Modelling post-implantation human development to yolk sac blood emergence.

Implantation of the human embryo begins a critical developmental stage that comprises profound events including axis formation, gastrulation and the emergence of haematopoietic system1,2. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons3-5. Stem cell models of human embryo have emerged to help unlock the mysteries of this stage6-16. Here we present a genetically inducible stem cell-derived embryoid model of early post-implantation human embryogenesis that captures the reciprocal codevelopment of embryonic tissue and the extra-embryonic endoderm and mesoderm niche with early haematopoiesis. This model is produced from induced pluripotent stem cells and shows unanticipated self-organizing cellular programmes similar to those that occur in embryogenesis, including the formation of amniotic cavity and bilaminar disc morphologies as well as the generation of an anterior hypoblast pole and posterior domain. The extra-embryonic layer in these embryoids lacks trophoblast and shows advanced multilineage yolk sac tissue-like morphogenesis that harbours a process similar to distinct waves of haematopoiesis, including the emergence of erythroid-, megakaryocyte-, myeloid- and lymphoid-like cells. This model presents an easy-to-use, high-throughput, reproducible and scalable platform to probe multifaceted aspects of human development and blood formation at the early post-implantation stage. It will provide a tractable human-based model for drug testing and disease modelling.

Pluripotent stem cell-derived model of the post-implantation human embryo.

The human embryo undergoes morphogenetic transformations following implantation into the uterus, but our knowledge of this crucial stage is limited by the inability to observe the embryo in vivo. Models of the embryo derived from stem cells are important tools for interrogating developmental events and tissue-tissue crosstalk during these stages1. Here we establish a model of the human post-implantation embryo, a human embryoid, comprising embryonic and extraembryonic tissues. We combine two types of extraembryonic-like cell generated by overexpression of transcription factors with wild-type embryonic stem cells and promote their self-organization into structures that mimic several aspects of the post-implantation human embryo. These self-organized aggregates contain a pluripotent epiblast-like domain surrounded by extraembryonic-like tissues. Our functional studies demonstrate that the epiblast-like domain robustly differentiates into amnion, extraembryonic mesenchyme and primordial germ cell-like cells in response to bone morphogenetic protein cues. In addition, we identify an inhibitory role for SOX17 in the specification of anterior hypoblast-like cells2. Modulation of the subpopulations in the hypoblast-like compartment demonstrates that extraembryonic-like cells influence epiblast-like domain differentiation, highlighting functional tissue-tissue crosstalk. In conclusion, we present a modular, tractable, integrated3 model of the human embryo that will enable us to probe key questions of human post-implantation development, a critical window during which substantial numbers of pregnancies fail.

Human embryoid bodies as a novel system for genomic studies of functionally diverse cell types.

Practically all studies of gene expression in humans to date have been performed in a relatively small number of adult tissues. Gene regulation is highly dynamic and context-dependent. In order to better understand the connection between gene regulation and complex phenotypes, including disease, we need to be able to study gene expression in more cell types, tissues, and states that are relevant to human phenotypes. In particular, we need to characterize gene expression in early development cell types, as mutations that affect developmental processes may be of particular relevance to complex traits. To address this challenge, we propose to use embryoid bodies (EBs), which are organoids that contain a multitude of cell types in dynamic states. EBs provide a system in which one can study dynamic regulatory processes at an unprecedentedly high resolution. To explore the utility of EBs, we systematically explored cellular and gene expression heterogeneity in EBs from multiple individuals. We characterized the various cell types that arise from EBs, the extent to which they recapitulate gene expression in vivo, and the relative contribution of technical and biological factors to variability in gene expression, cell composition, and differentiation efficiency. Our results highlight the utility of EBs as a new model system for mapping dynamic inter-individual regulatory differences in a large variety of cell types.

