Toward developing human organs via embryo models and chimeras.

Developing functional organs from stem cells remains a challenging goal in regenerative medicine. Existing methodologies, such as tissue engineering, bioprinting, and organoids, only offer partial solutions. This perspective focuses on two promising approaches emerging for engineering human organs from stem cells: stem cell-based embryo models and interspecies organogenesis. Both approaches exploit the premise of guiding stem cells to mimic natural development. We begin by summarizing what is known about early human development as a blueprint for recapitulating organogenesis in both embryo models and interspecies chimeras. The latest advances in both fields are discussed before highlighting the technological and knowledge gaps to be addressed before the goal of developing human organs could be achieved using the two approaches. We conclude by discussing challenges facing embryo modeling and interspecies organogenesis and outlining future prospects for advancing both fields toward the generation of human tissues and organs for basic research and translational applications.

An ethical framework for human embryology with embryo models.

A human embryo's legal definition and its entitlement to protection vary greatly worldwide. Recently, human pluripotent stem cells have been used to form in vitro models of early embryos that have challenged legal definitions and raised questions regarding their usage. In this light, we propose a refined legal definition of an embryo, suggest "tipping points" for when human embryo models could eventually be afforded similar protection to that of embryos, and then revisit basic ethical principles that might help to draft a roadmap for the gradual, justified usage of embryo models in a manner that aims to maximize benefits to society.

Why study human embryo development?

Understanding the processes and mechanisms underlying early human embryo development has become an increasingly active and important area of research. It has potential for insights into important clinical issues such as early pregnancy loss, origins of congenital anomalies and developmental origins of adult disease, as well as fundamental insights into human biology. Improved culture systems for preimplantation embryos, combined with the new tools of single cell genomics and live imaging, are providing new insights into the similarities and differences between human and mouse development. However, access to human embryo material is still restricted and extended culture of early embryos has regulatory and ethical concerns. Stem cell-derived models of different phases of human development can potentially overcome these limitations and provide a scalable source of material to explore the early postimplantation stages of human development. To date, such models are clearly incomplete replicas of normal development but future technological improvements can be envisaged. The ethical and regulatory environment for such studies remains to be fully resolved.

Species Choice and Model Use: Reviving Research on Human Development.

While model organisms have had many historians, this article places studies of humans, and particularly our development, in the politics of species choice. Human embryos, investigated directly rather than via animal surrogates, have gone through cycles of attention and neglect. In the past 60 years they moved from the sidelines to center stage. Research was resuscitated in anatomy, launched in reproductive biomedicine, molecular genetics, and stem-cell science, and made attractive in developmental biology. I explain this surge of interest in terms of rivalry with models and reliance on them. The greater involvement of medicine in human reproduction, especially through in vitro fertilization, gave access to fresh sources of material that fed critiques of extrapolation from mice and met demands for clinical relevance or "translation." Yet much of the revival depended on models. Supply infrastructures and digital standards, including biobanks and virtual atlases, emulated community resources for model organisms. Novel culture, imaging, molecular, and postgenomic methods were perfected on less precious samples. Toing and froing from the mouse affirmed the necessity of the exemplary mammal and its insufficiency justified inquiries into humans. Another kind of model-organoids and embryo-like structures derived from stem cells-enabled experiments that encouraged the organization of a new field, human developmental biology. Research on humans has competed with and counted on models.

Regulating embryo models in the UK.

One of this century's most dramatic scientific developments is the reprogramming of stem cells in order to create self-organizing embryo-like entities, known as stem cell based embryo models (SCBEMs). The science is moving very quickly, but if, as increasingly appears to be the case, scientists are capable of creating entities that are effectively indistinguishable from sperm and egg derived embryos, important legal questions arise. In countries like the UK, where a strict regulatory regime applies to research on embryos, should this be extended to SCBEM research, or would a different regulatory response be appropriate? Drawing on the 1984 Warnock Report, the Human Fertilisation and Embryology Act 1990 and the latest guidelines from the International Society for Stem Cell Research, this article considers principles for the regulation of the creation and use of SCBEMs.

Monochorionic Twinning in Bioengineered Human Embryo Models.

