Abstract
The thyroid gland captures iodide in order to synthesize hormones that act on almost all tissues and are essential for normal growth and metabolism. Low plasma levels of thyroid hormones lead to hypothyroidism, which is one of the most common disorder in humans and is not always satisfactorily treated by lifelong hormone replacement. Therefore, in addition to the lack of in vitro tractable models to study human thyroid development, differentiation and maturation, functional human thyroid organoids could pave the way to explore new therapeutic approaches. Here we report the first transplantable thyroid organoids derived from human embryonic stem cells capable of restoring plasma thyroid hormone to athyreotic mice as a proof of concept for future therapeutic development.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Additional data in revised version: -Co-immunostaining for NKX2-1/PAX8 at day 9 of the differentiation protocol (Fig. 1b, Page 41). -Quantification by flow cytometry of the ratio of NKX2-1 and PAX8 co-expressing cells at day 9 and 16 (Fig. 1c, Page 41). -Proliferation assay: NKX2-1/KI67 ratio assessment was replaced by BrdU assay throughout the protocol period (Fig. 1e, Page 41). -Immunostaining for TG was performed from day 30 onwards, every week (Fig. 1i, Page 41). -RNAseq was performed to assess protocol change at day 30 and to compare gene expression (thyroid genes, inflammation and TGF-beta effectors) between the cAMP condition and hrTSH+Dexa or SB conditions at days 38 and 45 (Supplementary Fig. 2a, b and m, Page 51). -qRT-PCR for thyroid genes was performed at days 38 and 45 using -Dox, cAMP (+/- Dexa and SB), and TSH (+/- Dexa and SB) as controls (Supplementary Fig. 2c-l, Page 51). -Immunostaining characterization of the thyroid organoids organization and function at day 45 (Fig. 2a-d, Page 43). -New scRNAseq experiment was performed using organoids at day 58 (Fig. 3a-c, Page 45 and Supplementary Fig. 3b, Page 53; Supplementary Fig. 4b,d,f,h,I, Page 54; Supplementary Fig. 5d-e, Page 56). -scRNAseq data at day 45 and 58 were compared by integration analysis between them, and to adult human thyroid tissue (Fig. 3d, f, Page 45). -Non-thyroid cells identified by scRNAseq were characterized by immunofluorescence (Supplementary Fig. 4j-m, Page 54). -Spatial demonstration of thyroid cells proximity to mesodermal cells (cardiovascular and fibroblasts) was evaluated by immunofluorescence (Supplementary Fig. 5c, f, Page 56). -Immunostaining for TG/F-actin was performed at differentiation day 58 (Fig. 3i, Page 45). -Immunostaining evaluation of organoids kept in culture until day 70 (Supplementary Fig. 3c, Page 53). -New in vivo experiment was performed with grafted mice. T4, T3 and TSH levels were evaluated. liver Dio1 mRNA levels were measured (Fig. 4g-j, Page 47 and Supplementary Fig. 6g-h, Page 57). -Adult thyroid tissue was used to compare protein expression and T4 accumulation to the grafted resulting tissue (Fig. 4d, Page 47). -The size of follicles was compared between grafted thyroid organoids and adult thyroid tissue (Supplementary Fig. 6e, Page 57). -Thyroid genes expression levels were compared among in vitro organoids (from day 58), grafted tissue (after 5 weeks) and adult human thyroid tissue (Fig. 4e, Page 47).