Stepwise rolling back of human pluripotent stem cells to an 8-cell-like stage

Human pluripotent stem cells (PSCs) are a good proxy for studying early developmental processes and hold tremendous promise for regenerative medicine. Yet, their spectrum and potency remain less well characterized than in rodents, thwarting application. Particularly, in contrast to their mouse counterparts, human totipotent-like cells corresponding to the time of zygote genome activation (ZGA) or eight-cell (8C) stage have not yet been generated. Here, we report a quick and controllable, exogenous factor-free, methodology for producing cells transcriptionally and epigenetically similar to 8C embryonic cells from human PSCs. Single-cell multiomics characterization identified key molecular events associated with this conversion, including a partial shutdown of the pluripotency network. Loss-of-function experiments also showed fundamental roles for two factors highly enriched in 8C human embryos, DPPA3/STELLA (a master regulator of DNA methylation in oocytes) and TPRX1 (an ETCHbox family transcription factor lacking in mouse). DPPA3/STELLA is necessary for inducing passive DNA demethylation throughout the entire conversion process, whereas TPRX1 is a key regulator of the 8C-like cell (8CLC) conversion. Our approach provides a unique experimental window into the early events of human embryogenesis, which may be instrumental for producing functional artificial blastocysts (blastoids) and high-contribution interspecies chimeras.