Stage-specific signaling modulation generates low-tumorigenicity thyroid progenitors from human pluripotent stem cells.

INTRODUCTION: Human pluripotent stem cell (hPSC)-derived thyroid cells hold great promise for thyroid disease modeling, drug screening, and cell replacement therapies. However, the direct differentiation of hPSCs into functional thyroid follicles remains challenging, suggesting deficient specification of hPSC-derived progenitor cells in conventional protocols. METHODS: In this study, we utilized a PAX8-mCherry hPSC reporter line to evaluate early differentiation and applied systematic pathway screening to optimize induction conditions. Furthermore, early-stage hPSC induction conditions were refined, and in vivo transplantation was performed to assess morphogenic capacity. RESULTS: We observed that inadequate FOXE1 induction may contribute to the limited maturation capacity of conventional protocols. Through our systematic screening, we successfully identified the optimized conditions required for generating NKX2-1/PAX8/FOXE1 triple-positive thyroid progenitors from hPSCs. Notably, these hPSC-derived progenitors are capable of forming thyroglobulin-positive follicular structures upon in vivo transplantation. Furthermore, we significantly reduced the inherent tumorigenic risk associated with residual hPSCs. DISCUSSION: In summary, we present an efficient approach to direct hPSCs into low-tumorigenicity thyroid progenitors capable of engraftment and early in vivo morphogenesis, establishing a robust platform for thyroid regenerative medicine.