphery, so as to fine tune motor output&#160;and coordinate motion. More recently, it has also been implicated in regulating cognition and emotions by potentially using similar information processing networks2,3,4.
The adult cerebellum is composed of an outer cerebellar cortex and inner white matter. Interspersed within these structures are deep intracerebellar nuclei. Similar to the rest of the nervous system, the development of the cerebellum is driven by the proliferation of multipotent progenitor cells (stem cells) that migrate and differentiate to yield this well-organized structure. In early development (E10.5&#8211;E13.5), a ventricular stem niche around the developing fourth ventricle generates GABAergic neurons (i.e., Purkinje cells, Lugaro cells, Golgi cells) along with Bergmann glia5,6,7,8.
Later in development (postnatal week one), a second stem cell niche in the rhombic lip generates MATH1- and Nestin-expressing progenitors that give rise to excitatory granule neurons9,10,11,12. Recently a third stem cell niche has been described13. These cells express prominin-1 (also known as CD133), a membrane-spanning glycoprotein that defines a subset of stem cells in the intestine and hematopoietic systems14,15,16. In vivo fate mapping shows that these stem cells generate key molecular layer interneurons (i.e., basket cells and stellate cells), along with astrocytes, during the first three postnatal weeks. In the past, it has been difficult to study these cells in vitro because prior methods have required costly and time-consuming techniques (i.e., fluorescence-activated cell sorting [FACS]) that are dependent on prominin-1 staining12,13,17. This protocol describes an immunomagnetic-based method for the isolation of these stem cells that can then be readily cultured and differentiated.

<table><tbody><tr><td>0.05%Trypsin</td><td>Thermo Fisher Scientific</td><td>25300054</td><td>0.05%</td></tr><tr><td>2% B27</td><td>Gibco; Thermo Fisher Scientific</td><td>17504001</td><td></td></tr><tr><td>2 mM EDTA solution</td><td>Corning</td><td>46-034-CI</td><td></td></tr><tr><td>Anti- Prominin-1 microbeads</td><td>Miltenyi Biote</td><td>130-092-333</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A9418</td><td></td></tr><tr><td>Column MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>Culture plates ultra - low attachment</td><td>Corning</td><td>3473</td><td></td></tr><tr><td>Cysteine</td><td>Sigma</td><td>C7880</td><td></td></tr><tr><td>DNase</td><td>Sigma</td><td>D4513-1VL</td><td>250 U/ml</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffer Saline</td><td>Thermo Fisher Scientific</td><td>14040141</td><td></td></tr><tr><td>Hank&#039;s balanced salt solution-HBSS</td><td>Gibco</td><td>14025-092</td><td></td></tr><tr><td>Human recombinant Basic Fibroblast Growth Factor</td><td>Promega</td><td>G507A</td><td>20 ng/mL</td></tr><tr><td>Human recombinant Epidermal Growth Factor</td><td>Promega</td><td>G502A</td><td>20 ng/mL</td></tr><tr><td>Leukemia Inhibitory Factor</td><td>Sigma</td><td>L5158</td><td></td></tr><tr><td>l-glutamine</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>Microscopy</td><td>Lieca TCS SP5 confocal microscopes</td><td></td><td></td></tr><tr><td>MiniMACS separator</td><td>Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>Mouse anti-Prominin-1</td><td>Affymetrix eBioscience</td><td>14-1331</td><td>1 in 100</td></tr><tr><td>Nestin</td><td>Abcam</td><td>ab27952</td><td>1 in 200</td></tr><tr><td>Neurobasal medium</td><td>Thermo Fisher</td><td>25030081</td><td></td></tr><tr><td>O4</td><td>Millopore</td><td>MAB345</td><td></td></tr><tr><td>Papain</td><td>Worthington</td><td>LS003126</td><td>(100 U/mL)</td></tr><tr><td>Platelet- Derived Growth Factor</td><td>Sigma</td><td>H8291</td><td>10 ng/mL</td></tr><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>P6407</td><td></td></tr><tr><td>Rabbit anti-tubulin, b-III</td><td>Sigma</td><td>T2200</td><td>1 in 500</td></tr><tr><td>Rabit anti-GFAP</td><td>Dako</td><td>Z0334</td><td>1 in 500</td></tr><tr><td>Separation columns-MS columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>Sterile cell strainer</td><td>Fisher Scientific</td><td>22363547</td><td>40 &amp;mu;m</td></tr></tbody></table>

