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 Préparation de milieux de culture, cytokines et plaques GELATINISE <ol><li> Préparer support de cellules ES en utilisant la modification de Dulbecco du milieu de Eagle (DMEM) avec l&#39;hyperglycémie et du pyruvate de sodium. Ajouter 20% ESC Qualifié de sérum bovin fœtal (FBS), 1% pénicilline / streptomycine, 1% de L-glutamine, 1% tampon HEPES, acides aminés 1% non essentiels, 0,1% de gentamicine (50 mg / ml) et 0,1% ( 55 pM) β-mercaptoéthanol. Stériliser les médias de cellules ES par filtration. </li><li> Préparation de supports en faisant OP9 1 L de milieu essentiel minimum alpha (α-MEM) de poudre selon les instructions du fabricant. Ajouter 20% de sérum bovin fœtal (FBS) et 1% de pénicilline / streptomycine. Mélanger par inversion. Stériliser par filtration en deux aliquotes de 500 ml. NOTE: L&#39;utilisation des médias à base de poudre est recommandé et susceptible de donner un meilleur entretien des cellules OP9 et dans les résultats de la différenciation in vitro. Cette approche est devenue notre procédure standard. Cependant, nous notons aussi that nous avons parfois utilisé liquides pré-faites α-MEM avec succès suffisant. </li><li> Préparation des milieux de congélation en ajoutant 10% de diméthylsulfoxyde (DMSO) à 90% de FBS. Agiter doucement et stériliser par filtration. Conserver à 4 ° C. </li><li> Préparer 2,000x ligand Flt-3 (10 ng / ml) en dissolvant recombinant ligand Flt-3 humain (Flt-3L) dans des milieux de OP9 complète à 10 pg / ml. Aliquote de 1,5 ml dans des microtubes et conserver à -80 ° C. Suivez les recommandations du fournisseur en ce qui concerne la stabilité et le stockage. REMARQUE: La stabilité de Flt-3L est court (1 mois à 4 ° C ou 3 mois à -80 ° C). </li><li> Préparer 1,000x IL-7 (1 ug / ml) en utilisant le support de OP9 complète. Aliquote de 1,5 ml dans des microtubes et conserver à -80 ° C. Suivre les recommandations du fournisseur sur la stabilité et le stockage (IL-7 est stable jusqu&#39;à 12 mois à -80 ° C). </li><li> Préparer facteur inhibiteur 1,000x leucémie (LIF) (10 pg / ml) par une dilution 1:10 de 10 7 unités de LIF in médias de cellules ES. aliquote de magasin à 4 ° C. N&#39;oubliez pas de noter la date d&#39;expiration prévue par le fabricant sur les flacons de parts. NOTE: Ce produit est stable dans la forme concentrée ou diluée au moins 18 mois à compter de la date de fabrication. </li><li> Préparer des plaques de 6 puits par gélatinisé ajouter 1,5 ml de solution à 0,1% de gélatine dans chaque puits d&#39;une plaque à 6 puits. Laisser la vaisselle dans la hotte stérile à température ambiante avec des couvercles sur les, ce qui permet au moins 30 min pour le revêtement. Les plats peuvent également être laissés dans un incubateur humidifié nuit. Éliminer toute solution de gélatine restes juste avant l&#39;ensemencement des cellules. Ne laissez pas la solution de gélatine sécher complètement dans le puits. </li></ol> 2 Préparation et maintenance de cellules nourricières et de la souris de cellules souches embryonnaires (MESC) Remarque: toutes les cellules à incuber dans un humidifié à 37 ° C incubateur avec 5% de CO 2. <ol><li> Décongélation fibroblastes embryonnaires de souris (MEF) <ol><li> Rapide décongeler un flacon congelé (~ 5 x 10 6 </sup&gt; cellules / flacon) de mitomycine C traités (ou non) la mitose arrêté MEF et les transfèrent à un tube de 15 ml. </li><li> Ajouter 8 ml de milieu de cellules ES à des cellules décongelées, dans un mode de goutte à goutte à diluer progressivement le DMSO du milieu de congélation. </li><li> Centrifuger les cellules à 400 x g à 4 ° C pendant 5 min. Retirez délicatement le surnageant et remettre en suspension le culot cellulaire dans 3 ml de