Thyroid/Other Endodermal Organ Lineages Derived from Pluripotent Stem Cells

Thyroid Hypothyroidism, Congenital Thyroid Developmental Defects, Brain–Lung–Thyroid Syndrome, and Hormone Dependence after Thyroid Surgery represent major unmet clinical needs in endocrine medicine. However, traditional thyroid research and drug development face significant technical challenges. Rodent models, such as mice and rats, exhibit substantial species differences in thyroid embryonic regulatory pathways compared with humans, making them unable to accurately recapitulate human thyroid differentiation, iodine metabolism, and hormone secretion regulation. In addition, primary human thyroid tissues are difficult to obtain and rapidly lose follicular architecture and hormone-producing functions during in vitro culture, preventing long-term stable maintenance. These limitations restrict applications in hypothyroidism repair, thyroid disease modeling, and endocrine drug evaluation. In 2015, a landmark study published in Cell Stem Cell established a standardized thyroid differentiation protocol for thyroid regeneration research. This study revealed that the coordinated action of BMP4 and FGF2 represents an essential and evolutionarily conserved signaling mechanism required for thyroid lineage specification. A serum-free, transgene-free directed differentiation system was developed using only sequential cytokine modulation, faithfully recapitulating the entire embryonic developmental trajectory from anterior foregut to mature thyroid follicles. The differentiation process consists of five sequential stages: definitive endoderm induction, foregut specification, thyroid progenitor specification, progenitor expansion, and three-dimensional follicular maturation. First, high-purity definitive endoderm is generated through Activin A stimulation. Subsequently, BMP/TGF-β pathway inhibitors are applied to establish and stabilize anterior foregut identity. BMP4 and FGF2 are then introduced to specifically activate the thyroid master transcription factor NKX2-1, while PAX8 co-expression is used to distinguish thyroid progenitors from lung progenitor populations. Finally, cells are transferred into a Matrigel-based three-dimensional culture system supplemented with maturation factors such as TSH and dexamethasone, enabling self-organization into mature thyroid follicle structures containing luminal cavities. Mature thyroid follicles exhibit robust expression of key thyroid functional genes, including Tg, TPO, NIS, and TSHR, and demonstrate essential physiological activities such as iodine uptake, thyroid hormone synthesis, and hormone secretion.

Gold-standard in vivo transplantation studies have demonstrated that purified mature thyroid follicles implanted into radioactive iodine-induced severe hypothyroid mice can survive long-term, integrate into host tissue, continuously secrete T3 and T4, and restore TSH negative-feedback regulation. The transplantation effectively corrects hypothyroidism-related pathological phenotypes, while exhibiting an extremely low risk of teratoma formation. Furthermore, this differentiation platform is compatible with healthy human ES/iPS cells as well as patient-specific iPS cells carrying NKX2-1 haploinsufficiency, enabling the establishment of congenital hypothyroidism disease models that directly reproduce patient-specific thyroid maturation defects. With clearly defined signaling sequences, high batch-to-batch consistency, and scalability for large-scale production, this standardized human thyroid follicle organoid platform can be broadly applied to congenital hypothyroidism mechanism studies, high-throughput screening of thyroid hormone and iodine metabolism-related drugs, preclinical development of thyroid replacement cell therapies, and fundamental research on human thyroid embryonic development. This technology provides a high-fidelity, traceable, and standardized human thyroid cell resource platform for endocrine disease research and regenerative medicine translation.

Differentiation process of pluripotent stem cell-derived thyroid lineage.

Through a five-stage serum-free directed differentiation system, pluripotent stem cells sequentially undergo definitive endoderm induction, foregut endoderm specification, thyroid progenitor specification, progenitor expansion, and three-dimensional follicular maturation, ultimately generating human thyroid follicular organoids with iodine uptake and thyroid hormone (T3/T4) synthesis functions. This platform recapitulates thyroid development and enables functional validation through in vivo transplantation and patient-derived iPSC models, providing a high-fidelity humanized model for studying congenital hypothyroidism mechanisms, drug screening, thyroid regeneration, and endocrine disease research.