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Under certain culture conditions, naive human pluripotent stem cells can generate human blastocyst-like structures (called human blastoids). Human blastoids serve as an accessible model for human blastocysts and are amenable for large-scale production. Here, we describe a detailed step-by-step protocol for the robust and high-efficient generation of human blastoids from naive human pluripotent stem cells., (© 2023. Springer Science+Business Media, LLC.)

In vitro stem cell models that replicate human gastrulation have been generated, but they lack the essential extraembryonic cells needed for embryonic development, morphogenesis, and patterning. Here, we describe a robust and efficient method that prompts human extended pluripotent stem cells to self-organize into embryo-like structures, termed peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. Although peri-gastruloids are not viable due to the exclusion of trophoblasts, they recapitulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and undergoing early neurulation and organogenesis. Single-cell RNA-sequencing unveiled transcriptomic similarities between advanced human peri-gastruloids and primary peri-gastrulation cell types found in humans and non-human primates. This peri-gastruloid platform allows for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine., Competing Interests: Declaration of interests J.W. and L.L. are named inventors on a patent application (applied through the Board of Regents of The University of Texas System, application number 63/510,612; status of application pending) entitled “SYSTEM AND METHODS FOR GENERATION OF HUMAN FETAL PROGENITORS, TISSUES, AND ORGANS FROM CULTURED STEM CELLS” arising from this work. All other authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Here, we report that a chemical cocktail (LCDM: leukemia inhibitory factor [LIF], CHIR99021, dimethinedene maleate [DiM], minocycline hydrochloride), previously developed for extended pluripotent stem cells (EPSCs) in mice and humans, enables de novo derivation and long-term culture of bovine trophoblast stem cells (TSCs). Bovine TSCs retain developmental potency to differentiate into mature trophoblast cells and exhibit transcriptomic and epigenetic (chromatin accessibility and DNA methylome) features characteristic of trophectoderm cells from early bovine embryos. The bovine TSCs established in this study will provide a model to study bovine placentation and early pregnancy failure., Competing Interests: Declaration of interests Z.J. and Y.W are co-inventors on US provisional patent application 63/413,798 relating to bTSCs and uses thereof., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dynamic pluripotent stem cell (PSC) states are in vitro adaptations of the pluripotency continuum in vivo. Previous studies have generated a number of PSCs with distinct properties. By modulating the FGF, TGF-β, and WNT pathways, we have derived intermediate PSCs (FTW-PSCs) that are permissive for direct primordial germ cell-like cell (PGC-LC) induction in vitro. Here, we describe the method for derivation and maintenance of mouse and human FTW-PSCs, as well as PGC-LC induction from FTW-PSCs., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cell competition involves a conserved fitness-sensing process during which fitter cells eliminate neighbouring less-fit but viable cells 1 . Cell competition has been proposed as a surveillance mechanism to ensure normal development and tissue homeostasis, and has also been suggested to act as a barrier to interspecies chimerism 2 . However, cell competition has not been studied in an interspecies context during early development owing to the lack of an in vitro model. Here we developed an interspecies pluripotent stem cell (PSC) co-culture strategy and uncovered a previously unknown mode of cell competition between species. Interspecies competition between PSCs occurred in primed but not naive pluripotent cells, and between evolutionarily distant species. By comparative transcriptome analysis, we found that genes related to the NF-κB signalling pathway, among others, were upregulated in less-fit 'loser' human cells. Genetic inactivation of a core component (P65, also known as RELA) and an upstream regulator (MYD88) of the NF-κB complex in human cells could overcome the competition between human and mouse PSCs, thereby improving the survival and chimerism of human cells in early mouse embryos. These insights into cell competition pave the way for the study of evolutionarily conserved mechanisms that underlie competitive cell interactions during early mammalian development. Suppression of interspecies PSC competition may facilitate the generation of human tissues in animals.

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.)