Generation of functional human thymic cells from induced pluripotent stem cells.

BACKGROUND: The thymus is a glandular organ that is essential for the formation of the adaptive immune system by educating developing T cells. The thymus is most active during childhood and involutes around the time of adolescence resulting in a severe reduction or absence of naïve T cell output. The ability to generate a patient derived human thymus would provide an attractive research platform and enable the development of novel cell therapies.

OBJECTIVE: We sought to systematically evaluate signaling pathways to develop a refined direct differentiation protocol that generates patient derived thymic epithelial progenitor cells (pTEPs) from multiple iPSCs, that can further differentiate into functional thymic epithelial cells (pTECs) upon transplantation into athymic nude mice.

METHODS: Directed differentiation of induced pluripotent stem cells (iPSC) generated thymic epithelial progenitors that were transplanted into nude mice. 14-19 weeks post-transplantation, grafts were removed and analyzed by flow cytometry, qPCR, bulk RNA sequencing, and single cell RNA sequencing (scRNAseq) for markers of thymic cell and T cell development.

RESULTS: Here, we describe a direct differentiation protocol that allows the generation of pTEPs from multiple iPSC lines. Upon transplantation into athymic nude mice, pTEPs further differentiate into functional pTECs that can facilitate the development of T cells. scRNAseq analysis of iPSC derived grafts shows characteristic thymic subpopulations and pTEC populations that are indistinguishable from TECs present in primary neonatal thymus tissue.

CONCLUSION: Our findings provide important insights and resources for researchers focusing on human thymus biology.