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.

1 Preparazione della Cultura Media, Citochine e piatti gelatinizzato <ol><li> Preparare supporti cellule ES utilizzando Modifica Dulbecco di Eagle Medium (DMEM) con alte concentrazioni di glucosio e piruvato di sodio. Aggiungere 20% ESC Qualificato Fetal Bovine Serum (FBS), 1% di penicillina / streptomicina, 1% L-glutammina, 1% HEPES Buffer, 1% non-aminoacidi essenziali, gentamicina 0,1% (50 mg / ml) e 0,1% ( 55 mM) β-mercaptoetanolo. Sterilizzare supporti cellule ES per filtrazione. </li><li> Preparare supporti OP9 facendo 1 L di Alpha Minimum Essential Medium (α-MEM) da polvere secondo le istruzioni del produttore. Aggiungere 20% di siero fetale bovino (FBS) e 1% di penicillina / streptomicina. Miscelarlo. Sterilizzare per filtrazione in due aliquote da 500 ml. NOTA: è consigliato e rischia di produrre una migliore manutenzione delle cellule OP9 e nei risultati di differenziazione in vitro L&#39;uso di supporti a base di polvere. Questo approccio è diventato la nostra procedura standard. Tuttavia, notiamo anche that abbiamo a volte usato liquido pre-fatte α-MEM con un adeguato successo. </li><li> Preparare il congelamento dei media con l&#39;aggiunta del 10% dimetilsolfossido (DMSO) al 90% FBS. Agitare delicatamente e sterilizzare per filtrazione. Conservare a 4 ° C. </li><li> Preparare 2,000x Flt-3 ligando (10 mg / ml) sciogliendo ricombinante umano Flt-3 ligando (Flt-3L) in mezzi OP9 completo a 10 mcg / ml. Aliquota in provette da 1,5 ml microcentrifuga e conservare a -80 ° C. Seguire le raccomandazioni del fornitore per quanto riguarda la stabilità e lo stoccaggio. NOTA: La stabilità di Flt-3L è breve (1 mese a 4 ° C o 3 mesi a -80 ° C). </li><li> Preparare 1,000x IL-7 (1 mg / ml) utilizzando mezzi OP9 completo. Aliquota in provette da 1,5 ml microcentrifuga e conservare a -80 ° C. Seguire le raccomandazioni del fornitore sulla stabilità e la conservazione (IL-7 è stabile fino a 12 mesi a -80 ° C). </li><li> Preparare 1,000x leucemia inibitorio Factor (LIF) (10 mg / ml) da una diluizione 1:10 del 10 7 unità di LIF in supporti cellule ES. Conservare aliquota a 4 ° C. Assicurarsi di annotare la data di scadenza prevista dal fabbricante sulle fiale aliquote. NOTA: Il prodotto è stabile in forma concentrata o diluita almeno 18 mesi dalla data di produzione. </li><li> Preparare 6 pozzetti gelatinizzati da aggiungere 1,5 ml di 0,1% soluzione di gelatina in ciascun pozzetto di una piastra da 6 pozzetti. Lasciare piatti nella cappa sterile a temperatura ambiente con coperchi su, permettendo almeno 30 minuti per il rivestimento. I piatti possono anche essere lasciati in un incubatore umidificato durante la notte. Rimuovere eventuali residui di soluzione di gelatina a destra prima semina delle cellule. Non lasciate che la soluzione di gelatina completamente asciugare nel pozzo. </li></ol> 2 Preparazione e manutenzione di cellule di alimentazione e di cellule staminali embrionali di topo (Mesc) NOTA: Incubare tutte le celle in un umidificata 37 ° C incubatore con il 5% di CO 2. <ol><li> Scongelamento fibroblasti embrionali di topo (MEF) <ol><li> Quick-scongelare una fiala congelata (~ 5 x 10 6 </sup&gt; cellule / fiala) di mitomicina C-trattati (o in altro modo mitoticamente arrestato MEF) e trasferirli in una provetta da 15 ml. </li><li> Aggiungere 8 ml di mezzi di cellule staminali alle cellule scongelati, in modo goccia a goccia per diluire gradualmente il DMSO dal mezzo congelamento. </li><li> Centrifugare le cellule a 400 x g a 4 ° C per 5 min. Rimuovere con cautela il surnatante e