Name: directed differentiation of induced pluripotent stem cells towards t lymphocytes
Uploaded: 2012-05-14T13:00:00
Description: Watch this Scientific Journal Video about Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes at JoVE.com

Ringraziamo il dottor Shinya Yamanaka (Kyoto University) per la fornitura di IPS-MEF-Ng-20D-17 della linea cellulare, Dr. Dario Vignali (Research Hospital di St. Jude Children) per sostenere l&#39;OT1-2A • pMig II costrutto, Dr. Juan Carlos Zuniga-Pflucker (Dipartimento di Immunologia, Università di Toronto) per sostenere il OP9-DL1 linea cellulare, e il Dr. Kent E Vrana (Dipartimento di Farmacologia, Penn State University College of Medicine) per aiutare la progettazione di questo studio. Questo progetto è finanziato, in base a concessioni con il numero di Grant K18CA151798 dal National Cancer Institute, il Trust Barsumian e il Melanoma Research Foundation (Song J.).

1. Coltura cellulare <ol><li> Preparazione delle cellule irradiate alimentazione SNL76 / 7 (irSNL76 / 7) per la cultura. SNL76 / 7 cellule sono generalmente mantenute in 10% di siero fetale bovino (FBS) Dulbeccòs Modified Eaglès Medium (DMEM) media. <ol><li> Un piatto di coltura o matraccio saranno rivestite con 0,1% soluzione di gelatina in 37 ° C; incubatore per 30 minuti prima di recuperare SNL76 / 7 cellule di azoto liquido. </li><li> Quando SNL76 / 7 cellule raggiungono la confluenza, le cellule saranno...

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<html> Per ACT-terapie, la generazione in vitro di un gran numero di cellule T reattive altamente Ag-specifiche in vivo reinfusione è un approccio ottimale. Sebbene il nostro metodo in vitro comporta funzionali di cellule T da cellule iPS, un gran numero di cellule iPS derivate cellule muoiono in quattro settimane, soprattutto nella quarta settimana. Si conclude che i segnali di sopravvivenza di Notch segnalazione mediata dalla DL1 e IL-7 e Flt3L non sono sufficienti a mantenere la sopravviven...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> C57BL/6J topi </td><td> Jackson Laboratory </td><td> 000664 </td></tr><tr><td> B6.129S7-RAG1 tm1Mom / J </td><td> Jackson Laboratory </td><td> 002216 </td></tr><tr><td> Anti-CD3 (2C11) anticorpi </td><td> BD PharMingen </td><td> 553058 </td></tr><tr><td> Anti-CD28 (37,51) anticorpi </td><td> BD PharMingen </td><td> 553295 </td></tr><tr><td> Anti-CD3 (17A2) anticorpi </td><td> BioLegend </td><td> 100202 </td></tr><tr><td> Anti-CD4 (GK1.5) anticorpi </td><td> BioLegend </td><td> 100417 </td></tr><tr><td> Anti-CD8 (53-6,7) anticorpi </td><td> BioLegend </td><td> 100714 </td></tr><tr><td> Anti-CD25 (3C7) anticorpi </td><td> BioLegend </td><td> 101912 </td></tr><tr><td> Unti-CD44 (1M7) anticorpi </td><td> BioLegend </td><td> 103012 </td></tr><tr><td> Anti-CD117 (2B8) anticorpi </td><td> BioLegend </td><td> 105812 </td></tr><tr><td> Anti-TCR-β (H57597) anticorpi </td><td> BioLegend </td><td> 109220 </td></tr><tr><td> Anti-IL-2 (JES6-5H4) anticorpo </td><td> BioLegend </td><td> 503810 </td></tr><tr><td> Anti-IFN-γ (XMG1.2) anticorpi </td><td> BioLegend </td><td> 505822 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> ABCD1234 </td></tr><tr><td> α-MEM </td><td> Invitrogen </td><td> A10490-01 </td></tr><tr><td> FBS </td><td> HyClone </td><td> SH3007.01 </td></tr><tr><td> A brefeldina </td><td> Sigma </td><td> B7651 </td></tr><tr><td> Polybrene </td><td> Sigma </td><td> 107689 </td></tr><tr><td> GeneJammer </td><td> Scienze integrate </td><td> 204130 </td></tr><tr><td> Kit di RNA &lt;/ Td&gt; <td> Qiagen </td><td> 74104 </td></tr><tr><td> DNA kit </td><td> Qiagen </td><td> 69504 </td></tr><tr><td> CD8 Kit di isolamento </td><td> Miltenyi Biotec </td><td> 130-095-236 </td></tr><tr><td> Tampone di lisi ACK </td><td> Lonza </td><td> 10-548E </td></tr><tr><td> mFlt-3L </td><td> Peprotech </td><td> 250-31L </td></tr><tr><td> mIL-7 </td><td> Peprotech </td><td> 217-17 </td></tr><tr><td> Gelatina </td><td> Sigma </td><td> G9391 </td></tr><tr><td> FITC-anticorpo anti-OVA </td><td> Rockland Immunochemicals </td><td> 200-4233 </td></tr><tr><td> Permeabilizzazione del buffer </td><td> Biolegend </td><td> 421002 </td></tr><tr><td> BSA </td><td> Sigma </td><td> A7906 </td></tr><tr><td> Formaldeide </td><td> Sigma </td><td> F8775 </td></tr><tr><td> 0,4 micron filtro </td><td> Millipore </td><td></td></tr><tr><td&gt; Moflo cella Sorter </td><td> Dake Cytomation </td><td></td></tr><tr><td> Calibur Citofluorimetro </td><td> BD </td><td></td></tr><tr><td> LSR II Citofluorimetro </td><td> BD </td><td></td></tr><tr><td> Mouse restrainer </td><td> Braintree Scientific </td><td></td></tr></tbody></table>

