Abstract
The production of mature functioning thyroid follicular cells (TFCs) from human induced pluripotent stem cells (iPSCs) is critical for potential novel therapeutic approaches to post-surgical and congenital hypothyroidism. To accomplish this, we developed a novel human iPSC line that expresses fluorophores targeted to the NKX2-1 and PAX8 loci, allowing for the identification and purification of cells destined to become TFCs. Optimizing a sequence of defined, serum-free media to promote stepwise developmental directed differentiation, we found that bone morphogenic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2) stimulated lineage specification into TFCs from multiple iPSC lines. Single-cell RNA sequencing demonstrated that BMP4 withdrawal after lineage specification promoted TFC maturation, with mature TFCs representing the majority of cells present within 1 month. After xenotransplantation into athyreotic immunodeficient mice, engrafted cells exhibited thyroid follicular organization with thyroglobulin protein detected in the lumens of NKX2-1-positive follicles. While our iPSC-derived TFCs presented durable expression of thyroid-specific proteins, they were unable to rescue hypothyroidism in vivo.