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

We thank Dr. Shinya Yamanaka (Kyoto University) for providing iPS-MEF-Ng-20D-17 cell line, Dr. Dario Vignali (St. Jude Children's Research Hospital) for supporting the OT1-2A&bull;pMig II construct, Dr. Juan Carlos Zuniga-Pflucker (Department of Immunology, University of Toronto) for supporting the OP9-DL1 cell line, and Dr. Kent E Vrana (Department of Pharmacology, Penn State University College of Medicine) for helping the design of this study. This project is funded, under grants with the Grant Number K18CA151798 from the National Cancer Institute, the Barsumian Trust and the Melanoma Research Foundation (J. Song).

1. Cell Culture
<ol>
 <li>Preparation of irradiated SNL76/7 (irSNL76/7) feeder cells for culture. 
 SNL76/7 cells are generally maintained in 10% fetal bovine serum (FBS) Dulbecco's Modified Eagle Medium (DMEM) media.
 <ol>
 <li>A culture dish or flask will be coated with 0.1% gelatin solution in 37 &deg;C; incubator for 30 minutes before recovering SNL76/7 cells from liquid nitrogen. </li>
 <li>When SNL76/7 cells reach confluency, cells will be trypsinized off, centrifuged at 400 g for 5 min and resuspen...

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For ACT-based therapies, the in vitro generation of large numbers of highly reactive Ag-specific T cells for in vivo re-infusion is an optimal approach. Although our in vitro method gives rise of functional T cells from iPS cells, large numbers of iPS cell-derived cells die in four weeks, especially in the fourth week. We conclude that the survival signals from Notch signaling mediated by the DL1 as well as IL-7 and FLt3L are not sufficient to maintain the survival of iPS cell-derived prog...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>C57BL/6J mice</td>
 <td>Jackson Laboratory</td>
 <td>000664</td>
 </tr>
 <tr>
 <td>B6.129S7-Rag1tm1Mom/J</td>
 <td>Jackson Laboratory</td>
 <td>002216</td>
 </tr>
 <tr>
 <td>Anti-CD3 (2C11) antibody</td>
 <td>BD PharMingen</td>
 <td>553058</td>
 </tr>
 <tr>
 <td>Anti-CD28 (37.51) antibody</td>
 <td>BD PharMingen</td>
 <td>553295</td>
 </tr>
 <tr>
 <td>Anti-CD3 (17A2) antibody </td>
 <td>BioLegend</td>
 <td>100202</td>
 </tr>
 <tr>
 <td>Anti-CD4 (GK1.5) antibody</td>
 <td>BioLegend</td>
 <td>100417</td>
 </tr>
 <tr>
 <td>Anti-CD8 (53-6.7) antibody</td>
 <td>BioLegend</td>
 <td>100714</td>
 </tr>
 <tr>
 <td>Anti-CD25 (3C7) antibody</td>
 <td>BioLegend</td>
 <td>101912</td>
 </tr>
 <tr>
 <td>Anti-CD44 (1M7) antibody</td>
 <td>BioLegend</td>
 <td>103012</td>
 </tr>
 <tr>
 <td>Anti-CD117 (2B8) antibody</td>
 <td>BioLegend</td>
 <td>105812</td>
 </tr>
 <tr>
 <td>Anti-TCR-&beta; (H57597) antibody</td>
 <td>BioLegend</td>
 <td>109220</td>
 </tr>
 <tr>
 <td>Anti-IL-2 (JES6-5H4) antibody</td>
 <td>BioLegend</td>
 <td>503810</td>
 </tr>
 <tr>
 <td>Anti-IFN-&gamma; (XMG1.2) antibody</td>
 <td>BioLegend</td>
 <td>505822</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>ABCD1234</td>
 </tr>
 <tr>
 <td>&alpha;-MEM</td>
 <td>Invitrogen</td>
 <td>A10490-01</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>HyClone</td>
 <td>SH3007.01</td>
 </tr>
 <tr>
 <td>Brefeldin A</td>
 <td>Sigma</td>
 <td>B7651</td>
 </tr>
 <tr>
 <td>Polybrene</td>
 <td>Sigma</td>
 <td>107689</td>
 </tr>
 <tr>
 <td>GeneJammer</td>
 <td>Integrated Sciences</td>
 <td>204130</td>
 </tr>
 <tr>
 <td>RNA kit</td>
 <td>Qiagen</td>
 <td>74104</td>
 </tr>
 <tr>
 <td>DNA kit</td>
 <td>Qiagen</td>
 <td>69504</td>
 </tr>
 <tr>
 <td>CD8 Isolation Kit</td>
 <td>Miltenyi Biotec</td>
 <td>130-095-236</td>
 </tr>
 <tr>
 <td>ACK lysis buffer</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>Gelatin</td>
 <td>Sigma</td>
 <td>G9391</td>
 </tr>
 <tr>
 <td>FITC-anti-OVA antibody</td>
 <td>Rockland Immunochemicals</td>
 <td>200-4233</td>
 </tr>
 <tr>
 <td>Permeabilization buffer</td>
 <td>Biolegend</td>
 <td>421002</td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma</td>
 <td>A7906</td>
 </tr>
 <tr>
 <td>Formaldehyde</td>
 <td>Sigma</td>
 <td>F8775</td>
 </tr>
 <tr>
 <td>0.4 &mu;m filter</td>
 <td>MIllipore</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Moflo Cell Sorter</td>
 <td>Dake Cytomation</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Calibur Flow Cytometer</td>
 <td>BD</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>LSR II Flow Cytometer</td>
 <td>BD</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mouse restrainer</td>
 <td>Braintree Scientific</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

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

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.

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

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