milieu de cellules ES. </li><li> Préparer un tube de centrifugation de 50 ml avec 33 ml de milieu préchauffé cellule ES. Ajouter 3 ml de MEF remis en suspension pour amener le volume final à 36 ml. </li><li> Distribuer les 3 ml de MEF remis en suspension dans chaque puits de deux plaques de 6 puits gélatinisé. Placer les plaques dans l&#39;incubateur pendant au moins six heures pour permettre aux cellules de se fixer et se propagent avant l&#39;ensemencement des mESCs sur eux. Ces couches nourricières mitotiquement arrêtés peuvent être utilisables pendant plusieurs jours après l&#39;ensemencement initial, si leurs médias sont changés tous les 2-3 jours. NOTE: MEF fraîchement arrêtés peuvent également être utilisés. </li> &lt;/ Ol&gt; </li><li> MESCs semis <ol><li> Quick-dégel d&#39;un flacon avec un clone MESC congelé dans un bain d&#39;eau à 37 C °, et de préparer les cellules comme décrit dans 2.1.1-2.1.3. NOTE: Nous geler 2 flacons de MESC d&#39;un puits confluent d&#39;une plaque à 6 puits. </li><li> Retirez le support d&#39;un puits (par MESC clone) de la plaque à 6 puits avec des monocouches arrêtées MEF (préparé dans l&#39;étape 2.1.5) et ensemencer 3 ml de la mESCs au-dessus de la monocouche MEF. Ajouter 3 pi de 1,000x LIF (10 ng / ml). Retour plats dans l&#39;incubateur. </li></ol></li><li> L&#39;entretien des cellules ES et de la trypsine médiation passage REMARQUE: les médias changent tous les jours, ou division sur la base confluent de MESC. Idéalement, la récolte et intermédiaires / re-plaque les cellules tous les deux jours. <ol><li> Retirez le support de cellules souches embryonnaires et laver mESCs avec 2 ml de PBS. Retirer du PBS. Ajouter 1 ml de trypsine à 0,25% et on incube à 37 ° C pendant 3-5 min. </li><li> Recueillir les cellules traitées à la trypsine et les transférer dans un tube à centrifuger de 15 ml. Vigoureusement pipette la suspension de cellules pour briser les mottes de mCES. Ajouter 2 ml de milieu complet de cellules ES à la suspension de cellules de neutraliser la trypsine. </li><li> cellules de tourner à 400 x g pendant 5 min à 4 ° C. Retirer le surnageant et remettre en suspension le culot dans 3 ml ES médias cellulaire. </li><li> Retirez le support de plaques de 6 puits contenant des monocouches préparés MEF arrêtés. Ajouter la quantité appropriée de la suspension de cellules (sur la base du rapport de division souhaité) dans 3 ml de milieu de cellules ES à chaque puits pour ensemencer. Ajouter 3 pi de 1,000x FRV. Un confluent MESC bien divisé 1: 6 sera prêt pour le passage en 2 jours. </li></ol></li><li> OP9 / entretien des cellules OP9-DL1 <ol><li> Décongeler une fiole de cellules OP9 (suivez les étapes 2.1.1-2.1.3 l&#39;utilisation des médias OP9) </li><li> Ajouter 7 ml de milieu de OP9 à boîte de 10 cm pour culture de tissus. Ajouter 3 ml de cellules remises en suspension d&#39;une manière goutte à goutte, la distribution des cellules tout au long de la plaque. Placer les cellules dans l&#39;incubateur pendant une nuit. </li><li> Vérifiez la confluence des cellules OP9 le lendemain. Si le plat contient de nombreuses cellules flottantes morts, remove les médias et ajouter 10 ml de milieu de OP9 frais. Remettre le plat dans l&#39;incubateur si presque pleine confluence n&#39;est pas observée. Split (utilisant de la trypsine) 1: 4 la monocouche près de la confluence de OP9. NOTE: Les plaques doivent encore atteindre un même niveau de confluence dans environ 2 jours. Prenez soin de ne pas laisser les cellules OP9 devenir plus confluentes. </li><li> Geler les premiers passages de cellules OP9 que les stocks d&#39;expansion. NOTE: Nous geler 2 flacons de cellules OP9 d&#39;un proche confluence de 10 cm. Chaque flacon d&#39;expansion stock peut