Figure 1.

Schematic diagram of the protocol used for in vitro differentiation of thyroid follicles from a tetracycline-inducible murine Nkx2-1-Pax8 ESC line. LIF, leukemia inhibitory factor; rhTSH, recombinant human thyroid stimulating hormone. Initial culturing of ESCs in hanging drops (without LIF supplementation) allows for embryoid body differentiation. Nkx2-1 and Pax8 are temporally induced by doxycycline, thereby directing differentiation toward a thyrocyte fate. Forced expression of Nkx2-1/Pax8 results in robust thyroid stimulating hormone receptor expression, suggesting cells are capable of responding to rhTSH. Therefore, the sequential treatment of embryoid bodies with Dox-rhTSH dramatically enhances thyrocyte lineage specification and the formation of follicle-like structures, which are subsequently isolated for transplantation [Ref. (4)].