WNT Inhibition and Increased FGF Signaling Promotes Derivation of Less Heterogeneous Primed Human Embryonic Stem Cells, Compatible with Differentiation.

Abstract

Human embryonic stem cells (hESCs) hold great value for future clinical applications. However, standard culture conditions maintain hESCs in a primed state, which bears heterogeneity in pluripotency and a tendency for spontaneous differentiation. To counter these drawbacks, primed hESCs have been converted to a naive state, but this has restricted the efficiency of existing directed differentiation protocols. In mouse, WNT inhibition by inhibitor of WNT production-2, together with a higher dose of fibroblast growth factor 2 (12 ng/mL) in DMEM/F12 basal medium (DhiFI), markedly improved derivation and maintenance of primed mouse epiblast stem cells. In this study, we show that DhiFI conditions similarly improved primed hESC traits, such as conferring a primed transcriptional signature with high levels of pluripotency markers and reduced levels of differentiation markers. When triggered to differentiate to neuronal and cardiac lineages, DhiFI hESCs and isogenic primed hESCs progressed similarly. Moreover, DhiFI conditions supported the derivation of hESC lines from a post-inner cell mass intermediate (PICMI). DhiFI-derived hESCs showed less spontaneous differentiation and expressed significantly lower levels of lineage-specific markers, compared to primed-derived lines from the same PICMI. Overall, DhiFI hESCs retained advantages of both primed and naive pluripotency and may ultimately represent a more favorable starting point for differentiation toward clinically desired cell types.