être encore propagée à constituer des stocks de travail qui sont ensuite utilisés dans Mesc expériences de co-culture. Gardez tous les stocks de OP9 congelés dans l&#39;azote liquide plutôt que dans le congélateur à -80 ° C, car les fluctuations de température peuvent influer négativement sur la qualité des gels. </li></ol></li></ol> 3. OP9-DL1 Co-culture procédure <ol><li> Préparations de co-culture <ol><li> Environ une semaine avant le début d&#39;une co-culture, décongeler MEF, mESCs et sto cellule OP9 travailcks. Comme les cellules OP9-DL1 ne sont pas nécessaires jusqu&#39;au jour de co-culture 8, décongeler les cellules OP9-DL1 pendant les trois premiers jours après le début de co-culture. Maintenir ou geler toutes les cellules que nécessaire. </li><li> Déterminer le nombre initial de plaques de 10 cm avec des monocouches de cellules OP9 préparés pour atteindre 80% de confluence sur la co-culture jour 0 fondé sur le barème de l&#39;expérience (par exemple, le nombre de clones Mesc à être différencié et le nombre de co-culture des moments de analyser). Pour se préparer à une co-culture, diviser près plat de confluence des cellules OP9 entre 1: 4 et 1: 6, deux jours avant lorsque les monocouches seront nécessaires. REMARQUE: Il faudra au moins une plaque par ESC clone. En règle générale, un seul clone ESC ensemencée sur une seule plaque (comme décrit ci-dessous) au jour 0 devrait donner suffisamment de cellules pour ensemencer six plaques à la journée 5 passage. Chaque plaque jour 5 permettra un ultérieur point de temps d&#39;analyse. </li><li> Depuis monocouches OP9 seront continuellement nécessaires pour le passage de co-culture, prenez soin de toujours maintadans un nombre suffisant de plaques de culture de OP9 supplémentaires en parallèle avec la co-culture. </li></ol></li><li> Jour 0: Initiation de co-culture <ol><li> Recueillir MESC comme dans 2.3.1-2.3.4. Compter les cellules. </li><li> Pour chaque clone MESC préparer 5 x 10 4 MESC dans 10 ml de milieu par boîte de OP9. NOTE: Bien que nous n&#39;avons pas utilisé, nous notons qu&#39;un moyen alternatif consistant en α-MEM, 10% de FBS, et 5 x 10 -5 M β-mercaptoéthanol a également été signalé pour une utilisation dans la différenciation des co-cultures 10. </li><li> Enlevez le vieux médias de OP9 plats et ajouter les 10 ml de suspension MESC les plats en prenant soin de distribuer les cellules uniformément dans toute la plaque. Placez les plats à la 37 ° C incubateur. </li></ol></li><li> Jour 3: Changement de média <ol><li> Retirer les plats de l&#39;incubateur et observer au microscope. Colonies devraient commencer à perdre brillance et semblent aplatie. </li><li> Retirez le papier de plats et ajouter doucement 10 ml de frais OP9médias. </li><li> Retour aux plats de co-culture de l&#39;incubateur. Contrôler visuellement la formation de colonies mésoderme comme tous les jours pour informer le calendrier de préparation OP9 alimentation monocouche pour le prochain passage (jour 5), qui ne devrait pas être effectuée jusqu&#39;à ~ 80-90% des colonies afficher visuellement mésoderme comme morphologie. Report maximum de cinq jours l&#39;étape de transfert de cellule est de 2 jours. </li></ol></li><li> Jour 5: trypsine passage médiation avec pré-placage. REMARQUE: Préparez à l&#39;avance un nombre suffisant de boîtes de 10 cm avec des cellules OP9 optimale confluentes. Procédez aux étapes suivantes que si les co-cultures afficher visuellement les caractéristiques décrites à l&#39;étape 3.3.3 (voir aussi résultats représentatifs). <ol><li> Retirez le support de la vaisselle de co-culture. Ajouter 4 ml de PBS à laver. Agiter le PBS et l&#39;enlever. Ajouter 4 ml de trypsine à 0,25% et placer les plats dans l&#39;incubateur pour ~ 5 min. </li><li> Perturber la couche de cellules traitées à la trypsine par pipetage vigoureux, en prenant soin de ne pas introduire de bulles d&#39;air excessifs, jusqu&#39;à unsuspension cellulaire homogène, la plupart du temps unique est obtenue. </li><li> Ajouter 4 ml de milieu OP9 complètes et mélanger par pipetage. Transfert des cellules dans un nouveau, vide de 10 cm et les placer dans l&#39;incubateur pendant 30 min pour permettre aux cellules OP9 à adhérer à l&#39;antenne. Cette étape de «pré-placage&quot; réduit le nombre de cellules transférées OP9 à la prochaine étape de co-culture. </li><li> Préparer 50 ml tubes de centrifugeuse avec tamis de cellules de 40 um. </li><li> Recueillir des cellules non-adhérentes de l&#39;antenne pré-plaqué et de les transmettre à travers le tamis de cellules dans le tube. Laver la capsule doucement avec 6 ml de PBS et passer à travers le même filtre, laissant les cellules OP9 attachées derrière. </li><li> Centrifuger les cellules à 400 x g 5 min à 4 ° C. Retirer le surnageant et remettre en suspension les cellules sédimentées dans 3 ml de milieu complet de OP9. Compter les cellules. </li><li> Retirez le support de plaques de 10 cm avec des cellules OP9 optimale confluentes. </li><li> Pour chaque point de temps d&#39;analyse, de semences 5 x 10 5 cellules sur la OP9 monocouche dans un volume final de 10 ml. </li><li> Ajouter 5 ng / ml recombinant Flt-3L humain et de placer les plats dans l&#39;incubateur. </li></ol></li><li> Jour 8: de cellules souches hématopoïétiques (HPC) collection REMARQUE: préparer à l&#39;avance un nombre suffisant de plaques de 6 puits avec des monocouches ou OP9 OP9-DL1 cellules de sorte qu&#39;elles seront ~ 80% de confluence dans le temps pour cette étape. <ol><li> Préparer 50 ml tubes de centrifugation avec 40 um tamis. </li><li> Retirez les plats de l&#39;incubateur et observer au microscope. Grappes brillantes de BHP devraient être plus ou moins attachés à la monocouche OP9. Recueillir le plus grand nombre de ces cellules que possible par lavage (mais pas trop perturber) la monocouche OP9 avec une pipette et les médias existant sur le plat. Pour éviter la formation de mousse, laissez toujours au moins 1 ml de médias dans la pointe de la pipette au cours de cette collection de HPC / étape de lavage. </li><li> Faire passer les cellules à travers le tamis de 40 um dans un tube. Ajouter 8 ml de PBS à la même plaque et vérifier sous le microscope si tous les CAH nousre recueillies. Répétez l&#39;étape de lavage / de collection avec du PBS et (si nécessaire) avec un lavage plus énergique. Filtrer les cellules dans le tube contenant déjà les cellules provenant de la même plaque. </li><li> Centrifuger les cellules à 400 g pendant 5 min à 4 ° C. </li><li> Préparer un mélange de maître de 3 ml (par plaque ensemencée au jour 5) des médias OP9 avec 5 ng / ml Flt-3L et 1 ng / ml d&#39;IL-7. </li><li> Retirer le surnageant et remettre le culot dans les médias OP9 avec Flt-3L + IL-7 master mix (3 ml pour chaque boîte de 10 cm traitées). Transférer les cellules recueillies à partir de chaque plaque dans un puits d&#39;une plaque à 6 puits avec des cellules OP9. <ol><li> Pour conduire les cellules vers monocytes, cellules B ou lignées érythroïdes, ensemencer les cellules prélevées sur des cellules OP9. Pour obtenir des cellules T, ensemencer les cellules sur des monocouches de cellules OP9-DL1. </li></ol></li><li> Placez les plaques de 6 puits en incubateur. </li></ol></li><li> Jour 10: Changement de média <ol><li> Préparer un tube à centrifuger de 15 ml pour chaque clone MESC-chargeur type de cellule distinct combination en co-culture. </li><li> Préparer un mélange maître de supports de OP9 avec 5 ng / ml de Flt-3L et 