directed differentiation of induced pluripotent stem cells towards t lymphocytes

Trasferimento adottivo di cellule (ACT) di antigene-specifica CD8 + linfociti T citotossici (CTL) è un trattamento promettente per una varietà di tumori maligni 1. CTL può riconoscere cellule maligne interagendo antigeni tumorali con i recettori delle cellule T (TCR), e citotossine rilascio nonché citochine per uccidere le cellule maligne. È noto che meno differenziati e centro-memory-simile (denominato altamente reattivo) CTL sono la popolazione ottimale per ACT-immunoterapia, perché questi CTL hanno un elevato potenziale proliferativo, sono meno inclini alla apoptosi di cellule più differenziate e hanno maggiore capacità di rispondere alle citochine omeostatiche 2-7. Tuttavia, a causa delle difficoltà nel reperire un numero elevato di CTL tali da pazienti, c&#39;è un bisogno urgente di trovare un nuovo approccio per la generazione altamente reattive CTL Ag-specifici per il successo terapie a base di ACT. TCR trasduzione del sé rinnovabile stelocellule per la ricostituzione immunitario ha un potenziale terapeutico per il trattamento di malattie 8-10. Tuttavia, l&#39;approccio per ottenere cellule staminali embrionali (CSE) di pazienti non è fattibile. Sebbene l&#39;uso di cellule staminali ematopoietiche (CSE) per scopi terapeutici è stato ampiamente applicato in clinica 11-13, CSE hanno ridotto la differenziazione e capacità proliferative, e CSE difficili da espandere in coltura cellulare in vitro 14-16. Recenti cellule iPS tecnologia e lo sviluppo di un sistema in vitro per il gene delivery sono in grado di generare cellule iPS da pazienti senza alcun approccio chirurgico. Inoltre, come CES, cellule iPS possiedono indefinita capacità proliferativa in vitro, e hanno dimostrato di differenziarsi in cellule ematopoietiche. In questo modo, le cellule iPS hanno maggiore potenziale per essere utilizzato in ACT-based immunoterapia rispetto al CES o CSE. Qui, presentiamo metodi per la generazione di linfociti Tcondrociti iPS da cellule in vitro e in vivo di programmazione antigene-specifici CTL di cellule iPS per promuovere sorveglianza immunitaria cancro. La stimolazione in vitro con un ligando Notch spinge la differenziazione delle cellule T da cellule iPS, e TCR risultati di trasduzione del gene nelle cellule iPS differenziarsi in cellule T antigene-specifiche in vivo, che impedisce la crescita del tumore. Così noi vogliamo dimostrare antigene-specifica differenziazione delle cellule T da cellule iPS. I nostri studi forniscono un approccio potenzialmente più efficiente per la generazione di CTL antigene-specifici per terapie a base di ACT e facilitare lo sviluppo di strategie terapeutiche per le malattie.

Watch this Scientific Journal Video about Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes at JoVE.com

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Generazione di linfociti T da staminali pluripotenti indotte (iPS), le cellule fornisce un approccio alternativo di utilizzare cellule staminali embrionali per T cell-based immunoterapia. Il metodo dimostra che utilizzando sia<em&gt; In vitro</em&gt; O<em&gt; In vivo</em&gt; Sistema ad induzione, le cellule iPS sono in grado di differenziarsi in linfociti T convenzionali e antigene-specifica.

Differenziazione Regia di cellule staminali pluripotenti indotte verso Linfociti T

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

Research

JoVE Journal

Immunology and Infection

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Metodi per valutare la morte delle cellule beta Mediata da linfociti T citotossici

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
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Ricerca

Immunologia e infezioni

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Differenziazione indotta di cellule M in monostrati Enteroidi Ileal Imbecilli derivati da cellule staminali umane

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...