CRM is a Damon Runyon-Genentech Clinical Investigator. This work was supported, in part, by grants to CRM from the Damon Runyon Cancer Research Foundation (CI-45-09), Department of Defense (W81XWH-09-2-0042), and University of North Carolina University Cancer Research Fund (UCRF). The authors wish to thank Daniel Roth for mouse husbandry assistance. The authors also wish to thank Hannah Chae, Carter McCormick, Demi Canoutas, Stephanie Gillette, and Susannah Krom for tissue culture and immunofluorescence assistance.

The authors have nothing to disclose.

En kritik adımlar, 2), 3) iyice hücreleri ayırmak, meninks kaldırmak için korpus kallosum altında doku almadan korteksi çıkarılması için uygun hasat ve kortikal astrositlerde kültürünü 1) sağlamak ve 4) GFAP zenginleştirmek için + astrositlerden salınmaktadır. Biz GAFP + astrositlere zenginleştirmek için yöntemler (GFAP promotör kontrolü altında (bir baskın dönüşen transgen bir Ad5GFAPCre vektörü veya kullanımı ile TgGZT 121) genetik re...

Astrositomlar en sık görülen primer beyin tümörü glioblastoma (GBM), bir sınıf IV astrositom vardır, 12-15 ay 1,2 medyan sağkalım en yaygın ve agresif bir alt tiptir. Yaygın astrositomlar, özellikle GBM, istilası, tam cerrahi rezeksiyon engellemektedir adjuvan tedavilerin etkinliğini sınırlayan ve kaçınılmaz tedavi sonrası nüks 3 yol açar. De novo (birincil) GBM ile ya da kaçınılmaz (ikincil) GBM 4 ilerledikçe daha düşük grade astrositom ile ya başlangıçta başvururlar. GBM genomik heterojen ve üç damarlı sinyal yolları yönlendiren genler birbirini dışlayan ve co-ortaya çıkan mutasyonlar ile karakterizedir: G 1 / S (Rb) hücre döngüsü kontrol noktası, reseptör tirozin kinaz (RTK) ve TP53 5-7 yolaklarıyla. GBM GBM alt tipi Grip Grip Ağrilari olduğunu düşündürmektedir, beyin hücre tiplerinin farklı benzer sentezleme profillerine sahip dört alt tipinin genomik oluşmaktadırköken 6,8,9 kendi hücre tarafından etkilemiştir. Iyi astrositom modelleri astrositomdur patogenezi sırasında belirli hücre tiplerine mutasyonların belirli kombinasyonları rolünü tanımlamak için gereklidir. Daha verimli preklinik ilaç geliştirme için bu modelleri yararlanan hasta sonuçlarını geliştirmemize yardımcı olacaktır. Mevcut astrositom modeller, insan hücre hatları, hasta türetilmiş ksenograftları (PDX) genetik olarak tadil edilmiş normal insan astrositlerin nöral kök hücreleri (MGK) ve genetik olarak fareler (GEM) 10-14 kurulmuş bulunmaktadır. Kortikal astrositleri ve NSC - - floxed onkojenik alleles çeşitli kombinasyonlarını barındıran GEM hasat Biz primer beyin hücrelerini kullanan bir alternatif olmayan germ GEM (nGEM) modeli 15 geliştirmiştir. Amaç, fenotipik i preklinik ilaç gelişimi için in vitro ve in vivo olarak karakterize edilmiş ve potansiyel olarak yararlanılabilir genetik olarak tanımlanmış hücrelerin astrositom modellerini üretmek için olanmmune-kompetan farelerde azaltılmıştır. Kurulan insan hücre çizgileri Bunlar düz ön teknik yaygın olarak mevcuttur. Astrositoma patogenezinde ve in vitro ve in vivo ilaç yanıtı en yaygın olarak kullanılan model, ve immün yetersiz farelerde 10,11,16-18, ortotopik Xenografting üzerine kinetik ve tümör oluşum nedenlerinin tanımlanmış . Onların dezavantajları sadece yüksek dereceli astrositom çalışma sınırlayıcı, düşük-dereceli astrositomlardan kurulan hücre hatları oluşturmak için yetersizlik içermektedir; Kalkış tanımlanmış bir hücre eksikliği; genellikle orijinal hasta numunesinden gelen belirgin bir şekilde farklıdır genomik profiller ile kompleks genomik anormalliklerin mevcudiyeti; ve duyarlılık serumdaki 11,17,19-22, seri kültürü sırasında fenotipik ve genotipik sürüklenme için. Kurulan insan GBM hücre hatlarında bireysel onkojenik mutasyonların fenotipik sonuçları genellikle eluc engeller aslında mevcut anomalilerin sayıda kamufle edilebilirDoğrudan genotip-fenotip sonuçları idation. PDX immün yetersiz farelerde veya bağışıklık yetersizliği olan farelerde 12,23 