We thank Joon Kim for expert flow cytometry assistance. Research in the authors&#8217; labs is supported by the SCORE program of the National Institutes of Health (grant SC1-GM095402 to B.D.O) and the Canadian Institutes of Health Research (to J.C.Z.P.). J.C.Z.P. is supported by a Canada Research Chair in Developmental Immunology. The biomedical research infrastructure of Hunter College is supported in part by the NIH Research Centers in Minority Institutions (RCMI) program via grant MD007599. We also acknowledge the New York State Stem Cell Science Program (NYSTEM) for its support of the initiation of stem cell research at Hunter College via grant C023048.

Kültür Medya, Sitokinlerle ve Jelleşmiş Plakaların 1. Hazırlık <ol><li> Yüksek glikoz ve sodyum piruvat ile Eagle Medium (DMEM) içinde Dulbecco modifikasyonu kullanılarak bir ES hücre ortamı hazırlayın. % 1 HEPES Tamponu,% 1 non-essential amino aist,% 0.1 gentamisin (50 mg / ml) ve% 0.1 (Fetal Sığır Serumu (FBS) Kalifiye% 20 ESC,% 1 Penisilin / Streptomisin,% 1 L-glutamin, ekleme 55 uM), β-mersaptoetanol eklenir. Süzme ile ES hücre ortamı sterilize edin. </li><li> Üreticinin talimatlarına göre toz Alfa Minimum Esansiyel Medium (α-MEM) 1 L yaparak OP9 ortamı hazırlayın. % 20 fetal sığır serumu (FBS) ve% 1 Penisilin / Streptomisin ekleyin. Karıştırmak için ters. Iki adet 500 ml&#39;lik alikota süzme ile sterilize edin. NOT: tozundan yapılan medya kullanımı tavsiye ve geliştirilmiş OP9 hücre bakım ve in vitro farklılaşma sonuçları elde etmek muhtemeldir. Bu yaklaşım standart bir prosedür haline gelmiştir. Ancak, biz de tha dikkatt biz bazen yeterli başarı ile önceden yapılmış sıvı α-MEM kullandık. </li><li> % 90 FBS,% 10 dimetil sülfoksit (DMSO) eklenerek ortam dondurma hazırlayın. Yavaşça karıştırınız ve süzülerek sterilize. 4 ° C&#39;de saklayın. </li><li> 10 ug / ml tam ortam içinde OP9 insan rekombinant Flt-3, ligand (Flt-3 L) içinde çözülmesiyle 2,000x Flt-3, ligand (10 ug / ml) hazırlayın. -80 ° C&#39;de 1.5 mL mikrosantrifüj tüpleri içine kısım saklayın. Istikrar ve depolama ile ilgili tedarikçinin tavsiyelerine uyun. Not: Flt-3L stabilitesi (4 ° C &#39;de 3 ay -80 ° C de 1 ay) kısadır. </li><li> Tam OP9 ortamını kullanarak 1,000x, IL-7 (1 ug / ml) hazırlayın. -80 ° C&#39;de 1.5 mL mikrosantrifüj tüpleri içine kısım saklayın. Istikrar ve depolama tedarikçinin önerileri izleyin (IL-7 -80 ° C&#39;de 12 aya kadar dayanır). </li><li> LİF i 7 10 adet 1:10 seyreltme ile 1,000x, lösemi önleyici faktörü (LİF) ya da (10 ug / ml) hazırlayınn, ES hücre ortamı. 4 ° C&#39;de muhafaza kısım. Kısım şişeler üzerinde üretici tarafından sağlanan son kullanma tarihini not ettiğinizden emin olun. NOT: Ürün üretim tarihinden itibaren konsantre veya seyreltilmiş formu en az 18 ay stabildir. </li><li> Jelatinleştirilmiş 6 oyuklu plakalar hazırlamak ile 6 oyuklu plakanın her bir oyuğuna 1.5 ml% 0.1 jelatin solüsyonunu ilave edin. Kaplama için en az 30 dakika izin kapakları ile oda sıcaklığında steril bir muhafaza içinde yemekler bırakın. Yemekler de nemlendirilmiş bir inkübatör içinde bir gece boyunca kalabilir. Sağ cep tohumlamadan önce herhangi bir artık jelatin çözüm çıkarın. Jelatin çözüm tamamen kuyuda kurumasına izin vermeyin. </li></ol> 2. Hazırlık ve Besleyici Hücreleri ve Fare Embriyonik Kök Hücreleri Bakım (Mesc) NOT:% 5 CO2 ile nemlendirilmiş 37 ° C inkübatör tüm hücreleri inkübe edin. <ol><li> Çözülme Fare Embriyonik Fibroblastlar (MEF) <ol><li> Donmuş bir flakon (~ 5 x 10 6 Hızlı-çözülme </sup&gt; hücre / mitomisin C ile tedavi (veya başka mitotikal tutuklandı) MEF&#39;lerin flakon) ve onları bir 15 ml tüp transfer. </li><li> Yavaş yavaş dondurma ortamından DMSO sulandırmak için damla damla olarak, çözülmüş hücrelere ES hücre ortamı 8 ml ilave edilir. </li><li> 5 dakika boyunca 4 ° C&#39;de 400 x g&#39;de santrifüjleyin hücreleri. Süpernatantı dikkatlice çıkarın ve ES hücre ortamı, 3 ml hücre pelletini. </li><li> Önceden ısıtılmış ES hücre ortamı 33 ml 50 ml&#39;lik bir santrifüj tüpü hazırlayın. 