Upon removal of culture conditions that maintain an undifferentiated state, mouse embryonic stem cells (ESCs) differentiate into various cell types. Differentiation can be facilitated by forced expression of certain transcription factors (TFs), each of which can generally specify a particular developmental lineage. We previously established 137 mouse ESC lines, each of which carried a doxycycline-controllable TF. Among them, Sox9 has unique capacity: its forced expression accelerates differentiation of mouse ESCs into cells of all three germ layers. With the additional use of specific culture conditions, overexpression of Sox9 facilitated the generation of endothelial cells, hepatocytes and neurons from ESCs. Furthermore, Sox9 action increases formation of p21 (WAF1/CIP1), which then binds to the SRR2 enhancer of pluripotency marker Sox2 and inhibits its expression. Knockdown of p21 abolishes inhibition of Sox2 and Sox9-accelerated differentiation, and reduction of Sox2 2 days after the beginning of ESC differentiation can comparably accelerate mouse ESC formation of cells of three germ layers. These data implicate the involvement of the p21-Sox2 pathway in the mechanism of accelerated ESC differentiation by Sox9 overexpression. The molecular cascade could be among the first steps to program ESC differentiation.
Author contributions
K.Y. performed all experiments and wrote the manuscript; D.S. supervised the project; M.S.H.K. supervised all experiments and wrote the manuscript.
Funding
This research was supported in part by the Intramural Research Program of the National Institutes of Health (NIH), National Institute on Aging. This research was also supported in part by the Japan Science and Technology Agency (JST), CREST program and Research Center Network for Realization of Regenerative Medicine program. K.Y. was supported by the postdoctoral fellowships from the Kanae Foundation (Japan), Uehara Memorial Foundation (Japan), Naito Foundation (Japan) and the Japan Society for Promotion of Science (JSPS). Deposited in PMC for release after 12 months.
