Biz video yardım için Marc Wozniak kabul etmek istiyorum. Biz de kültür sıçan NK hücreleri için orijinal protokol için UT Southwestern az Sean Morrison kabul etmek istiyorum. Bu çalışma Vanderbilt Üniversitesi Tıp Merkezi Akademik Destek Programı desteklenen ve NIH (HD36720 ve HD036720-11S109) ve PAL için AHA 11GRNT7690040, NAM için AHA (0615209B) ve NIH (NS065604) den predoctoral burslar, ve ERP gelen hibelerle oldu Bir NIH eğitim bursu T32HD007502 tarafından desteklenmektedir.

1. Hazırlanması Tabaklar <ol><li> Her zaman steril tekniği kullanın. </li><li> Dulbecco PBS (DPBS) içerisinde 3.3 mL &#39;lik bir son hacim içine insan plazmasından FN stoğu 100 uL seyreltilmesiyle fibronektin (FN) hazırlayın. Nihai konsantrasyon 30 ug / mL &#39;dir ve bu 1 hafta süreyle 4 ° C&#39;de saklanabilir. </li><li> FN çözümü ile steril doku kültürü dört sıra plakanın her alt örtün ve 15 dakika bekletin. Tüm yüzeyi kaplı olduğundan emin olun. Bu kez (2. ve 3. adımları) sırasında ortam olun. </li><li> Çözüm FN ve plakaları kurumasını bekleyin çıkarın. Yavaşça 500 uL DMEM, kaldır DMEM ile kuyu durulayın, ve Öz-Yenileme 500 uL (SR), orta (aşağıya bakınız) ekleyin. CO2 5 oranında içeren nemlendirilmiş inkübatöründe 37 ° C&#39;de inkübe edilir. Plakalar kullanımdan önce kuru olacak şekilde Diseksiyon önce yaklaşık bir saat tamamlar. </li></ol> 2. SR Orta hazırlanması <ol><li> Vagal ve gövde NC kültürü içinon embriyolar (yaklaşık bir kumu, fare hattı genetik arka plan bağlı olarak), SR orta 25 mL hazırlamak. 12,5 mL, düşük glukoz DMEM, 7.5 mL Neurobasal Orta, 25 uL retinoik asit (117 uM son konsantrasyon) ve 25 ul 2-mercaptoethanol (50 mM nihai konsantrasyon) birleştirir. İyice karıştırın. </li><li> 3.75 ml Civciv Embriyo Özü, 250 uL N 2 tuz takviyesi, 500 uL B27 takviyesi ve 250 uL penisilin streptomisin (% 1 final konsantrasyon) ekleyin. 0.22 um bir filtreden orta filtre. </li><li> 10 uL steril IGF1 (20 ug / mL son konsantrasyon) ve 20 ul steril bFGF (20 ug / mL son konsantrasyon) eklenir. Tersine çevirerek karıştırın. 4 ° C&#39;de depolamak </li></ol> 3. Yıkama Orta hazırlanması <ol><li> Için yaklaşık 10 embriyo yıkama orta 50 ml hazırlamak. 35 mL düşük glikoz DMEM, 15 mL Neurobasal Orta ve 500 uL penisilin streptomisin (% 1 final konsantrasyon) ile 50 mg BSA birleştirin. 0.22 ile Steril filtremikron filtre. </li></ol> 4. Kollajenaz / dispase hazırlanması <ol><li> 100 mg / mL kollajenaz / dispase için 5 mL DPBS 50 uL ekleyin. İyice karıştırın. </li><li> 0.2 um bir şırınga ile filtre on iki iyi plaka üç kuyu her birine 1.5 mL filtre ve ekleyin. Kalan kuyulara pipetle yaklaşık 1 ml yıkama ortamı. Disseke doku sindirmek için hazır olana kadar buz üzerinde tüm plaka saklayın. </li><li> Steril bir jilet ile P20 ve P1000 pipet filtre ucu ipuçları kesin. Sadece ucu eğimli kenar aşağıda kesin. P1000 cut off P20 izole doku parçalarını aktarmak için kullanılacak ise tüm embriyo transfer etmek için kullanılacaktır. </li></ol> 5.. 9.5 DPC Embriyolar gelen vagal ve Trunk Nöral Tüp Ayırma <ol><li> Zamanlanmış gebelikler için, vajinal fişi ile baraj sabah fişi görülmektedir öğle saatlerinde 0.5 DPC kabul edilir. 