SUMMARY Self-renewal of human embryonic stem (ES) cells is promoted by FGF and TGFβ/Activin signaling, and differentiation is promoted by BMP signaling, but how these signals regulate genes critical to the maintenance of pluripotency has been unclear. Using a defined medium, we show here that both TGFβ and FGF signals synergize to inhibit BMP signaling, sustain expression of pluripotency-associated genes such as NANOG, OCT4, and SOX2, and promote long-term undifferentiated proliferation of human ES cells. We also show that both TGFβ- and BMP-responsive SMADs can bind with the NANOG proximal promoter. NANOG promoter activity is enhanced by TGFβ/Activin and FGF signaling, and is decreased by BMP signaling. Mutation of putative SMAD binding elements reduces NANOG promoter activity to basal levels, and makes NANOG unresponsive to BMP and TGFβ signaling. These results suggest that direct binding of TGFβ/Activin-responsive SMADs to the NANOG promoter plays an essential role in sustaining human ES cell self-renewal.

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Human pluripotent stem cells (hPSCs), including embryonic stem (ES) and induced pluripotent stem (iPS) cells, have been historically cultured in media containing xenogeneic animal components. As hPSC-derived cells and tissues are being developed for human therapies, the application of culture systems to reduce potential immunoreactivity and improve reproducibility becomes increasingly vital. Methods for directly adapting hPSCs to a commercially available culture system free of nonhuman proteins (xeno-free) are described in this chapter.