Name: isolation and culture of adult neural stem cells from the mouse subcallosal zone
Uploaded: 2016-12-15T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone at JoVE.com

This work was supported by the Brain Research Program through the National Research Foundation (NRF), funded by the Korean Ministry of Science, ICT, and Future Planning (Grants NRF-2015M3C7A1028790); and by a grant from the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (HI14C3347).

1. Preparation of Materials and Culture Medium
<ol>
	<li>For the dissection and dissociation of the SCZ, wrap the brain matrix, double-edged razor blade, and forceps with aluminum foil, and then sterilize them by autoclaving.</li>
	<li>Prepare 50 ml of cold PBS buffer to wash the whole mouse brain.</li>
	<li>Set up a dissection microscope and prepare the surgical tools required for the dissection of the brain (autoclaved scissors and forceps) and the isolation of the SCZ (1 ml syringe, 30 G needle, brain matrix, and fi...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+generation+of+neuronal+cells+from+the+adult+mouse+brain.">De novo generation of neuronal cells from the adult mouse brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 89, 8591-8595 (1992).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Gritti, 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=Epidermal+and+fibroblast+growth+factors+behave+as+mitogenic+regulators+for+a+single+multipotent+stem+cell-like+population+from+the+subventricular+region+of+the+adult+mouse+forebrain.">Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 19, 3287-3297 (1999).</li><li>Wachs, F. 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=High+efficacy+of+clonal+growth+and+expansion+of+adult+neural+stem+cells.">High efficacy of clonal growth and expansion of adult neural stem cells.</a> Laboratory investigation; a journal of technical methods and pathology. 83, 949-962 (2003).</li><li>Xiong, 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=Optimal+time+for+passaging+neurospheres+based+on+primary+neural+stem+cell+cultures.">Optimal time for passaging neurospheres based on primary neural stem cell cultures.</a> Cytotechnology. 63, 621-631 (2011).</li><li>Lee, S. W., 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=Optimization+of+Matrigel-based+culture+for+expansion+of+neural+stem+cells.">Optimization of Matrigel-based culture for expansion of neural stem cells.</a> Animal Cells and Systems. 19, 175-180 (2015).</li><li>Guo, W., Patzlaff, N. E., Jobe, E. M., Zhao, X. <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+multipotent+neural+stem+or+progenitor+cells+from+both+the+dentate+gyrus+and+subventricular+zone+of+a+single+adult+mouse.">Isolation of multipotent neural stem or progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse.</a> Nature protocols. 7, 2005-2012 (2012).</li><li>Walker, T. 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This paper describes a detailed protocol to generate NSCs from the adult mouse SCZ and to maintain them for various applications. There are three critical steps for establishing the in vitro culture system needed to purify and expand SCZ-NSCs. First, it is important to ensure that the SCZ region is precisely dissected out from other potential neurogenic regions (Figure 1B). Thick and precise sections containing the SCZ regions were obtained with a 1 mm interval brain matrix, and then a fine need...

