Three-Dimensional Thymic Culture System to Generate iPSC-Derived Thymic Emigrants

In This Article

Abstract

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).

Protocol

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

Harvest mouse fetal thymic lobes and deploy endogenous lymphocytes by deoxyguanosine (dGUO) treatment.
On day 7 of dGUO treatment, take four new 10 cm dishes and fill each with 20 mL of complete media (Roswell Park Memorial Institute Media 1640 [RPMI 1640] + 10% FBS + 1x L-alanyl-L-glutamine + 1x sodium pyruvate + 1x minimum essential medium with non-essential amino acids (MEM-NEAA) + 1x penicillin-streptomycin + [1:1....

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Results

Thymus histology, DAPI, CD3 fluorescence assay; H&E stain, T cell imaging, fluorescence microscopy.
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.......