Bu çalışma, California Rejeneratif Tıp Enstitüsü (RC1-00104), Kamu Sağlık Hizmetleri NHLBI Grant (HL085377) ve Pollin&#39;in Vakfı HSB bir hediye Kapsamlı Araştırma Fonu tarafından desteklenen

Aşağıda yazılı protokol, bazı değişikliklerle birlikte, yazarlar tarafından bildirilen yayınlanmış parametreler üzerine dayanır. Protokol UCSF Kurumsal Hayvan Bakım ve Kullanımı (IACUC) ve Kök Hücre Araştırma Gözetim (SCRO) Komiteleri tarafından onayı ile yapılır. Insan embriyonik kök hücreleri (hESC) I. Kültür <ol><li> Standart 6 (3.5 cm çapında kuyular) plakaları hESC büyütün. </li><li> Plakalar üzerine besleyici hücrelerin% 1 jelatin (6,7) ile kaplı, en az 12 saat önce hücreleri, tohum fare embriyonik fibroblast (MEF), CF1 gün 1000 Ci az ışınlanmış e12.5 zaman-evlendirilen fareler, elde Pasajlanması. MEFS% 50-60 izdiham, ya da 4 kaplama olmalıdır de 3,5 cm çapına göre x 10 5 hücreleri (Şekil 1). </li><li> Passage koloniler ~% 70 izdiham (Şekil 2) ulaşmıştır hücreleri. </li><li> Geçişi için hücreler aspirat KSR büyüme kuyulardan orta (6) ve 0.5 ml orta 1 mg / ml konsantrasyonda KSR kollajenaz IV içeren ile değiştirin. </li><li> 37 kollajenaz IV plakaları inkübe ° C /% 5, 5 dakika veya koloniler kenarlarına kadar CO 2 plakadan kaldırmaya başlar . </li><li> Plakadan kollajenaz IV çıkarın, her bir kuyu için 0.5 ml KSR ekleyin ve elle plaka tamamen kaldırmak için hücreleri kazıyın. </li><li> 15 ml konik bir tüp içinde, her iyi, yerden hücre süspansiyonu toplayın ve hücreleri 5 dakika boyunca ağırlık yerleşmek için izin. </li><li> ~ 300μl toplam hacmi hücreler ve orta bırakarak, tüpten süpernatantı. </li><li> Tam hacmi 200 ul mikro-pipet seti kullanarak, hafifçe koloniler break up hücre süspansiyonu 5-6 kez pipetle. </li><li> Toplam hacim başına 9 ml KSR orta (yani, bir de hasat ise, tüp 9 ml içermelidir) kullanarak hücrelerin orijinal iyi bir hücre süspansiyonu getirin. 12.5 ng / ml &#39;lik bir konsantrasyon rekombinant insan temel fibroblast büyüme faktörü (bFGF) ile orta tamamlayın. </li><li> Dulbecco&#39;nun fosfat ile MEF hücrelerinin (bkz. adım 2) içeren yeni kuyular yıkandıktan sonra, bütün serum kaldırmak 3 ml hücre süspansiyonu, her bir kuyunun ekleyin ve 37 ° C / 5% CO 2 plakalar inkübe için (DPBS) tuzlu tamponlu . </li><li> BFGF 24 saat geçtikten sonra ve her 24-48 saat hücreleri yeniden geçişli olarak hazır olana kadar KSR orta değiştirin. </li></ol> II. HESC pelet aşılama için hazırlanması <ol><li> Aşılama öncesi 24 saat 10μM nihai konsantrasyonu, hücre canlılığı artırmak için (8) KSR orta ROCK inhibitörü. ~% 70 izdiham (5 x 10 5 hücreleri) hücreler, böbrek kapsül başına eklenen iki pelet iki pelet yaratacaktır. </li><li> Aşılama (9) hücre pelletleri hazırlamak için 5 dakika kollajenaz IV ROCK inhibitörü ile tedavi edilen kültürler, 37 ° C /% 5 CO 2 inkübe. </li><li> Iyi kollajenaz IV aspire edin ve% 10 penisilin / streptomisin (pen / strep) 1 ml DPBS ile değiştirin. </li><li> El plaka koloniler kazımak ile ek bir kalem / strep 1 ml DPBS iyi durulama ve 1,5 ml mikrofuge&#39;de tüp hücre süspansiyonu her bir 1ml kısım transfer. </li><li> 8.000 x g kısaca mikrofuge&#39;de tüpler Spin süpernatantı aspirat ve pelet pen / strep 500μl DPBS tekrar süspansiyon. </li><li> Hücrelerin nakli için bir araya bağlamak yardımcı olmak için, her tüp DPBS içinde 100μl 1 mg / ml Phaseolus vulgaris lektin (PHA) ekleyin. </li><li> Hücre / PHA süspansiyon oda sıcaklığında 5 dakika inkübe edin, sonra 8.000 x g az 2 dakika boyunca dönerler. Rotor kuyu içinde borular 180 ° döndürün ve 8.000 x g 2 dakika için tekrar döndürün. </li><li> Süpernatant pelet zedelemeden dikkatlice aspire. </li><li> Yavaş yavaş yukarı kalkana kadar yavaşça pelet kenarına sonraki 200μl KSR pipetleme tüplerinden hücre pelletleri yerinden oynatın. </li><li> Bir kez yerinden hemen 3 ml KSR içeren 3.5 cm çapında kuyular yerleştirilir 0.4μm MILLICELL ekler pelet transferi, ve 37 ° C /% 5 CO 2 gece konaklama 2 saat inkübe edin. </li></ol> III. Greft renal kapsül <ol><li> Uygun aseptik teknik kullanılarak kurumsal kurallarına uygun olarak tüm hayvan işlemleri gerçekleştirin. </li><li> Inhale isoflurane/0.5%% 3 O2 ve intraperitoneal tribromoethanol (25 mg / taze hazırlanmış kg Avertin) bir kombinasyonu kullanılarak, 8-12 hafta arası SCID bej fareler (CB17.Cg Prkdc SCID LYST bg / CRL), narkoz . Farenin başına iki hücre pelletleri açıklanan teknikler kullanarak (10) engrafted olacaktır. </li><li> Hipotermi önlemek için bir ısıtma yastığı üzerinde fare yerleştirildikten sonra, kesi bölgesi tıraş sonra klorheksidin ile dezenfekte edin. Sadece merkezden cilt ama omurga paralel yoluyla 2,5 cm&#39;lik kesi olun. </li><li> Steril Salin ile kesi bölgesi nemli tutmake, sadece omurga, kaburga ve yaklaşık 1 cm altında, kas üzerinden küçük bir kesi yapmak. Kesi ile böbrek çekmeye yetecek büyüklükte, ancak kuvvet olmadan böbrek karın boşluğuna geri kayma olmaz bu kadar küçük olmalıdır. </li><li> Künt, hafif kavisli bir kanca şeklini içine çekti bir bardak Pasteur pipeti kullanarak kas kesiden böbrek yavaşça çekin. </li><li> Kapsül kurumasını önlemek için steril DPBS böbrek ıslatın. Bu kapsül, çok kırılgan olduğu gibi, işlem