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.

Keywords

mouse pluripotent stem cells / lipids / pluripotency transition / genomic stability / developmental potency / nucleotide pool depletion / 2i medium / X chromosome loss / female all-ESC mice

References

[1] Anderson DH. Role of lipids in the MAPK signaling pathway. Prog Lipid Res 2006;45:102–119.

[2] Arnold PK, Jackson BT, Paras KI, et al. A non-canonical tricarboxylic acid cycle underlies cellular identity. Nature 2022;603:477–481.

[3] Brind’Amour J, Liu S, Hudson M, et al. An ultra-low-input native ChIP-seq protocol for genome-wide profiling of rare cell populations. Nat Commun 2015;6:6033.

[4] Buehr M, Meek S, Blair K, et al. Capture of authentic embryonic stem cells from rat blastocysts. Cell 2008;135:1287–1298.

[5] Chen HX, Guo RP, Zhang Q, et al. Erk signaling is indispensable for genomic stability and self-renewal of mouse embryonic stem cells. P Natl Acad Sci USA 2015;112:E5936–E5943.

[6] Choi J, Huebner AJ, Clement K, et al. Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells. Nature 2017;548:219–223.

[7] Cornacchia D, Zhang C, Zimmer B, et al. Lipid deprivation induces a stable, naive-to-primed intermediate state of pluripotency in human PSCs. Cell Stem Cell 2019;25:120–136.e10.

[8] Czechanski A, Byers C, Greenstein I, et al. Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains. Nat Protoc 2014;9:559–574.

[9] Eggan K, Akutsu H, Loring J, et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci USA 2001;98:6209–6214.

[10] Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154–156.

[11] Fasullo M, Endres L. Nucleotide salvage deficiencies, DNA damage and neurodegeneration. Int J Mol Sci 2015;16:9431–9449.

[12] Garcia-Gonzalo FR, Belmonte JCI. Albumin-associated lipids regulate human embryonic stem cell self-renewal. PLoS One 2008;3.

[13] George SHL, Gertsenstein M, Vintersten K, et al. Developmental and adult phenotyping directly from mutant embryonic stem cells. Proc Natl Acad Sci USA 2007;104:4455–4460.

[14] Guo G, von Meyenn F, Santos F, et al. Naive pluripotent stem cells derived directly from isolated cells of the human inner cell mass. Stem Cell Rep 2016;6:437–446.

[15] Gupta A, Sharma S, Reichenbach P, et al. Telomere length homeostasis responds to changes in intracellular dNTP pools. Genetics 2013;193:10951095–1095101105.

[16] Hackett JA, Surani MA. Regulatory principles of pluripotency: from the ground state up. Cell Stem Cell 2014;15:416–430.

[17] Halliwell JA, Frith TJR, Laing O, et al. Nucleosides rescue replication- mediated genome instability of human pluripotent stem cells. Stem Cell Rep 2020;14:1009–1017.

[18] Hamilton WB, Brickman JM. Erk signaling suppresses embryonic stem cell self-renewal to specify endoderm. Cell Rep 2014;9:2056–2070.

[19] Han NR, Baek S, Kim HY, et al. Generation of embryonic stem cells derived from the inner cell mass of blastocysts of outbred ICR mice. Anim Cells Syst 2020;24:91–98.

[20] Kantor PF, Lucien A, Kozak R, et al. The antianginal drug trimetazidine shifts cardiac energy metabolism from fatty acid oxidation to glucose oxidation by inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase. Circ Res 2000;86:580–588.

[21] Kruszynska YT, Sherratt HSA. Glucose kinetics during acute and chronic treatment of rats with 2[6(4-chlorophenoxy) hexyl]oxirane-2-carboxylate, Etomoxir. Biochem Pharmacol 1987;36:3917–3921.

[22] Kunath T, Saba-El-Leil MK, Almousailleakh M, et al. FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development 2007;134:2895–2902.

[23] Lee KH, Chuang CK, Guo SF, et al. Simple and efficient derivation of mouse embryonic stem cell lines using differentiation inhibitors or proliferation stimulators. Stem Cells Dev 2012;21:373–383.