NSCs have characteristics of self-renewal and multiple-lineage differentiation. To confirm these properties, a neurosphere culture system has widely been used. The neurosphere culture system was developed in the early 1990s and served as a standard stem cell culture system1. Depending on self-renewal potency, NSCs continuously proliferate and generate a cell mass in a suspension culture. The number of cells and the size of the neurosphere are considered to be closely associated with the proliferation properties of the NSCs. Monolayer cultures are also widely used for the maintenance and differentiation of NSCs. Compared to the neurosphere culture, the monolayer culture system provides better homogenous maintenance and expansion of NSCs2. These two well-developed culture systems have contributed to the characterization of aNSCs in vitro. 
NSCs reside in different brain regions, such as the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus3-5. The subcallosal zone (SCZ) of the caudal subcortical white matter is recognized as a novel neurogenic region6-8. It was recently reported that the SCZ-aNSCs have therapeutic potential in traumatic brain injury9. Compared to other neurogenic regions, the SCZ resides along the subcortical white matter. In the human brain, subcortical white matter occupies a larger region than in the mouse brain10. Therefore, an understanding of the characteristics of SCZ-aNSCs using an in vitro culture system is important in order to promote the potential use of these cells for neural regeneration. Precise dissection of the desired brain region is required to rule out possible contamination by unwanted regions containing active or quiescent NSCs. For instance, aNSCs in non-neurogenic regions can be activated and produce new neural cells in injured brains or during in vitro culturing11. To obtain NSCs from the SCZ, cells were collected from brain slices containing the SCZ. Then, a careful micro-dissection of the SCZ region was performed using a fine needle. To generate the neurosphere from the SCZ, micro-dissected SCZ tissue chunks were dissociated into single cells and then cultured as a suspension in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After SCZ-aNSCs form neurospheres, they also can be maintained as neurospheres or monolayers for expansion. This procedure also demonstrates immunostaining processes with various markers for the detection of NSCs and their progeny after their expansion and differentiation in a monolayer culture. Here, a visual protocol of the SCZ-aNSC culture system is presented. This protocol contains detailed instructions for the micro-dissection of the SCZ region and for the maintenance and passaging of the cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medium components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12&#160;</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td>+L-glutamin, +Sodium bicarbonate</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth factor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Gibco</td>
			<td>PHG0313</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>BIOSOLUTION</td>
			<td>BP007a</td>
			<td>1X dilution</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociattion buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche</td>
			<td>04-942-078-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>3126</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>ICT</td>
			<td>AT-104</td>
			<td></td>
		</tr>
		<tr>
			<td>Tool</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>WPI</td>
			<td>555229F</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Storz</td>
			<td>E3321-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain matrix (1mm)</td>
			<td>RWD</td>
			<td>68707</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-edged razor</td>