risospendere il pellet cellulare in 3 ml di media cellulare ES. </li><li> Preparare una provetta da centrifuga da 50 ml con 33 ml di supporti pre-riscaldato cellule ES. Aggiungere 3 ml di MEF risospeso a portare il volume finale di 36 ml. </li><li> Distribuire 3 ml del MEF risospeso in ciascun pozzetto di due piastre gelatinizzato 6 pozzetti. Posizionare le piastre in incubatore per almeno sei ore per consentire alle cellule di fissare e sparsi prima della semina i mESCs su di loro. Questi strati alimentatore mitoticamente arrestati potrebbero essere utilizzabili per diversi giorni dopo la semina iniziale, se i loro media viene cambiato ogni 2-3 giorni. NOTA: MEF di recente arrestati possono anche essere utilizzati. </li> &lt;/ Ol&gt; </li><li> MESCs Semina <ol><li> Quick-scongelare un flaconcino con un clone Mesc congelato nel bagno di 37 ° C l&#39;acqua e preparare le celle come descritto in 2.1.1-2.1.3. NOTA: congelare 2 fiale di Mesc da un pozzo confluente di un 6-pozzetti. </li><li> Rimuovere i supporti da un pozzetto (per Mesc clone) del 6 pozzetti con monostrati MEF arrestati (preparati nel passaggio 2.1.5) e seminare il 3 ml di mESCs sulla parte superiore del monostrato MEF. Aggiungere 3 ml di 1,000x LIF (10 ng / ml). Piatti Rientro in incubatrice. </li></ol></li><li> ES manutenzione delle cellule e il passaggio tripsina mediata NOTA: Modificare supporti quotidiani, o Spalato sulla base di confluenza di Mesc. In modo ottimale, la raccolta e split / re-piastra le cellule a giorni alterni. <ol><li> Rimuovere i supporti di cellule ES e lavare mESCs con 2 ml di PBS. Rimuovere PBS. Aggiungere 1 ml di 0,25% tripsina e incubare a 37 ° C per 3-5 min. </li><li> Raccogliere le cellule trypsinized e trasferirli in una provetta da centrifuga 15 ml. Pipettare vigorosamente la sospensione cellulare per rompere ciuffi di mCES. Aggiungere 2 ml di completo supporto cellula ES alla sospensione cellulare per neutralizzare la tripsina. </li><li> Cellule Spin a 400 x g per 5 minuti a 4 ° C. Rimuovere il surnatante e risospendere il pellet in 3 ml ES supporto cellulare. </li><li> Rimuovere i supporti da 6 pozzetti contenenti preparati monostrati MEF arrestati. Aggiungere la quantità appropriata di sospensione cellulare (in base al rapporto di divisione desiderato) in 3 ml di supporti cellule ES in ciascun pozzetto per essere seminati. Aggiungere 3 ml di 1,000x LIF. Un confluenti Mesc ben diviso 1: 6 sarà pronto per il passaggio in 2 giorni. </li></ol></li><li> OP9 / mantenimento delle cellule OP9-DL1 <ol><li> Scongelare una fiala di cellule OP9 (seguire la procedura 2.1.1-2.1.3 utilizzando supporti OP9) </li><li> Aggiungere 7 ml OP9 media per 10 centimetri piatto di coltura di tessuti. Aggiungere 3 ml di cellule risospese in maniera goccia a goccia, la distribuzione di cellule in tutta la piastra. Posizionare le cellule durante la notte in incubatrice. </li><li> Controllare la confluenza delle cellule OP9 il giorno successivo. Se il piatto contiene molte cellule galleggianti morte, remove media e aggiungere 10 ml di media OP9 fresco. Riportare il piatto per l&#39;incubatore se quasi piena confluenza non si osserva. Split (utilizzando tripsina) 1: 4 prossimo monostrato confluente OP9. NOTA: Le piastre devono raggiungere di nuovo allo stesso livello confluenza in circa 2 giorni. Fare attenzione a non lasciare che le cellule OP9 diventare over-confluenti. </li><li> Congelare i primi passaggi di cellule OP9 come scorte di espansione. NOTA: congelare 2 fiale di cellule OP9 da un vicino confluenti 10 centimetri piatto. Ogni flaconcino di espansione magazzino può