Pyruvate metabolism modulates cell fates in naïve human pluripotent stem cell culture and blastoid formation

Naïve human pluripotent stem cells (nhPSCs) have advantages over primed hPSCs (phPSCs) in modeling early human embryogenesis and constructing blastoids. However, current protocols of nhPSC derivation and maintenance are not optimal, mainly due to poor understanding of molecular basis of hPSC primed-to-naïve conversion. Here, we investigate the dynamic process of the conversion at a single cell level, focusing on the early stage. Two sequential cell fate bifurcations are identified. An alive/dead cell fate bifurcation happens at the earliest stage. Pyruvate supplementation significantly enhances hPSC survival. The second bifurcation occurs afterwards and determines the choice between naïve pluripotency and extra-embryonic lineage-like fates. Inhibiting pyruvate uptake into mitochondria yields significantly more naïve colonies. Based on these findings, we develop optimized protocols for nhPSC derivation and maintenance as well as human blastoid formation. Together, this study comprehensively outlines a cell fate trajectory of hPSC primed-to-naïve conversion, improving nhPSC culture and human blastoid formation. Pyruvate-related metabolism is a pivotal factor in regulating the cell fate determinations of the two cell fate bifurcations during human pluripotent stem cell primed-to-naïve conversion.