Single-cell RNA-Seq of mouse thyroid organoid

The thyroid gland regulates metabolism and growth via secretion of thyroid hormone 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 molecular pathways that regulate thyroid maturation in vitro. 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. Overall design: One sample, containing a mixture of four different stages of differentiation protocol, was profiled using 10x Chromium pipeline and sequenced to a depth of 100 million reads. Cells from all stages were mixed in a single run and cannot be de-multiplexed. The mixed cells are separated in silico using known markers of the corresponding stages. This is part of the downstream analysis and will be provided with the manuscript.