One major goal of human genetics is to understand how changes in the way genes are regulated affect human traits, including disease susceptibility. To date, most studies of gene regulation have been performed in adult tissues, such as liver or kidney tissue, that were collected at a single time point. Yet, gene regulation is highly dynamic and context-dependent, meaning that it is important to gather data from a greater variety of cell types at different stages of their development. Additionally, observing which genes switch on and off in response to external treatments can shed light on how genetic variation can drive errors in gene regulation and cause diseases. Stem cells can produce more cells like themselves or differentiate ­ acquire the characteristics ­ of many cell types. These cells have been used in the laboratory to research gene regulation. Unfortunately, these studies often fail to capture the complex spatial and temporal dynamics of stem cell differentiation; in particular, these studies are unable to observe gene regulation in the transient cell types that appear early in embryonic development. To overcome these limitations, scientists developed systems such as embryoid bodies: three-dimensional aggregates of stem cells that, when grown under certain conditions, spontaneously develop into a variety of cell types. Rhodes, Barr et al. wanted to assess the utility of embryoid bodies as a model to study how genes are dynamically regulated in different cell types, by different individuals who have distinct genetic makeups. To do this, they grew embryoid bodies made from human stem cells from different individuals to examine which genes switched on and off as the stem cells that formed the embryoid bodies differentiated into different types of cells. The results showed that it was possible to grow embryoid bodies derived from genetically distinct individuals that consistently produce diverse cell types, similar to those found during human fetal development. Rhodes, Barr et al.'s findings suggest that embryoid bodies are a useful model to study gene regulation across individuals with different genetic backgrounds. This could accelerate research into how genetics are associated with disease by capturing gene regulatory dynamics at an unprecedentedly high spatial and temporal resolution. Additionally, embryoid bodies could be used to explore how exposure to different environmental factors during early development affect disease-related outcomes in adulthood in different individuals.

Scalable in vitro production of defined mouse erythroblasts.

Mouse embryonic stem cells (mESCs) can be manipulated in vitro to recapitulate the process of erythropoiesis, during which multipotent cells undergo lineage specification, differentiation and maturation to produce erythroid cells. Although useful for identifying specific progenitors and precursors, this system has not been fully exploited as a source of cells to analyse erythropoiesis. Here, we establish a protocol in which characterised erythroblasts can be isolated in a scalable manner from differentiated embryoid bodies (EBs). Using transcriptional and epigenetic analysis, we demonstrate that this system faithfully recapitulates normal primitive erythropoiesis and fully reproduces the effects of natural and engineered mutations seen in primary cells obtained from mouse models. We anticipate this system to be of great value in reducing the time and costs of generating and maintaining mouse lines in a number of research scenarios.

SEPHS1 is dispensable for pluripotency maintenance but indispensable for cardiac differentiation in mouse embryonic stem cells.

Embryonic stem cells (ESCs) are derived from the inner cell mass of developing blastocysts, which have self-renewal ability and have the potential to develop or reconstitute into all embryonic lineages. Selenophosphate synthetase 1 (SEPHS1) is an essential protein in mouse early embryo development. However, the role of SEPHS1 in mouse ESCs remains to be elucidated. In this study, we generated Sephs1 KO ESCs and found that deficiency of SEPSH1 has little effect on pluripotency maintenance and proliferation. Notably, SEPHS1 deficiency impaired differentiation into three germ layers and gastruloid aggregation in vitro. RNA-seq analysis showed SEPHS1 is involved in cardiogenesis, verified by no beating signal in Sephs1 KO embryoid body at d10 and low expression of cardiac-related and contraction markers. Taken together, our results suggest that SPEHS1 is dispensable in ESC self-renewal, but indispensable in subsequent germ layer differentiation especially for functional cardiac lineage.

Induction of Rosette-to-Lumen stage embryoids using reprogramming paradigms in ESCs.

Blastocyst-derived stem cell lines were shown to self-organize into embryo-like structures in 3D cell culture environments. Here, we provide evidence that embryo-like structures can be generated solely based on transcription factor-mediated reprogramming of embryonic stem cells in a simple 3D co-culture system. Embryonic stem cells in these cultures self-organize into elongated, compartmentalized embryo-like structures reflecting aspects of the inner regions of the early post-implantation embryo. Single-cell RNA-sequencing reveals transcriptional profiles resembling epiblast, primitive-/visceral endoderm, and extraembryonic ectoderm of early murine embryos around E4.5-E5.5. In this stem cell-based embryo model, progression from rosette formation to lumenogenesis accompanied by progression from naïve- to primed pluripotency was observed within Epi-like cells. Additionally, lineage specification of primordial germ cells and distal/anterior visceral endoderm-like cells was observed in epiblast- or visceral endoderm-like compartments, respectively. The system presented in this study allows for fast and reproducible generation of embryo-like structures, providing an additional tool to study aspects of early embryogenesis.

