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Summary: Inhibitors of Mek1/2 and Gsk3β, known as 2i, and, together with leukemia inhibitory factor, enhance the derivation of embryonic stem cells (ESCs) and promote ground-state pluripotency (2i/L-ESCs). However, recent reports show that prolonged Mek1/2 suppression impairs developmental potential of ESCs, and is rescued by serum (S/L-ESCs). Here, we show that culturing ESCs in Activin A and BMP4, and in the absence of MEK1/2 inhibitor (ABC/L medium), establishes advanced stem cells derived from ESCs (esASCs). We demonstrate that esASCs contributed to germline lineages, full-term chimeras and generated esASC-derived mice by tetraploid complementation. We show that, in contrast to 2i/L-ESCs, esASCs display distinct molecular signatures and a stable hypermethylated epigenome, which is reversible and similar to serum-cultured ESCs. Importantly, we also derived novel ASCs (blASCs) from blastocysts in ABC/L medium. Our results provide insights into the derivation of novel ESCs with DNA hypermethylation from blastocysts in chemically defined medium. : In this article, Bao and colleagues show that ASCs cultured in Activin A and BMP4, and in the absence of MEK1/2 inhibitor, possess reversible epigenetic changes that extend their developmental potency, distinct transcriptome pattern, and a stable hypermethylated epigenome. Importantly, they also derived novel hypermethylated ASCs from blastocysts in ABC/L medium. Keywords: mouse, embryonic stem cells, epigenetics, hypermethylation, development, chemically defined, genomic imprinting, blastocyst, pluripotency, implantation