Why researchers should use human embryo models with caution

Karvas R.M., Zemke J.E., Ali S.S., Upton E., Sane E., Fischer L.A., Dong C., Park K., Wang F., Park K., Hao S., Chew B., Meyer B., Zhou C., Dietmann S., et. al.

Naive human pluripotent stem cells have the remarkable ability to self-organize into blastocyst-like structures ("blastoids") that model lineage segregation in the pre-implantation embryo. However, the extent to which blastoids can recapitulate the defining features of human post-implantation development remains unexplored. Here, we report that blastoids cultured on thick three-dimensional (3D) extracellular matrices capture hallmarks of early post-implantation development, including epiblast lumenogenesis, rapid expansion and diversification of trophoblast lineages, and robust invasion of extravillous trophoblast cells by day 14. Extended blastoid culture results in the localized activation of primitive streak marker TBXT and the emergence of embryonic germ layers by day 21. We also show that the modulation of WNT signaling alters the balance between epiblast and trophoblast fates in post-implantation blastoids. This work demonstrates that 3D-cultured blastoids offer a continuous and integrated in vitro model system of human embryonic and extraembryonic development from pre-implantation to early gastrulation stages.

Rivron N.C., Martinez Arias A., Pera M.F., Moris N., M’hamdi H.I.

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.

Liu L., Oura S., Markham Z., Hamilton J.N., Skory R.M., Li L., Sakurai M., Wang L., Pinzon-Arteaga C.A., Plachta N., Hon G.C., Wu J.

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.

Ai Z., Niu B., Yin Y., Xiang L., Shi G., Duan K., Wang S., Hu Y., Zhang C., Zhang C., Rong L., Kong R., Chen T., Guo Y., Liu W., et. al.

AbstractStudies of cultured embryos have provided insights into human peri-implantation development. However, detailed knowledge of peri-implantation lineage development as well as underlying mechanisms remains obscure. Using 3D-cultured human embryos, herein we report a complete cell atlas of the early post-implantation lineages and decipher cellular composition and gene signatures of the epiblast and hypoblast derivatives. In addition, we develop an embryo-like assembloid (E-assembloid) by assembling naive hESCs and extraembryonic cells. Using human embryos and E-assembloids, we reveal that WNT, BMP and Nodal signaling pathways synergistically, but functionally differently, orchestrate human peri-implantation lineage development. Specially, we dissect mechanisms underlying extraembryonic mesoderm and extraembryonic endoderm specifications. Finally, an improved E-assembloid is developed to recapitulate the epiblast and hypoblast development and tissue architectures in the pre-gastrulation human embryo. Our findings provide insights into human peri-implantation development, and the E-assembloid offers a useful model to disentangle cellular behaviors and signaling interactions that drive human embryogenesis.

Terhune A.H., Bok J., Sun S., Fu J.

ABSTRACT The complex process by which a single-celled zygote develops into a viable embryo is nothing short of a miraculous wonder of the natural world. Elucidating how this process is orchestrated in humans has long eluded the grasp of scientists due to ethical and practical limitations. Thankfully, pluripotent stem cells that resemble early developmental cell types possess the ability to mimic specific embryonic events. As such, murine and human stem cells have been leveraged by scientists to create in vitro models that aim to recapitulate different stages of early mammalian development. Here, we examine the wide variety of stem cell-based embryo models that have been developed to recapitulate and study embryonic events, from pre-implantation development through to early organogenesis. We discuss the applications of these models, key considerations regarding their importance within the field, and how such models are expected to grow and evolve to achieve exciting new milestones in the future.

Tarazi S., Aguilera-Castrejon A., Joubran C., Ghanem N., Ashouokhi S., Roncato F., Wildschutz E., Haddad M., Oldak B., Gomez-Cesar E., Livnat N., Viukov S., Lokshtanov D., Naveh-Tassa S., Rose M., et. al.

SummaryIn vitro cultured stem cells with distinct developmental capacities can contribute to embryonic or extraembryonic tissues after microinjection into pre-implantation mammalian embryos. However, whether cultured stem cells can independently give rise to entire gastrulating embryo-like structures with embryonic and extraembryonic compartments remains unknown. Here, we adapt a recently established platform for prolonged ex utero growth of natural embryos to generate mouse post-gastrulation synthetic whole embryo models (sEmbryos), with both embryonic and extraembryonic compartments, starting solely from naive ESCs. This was achieved by co-aggregating non-transduced ESCs, with naive ESCs transiently expressing Cdx2 or Gata4 to promote their priming toward trophectoderm and primitive endoderm lineages, respectively. sEmbryos adequately accomplish gastrulation, advance through key developmental milestones, and develop organ progenitors within complex extraembryonic compartments similar to E8.5 stage mouse embryos. Our findings highlight the plastic potential of naive pluripotent cells to self-organize and functionally reconstitute and model the entire mammalian embryo beyond gastrulation.