boyunca tekrarlamak gerekli olabilir. </li><li> Dumont # 5 forseps kullanarak hafifçe böbrek kapsülü yukarı çekin ve Vannas yaylı makas kullanılarak 2 mm kesi yapmak. </li><li> Çok ince, körelmiş bir prob içine çekilmiş olan başka bir cam pipet ile, kapsül ve böbrek arasında küçük bir cep olun. </li><li> MILLICELL eklemek, tek bir transferi pipet yerleştirilen büyük orifis pipet kullanarak, tek bir hücre pelletini. Fazla medya pelet transferi ile müdahale gibi, mümkün olduğunca birlikte biraz ekstra medya olarak emin olun. </li><li> Cebine oluşturmak için kesi kenarını tutarken, nazikçe açılması yanında pelet yeri ve tek kutuplu doğru çekti pipet ile böbrek cebine doğru itin. </li><li> Ikinci bir pelet işlemi tekrarlayın. Bu aynı böbrek kapsülü altında yerleştirin, fakat zıt kutup doğru. </li><li> Pelet üzerine geri aşağı kapsül dikkatlice yerleştirin. Pelet cebi içinde kalmalıdır ve hiçbir sütür kapsül kapatmak için gereklidir. </li><li> Böbrek karın boşluğuna yavaşça geri itin ve ekli ters kesme iğne ile 4-0 ipekle (veya daha küçük) kullanarak kas duvarı kapatın. Kesi, sadece tek bir sütür gerekli olduğu kadar küçük olmalıdır. </li><li> Aynı sütür materyali kullanarak, bir dizi 4-5 kesintiye dikiş cilt kapatın. </li><li> Fare kendi kafesine yerleştirmeden önce, sırasıyla 0.05 mg / kg buprenorfin (11,12) ve 5 mg / kg Ketoprofen (12,13), kısa vadeli ve uzun vadeli analjezikler olarak yönetmek. Kontrollü maddeler sadece kurumsal kurallarına uygun olarak kullanılmalıdır. </li><li> Hayvan kendi kafesine geri aktarın ve bir ısıtma yastığı anestezi kurtarmak için izin verir. Bu yaklaşık 20-30 dakika sürer. </li><li> Fare tam ayaktan sonra, hayvan barınağı tesisi iade edilebilir. </li></ol> IV. Teratom hasat, fiksasyon ve kesit <ol><li> 8-12 hafta ameliyat sonrası, böbrek yakınlarındaki bir zorlukla palpabl teratom olması gerekir. Büyük, içi sıvı dolu kistler genellikle 6-8 hafta başlayan, hem de gelişecektir. Hayvana sıkıntı neden oluyorsa bu bir erken hasat gerekebilir. Teratomlar kistler daha yavaş büyüdüğünden, ancak analiz için yeterli büyüklükte (yani, en az 8 hafta) teratom elde etmek için mümkün olduğunca uzun süre beklemek isteyeceksiniz. </li><li> Böbrek, teratom ve çevresindeki kistleri, böbrek ilk erişilen aynı cerrahi yaklaşım ile karın boşluğuna doku bir blok olarak herhangi bir çıkarın (bkz. Bölüm III, yukarıdaki adımları 1-4). </li><li> Eksize edilen doku tüm blok% 10 formalin solüsyonu içeren bir tüp içine yerleştirin. </li><li> Teratom 4 gece düzeltmek için izin ver ° C </li><li> Ertesi gün, tüp teratom çıkarın. (Şekil 3) teratom kendisi zarar vermeden, mümkün olduğu kadar herhangi bir kist ve çok böbrek olarak parçalara ayır. Büyük teratomlar diseksiyon ve gömme kolaylaştırmak için yarım kesilebilir. </li><li> Standart bir parafin fiksasyon tekniği kullanılarak teratom işleyin. Otomatik üniteler (örneğin, Shandon Hypercenter) mevcuttur: <table border="0"><tbody><tr><td> I. </td><td> % 80 Flex alkol </td><td> 2 saat </td></tr><tr><td> II. </td><td> % 80 Flex alkol </td><td> 1 saat </td></tr><tr><td> III. </td><td> % 85 Flex alkol </td><td> 1.5 saat </td></tr><tr><td> IV. </td><td> % 95 Flex alkol </td><td> 1 saat </td></tr><tr><td> V. </td><td> % 100 Flex alkol </td><td> 1 saat </td></tr><tr><td> VI. </td><td> % 100 Flex alkol </td><td> 1 saat </td></tr><tr><td> VII. </td><td> % 100 Flex alkol </td><td> 1 saat </td></tr><tr><td> VIII. </td><td> Clear-Rite 3 </td><td> 1 saat </td></tr><tr><td> IX. </td><td> Clear-Rite 3 </td><td> 1 saat </td></tr><tr><td> X </td><td> Clear-Rite 3 </td><td> 1 saat </td&gt; </tr><tr><td> XI. </td><td> Parafin (TissuePrep) </td><td> 2 saat </td></tr><tr><td> XII. </td><td> Parafin (TissuePrep) </td><td> 2 saat </td></tr></tbody></table></li></ol> V. İmmünohistokimya ve analiz <ol><li> Bir zamanlar üzerine parafin, slaytlar bölümünde döner mikrotom kullanarak 5μm seri dilimleri teratom ve float bölümleri gömülü. </li><li> Boyama önce slaytlar, en az bir saat pişirin. </li><li> Her üç embriyonik germ (Şekil 4) temsilcisi doku yapıları belirlemek için standart yöntemler kullanılarak hematoksilen ve eozin ile slaytlar Leke: <table border="0"><tbody><tr><td> I. </td><td> Ksilen yıkama </td><td> 3-5 dakika </td></tr><tr><td> II. </td><td> Ksilen </td><td> 3-5 dakika </td></tr><tr><td> III. </td><td> % 100 alkol </td><td> 3-5 dakika </td></tr><tr><td> IV. </td><td> % 100 alkol </td><td> 3-5 dakika </td></tr><tr><td> V. </td><td> % 95 alkol </td><td> 3-5 dakika </td></tr><tr><td> VI. </td><td> % 80 alkol </td><td> 3-5 dakika </td></tr><tr><td> VII. </td><td> Suyla yıkayın (çeşitli değişiklikler) </td><td> 2 dakika </td></tr><tr><td> VIII. </td><td> Gül&#39;ün Hematoksilen # 3 </td><td> 2-5 dakika </td></tr><tr><td> IX. </td><td> Suyla yıkayın (çeşitli değişiklikler) </td><td> suyu temiz çalışır kadar </td></tr><tr><td> X </td><td> Scott&#39;ın su (mavi çekirdekleri) </td><td> 3 dakika veya daha fazla </td></tr><tr><td> XI. </td><td> Suyla yıkayın (çeşitli değişiklikler) </td><td> 1-2 dakika </td></tr><tr><td> XII. </td><td> Eosin Y </td><td> 1 dakika </td></tr><tr><td> XIII. </td><td> Suyla yıkayın (çeşitli değişiklikler) </td><td> 30 saniye </td></tr><tr><td> XIV. </td><td> % 80 alkol </td><td> 1 dakika </td></tr><tr><td> XV. </td><td> % 95 alkol </td><td> 2 dakika </td></tr><tr><td> XVI. </td><td> % 95 alkol </td><td> 2 dakika </td></tr><tr><td> XVII. </td><td> % 100 alkol </td><td> 3 dakika </td></tr><tr><td> XVIII. </td><td> % 100 alkol (temiz) </td><td> 3 dakika </td></tr><tr><td> XIX. </td><td> Ksilen </td><td> 2 dakika veya daha fazla </td></tr><tr><td> XX. </td><td> Ksilen </td><td> 2 dakika veya daha fazla </td></tr></tbody></table></li><li> Permount her slayt bir lamel monte edin. </li></ol> VI. