Derivation of thyroid progenitors from human induced pluripotent stem cells

Abstract

Abstract Objective 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. Methods 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. Results We adapted our published in vitro differentiation protocol previously used to differentiate mouse PSCs into 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. Thus, we profiled all cells deriving from hiPSCs by single-cell RNA sequencing >6,000 cells captured on days 12 and 29 of in vitro differentiation. At day 12, tdTomato/GFP co-expression was observed in 12% 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. By day 29 tdTomato+/GFP+ cells represented 69% 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. Conclusion 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.