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.

1. Culturing MEF
   1. Coat a 10 cm cell culture Petri dish with 4 mL of 0.1% gelatin and incubate for 30 min at 37 °C.
   2. Thaw a vial of 4 x ....

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 o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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