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2036/2036fig1.jpg" alt="Şekil 1" /> Şekil 1. Kültür farklılaşmamış hESC için besleyici bir katman olarak ışınlanmış CF1 fare embriyonik fibroblastlar Kaplama. MEFS ~% 50 birleştiği noktada yer kaplama, burada gösterildiği gibi, ya da başına 4 x 6 kuyu kültür çanak iyi 3.5 cm çapında 10 5 hücre. Bar, 100 mm. <img src="/files/ftp_upload/2036/2036fig2.jpg" alt="Şekil 2" /> Şekil 2. MEF besleyici tabaka farklılaşmamış hESC akışkan kültür örneği. HESC sonra geçişli olarak tanımlanan, burada görüldüğü gibi, ~% 70 konfluent kadar MEF besleyici katmanlar üzerinde yetiştirilmektedir. Bar, 100 mm. <img src="/files/ftp_upload/2036/2036fig3.jpg" alt="Şekil 3" /> Şekil 3. Eksplante teratomlar Örnek eksplantasyonu ardından, teratom, böbrek ve herhangi bir aksesuar doku formalin bir blok olarak sabit, sonra böbrek ve aksesuar doku dikkatlice parafin içine gömmeden önce kesilmiş bulunmaktadır . <img src="/files/ftp_upload/2036/2036fig4.jpg" alt="Şekil 4" /> Şekil 4. Farklılaşmamış hESC türetilen Teratomlar her üç germ in vivo doku temsilcisi içerir. Teratom, Şekil 3&#39;te gibi embriyonik dokular tespit kesitli ve hematoksilen ve eozin ile boyandı. hESC üç embriyonik germ (ektoderm (A, B), mezoderm (C), endoderm türetilen dokulara doğuran (D). (A) Doğan nöral tüp yapısı. (B) İlkel skuamöz epitel. (C) Kıkırdak yoğunlaştırılmış mezenşimin kapsül ile çevrilidir. (D) Salgı bağırsak yapısı. Bar, 100 mm. Görüntüler yazarın izniyle yeniden basıldı. <img src="/files/ftp_upload/2036/2036fig5.jpg" alt="Şekil 5" /> Şekil 5. Teratom analiz etiketli, farklılaşmamış hESC kaderi eşlemek için kullanılır. Daha önce yayınlanmış gibi (9), ile özel etiketli etiketsiz hESC hESC bir karışım teratom oluşum denemesi etiketli hücrelerinin kaderi harita için kullanılabilir. Burada gösterildiği gibi, yüzey işaretleyici, CD133, ve gelişmiş yeşil flüoresan protein (eGFP) etiketli ifade farklılaşmamış hESC etiketsiz, farklılaşmamış hESC ile karıştırıldı. Bu aşılama renal kapsül tarafından teratomlar oluşturmak için kullanılır. Anti-eGFP antikoru ile immünohistokimyasal analizi, hematoksilen ve eozin lekeli teratom bölümleri CD133 + hESC nöroektodermal kaderi gösteriyor. Teratomlar Şekil 4 gibi işlenmiş ve dokular içinde CD133 + GFP + hücreler türevleri lokalize anti-eGFP antikor (kahverengi) ile zıt. CD133 + elde edilen hücreler (ok) embriyonik ektoderm, özellikle sinir epiteli (solda) ve doğmakta olan nöral tüp benzeri yapılar (sağda) kaynaklanan dokularda gözlendi . Bar, 100 mm. Görüntüler yazarın izniyle yeniden basıldı. Jelâtin <ul><li> % 0,1 &#39;Domuz jelatin </li><li> % 99.9 DPBS <ul><li> 37 ° C (0.22μm) bir saat veya tüm jelatin solüsyonu içine girene kadar, ve sterilize kullanmadan önce inkübe DPBS, jelatin ekleyin. </li></ul></li></ul> MEF orta <ul><li> % 10 fetal sığır serumu (Nitelikli) </li><li> % 90 Dulbecco&#39;nun (D-MEM) Esansiyel Orta Modifiye bulunuyor </li><li> 1x L-Glutamine </li><li> 1x Pen / Strep <ul><li> Kullanımdan önce sterilize Filtre. </li></ul></li></ul> KSR (Knockout Serum Değiştirme) orta <ul><li> % 20 nakavt Serum Değiştirme </li><li> % 80 nakavt D-MEM </li><li> 1x L-Glutamine </li><li> 1x gerekli olmayan Amino Asitler </li><li> 0.1 mM β-mercaptoethanol / </li><li> 12.5 ng / ml bFGF <ul><li> Sterilize Filtresi. </li><li> 37 ° C, ama sıcak ° C hücrelere eklemeden önce 4 saklayın. </li><li> BFGF kullanımdan hemen önce ekleyin. </li></ul></li></ul> Kollajenaz IV. <ul><li> 1 mg / ml KSR ortamda tüm toz çözüm girene kadar 1-2 saat ya da 37 ° C yerleştirerek eritin. </li><li> Sterilize Filtresi. </li></ul> Avertin <ul><li> 0,25 g 2,2,2-tribromoethanol </li><li> 0.25 ml 2-metil-2-bütanol <ul><li> 37 ° ° C&#39;de% 100 çözüm oluşturmak için bir gecede. </li><li> Ameliyat öncesi birkaç saat, 37 DPBS ve inkübe kullanarak sulandırmak için% 2,5 &#39;çalışma çözüm ° C en az 1 saat. </li><li> Kısım sterilize ve Filtre. </li><li> Tribromoethanol yüksek ışığa duyarlı hazırlanması ve kullanımının her aşamasında, bu yüzden ışıktan korur. </li><li> Kullanmadan önce 12-24 saatten fazla hazırlayın. </li></ul></li></ul> Buprenorfin <ul><li> Metanol içinde 1 mg / ml stok solüsyonu <ul><li> 0.015 mg / ml steril H 2 O. kullanarak çalışma çözeltisi ile seyreltilir </li><li> 1 aya kadar -20 ° C&#39;de saklayın. </li></ul></li></ul> Ketoprofen <ul><li> DPBS 0.5 mg / ml çalışma çözeltisi <ul><li> DPBS 37 o gecede inkübe ° C&#39;de 1-2 saat veya tüm toz çözüm içine girene kadar. </li><li> Sterilize Filtre ve 4 saklamak ° C </li></ul></li></ul>