[24] Leeb M, Dietmann S, Paramor M, et al. Genetic exploration of the exit from self-renewal using haploid embryonic stem cells. Cell Stem Cell 2014;14:385–393.

[25] Li S, Lu YC, Peng BZ, et al. Crystal structure of human phosphoribosylpyrophosphate synthetase 1 reveals a novel allosteric site. Biochem J 2007;401:39–47.

[26] Li P, Tong C, Mehrian-Shai R, et al. Germline competent embryonic stem cells derived from rat blastocysts. Cell 2008;135:1299–1310.

[27] Maciejowski J, de Lange T. Telomeres in cancer: tumour suppression and genome instability (vol 18, pg 175, 2017). Nat Rev Mol Cell Biol 2019;20:259–259.

[28] Magdalou I, Lopez BS, Pasero P, et al. The causes of replication stress and their consequences on genome stability and cell fate. Semin Cell Dev Biol 2014;30:154–164.

[29] Martin GR. Isolation of a pluripotent cell-line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem-cells. Proc Natl Acad Sci Biol 1981;78:7634–7638.

[30] Nagy A, Rossant J, Nagy R, et al. Derivation of completely cell culture- derived mice from early-passage embryonic stem-cells. Proc Natl Acad Sci USA 1993;90:8424–8428.

[31] Pai CC, Kearsey SE. A critical balance: dNTPs and the maintenance of genome stability. Genes Basel 2017;8:57.

[32] Shimizu T, Ueda J, Ho JC, et al. Dual inhibition of Src and GSK3 maintains mouse embryonic stem cells, whose differentiation is mechanically regulated by Src signaling. Stem Cells 2012;30:1394–1404.

[33] Smith A. Formative pluripotency: the executive phase in a developmental continuum. Development 2017;144:365–373.

[34] Tee WW, Shen SS, Oksuz O, et al. Erk1/2 activity promotes chromatin features and RNAPII phosphorylation at developmental promoters in mouse ESCs. Cell 2014;156:678–690.

[35] Tsogtbaatar E, Landin C, Minter-Dykhouse K, et al. Energy metabolism regulates stem cell pluripotency. Front Cell Dev Biol 2020;8.

[36] Van der Jeught M, Taelman J, Duggal G, et al. Application of small molecules favoring naive pluripotency during human embryonic stem cell derivation. Cell Reprogram 2015;17:170–180.

[37] Visentin LP, Hasnain S, Gallin W, et al. Ribosomal-protein S1-S1a in bacteria. FEBS Lett 1977;79:258–263.

[38] Ware CB, Nelson AM, Mecham B, et al. Derivation of naive human embryonic stem cells. Proc Natl Acad Sci USA 2014;111:4484–4489.

[39] Wen D, Saiz N, Rosenwaks Z, et al. Completely ES cell-derived mice produced by tetraploid complementation using inner cell mass (ICM) deficient blastocysts. PLoS One 2014;9:e94730.

[40] Xu F, Deng C, Ren Z, et al. Lysophosphatidic acid shifts metabolic and transcriptional landscapes to induce a distinct cellular state in human pluripotent stem cells. Cell Rep 2021;37:110063.

[41] Yagi M, Kishigami S, Tanaka A, et al. Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation. Nature 2017;548:224–227.

[42] Ying QL, Wray J, Nichols J, et al. The ground state of embryonic stem cell self-renewal. Nature 2008;453:519–523.

[43] Yuan L, Liu JG, Hoja MR, et al. Female germ cell aneuploidy and embryo death in mice lacking the meiosis-specific protein SCP3. Science 2002;296:1115–1118.

[44] Zhang HL, Li YY, Ma YJ, et al. Epigenetic integrity of paternal imprints enhances the developmental potential of androgenetic haploid embryonic stem cells. Protein Cell 2022;13:102–119.

RIGHTS & PERMISSIONS

2023 The Author(s) 2023. Published by Oxford University Press on behalf of Higher Education Press.