Rapid and efficient generation of human 8-cell-like cells for embryo modelling

Abstract

8-cell blastomeres of human embryos possess broad lineage potential and undergo major zygotic genome activation (ZGA), yet experimental access to this transient cell state remains limited. Rare 8-cell-like cells (8CLCs) arise spontaneously in naive pluripotent stem cell cultures, but their low abundance has constrained mechanistic and functional studies. Here, we develop a chemically defined strategy for rapid and robust induction of 8CLCs. Through sequential small-molecule screens focused on chromatin regulators, we identify five compounds acting through distinct pathways that generate up to 40% 8CLCs within 48 hours. The resulting cells, which we term rapidly induced 8CLCs (ri8CLCs), recapitulate key molecular features of 8-cell blastomeres, including induction of ZGA-associated genes, cleavage-stage transposable elements, and 8-cell-stage transcriptional signatures in bulk and single-cell transcriptomic analyses. Functionally, ri8CLCs exhibit enhanced developmental competence, acquiring the ability for spontaneous extraembryonic differentiation and assembly into well-cavitated blastoids on an accelerated 72-hour timeline. Notably, ri8CLC induction enables blastoid formation even in the absence of MEK inhibition, TGF-{beta}/Activin/Nodal inhibition and exogenous LIF, revealing a developmental competence consistent with an early embryonic state. Together, these findings establish a rapid, defined, and highly efficient platform for generating human ri8CLCs and provide a tractable model for studying early human embryogenesis.