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B., Nishikawa, S., Muguruma, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat Biotechnol. 25, 681-686 (2007).</li><li>King, F. W., Ritner, C., Liszewski, W., Kwan, H. C., Pedersen, A., Leavitt, A. D., Bernstein, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subpopulations+of+human+embryonic+stem+cells+with+distinct+tissue-specific+fates+can+be+selected+from+pluripotent+cultures.">Subpopulations of human embryonic stem cells with distinct tissue-specific fates can be selected from pluripotent cultures.</a> Stem Cells Dev. 18, 1441-1450 (2009).</li><li>Cunha, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelio-mesenchymal+interactions+in+primordial+gland+structures+which+become+responsive+to+androgenic+stimulation.">Epithelio-mesenchymal interactions in primordial gland structures which become responsive to androgenic stimulation.</a> Anat Rec. 172, 179-195 (1972).</li><li>Kirsch, J. H., Klaus, J. A., Blizzard, K. K., Hurn, P. D., Murphy, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pain+evaluation+and+response+to+buprenorphine+in+rats+subjected+to+sham+middle+cerebral+artery+occlusion.">Pain evaluation and response to buprenorphine in rats subjected to sham middle cerebral artery occlusion.</a> Contemp Top Lab Anim Sci. 41, 9-14 (2002).</li><li>Lee-Parritz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analgesia+for+rodent+experimental+surgery.">Analgesia for rodent experimental surgery.</a> Isr J Vet Med. 62, 74-78 (2007).</li><li>Julou, L., Guyonnet, J. C., Ducrot, R., Pasquet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+pharmacological+and+toxicological+studies+on+ketoprofen.">Some pharmacological and toxicological studies on ketoprofen.</a> Rheumatol Rehabil. , (1976).</li></ol>