essere ulteriormente propagato per creare scorte di lavoro che vengono poi utilizzati in Mesc esperimenti di co-coltura. Tenere tutti gli stock OP9 congelati in azoto liquido piuttosto che nel -80 ° C freezer, dal momento che le temperature fluttuanti possono influenzare negativamente la qualità delle blocca. </li></ol></li></ol> 3. OP9-DL1 Co-cultura Procedura <ol><li> Preparazioni di co-coltura <ol><li> Circa una settimana prima dell&#39;inizio di una co-coltura, scongelare MEF, mESCs e sto lavorando cellulare OP9CKS. Poiché le cellule OP9-DL1 non sono necessari fino a co-coltura giorno 8, scongelare le cellule OP9-DL1 durante i primi tre giorni dopo co-coltura iniziazione. Mantenere o congelare tutte le cellule, se necessario. </li><li> Determinare il numero iniziale di 10 cm piastre con monostrati cellulari OP9 pronti a raggiungere l&#39;80% di confluenza in co-coltura giorno 0 in base alla scala dell&#39;esperimento (ad esempio, il numero di cloni Mesc per differenziare e il numero di co-coltura punti di tempo a da analizzare). Per preparare una co-coltura, diviso nei pressi di piatto confluenti di cellule OP9 tra 1: 4 e 1: 6 due giorni prima di quando saranno necessari i monostrati. NOTA: Uno sarà bisogno di almeno una piastra per ogni ESC clone. Tipicamente, un singolo clone ESC seminato su un singolo piatto (come descritto sotto) al giorno 0 dovrebbe produrre abbastanza cellule per seminare sei piatti al giorno 5 di passaggio. Ogni piastra giorno 5 consentirà un certo punto volta successiva analisi. </li><li> Dal monostrati OP9 saranno continuamente necessari per il passaggio co-coltura, aver cura di sempre maintain un numero adeguato di ulteriori piastre di coltura OP9 in parallelo con la co-coltura. </li></ol></li><li> Giorno 0: Avvio di co-coltura <ol><li> Raccogliere Mesc come in 2.3.1-2.3.4. Contare le cellule. </li><li> Per ogni clone Mesc preparare 5 x 10 4 Mesc in 10 ml di OP9 supporti per piastra. NOTA: Anche se non abbiamo usato, notiamo che un mezzo alternativo composto da α-MEM, 10% FBS, e 5 x 10 -5 M β-mercaptoetanolo è stato segnalato anche per l&#39;uso in differenziazione co-colture 10. </li><li> Rimuovere vecchi media da OP9 piatti e aggiungere 10 ml di sospensione Mesc ai piatti avendo cura di distribuire le cellule in modo uniforme in tutta la piastra. Porre le capsule nella C incubatore 37 °. </li></ol></li><li> 3 ° giorno: Cambio media <ol><li> Togliere i piatti dal termostato e osservare al microscopio. Colonie dovrebbero cominciare a perdere lucentezza e appaiono appiattite. </li><li> Rimuovere i supporti dai piatti e aggiungere delicatamente 10 ml di fresca OP9media. </li><li> Restituire i piatti co-coltura per l&#39;incubatrice. Visivamente monitorare la formazione di colonie mesoderma-come ogni giorno per informare i tempi di preparazione OP9 alimentatore monostrato per il passaggio successivo (giorno 5), che non deve essere effettuata fino a ~ 80-90% delle colonie visualizzare visivamente mesoderma simile morfologia. Massima rinvio della fase di trasferimento di cellule giorno 5 è di 2 giorni. </li></ol></li><li> Giorno 5: tripsina passaggio mediato con pre-placcatura. NOTA: Preparare in anticipo un numero adeguato di 10 cm piatti con le cellule OP9 ottimale confluenti. Procedere con le fasi successive solo se le co-colture mostrano visivamente le caratteristiche descritte al punto 3.3.3 (vedi risultati anche Rappresentante). <ol><li> Rimuovere i supporti dai piatti di co-coltura. Aggiungere 4 ml di PBS per lavare. Agitare il PBS e rimuovere. Aggiungere 4 ml di 0,25% tripsina e posizionare i piatti in incubatrice