9.5 DPC de rahim kurban ve kaldırın. </li><li> Uterustan desidua kaldırın ve yavaşça rdesidua gelen embriyo aldır. Sonra bu protokol ile deneyimli, steril DPBS bir defada 3-4 9.5 DPC embriyo izole. Steril teknik ve sterilize diseksiyon aletler kullanın. Instruments otoklav, ısı sterilizasyon veya etanol içinde inkübasyonu ile sterilize edilebilir. </li><li> Anterior vagal NC için: insülin iğneleri kullanarak, orta kulak taslakları da nöral tüp kesti. 4. hücre gruplarının arka kenarı tekrar kesin. Faringeal kemerler ve kalp kaldırmak için nöral tüp ventral doku kesin. </li><li> Gövde NC için: insülin iğneleri kullanılarak, hücre grubu 16-22 (ya da embriyo önceki 22 hücre gruplarının sahne daha gelişimsel olarak ise son hücre gruplarının) arasındaki nöral tüp kısmını kaldırın. </li><li> Yolk kesesi ve genotiplendirme için kalan embriyonik doku tutun. </li></ol> 6. Sigara nöral Ektoderm ve Mezodermin Kaldırma <ol><li> 10 dakika için oda sıcaklığında kollajenaz / dispase içine bölümleri içeren sinirsel tüp yeris. Hemen yıkama ortamda yıkayın. </li><li> Steril DPBS doku dönün. Steril insülin iğneleri kullanarak, yavaşça dokusundan olmayan nöral ektoderm kaldırmak ve uzak nöral tüp Somitlerin ayırın. Hücre gruplarının doku mezodermin son kalan parçaları bir cut-down P20 ucu ile hafifçe triturating tarafından kaldırılabilir. Nöral tüp zarar için dikkatli olun. Öğütme sırasında yakından gözlemleyin. </li><li> Yıkama orta bir ikinci ve üçüncü 30 saniye yıkama yoluyla izole sinir yerleştirilir. </li><li> SR orta bir kez yıkayın ve (Adım 1.3) daha önce hazırlanan bir FN kaplı kuyunun merkezi haline izole nöral tüp yerleştirin. Steril su bir tabak ile hipoksi odası Nemlendirme. Hipoksi odasına çanak yerleştirin ve% 3 O 2 (1% karışımı O 2,% 6 CO 2,% 93 N 2 içeren bir tank kullanmak) karışık gaz odası yıkayın. </li><li> Daima tha sağlamak için son derece dikkatli odası elet eksplantları rahatsız olan ve kuyularının merkezi kalır. </li><li> 37 ° C&#39;de inkübe Adımları 5-6 Özeti Şekil 1 &#39;de gösterilmiştir. </li></ol> 7. Nöral Tüp çıkarılması <ol><li> Inkübasyondan 24 saat sonra, nazikçe uzak steril bir insülin iğnesi kullanarak göç eden hücrelerin nöral tüpün kenarına alay tarafından nöral tüp kaldırın. Çıkarın ve adım 6.2 gibi steril bir P20 kesim kullanarak ortamdan nöral tüp atmak ve taze SR orta (Şekil 2) ile orta yerine. Bu en kolay ters bir mikroskop kullanılarak yapılır. </li></ol> 8. Temsilcisi Sonuçlar Hipoksik koşullarda ° C, NK hücreleri neredeyse saf nüfus (Şekil 3a) nöral tüp uzak göç etmiş 37 inkübasyondan 24 saat sonra. Bazen, ideal kültürler daha az güçlü çıkıntılar vermeyecektir. Örneğin, bu, 24 saat sonra t mümkündürdiye nöral tüp kendi üzerine kıvrılmış olacak ve NC nöral tüp (Şekil 3b) uzağa göç olmaz. Bazen nöral tüp fibronektin kaplı plakaları takmak değil. Bizim deneyimlerimize göre, sub-optimal NC göç ya da nöral tüp eki ile ilgili sorunlar olumsuz sırasıyla normoksi koşulları veya fibronektin konsantrasyonu etkilenebilir. Kollajenaz / dispase Enzimatik aktivite toplu ve sindirim zaman biraz değişir uygun şekilde ayarlanması gerekir, ancak on beş dakika daha uzun doku sindirmek değil. Kollajenaz / dispase doku içeren sinirsel tüpün Overdigestion da eksik çıkıntılar ile sonuçlanacaktır. Hücre