Self-renewing human naïve pluripotent stem cells dedifferentiate in 3D culture and form blastoids spontaneously
Human naïve pluripotent stem cells (hnPSCs) can generate integrated models of blastocysts termed blastoids upon switch to inductive medium. However, the underlying mechanisms remain obscure. Here we report that self-renewing hnPSCs spontaneously and efficiently give rise to blastoids upon three dimensional (3D) suspension  culture. The spontaneous blastoids mimic early stage human blastocysts in terms of structure, size, and transcriptome characteristics and are capable of progressing to post-implantation stages. This property is conferred by the glycogen synthase kinase-3 (GSK3) signalling inhibitor IM-12 present in 5iLAF self-renewing medium. IM-12 upregulates oxidative phosphorylation-associated genes that underly the capacity of hnPSCs to generate blastoids spontaneously. Starting from day one of self-organization, hnPSCs at the boundary of all 3D aggregates dedifferentiate into E5 embryo-like intermediates. Intermediates co-express SOX2/OCT4 and GATA6 and by day 3 specify trophoblast fate, which coincides with cavity and blastoid formation. In summary, spontaneous blastoid formation results from 3D culture triggering dedifferentiation of hnPSCs into earlier embryo-like intermediates which are then competent to segregate blastocyst fates. Overall design: We convert human primed pluripotent stem cells to a naïve state by GSK3 inhibitor CHIR99021 to IM-12 switch (CI-hnPSCs). We find that CI-hnPSCs spontaneously and efficiently generate blastocyst-like structures (blastoids) in the 5iLAF self-renewing medium. Removing BRAF and MEK inhibitors from 5iLAF, using 3iLAF from day 3 of blastoid formation results in hypoblast specification.