WNT inhibition blocks derivation of human embryonic stem cells, but confers a unique transcriptional state to established hESCs compatible with differentiation.

Human embryonic stem cells (hESCs) and their ability to differentiate to specific cell types hold great value for future clinical application. However, standard culture conditions maintain hESCs in a primed state, which bears heterogeneity and a tendency for spontaneous differentiation. To counter these drawbacks hESCs have been adapted to a naive state, but this has in turn restricted the efficiency of existing directed differentiation protocols.We show that inhibition of Wnt signaling (Wnt-i) during prolonged maintenance of hESCs resulted in a unique transcriptional profile with high levels of pluripotency markers, but reduced expression of differentiation markers. Moreover, neuronal differentiation of Wnt-i hESCs progressed in a similar manner to primed hESCs, unlike naive hESCs. Surprisingly, direct derivation of hESCs under Wnt-i conditions failed, suggesting that active Wnt signaling is necessary during the transition from inner cell mass to hESCs.We demonstrate that Wnt-i hESCs have a unique pluripotency state, with advantages to both primed and naïve state and may therefore be a more suitable starting point to further applications.