Monochorionic twinning of human embryos increases the risk of complications during pregnancy. The rarity of such twinning events, combined with ethical constraints in human embryo research, makes investigating the mechanisms behind twinning practically infeasible. As a result, there is a significant knowledge gap regarding the origins and early phenotypic presentation of monochorionic twin embryos. In this study, a microthermoformed-based microwell screening platform is used to identify conditions that efficiently induce monochorionic twins in human stem cell-based blastocyst models, termed "twin blastoids". These twin blastoids contain a cystic GATA3+ trophectoderm-like epithelium encasing two distinct inner cell masses (ICMs). Morphological and morphokinetic analyses reveal that twinning occurs during the cavitation phase via splitting of the OCT4+ pluripotent core. Notably, each ICM in twin blastoids contains its own NR2F2+ polar trophectoderm-like region, ready for implantation. This is functionally tested in a microfluidic chip-based implantation assay with epithelial endometrium cells. Under defined flow regimes, twin blastoids show increased adhesion capacity compared to singleton blastoids, suggestive of increased implantation potential. In conclusion, the development of technology enabling large-scale formation of twin blastoids, coupled with high-sensitivity readout capabilities, presents an unprecedented opportunity for systematically exploring monochorionic twin formation and its impact on embryonic development.

A comprehensive review: synergizing stem cell and embryonic development knowledge in mouse and human integrated stem cell-based embryo models.

Mammalian stem cell-based embryo models have emerged as innovative tools for investigating early embryogenesis in both mice and primates. They not only reduce the need for sacrificing mice but also overcome ethical limitations associated with human embryo research. Furthermore, they provide a platform to address scientific questions that are otherwise challenging to explore in vivo. The usefulness of a stem cell-based embryo model depends on its fidelity in replicating development, efficiency and reproducibility; all essential for addressing biological queries in a quantitative manner, enabling statistical analysis. Achieving such fidelity and efficiency requires robust systems that demand extensive optimization efforts. A profound understanding of pre- and post-implantation development, cellular plasticity, lineage specification, and existing models is imperative for making informed decisions in constructing these models. This review aims to highlight essential differences in embryo development and stem cell biology between mice and humans, assess how these variances influence the formation of partially and fully integrated stem cell models, and identify critical challenges in the field.

Automated, High-Throughput Phenotypic Screening and Analysis Platform to Study Pre- and Post-Implantation Morphogenesis in Stem Cell-Derived Embryo-Like Structures.

Combining high-throughput generation and high-content imaging of embryo models will enable large-scale screening assays in the fields of (embryo) toxicity, drug development, embryogenesis, and reproductive medicine. This study shows the continuous culture and in situ (i.e., in microwell) imaging-based readout of a 3D stem cell-based model of peri-implantation epiblast (Epi)/extraembryonic endoderm (XEn) development with an expanded pro-amniotic cavity (PAC) (E3.5 E5.5), namely XEn/EPiCs. Automated image analysis and supervised machine learning permit the identification of embryonic morphogenesis, tissue compartmentalization, cell differentiation, and consecutive classification. Screens with signaling pathway modulators at different time windows provide spatiotemporal information on their phenotypic effect on developmental processes leading to the formation of XEn/EPiCs. Exposure of the biological model in the microwell platform to pathway modulators at two time windows, namely 0-72 h and 48-120 h, show that Wnt and Fgf/MAPK pathway modulators affect Epi differentiation and its polarization, while modulation of BMP and Tgfß/Nodal pathway affects XEn specification and epithelialization. Further, their collective role is identified in the timing of the formation and expansion of PAC. The newly developed, scalable culture and analysis platform, thereby, provides a unique opportunity to quantitatively and systematically study effects of pathway modulators on early embryonic development.

The fusion of physics and biology in early mammalian embryogenesis.

Biomechanics in embryogenesis is a dynamic field intertwining the physical forces and biological processes that shape the first days of a mammalian embryo. From the first cell fate bifurcation during blastulation to the complex symmetry breaking and tissue remodeling in gastrulation, mechanical cues appear critical in cell fate decisions and tissue patterning. Recent strides in mouse and human embryo culture, stem cell modeling of mammalian embryos, and biomaterial design have shed light on the role of cellular forces, cell polarization, and the extracellular matrix in influencing cell differentiation and morphogenesis. This chapter highlights the essential functions of biophysical mechanisms in blastocyst formation, embryo implantation, and early gastrulation where the interplay between the cytoskeleton and extracellular matrix stiffness orchestrates the intricacies of embryogenesis and placenta specification. The advancement of in vitro models like blastoids, gastruloids, and other types of embryoids, has begun to faithfully recapitulate human development stages, offering new avenues for exploring the biophysical underpinnings of early development. The integration of synthetic biology and advanced biomaterials is enhancing the precision with which we can mimic and study these processes. Looking ahead, we emphasize the potential of CRISPR-mediated genomic perturbations coupled with live imaging to uncover new mechanosensitive pathways and the application of engineered biomaterials to fine-tune the mechanical conditions conducive to embryonic development. This synthesis not only bridges the gap between experimental models and in vivo conditions to advancing fundamental developmental biology of mammalian embryogenesis, but also sets the stage for leveraging biomechanical insights to inform regenerative medicine.