beyinlerine ortotopik enjeksiyondan önce tanımlandığı gibidir, serumsuz ortam içinde yapışkan olmayan küremsi olarak kültür yoluyla hastaya izole astrositom hücrelerinin deri altına geçişi elde edilmektedir. PDX daha doğru bir insan astrositomas genomik manzara eder, fakat bilinen insan hücre çizgilerine benzer, tek tek onkojenik mutasyonların fenotipik etkisinin genomik karmaşıklığı nedeniyle 19,24 maskelenebilir. Özellikle yeni tedavilere karşılık olarak, belirli onkojenik mutasyonların fenotipik sonuçlarını tanımlamak için, bilinen insan hücre çizgileri veya PDX panelleri genellikle, genotip-fenotip bağlantıları ortaya genelleştirilebilirliğini gösterir ve hücre çizgisi özel etkilerin ihtimalini en aza indirmek için kullanılmaktadır. PDX doğru insan astrositomaların, inc histopatolojik izlerini recapitulate ikeninvazyon luding, bilinen insan hücre çizgilerinin ortotopik ksenogrefleri genelde 21,23,25 yok. Buna ek olarak, normal insan astrositler ve NSC in vitro ve in vivo olarak 13,14,26 astrositom tümörogenez modellemek için tanımlandığı onkojenik mutasyonu olan genetik olarak tasarlanmış edilmiştir. Bu hücreler, bilinen insan hücre çizgileri ve PDX genomik karmaşıklığı eksikliği ve doğru bir şekilde insan astrositoma histopatoloji özetlemek, fakat in vivo olarak bağışıklık eksikliği, kemirgenlerde yabancı-doku naklinde gerektirir. Tüm insan hücre modelleri immün-aracılı ksenogreft reddini önlemek için bağışıklık yetersizliği olan kemirgen ana gerektirdiğinden, bu modeller stroma hedefli ve immün-modülatör preklinik soruşturma sınırlayıcı, bir singenik sisteminin yerel tümör-stroma etkileşimleri özetlemek başarısız ve sağlam bir bağışıklık sistemi eksikliği 10,11 terapiler. Onkojenik Muta önceden belirlenmiş kombinasyonları fenotipik sonuçları GEM izni incelenmesiin situ tümörigenezinde sırasında in vivo leri. -Çıkışlı şartsız GEM gelişimi boyunca bütün dokular içinde mutasyonlara sahip ise, koşullu GEM hücre tipine özgü promoterler 10,11,15,18 kullanımı yoluyla, özel hücre tipleri Cre-aracılı rekombinasyon sınırlayan mutasyonların hedefleme olanak onkojenik allelleri floxed var. Koşullu astrositom GEM sağlam bir beyin içinde 11 farklı hücre tiplerinde onkojenik mutasyonların işlevsel roller ortaya çıkarmak için kullanılmaktadır. Koşullu GEM kullanılarak in situ gliom klinik öncesi programı 1) bir in vitro bir korelasyon eksikliği, kompleks genotipler ile, in situ tümör gelişiminde, 3) uzun gecikme farelerin büyük gruplarının bir üreten 2) zorluk içeren bir dizi faktöre ile sınırlıdır , 4) ve stokastik tümör ilerlemesi. In situ uygun bir tümör oluşumu in vitro bir model eksik olduğundan, in vitro ilaç testi ağırlık gerçekleştirilemezi&#39;inci geleneksel koşullu GEM modelleri. Diğer kanserlerin tersine, astrositom şartlı gem modelleri nadiren tek onkogenik mutasyon 11 ile uyarılmaktadır. Böylece, karmaşık ıslah programları çoklu onkojenik mutasyonlar ile koşullu GEM oluşturmak için gereklidir. Yüksek dereceli astrositom ilerleme genellikle tekdüze olmayan, stokastik bir şekilde ortaya çıkar ve sonuçta karmaşık genomik manzaralar ve hızlı büyüme kinetiği 27 ile tümörlere yol açar Dahası, astrositom başlatma, bu modellerde uzun bir kuluçka döneminden sonra penetransı değişken oluşur, 28. Koşullu GEM modellerinde değişken Penetrans ve malign ilerlemesi stokastik doğası bireysel farenin preklinik ilaç araştırmaları yaptığını kayıt önce yüksek dereceli astrositomaların varlığını ve yerini belirlemek için radyografik görüntüleme ile taranmalıdır gerektirir. Birlikte ele alındığında, bu sınırlamalar pr için gerekli üretim ve koşullu GEM büyük kohortlarda test engeleclinical uyuşturucu testi. Belirli bir nöral hücre tipleri üzerinde bulunan viral reseptörü (tva) ifade etmek üzere geliştirilen GEM enfekte etmek için kuş retroviral (RCAS) vektör kullanır RCAS-tva GEM sistemi,, geniş bir astrositoma tümörogenez 11 modellemek için kullanılmıştır. Koşullu GEM aksine, bu model