per ~ 5 min. </li><li> Interrompere lo strato di cellule trypsinized di pipettaggio vigoroso, facendo attenzione a non introdurre eccessive bolle d&#39;aria, fino a quando un, sospensione di cellule per lo più unico omogeneo si ottiene. </li><li> Aggiungere 4 ml di media OP9 completi e mescolare pipettaggio. Trasferire le cellule in un nuovo, piatto 10 centimetri vuoto e posto in incubatrice per 30 minuti per permettere alle cellule OP9 di aderire al piatto. Questa fase &quot;pre-plating&quot; riduce il numero di cellule OP9 trasferiti alla fase successiva di co-coltura. </li><li> Preparare 50 ml provette con filtri di cella di 40 micron. </li><li> Raccogliere le cellule non aderenti dal piatto pre-plated e farli passare attraverso il filtro delle cellule nel tubo. Lavare il piatto delicatamente con 6 ml di PBS e farla passare attraverso lo stesso filtro, lasciando le cellule OP9 attaccate dietro. </li><li> Centrifugare le cellule a 400 x g 5 minuti a 4 ° C. Rimuovere il surnatante e sospendere nuovamente le cellule pellet in 3 ml di media OP9 completo. Contare le cellule. </li><li> Rimuovere i supporti da 10 cm piastre con cellule OP9 ottimale confluenti. </li><li> Per ogni punto di tempo di analisi, seminare 5 x 10 5 cellule in mono OP9strato in 10 ml di volume finale. </li><li> Aggiungere 5 ng / ml umana ricombinante Flt-3L e posizionare i piatti in incubatrice. </li></ol></li><li> 8 ° giorno: emopoietiche progenitrici delle cellule (HPC) di raccolta NOTA: Preparare in anticipo un numero adeguato di 6 pozzetti con monostrati OP9 o OP9-DL1 cellule in modo che essi saranno in ~ 80% confluenti in tempo per questa fase. <ol><li> Preparare 50 ml provette da centrifuga con 40 micron filtri. </li><li> Togliere i piatti dal termostato e osservare al microscopio. Cluster Shiny di HPC devono essere liberamente fissati alla monostrato OP9. Raccogliere il maggior numero di queste cellule come possibile lavando (ma non eccessivamente interrompere) il monostrato OP9 con una pipetta e la media esistenti sul piatto. Per evitare la formazione di schiuma, lasciare sempre almeno 1 ml di media nella punta della pipetta durante questa collezione HPC / fase di lavaggio. </li><li> Far passare le cellule attraverso il filtro 40 micron in un tubo. Aggiungere 8 ml di PBS alla stessa piastra e controllare al microscopio se tutti HPC noiri raccolti. Ripetere la fase di lavaggio / collezione con PBS e (se necessario) con il lavaggio più forte. Scolare le cellule in provetta già contenente le cellule dallo stesso piatto. </li><li> Centrifugare le cellule a 400 x g per 5 minuti a 4 ° C. </li><li> Preparare una master mix di 3 ml (per piastra seminata il giorno 5), dei mezzi di comunicazione OP9 con 5 ng / ml Flt-3L e 1 ng / ml di IL-7. </li><li> Rimuovere il surnatante e risospendere pellet in mezzi OP9 con Flt-3L + IL-7 Master Mix (3 ml per ogni piatto 10 centimetri trasformati). Trasferire le cellule raccolte da ogni piatto in un pozzetto di una piastra da 6 pozzetti con cellule OP9. <ol><li> Al fine di guidare le cellule verso monociti, cellule B o lignaggi eritroidi, seminare le cellule raccolte sulle cellule OP9. Per ricavare cellule T, seminare le cellule su monostrati di cellule OP9-DL1. </li></ol></li><li> Posizionare le piastre da 6 pozzetti in incubatrice. </li></ol></li><li> Giorno 10: Cambio media <ol><li> Preparare una provetta da centrifuga da 15 ml per ogni distinto mesc clone-alimentatore tipo di cellula combinazionizione in co-coltura. </li><li> Preparare una master mix di mezzi OP9 con 5 ng / ml di Flt-3L e 1 ng / ml di IL-7. Preparare 3 ml per