purification of prominin-1+ stem cells from postnatal mouse cerebellum

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, and a ventricular zone niche, which generates the inhibitory GABAergic Purkinje cells, which are neurons that constitute the deep cerebellar nuclei and Bergman glia. Recently, a third stem cell niche has been described that arises as a secondary germinal zone from the ventricular zone niche. The cells of this niche are defined by the cell surface marker prominin-1 and are localized to the developing white matter of the postnatal cerebellum. This niche accounts for the late born molecular layer GABAergic interneurons along with postnatally generated cerebellar astrocytes. In addition to their developmental role, this niche is gaining translational importance in regards to its involvement in neurodegeneration and tumorigenesis. The biology of these cells has been difficult to decipher because of a lack of efficient techniques for their purification. Demonstrated here are efficient methods to purify, culture, and differentiate these postnatal cerebellar stem cells.

Prominin-1-positive postnatal cerebellar stem cells formed neurospheres in neurosphere medium rich in growth factors (EGF and bFGF). These neurospheres were positive for prominin-1-staining, the marker used for isolation, and also as a stain for other stem cell markers such as Nestin and GFAP13 (Figure 1). The stem cell marker expression was maintained throughout culture and for up to at least eight passages20. Upon withdrawal of growth factors...

Watch this Scientific Journal Video about Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum at JoVE.com

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Demonstrated here is an efficient and cost-effective method to purify, culture, and differentiate white matter stem cells from postnatal mouse cerebellum.

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, ...

purification-of-prominin-1-stem-cells-from-postnatal-mouse-cerebellum

Research

JoVE Journal

Neuroscience

5.9K Views.  Northwestern University Feinberg School of Medicine Chicago. Demonstrated here is an efficient and cost-effective method to purify, culture, and differentiate white matter stem cells from postnatal mouse cerebellum.

Video: Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Biology

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

Extraction and Enzymatic Purification of Microglia from the Mouse Spinal Cord

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and function of the mammalian central nervous system rely significantly on the activity of resident macrophages known as microglia. Microglia display heterogeneity and multifunctionality, enabling distinct gene expression and behavior within the spinal cord and brain. Numerous studies have explored cerebral microglia function, detailing purification methods extensively. However, the purification of microglia from the spinal cord in mice lacks a comprehensive description. In contrast, the utilization of a highly purified collagenase, as opposed to an unrefined extract, lacks reporting within central nervous system tissues. In this study, the vertebral column and spinal cord were excised from 8-10 week-old C57BL/6 mice. Subsequent digestion employed a highly purified collagenase, and microglia purification utilized a density gradient. Cells underwent staining for flow cytometry, assessing viability and purity through CD11b and CD45 staining. Results yielded an average viability of 80% and a mean purity of 95%. In conclusion, manipulation of mouse microglia involved digestion with a highly purified collagenase, followed by a density gradient. This approach effectively produced substantial spinal cord microglia populations.

Author Spotlight: Microglia Research on Spinal Cord Heterogeneity and Purification 

Microglia are regarded as some of the most versatile cells in the body, capable of morphological and functional adaptation. Their heterogeneity and multifunctionality enable the maintenance of brain homeostasis, while also being linked to various neurological pathologies. Here, a technique for purifying spinal cord microglia is described.

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

Source: Bovio, P., et al., <a href="https://app.jove.com/v/56631">Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark.</a> J. Vis. Exp. (2018)
This video demonstrates the isolation and cultivation of cerebellar neural progenitor cells from a mouse pup&#39;s cerebellum. Cerebellar granule neuron progenitors are isolated and purified through differential adhesion, effectively removing astrocytes. These cultured progenitors subsequently differentiate into cerebellar granule neurons.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1.&#160;Preparations

	Preparations ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Isolation of Stem Cells from Postnatal Mouse Cerebellum and Differentiation into Neural Cells

Source: Edamakanti, C. R., et al. <a href="https://app.jove.com/v/60554">Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum.</a> J. Vis. Exp. (2020).
This video demonstrates a method for isolating stem cells from the postnatal mouse cerebellum and differentiating them into neural cells. Stem cells from the isolated cell suspension are purified using antibody-coated magnetic beads and are cultured to form neurospheres. The neurospheres are then maintained in a differentiation medium to generate neural cells.

1. Preparation of Solutions

	Prepare tissue dissociation solution made from sterile phenol red-containing Dulbecco&#39;s phosphate-buffered saline (DPBS) ...

Purification of Prominin-1⁺ Stem Cells from Postnatal Mouse Cerebellum

Summary

Explore More Videos

Chapters in this video

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

A High Output Method to Isolate Cerebral Pericytes from Mouse

Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

Isolation of Stem Cells from Postnatal Mouse Cerebellum and Differentiation into Neural Cells