Behind the developing brains and beating hearts of stem cell-derived embryo models.

Studies over the past decade have shown how stem cells representing embryonic and extra-embryonic tissues of the mouse can self-assemble in the culture dish to recapitulate an astonishing part of early embryonic development. A systematic analysis has demonstrated how pluripotent embryonic stem cells can be induced to behave like the implanting epiblast; how they can interact with trophectoderm stem cells to form a patterned structure resembling the implanting embryo prior to gastrulation; and how the third stem cell type-extra-embryonic endoderm cells-can be incorporated to generate structures that undergo the cell movements and gene expression patterns of gastrulation. Moreover, such stem cell-derived embryo models can proceed to neurulation and establish progenitors for all parts of the brain and neural tube, somites, beating heart structures and gut tube. They develop within extra-embryonic yolk sacs that initiate haematopoiesis. Here we trace this journey of discovery.

Stem cell-derived embryo models: a frontier of human embryology.

Studying human development remains difficult due to limited accessibility to human embryonic tissues. Prompted by the availability of human stem cells that share molecular and cellular similarities with embryonic and extraembryonic cells in peri-implantation human embryos, researchers have now successfully developed stem cell-based human embryo models that are promising as experimental tools for studying early human development. In this Perspective, we discuss the current progress in mouse and human stem cell-derived embryo models and highlight their promising applications in advancing the fundamental understanding of mammalian development.

Epigenetic regulation of early human embryo development.

Studies of mammalian development have advanced our understanding of the genetic, epigenetic, and cellular processes that orchestrate embryogenesis and have uncovered new insights into the unique aspects of human embryogenesis. Recent studies have now produced the first epigenetic maps of early human embryogenesis, stimulating new ideas about epigenetic reprogramming, cell fate control, and the potential mechanisms underpinning developmental plasticity in human embryos. In this review, we discuss these new insights into the epigenetic regulation of early human development and the importance of these processes for safeguarding development. We also highlight unanswered questions and key challenges that remain to be addressed.

Gastruloids: A Novel System for Disease Modelling and Drug Testing.

By virtue of its inaccessible nature, mammalian implantation stage development has remained one of the most enigmatic and hard to investigate periods of embryogenesis. Derived from pluripotent stem cells, gastruloids recapitulate key aspects of gastrula-stage embryos and have emerged as a powerful in vitro tool to study the architectural features of early post-implantation embryos. While the majority of the work in this emerging field has focused on the use of gastruloids to model embryogenesis, their tractable nature and suitability for high-throughput scaling, has presented an unprecedented opportunity to investigate both developmental and environmental aberrations to the embryo as they occur in vitro. This review summarises the recent developments in the use of gastruloids to model congenital anomalies, their usage in teratogenicity testing, and the current limitations of this emerging field.

Human embryos in a dish - modeling early embryonic development with pluripotent stem cells.

Stem cell-based embryo models present new opportunities to study early embryonic development. In a recent study, Kagawa et al. identified an approach to create human pluripotent stem cell-based blastoids that resemble the human blastocysts. These blastoids efficiently generated analogs of the EPI, TE, PrE lineages with transcriptomes highly similar to those found in vivo. Furthermore, the formation of these lineages followed the same sequence and pace of blastocyst development, and was also dependent on the same pathways required for lineage specification. Finally, the blastoids were capable of attaching to stimulated endometrial cells to mimic the process of implantation. While more comprehensive analysis is needed to confirm its validity and usefulness, this new blastoid system presents the latest development in the attempt to model early human embryogenesis in vitro.

Stem Cell-Based Embryo Models: En Route to a Programmable Future.

Early-stage human embryogenesis, such as implantation, gastrulation, and neurulation, are critical for successful pregnancy. For decades, our knowledge about these stages has been limited by the inaccessibility to such embryo specimens in vivo and the difficulty in rebuilding them in vitro. Although human embryos could be cultured in vitro beyond implantation, it remains challenging for the cultured embryos to recapitulate the continuous, coordinated morphogenesis and cytodifferentiation as seen in vivo. Stem cell-based embryo models, mainly derived from human pluripotent stem cells, are organized structures mimicking essential developmental processes in the early-stage human embryos. Despite their invaluable potentials, most embryo models are based on the self-organization of human pluripotent stem cells, which are limited in controllability, reproducibility, and developmental fidelity. Recently, the integration of bioengineered tools and stem cell biology has fueled a technological transformation towards programmable, highly complex, high-fidelity stem cell-based embryo models. Given its scientific and clinical significance, we present an overview of recent paradigm-shifting advances as well as historical perspectives regarding the past, present, and future of synthetic human embryology. Following the developmental roadmap of human embryogenesis, we critically review existing stem cell-based models for implantation, gastrulation, and neurulation, respectively. We highlight the limitations encountered by autonomous self-organization strategy and discuss the concept and application of guided cell organization as a game-changer for innovating next-generation embryo models. Future endeavors in synthetic human embryology should rationally leverage both the self-organizing power and programmable microenvironmental guidance to secure faithful reconstructions of the hierarchical orders of human embryogenesis in vitro.

