Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells

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…

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