Abstract
Introduction: Human blastoids, blastocyst-like structures assembled in vitro, hold great promise for decoding the earliest steps of human development. However, most existing protocols rely on naïve human pluripotent stem cells (hPSCs), which are chromosomally unstable and technically demanding. 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 in vitro implantation assays. 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 in vitro implantation potential, trophoblast differentiation, and secretion of human chorionic gonadotropin. 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.