Endocrine Abstracts

Despite the availability of synthetic thyroid hormone for therapeutic use, a significant number of patients with hypothyroidism do not feel well on replacement doses of thyroid hormones suggesting that better individualized therapy is needed. For this reason, hypothyroidism resulting from congenital lack of functional thyrocytes, surgical tissue removal, or gland ablation, represents a particularly attractive endocrine disease target that may be conceivably cured by transplantation of long-lived functional thyroid progenitors or mature follicular epithelial cells, provided a source of autologous cells can be generated and a variety of technical or biological challenges can be surmounted. To generate thyroid follicular progenitors from human induced pluripotent stem cells (hiPSCs), we sought to develop a directed differentiation approach by activating or inhibiting endogenous developmental signaling pathways previously identified in the mouse. To facilitate tracking and purification of candidate human thyroid progenitors, we engineered a hiPSC-line carrying a tdTomato reporter targeted to the PAX8 locus and a GFP reporter targeted to the NKX2–1 locus. We adapted our published in vitro differentiation protocol previously used to differentiate mouse PSCs into endoderm, foregut endoderm and then thyroid progenitors and observed tdTomato/GFP co-expressing cells first emerging from our hiPSC line by day 12 of culture and persisting for at least 2 months of further culture in thyroid maturation media, supplemented with TSH. We profiled all cells (regardless of fluorochrome expression) deriving from hiPSCs using this protocol by single cell RNA sequencing >6.000 cells captured on days 12 and 29 of in vitro differentiation. At day 12 of differentiation, tdTomato/GFP co-expression was observed in 17% of all cells and these cells appeared to be early thyroid follicular progenitors as they uniquely co-expressed the tetrad of thyroid lineage selective transcription factors, NKX2–1, PAX8, FOXE1, and HHEX, and already expressed thyroid maturation markers, TG or TPO. By day 29 tdTomato+/GFP+ cells represented 72.1% of all cells and had upregulated TG, TSHR, NIS and TPO expression in addition to the previously described four thyroid lineage markers, suggesting time dependent differentiation and maturation of thyroid follicular epithelial cells. Thus, we have employed a novel hiPSC line to optimize a protocol able to generate human thyroid progenitors and mature follicular epithelial cells, representing a purifiable source of human thyroid lineage cells whose functional and thyroid reconstituting potential can be tested in vivo in animal models of hypothyroidism.