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Recent advances in human blastoids have opened new avenues for modeling early human development and implantation. One limitation of our first protocol for human blastoid generation was relatively low efficiency. We now report an optimized protocol for the efficient generation of large quantities of high-fidelity human blastoids from naive pluripotent stem cells. This enabled proteomics analysis that identified phosphosite-specific signatures potentially involved in the derivation and/or maintenance of the signaling states in human blastoids. Additionally, we uncovered endometrial stromal effects in promoting trophoblast cell survival, proliferation, and syncytialization during co-culture with blastoids and blastocysts. Side-by-side single-cell RNA sequencing revealed similarities and differences in transcriptome profiles between pre-implantation blastoids and blastocysts, as well as post-implantation cultures, and uncovered a population resembling early migratory trophoblasts during co-culture with endometrial stromal cells. Our optimized protocol will facilitate broader use of human blastoids as an accessible, perturbable, scalable, and tractable model for human blastocysts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Dynamic pluripotent stem cell (PSC) states are in vitro adaptations of pluripotency continuum in vivo. Previous studies have generated a number of PSCs with distinct properties. To date, however, no known PSCs have demonstrated dual competency for chimera formation and direct responsiveness to primordial germ cell (PGC) specification, a unique functional feature of formative pluripotency. Here, by modulating fibroblast growth factor (FGF), transforming growth factor β (TGF-β), and WNT pathways, we derived PSCs from mice, horses, and humans (designated as XPSCs) that are permissive for direct PGC-like cell induction in vitro and are capable of contributing to intra- or inter-species chimeras in vivo. XPSCs represent a pluripotency state between naive and primed pluripotency and harbor molecular, cellular, and phenotypic features characteristic of formative pluripotency. XPSCs open new avenues for studying mammalian pluripotency and dissecting the molecular mechanisms governing PGC specification. Our method may be broadly applicable for the derivation of analogous stem cells from other mammalian species., Competing Interests: Declaration of Interests L.Y. and J.W. are inventors on a patent application arising from this work. The other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)