Source: Vizcardo, R., et al. A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants. J. Vis. Exp. (2019).
This video demonstrates a protocol for generating functional antigen-specific T cells from iPSC-derived immature T cells using a three-dimensional thymic culture system. Co-culturing the iPSC-derived immature T cells with endogenous lymphocyte-depleted mice fetal thymic lobes resulted in successful maturation of the T cells to form CD8αβ+ MHC class I+ iPSC-derived thymic emigrants (iTEs).
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. 3D Thymic Organ Culture to Generate iTE
Figure 1: Immunohistochemistry of thymic lobes seeded with iPSC-derived immature T cells. Top: H&E staining of a thymic lobe with and without seeding of iPSC-derived immature T cells. From second top to bottom: confocal images of the sectioned lobes stained with DAPI (nucleus), CD3 (T cell), and merge. Scale bars = 100 µm.
Figure 2: iTEs show a post-thymic T cell phenotype. (A) FACS analyses of thymocytes, extrathymic T cells (OP9/DLL1 co-culture system), and Pmel-iTE. Live cells were gated on congenic CD45+. CD8 SP populations were further analyzed for CD62L and MHC-I expression. (B) Average number of CD8SP CD45.1 iTE produced overnight per lobe 7 days after pre-seeding. Data were collected from 12 independent experiments.
Figure 3: iTEs produce various cytokines by antigen-specific stimulation. FACS analyses of intracellular production of cytokines by iTE. iTE were co-cultured with APCs pre-loaded with irrelevant (nucleoprotein) or cognate (hgp100) peptide for three days. The numbers shown in the upper right quadrants indicate the percentages of iTEs producing cytokine.
Figure 4: Whole-transcriptome analysis reveals a shift in iTE gene expression toward a naïve CD8+ T cell program. Principle component analysis (PCA) of RNA-seq data from DP, extrathymic CD8 SP, iTE, and naïve T cells. (Analysis of 102 genes related to thymic differentiation using public database GSE105110).
The authors have nothing to disclose.
2-deoxyguanosine | Sigma-Aldrich | 312693-72-4 | |
2-Mercaptoethanol (1,000x) | Thermo Fisher Scientific | 21985-023 | |
FBS | Gemini | 100-500 | |
Flt-3 ligand | R&D Systems | 427-FL | |
Interleukin-7 | R&D Systems | 407-ML | |
MEM Non-Essential Amino Acids Solution | Gibco | 11140050 | |
MEM powder | Gibco | 61100061 | |
Sodium Pyruvate | Thermo Fisher Scientific | 11360-070 | |
Stem Cell Factor (SCF) | R&D Systems | 455-MC | |
Penicillin/streptomycin | Thermo Fisher Scientific | 15140-122 | |
Phosphate-buffered saline pH 7.4 (1x) | Thermo Fisher Scientific | 10010-023 | |
RPMI 1640 | Gibco | 11875093 | |
10 cm dish | Corning, Inc | 353003 | |
Perfecta3D Hanging Drop Plate | Sigma-Aldrich | HDP1096 | |
Forceps | DUMONT | 0108-5PO | |
OP9/N-DLL1 | Riken Bioresource center | Cat# RCB2927; RRID:CVCL_B220 | |
GlutaMAX (100x) | Thermo Fisher Scientific | 35050-061 |