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).

Malzemeler ve kültür besiyeri hazırlanması 1. <ol><li> SCz diseksiyonu ve ayrışma için, alüminyum folyo ile beyin matrisi, iki ucu keskin jilet ve forseps sarın ve daha sonra otoklav ile sterilize. </li><li> Bütün fare beyin yıkamak için soğuk PBS tamponu 50 ml hazırlayın. </li><li> Bir diseksiyon mikroskobu kurmak ve beyin (otoklav makas ve forseps) diseksiyonu için gerekli cerrahi aletler ve SCZ (1 ml şırınga, 30 G iğne, beyin matris ve ince forseps) izolasyonuna hazırlar. </li><li> N2 ortamında hazırlanması: <ol><li> F-12 / DMEM (+ L-glutamin + sodyum bikarbonat)% 2 B27,% 1, N2 takviyeleri ve% 1 Penisilin-Streptomisin ile orta hazırlayın. Not: büyüme ortamı N2 ortamı ve büyüme faktörleri oluşur (20 ng / ml saflaştırılmış epidermal büyüme faktörü (EGF) ve 20 ng / ml temel fibroblast büyüme faktörü (bFGF2)). </li></ol></li><li> Bir sindirim tampon hazırlayın (40 ünite / ml papain, 2.4 ünite / ml dispase II ve% 2 penisilin-streptomisin, PBS içinde), Doku sindirimi için. </li><li> Kaplama Plakası ve lamel hazırlanması: <ol><li> (0.01,% PLO) ve laminin (DH 2 O içinde çözülmüş 10 mg / ml), poli-L-ornitin hazırlayın. bir tek tabaka ya da immün 18 mm lamelleri olarak SCZ-aNSCs bakımı için kaplama 6 oyuklu plakalar, 4 ° C&#39;de gece boyunca PLO ile inkübe edin. Sonra O. tabak ve lamelleri son yıkandıktan sonra kurumaya bırakın DH 2 ile onları 3 kez yıkayın. </li><li> Daha sonra, gece boyunca 4 ° C &#39;de laminin inkübe edin. Sonra, DH 2 O. ile onları 3 kere yıkayın Dikkat: hücre eki etkileyecek olan, laminin kurutmayın. Not: kaplama çözeltileri 3 kez tekrar edilebilir. </li></ol></li></ol> 2. İzolasyon ve Yetişkin SCz ve Ayrılma <ol><li> kültür hazırlama öncesinde, buz üzerinde beyin matrisi ve iki ucu keskin jilet yerleştirin. Not: beyin matris dondurmak br çünkü etmeyinain buna eklemek olabilir. </li><li> CO 2 boğulma ya da servikal dislokasyon (8 haftalık) bir fare Kurban. <ol><li> % 70 etanol püskürtme sonra keskin bir makas ile kafasını kesti. kafasını hareketsiz kılmak, sıkıca başın her iki tarafını tutun. Bir kuyruk-rostral yönde orta hatta makas ile cilt kesin. Bu kafatası derinin tamamen kaldırılması teşvik etmektedir. </li><li> dorsal orta hat konumunda kafatası ve beyin arasındaki forseps yerleştirin daha sonra ilk kafatasına (lambda posterior) kaudal parçası kaldırmak ve. sol (veya sağ) parietal kemik kapmak ve dikkatlice sökün. Diğer parietal kemiğin çıkarılması için bu yordamı yineleyin. Not: Optik sinir ve beyin menenjlerin çıkarılması Ayırma daha kolay kafatası beyin ayırmak için yapmak. </li><li> Soğuk PBS tampon (25 ml) beyin aktarın ve aşırı kan çıkarmak için iki kez yıkayın. </li></ol></li><li> buz ve ma beyin matris içine beyin yerleştirinke koronal kesimler 1 mm kalınlığında dilimler elde etmek. 