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Forkhead box D3 (FOXD3) is a key transcription factor maintaining pluripotency in mouse embryonic stem cells (ESCs). Yet to date studies on its role in human ESCs are quite limited. In this study, we report that deletion of FOXD3 in human ESCs results in loss of pluripotency and spontaneous differentiation toward meso-endoderm. Ectopic overexpression of FOXD3 in hESCs leads to two different phenotypes: Human ESCs expressing high levels of FOXD3 undergo spontaneous meso-endoderm differentiation, whereas those with lower levels of FOXD3 maintain pluripotency. Next we deleted endogenous FOXD3 in the low ectopic expression model and find that addition of exogenous FOXD3 at a low level could rescue FOXD3-deficiency phenotype in hESCs. In summary, our findings suggest that FOXD3 dose-dependently regulates the balance of human ESCs between pluripotency and meso-endoderm fates, which adds to our understanding of the role of FOXD3 in humans. • FOXD3 is required to maintain pluripotency in hESCs, and deletion of FOXD3 in hESCs induces spontaneous differentiation toward meso-endoderm lineages. • Overexpression of exogenous FOXD3 leads to two distinct dose-dependent phenotypes: 1) meso-endoderm differentiated cells associated with higher expression levels of FOXD3 and 2) undifferentiated colonies associated with lower expression levels of FOXD3 • FOXD3 functions in a dose-dependent manner to balance the hESCs between pluripotency and meso-endoderm differentiation. [ABSTRACT FROM AUTHOR]

The placenta is an essential organ for the fetus, but its regulatory mechanism for formation of functional trophoblast lineage remains elusive in humans. Although widely known in mice, TEAD4 and its downstream targets CDX2 and GATA3 have not been determined in human models. In this work, we used a human model of trophoblast transition from BAP (BMP4, A83-01 and PD173074)-treated human embryonic stem cells (hESCs) and performed multiple gain- and loss-of-function tests of TEAD4, CDX2 or GATA3 to study their roles during this process. Although hESCs with TEAD4 deletion maintain pluripotency, their trophoblast transition potentials are attenuated. This impaired trophoblast transition could be rescued by separately overexpressing TEAD4 , CDX2 or GATA3. Furthermore, trophoblast transition from hESCs is also attenuated by knockout of CDX2 but remains unaffected with deletion of GATA 3. However, CDX2 -overexpressed hESCs maintain pluripotency, whereas overexpression of GATA3 in hESCs leads to spontaneous differentiation including trophoblast lineage. In brief, our findings using a human model of trophoblast transition from BAP-treated hESCs reveal transcription roles of TEAD4 , CDX2 and GATA in humans that are different from those in mice. We hope that this evidence can aid in understanding the distinct transcriptional network regulating trophoblast development in humans. • Although hESCs with TEAD4 deletion maintain in pluripotency, their potential of trophoblast transition is attenuate. • Moreover, overexpressing TEAD4, CDX2 or GATA3 rescues this defect phenotype. • hESCs show impaired potential of trophoblast transition with deletion of CDX2 but retain normally lacking GATA3. • Overexpression of GATA3 not CDX2 leads to spontaneous and non-directional trophoblast transition from hESCs without BMP4. [ABSTRACT FROM AUTHOR]