Lipids Maintain Genomic Stability and Developmental Potency of Murine Pluripotent Stem Cells

Abstract

Lipids play vital roles in cellular homeostasis and regulate pluripotency of human stem cells. However, the impact of lipids on murine pluripotent stem cells is unclear. While Mek and Gsk3 inhibition supports the maintenance of murine embryonic stem cells in a homogenous naive 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 ZCAN4 and DNMT3s that prevent telomere shortening and DNA hypomethylation. In concert with 2i, lipids induce a formative-like pluripotent state through direct stimulation of Mek-mediated 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.

The role of lipids in genome integrity and pluripotency

“…However, extended Mek1/2 suppression of ESCs in this medium leads to aneuploidy, global DNA hypomethylation and loss of imprinting. This is typically more prominent for female ESCs, leading to rapid loss of pluripotency in early passages [ 15 , 16 , 23 , 24 ]. Our recent study has shown that supplementing the 2i/LIF medium with AX, can mitigate DNA hypomethylation and prevent loss of imprinting, thereby supporting the long-term culture of mESCs while preserving pluripotency [ 23 , 24 ].…”

“…This is typically more prominent for female ESCs, leading to rapid loss of pluripotency in early passages [ 15 , 16 , 23 , 24 ]. Our recent study has shown that supplementing the 2i/LIF medium with AX, can mitigate DNA hypomethylation and prevent loss of imprinting, thereby supporting the long-term culture of mESCs while preserving pluripotency [ 23 , 24 ]. In 2i/LIF cultures, the DNA methyltransferase 3 enzymes (DNMT3A, DNMT3B, DNMT3L), which add a methyl group to the carbon 5 position of cytosine residues in DNA — a process known as DNA methylation — are suppressed in ESCs.…”

“…In 2i/LIF cultures, the DNA methyltransferase 3 enzymes (DNMT3A, DNMT3B, DNMT3L), which add a methyl group to the carbon 5 position of cytosine residues in DNA — a process known as DNA methylation — are suppressed in ESCs. However, we found that the expression levels of these genes significantly increase when lipids are introduced to the 2i/LIF medium [ 23 , 24 ]. This observation is consistent with findings in human PSCs cultured in an AX-supplemented medium [ 27 ].…”

“…We recently discovered that adding lipid-rich albumin, specifically AlbuMAX (AX), to the standard 2i/LIF medium can significantly improve the genome stability and developmental potency. This addition also induces a pluripotency transition from naive to a formative-like state and enhances responsiveness to differentiation clues in mESCs [ 23 , 24 ]. We further demonstrated that it is the lipids, rather than the albumin in AX, that are responsible for these effects [ 23 , 24 ].…”

“…This addition also induces a pluripotency transition from naive to a formative-like state and enhances responsiveness to differentiation clues in mESCs [ 23 , 24 ]. We further demonstrated that it is the lipids, rather than the albumin in AX, that are responsible for these effects [ 23 , 24 ]. Similar to naive 2i/LIF-ESCs, AX-ESCs (ESCs cultured in AX-containing 2i/LIF medium) can generate all-ESC mice but possess distinct properties compared with both naive 2iL-ESCs and reported formative PSCs (fPSCs) [ 2 , 3 ].…”