Modulation of FOXD3 activity in human embryonic stem cells directs pluripotency and paraxial mesoderm fates

Transcription factor Foxd3 has been described in model systems as a key member of the pluripotency network in mice as well as being involved in the formation of many critical vertebrate cell types in vivo. Yet virtually nothing is known about roles of FOXD3 in human development and conflicting reports exist regarding its expression in human embryonic stem cells (hESCs). We find that FOXD3 is expressed at both the RNA and protein levels in undifferentiated hESCs and report a Foxd3 expression domain in paraxial mesoderm derivatives of wild-type mouse embryos. Furthermore, increasing FOXD3 activity in hESCs is sufficient for rapid and specific generation of mesenchymal cell types of the paraxial mesoderm, even under pluripotency maintenance conditions. Gene expression diagnostic of chondroblasts, skeletal myoblasts, osteoblasts, and adipoblast is observed within 48 hours of FOXD3 induction, as are morphological and genetic hallmarks of epithelial-to-mesenchymal transition. FOXD3-overexpressing cells can be maintained for several passages, while downregulation of the transgene leads to further differentiation. Loss-of-function also leads to differentiation, toward endoderm and mesoderm. Taken together, these data indicate that a balance of FOXD3 activity is required to maintain pluripotency.