Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells
Published: May 31, 2024
Abstract
Source: Good, M. L., et al. Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells. J. Vis. Exp. (2019).
This video demonstrates an assay to generate tumor antigen-specific induced pluripotent stem cell (iPSC)-derived CD8αβ+ T Cells. Tumor antigen-specific iPSCs are grown on a stromal cell monolayer and differentiated into hematopoietic progenitor cells (HPCs). The HPCs are differentiated into CD4+ CD8+ double-positive T cells using a differentiation medium. These cells are then stimulated to generate single-positive T cells expressing CD8.
Protocol
1. Culturing Human iPSCs (hiPSCs) on Mouse Embryonic Fibroblasts (MEF) NOTE: Alternative methods for culturing hiPSCs can also be used, including but not limited to seeding onto a 6-well plate pre-coated with gelatin, a gelatinous protein mixture, recombinant laminin 511, or any other extracellular matrix used in hiPSC expansion, and cultured using defined media specially formulated for human pluripotent stem cell culture. Culturing MEF</st…
Representative Results
Figure 1: Generation of hiPSC-derived hematopoietic progenitor cells. (A) Schematic overview of the differentiation of hiPSCs to hematopoietic lineage using OP9/DLL1 co-culture. (B) Appearance of hiPSC-derived structures on days 1 (top left), 3 (top right), 7 (bottom left), and 13 (bottom right). Scale bars = 100 µm. (C) Flow cytometric anal…
Divulgazioni
The authors have nothing to disclose.
Materials
| 10 cm dish | Corning, Inc. | 353003 | |
| Anti-CD3, human | BD Biosciences | Cat# 561812, RRID:AB_1089628 | |
| Anti-CD34, human | BD Biosciences | Cat# 348791, RRID:AB_400381 | |
| Anti-CD4, human | Biolegend | Cat# 344612, RRID:AB_2028479 | |
| Anti-CD43, human | BD Biosciences | Cat# 560198, RRID:AB_1645460 | |
| Anti-CD7, human | BD Biosciences | Cat# 555361, RRID:AB_395764 | |
| Anti-CD8a, human | BD Biosciences | Cat# 555369, RRID:AB_398595 | |
| Anti-CD8b, human | BD Biosciences | Cat# 641057, RRID:AB_1645747 | |
| Anti-TCRb, human | BD Biosciences | Cat# 555548, RRID:AB_395932 | |
| CD28 human monoclonal antibody (15E8), pure functional grade | Miltenyl Biotec | 130-093-375 | |
| CD3 human monoclonal antibody (OKT3), pure functional grade | Miltenyl Biotec | 130-093-387 | |
| CD4 Microbeads, human | Miltenyl Biotec | 130-045-101 | |
| Cell strainer 100 um | Fisher Scientific | 22-363-549 | |
| Fetal Bovine Serum (FBS) | Gemini | 100-500 | |
| Flt-3 ligand | R&D Systems | 427-FL | |
| Gelatin Solution 2% | SIGMA-Aldritch | G1393-100ML | |
| GlutaMAX (100X) | Thermo Fisher Scientific | 35050-061 | L-Glutamine supplement |
| HBSS Mg+Ca+ Phenol-Red Free | Gibco | 14025-092 | |
| Interleukin-2 | R&D Systems | 202-IL | |
| Interleukin-7 | R&D Systems | 407-ML | |
| iTAG MHC Tetramer HLA-A*0201 Mart1 Tetramer -ELAGIGILTV | MBL | Cat#TB-0009-2 | |
| Mart1-hiPSC | Vizcardo et al., Cell Stem Cell 2013 | RIKEN-IMS | |
| Melan-A, MART 1 (26-35) | InnoPep | 3146-0100 | |
| MEM Non-Essential Amino Acids Solution | Gibco | 11140050 | |
| αMEM powder | Gibco | 61100061 | |
| Mouse Embryonic Fibroblasts (MEF) | Thermo Fisher Scientific | C57BL/6 MEF MITC-TREATED 4M EACH; A34962 | |
| OP9/N-DLL1 | Riken Bioresource center | Cat# RCB2927; RRID:CVCL_B220 | OP9/DLL1 |
| Penicillin/streptomycin | Thermo Fisher Scientific | 15140-122 | |
| Phosphate buffered saline pH 7.4 (1x) | Thermo Fisher Scientific | 10010-023 | |
| Primate ES Cell Medium | Reprocell | RCHEMD001 | Human ESC Culture Media |
| Rhok inhibitor (Y-27632 dihydrochloride) | Tocris | 1254 | |
| RPMI 1640 | Gibco | 11875093 | |
| Stem Cell Factor (SCF) | R&D Systems | 455-MC | |
| StemPro | EZPassage | 23181-010 | |
| T2-tumor | ATCC | T2 (174 x CEM.T2) (ATCC® CRL-1992™) | |
| Trypsin-EDTA (0.05%), phenol red | Thermo Fisher Scientific | 25300-062 | |
| Trypsin-EDTA (0.25%), phenol red | Thermo Fisher Scientific | 25200-072 | |
| U Bottom 96 well plate | Corning, Inc. | 3799 |
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Citazione di questo articolo
Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells. J. Vis. Exp. (Pending Publication), e22280, doi: (2024).