Sequential TPRX1 and HERVL activation delineates a progression toward the totipotent-like state.

The in vitro induction of 8-cell-like cells (8CLCs) from human pluripotent stem cells (hPSCs) provides a model to study the regulatory principles of totipotency and zygotic genome activation (ZGA). However, the cellular dynamics underlying 8CLC emergence remain poorly understood. Here, we generate a TPRX1-EGFP/HERVL-tdTomato dual-reporter hPSC line to resolve the stepwise acquisition of totipotent-like identity. We show that 8CLC induction follows an ordered trajectory in which TPRX1 activation precedes the emergence of TPRX1+HERVL+ cells, which most closely resemble the in vivo 8-cell embryo transcriptionally. Single-cell transcriptomics and time-resolved flow cytometry further define a continuous transition from the primed state through a TPRX1+ intermediate to the HERVL+ 8CLC state. CRISPRi and RNA-directed perturbations support functional contributions of HERVL elements and HERVL-derived RNA to this transition, while potential locus-dependent mechanisms remain unresolved. Together, these findings establish sequential TPRX1 and HERVL activation as a defining feature of human 8CLC induction.