Robotic high-throughput biomanufacturing and functional differentiation of human pluripotent stem cells.

Efficient translation of human induced pluripotent stem cells (hiPSCs) requires scalable cell manufacturing strategies for optimal self-renewal and functional differentiation. Traditional manual cell culture is variable and labor intensive, posing challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient- and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation and generated functional neurons, cardiomyocytes, and hepatocytes. To validate our approach, we compared robotic and manual cell culture operations and performed comprehensive molecular and cellular characterizations (e.g., single-cell transcriptomics, mass cytometry, metabolism, electrophysiology) to benchmark industrial-scale cell culture operations toward building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy.

Generation of Primordial Germ Cell-like Cells from iPSCs Derived from Turner Syndrome Patients.

Turner syndrome (TS) is a genetic disorder in females with X Chromosome monosomy associated with highly variable clinical features, including premature primary gonadal failure leading to ovarian dysfunction and infertility. The mechanism of development of primordial germ cells (PGCs) and their connection with ovarian failure in TS is poorly understood. An in vitro model of PGCs from TS would be beneficial for investigating genetic and epigenetic factors that influence germ cell specification. Here we investigated the potential of reprogramming peripheral mononuclear blood cells from TS women (PBMCs-TS) into iPSCs following in vitro differentiation in hPGCLCs. All hiPSCs-TS lines demonstrated pluripotency state and were capable of differentiation into three embryonic layers (ectoderm, endoderm, and mesoderm). The PGCLCs-TS recapitulated the initial germline development period regarding transcripts and protein marks, including the epigenetic profile. Overall, our results highlighted the feasibility of producing in vitro models to help the understanding of the mechanisms associated with germ cell formation in TS.

The Use of Induced Pluripotent Stem Cells to Study the Effects of Adenosine Deaminase Deficiency on Human Neutrophil Development.

Inherited defects that abrogate the function of the adenosine deaminase (ADA) enzyme and consequently lead to the accumulation of toxic purine metabolites cause profound lymphopenia and severe combined immune deficiency. Additionally, neutropenia and impaired neutrophil function have been reported among ADA-deficient patients. However, due to the rarity of the disorder, the neutrophil developmental abnormalities and the mechanisms contributing to them have not been characterized. Induced pluripotent stem cells (iPSC) generated from two unrelated ADA-deficient patients and from healthy controls were differentiated through embryoid bodies into neutrophils. ADA deficiency led to a significant reduction in the number of all early multipotent hematopoietic progenitors. At later stages of differentiation, ADA deficiency impeded the formation of granulocyte colonies in methylcellulose cultures, leading to a significant decrease in the number of neutrophils generated from ADA-deficient iPSCs. The viability and apoptosis of ADA-deficient neutrophils isolated from methylcellulose cultures were unaffected, suggesting that the abnormal purine homeostasis in this condition interferes with differentiation or proliferation. Additionally, there was a significant increase in the percentage of hyperlobular ADA-deficient neutrophils, and these neutrophils demonstrated significantly reduced ability to phagocytize fluorescent microspheres. Supplementing iPSCs and methylcellulose cultures with exogenous ADA, which can correct adenosine metabolism, reversed all abnormalities, cementing the critical role of ADA in neutrophil development. Moreover, chemical inhibition of the ribonucleotide reductase (RNR) enzyme, using hydroxyurea or a combination of nicotinamide and trichostatin A in iPSCs from healthy controls, led to abnormal neutrophil differentiation similar to that observed in ADA deficiency, implicating RNR inhibition as a potential mechanism for the neutrophil abnormalities. In conclusion, the findings presented here demonstrate the important role of ADA in the development and function of neutrophils while clarifying the mechanisms responsible for the neutrophil abnormalities in ADA-deficient patients.

Comparison of Differentiation Pattern and WNT/SHH Signaling in Pluripotent Stem Cells Cultured under Different Conditions.

