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. 2021 May 31;12:657195. doi: 10.3389/fendo.2021.657195

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Copyright © 2021 Romitti, Eski, Fonseca, Gillotay, Singh and Costagliola

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Enhancement of maturation efficiency by pharmacological inhibition of TGF-beta. (A) UMAPs demonstrate TGF-beta receptors (Tgfbr1 and Tgfbr2), (B) activators (Smad2 and Smad3) and (C) ligands (Tgfb1 and Tgfb2) are expressed in mesoderm cells and in thyroid cluster. (D) Expression trends of TGF-beta receptors and ligands along pseudotime trajectory in thyroid cells. (E) Schematic representing the thyroid differentiation protocol to test the impact of TGF-beta inhibition on thyroid organoid maturation. TGF-beta inhibition was induced by co-treatment with cAMP+SB431542 from day 22 to day 29. (F) qPCR analysis demonstrates significant upregulation in Pax8, Tg and Nis gene expression after 7 days of TGF-beta pathway inhibition. *p<0.05, ****p<0.001 by Mann-Whitney test (G–I) Functional organification assay confirms significant improvement in 125I uptake (G) and protein-bound 125I (H) under SB431542 treatment, which results in a higher percentage of cells with capacity of 125I organification (I). **p< 0.01, *p<0.05 by Student’s t-test. (J) Immunofluorescence staining demonstrating that SB431542 treatment does not affect thyroid follicular organization evidenced by Nis specific expression and Tg-I presence in the lumen space Scale bar: 20 µm and 10 µm (inset).