gruplarının doku kolayca kollajenaz / dispase inkübe edildikten sonra nöral tüp kaldırılmaz ise, nöral tüp on dakika daha uzun süre inkübe edilebilir. Bazen nöral tüp substrat eklemek olmayacak. Bu durumda, fibronec çift kontrolkalay konsantrasyonu ve hipoksi koşulları. Normoksik koşullar kültürü vahşi tip NC için kullanılabilmesine rağmen, hipoksik koşulları daha yakından in vivo ortamda 29,30 in taklit edebilirler. Deneyimlerimize göre, hipoksik koşullarda zaman kültür mutant NC kritik hale geldi. Örneğin, Foxd3 mutant NC normoksik koşullar kültive edildiğinde, gövde NC kontroller ile karşılaştırıldığında önemli ölçüde azalır hücre outgrowth sahipti. Eksplantları hipoksik koşullar (Şekil 4) kültive edildiğinde, outgrowth boyutunda bu farkı çıkarıldı. Ayrıca, yabani tip nöral tüp eksplantları normoksi kültüre edildiğinde, kaspaz-pozitif hücre sayısı (veriler gösterilmemiştir) hipoksi kültüre benzer eksplantlarının o zaman daha büyüktü. Hipoksi tüm NC kültürünü koruyarak, karşılaştırmaları daha kolay kontrol dinamikleri ve mutant kültürler arasında yapılabilir. <img src="/files/ftp_upload/4134/4134fig1.jpg" alt="Şekil 1" /> <strong&gt; Şekil 1. NC izolasyon Genel şematik. A) embriyodan ilgi bölgeleri parçalara ayır. B) kollajenaz / on dakika (on beş dakikayı aşmayan) için dispase nöral tüp Digest. C) yıkama ortamda yıkayın. D) non-nöral ektoderm ve mezoderm uzak parçalara ayır. EF) yıkama ortamda iki kez yıkayın. Kendini yenileme ortamda G) Levha. % 3 O 2 hipoksik koşullarda 37 ° C&#39;de inkübe edilir. <img src="/files/ftp_upload/4134/4134fig2.jpg" alt="Şekil 2" /> Şekil 2. Eksplant gelen nöral tüp adımsal kaldırılması. Nöral tüp olmayan NC hücreleri ile kirlenmesini önlemek için 24-48 saat sonra uzaklaştırılmalıdır. A) nöral tüp ve NC akıbet (düz çizgi) arasındaki sınır dikkat edin. B) bir insülin iğnesi ile nöral tüpün kenarı boyunca kesin. C) nöral tüp atın ve taze kendini yenileme orta ile orta yerine. Kesikli çizgi outgrowth kapsamını göstermektedir. Kısaltma: NT, nöral tüp. Şekil 3. Temsilcisi sonuçları örnekleri. Hipoksik koşullarda kendinden yenilenmesi ortam içinde 24 saat inkübasyondan sonra A) Tipik eksplant outgrowth (tireli çizgi) outgrowth kapsamını göstermektedir. B) kültüründe 48 saat sonra NC akıbet açısından Büyütülmüş. Düşük outgrowth verim C) Daha az ideal bir kültür. A ve C, aynı koşullar altında kültüre edildi. Görüntüler kültür sağlamlık doğal geniş olduğunu gösterir. Bu kollajenaz / dispase sindirimler nöral tüp izolasyon, FN konsantrasyonu, hipoksik koşullarda ve zaman verimliliği etkilenebilir. <img src="/files/ftp_upload/4134/4134fig4.jpg" alt="Şekil 4" /> Şekil 4. Normoksi karşı hipoksi NC eksplant kültürlerinde in vitro inceleme. Normoksik kültür koşullarında 48 saat sonra nöral tüp eksplantları göç Kontrolü (yabani tip) NK hücreleri. Buna karşılık, mutant Foxd3NC büyük normoksi hücre çıkıntılar (NC çıkıntılar kırmızı hatlarını işareti kenarları) azalmıştı. Karşılaştırılabilir eksplantlarında hipoksik koşullarda yetiştirilen alındığında, Foxd3 mutant NC eksplantları sonraki analizlerde izin kontrollerine nispeten büyüdü. Not Bu davranış, in vivo Foxd3 mutant NC davranışı ile ilişkili olduğu bulunmuştur.