			<td>DORCO</td>
			<td>ST-300</td>
			<td></td>
		</tr>
		<tr>
			<td>30 gage needle</td>
			<td>SUNGSHIM</td>
			<td>N1300</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm dish</td>
			<td>SPL</td>
			<td>10035</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>100mm dish</td>
			<td>SPL</td>
			<td>10090</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>Cover slip (18mm)</td>
			<td>Deckglaser</td>
			<td>111580</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well dish</td>
			<td>SPL</td>
			<td>32012</td>
			<td>non-coating</td>
		</tr>
		<tr>
			<td>6 well dish</td>
			<td>SPL</td>
			<td>32006</td>
			<td>non-coating</td>
		</tr>
		<tr>
			<td>Coating materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PLO</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td>0.01%</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>Primary antibodies&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin</td>
			<td>Millipore</td>
			<td>MAB353</td>
			<td>mouse (1:1000)</td>
		</tr>
		<tr>
			<td>EGFR</td>
			<td>Abcam</td>
			<td>ab2430</td>
			<td>rabbit (1:1000)</td>
		</tr>
		<tr>
			<td>DCX</td>
			<td>Santa Cruz</td>
			<td>SC8066</td>
			<td>goat (1:500)</td>
		</tr>
		<tr>
			<td>Tuj1</td>
			<td>Sigma</td>
			<td>T2200</td>
			<td>rabbit (1:2000)</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Invitrogen</td>
			<td>13-0300</td>
			<td>rat (1:1000)</td>
		</tr>
		<tr>
			<td>O4</td>
			<td>Millipore</td>
			<td>MAB345</td>
			<td>mouse (1:500)</td>
		</tr>
		<tr>
			<td>BrdU</td>
			<td>Abcam</td>
			<td>ab6326</td>
			<td>Rat (1:500)</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse 488</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td rowspan="12">1:500</td>
		</tr>
		<tr>
			<td>anti-mouse cy3</td>
			<td>Jackson</td>
			<td>715-165-151</td>
		</tr>
		<tr>
			<td>anti-mouse 647</td>
			<td>Jackson</td>
			<td>715-606-150</td>
		</tr>
		<tr>
			<td>anti-rabbit 488</td>
			<td>Alexa</td>
			<td>A21206</td>
		</tr>
		<tr>
			<td>anti-rabbit cy3</td>
			<td>Jackson</td>
			<td>711-165-152</td>
		</tr>
		<tr>
			<td>anti-rabbit 647</td>
			<td>Jackson</td>
			<td>711-605-152</td>
		</tr>
		<tr>
			<td>anti-goat 488</td>
			<td>Alexa</td>
			<td>A11055</td>
		</tr>
		<tr>
			<td>anti-goat cy3</td>
			<td>Jackson</td>
			<td>705-165-147</td>
		</tr>
		<tr>
			<td>anti-goat 647</td>
			<td>Invitrogen</td>
			<td>A21447</td>
		</tr>
		<tr>
			<td>anti-rat 488</td>
			<td>Invitrogen</td>
			<td>A21208</td>
		</tr>
		<tr>
			<td>anti-rat cy3</td>
			<td>Jackson</td>
			<td>712-166-150</td>
		</tr>
		<tr>
			<td>anti-rat 647</td>
			<td>Jackson</td>
			<td>712-605-153</td>
		</tr>
		<tr>
			<td>Immunostaining materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Millipore</td>
			<td>82-100-6</td>
			<td>3% BSA with 0.1% Triton X-100 in PBS</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>usb</td>
			<td>22686</td>
			<td></td>
		</tr>
		<tr>
			<td>4% PFA</td>
			<td>Biosesang</td>
			<td>P2031</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechest33342</td>
			<td>Life Technology</td>
			<td>H3570</td>
			<td>Dye for staining nuclei</td>
		</tr>
	</tbody>
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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 into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Defining the culture system for aNSCs from the unknown neurogenic region is essential for understanding these cells and for developing their potential use in brain repair12. It is known that NSCs in different developmental stages or in different regions behave differently3,4. Recently, it was reported that SCZ-derived cells exhibit differential potentials for neuronal differentiation in vivo and in vitro compared to SVZ-derived cells7-9. ...