35 mm plastik Petri tabağı içine soğuk PBS beynin arka kısmında yer almaktadır SCZ bölgeler içeren Beyin dilimleri (1 mm) aktarın. Dikkat: beyin fissür beyin matris orta hatta yerleştirilir emin olun, koronal kesitlerin paralel düzlemi elde etmek için; bölümlerde gereksiz değişimleri önlemek için önemlidir. Not: Bregma 2-3 mm posterior beyin dilimleri alın; Bu SVZ&#39;unda gelen SCZ ayrılmasını sağlar. </li><li> Düşük büyütme ile mikroskop altında, bir bükülmüş 30G iğne 6,9 ile korteks ve hipokampus beyaz cevher alanından SCZ mikro-teşrih. Ardından, SCZ yukarıdaki korteks bölgeleri kaldırın. soğuk PBS olmadan buz üzerinde bir 35 mm plastik petri içine dilim disseke SCZ bölgesini yerleştirin. Not: olgun nöron içeren ekstra bölgelerin Kirlilik aNSCs ve neurosphere oluşum b canlılığını etkileyebilirçünkü ara bunlar ANSC kültür koşullarında hücre ölümüne uğrarlar. </li><li> bükülmüş 30 G iğne kullanılarak, hemen küçük parçalar halinde disseke dokuların doğrayın. Not: daha uzun bir zaman SCZ doku kesme için gerekli ise, kesme işleminden önce soğuk PBS içinde parçalara dokuların bırakın. </li><li> Yeniden askıya sindirim tamponu 1 ml doğranmış doku, sindirim tamponu 2 mi ihtiva eden bir 15 ml tüp doku aktarın ve 37 ° C su banyosu içinde 30 dakika için inkübe edilir. Not: iyice karıştırın etmek için tüpü her 10 dakikada sallayın. </li></ol> 3. Subcallosal Bölge türetilmiş Yetişkin Sinir Kök Hücre Kültürü <ol><li> sindirilmiş doku ayırmak hafif tüp dokunun ve ardından 5 dakika boyunca 145 xg&#39;de tüp santrifüj. sindirim tamponu yıkamak için önceden ısıtılmış N2 ortamı 1 mL süpernatan, yeniden süspansiyon haline Sindirilmiş doku atın ve yavaşça örnek çözelti P1000 pipet kullanarak 5 kez fazla pipetleyin. Not: Aşırı triturating dar olanpipetlemeyin ucu hücre canlılığı ve sonraki büyüme azaltabilir. </li><li> 5 dakika boyunca 145 x g&#39;de tüp santrifüjleyin. Süpernatant atıldıktan sonra, N2 ortamı, 1 ml hücre pelletini yeniden askıya. </li><li> olmayan bir kaplanmış 6-çukurlu plaka içindeki N2 ortamı içinde 1 ml hazırlayın ve 2 ml nihai bir hacimde süspanse hücrelerin 1 ml. </li><li> Her kuyuya EGF (20 ng / ml) ve bFGF (20 ng / ml) eklenir. Yavaşça, tabakalanmış hücrelere ilave büyüme faktörleri karıştırmak için elle 6 oyuklu kültür çanak sallayın. 37 ° C ve% 5 CO2 inkübatöründe 6 oyuklu plaka tutun. </li><li> 8 gün boyunca her gün, her bir kuyucuğa EGF (20 ng / ml) ve bFGF (20 ng / ml) eklenir. Her üç gün ortam, yaklaşık 2 mi hacmını muhafaza etmek için N2 ortamı 200 ul ekle. </li></ol> Neurospheres olarak ve Tek tabakalı Kültürler için NSC&#39;lerde 4. Pasajlanması <ol><li> neurospheres toplayın ve yeni bir 15 ml konik tüp aktarabilirsiniz. Not: neurospheres sayısını (&gt; 50 mikron çapında) pede tek bir fare beyin SCz gelen R SVZ&#39;unda daha azdı 64.3 ± 7.31 idi (190.5 ± 6.33) 9. </li><li> Tek hücrelere Neurospheres ayırmak için bir 37 ° C su banyosu içinde 5 dakika için ayrılma tamponu, 0.5 ml Neurospheres inkübe edin. Birincil Neurospheres tek hücre halinde ayrılmış ve neurosphere ya tek tabakalı kültürler halinde birçok pasaj boyunca muhafaza edilebilir. Not: SCZ-aNSCs bir neurosphere kültürü biçiminde bir tek tabakalı kültürü biçimde&gt; 10 kat, fakat &lt;5 kere daha pasaj yapıldı, çünkü tek-katman olarak SCZ-aNSCs Genişleyen neurospheres üstündür. Dikkat: SCZ-aNSCs kuvvetli toplanmayı göstermektedir ve tek bir hücre içine, çözünmelerini zordur. Bu nedenle, neurosphere kültürü biçimi, rutin hücre genişlemesi için uygun değildir. </li><li> Yavaşça 5 dakika boyunca 145 