Pluripotent stem cells (PSCs) are characterized by the ability to self-renew as well as undergo multidirectional differentiation. Culture conditions have a pivotal influence on differentiation pattern. In the current study, we compared the fate of mouse PSCs using two culture media: (1) chemically defined, free of animal reagents, and (2) standard one relying on the serum supplementation. Moreover, we assessed the influence of selected regulators (WNTs, SHH) on PSC differentiation. We showed that the differentiation pattern of PSCs cultured in both systems differed significantly: cells cultured in chemically defined medium preferentially underwent ectodermal conversion while their endo- and mesodermal differentiation was limited, contrary to cells cultured in serum-supplemented medium. More efficient ectodermal differentiation of PSCs cultured in chemically defined medium correlated with higher activity of SHH pathway while endodermal and mesodermal conversion of cells cultured in serum-supplemented medium with higher activity of WNT/JNK pathway. However, inhibition of either canonical or noncanonical WNT pathway resulted in the limitation of endo- and mesodermal conversion of PSCs. In addition, blocking WNT secretion led to the inhibition of PSC mesodermal differentiation, confirming the pivotal role of WNT signaling in this process. In contrast, SHH turned out to be an inducer of PSC ectodermal, not mesodermal differentiation.

Variant PCGF1-PRC1 links PRC2 recruitment with differentiation-associated transcriptional inactivation at target genes.

Polycomb repressive complexes-1 and -2 (PRC1 and 2) silence developmental genes in a spatiotemporal manner during embryogenesis. How Polycomb group (PcG) proteins orchestrate down-regulation of target genes upon differentiation, however, remains elusive. Here, by differentiating embryonic stem cells into embryoid bodies, we reveal a crucial role for the PCGF1-containing variant PRC1 complex (PCGF1-PRC1) to mediate differentiation-associated down-regulation of a group of genes. Upon differentiation cues, transcription is down-regulated at these genes, in association with PCGF1-PRC1-mediated deposition of histone H2AK119 mono-ubiquitination (H2AK119ub1) and PRC2 recruitment. In the absence of PCGF1-PRC1, both H2AK119ub1 deposition and PRC2 recruitment are disrupted, leading to aberrant expression of target genes. PCGF1-PRC1 is, therefore, required for initiation and consolidation of PcG-mediated gene repression during differentiation.

Tissue of Origin, but Not XCI State, Influences Germ Cell Differentiation from Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) are not only a promising tool to investigate differentiation to many cell types, including the germline, but are also a potential source of cells to use for regenerative medicine purposes in the future. However, current in vitro models to generate human primordial germ cell-like cells (hPGCLCs) have revealed high variability regarding differentiation efficiency depending on the hPSC lines used. Here, we investigated whether differences in X chromosome inactivation (XCI) in female hPSCs could contribute to the variability of hPGCLC differentiation efficiency during embryoid body (EB) formation. For this, we first characterized the XCI state in different hPSC lines by investigating the expression of XIST and H3K27me3, followed by differentiation and quantification of hPGCLCs. We observed that the XCI state did not influence the efficiency to differentiate to hPGCLCs; rather, hPSCs derived from cells isolated from urine showed an increased trend towards hPGCLCs differentiation compared to skin-derived hPSCs. In addition, we also characterized the XCI state in the generated hPGCLCs. Interestingly, we observed that independent of the XCI state of the hPSCs used, both hPGCLCs and soma cells in the EBs acquired XIST expression, indicative of an inactive X chromosome. In fact, culture conditions for EB formation seemed to promote XIST expression. Together, our results contribute to understanding how epigenetic properties of hPSCs influence differentiation and to optimize differentiation methods to obtain higher numbers of hPGCLCs, the first step to achieve human in vitro gametogenesis.

Myogenic Differentiation of iPS Cells Shows Different Efficiency in Simultaneous Comparison of Protocols.

