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

Overview

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

1. Harvest mouse fetal thymic lobes and deploy endogenous lymphocytes by deoxyguanosine (dGUO) treatment.
2. 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 + [1:1000] 2-mercapto ethanol).
3. Transfer all nitrocellulose membranes with thymic lobes into one 10 cm dish. Detach the individual lobes from the membrane with forceps, allowing them to be submerged in media. Discard the membranes. Incubate for 1 h at RT.
4. Transfer the thymic lobes to a new 10 cm dish with complete media and incubate for 1 h at RT. Repeat this step 2 more times.
5. Using forceps, fix the thymic lobes to the dish (one at a time), and with the other hand make a 100–200 µm deep incision in the center and extend half the diameter of the lobe to facilitate T cell progenitor migration into the lobe.
6. Transfer the thymic lobes to a new 10 cm dish filled with complete differentiation media (complete media + 5 ng/mL mouse interleukin-7 (IL-7) + 5 ng/mL mouse Flt3 ligand (FLT3L) + 5 ng/mL stem cell factor (SCF)).
7. Optionally, if using 3D culture plates with lower and upper-level grids, fill both grids with sterile PBS to prevent the evaporation and drying of the hanging drops.
8. Transfer 30 µL of complete media containing one dGuo-treated thymic lobe from step 1.6 into each well of the 3D culture plate.
9. Collect non-adherent T lineage cells (iPSC-derived immature T cells) from OP9/DLL1 co-culture (days 16-21) and resuspend at 2–5 x 10³ T lineage cells per 20 µL media.
10. Add 20 µL of T lineage cell suspension to each thymic lobe in the 3D culture plate. Incubate overnight at 37 °C with 5% CO₂.
11. Set the P200 pipet to 30 µL and aspirate the media after pipetting several times from each well to remove all the cells surrounding the thymic lobes. Discard media and add 30 µL of complete media. Repeat this procedure 5–7 times to remove any extra immature T cells which do not migrate into the lobes. Change 25–30 µL of media daily thereafter.
12. Confirm the formation of a halo of iPSC-derived thymic emigrants (iTE) around the lobes beginning on days 4–5 by light microscopy.
13. Collect iTE daily by pipetting media without lobe disruption. Change media every day and continue collection for up to approximately 12 days.
14. Harvested iTE are ready to use for molecular analyses (Figure 1, Figure 2, Figure 3, and Figure 4) or in vivo transplantation experiments.

Representative Results

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).

Materials

Name	Company	Catalog Number	Comments
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