Name: purification of prominin-1+ stem cells from postnatal mouse cerebellum
Uploaded: 2020-04-12T13:00:00
Description: Watch this Scientific Journal Video about Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum at JoVE.com

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...

<ol>
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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>

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>2mM 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&#8217;s Phosphate Buffer Saline</td>
			<td>Thermo Fisher Scientific</td>
			<td>14040141</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;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>40um</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, ...

This protocol describes an easy and cost effective method to purify stem cells from the postnatal cerebellum. These stem cells account for the late-born molecularly garbled interneurons, but they also account for postnatally-derived cerebellar astrocytes. Thus, studying these cells is clearly important for understanding the development of the cerebellum.Using this protocol, the yield of stem cell is comparable to that of FACS-based strategy which is typically 10 times more expensive. This protocol can be generalized to extract prominin-1 expressing stem cells from other tissues such as intestine and bone marrow. Begin by placing the dish with the brain under a dissection microscope.Use fine number five forceps to remove the meninges and large blood vessels from the cerebellum, then separate the cerebellum from the brainstem using the spatula. Transfer the cerebellum into a 15 milliliter centrifuge tube containing five milliliters of ice cold HBSS solution. Rinse it then decant the HBSS.Repeat the wash two more times. After the last rinse, add five milliliters of papain-based tissue dissociation solution that has been pre-warmed to 37 degrees Celsius. Incubate the tissue for 15 minutes in a 37 degree Celsius water bath and slowly mix the contents of the tube by inverting it three to five times every three minutes.Prepare a wide diameter and narrow diameter glass Pasteur pipette by fire polishing over a Bunsen burner. After the incubation, wash the tissue three times with five milliliters of HBSS solution making sure to avoid losing tissue while decanting. After the last HBSS wash, add five milliliters of DPBS with 250 microliters of DNAse to the tissue and dissociate it by gently triturating 10 to 15 times with the wide diameter Pasteur pipette.Then use the narrow Pasteur pipette to further triturate the tissue slurry 10 times. If larger pieces of tissue remain, press them against the bottom of the tube with the pipette tip and continue pipetting until a fine suspension is achieved. Place the centrifuge tubes on ice.Then strain the dissociated cells through a 40 micrometer cell strainer into a 50 milliliter tube. Top the filter with 10 milliliters of ice cold HBSS solution to ensure that the cells pass through the mesh. Transfer the filtered cells to a fresh 15 milliliter centrifuge tube and centrifuge the suspension at 300 times g for 10 minutes at four degrees Celsius.Then use a vacuum aspirator to remove the supernatant completely. Resuspend the cell pellet in 160 microliters of ice cold magnetic column buffer. Add 40 microliters of anti-prominin-1 microbeads to the cell suspension.Then incubate the tubes in a refrigerator for 15 minutes to allow the antibody to bind to the prominin-1 expressing cells. Wash the cells with one to two milliliters of column buffer X and centrifuge them at 300 times g for 10 minutes. Aspirate the supernatant and resuspend the pellet in one milliliter of column buffer X.Place the magnetic columns on the magnetic stand and rinse them once with 500 microliters of buffer X.Apply the labeled cell suspension onto the column and collect the flowthrough in fresh 15 milliliter tubes.Wash the column three times with 500 microliters of buffer X.Then remove the columns from the magnetic field and place them in 1.5 milliliter tubes. Add one milliliter of culture medium and push the plunger into the column to flush out the cells tagged with prominin-1 beads. To passage the neurospheres, transfer them along with the culture medium into a sterile 15 milliliter centrifuge tube.Pellet the neurospheres by centrifugation at 300 times g for five minutes and discard the supernatant. Resuspend the pellet in five milliliters of dissociation media with papain or 0.05%trypsin solution and incubate the cells at 37 degrees Celsius for 10 minutes. Centrifuge the cell suspension again.Then resuspend the cells in five milliliters of neurosphere medium and dissociate them by slowly pipetting them up and down 10 times with a Pasteur pipette. Plate the cells in neurosphere media as described in the text protocol and enrich them with secondary neurospheres after seven to 10 days in culture as previously described. Count the cells on a hemocytometer and plate the optimal number of cells on poly-D-lysine coated plates for future experiments.Column purified prominin-1 positive postnatal cerebellar stem cells formed neurospheres in growth factor rich neurosphere medium. These neurospheres were positive for prominin-1, their marker used for isolation and for other stem cell markers such as nestin and GFAP. Cells in the flowthrough stained negative for stem cell markers prominin-1 and nestin and positive for neuronal marker beta-III tubulin.Upon withdrawal of growth factors and in the presence of leukemia inhibitory factor or PDGF-AA, the prominin-1 positive neurospheres differentiated into neurons, astrocytes and oligodendrocytes. When attempting this technique, it is important to make sure the neurospheres are pushed through for prominin-1 antibody and they can be passaged because normal cells also tend to form clumps which may look like neurospheres. Using this isolation method, one can characterize its derivative lineages like neurons and glia in health and disease condition.This method is valuable and provides novel insight into role of cerebellar stem cells in cerebellar development and disease conditions like spinocerebellar ataxia and cancer.

Research

JoVE Journal

Neuroscience

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

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. ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

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 ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...