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semaphorin/neuropilin+signaling+influences+the+positioning+of+migratory+neural+crest+cells+within+the+hindbrain+region+of+the+the+chick.">Semaphorin/neuropilin signaling influences the positioning of migratory neural crest cells within the hindbrain region of the the chick.</a> Dev. Dyn. 232, 939-949 (2005).</li><li>Schwarz, Q., Maden, C. H., Vieira, J. M., Ruhrberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropilin+1+signaling+guides+neural+crest+cells+to+coordinate+pathway+choice+with+cell+specification.">Neuropilin 1 signaling guides neural crest cells to coordinate pathway choice with cell specification.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 6164-6169 (2009).</li><li>Simon, M. C., Keith, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+oxygen+availability+in+embryonic+development+and+stem+cell+function.">The role of oxygen availability in embryonic development and stem cell function.</a> Nat. Rev. Mol. Cell Biol. 9, 285-296 (2008).</li><li>Ivanovic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+or+in+situ+normoxia:+The+stem+cell+paradigm.">Hypoxia or in situ normoxia: The stem cell paradigm.</a> J. Cell Physiol. 219, 271-275 (2009).</li></ol>

Biz ifşa hiçbir şey yok.

Dikkat dikkat bu yaklaşımın başarısı için embriyonun gelişim aşamasında dikkat edilmelidir. Erken fare embriyo Somitlerin sayan bir çöp içinde embriyo eşleme ve izolasyon için nöral tüpün doğru bölgelerin belirlenmesinde aşaması için hem kritik önem taşımaktadır. Embriyo arasındaki bir ya da iki Somitlerin bir varyasyonu yapılan deney çözünürlüğü bağlı olarak, gelişim zamanlama makul bir aralığı içindedir. 9 ve 9.5 DPC arasında bir embriyo 17 ve 25 Somitlerin arasında olacakt...

<table border="1"><tbody><tr><td> Reaktif </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> DMEM (düşük glukoz) </td><td> Gibco / Invitrogen </td><td> 11885 </td><td></td></tr><tr><td> Neurobasal Orta </td><td> Gibco </td><td> 21103 </td><td></td></tr><tr><td> BSA </td><td> Sigma </td><td> A3912-10G </td><td></td></tr><tr><td> DPBS </td><td> Gibco </td><td> 14190-144 </td><td></td></tr><tr><td> IGF1 </td><td> BD Biosciences </td><td> 354037 </td><td> -20 ° C&#39;de 50 mcg / ml hacimde Mağaza </td></tr><tr><td> bFGF </td><td> BD Biosciences </td><td> 354060 </td><td> -20 ° C&#39;de 25 mcg / ml hacimde Mağaza </td></tr><tr><td> Fibronektin </td><td> Gibco </td><td> 33016-015 </td><td> 1mg/ml al Saklanan-20 ° C&#39;de iquots </td></tr><tr><td> Retinoik asit </td><td> Sigma </td><td> R2625 </td><td> -20 ° C&#39;de etanol içerisinde yeniden yapılandırılması sonra, 35 ug / ml alikots içinde deposunda </td></tr><tr><td> 2-merkaptoetanol </td><td> Sigma </td><td> D-5637 </td><td></td></tr><tr><td> N 2 takviyesi </td><td> Gibco </td><td> 17502-048 </td><td></td></tr><tr><td> B27 takviyesi </td><td> Gibco </td><td> 17504-044 </td><td></td></tr><tr><td> Steriflip 0,22 um filtreler </td><td> Millipore </td><td> SCGP00525 </td><td></td></tr><tr><td> Penisilin-streptomisin </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> 0.20 mikron filtreleri </td><td> Corning </td><td> 431219 </td><td></td></tr><tr><td> Şırınga (filtrasyon için) </td><td> BD Biosciences </td><td> 301604 </td><td></td></tr&gt; <tr><td> Dört kuyucuğu </td><td> Thermo Fisher Scientific </td><td> 176740 </td><td></td></tr><tr><td> Kollajenaz / Dispase </td><td> Roche </td><td> 269 ​​638 </td><td> Etkinlik toplu göre değişir. -20 ° C&#39;de 100 mg / mL alikots içinde deposunda </td></tr><tr><td> İnsülin iğneleri (29 ½ ölçer) </td><td> Becton Dickson </td><td> 309306 </td><td></td></tr><tr><td> Hipoksi Odası </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Oksijen Analiz Cihazı </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Forseps # 5 </td><td> Güzel Bilim Araçları </td><td></td><td> Rahim ve desidua kaldırmak için. </td></tr><tr><td> Tripsin-EDTA (% 0.25) </td><td> Gibco </td><td> 25200 </td><td></td></tr></tbody></table>