Induced pluripotent stem (iPS) cells constitute a perfect tool to study human embryo development processes such as myogenesis, thanks to their ability to differentiate into three germ layers. Currently, many protocols to obtain myogenic cells have been described in the literature. They differ in many aspects, such as media components, including signaling modulators, feeder layer constituents, and duration of culture. In our study, we compared three different myogenic differentiation protocols to verify, side by side, their efficiency. Protocol I was based on embryonic bodies differentiation induction, ITS addition, and selection with adhesion to collagen I type. Protocol II was based on strong myogenic induction at the embryonic bodies step with BIO, forskolin, and bFGF, whereas cells in Protocol III were cultured in monolayers in three special media, leading to WNT activation and TGF-ß and BMP signaling inhibition. Myogenic induction was confirmed by the hierarchical expression of myogenic regulatory factors MYF5, MYOD, MYF6 and MYOG, as well as the expression of myotubes markers MYH3 and MYH2, in each protocol. Our results revealed that Protocol III is the most efficient in obtaining myogenic cells. Furthermore, our results indicated that CD56 is not a specific marker for the evaluation of myogenic differentiation.

Bioengineered embryoids mimic post-implantation development in vitro.

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. We employ a high-throughput cell aggregation approach to simultaneously coax mouse embryonic stem cells into hundreds of uniform epiblast-like aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro.

miRNAs as cell fate determinants of lateral and paraxial mesoderm differentiation from embryonic stem cells.

To date, the role of miRNAs on pluripotency and differentiation of ESCs into specific lineages has been studied extensively. However, the specific role of miRNAs during lateral and paraxial mesoderm cell fate decision is still unclear. To address this, we firstly determined miRNA profile of mouse ESCs differentiating towards lateral and paraxial lineages which were detected using Flk1 and PDGFαR antibodies, and of myogenic and hematopoietic differentiation potential of purified paraxial and lateral mesodermal cells within these populations. miRNAs associated with lateral and paraxial mesoderm, and their targets were identified using bioinformatics tools. The targets of the corresponding miRNAs were validated after transfection into mouse ESCs. The roles of the selected miRNAs in lateral, and paraxial mesoderm formation were assessed along with hematopoietic and myogenic differentiation capacity. Among the miRNAs, mmu-miR-126a-3p, mmu-miR-335-5p and mmu-miR-672-5p, upregulated in lateral mesoderm cells, and mmu-miR-10b-5p, mmu-miR-196a-5p and mmu-miR-615-3p, upregulated in paraxial mesoderm cells. While transient co-transfection of mmu-miR-126a-3p, mmu-miR-335-5p and mmu-miR-672-5p increased the number of lateral mesodermal cells, co-transfection of mmu-miR-10b-5p, mmu-miR-196a-5p and mmu-miR-615-3p increased the number of paraxial mesodermal cells. Moreover, differentiation potential of the lateral mesodermal cells into hematopoietic cell lineage increased upon co-transfection of mmu-miR-126a-3p, mmu-miR-335-5p and mmu-miR-672-5p and differentiation potential of the paraxial mesodermal cells into skeletal muscle lineage were increased upon co-transfection of mmu-miR-10b-5p, mmu-miR-196a-5p and mmu-miR-615-3p. In conclusion, we determined the miRNA profile of lateral and paraxial mesodermal cells and co-transfection of miRNAs increased differentiation potential of both lateral and paraxial mesodermal cells transiently.

The Presence or Absence of Alkaline Phosphatase Activity to Discriminate Pluripotency Characteristics in Porcine Epiblast Stem Cell-Like Cells.

Porcine embryonic stem cells (pESCs) would provide potentials for agricultural- and biotechnological-related applications. However, authentic pESCs have not been established yet because standards for porcine stem cell-specific markers and culture conditions are not clear. Therefore, the present study reports attempts to derive pluripotent epiblast stem cells either from in vitro or in vivo derived porcine embryos. Nine epiblast cell lines (seven lines from Berkshire and two lines from Duroc) could only be isolated from day 9- to 9.5-old in vivo derived early conceptuses. Pluripotency features were analyzed in relation to the presence or absence of alkaline phosphatase (AP) activity. Interestingly, the mRNA expression of several marker genes for pluripotency or epiblast was different between putative epiblast stem cells of the two groups [AP-positive (+) pEpiSC-like cell 2 line and AP-negative (-) pEpiSC-like cell 8 line]. For example, expressions of OCT-3/4, NANOG, SOX2, c-MYC, FGF2, and NODAL in AP-negative (-) porcine epiblast stem cell (pEpiSC)-like cells were higher than those in AP-positive (+) pEpiSC-like cells. Expression of surface markers differed between the two groups to some extent. SSEA-1 was strongly expressed only in AP-negative (-) pEpiSC-like cells, whereas AP-positive (+) pEpiSC-like cells did not express. In addition, we report to have some differences in the in vitro differentiation capacity between AP-positive (+) and AP-negative (-) epiblast cell lines. Primary embryonic germ layer markers (cardiac actin, nestin, and GATA 6) and primordial germ cell markers (Dazl and Vasa) were strongly expressed in embryoid bodies (EBs) aggregated from AP-negative (-) pEpiSC-like cells, whereas EBs aggregated from AP-positive (+) pEpiSCs did not show expression of primary embryonic germ layers and primordial germ cell markers except GATA 6. These results indicate that pEpiSC-like cells display different pluripotency characteristics in relation to AP activity.