36 ml nihai hacme getirmek üzere yeniden süspansiyon haline getirilmiş MEF&#39;lerin 3 ml ilave edilir. </li><li> İki jelatinleştirilmiş 6 oyuklu plakaların her bir kuyunun içine yeniden süspanse MEF&#39;lerin 3 ml dağıtın. Hücreler takın ve onlara mESCs, tohumlamadan önce yaymak için izin vermek üzere en az altı saat süreyle tekrar inkübatör içine tabak yerleştirin. Medya her 2-3 günde bir değiştirilir ise bu mitotikal tutuklandı besleyici katmanlar, ilk ekimden birkaç gün boyunca kullanılabilir olabilir. NOT: Taze tutuklandı MEF&#39;ler da kullanılabilir. </li> &lt;/ Ol&gt; </li><li> Tohumlama mESCs <ol><li> 37 ° C su banyosu içinde dondurulmuş Mesc klon ile bir şişe hızlı çözünmesi ve 2.1.1-2.1.3 tarif edildiği gibi hücrelerini hazırlamak. NOT: Bir 6-çukurlu plaka bir birleşik kuyusundan Mesc 2 şişe dondurma. </li><li> (Adım 2.1.5 hazırlandı) tutuklandı MEF tek tabakasıyla 6-plaka (Mesc klonu itibarıyla) bir kuyudan ortamları çıkarın ve MEF tek tabaka üstüne mESCs ve 3 ml tohum. 1,000x LİF (10 ng / ml), 3 ul ekleyin. Kuvöz içine yemekleri dönün. </li></ol></li><li> ES hücre bakım ve tripsin aracılı geçit NOT: Mesc izdiham göre değiştir günlük medya, ya da bölünmüş. Optimal, hasat ve split / yeniden-plaka hücreleri her gün. <ol><li> ES hücre ortamı çıkarın ve 2 ml PBS ile mESCs yıkayın. PBS çıkarın. % 0.25 tripsin 1 ml ilave edilir ve 3-5 dakika 37 ° C&#39;de inkübe edin. </li><li> Tripsinize hücreleri toplamak ve 15 ml&#39;lik bir santrifüj tüpü içine transfer edin. Şiddetle m topaklannı kırmaya hücre süspansiyonu pipetleyinEKH. Tripsin nötralize etmek için hücre süspansiyonu tam ES hücre ortamı 2 ml ilave edilir. </li><li> 4 ° C&#39;de 5 dakika boyunca 400 x g&#39;de santrifüj hücreleri. Süpernatantı ve 3 ml ES hücre medya pelletini. </li><li> Hazırlanan tutuklandı MEF tek tabakaları ihtiva eden 6 oyuklu plakalar ortamı çıkarın. Her bir oyuğa ES hücre ortamı, 3 ml (istenilen bölme oranına göre) hücre süspansiyonu uygun miktarda ekilmiş edin. 1,000x LIF 3 ul ekleyin. 2 gün içinde geçiş için hazır olacak 6: Birleşik bir Mesc de 1 bölün. </li></ol></li><li> OP9 / OP9-DL1 hücrelerin bakım <ol><li> OP9 hücrelerin bir şişe çözülme (adımları izleyin 2.1.1-2.1.3 OP9 medyayı kullanarak) </li><li> 10 cm doku kültürü çanak 7 mi OP9 ortam ekleyin. Plaka boyunca hücrelerin dağıtılması, damla damla bir şekilde yeniden süspansiyon haline getirilmiş hücreler 3 ml ilave edilir. Gece boyunca inkübatör içinde hücreleri yerleştirin. </li><li> Ertesi gün hücrelerin OP9 izdiham kontrol edin. Çanak birçok ölü yüzen hücreleri içeriyorsa, remoOrtamı ettik ve taze ortam OP9 10 ml ekleyin. Neredeyse tam izdiham görülmez ise inkübatör tabak dönmek. 4 yakın birleşik OP9 monolayer: Split 1 (tripsin kullanarak). NOT: Tabaklar yaklaşık 2 gün içinde tekrar aynı izdiham düzeyine ulaşması gerekir. Aşırı yapışık hale OP9 hücreleri bırakmamaya özen gösterin. </li><li> Genişleme hisse senetleri gibi OP9 hücrelerin erken pasajlar dondurun. NOT: konfluen 10 cm çanak yakın birinden OP9 hücrelerin 2 şişe dondurma. Genişletme stoklar Her bir ampul, aşağıda daha ayrıntılı olarak Mesc ko-kültür deneyleri kullanılan çalışma stoğu oluşturmak için çoğaltılabilir. Değişen sıcaklıklara olumsuz donar kalitesini etkileyebilir, çünkü daha -80 ° C&#39;de derin dondurucuda daha sıvı azot içinde dondurulur, tüm OP9 stokları tutun. </li></ol></li></ol> 3. OP9-DL1 Ortak kültür Prosedür <ol><li> Eş-kültür preparatlar <ol><li> Yaklaşık bir hafta önce, bir ortak-kültürünün başlatılmasından MEF&#39;ler, mESCs ve OP9 Hücre çalışma STO çözülmecks. OP9-DL1 hücrelerinin ko-kültür gününde 8 kadar gerekli olmadığından, ko-kültür başlangıcından sonraki ilk üç gün boyunca OP9-DL1 hücrelerini eritin. Bakım ya da gerektiği gibi tüm hücreleri dondurmak. </li><li> Deneyin ölçeği dayalı gün 0 ko-kültür ile% 80 kaynaşmaya eriştikten hazır OP9 hücre tekli katmanları, 10 cm tabaklar başlangıç ​​sayısını belirlemek (örneğin, Mesc klonların sayısı ve ayırt edilmesi için ortak-kültür zaman noktalarının sayısı ile ) analiz edilebilir. 