Optimized and efficient generation of cynomolgus monkey blastoids recapitulating blastocyst phenotype and molecular features

Cynomolgus monkey blastoids are promising models for investigating early primate embryogenesis, yet previous generation systems have suffered from low efficiency and the presence of undefined cell clusters, limiting their broader application due to high costs, time consumption, and ethical concerns. In this study, we established an optimized protocol for efficiently inducing cynomolgus monkey blastoids by systematically modifying key parameters based on established human blastoid protocols. The resulting cyBlastoids consistently exhibited an induction efficiency exceeding 70%, with morphology and cell lineage allocation closely resembling those of natural blastocysts. Immunofluorescence staining and transcriptomic analyses revealed that the ICM-like and TE-like cells within cyBlastoids shared high molecular similarity to their counterparts in natural embryos and previously reported cynomolgus monkey blastoids. Extended in vitro culture further demonstrated that cyBlastoids displayed typical morphological and lineage characteristics of post-implantation embryos. Our stable and efficient protocol provides a robust alternative for primate embryo research, facilitating reproducible studies on early embryonic development in non-human primates.