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

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

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

The overall goal of this adult neural cell culture system is to isolate adult neural stem cells from the subcallosal zone and expand the cells in nonadherent and adherent culture systems. This message can help answer key questions in the adult neural stem cell field such as isolation and culturization of neural stem cells from small brain lesions such as subcallosal zone. The main advantage of this technique is the isolation of adult neural stem cells from the subcallosal zone.It's simple, but precise. To begin this procedure, autoclave the brain matrix, a double edged razor blade, and forceps. Next, prepare 50 milliliters of cold PBS for washing the mouse brain.Then, set up a dissection microscope and prepare the surgical tools required for brain dissection and SCZ isolation. Prepare F-12 DMEM medium with 2%B-27 and 1%N-2 supplements and 1%penicillin-streptomycin. Then, prepare the digestion buffer for tissue digestion.After that, prepare 0.01%Poly-L-Ornithine and laminin. Following that, coat six small plates for the maintenance of SCZ adult neural stem cells as a monolayer, or 18 millimeter cover slips for immunostaining. Then, incubate them with Poly-L-Ornithine overnight at four degrees Celsius.Afterwards, wash them three times with DH20 and allow them to dry after the last wash. After that, incubate the plates with laminin overnight at four degrees Celsius. Once dissected, transfer the mouse brain to cold PBS buffer and rinse it twice to remove excess blood.Next, place the brain in the brain matrix on ice and make coronal cuts to obtain one millimeter thick slices. Subsequently, transfer the posterior brain slices containing SCZ regions to cold PBS. Under a dissecting microscope with low magnification, micro-dissect the SCZ from the white matter area of the cortex and the hippocampus with a bent 30 gauge needle.Then, remove the cortex regions above the SCZ and place the dissected SCZ into cold PBS in a 35 millimeter Petri dish on ice. The region of subcallosal zone can be dissected precisely from the whole tissue by using brain metrics and bent 30 gauge needle. Following that, remove the PBS and then immediately chop the dissected tissues into small pieces.Resuspend them with one milliliter of digestion buffer. Afterward, transfer the sample to a 15 milliliter tube containing two milliliters of digestion buffer. Then, incubate it for 30 minutes in a 37 degree Celsius water bath.In this step, tap the tube mildly to dissociate the digested tissue. And then centrifuge the tube at 145 x g for five minutes. After five minutes, discard the supernatant, resuspend the digested tissue with 1 milliliter of pre-warmed N-2 medium to wash out the digestion buffer and gently pipet the sample solution a maximum of five times.Next, centrifuge the sample again at 145 x g for five minutes. After discarding the supernatant, resuspend the cell pellet in one milliliter of N-2 medium. Place one milliliter of N-2 medium in a non-coated six-well plate.And add one milliliter of the suspended cells to make a final volume of two milliliters. Next, add 20 nanograms per milliliter EGF and 20 nanograms per milliliter bFGF into each well. Gently shake the six-well culture dish by hand, to mix the added growth factors with the plated cells.Then, keep the six-well plate in a 37 degree Celsius and 5%carbon dioxide incubator. Add 20 nanograms per milliliter EGF and 20 nanograms per milliliter bFGF to each well every day for eight days. Every third day, add 200 microliters of N-2 media to maintain approximately two milliliters of the medium.Here is an image of the neurospheres after eight days. In this procedure, gather the neurospheres and transfer them into a new 15 milliliter conical tube. Centrifuge the tube at 145 x g for five minutes.Subsequently, discard the supernatant and resuspend the neurospheres with 0.5 milliliters of dissociation buffer. Incubate them with 0.5 milliliters of dissociation buffer for five minutes in a 37 degree Celsius water bath, to dissociate the neurospheres into single cells. Primary neurospheres can be dissociated into single cells and maintained over several passages as a neurosphere or monolayer culture.Gently pipet the sample solution up and down less than five times and centrifuge the tube at 145 x g for five minutes. Subsequently, discard the supernatant and resuspend the neurospheres with one milliliter of N-2 medium. To count the cells, make 10 microliters of a cell suspension and 0.4%Trypan blue solution mixture, and count the number of live cells on a hemocytometer.After coating a six-well plate with PLO/Laminin, plate the cells at 2.5 x 10 to the fifth cells per milliliter, with two milliliters of N-2 medium per well. Maintain the SCZ adult neural stem cells with a daily treatment of growth factors for five days before passaging them. To differentiate SCZ adult neural stem cells, plate adult neural stem cells onto the PLO/Laminin coated 18 millimeter cover slips with 20 nanograms per milliliter growth factors.The next day, when the cells are firmly attached to the cover slips, replace the growth medium with N-2 to remove the growth factors. After six days, wash the differentiated cells with one milliliter of PBS to remove the cell debris and fix them for immunostaining. Shown here are the representative images of SCZ adult neural stem cells in the monolayer culture system, which shows the increase of the number of SCZ adult neural stem cells after three days in vitro.Here are the representative images of SCZ adult neural stem cell proliferation using the BrdU essay. Cells maintained in the monolayer culture system were not stained with markers for type C transient amplifying cells and type A neuroblasts. To confirm the differentiation capacity of SCZ adult neural stem cells, growth factors were removed from the culture media.After six days, cells were immunostained with markers for neurons, astrocytes, and oligodendrocytes. Once mastered, this technique can be done in 1.5 hours if it is performed properly. While attempting this procedure, it's important to remember to maintain the dissected tissue on ice and perform the procedure with precision.Following this procedure, other methods like effects can be performed in order to answer additional questions, like analyzing the intrinsic properties of subcallosal zone derived adult neural stem cells. After its development, this technique paved the way for researchers in the field of other neural stem cells to explore their potential for regeneration in brain damages. After watching this video, you should have a good understanding of how to culture other neural stem cells precisely from the subcallosal zone and maintain them in vitro.

Research

JoVE Journal

Neuroscience

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