4 ve 1: bir ko-kültürü hazırlamak için, 1 ila OP9 hücrelerinin konfluent çanak yakınındaki bölme 6 iki gün önce mono tabakalar gerekli olacaktır ne zaman. NOT: Bir ÇIK klon başına en az bir levha gerekir. Gün 5 pasaj altı tabak tohum için yeterli hücrelerin vermelidir 0. günde (aşağıda tarif edildiği gibi), genellikle, tek bir ESC klon, tek bir plaka üzerinde tohumlandı. Her gün 5 tabak bir sonraki analiz zaman noktasını sağlayacaktır. </li><li> OP9 monokatmanlar sürekli ko-kültür geçişi için gerekli olacak bu yana, bakımı her zaman HAYATIMIN almakko-kültür ile paralel olarak ilave OP9 kültür plakalarının yeterli sayıda. </li></ol></li><li> Gün 0: ortak-kültürünün başlatılması <ol><li> 2.3.1-2.3.4 gibi Mesc toplayın. Hücreleri saymak. </li><li> Her bir klon için Mesc plaka başına OP9 ortam, 10 ml 5 x 10 4 Mesc hazırlar. NOT:. Biz kullanılan olmasına rağmen, biz alternatif bir orta α-MEM,% 10 FBS oluşan ve 5 x 10 β-mercaptoethanol da farklılaşma ko-kültürlerinde kullanılmak üzere rapor edilmiştir -5 M 10 unutmayın </li><li> OP9 yemekleri eski ortamları çıkarın ve plaka boyunca eşit hücrelerini dağıtmak için özen yemekleri Mesc süspansiyonu 10 ml ekleyin. 37 ° C inkübatör yemekler yerleştirin. </li></ol></li><li> 3. Gün: Medya değişiklik <ol><li> Inkübatör yemekleri çıkarın ve mikroskop altında gözlemlemek. Koloniler üzere parlaklık kaybetmek ve basık görünür başlamalıdır. </li><li> Yemekler ortamı kaldırın ve yavaşça taze OP9 10 ml ekleyinmedya. </li><li> Inkübatör ko-kültür yemekleri dönün. Görsel kolonilerin% 80-90 görsel mezoderm-benzeri morfolojiye sahiptir ~ kadar gerçekleştirilemeyen gereken bir sonraki geçidin (gün 5) için, besleyici OP9 tek tabaka hazırlama zamanlama bilgi günlük mezoderm gibi koloni oluşumunu izlemek. 5 gün hücre transferi, aşama fazla erteleme 2 gündür. </li></ol></li><li> 5. Gün: ön-kaplama ile Tripsin aracılı geçit. NOT: optimal konfluen OP9 hücreleri ile önceden 10 cm yemekleri yeterli sayıda hazırlayın. Ko-kültürler görsel aşama 3.3.3 açıklanan özellikler gösterir yalnızca sonraki adımlara devam (ayrıca Temsilcisi Sonuçlar bakınız). <ol><li> Ko-kültür yemekleri ortamı çıkarın. Yıkamak için 4 ml PBS ilave edin. PBS girdap ve çıkarın. % 0.25 tripsin 4 ml ilave edilir ve ~ 5 dakika boyunca inkübatör yemekler yerleştirin. </li><li> Dikkat ederek kadar, aşırı hava kabarcıkları tanıtmak için, güçlü pipetleme ile tripsinize edilmiş hücre katmanı bozabilirHomojen, çoğunlukla tek bir hücre süspansiyonu elde edilir. </li><li> Tam OP9 medya 4 ml ekleyin ve pipetleme karıştırın. 30 dakika boyunca inkübatör yeni, boş bir 10 cm çanak ve yerine hücreleri aktarın OP9 hücreleri çanak uymak için izin vermek. Bu &quot;ön kaplama&quot; aşaması eş-kültür bir sonraki adıma aktarılmıştır OP9 hücrelerin sayısını azaltır. </li><li> 40 mikron hücre süzgeçler ile 50 ml santrifüj tüpleri hazırlayın. </li><li> Ön kaplama çanak yapışkan olmayan hücreler toplanır ve daha sonra tüpe hücre süzgecinden yoluyla geçmektedir. 6 ml PBS&#39;de hafifçe bulaşık yıkama ve arkasında bağlı OP9 hücreleri bırakarak aynı süzgecinden geçirin. </li><li> 4 ° C&#39;de 400 x g&#39;de 5 dakika santrifüjleyin hücreleri. Süpernatantı ve tam OP9 medya 3 ml pelet hücreleri tekrar süspansiyon. Hücreleri saymak. </li><li> Optimal Konfluent OP9 hücreleri ile 10 cm plakalardan ortamı çıkarın. </li><li> Her analiz zaman noktası için, OP9 mono 5 x 10 5 hücre tohum10 ml nihai hacim içinde katmanı. </li><li> 5 ng / ml insan rekombinan Fit-3L ekleyin ve inkübatör yemekler yerleştirin. </li></ol></li><li> 8. Gün: Hematopoetik progenitör Hücre (HPC) koleksiyonu NOT: Bu aşama için süre içinde bu olacağı şekilde önceden OP9 veya OP9-DL1 hücre mono tabakaları ile 6 oyuklu plakaların yeterli sayıda Hazırlama ~% 80 konfluent. <ol><li> 40 mikron filtreli, 50 ml santrifüj tüpleri hazırlayın. </li><li> Inkübatör yemekleri çıkarın ve mikroskop altında gözlemlemek. Yüksek basınçlı bir parlak kümeleri gevşek OP9 tek tabaka eklenmesi gerekmektedir. Bir pipet ile OP9 tek tabaka ve yemeğin üzerine mevcut medya yıkanması (ama aşırı rahatsız değil) mümkün olduğunca bu hücrelerin birçok toplayın. Köpük oluşumu önlemek için, her zaman bu HPC toplama / yıkama aşaması sırasında pipet ucuna ortamın en az 1 ml bırakın. </li><li> Bir tüp içine 40 um süzgecinden hücreleri geçirin. , Tüm HPC aynı plaka 8 ml PBS ilave edin ve bir mikroskop altında kontrol bizyeniden toplandı. Daha kuvvetli bir yıkama ile (eğer gerekli ise) PBS Yıkama / toplama aşamasından tekrar edilmelidir. Zaten aynı Hücreleri