Differentiation of embryonic stem cells into a putative hair cell-progenitor cells via co-culture with HEI-OC1 cells.

Several studies have shown how different cell lines can influence the differentiation of stem cells through co-culture systems. The House Ear Institute-Organ of Corti 1 (HEI-OC1) is considered an important cell line for in vitro auditory research. However, it is unknown if HEI-OC1 cells can promote the differentiation of embryonic stem cells (ESCs). In this study, we investigated whether co-culture of ESCs with HEI-OC1 cells promotes differentiation. To this end, we developed a co-culture system of mouse ESCs with HEI-OC1 cells. Dissociated or embryonic bodies (EBs) of ESCs were introduced to a conditioned and inactivated confluent layer of HEI-OC1 cells for 14 days. The dissociated ESCs coalesced into an EB-like form that was smaller than the co-cultured EBs. Contact co-culture generated cells expressing several otic progenitor markers as well as hair cell specific markers. ESCs and EBs were also cultured in non-contact setup but using conditioned medium from HEI-OC1 cells, indicating that soluble factors alone could have a similar effect. The ESCs did not form into aggregates but were still Myo7a-positive, while the EBs degenerated. However, in the fully differentiated EBs, evidence to prove mature differentiation of inner ear hair cell was still rudimentary. Nevertheless, these results suggest that cellular interactions between ESCs and HEI-OC1 cells may both stimulate ESC differentiation.

Application of FluoVolt Membrane Potential Dye for Induced Pluripotent Stem Cell-Derived Cardiac Single Cells and Monolayers Differentiated via Embryoid Bodies.

FluoVolt, a membrane potential dye, has been used to depict the action potentials of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) in order to classify the cardiac cell subtype, evaluate long QT syndrome, and conduct cardiotoxic drug-responsive tests. To apply FluoVolt, users must prepare the hiPSC-CMs, assess the dye loadings, and apply the excitation light. Here we describe the steps to measure action potentials from single hiPSC-CMs and hiPSC-CM monolayers using this dye.

Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre.

Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.

The ethics of human-embryoids model: a call for consistency.

In this article, we discuss the ethics of human embryoids, i.e., embryo-like structures made from pluripotent stem cells for modeling natural embryos. We argue that defining our social priorities is critical to design a consistent ethical guideline for research on those new entities. The absence of clear regulations on these emerging technologies stems from an unresolved debate surrounding natural human embryo research and one common opinion that one needs to solve the question of the moral status of the human embryo before regulating their surrogate. The recent NIH funding restrictions for research on human embryoids have made scientists even more unlikely to raise their voices. As a result, the scientific community has maintained a low profile while longing for a more favorable socio-political climate for their research. This article is a call for consistency among biomedical research on human materials, trying to position human embryoids within a spectrum of existing practice from stem cell research or IVF to research involving human subjects. We specifically note that the current practices in infertility clinics of freezing human embryos or disposing of them without any consideration for their potential benefits contradicts the assumption of special consideration for human material. Conversely, creating human embryoids for research purposes could ensure that no human material be used in vain, always serving humankind. We argue here that it is time to reconsider the full ban on embryo research (human embryos and embryoids) beyond the 14-day rule and that research on those entities should obey a sliding scale combining the completeness of the model (e.g., complete vs. partial) and the developmental stage: with more advanced completeness and developmental stage of the considered entity, being associated with more rigorous evaluation of societal benefits, statements of intention, and necessity of such research.