xg&#39;de yukarı ve aşağı P1000 pipet ile en az 5 kez ve santrifüj tüpü örnek çözümü pipetlemeyin. Süpernatantı ve yeniden-su atınN2 ortamı içinde 1 ml Neurospheres geçirirler. Not: hücre canlılığı ve sonraki büyüme azaltabilir dar pipet ile öğütülerek üzerinde. </li><li> Hücre süspansiyonu (10 ul) ve% 0.4 tripan mavisi çözeltisi 1 karışımı, ve daha sonra bir hematositometre ile canlı hücre sayısını: hücre sayımı için, 1 olun. PLO / laminin ile 6 çukurlu bir levha kaplandıktan sonra, her bir N2 ortamında, 2 ml 2.5 x 10 5 hücre / ml&#39;de hücreleri plaka. Not: hücre yoğunluğu değişiklikler durumlarını ve farklılaşma potansiyelinin etkileyebilir. </li><li> 5 gün boyunca büyüme faktörleri günlük tedaviden SCZ-aNSCs (2 mi, 20 ng / ml) korumak ve bunları kanalı bunları. </li></ol> Subcallosal Bölgesi kaynaklı Yetişkin Sinir Kök Hücre 5. Farklılaşma <ol><li> SCZ-aNSCs farklılaşması için büyüme faktörleri ile 1 mi N2 (20 ng / ml), 1 x 10 5 hücre / ml bir PLO / laminin ile kaplanmış 18 mm örtücü kısım üzerine Levha aNSCs. </li><li> Sonraki gün,Hücreler sıkıca lamel bağlandıkları zaman, büyüme faktörleri kaldırmak için N2 ile büyüme ortam değişikliği. </li><li> 6 gün sonra, hücre artıklarının çıkarılması ve immün giderilmesi için 1 ml PBS ile farklılaşmış hücreleri yıkayın. Not: BrdU çoğalan hücrelerin yeni sentezlenmiş DNA içine dahil edilebilir. arzu edildiği takdirde, bu nedenle, BrdU (10 ug / ml), hücrelerin tespit öncesi hücrelerin yaşama eklenebilir. </li></ol> 6. İmmünoboyama Erişkin Nöral Kök Hücreler ve Farklılaşmış Progenitörler <ol><li> immün için, hücrelerin PBS ile yıkayın ve oda sıcaklığında 20 dakika süreyle% 4 PFA sabitleyin. Dikkat: PFA son derece zehirli olduğu; Göz ve cilt ile temasından kaçının. </li><li> % 4 PFA kaldır 3 defa PBS ile sabit hücreleri durulanır ve daha sonra 4 ° Cuntil da immün için gerekli saklayın. </li><li> (% 3 sığır serum albumin ve% 0.1 Triton X-100 1x P çözüm engelleme ile lamel hücreleri inkübeOda sıcaklığında, en az 30 dakika boyunca bs). </li><li> Taze bloke çözeltisi içinde primer antikorların hazırlanması ve 4 ° C&#39;de gece boyunca inkübe örnekleri. <ol><li> aNSCs immün <ol><li> Anti-Nestin ve anti-BrdU antikoru ile aNSCs Leke. tespit önce aNSCs için BrdU 20 ug ilave edin ve 2 saat boyunca inkübe. bloke etme adımı gerçekleştirmek 37 ° Cbefore 20 dakika süreyle 2 N HCI kullanılarak denatüre aşamasını gerçekleştirir. </li></ol></li><li> farklılaşmış atalarıdır immün: <ol><li> farklı progenitörlerin etiketlemek için, anti-O4, anti-BIII-tubulin, ve anti-glial fibriler asidik protein (GFAP) antikorları kullanın. </li></ol></li></ol></li><li> PBS ile 3 kez örnekleri yıkayıp floresan boyalar (1: 500) ile konjuge sekonder antikor ile inkübe oda sıcaklığında 30 dakika süre ile çözelti bloke. Sonra, PBS ile numuneleri 3 kere yıkayın. Not: birincil ana maç Kullanım sekonder antikorlarantikorlardır. Hoechest33343 (1: 2,000) nükleer boyama için kullanılmaktadır. </li><li> bir slayt cam monte çözüm ekleyin ve montaj ile devam edin. Birden fazla dalga boylarında bir konfokal mikroskop örnek gözlemlemek ve görüntü: FITC (488 nm), Cy3 (543 nm), Cy5 (647 nm) ve Hoechest33343 (405 nm). </li></ol>