plakadan içeren tüp içine hücreleri süzün. </li><li> 4 ° C&#39;de 5 dakika boyunca 400 x g&#39;de santrifüje hücreleri. </li><li> 5 ng OP9 ortam (levha, 5. günde tohumlanır başına) 3 ml &#39;lik bir ana karışımı hazırlayın / ml Flt-3L ve 1 ng / ml IL-7. </li><li> Fit-3L + IL-7 ana karışımı ile OP9 medya (işlenen her 10 cm çanak için 3 ml) süpernatant ve tekrar süspansiyon pelet çıkarın. OP9 hücrelerine sahip bir 6-yuvalı plakanın bir oyuğuna Her levhadan toplanan hücreler aktarın. <ol><li> , Monositik, B hücreleri ya da eritroid soylar doğru pillerinin OP9 hücreler üzerine hücreler toplandı tohum için. T hücreleri elde etmek için, OP9-DL1 hücre mono tabakaları üzerine hücreleri tohum. </li></ol></li><li> Inkübatör içine 6 oyuklu plakalar yerleştirin. </li></ol></li><li> Gün 10: Medya değişiklik <ol><li> Her biri farklı klon Mesc besleyici hücre tipi Combina bir 15 ml santrifüj tüpü hazırlayınko-kültür tion. </li><li> Flt-3L 5 ng / ml IL-7 ve 1 ng / ml &#39;si ile OP9 ortamın bir ana karışımı hazırlayın. Her bir oyuk için 3 ml hazırlayın. </li><li> Yavaşça aynı ESC klon besleyici hücre tipi kombinasyonunu içeren çoklu ortam çukurlardan birleştirerek 6 oyuklu plakanın her bir medya toplar. </li><li> Her bir kuyuya OP9 ortam ana karışımı 2 ml (3.6.2) ekleyin. </li><li> Santrifüj, 4 ° C&#39;de 5 dakika boyunca 400 x g&#39;de toplanmıştır ortam. Kuyu başına OP9 medya ana karışımı 1 ml her pelet (görünür eğer çok küçük) (3.6.2 bakınız) başlangıçta adımda 3.6.3 toplanan süpernatant kaldırmak ve tekrar süspansiyon. </li><li> Lütfen, her oyuğa ortam başlangıçta 3 ml her bir hazne içinde toplanmıştır hacmi getiren başka içine hücre 1 ml dağıtın. Inkübatör yemekleri dönün. </li></ol></li><li> Gün 12: Hayır tripsin pasaj Not: Bu s onlar optimal zamanında birleştirilebilmeli böylece önceden OP9 veya OP9-DL1 hücreleri ile 6 oyuklu tabaklarda yeterli sayıda hazırlayındım. Gün 12 geliştirme eritrosit, monositik ve erken evre T hücreleri akış sitometrisi analizleri için idealdir. <ol><li> Bir ana karışımı Fit-3L 5 ng / ml IL-7 ve 1 ng / ml &#39;si ile OP9 ortam (ko-kültür içinde devam edecek her çukuruna 3 ml) hazırlayın. </li><li> 40 um süzgeçler (ko-kültür, her klon-ESC besleyici hücre tipi kombinasyon için bir adet) ile 50 ml&#39;lik bir santrifüj tüpü hazırlayın. </li><li> Kuvvetlice kuyuda (tek tabaka da dahil olmak üzere) tüm hücreleri parçalamak için her bir oyuğa mevcut ortamı pipetle. Tek bir hücre süspansiyonu andıran bir kıvama gelinceye kadar güçlü pipetlenmesiyle devam edin. </li><li> Tüp içine süzgecinden toplanan hücreler geçirin. Kalan tüm yıkama hücreleri ve toplamak için, her bir oyuğa PBS 3 ml ilave edilir. Aynı süzgecinden hücrelere geçerler. Aynı ESC klon-besleyici hücre tipi kombinasyonu içeren çoklu kuyu hücreleri birleştirin. </li><li> Kullanılan hücre süzgeç atın ve bir kuyuya bir kısım eşdeğer kaldırmak (~ 6 ml) İstenilen için(ya da başka) akış sitometrisi o gün yürütülecek analiz eder. Kullanımdan önce buz üzerinde, bu alikotları saklayın. </li><li> 4 ° C&#39;de 5 dakika boyunca 400 x g&#39;de santrifüje hücreleri. Süpernatantı. Oyuk başına ana karışımı 3 ml, 50 ml santrifüj tüpünden elde edilen pelet yeniden tohumlu edilmesi. Uygun besleyici bir hücre tipi için her hücre süspansiyonu, göz başına 3 ml ilave edilir ve kuluçka makinesi içindeki yemekler yerleştirin. </li></ol></li><li> Gün 14: Medya değişiklik <ol><li> Bölüm 3.6 özetlenen adımları izleyin. </li></ol></li><li> Gün 16: Hayır tripsin pasaj NOT: Bu aşama için önceden en uygun konfluent OP9 veya OP9-DL1 hücreleri, 6 oyuklu kapları hazırlayın. NOT: Flow sitometri B lenfositleri ve orta evre T hücrelerinin analizleri için günlük 16 idealdir. <ol><li> Bölüm 3.7 özetlenen adımları izleyin. </li></ol></li><li> Gün 18: Medya değişiklik <ol><li> Bölüm 3.6 özetlenen adımları izleyin. </li></ol></li><li> Gün 20: Hayır tripsin pasaj NOT: Gün 20 iAkış sitometri için s ideal bir T hücrelerini gelişmekte geç sahneye orta analizleri. <ol><li> Bölüm 3.7 özetlenen adımları izleyin. İstenirse, hücreleri son gün 20 alternatif medya değişikliği ayırt taşımak ve hiçbir tripsin lenfosit genişleme oranı günlük izlenmesi ile, her iki gün geçitleri. </li></ol></li></ol>

