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

To generate iPSC-derived thymic emigrants, iTEs from immature T cells in a 3D thymic organ culture system, take deoxyguanosine, dGUO-treated mouse fetal thymic lobes in suitable media.

dGUO enters the thymic environment and destroys endogenous thymic lymphocytes, preventing their interference with iPSC-derived immature T cells during co-culture.

Make a deep incision at the center of each thymic lobe. Transfer the lobes to a fresh culture dish containing medium supplemented with the growth factors stem cell factor, FLT3 ligand, and interleukin-7.

Pipette the thymic lobe-containing media into the wells of a 3D culture plate. Add iPSC-derived immature T cells. Incubate. The T cells migrate into the thymic lobes through the incision.

Within the thymic environment, the growth factors FLT3 ligand and IL-7 bind to their respective receptors on immature T cells leading to the activation of transcription factors and the expression of specific genes.

Consequently, the immature T cells undergo a series of changes and acquire specific characteristics, ultimately maturing into CD8αβ+ T cells which express CD8 molecules, T cell receptors, and MHC class I molecules on their surface.

Remove the immature T cells that have failed to migrate into the lobes.

The mature iTEs migrate out of the thymic lobes which can be visualized as a halo-like pattern surrounding the lobes under a light microscope.