Name: derivation of t cells in vitro from mouse embryonic stem cells
Uploaded: 2014-10-14T15:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells at JoVE.com

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>

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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><tbody><tr><td>DMEM</td><td>Corning</td><td>15-013-CV</td><td></td></tr><tr><td>Stem cell qualified FBS</td><td>Gemini</td><td>100-125</td><td>Heat inactivated</td></tr><tr><td>Penicillin/Streptomycin</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>L-alanyl L-glutamine</td><td>Corning</td><td>25-015-CI</td><td></td></tr><tr><td>HEPES buffer&amp;nbsp;</td><td>Millipore</td><td>TMS-003-C</td><td></td></tr><tr><td>Non-Essential Amino Acids</td><td>ThermoScientific</td><td>SH30853.01</td><td></td></tr><tr><td>Gentamicin Regent Solution (50 mg/ml)</td><td>Life Technologies&amp;nbsp;</td><td>15750-060</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol (55 mM) in DPBS</td><td>Life Technologies&amp;nbsp;</td><td>21985-023</td><td></td></tr><tr><td>Filter Unit</td><td>Millipore</td><td>SCGPU05RE</td><td>0.22 &amp;mu;m PES membrane</td></tr><tr><td>Cell Culture Grade Water</td><td>Corning</td><td>25-055-CM</td><td></td></tr><tr><td>&amp;alpha;-MEM&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>12000-022</td><td>Powder, reconstitute per manufacture recommendation</td></tr><tr><td>Sodium bicarbonate&amp;nbsp;</td><td>Sigma</td><td>S5761-500G</td><td></td></tr><tr><td>FBS</td><td>ThermoScientific</td><td>SH 30396.03</td><td>Testing of individual lots required&amp;nbsp;</td></tr><tr><td>Dimethyl Sulphoxide&amp;nbsp;</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Recombinant Human Flt-3 Ligand</td><td>R&amp;amp;D Systems</td><td>308-FK</td><td></td></tr><tr><td>Recombinant Murine IL-7</td><td>PeproTech</td><td>217-17</td><td></td></tr><tr><td>LIF&amp;nbsp;</td><td>Millipore</td><td>ESG1107</td><td></td></tr><tr><td>Utrapure water with 0.1% gelatin</td><td>Millipore</td><td>ES-006-B</td><td></td></tr><tr><td>MEFs mitomycin C treated</td><td>Millipore</td><td>PMEF-CF</td><td>Any mitotically arresteded MEFs can be used</td></tr><tr><td>DPBS</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Cell strainer (40 &amp;mu;m)</td><td>Fisher</td><td>22363547</td><td></td></tr><tr><td>Tissue culture dish 100 x 20 mm</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>Multiwell 6-well</td><td>BD Falcon</td><td>353046</td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>BD Falcon</td><td>352096</td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>BD Falcon</td><td>352070</td><td></td></tr><tr><td>5 ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap</td><td>BD Falcon</td><td>352235</td><td>Tubes for FACS&amp;nbsp;</td></tr><tr><td>ES R1 cells</td><td>ATCC</td><td>SCRC-1011</td><td></td></tr><tr><td>OP9 cells</td><td></td><td></td><td>Cells can be obtained from the Riken Laboratory Cell Repository (Japan).</td></tr><tr><td>OP9-DL1 cells</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td>Cells can be requested from the Z&amp;uacute;&amp;ntilde;iga-Pfl&amp;uuml;cker laboratory.</td></tr><tr><td>FlowJo software&amp;nbsp;</td><td>Tree Star</td><td></td><td>FACS data analyses</td></tr><tr><td>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

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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

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

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

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

Ricerca

Biologia

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 ...

Immunologia e infezioni

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Caratterizzazione di timica assestamento Progenitori nell&#39;embrione mouse Uso<em&gt; In Vivo</em&gt; E<em&gt; In Vitro</em&gt; Saggi

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Biologia dello sviluppo

Derivation of Human Embryonic Stem Cells by Immunosurgery

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


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

Derivazione di cellule staminali embrionali umane da Immunosurgery

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

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

Un sistema di coltura timica tridimensionale per generare emigrati timici derivati da murine

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Ricerca sul cancro

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Derivazione di linee di cellule staminali da embrioni di Preimplantation Mouse

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Mouse Naïve CD4<sup&gt; +</sup&gt; Isolamento T cellulare e<em&gt; In vitro</em&gt; Differenziazione in T sottopopolazioni cellulari

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Derivazione di cellule staminali trofoblasto mouse da blastocisti

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

<meta charset="utf8"></head><body>Differenziazione cellulare Th17 da cellule T CD4+ naive mediante citometria a flusso

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

<meta charset="utf8"></head><body>Autore in primo piano: Ottenere la differenziazione in vitro ad alta purezza delle cellule Th17 utilizzando la modulazione della concentrazione di citochine

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

Differenziazione in vitro di cellule T CD4+ naive in cellule Th17 patogene nel topo

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

Derivazione di cellule T

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

Caratterizzazione di timica assestamento Progenitori nell'embrione mouse Uso

Derivazione di cellule staminali embrionali umane da Immunosurgery

Un sistema di coltura timica tridimensionale per generare emigrati timici derivati da murine

Derivazione di linee di cellule staminali da embrioni di Preimplantation Mouse

Mouse Naïve CD4

Derivazione di cellule staminali trofoblasto mouse da blastocisti

Generazione di topi derivati da cellule staminali pluripotenti indotte

Differenziazione in vitro di cellule T CD4⁺ naive in cellule Th17 patogene nel topo

Derivazione di cellule T

Riepilogo

Esplora Altri Video

Capitoli in questo video

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

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

Caratterizzazione di timica assestamento Progenitori nell'embrione mouse Uso

Derivazione di cellule staminali embrionali umane da Immunosurgery

Un sistema di coltura timica tridimensionale per generare emigrati timici derivati da murine

Derivazione di linee di cellule staminali da embrioni di Preimplantation Mouse

Mouse Naïve CD4

Derivazione di cellule staminali trofoblasto mouse da blastocisti

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

Differenziazione in vitro di cellule T CD4+ naive in cellule Th17 patogene nel topo

Generazione di topi derivati da cellule staminali pluripotenti indotte

Differenziazione in vitro di cellule T CD4⁺ naive in cellule Th17 patogene nel topo