1 ng / ml d&#39;IL-7. Préparez 3 ml pour chaque puits. </li><li> Recueillir délicatement les médias de chaque puits de la plaque de 6 puits combiner les médias à partir de plusieurs puits contenant le même clone-alimentation combinaison de type de cellule ESC. </li><li> Ajouter 2 ml de mélange maître de médias OP9 (voir 3.6.2) dans chaque puits. </li><li> Centrifuger la presse collectées à 400 x g pendant 5 min à 4 ° C. Retirer le surnageant et remettre en suspension chaque culot (très faible si visible) dans 1 ml de OP9 maître médiatique mélange (voir 3.6.2) par puits collectées à l&#39;origine à l&#39;étape 3.6.3. </li><li> Distribuer 1 ml de cellules de nouveau dans chaque puits à partir de laquelle les médias ont été recueillis portant le volume dans chaque puits de 3 ml. Remettre les boîtes dans l&#39;étuve. </li></ol></li><li> Jour 12: Pas de passage de la trypsine REMARQUE: préparer à l&#39;avance un nombre suffisant de boîtes à 6 puits avec des cellules OP9 ou OP9-DL1 afin qu&#39;ils soient confluentes de façon optimale dans le temps pour ce step. Jour 12 est idéal pour la cytométrie en flux analyses de érythroïde en développement, monocytes et lymphocytes T à un stade précoce. <ol><li> Préparer un mélange maître (3 ml pour chaque bien qui se poursuivra en co-culture) des médias OP9 avec 5 ng / ml de Flt-3L et 1 ng / ml d&#39;IL-7. </li><li> Préparer 50 ml tubes à centrifuger de 40 um tamis (un pour chaque clone-alimentation combinaison de type de cellule ESC en co-culture). </li><li> Pipette vigoureusement les médias existants dans chaque puits de ventiler toutes les cellules (y compris la monocouche) dans le puits. Continuer avec pipetage vigoureux jusqu&#39;à un état ressemblant à une suspension de cellule unique est obtenue. </li><li> Passez les cellules recueillies à travers le tamis dans le tube. Ajouter 3 ml de PBS dans chaque puits pour laver et de recueillir toutes les cellules restantes. Passez cellules à travers le même filtre. Mélanger les cellules à partir de plusieurs puits contenant le même clone-alimentation combinaison de type de cellule ESC. </li><li> Jeter le filtre cellulaire et retirer une aliquote l&#39;équivalent d&#39;un bien (~ 6 ml) pour toute désirécytométrie de flux (ou autre) des analyses à effectuer ce jour-là. Conserver les échantillons sur la glace avant utilisation. </li><li> Centrifuger les cellules à 400 g pendant 5 min à 4 ° C. Retirer le surnageant. Reprendre le culot des tubes de 50 ml de centrifugeuses dans 3 ml de mélange maître par puits pour être re-graine. Ajouter 3 ml par puits de chaque suspension cellulaire sur le type de cellules nourricières appropriées et placer les boîtes dans l&#39;incubateur. </li></ol></li><li> Jour 14: Changement de média <ol><li> Suivez les étapes décrites à la section 3.6. </li></ol></li><li> Jour 16: Pas de passage de la trypsine REMARQUE: préparer à l&#39;avance des boîtes de 6 puits de cellules OP9 ou OP9-DL1 confluentes de façon optimale pour cette étape. REMARQUE: Jour 16 est idéal pour les analyses de flux cytométrie de lymphocytes B et les lymphocytes T de la scène centrale. <ol><li> Suivez les étapes décrites dans la section 3.7. </li></ol></li><li> Jour 18: Changement de média <ol><li> Suivez les étapes décrites à la section 3.6. </li></ol></li><li> Jour 20: n passage de la trypsine REMARQUE: Jour 20 is idéal pour la cytométrie en flux analyse de mi à fin phase de développement des cellules T. <ol><li> Suivez les étapes décrites dans la section 3.7. Si vous le souhaitez, effectuer la différenciation des cellules passé 20 jours changement de support alternatif et pas trypsine passages tous les deux jours, avec un suivi quotidien du taux d&#39;expansion des lymphocytes. </li></ol></li></ol>

