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The precise relationship of embryonic stem cells (ESCs) to cells in the mouse embryo remains controversial. We present transcriptional and functional data to identify the embryonic counterpart of ESCs. Marker profiling shows that ESCs are distinct from early inner cell mass (ICM) and closely resemble pre-implantation epiblast. A characteristic feature of mouse ESCs is propagation without ERK signalling. Single-cell culture reveals that cell-autonomous capacity to thrive when the ERK pathway is inhibited arises late during blastocyst development and is lost after implantation. The frequency of deriving clonal ESC lines suggests that all E4.5 epiblast cells can become ESCs. We further show that ICM cells from early blastocysts can progress to ERK independence if provided with a specific laminin substrate. These findings suggest that formation of the epiblast coincides with competence for ERK-independent self-renewal in vitro and consequent propagation as ESC lines.

Human naïve pluripotent stem cells (PSCs) share features with the pre-implantation epiblast. They therefore provide an unmatched opportunity for characterising the developmental programme of pluripotency in Homo sapiens. Here, we confirm that naïve PSCs do not respond directly to germ layer induction, but must first acquire competence. Capacitation for multi-lineage differentiation occurs without exogenous growth factor stimulation and is facilitated by inhibition of Wnt signalling. Whole-transcriptome profiling during this formative transition highlights dynamic changes in gene expression, which affect many cellular properties including metabolism and epithelial features. Notably, naïve pluripotency factors are exchanged for postimplantation factors, but competent cells remain devoid of lineage-specific transcription. The gradual pace of transition for human naïve PSCs is consistent with the timespan of primate development from blastocyst to gastrulation. Transcriptome trajectory during in vitro capacitation of human naïve cells tracks the progression of the epiblast during embryogenesis in Macaca fascicularis, but shows greater divergence from mouse development. Thus, the formative transition of naïve PSCs in a simple culture system may recapitulate essential and specific features of pluripotency dynamics during an inaccessible period of human embryogenesis., Highlighted Article: Human naïve pluripotent stem cells acquire competence for multilineage differentiation following a developmental trajectory that is comparable with the primate embryonic epiblast, and is facilitated by inhibition of the Wnt pathway.

The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation., Development, 146, ISSN:0950-1991, ISSN:1477-9129

In the mammalian embryo, epiblast cells must exit their naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro . Here we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large-scale transcriptomics and computational systems-biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 knockouts (KOs) predominantly manifest delays on the trajectory from naive to formative epiblast. We identified 374 naïve-associated genes (NAGs), which are tightly connected to the epiblast state and largely conserved in human stem cells and primate embryos. Integrated analysis of mutant transcriptomes revealed that the activity of multiple genes promoting pluripotency progression is funneled into discrete regulatory modules. We demonstrate that these modules are under control of five signaling pathways that operate in parallel to direct this pivotal mammalian cell state transition.

SUMMARY Classical mouse embryology has established a sequence of lineage bifurcations underpinning early mammalian development. Consistent with this paradigm, mouse embryonic stem cells have lost the capacity to generate extraembryonic trophectoderm. We show here, however, that human naïve epiblast stem cells readily produce this lineage. Inhibition of ERK signalling, fundamental to naïve cell propagation, is unexpectedly instrumental in trophectoderm induction. Transcriptome analyses authenticate trophoblast fate and expose a trajectory via reversion to inner cell mass (ICM). Nodal inhibition enhances differentiation and BMP signalling is not engaged. Strikingly, after formative transition primed stem cells cannot make trophectoderm but respond to BMP and form amnion. Gene perturbations in naïve cells reveal that YAP and TFAP2C promote trophectoderm as in mouse, while NANOG suppresses distinctively in human. Finally, ICMs from expanded human blastocysts efficiently regenerate trophectoderm. Thus in human, retained trophectoderm potential is an integral feature of emergent pluripotency that confers higher regulative plasticity.