Figure 1.

Establishment and Characterization of Murine and Human Thyroid Gland Organoid Cultures

(A) Schematic representation of murine and human thyroid primary cell culture. Thyroid gland tissue was mechanically and enzymatically digested and resuspended in culture medium or seeded in Matrigel.

(B) Primary murine thyroid spheres after 1 day in floating culture, and primary human thyroid organoids after 7 days in culture in Matrigel. Scale bars, 100 μm.

(C) NKX2.1, thyroglobulin, and T4 staining of murine and human tissue and primary murine spheres after 1 day in floating culture and primary human thyroid organoids after 7 days in culture in Matrigel shows a nuclear staining for NKX2.1 and staining for thyroglobulin and T4. Scale bars, 50 μm for tissue and 25 μm for organoids.

(D) Schematic representation of the self-renewal assay. Primary spheres (murine and human) were digested into single cells and replated in Matrigel. Organoids were passaged every 7 (murine) or 14 days (human).

(E) Murine thyroid gland organoids at passages 1, 3, and 5 cultured in thyroid gland medium (TGM) or cultured in TGM supplemented with Wnt and R-spondin1 (TGM + WR) and human thyroid gland organoids at passages 1, 3, and 5. Scale bars, 100 μm.

(F) Organoid-forming efficiency of murine thyroid gland cells in TGM (n = 15 independent self-renewal assays) and in TGM + WR (n = 6 independent self-renewal assays) up to passage 10.

(G) Organoid-forming efficiency of human thyroid gland cells during multiple passages (n = 17 independent donor biopsies used for the self-renewal assay).

(H) Representative confocal images of immunofluorescence staining for NKX2.1, PAX8, NIS, and ZO-1 in murine and human organoids. All organoids were from passage 2; scale bars, 20 μm.

(I) Quantification indicating percentage of cells expressing thyroid-specific genes NKX2.1, PAX8, and NIS in murine and human organoids from passage 2.

(J) Dual-pulse labeling using EdU and BrdU in passage 3 murine thyroid organoids to identify DNA template strand segregation during cell division.

All data are represented as mean ± SEM. See also Figure S1 and Video S1.