ogni bene. </li><li> Raccogliere delicatamente i media da ciascun pozzetto della piastra da 6 pozzetti che combina supporti da più pozzetti contenenti la stessa combinazione di tipo cellulare ESC clone-alimentatore. </li><li> Aggiungere 2 ml di master mix multimediale OP9 (vedi 3.6.2) in ogni pozzetto. </li><li> Centrifugare la media raccolti a 400 x g per 5 minuti a 4 ° C. Rimuovere il surnatante e risospendere ogni pellet (molto piccola se visibile) in 1 ml di OP9 master mix dei media (vedi 3.6.2) per pozzetto originariamente raccolti nella fase 3.6.3. </li><li> Distribuire 1 ml di cellule di nuovo in ogni pozzetto da cui il supporto è stato originariamente raccolti portando il volume in ogni pozzetto a 3 ml. Riportare i piatti per l&#39;incubatrice. </li></ol></li><li> Giorno 12: Nessun passaggio tripsina NOTA: Preparare in anticipo un numero adeguato di piatti 6 pozzetti con cellule OP9 o OP9-DL1 in modo che essi saranno in modo ottimale confluenti in tempo per questo step. Giorno 12 è ideale per la citometria di flusso analisi di sviluppo eritroide, monociti e cellule T early stage. <ol><li> Preparare una master mix (3 ml per ogni bene che continuerà in co-coltura) dei media OP9 con 5 ng / ml di Flt-3L e 1 ng / ml di IL-7. </li><li> Preparare 50 ml provette da centrifuga con 40 micron filtri (uno per ogni ESC clone-alimentatore tipo di cellula combinazione in co-coltura). </li><li> Pipettare vigorosamente i media esistenti in ciascun pozzetto di disaggregare tutte le cellule (tra cui il monostrato) nel pozzo. Continuare con pipettaggio forte fino a raggiungere uno stato simile ad una sospensione di cellule singole. </li><li> Passare le cellule raccolte attraverso il filtro nel tubo. Aggiungere 3 ml di PBS in ogni pozzetto per lavare e raccogliere tutte le cellule rimanenti. Passare cellule attraverso lo stesso filtro. Combinare le cellule da più pozzetti contenenti la stessa combinazione di tipo cellulare ESC clone-alimentatore. </li><li> Eliminare il filtro cella utilizzata e rimuovere un equivalente aliquota da un pozzetto (~ 6 ml) per ogni desideratocitometria a flusso (o altro) analisi da effettuare in quel giorno. Conservare queste aliquote sul ghiaccio prima dell&#39;uso. </li><li> Centrifugare le cellule a 400 x g per 5 minuti a 4 ° C. Rimuovere il surnatante. Risospendere il pellet dai tubi centrifuga da 50 ml a 3 ml di Master Mix per pozzetto di essere ri-seminato. Aggiungere 3 ml per pozzetto di ciascuna sospensione cellulare al tipo di cellula alimentatore appropriato e mettere i piatti in incubatrice. </li></ol></li><li> Giorno 14: Cambio media <ol><li> Seguire la procedura descritta nella sezione 3.6. </li></ol></li><li> Giorno 16: Nessun passaggio tripsina NOTA: Preparare in anticipo piatti da 6 pozzetti di cellule OP9 o OP9-DL1 confluenti in modo ottimale per questo passo. NOTA: Giorno 16 è ideale per citometria a flusso di analisi dei linfociti B e le cellule T fase centrale. <ol><li> Seguire la procedura descritta nella sezione 3.7. </li></ol></li><li> Giorno 18: Cambio media <ol><li> Seguire la procedura descritta nella sezione 3.6. </li></ol></li><li> Giorno 20: Nessun passaggio tripsina NOTA: Giorno 20 is ideale per citometria a flusso analisi di metà a fine fase di sviluppo le cellule T. <ol><li> Seguire la procedura descritta nella sezione 3.7. Se lo si desidera, effettuare differenziazione delle cellule passato giorno 20 cambiamento alternata media e non tripsina passaggi ogni due giorni, con monitoraggio giornaliero del tasso di espansione dei linfociti. </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.

