Generating Human Blastoids Following Transient Naïve Resetting of Primed Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) exist in at least two distinct states of pluripotency: naïve and primed. While naïve hPSCs possess the unique ability to generate blastocyst-like structures, they are often genetically and epigenetically unstable, which compromises the quality and developmental potential of naïve hPSC-derived blastoids. This protocol presents an optimized human blastoid protocol through a transient resetting method that converts primed hPSCs into a naïve-like state, addressing the stability issues associated with long-term naïve hPSC maintenance. The approach is compatible with both feeder-free and feeder-based culture systems and demonstrates high efficiency in generating human blastoids directly from primed hPSCs. This advancement provides a more robust and reliable strategy for blastoid formation, circumventing the limitations of suboptimal naïve hPSC cultures.