First, we generated DPY30 OPTiKD hESC sublines (Fig. S6A), and confirmed that inducible DPY30 knockdown in undifferentiated hESCs impaired the expression of pluripotency genes and triggered neuroectoderm differentiation (Fig. 6B, Fig. S6B), as shown previously (Bertero et al., 2015). We then analyzed the function of DPY30 during lineage specification by differentiating DPY30 OPTiKD hESCs into five different cell types while inducing DPY30 knockdown from the induction, specification or maturation stages (Fig. 6A). qPCR confirmed the decrease in DPY30 expression in all the cells generated (Fig. S6C-H). Interestingly, phenotypic analyses demonstrated that DPY30 knockdown from the early induction of cardiac specification impaired cardiomyocyte differentiation, as shown by the decrease in contractile markers (Fig. 6C). However, knockdown at later stages had no significant effects (Fig. 6C). A similar result was observed for the hepatocyte lineage, since decrease of DPY30 expression in endoderm progenitors led to extensive cell death at the anterior foregut stage thereby preventing further differentiation (Fig. S6I). Similarly, specification of pancreatic endocrine cells was also impaired by knockdown of DPY30 in the initial stage of differentiation (Fig. 6D). However, neither