Can we do that here? An analysis of US federal and state policies guiding human embryo and embryoid research.

Recent technological advances have helped scientists understand early human development. However, scientists' ability to fully explore their potential comes in conflict with national and state-level policies in the USA. In 2016, for the first time, researchers were able to grow human embryos in culture up to 14 days but stopped because of scientific and legal limits. Other researchers have used stem cells in culture to create organized models of early human development, known as embryoids or cell-based embryo models. In this paper, we review federal and state laws that affect US human embryo and embryoid research. While federal policies focus on funding, state laws are often associated with human embryonic stem cells, abortion, fetal tissue research, and reproductive cloning. Of the 29 states with laws impacting human embryo research, only 11 states ban it, and none address embryoids directly, although five states limit aspects of this research. Overall, this complicated landscape suggests that additional national guidance would help scientists and the public navigate these controversial areas of research, however, it is unlikely to happen, considering the lack of past progress determining embryo research policy.

Early human embryonic development: Blastocyst formation to gastrulation.

There has been recent renewed interest in studying human early embryonic development. The advent of improved culture conditions to maintain blastocysts in vitro for an extended period and the emerging stem-cell-based models of the blastocyst and peri-implantation embryos have provided new information that is relevant to early human embryogenesis. However, the mechanism of lineage development and embryonic patterning, and the molecular pathways involved in their regulation, are still not well understood. Interest in human embryonic development has been reinvigorated recently given numerous technical advances. In this review, Rossant and Tam discuss new insights into human embryogenesis gathered from successes in culturing human embryos in vitro and stem-cell-based embryo models. Then they outline what questions still need answering.

Human primed and naïve PSCs are both able to differentiate into trophoblast stem cells.

The recent derivation of human trophoblast stem cells (TSCs) from placental cytotrophoblasts and blastocysts opened opportunities for studying the development and function of the human placenta. Recent reports have suggested that human naïve, but not primed, pluripotent stem cells (PSCs) retain an exclusive potential to generate TSCs. Here we report that, in the absence of WNT stimulation, transforming growth factor ß (TGF-ß) pathway inhibition leads to direct and robust conversion of primed human PSCs into TSCs. The resulting primed PSC-derived TSC lines exhibit self-renewal, can differentiate into the main trophoblast lineages, and present RNA and epigenetic profiles that are indistinguishable from recently established TSC lines derived from human placenta, blastocysts, or isogenic human naïve PSCs expanded under human enhanced naïve stem cell medium (HENSM) conditions. Activation of nuclear Yes-associated protein (YAP) signaling is sufficient for this conversion and necessary for human TSC maintenance. Our findings underscore a residual plasticity in primed human PSCs that allows their in vitro conversion into extra-embryonic trophoblast lineages.

In vitro attachment and symmetry breaking of a human embryo model assembled from primed embryonic stem cells.

Our knowledge of the molecular mechanisms surrounding human embryo implantation and gastrulation is lacking, largely due to technical and ethical limitations of experimenting with human embryos. Alternatives to human embryos have been reported, in which 3D clusters of embryonic stem cells are differentiated in a stepwise manner to model aspects of human embryogenesis. Yet it remains challenging to model the events past attachment. We propose a strategy of modeling the post-attachment human embryo by assembling a pre-formed polarized epithelial epiblast and extraembryonic cells, allowing them to self-organize into a structure that mimics the dish-attached human embryo. The model attaches in vitro and, in the absence of exogenous morphogens, breaks anteroposterior symmetry, giving rise to early gastrulation cell types. Our assembloid approach enables in a modular way to upgrade or exchange extraembryonic tissues to access more advanced stages of post-attachment development while complying with ethical policies.

All models are wrong, but some are useful: Establishing standards for stem cell-based embryo models.

Detailed studies of the embryo allow an increasingly mechanistic understanding of development, which has proved of profound relevance to human disease. The last decade has seen in vitro cultured stem cell-based models of embryo development flourish, which provide an alternative to the embryo for accessible experimentation. However, the usefulness of any stem cell-based embryo model will be determined by how accurately it reflects in vivo embryonic development, and/or the extent to which it facilitates new discoveries. Stringent benchmarking of embryo models is thus an important consideration for this growing field. Here we provide an overview of means to evaluate both the properties of stem cells, the building blocks of most embryo models, as well as the usefulness of current and future in vitro embryo models.