Human blastocysts are comprised of the first three cell lineages of the embryo: trophectoderm, epiblast and primitive endoderm, all of which are essential for early development and organ formation. However, due to ethical concerns and restricted access to human blastocysts, a comprehensive understanding of early human embryogenesis is still lacking. To bridge this knowledge gap, a reliable model system that recapitulates early stages of human embryogenesis is needed. Here we developed a three-dimensional (3D), two-step induction protocol for generating blastocyst-like structures (EPS-blastoids) from human extended pluripotent stem (EPS) cells. Morphological and single-cell transcriptomic analyses revealed that EPS-blastoids contain key cell lineages and are transcriptionally similar to human blastocysts. Furthermore, EPS-blastoids are similar with human embryos that were cultured for 8 or 10 days in vitro, in terms of embryonic structures, cell lineages and transcriptomic profiles. In conclusion, we developed a scalable system to mimic human blastocyst development, which can potentially facilitate the study of early implantation failure that induced by developmental defects at early stage.

Human cell lines, first cultured in the 1950s 1 , are indispensable in biomedical research. Today, a wide range of cell types are available, and sophisticated advanced 'omics' and visualization techniques allow for the routine assessment of cell identity and cellular responses 2 . However, the culture methods have remained relatively unchanged. Major advances in culture systems were made over three decades ago 3,4 , yet the old standard approach of batch cell culturethe culture of cells either in suspension or as adherent monolayers of cells in standard media [5][6][7] remains the predominant method in biomedical research.Culture media provides crucial nutrients, signalling molecules (such as growth factors), and suitable osmotic conditions. The gaseous and thermal environments of cell cultures are typically controlled by the incubator. The initial media conditions are generally stabilized by adjusting them to 18.6% O2 and a standard pH of 7.4, and this adjustment is achieved by adding a given amount of HCO3 − salt (a base), and by enriching the media with CO2 to a given percentage in the air (usually 5% or 10%). However, cell metabolism involves the exchange of gasesspecifically the release of CO2 and the consumption of O2and this can affect cellular growth via the alteration of, for example, the pH and the level of dissolved O2 (dO2) in the cellular microenvironment 8 . In theory, the equilibration of the medium with the gaseous and thermal environments of the incubator provides a way to reliably mimic O2, CO2 and HCO3 − homeostasis in metazoan body fluids. Yet this doesn't take into account the fact that homeostasis in a living mammal is supported by the active exchange of gases with the atmosphere. The absence of such active gas exchange in cell cultures suggests that, over time, cellular metabolic activities might acidify and deoxygenate the cellular microenvironment 8-10 , if intermittent monitoring and (when necessary) corrective action are not carried out.To mimic a physiological environment when using cell cultures, careful control over environmental factors (such as pH, CO2 and O2) is typically needed, in particular because even small deviations of environmental parameters from physiological levels may impair cellular function. For instance, in human blood, pH values below 7.2 (acidaemic conditions) and above 7.44 (alkalaemic conditions) can be fatal [11][12][13] . In cell cultures, the optimal growth of normal cells (that is, non-cancerous cells and non-transformed cells) occurs within a specific alkaline pH range, whereas cancer cells grow in a broader pH range that is shifted towards acidic values [14][15][16][17][18] . Cells have evolved mechanisms, including the use of Na + /H + antiporters or histone deacetylation, that restore the alkaline pH of the cytoplasm when the extracellular pH deviates from physiological levels [19][20][21][22][23][24][25] . However, such regulatory mechanisms require cellular energy, and changes in the acetylation state of chromatin can alter gene transcription and reduc...

Mammalian cell cultures are a keystone resource in biomedical research, but the results of published experiments often suffer from reproducibility challenges. This has led to a focus on the influence of cell culture conditions on cellular responses and reproducibility of experimental findings. Here, we perform frequent in situ monitoring of dissolved O2 and CO2 with optical sensor spots and contemporaneous evaluation of cell proliferation and medium pH in standard batch cultures of three widely used human somatic and pluripotent stem cell lines. We collate data from the literature to demonstrate that standard cell cultures consistently exhibit environmental instability, indicating that this may be a pervasive issue affecting experimental findings. Our results show that in vitro cell cultures consistently undergo large departures of environmental parameters during standard batch culture. These findings should catalyze further efforts to increase the relevance of experimental results to the in vivo physiology and enhance reproducibility.

The characterization, control, and reporting of environmental conditions in mammalian cell cultures is fundamental to ensure physiological relevance and reproducibility in basic and preclinical biomedical research. The potential issue of environment instability in routine cell cultures in affecting biomedical experiments was identified many decades ago. Despite existing evidence showing variable environmental conditions can affect a suite of cellular responses and key experimental readouts, the underreporting of critical parameters affecting cell culture environments in published experiments remains a serious problem. Here, we outline the main sources of potential problems, improved guidelines for reporting, and deliver recommendations to facilitate improved culture-system based research. Addressing the lack of attention paid to culture environments is critical to improve the reproducibility and translation of preclinical research, but constitutes only an initial step towards enhancing the relevance of in vitro cell cultures towards in vivo physiology.

Human blastocysts are comprised of the first three cell lineages of the embryo: trophectoderm, epiblast, and primitive endoderm, all of which are essential for early development and organ formation1,2. However, due to ethical concerns and restricted access to human blastocysts, we lack a comprehensive understanding of early human embryogenesis. To bridge this knowledge gap, we need a reliable model system that recapitulates early stages of human embryogenesis. Here we report a ~three-dimensional (3D), two-step induction protocol for generating blastocyst-like structures (EPS-blastoids) from human extended pluripotent stem (EPS) cells. Morphological and single-cell transcriptomic analyses revealed that EPS-blastoids contain key cell lineages and are transcriptionally similar to human blastocysts. Furthermore, EPS-blastoids also exhibited the developmental potential to undergo post-implantation morphogenesis in vitro to form structures with a cellular composition and transcriptome signature similar to human embryos that had been cultured in vitro for 8 or 10 days. In conclusion, human EPS-blastoids provide a new experimental platform for studying early developmental stages of the human embryo.