TCR repertoire analysis of donor-derived and iTEC-supported thymocytes

We analyzed the TCRb repertoires of CD4 and CD8 single-positive thymocytes isolated from a pediatric donor, as well as CD4 and CD8 single-positive thymocytes matured in vitro from the double-positive stage in 3D organoid co-culture with iPSC-derived thymic epithelial cells (iTECs). Our analysis shows that iTECs are capable of positively selecting thymocytes with diverse TCR repertoires in vitro. CD4/CD8 double-positive thymocytes were isolated from a pediatric donor and co-cultured with iTECs in 3D organoid culture for two weeks. The resulting CD4 and CD8 single-positive thymocytes were subjected to TCRb repertoire analysis using RNA-seq. CD4 and CD8 single-positive thymocytes isolated from the same donor were used as controls.