<ol>
	<li>Schmitt, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+T+cell+development+and+establishment+of+T+cell+competence+from+embryonic+stem+cells+differentiated+in+vitro.">Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro.</a> Nat Immunol. 5, 410-417 (2004).</li><li>Pui, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+expression+in+early+lymphopoiesis+influences+B+versus+T+lineage+determination.">Notch1 expression in early lymphopoiesis influences B versus T lineage determination.</a> Immunity. 11, 299-308 (1999).</li><li>Nakano, T., Kodama, H., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+lymphohematopoietic+cells+from+embryonic+stem+cells+in+culture.">Generation of lymphohematopoietic cells from embryonic stem cells in culture.</a> Science. 265, 1098-1101 (1994).</li><li>Schmitt, T. M., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+T+cell+development+from+hematopoietic+progenitor+cells+by+delta-like-1+in+vitro.">Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro.</a> Immunity. 17, 749-756 (2002).</li><li>Chen, J., Lansford, R., Stewart, V., Young, F., Alt, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAG-2-deficient+blastocyst+complementation:+an+assay+of+gene+function+in+lymphocyte+development.">RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development.</a> Proceedings of the National Academy of Sciences of the United States of America. 90, 4528-4532 (1993).</li><li>de Pooter, R. F., Cho, S. K., Carlyle, J. R., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+generation+of+T+lymphocytes+from+embryonic+stem+cell-derived+prehematopoietic+progenitors.">In vitro generation of T lymphocytes from embryonic stem cell-derived prehematopoietic progenitors.</a> Blood. 102, 1649-1653 (2003).</li><li>Holmes, R., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OP9-DL1+system:+generation+of+T-lymphocytes+from+embryonic+or+hematopoietic+stem+cells+in+vitro.">The OP9-DL1 system: generation of T-lymphocytes from embryonic or hematopoietic stem cells in vitro.</a> Cold Spring Harb Protoc. 2009, (2009).</li><li>Arsov, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+autophagic+protein+beclin+1+early+in+lymphocyte+development.">A role for autophagic protein beclin 1 early in lymphocyte development.</a> J Immunol. 186, 2201-2209 (2011).</li><li>Lahiji, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+TCR-alpha+gene+locus+control+region+activity+in+T+cells+derived+in+vitro+from+embryonic+stem+cells.">Complete TCR-alpha gene locus control region activity in T cells derived in vitro from embryonic stem cells.</a> J Immunol. 191, 472-479 (2013).</li><li>Hirashima, M., Kataoka, H., Nishikawa, S., Matsuyoshi, N., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+embryonic+stem+cells+into+endothelial+cells+in+an+in+vitro+model+of+vasculogenesis.">Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis.</a> Blood. 93, 1253-1263 (1999).</li><li>Nishikawa, S. I., Nishikawa, S., Hirashima, M., Matsuyoshi, N., Kodama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+lineage+analysis+by+cell+sorting+and+culture+identifies+FLK1+VE-cadherin++cells+at+a+diverging+point+of+endothelial+and+hemopoietic+lineages.">Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages.</a> Development. 125, 1747-1757 (1998).</li><li>de Pooter, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+requires+GATA-2+to+inhibit+myelopoiesis+from+embryonic+stem+cells+and+primary+hemopoietic+progenitors.">Notch signaling requires GATA-2 to inhibit myelopoiesis from embryonic stem cells and primary hemopoietic progenitors.</a> J Immunol. 176, 5267-5275 (2006).</li><li>Watarai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+NKT+cells+in+vitro+from+embryonic+stem+cells+bearing+rearranged+invariant+Valpha14-Jalpha18+TCRalpha.">Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant Valpha14-Jalpha18 TCRalpha.</a> 115, 230-237 (2010).</li><li>Pipkin, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+transfer+activates+and+delineates+a+locus+control+region+for+perforin.">Chromosome transfer activates and delineates a locus control region for perforin.</a> Immunity. 26, 29-41 (2007).</li></ol>