Biz, in vivo ortamda bir hESC ayırt kaderi haritalama amaç için son derece verimli teratom oluşum denemesi adapte var. HESC az sayıda özel olarak seçilmiş, farklılaşmamış vahşi tip hESC ve bir hücre granüle Phaseolus vulgaris lektin ile karıştırılır. Bu immünyetmezligi bir fare böbrek kapsül altında aşılı. Başlangıçta seçilen hESC kaderi immünohistokimya tarafından belirlenir (Şekil 5), daha önce de bildirilmiştir (9). Bu yöntem, doku, hücre tabanlı tedavi için özel reaktifler belirlenmesi ve üretmek için değerli bir araç sağlar.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2-Methyl-2-butanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>240486</td>
<td>Avertin</td>
</tr>
<tr>
<td>2,2,2-Tribromoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T48402</td>
<td>Avertin</td>
</tr>
<tr>
<td>4-0 silk sutures</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>641G</td>
<td>&nbsp;</td>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>233FB</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>B7536</td>
<td>&nbsp;</td>
<tr>
<td>Clear-Rite 3</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-046-341</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C5138</td>
<td>&nbsp;</td>
<tr>
<td>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>Dupont #5 forceps</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>500233</td>
<td>Kidney capsule</td>
</tr>
<tr>
<td>Eosin Y</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>23-245-658</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (Qualified)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>26140-079</td>
<td>&nbsp;</td>
<tr>
<td>Flex alcohol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-046344</td>
<td>&nbsp;</td>
<tr>
<td>Gill&#x2019;s Hematoxylin #3</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-050-204</td>
<td>&nbsp;</td>
<tr>
<td>Hemostat, straight</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>501241</td>
<td>General surgery</td>
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<tr>
<td>Iris forceps</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>15914</td>
<td>General surgery</td>
</tr>
<tr>
<td>Ketoprofen</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>K2012</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10829</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25020-081</td>
<td>&nbsp;</td>
<tr>
<td>MILLICELL inserts</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICM01250</td>
<td>&nbsp;</td>
<tr>
<td>Nonessential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>Operating scissors</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>501754</td>
<td>General surgery</td>
</tr>
<tr>
<td>Paraffin (TissuePrep)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>8002-74-02</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Permount</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SP15-100</td>
<td>&nbsp;</td>
<tr>
<td>PHA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>L1668</td>
<td>&nbsp;</td>
<tr>
<td>Porcine gelatin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G6144</td>
<td>&nbsp;</td>
<tr>
<td>ROCK inhibitor</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>Y-27632</td>
<td>&nbsp;</td>
<tr>
<td>SCID beige mice</td>
<td>&nbsp;</td>
<td>Charles River </td>
<td>250</td>
<td>&nbsp;</td>
<tr>
<td>Scott&#x2019;s Wash</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>6697-32</td>
<td>Ricca Chemicals</td>
</tr>
<tr>
<td>Vannas spring scissors</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>14003</td>
<td>Kidney capsule</td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M7522</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fate mapping of human embryonic stem cells by teratoma formation 