Amadei G., Handford C.E., Qiu C., De Jonghe J., Greenfeld H., Tran M., Martin B.K., Chen D., Aguilera-Castrejon A., Hanna J.H., Elowitz M.B., Hollfelder F., Shendure J., Glover D.M., Zernicka-Goetz M.

Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro1–5, but their developmental potential is substantially extended by interactions with extraembryonic stem cells, including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells6–11. Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis. Synthetic mouse embryos assembled from embryonic stem cells, trophoblast stem cells and induced extraembryonic endoderm stem cells closely recapitulate the development of wild-type and mutant natural mouse embryos up to embryonic day 8.5.

Zhao C., Biondic S., Vandal K., Björklund Å.K., Hagemann-Jensen M., Sommer T.M., Canizo J., Clark S., Raymond P., Zenklusen D.R., Rivron N., Reik W., Petropoulos S.

The preconceptual, intrauterine, and early life environments can have a profound and long-lasting impact on the developmental trajectories and health outcomes of the offspring. Given the relatively low success rates of assisted reproductive technologies (ART; ∼25%), additives and adjuvants, such as glucocorticoids, are used to improve the success rate. Considering the dynamic developmental events that occur during this window, these exposures may alter blastocyst formation at a molecular level, and as such, affect not only the viability of the embryo and the ability of the blastocyst to implant, but also the developmental trajectory of the first three cell lineages, ultimately influencing the physiology of the embryo. In this study, we present a comprehensive single-cell transcriptome, methylome, and small RNA atlas in the day 7 human embryo. We show that, despite no change in morphology and developmental features, preimplantation glucocorticoid exposure reprograms the molecular profile of the trophectoderm (TE) lineage, and these changes are associated with an altered metabolic and inflammatory response. Our data also suggest that glucocorticoids can precociously mature the TE sublineages, supported by the presence of extravillous trophoblast markers in the polar sublineage and presence of X Chromosome dosage compensation. Further, we have elucidated that epigenetic regulation—DNA methylation and microRNAs (miRNAs)—likely underlies the transcriptional changes observed. This study suggests that exposures to exogenous compounds during preimplantation may unintentionally reprogram the human embryo, possibly leading to suboptimal development and longer-term health outcomes.

Karzbrun E., Khankhel A.H., Megale H.C., Glasauer S.M., Wyle Y., Britton G., Warmflash A., Kosik K.S., Siggia E.D., Shraiman B.I., Streichan S.J.

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior–posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease. Stem cells cultured in a micropattern-constrained platform  form a quantitative and robust model of human neural tube morphogenesis.

Olmsted Z.T., Paluh J.L.

Stem cell technologies including self-assembling 3D tissue models provide access to early human neurodevelopment and fundamental insights into neuropathologies. Gastruloid models have not been used to investigate co-developing central and peripheral neuronal systems with trunk mesendoderm which we achieve here in elongating multi-lineage organized (EMLO) gastruloids. We evaluate EMLOs over a forty-day period, applying immunofluorescence of multi-lineage and functional biomarkers, including day 16 single-cell RNA-Seq, and evaluation of ectodermal and non-ectodermal neural crest cells (NCCs). We identify NCCs that differentiate to form peripheral neurons integrated with an upstream spinal cord region after day 8. This follows initial EMLO polarization events that coordinate with endoderm differentiation and primitive gut tube formation during multicellular spatial reorganization. This combined human central-peripheral nervous system model of early organogenesis highlights developmental events of mesendoderm and neuromuscular trunk regions and enables systemic studies of tissue interactions and innervation of neuromuscular, enteric and cardiac relevance. The authors generate EMLOs (elongating multi-lineage organized gastruloids): organoids that self-organize to form compartments with characteristics of the central nervous system, peripheral nervous system, mesenchyme, and gut tube.

Rossant J., Tam P.P.

Stem cell-based embryo models open an unprecedented avenue for modeling embryogenesis, cell lineage differentiation, tissue morphogenesis, and organogenesis in mammalian development. Experimentation on these embryo models can lead to a better understanding of the mechanisms of development and offers opportunities for functional genomic studies of disease-causing mechanisms, identification of therapeutic targets, and preclinical modeling of advanced therapeutics for precision medicine. An immediate challenge is to create embryo models of high fidelity to embryogenesis and organogenesis in vivo, to ensure that the knowledge gleaned is biologically meaningful and clinically relevant.