The authors have nothing to disclose.

OP9-DL1 ko-kültür sistemi, kök hücreleri, kan hücre tiplerinin gelişimi esnasında, gen rolünü incelemek için kullanılmıştır. 8,12,13 Ayrıca gen düzenleyici DNA fonksiyonlarını incelemek için etkin bir modeli olduğu kanıtlanmıştır hücre farklılaşması sırasında. 9,14 zaman ve hematopozun birçok temel soruları deneylerin maliyeti önemli ölçüde tasarruf elde edebilirsiniz tüm fare modellerine alternatif olarak bu yaklaşımı kullanarak. Bununla birlikte, bu amaçla...

A cell culture system has been established in which mouse embryonic stem cells (mESC) are differentiated to T cells in vitro.1 This system exploits the ability of Notch signaling to drive T cell differentiation.2 The OP9-DL1 cell line was created by transducing bone marrow-derived OP9 cells3 with a Notch ligand, Delta-like 1 (DL1).4 Activation of the Notch signaling cascade in vitro facilitates T cell development to the exclusion of other cell lineages. With the inclusion of appropriate cytokines, this system provides a cell culture &#8220;microenvironment&#8221; that supports the sequential advancement of mESC toward hematopoietic and ultimately T cell lineages. This system supports the flow cytometric identification of T cells at the various developmental stages seen during normal T cell ontogeny in the thymus. For investigating selected questions relating to T cell development, this procedure has become an attractive alternative to in vivo whole mouse models5 and in vitro fetal thymic organ culture methods used to elicit T cell development from mouse embryonic stem cell derived hematopoietic precursors.6 The major advantage of the OP9 co-culture system is that it involves standard and straightforward cell culture techniques and does not depend on the continual use of experimental animals. 
We follow a detailed, previously published protocol in our experiments using this approach.7 We have utilized this technology to examine the hematopoietic differentiation products of non-manipulated mESC clones, high quality mESC clones handpicked to make chimeric embryos8 and stably-transfected ESC clones coming directly out of drug selection.9 We have noted that the temporal kinetics of initial in vitro differentiation from mESC to mesoderm-like colonies in this model can be variable among individual clones. The mESC-OP9 co-cultures can be visually assessed for progression to mesoderm. While this will usually be completed by the fifth day of co-culture, among individual clones, completion can be delayed for one or two days. Quantitative (~80-90%) mesoderm formation must be achieved prior to transfer in order to obtain optimal hematopoietic progenitor cell (HPC) formation and robust lymphopoiesis. Thus, when working with multiple mESC clones, this &#8220;day 5&#8221; passage is best delayed until all clones complete the transition to mesoderm-like colonies. This enables synchrony of subsequent development among the clones after their transfer into hematopoietic differentiation conditions. Three days after the passaging of the 80-90% mesodermal formations, HPCs are collected from the OP9 monolayers. HPCs can be seeded on new OP9 cells to allow differentiation of monocytic, erythroid and B cell lineages. Alternatively, HPCs can be seeded on OP9-DL1 cells and driven towards T cell development. All in vitro differentiation cultures are provided Flt-3L beginning at day 5, with further addition of IL-7 beginning at day 8. Flow cytometry analyses performed at various time points during the experiment enable monitoring of progress through the stages and lineages of hematopoietic differentiation and T cell development. CD4/CD8 double positive (DP) T cells begin emerging by day 16 of the co-culture, and both DP and CD8 single positive (SP) cells are abundant by day 20. The general outcome and robustness of co-culture is greatly dependent on the ability to visually ascertain the completion of the significant developmental turning points that occur. This protocol aims to be a guide to the recognition of these milestones, as well as the other critical parameters, that are key to successful differentiation.

