To investigate the role of T during the differentiation of hPSCs we combined T sOPTiKD sublines with culture conditions known to drive the differentiation of hPSCs into subpopulations that recapitulate different portions of the primitive streak and their derived lineages (Fig. 5A) (Bernardo et al., 2011; Cheung et al., 2012; Mendjan et al., 2014; Touboul et al., 2010). Inducible knockdown of T was robust in all cell types analyzed (Fig. 5B,C, Fig. S5A,B), confirming the efficiency of sOPTiKD to knock down developmental genes. Decrease in T expression did not affect definitive endoderm specification, while differentiation into posterior primitive streak cells, cardiac mesoderm and lateral plate mesoderm was mildly impaired (Fig. 5D, Fig. S5C). By contrast, the generation of late primitive streak progenitors (recapitulating the onset of somitogenesis) and their further specification into presomitic mesoderm and chondrocytes were severely affected following inducible knockdown of T (Fig. 5D-G). In particular, induction of TBX6, MSGN1 and MEOX1 was nearly abolished, in agreement with the established role of T in the expression of such genes (Chapman et al., 1996; Faial et al., 2015; Martin, 2015).
