The generation of FOXN1+ thymic epithelial progenitors from human pluripotent stem cells

Thymic epithelial cells (TECs) form the main constituent of the thymus microenvironment and play an essential role in the maturation of thymocytes and the development of a diverse self-tolerant T-cell repertoire. Critical to the differentiation of both fetal and postnatal TECs is the expression of the forkhead-box transcription factor Foxn1. Accordingly, mice deficient in Foxn1 (nude mice) are athymic due to aberrant morphogenesis of the thymic epithelium, which in turn cannot support the production of functional T-cells. A phenotypically equivalent human Nude/SCID syndrome, in which affected individuals present with severe T-cell immunodeficiency, has been attributed to a mutation in the human FOXN1 gene. A decrease in thymic function may also arise from other congenital thymic hypoplasias, ageing-associated thymic involution, cytoablative therapies, and autoimmune disease. The generation of TECs from human pluripotent stem cells (hPSCs) could represent an attractive avenue to replenish the thymic microenvironment and reconstitute overall thymus function. This study combines knowledge gained from thymus ontogeny, with technologies for stem cell genetic manipulation and differentiation, to develop a strategy for the in vitro differentiation of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) towards the thymic lineage. To facilitate monitoring of thymic epithelial cell differentiation in vitro, we developed a hESC reporter (FOXN1GFP/w) line in which the green fluorescent protein (GFP) gene is targeted to the endogenous FOXN1 locus. The FOXN1GFP/w hESCs were used in conjunction with a serum-free spin embryoid body system to develop and optimise a robust and reproducible protocol for the generation of FOXN1+ thymic epithelial progenitors. Transcriptional profiling of FOXN1-GFP+ cells assisted in the identification of specific cell surface antibody combinations that permitted the isolation of TEC progenitors from unmodified hPSCs. The ability to purify TEC progenitors provides a novel platform to investigate the derivation of thymic endoderm from patient-specific stem cells, an ultimate goal that would allow therapeutic application of these cells within a clinical setting.