<table border="0" cellpadding="0" cellspacing="0" width="1055">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:355px;">MATERIALS</td>
			<td style="width:127px;">COMPANY</td>
			<td style="width:141px;">CATALOG NUMBER</td>
			<td style="width:432px;">COMMENTS</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Stem cell qualified FBS</td>
			<td>Gemini</td>
			<td>100-125</td>
			<td>Heat inactivated</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Penicillin/Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-alanyl L-glutamine</td>
			<td>Corning</td>
			<td>25-015-CI</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HEPES buffer&#160;</td>
			<td>Millipore</td>
			<td>TMS-003-C</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Non-Essential Amino Acids</td>
			<td>ThermoScientific</td>
			<td>SH30853.01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Gentamicin Regent Solution (50 mg/mL)</td>
			<td>Life Technologies&#160;</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#946;-mercaptoethanol (55 mM) in DPBS</td>
			<td>Life Technologies&#160;</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Filter Unit</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td>0.22&#956;m PES membrane</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell Culture Grade Water</td>
			<td>Corning</td>
			<td>25-055-CM</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#945;-MEM&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>12000-022</td>
			<td>Powder, reconstitute per manufacture recommendation</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">FBS</td>
			<td>ThermoScientific</td>
			<td>SH 30396.03</td>
			<td>Testing of individual lots required&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dimethyl Sulphoxide&#160;</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Recombinant Human Flt-3 Ligand</td>
			<td>R&#38;D Systems</td>
			<td>308-FK</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Recombinant Murine IL-7</td>
			<td>PeproTech</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LIF&#160;</td>
			<td>Millipore</td>
			<td>ESG1107</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Utrapure water with 0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MEFs mitomycin C treated</td>
			<td>Millipore</td>
			<td>PMEF-CF</td>
			<td>Any mitotically arresteded MEFs can be used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DPBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Trypsin EDTA, 1X</td>
			<td>Corning</td>
			<td>25-053-Cl</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell strainer (40 &#956;m)</td>
			<td>Fisher</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue culture dish 100 X 20 mm</td>
			<td>BD Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Multiwell 6-well</td>
			<td>BD Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 ml microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">15 ml centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">50 ml centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">5 ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td>Tubes for FACS&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ES R1 cells</td>
			<td>ATCC</td>
			<td>SCRC-1011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">OP9 cells</td>
			<td></td>
			<td></td>
			<td>Cells can be obtained from the Riken Laboratory Cell Repository (Japan).</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">OP9-DL1 cells</td>
			<td>&#160;&#160;&#160;&#160;</td>
			<td></td>
			<td>Cells can be requested from the Z&#250;&#241;iga-Pfl&#252;cker laboratory.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">FlowJo software&#160;</td>
			<td>Tree Star</td>
			<td></td>
			<td>FACS data analyses</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Flow Cytometer</td>
			<td>BD</td>
			<td></td>
			<td>FACScan, FACSCalibur and FACSVantage have been used in our lab</td>
		</tr>
	</tbody>
</table>

derivation of t cells in vitro from mouse embryonic stem cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

LİF mevcudiyetinde MEF&#39;ler üzerinde yetiştirildiğinde, mESCs farklılaşmamış bir halde muhafaza edilebilir. İdeal koşullar altında, bunlar faz kontrast mikroskopisi parlak bir halosu ile çevrili olan hücrelerin yoğun kolonileri (Şekil 1) olarak görünür. Bu kültürler günlük olarak takip edilmelidir. Hücrelerin birleştiği bağlı olarak, ortam değiştirilebilir ya da hücre ayrılabilir. Komşu Mesc koloniler birbirleri ile temas noktasına gelmemeli. Farklılaşmamış mESCs ...