İnsan embriyonik kök hücreleri (hESC) için sınırsız bir kendini yenileme kapasitesi ve her üç embriyonik germ (1) türetilen hücrelerin içine ayırt etme yeteneği var. Spesifik hücre tiplerine hESC Yönetmen farklılaşma, rejeneratif tıp alanında (örneğin, (2-5)) çok fazla ilgi ve belirlenmesi için yöntemler vardır<em&gt; In vivo</em&gt; Seçilen ya da manipüle hESC kaderi bu çaba için gerekli. Biz, bu amaç için son derece verimli bir teratom oluşum denemesi adapte var. HESC az sayıda özel olarak seçilmiş, farklılaşmamış vahşi tip hESC ve bir hücre granüle Phaseolus vulgaris lektin ile karıştırılır. Bu immünyetmezligi bir fare böbrek kapsül altında aşılı. 2.5 x 10 gibi az bir<sup&gt; 5</sup&gt; HESC 16 cm oluşturmak için gerekli<sup&gt; 3</sup8-12 hafta içinde teratom. Başlangıçta seçilen hESC kaderi sonra immünohistokimyasal olarak tespit edilebilir. Bu yöntem, doku, hücre tabanlı tedavi için özel reaktifler tanımlamak için değerli bir araç sağlar.

Watch this Scientific Journal Video about Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation at JoVE.com

