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Mammalian pre-implantation development is associated with striking metabolic robustness, as embryos can develop in a wide variety of nutrient conditions including even the complete absence of soluble amino acids. Here, we show that mouse embryonic stem cells (ESCs) capture the unique metabolic state of pre-implantation embryos and proliferate in the absence of several essential amino acids. Amino acid independence is enabled by constitutive uptake of exogenous protein through macropinocytosis alongside a robust lysosomal digestive system. Upon transition to more committed states, ESCs reduce digestion of extracellular protein and instead become reliant upon exogenous amino acids. Accordingly, amino acid withdrawal selects for ESCs that mimic the pre-implantation epiblast. More broadly, we find that all lineages of the pre-implantation blastocysts exhibit constitutive macropinocytotic protein uptake and digestion. Together, these results highlight exogenous protein uptake and digestion as an intrinsic feature of pre-implantation development and provide insight into the catabolic strategies that enable embryos to sustain viability prior to implantation.

The tricarboxylic acid (TCA) cycle is a central hub of cellular metabolism, oxidizing nutrients to generate reducing equivalents for energy production and critical metabolites for biosynthetic reactions. Despite the importance of TCA cycle products for cell viability and proliferation, mammalian cells display diversity in TCA cycle activity(1,2). How this diversity is achieved, and whether it is critical for establishing cell fate, remains poorly understood. Here we identify a non-canonical TCA cycle required for changes in cell state. Genetic co-essentiality mapping revealed a cluster of genes sufficient to compose a biochemical alternative to the canonical TCA cycle, wherein mitochondrially-derived citrate exported to the cytoplasm is metabolized via ATP citrate lyase (ACL), ultimately regenerating mitochondrial oxaloacetate to complete this non-canonical TCA cycle. Manipulating expression of ACL or the canonical TCA cycle enzyme aconitase 2 in mouse myoblasts and embryonic stem cells (ESCs) revealed that changes in TCA cycle configuration accompany cell fate transitions. During exit from pluripotency, ESCs switch from canonical to non-canonical TCA cycle metabolism; accordingly, blocking the non-canonical TCA cycle prevents cells from exiting pluripotency. These results establish a context-dependent alternative to the traditional TCA cycle and reveal that appropriate TCA cycle engagement is required for changes in cell state.