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The authors declare no conflicts of interest.

Bu yazıda erişkin fare SCZ gelen NSCs oluşturmak ve çeşitli uygulamalar için onları korumak için ayrıntılı bir protokol açıklar. SCZ-NSCs arındırmak ve genişletmek için gerekli in vitro kültür sistemi kurulması için üç kritik adımlar vardır. İlk olarak, SCZ bölgesi hassas diğer potansiyel nörojenik bölgeler (Şekil 1B) dışarı parçalara sağlamak için önemlidir. SCZ bölgeler içeren kalın ve hassas Bölümler 1 mm&#39;lik aralıklı beyin matriksi ile elde e...

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>
</table>

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.

Bilinmeyen nörojenik bölgeden aNSCs için kültür sistemi tanımlayan bu hücrelerin anlamak için beyin tamir 12 potansiyel kullanımını geliştirmek için gereklidir. Farklı gelişim evrelerindeki ya da farklı bölgelerde NSC&#39;lerde farklı 3,4 davranır bilinmektedir. Son zamanlarda, bu SCZ türetilmiş hücreler SVZ türetilmiş hücreler 7-9 ile karşılaştırıldığında in vivo ve in vitro nöronal farklılaşma için ...

Watch this Scientific Journal Video about Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone at JoVE.com

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.

Fare Subcallosal Zone İzolasyon ve Yetişkin Nöral Kök Hücre Kültürü

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

isolation-culture-adult-neural-stem-cells-from-mouse-subcallosal

Research

JoVE Journal

Neuroscience

15.0K Görüntüleme.  Korea University College of Medicine. 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. 

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

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Neurosphere Testi Yetişkin Fare Nöral Kök Hücre İzolasyonu ve Genişleme

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Nörobilim

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Neurosphere Testi kullanarak Embriyonik Fare Nöral Kök Hücre Kültürü oluşturulması

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

İnsan Saç köklerinin arasından Sinir Crest Kök Hücre İzolasyonu ve Kültür

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

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 mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Embriyonik Mürin Nöral Tüp çıkan sinirsel Crest Hücre İzolasyonu ve Kültür

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

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Yetişkin Kemirgen Beyin konvansiyonel ve konvansiyonel olmayan Bölgelerinden Nöral Kök Hücreler Büyüyen

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embriyonik ön beyin gelen Endotel Hücreleri İzolasyon ve Kültürü

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

Bir Fare, İki Kültür: İzolasyon ve Yetişkin Nöral Kültür Bireysel Farelerde iki Nörojenik Bölgeler&#39;den Kök Hücre

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

İnsan Pluripotent dan Sinir Crest Progenitör Hücreler besleyici ücretsiz Derivasyon Kök Hücre

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Hücre Yetişkin Fare subventricular Bölgesinden Nöral Kök ve Progenitör Hücreler Sıralama ve Hücre Döngüsü Dynamics Canlı görüntüleme

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

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 expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.