Watch this Scientific Journal Video about Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells at JoVE.com

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

T hücrelerinin türetilmesi<em&gt; İn Vitro</em&gt; Fare Embriyonik Kök Hücreleri

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

derivation-of-t-cells-in-vitro-from-mouse-embryonic-stem-cells

Research

JoVE Journal

Immunology and Infection

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

10.9K Görüntüleme.  City University of New York. Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Video: Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Fare T-hücrelerinin, T-hücre reseptörleri retroviral İletimi

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

İmmünoloji ve Enfeksiyon

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

Timik Lenfoma T-hücre Hatları türetilmesi Atm - / -</sup Ve P53 - / -</sup Fare

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

İlköğretim İnsan naif ve Bellek T Hücreleri gelen İsteyerek Düzenleyici T Hücreleri Üretimi

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

T Lenfositler doğru İsteyerek Pluripotent Kök Hücrelerinin Yönetmen Farklılaşma

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

Primer insan T hücrelerinin piggyBac Transposon Sistem Modifikasyon

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Klinik Uygulama<em&gt; Sleeping Beauty</emGenetiği Periferik ve Göbek Kordon Kanı alınan T hücreleri Değiştir Sunan Hücreler&gt; ve Yapay Antijen

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Etkin Killer yapay antijen sunan hücreler (KaAPC)<em&gt; İn Vitro</em&gt; İnsan Antijen-spesifik T hücrelerinin tükenmesi

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Periferik Kan İnsan alloantijen-özgü T hücrelerinin üretilmesi

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T hücreleri dendritik hücreler gelen Transinfection tarafından Bakteriler Yakalama

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Bir TGF-β-içeren Protokolü kullanılarak naif CD4 + T hücrelerinden insan CD4 + FoxP3 + Bağlı regülatör T hücreleri (iTregs) in vitro ayrımında

T hücrelerinin türetilmesi

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Fare T-hücrelerinin, T-hücre reseptörleri retroviral İletimi

Timik Lenfoma T-hücre Hatları türetilmesi Atm^{- / - Ve P53^{- / - Fare}}

İlköğretim İnsan naif ve Bellek T Hücreleri gelen İsteyerek Düzenleyici T Hücreleri Üretimi

T Lenfositler doğru İsteyerek Pluripotent Kök Hücrelerinin Yönetmen Farklılaşma

Primer insan T hücrelerinin piggyBac Transposon Sistem Modifikasyon

Klinik Uygulama

Etkin Killer yapay antijen sunan hücreler (KaAPC)

Periferik Kan İnsan alloantijen-özgü T hücrelerinin üretilmesi

T hücreleri dendritik hücreler gelen Transinfection tarafından Bakteriler Yakalama

Bir TGF-β-içeren Protokolü kullanılarak naif CD4 ⁺ T hücrelerinden insan CD4 ⁺ FoxP3 ⁺ Bağlı regülatör T hücreleri (iTregs) in vitro ayrımında

T hücrelerinin türetilmesi

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Fare T-hücrelerinin, T-hücre reseptörleri retroviral İletimi

Timik Lenfoma T-hücre Hatları türetilmesi Atm - / - Ve P53 - / - Fare

İlköğretim İnsan naif ve Bellek T Hücreleri gelen İsteyerek Düzenleyici T Hücreleri Üretimi

T Lenfositler doğru İsteyerek Pluripotent Kök Hücrelerinin Yönetmen Farklılaşma

Primer insan T hücrelerinin piggyBac Transposon Sistem Modifikasyon

Klinik Uygulama

Etkin Killer yapay antijen sunan hücreler (KaAPC)

Periferik Kan İnsan alloantijen-özgü T hücrelerinin üretilmesi

T hücreleri dendritik hücreler gelen Transinfection tarafından Bakteriler Yakalama

Bir TGF-β-içeren Protokolü kullanılarak naif CD4 + T hücrelerinden insan CD4 + FoxP3 + Bağlı regülatör T hücreleri (iTregs) in vitro ayrımında

Timik Lenfoma T-hücre Hatları türetilmesi Atm^{- / - Ve P53^{- / - Fare}}

Bir TGF-β-içeren Protokolü kullanılarak naif CD4 ⁺ T hücrelerinden insan CD4 ⁺ FoxP3 ⁺ Bağlı regülatör T hücreleri (iTregs) in vitro ayrımında