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Belirli hücrelerin içine hESC Yönetmen farklılaşma, rejeneratif tıp alanında büyük ilgi yarattı. Biz belirlemek için bir özlü, adım-adım protokolü<em&gt; In vivo</em&gt;, Doku, hücre tabanlı tedavi için özel reaktifler karakterize için çok değerli bir araç sağlar seçilen hESC kaderi.

Teratom Oluşumu İnsan Embriyonik Kök Hücre Kader Haritalama

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

fate-mapping-of-human-embryonic-stem-cells-by-teratoma-formation

Research

JoVE Journal

Biology

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

17.1K Görüntüleme.  University of California San Francisco. Belirli hücrelerin içine hESC Yönetmen farklılaşma, rejeneratif tıp alanında büyük ilgi yarattı. Biz belirlemek için bir özlü, adım-adım protokolü In vivo, Doku, hücre tabanlı tedavi için özel reaktifler karakterize için çok değerli bir araç sağlar seçilen hESC kaderi.

Video: Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

İnsan Embriyonik Kök Hücre (ES) yayılması

Biyoloji

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murin Embriyonik Kök Hücre Hematopoetik Kök Hücre türetilmesi

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery İnsan Embriyonik Kök Hücreleri türetilmesi

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

İnsan Embriyonik Kök Hücreleri Mitokondrial Kompleksi Aktivite araştırmayı

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

İnsan Embriyonik Kök Hücre Kromatin Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

İnsan Embriyonik Kök Hücreleri Manyetik Rezonans Görüntüleme için in vitro Etiketleme

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

İnsan Embriyonik Kök Hücre Kültürü ve Bakım

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Karyotipik İnsan Embriyonik Kök Hücreleri kromozom Yayılı Hazırlık

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

İnsan Embriyonik Kök Hücreler Donma ve Çözülme

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Embriyonik Kök Hücreler farklılaşma oligodendrosit Öncüleri

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...