sistem karmaşık yetiştirme programları için gerek olmaksızın, belirli hücre tiplerinde çoklu onkojenik mutasyonun ortaya çıkmasına olanak tanır. Bununla birlikte, değişken penetrans aktif viral entegrasyonu sağlamak için bölünen hücreler için ihtiyaç ve bunlar, konakçı genomu 29 içine transgenlerin rastgele yerleştirilmesi ile sınırlıdır. Diğer model sistemler 15 sınırlamaları birçok üstesinden çünkü GEM hasat hücreleri kullanması Sigara germ GEM (nGEM) modelleri, giderek daha önemli hale gelmektedir. Astrositoma patogenezinde hücre tipinin ve birlikte görülen mutasyon başlatma rolü engellemek için zordurbu habis ilerleyişi sırasında belirsiz hücre tipinde yaygın genetik mutasyonlar birikmiş son aşama tümörlerinden türetilmiştir, çünkü maden insan GBM hücresi türleri veya PDX tesis kullanılarak. Buna karşılık, astrositomaların tüm sınıflarda spesifik saflaştırılmış beyin hücre tipleri 11,30 içindeki genetik mutasyonlar tanımlanmış indükleyerek nGEM kullanılarak modellenebilir. Bu nedenle, belirli genetik mutasyon ve hücresel ve moleküler fenotipe hücre tipinin etkisi in vitro ve in vivo olarak belirlenebilir. Kurulan insan GBM hücre çizgilerine benzer bir şekilde, başlangıç ​​nGEM kullanılarak in vitro ilaç testleri aynı hücreleri kullanılarak, in vivo testler için ilaç öncelik için kullanılabilir. In vivo olarak tümör oluşumu daha sonra ortotopikal bağışıklık-kompetan singeneik litermatlarından 30 beyinlerine nGEM hücreleri allogeneik belirlenebilir. Bu ortotopik alograft modelleridir nedenle, sadece sitotoksik a in vivo testler izinnd terapileri hedefli, ancak immün-modülatör ve stroma hedefli tedaviler de. Son olarak, tümör başlaması ve ilerlemesi üzerinde mikro-rolü, aynı hücre tipinde aynı mutasyonları kullanılarak, nGEM konvansiyonel GEM modelleri arasında sonuçları karşılaştırarak belirlenebilir. Biz ve diğerleri birincil hücreler kullanılarak astrositomdur nGEM geliştirdik - astrositleri, MGK, ya olgodendrosit öncü hücrelerini (OPC) - GEM 30-34 hasat. Astrositom nGEM geliştirilmesi arkasındaki mantık, potansiyel olarak, bağışıklık-kompetan hayvanlarda klinik öncesi in vitro ilaç testleri için ve in vivo kullanılabilecek belirli hücre tiplerinde onkojenik mutasyonların fenotipik sonuçlarını belirlemek için bir model oluşturmak için olmuştur. Rb1 loxp / loxp veya TgGZT - Biz ifade fenotipik WT kortikal astrositleri ve non-Cre gelen MGK, floxed RB yolun bir&gt;% 94 C57 / BL6 arka plan üzerinde muhafaza koşullu GEM hasat121 - çeşitli kombinasyonlarda 35-39 genleri - Nf1 LoxP / LoxP, Kras G12D, PTEN LoxP / loxp - ve RTK / RAS / PI3K- yolu floxed. Bu Cre rekombinazı kodlayan adenoviral vektörler kullanılarak in vitro genetik yeniden neden oldu. Kortikal astrosit hasat hücre tiplerinin bir karışımını içerdiği için, bu kültürlerde GFAP + kortikal astrositler zenginleştirmek için, bu tür TgGZT insan GFAP promotöründen 121 olarak Ad5GFAPCre vektörler veya baskın onkojenik transgenleri, ikinci. Bu in vitro ve in vivo olarak kortikal astrositler ve NSC fenotipik G1 / S sonuçlarını (Rb) için, MAPK ve PI3K yolu tanımlanmış mutasyonlar. MAPK ve PI3K yolu ile aktive olan G1 / S hatalı olarak taklit insan astrosit moleküler proneural GBM ve ortotopik enjeksiyonu üzerine, düzgün büyüme kinetikleri, kısa süreye göre önceden belirlenmiş bir konumda oluşan tümörler, ve histopatolojikinsan GBM 30 al işaretlerinden. In vivo tümör büyümesinin tedavisine boyuna izlenmesi 40 cevaben tedavi gruplarına ya da tümör büyümesinin kantitatif analiz normalizasyonu ile klinik öncesi bir ilaç test yardımcı olur. Biz, lusiferaz sentezleyen kortikal astrositler enjekte edilmiş farelerin uzunlamasına biyolüminesans görüntüleme ile tümör büyüme kinetiklerini belirlenmiştir. Bu nedenle, elde edilen koşullu GEM kortikal astrositler ve NSC astrositom ve ilişkili mutasyonların işlevsel sonuçları ve klinik öncesi bir ilaç gelişimi için olası bir model sisteminin tanımı için izlenebilir model sistem sağlar.