Il sistema OP9-DL1 co-coltura è stata utilizzata per studiare il ruolo dei vari prodotti genici durante lo sviluppo di tipi di cellule del sangue da cellule staminali. 8,12,13 Ha anche dimostrato un modello efficace per studiare la funzione del gene DNA normativo durante il differenziamento cellulare. 9,14 Usando questo approccio come alternativa ai modelli interi mouse possono produrre un notevole risparmio di tempo e costi di esperimenti che affrontano molte questioni fondamentali in ematopoiesi...

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.

Quando coltivate su MEF, in presenza di LIF, mESCs possono essere mantenuti in uno stato indifferenziato. In condizioni ideali, appaiono come colonie compatti di cellule circondate da un alone lucido in microscopia a contrasto di fase (Figura 1). Tali colture devono essere monitorati quotidianamente. In base alla confluenza delle cellule, i media possono essere cambiati o cellule possono essere divise. Limitrofe colonie Mesc non dovrebbero venire al punto di contatto uno con l&#39;altro. Un normale, san...

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.

Derivazione di cellule T<em&gt; In Vitro</em&gt; Da cellule staminali embrionali di topo

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 Views.  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

Trasduzione retrovirale di recettori delle cellule T nel topo le cellule T

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 ...

Ricerca

Immunologia e infezioni

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.

Derivazione di timica linfoma a cellule T Linee da Atm - / -</sup E P53 - / -</sup Mouse

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&amp;#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.

Generazione di cellule T regolatrici indotte da cellule primarie umane T naive e memoria

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.

Differenziazione Regia di cellule staminali pluripotenti indotte verso Linfociti T

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

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.

Applicazione clinica della<em&gt; Bella Addormentata</em&gt; Artificiale e cellule presentanti l&#39;antigene per modificare geneticamente le cellule T da sangue del cordone ombelicale e periferico

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 ...

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.

Cellule T Capture batteri da Transinfection da cellule dendritiche

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.

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

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.

La differenziazione in vitro di cellule CD4 + umana FOXP3 + indotta da T regolatorie (iTregs) da cellule T CD4 Naïve utilizzando un protocollo TGF-β contenenti

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

In Vitro Differenziazione dei Mouse del Granulocyte-macrofago-colony-stimulating Factor (GM-CSF)-producendo le cellule T Helper (THGM)

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Purificazione ed espansione di cellule T Natural Killer invarianti di topo per studi in vitro e in vivo

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4&#945; nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

Un metodo efficiente per l'induzione di cellule simili a epatociti diretti da cellule staminali embrionali umane

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug ...