DataSheet_1_Single-Cell Trajectory Inference Guided Enhancement of Thyroid Maturation In Vitro Using TGF-Beta Inhibition.pdf

The thyroid gland regulates metabolism and growth via secretion of thyroid hormones by thyroid follicular cells (TFCs). Loss of TFCs, by cellular dysfunction, autoimmune destruction or surgical resection, underlies hypothyroidism. Recovery of thyroid hormone levels by transplantation of mature TFCs derived from stem cells in vitro holds great therapeutic promise. However, the utilization of in vitro derived tissue for regenerative medicine is restricted by the efficiency of differentiation protocols to generate mature organoids. Here, to improve the differentiation efficiency for thyroid organoids, we utilized single-cell RNA-Seq to chart the molecular steps undertaken by individual cells during the in vitro transformation of mouse embryonic stem cells to TFCs. Our single-cell atlas of mouse organoid systematically and comprehensively identifies, for the first time, the cell types generated during production of thyroid organoids. Using pseudotime analysis, we identify TGF-beta as a negative regulator of thyroid maturation in vitro. Using pharmacological inhibition of TGF-beta pathway, we improve the level of thyroid maturation, in particular the induction of Nis expression. This in turn, leads to an enhancement of iodide organification in vitro, suggesting functional improvement of the thyroid organoid. Our study highlights the potential of single-cell molecular characterization in understanding and improving thyroid maturation and paves the way for identification of therapeutic targets against thyroid disorders.

甲状腺通过甲状腺滤泡细胞（thyroid follicular cells, TFCs）分泌甲状腺激素，调控机体新陈代谢与生长发育。因细胞功能异常、自身免疫破坏或手术切除导致的甲状腺滤泡细胞丢失，是甲状腺功能减退症的核心病因。利用体外诱导干细胞分化得到的成熟甲状腺滤泡细胞恢复甲状腺激素水平，具备极高的治疗潜力。然而，体外衍生组织用于再生医学的应用，受限于分化方案生成成熟类器官的效率。为提升甲状腺类器官的分化效率，本研究采用单细胞RNA测序（single-cell RNA-Seq）解析了小鼠胚胎干细胞向甲状腺滤泡细胞体外转化过程中单个细胞的分子调控步骤。我们首次通过小鼠甲状腺类器官的单细胞转录组图谱，系统且全面地鉴定了甲状腺类器官构建过程中产生的各类细胞类型。借助拟时间分析，我们鉴定出转化生长因子-β（TGF-beta）是体外甲状腺成熟过程的负调控因子。通过对TGF-beta通路进行药物抑制，我们提升了甲状腺成熟水平，尤其是钠碘同向转运体（Nis）的表达诱导效果。这进而增强了体外碘有机化能力，提示甲状腺类器官的功能得到实质性改善。本研究彰显了单细胞分子表征在解析与优化甲状腺成熟过程中的应用潜力，为靶向甲状腺疾病的治疗靶点发掘铺平了道路。