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			<td height="60" style="height:60px;width:241px;">Dulbecco&#39;s Modified Eagle Medium (DMEM) (1X)</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">11995065</td>
			<td style="width:233px;">DMEM can also be purchased from other suppliers including GIBCOSigma and Cellgro</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Fetal Bovine Serum, Regular</td>
			<td style="width:191px;">Cellgro</td>
			<td style="width:119px;">35-010-CV</td>
			<td style="width:233px;"></td>
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			<td height="20" style="height:20px;width:241px;">Penicillin-Streptomycin</td>
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			<td height="40" style="height:40px;width:241px;">Cartilage Thumb Forceps, Curved</td>
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			<td style="width:119px;">1631</td>
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			<td height="40" style="height:40px;width:241px;">Miltex 17-301 Style 1 Jeweler Style Forceps, Fine, 4</td>
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			<td style="width:119px;">17-301</td>
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			<td height="20" style="height:20px;width:241px;">Ethanol (200 proof)</td>
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			<td style="width:119px;">2710</td>
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			<td height="40" style="height:40px;width:241px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid</td>
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			<td style="width:119px;">14175-095</td>
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			<td height="60" style="height:60px;width:241px;">TrypLE Express (1X), Phenol Red</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">12605-010</td>
			<td style="width:233px;">Other Trypsin solutions are also suitable for cortical astrocyte havest and culture</td>
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			<td height="20" style="height:20px;width:241px;">CULTURE DISH, 60 x 15 mm</td>
			<td style="width:191px;">Thomas Scientific</td>
			<td style="width:119px;">9380H77</td>
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			<td height="20" style="height:20px;width:241px;">15 ml tubes</td>
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			<td style="width:119px;">352096</td>
			<td style="width:233px;"></td>
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			<td height="20" style="height:20px;width:241px;">50 ml tubes</td>
			<td style="width:191px;">BD Biosciences</td>
			<td style="width:119px;">352070</td>
			<td style="width:233px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:241px;">Adenovirus stock</td>
			<td style="width:191px;">Gene Transfer Vector Core, U. Iowa</td>
			<td style="width:119px;">Ad5CMVCre</td>
			<td style="width:233px;">Store in 5 &#181;l aliquots at -80 C. Hazardous.&#160; Use Bsl2 safetyy&#160; precautions</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Sodium sulfate (Na2SO4)&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">238597</td>
			<td style="width:233px;"></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Potassium sulfate (K2SO4)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">221325</td>
			<td style="width:233px;"></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Magnesium chloride (MgCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td style="width:233px;"></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Calcium chloride (CaCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">746495</td>
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			<td height="20" style="height:20px;width:241px;">HEPES potassium salt</td>
			<td style="width:191px;">Sigma-Aldrich</td>
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			<td height="20" style="height:20px;width:241px;">Phenol Red</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">P3532</td>
			<td style="width:233px;"></td>
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			<td height="20" style="height:20px;width:241px;">Sodium hydroxide (NaOH)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5881</td>
			<td style="width:233px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Papain</td>
			<td style="width:191px;">Worthington</td>
			<td style="width:119px;">LS003127</td>
			<td style="width:233px;"></td>
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			<td height="20" style="height:20px;width:241px;">L-Cysteine-HCl</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1276</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Syringe filter (0.22 &#181;m pore size)</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">SLGP033NS</td>
			<td style="width:233px;"></td>
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		<tr height="80">
			<td height="80" style="height:80px;width:241px;">Neurocult proliferation kit, mouse</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">5702</td>