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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.

Le système OP9-DL1 co-culture a été utilisée pour étudier le rôle des différents produits de gènes au cours du développement de types de cellules sanguines à partir de cellules souches. 8,12,13 Il s&#39;est également avéré un modèle efficace pour étudier la fonction de l&#39;ADN de régulation de gènes cours de la différenciation cellulaire 9,14. Grâce à cette approche comme une alternative aux modèles entiers de la souris peut produire des économies considérables en temps et ...

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.

Lorsqu&#39;il est cultivé sur MEF en présence de LIF, mESCs peuvent être maintenues dans un état indifférencié. Dans des conditions idéales, ils apparaissent sous forme de colonies compactes de cellules entourées par un halo lumineux en microscopie à contraste de phase (figure 1). Ces cultures doivent être contrôlés quotidiennement. Selon la confluence des cellules, les médias peuvent être modifiés ou cellules peuvent être divisées. Voisins colonies de Mesc ne doivent pas venir au point...

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.

Dérivation des cellules T<em&gt; In Vitro</em&gt; De la souris les cellules souches embryonnaires

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 vues.  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.

Vidéo: 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

Transduction rétrovirale des récepteurs de cellules T dans les lymphocytes T de souris

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 ...

Recherche

Immunologie et infection

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.

Dérivation des thymique lymphome à cellules T à partir des lignes Atm - / -</sup Et<emP53> - / -</sup Souris

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.

Génération de cellules T régulatrices induites de l&#39;homme primaires cellules T naïves et mémoire

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.

Différenciation dirigée des cellules souches pluripotentes induites à l&#39;égard des lymphocytes 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.

Application clinique de<em&gt; Belle au Bois Dormant</em&gt; Et artificielle cellules présentatrices d&#39;antigènes pour modifier génétiquement des cellules T du sang de cordon ombilical et périphériques

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.

Cellules T Capturez bactéries par Transinfection de cellules dendritiques

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.

In Vitro Differentiation of Human CD4 + FOXP3 cellules + Induced T régulatrices (de iTregs) de Naïf cellules T CD4 + en utilisant un protocole TGF-β contenant

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 Différenciation des souris Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-production de cellules T auxiliaires (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.

Purification et expansion de cellules T tueuses naturelles invariantes de souris pour des études in vitro et 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).

Une méthode efficace pour l’induction de cellules dirigées de type hépatocytes à partir de cellules souches embryonnaires humaines

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