isolation and culture of neural crest cells from embryonic murine neural tube

Embriyonik nöral krest (NC), nöral tüpün dorsalinde kaynaklanan multipotent progenitör nüfusu mezenkimal geçiş (EMT) bir epitel uğrar ve farklı hücre tipleri 1-3 sebebiyet veren, embriyo boyunca göç eder. NC de hedef organlar 4-6 farklılaşması ve olgunlaşması etkilemek için eşsiz bir yeteneği vardır. In vitro eksplante zaman, NC ataları kendini yenileme geçmesi, göç ve nöronlar, glia, düz kas hücreleri, kıkırdak ve kemik dokusu da dahil olmak üzere değişik türde farklılaşırlar. NC multipotency ilk. Kuş nöral tüp 7-9 eksplant tanımlanmıştır NK hücrelerinin in vitro izolasyonunda proliferasyon, göç ve multipotency dahil NC dinamikleri çalışmaya kolaylaştırır edildi. Kuş ve sıçan sistemlerinde daha fazla çalışmalar embriyo 10 geri nakledilen zaman eksplante NK hücreleri, NK potansiyelini korumak olduğunu göstermiştir-13. Bu doğal hücresel özellikleri eksplante NC ataları korunur Çünkü nöral tüp eksplant testi in vitro NC eğitim için cazip bir seçenek sunar. Memeli NC daha iyi anlaşılmasını sağlamak amacıyla, birçok yöntem NC nüfus yalıtmak için istihdam edilmiştir. NC kökenli ataları sonrası göçmen NC progenitörlerin 11,14-20 dinamiklerini incelemek için embriyo ve yetişkin hem de post-göçmen konumları kültüre edilebilir, ancak bunlar nöral tüp göç gibi NC progenitörlerin izolasyon optimum muhafaza sağlar NC potansiyeli hücre ve göçmen özellikleri 13,21,22. Bazı protokoller, özellikle ataları 11,13,14,17 için zenginleştirilmiş bir NC nüfusu izole etmeye fluorescence activated cell sorting (FACS) kullanır. Ancak, erken evre embriyo ile başlatırken, analizler için yeterli hücre sayıları erken NC alan nüfus izolasyonu karmaşıklaştıran, FACS ile elde etmek zorbireysel embriyolardan ns. Burada, bir Wnt1-Cre aktive soy muhabiri 23 dayanan yaklaşık% 96 oranında saf NC nüfus FACS ve sonuçları dayanmayan bir yaklaşım açıklanmaktadır. Burada sunulan yöntemi sıçan NC 11,13 kültürü için optimize protokolleri uyarlanmıştır. Bu protokol daha önceki yöntemlere göre avantajları 1) hücre FACS)) göreceli olarak saf NC nüfusu, 3 elde etmek için gerekli değildir premigratory NC hücreleri izole edilir ve 4), bir besleyici tabakası, 2 yetiştirilen değildir sonuçlar kolayca olmalarıdır sayılabilir. Bundan başka, bu protokolü farklı genetik manipülasyon ile NC özelliklerinin çalışma kolaylaştırıcı, herhangi bir mutant fare modelinde gelen NC izole edilmesi için kullanılabilir. Bu yaklaşımın sınırlaması NC NC 2,24-28 hayatta kalması, göç ve farklılaşmayı etkileyen bilinmektedir embriyo bağlamında kaldırılır olmasıdır.

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube at JoVE.com

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

Nöral tüpten embriyonik nöral krest izolasyonu kullanımını kolaylaştırır<em&gt; In vitro</em&gt; Göç, kendini yenileme ve nöral krest multipotency çalışmak için yöntemler.

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 ...

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Research

JoVE Journal

Neuroscience

17.1K Görüntüleme.  Vanderbilt University Medical Center. Nöral tüpten embriyonik nöral krest izolasyonu kullanımını kolaylaştırır In vitro Göç, kendini yenileme ve nöral krest multipotency çalışmak için yöntemler.

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Nörobilim

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

Civciv Embriyo Özü murin Sinir Crest Kök Hücre Yetiştiriciliği Üretimi

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

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 ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

Hareketi tarafından Modülasyonlu Nöronlar Fizyolojik, Morfolojik ve nörokimyasal Karakterizasyonu

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

Nöral Tüp Kapatma Fare Tüm Embriyo Kültürü

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

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 ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Tüm Dağı Muscle Analizi ile tekrarlanabilir Fare Siyatik Sinir Ezilme ve Rejenerasyon daha sonraki Değerlendirme

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Çapraz türler Nakli Sinir Crest Göç ve Farklılaşma Analiz

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Sıçan Embriyonik Nöral Hücre İzolasyonu ve Kültür: Bir Hızlı Protokol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

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 ...