			<td style="width:233px;">This kit contains the NeuroCult NSC Basal Medium and NeuroCult NSC supplement needed for NSC culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">0.2% Heparin solution</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">7980</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">EGF&#160;</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">PMG8041</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">bFGF&#160;</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">PHG0261</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hanks&#39; Balanced Salt Solution (HBSS) (10X)</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">14185-052</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Magnesium sulfate (MgSO4)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Sodium bicarbonate (NaHCO3)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">E-64</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">E3132</td>
			<td style="width:233px;">Make 10 mM stock in DMSO, store at -20 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">6-well plates</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-83</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Cell strainer (40 &#181;m pore size)</td>
			<td style="width:191px;">Corning</td>
			<td style="width:119px;">352340</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Stem cell dissociation solution</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">5707</td>
			<td style="width:233px;">Alternatively, use gentle enzyme solutions such as Accutase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Methyl cellulose 15 cP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7027</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Dulbecco&#39;s Modified Eagle&#39;s Media 2X</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">SLM-202-B</td>
			<td style="width:233px;">For making 5% methyl cellulose solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">1.7mL Snap Cap Microcentrifuge Tube</td>
			<td style="width:191px;">Corning</td>
			<td align="right" style="width:119px;">3620</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hamilton syringe, 250 &#181;l LT no needle</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">14-815-92</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">PB600-1 Antigen Dispenser</td>
			<td style="width:191px;">Hamilton</td>
			<td style="width:119px;">&#160;83700</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Disposable 18 ga needles&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9015638&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">27 ga 1/2&#34; luer tip needle</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">14-826-48</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">2,2,2-Tribromoethanol (Avertin)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">T48402</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Betadine</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9386574</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Puralube Opthalmic Ointment</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9689910</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">&#160;Model 900 Stereotaxic frame</td>
			<td style="width:191px;">Kopf Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">VETBOND</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9259532&#160;</td>
			<td style="width:233px;">Tissue adhesive&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Lidocaine&#160;</td>
			<td style="width:191px;">ShopMedVet</td>
			<td style="width:119px;">RXLIDO-EPI</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)</td>
			<td style="width:191px;">Promega</td>
			<td style="width:119px;">G3580</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Anti-Sox2</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">AB5603</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Polyclonal Rabbit Anti-Glial Fibrillary Acidic Protein (GFAP)</td>
			<td style="width:191px;">Dako</td>
			<td style="width:119px;">Z0334&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">IVIS Kinetic</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td style="width:233px;">For in vivo imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">D-Luciferin - K+ Salt Bioluminescent Substrate</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;">122796</td>
			<td style="width:233px;">For in vivo bioluminescence imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">EdU Imaging Kit</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">C10340</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">MSCV Luciferase PGK-hygro</td>
			<td style="width:191px;">Addgene</td>
			<td style="width:119px;">18782</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling astrocytoma pathogenesis in vitro and in vivo using cortical astrocytes or neural stem cells from conditional, genetically engineered mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Bu, fenotipik olarak in vitro ve in vivo olarak karakterize edilebilir koşullu onkojenik alelleri (Şekil 1) floxed yenidoğan GEM barınağı hasat kortikal astrositler ve MGK bir nGEM model sistemi geliştirmiştir. Özellikle in vitro kortikal astrositler onkojenik mutasyonların sonuçlarını incelemek için, ilk olarak astrositleri zenginleştirmek için kritik öneme sahiptir. Kortikal astrosit hasat mikroglia, astrositler, oligodendrositler, OPC ve nöronların bir ka...

