AMPK-p38 axis converts human pluripotent stem cells to naive state

Pluripotent stem cells (PSCs) exist in either a "primed" state or a "naive" state. While several protocols are available to convert primed human PSCs (hPSCs) to the naive-state hPSCs, they often require multiple exogenous factors. Here, we show that the activation of AMP-activated protein kinase (AMPK) or its downstream p38 alone induces naive conversion. Primed hPSCs cultured with activated AMPK-p38 displayed key naive features, including naive marker expression, three-germ-layer differentiation, epigenomic resetting, and increased mitochondrial activity. An AMPK activator-5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR)-synergistically enhances the naive conversion efficiency of reported conversion protocols. Single-cell RNA sequencing (scRNA-seq) with RNA velocity analyses and Totem trajectory mapping identified an intermediate state bridging the primed and naive states. These cells showed three upregulated gene groups: (1) pluripotency genes (e.g., Pou5f1 and Nanog), (2) naive state-related genes (e.g., Dnmlt3l and Alpg), and (3) differentiation-suppressive genes (e.g., Rest and Hhla1). These findings establish a simple induction method that illuminates underlying mechanisms and enables broad applications through efficient naive conversion.