Primed human pluripotent stem cell-derived blastocyst-like cell aggregates with partial lineage specification.

Introduction: Human blastoids, blastocyst-like structures assembled Methods: We developed a method to generate human blastocyst-like cell aggregates directly from primed hPSCs using a thermoresponsive hydrogel. Generated blastocyst-like cell aggregates were evaluated for morphological features, lineage marker expression via immunocytochemistry, transcriptional profiles using single-cell RNA sequencing, and functional capacity through Results: Primed hPSC-derived blastocyst-like cell aggregates recapitulated key morphological features of human blastocysts, including cyst formation and spatial expression of epiblast, trophectoderm, and primitive endoderm markers. Single-cell RNA sequencing revealed that a subset of cells showed transcriptional profiles resembling epiblast-, trophectoderm-, and primitive endoderm-like cells although a substantial proportion remained undifferentiated. Functionally, blastocyst-like cell aggregates demonstrated Conclusions: This work introduces a more accessible platform for generating human blastocyst-like cell aggregates from primed hPSCs, broadening their utility for investigating early development events. Insights gained from blastocyst-like cell aggregates have the potential to advance the modelling of early-onset diseases, drive innovations in regenerative therapies, and contribute to the development of assisted reproductive technologies.