Watch this Scientific Journal Video about Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice at JoVE.co

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modelleme Astrositom Patogenez<em&gt; İn Vitro</em&gt; Ve<em&gt; In Vivo</em&gt; Kortikal Astrositler veya Şartlı, Genetiği fareler ile Nöral Kök Hücreler kullanma

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

modeling-astrocytoma-pathogenesis-vitro-vivo-using-cortical

Research

JoVE Journal

Neuroscience

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

13.3K Görüntüleme.  University of North Carolina School of Medicine. Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Video: Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Atılan İnsan Fetal Kortikal Doku Sinir Kök Hücre Üretimi

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Nörobilim

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Yayılma Depresyon Episodik ve Kronik Migren Sinir Bağışıklık Sinyalizasyonu Modelleme In Vitro</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Ultrasonik Kan-beyin bariyeri bozulması ve Manganez kontrastlı MRG kullanma İşlevsel beyin görüntüleme

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Tarafından Embriyonik ve Erişkin Nöral Kök Hücre Genişletme<em&gt; In Utero</em&gt; Elektroporasyon veya Viral Stereotaksik Enjeksiyon

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

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 ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

Kortikal Nöronlar In Vivo İki foton Of Görüntüleme Deneyimi bağımlı Moleküler Değişiklikler

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

Görüntüleme hücre içi Ca<sup&gt; 2+</sup&gt; Genetik olarak kodlanmış Kalsiyum Göstergelerini Kullanma Yetişkin fareler ile Striatal Astrositler Sinyalleri

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Farelerin Nöral Kök Hücreleri ve Beyinlerinde 5-Hidroksimetilsitozozin Insaptama

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

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 potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Fare Embriyonik Kök Hücre in vitro nöronal farklılaşma

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

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

Fibroblastlardan Nöronal Progenitör Hücrelere Doğrudan Dönüşüm ve Astrositlere Farklılaşma Kullanarak Nörolojik Hastalıklar için İn vitro Modelleme

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...