Dual role of lipids for genome stability and pluripotency facilitates full potency of mouse embryonic stem cells

Abstract

While Mek1/2 and Gsk3β inhibition (“2i”) supports the maintenance of murine embryonic stem cells (ESCs) in a homogenous naïve state, prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential. Additionally, 2i fails to support derivation and culture of fully potent female ESCs. Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin (AlbuMAX) undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture. Mechanistically, lipids in AlbuMAX impact intracellular metabolism including nucleotide biosynthesis, lipid biogenesis, and TCA cycle intermediates, with enhanced expression of DNMT3s that prevent DNA hypomethylation. Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation, which also alleviates X chromosome loss in female ESCs. Importantly, both male and female “all-ESC” mice can be generated from de novo derived ESCs using AlbuMAX-based media. Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.

ERas-Null Mice Generated Directly from Embryonic Stem Cells in a Lipid-rich Medium Enable the Discovery of A Novel Role in Craniofacial Development

“…Supplementing the standard 2i/LIF medium with lipid-rich albumin, AlbuMAX, enhances long-term genome stability while promoting a transition in pluripotency to a formative-like state (11). In line with previous study, the ERas gene exhibits a notable increase in expression in formative-like ESCs cultured in the 2iLA medium ( Fig.…”

“…Our system, which enables the generation of both male and female isogenic all-ESC mice, offers a valuable approach for studying the function of genes involved in early embryonic development and provides an alternative method for expediting the construction of GM mouse models. ERas is particularly upregulated in ESCs cultured in lipid-rich 2iLA medium (11), which is associated with lipid-induced pluripotency transitions. Both ERas-null male and female all-ESC pups exhibit a non-Mendelian phenotype characterized by fatal craniofacial abnormalities.…”

“…However, prolonged culture of mouse ESCs in 2i/LIF unfortunately leads to aneuploidy, DNA hypomethylation, and loss of imprinting, which impairs the 4n competency (9)(10)(11).…”

“…However, prolonged culture of mouse ESCs in 2i/LIF unfortunately leads to aneuploidy, DNA hypomethylation, and loss of imprinting, which impairs the 4n competency (9–11). We recently reported that supplementing lipid-rich albumin (AlbuMAX) into 2i/LIF medium (2iLA) can significantly improve the genomic stability and 4n competency of mouse ESCs over long-term culture in vitro (11).…”

“…However, prolonged culture of mouse ESCs in 2i/LIF unfortunately leads to aneuploidy, DNA hypomethylation, and loss of imprinting, which impairs the 4n competency (9–11). We recently reported that supplementing lipid-rich albumin (AlbuMAX) into 2i/LIF medium (2iLA) can significantly improve the genomic stability and 4n competency of mouse ESCs over long-term culture in vitro (11). Our innovative culture medium, combined with the technology to generate isogenic male and female all-ESC mice, provides a feasible approach to expedite the generation of GM mouse model from ESCs.…”