Sawai T., Minakawa T., Pugh J., Akatsuka K., Yamashita J.K., Fujita M.

Moris N., Anlas K., van den Brink S.C., Alemany A., Schröder J., Ghimire S., Balayo T., van Oudenaarden A., Martinez Arias A.

The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids—three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development. Human gastruloids—three-dimensional aggregates derived from human embryonic stem cells—show features of human embryos at around 19–21 days, and provide a model for the study of early human development.

Zheng Y., Xue X., Shao Y., Wang S., Esfahani S.N., Li Z., Muncie J.M., Lakins J.N., Weaver V.M., Gumucio D.L., Fu J.

Early human embryonic development involves extensive lineage diversification, cell-fate specification and tissue patterning1. Despite its basic and clinical importance, early human embryonic development remains relatively unexplained owing to interspecies divergence2,3 and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast and amniotic ectoderm parts of the conceptus, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells. We further show that amniotic ectoderm-like cells function as a signalling centre to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model could assist in the rational design of differentiation protocols of hPSCs for disease modelling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects. Landmarks of early stages of human embryogenesis can be recapitulated in a highly controllable and scalable fashion by culturing human pluripotent stem cells in a microfluidic device.

Turco M.Y., Gardner L., Kay R.G., Hamilton R.S., Prater M., Hollinshead M.S., McWhinnie A., Esposito L., Fernando R., Skelton H., Reimann F., Gribble F.M., Sharkey A., Marsh S.G., O’Rahilly S., et. al.

The placenta is the extraembryonic organ that supports the fetus during intrauterine life. Although placental dysfunction results in major disorders of pregnancy with immediate and lifelong consequences for the mother and child, our knowledge of the human placenta is limited owing to a lack of functional experimental models1. After implantation, the trophectoderm of the blastocyst rapidly proliferates and generates the trophoblast, the unique cell type of the placenta. In vivo, proliferative villous cytotrophoblast cells differentiate into two main sub-populations: syncytiotrophoblast, the multinucleated epithelium of the villi responsible for nutrient exchange and hormone production, and extravillous trophoblast cells, which anchor the placenta to the maternal decidua and transform the maternal spiral arteries2. Here we describe the generation of long-term, genetically stable organoid cultures of trophoblast that can differentiate into both syncytiotrophoblast and extravillous trophoblast. We used human leukocyte antigen (HLA) typing to confirm that the organoids were derived from the fetus, and verified their identities against four trophoblast-specific criteria3. The cultures organize into villous-like structures, and we detected the secretion of placental-specific peptides and hormones, including human chorionic gonadotropin (hCG), growth differentiation factor 15 (GDF15) and pregnancy-specific glycoprotein (PSG) by mass spectrometry. The organoids also differentiate into HLA-G+ extravillous trophoblast cells, which vigorously invade in three-dimensional cultures. Analysis of the methylome reveals that the organoids closely resemble normal first trimester placentas. This organoid model will be transformative for studying human placental development and for investigating trophoblast interactions with the local and systemic maternal environment.An in vitro system that generates three-dimensional cultures of extraembryonic fetal trophoblast cells that differentiate into the two main types of trophoblast can be used to study human placental development.

Choi H.K., Moon S.

Sozen B.

Rapid advances in stem cell and bioengineering technologies have sparked a revolution in developmental biology, with stem cell-based embryo models emerging as crucial tools to uncover the intricacies of human embryogenesis. However, making progress relies on precisely posing our questions and selecting our models.

Xue X., Liu Y., Fu J.

Stem cell-based embryo models, which recapitulate symmetry breaking, pattern formation and tissue morphogenesis during early development, provide promising experimental tools to study the development of mammalian species, including humans. Despite considerable progress in embryo modelling using cultured stem cells, generating embryo models with high fidelity, efficiency, controllability, and in vivo-like cellular organization and tissue architecture remains challenging. This is largely due to intrinsic variabilities in self-organization and differentiation of mammalian stem cells in uncontrolled culture environments. In this Review, we argue that bioengineering tools, which are powerful for controlling topological boundaries and dynamic chemical and mechanical signals, can efficiently guide symmetry breaking, pattern formation, tissue morphogenesis and tissue–tissue interactions. We discuss pattern formation and morphogenesis during embryonic development and examine different embryo models to highlight the importance of bioengineering strategies in developing models with improved efficiency, reproducibility, controllability, complexity and in vivo relevance. Stem cell-based embryo models can recapitulate developmental processes such as tissue patterning and morphogenetic events from pre-implantation to early organogenesis. This Review discusses how bioengineering approaches can be used to generate more efficient, controllable, reproducible and scalable embryo models.

Pennings G., Dondorp W., Popovic M., Chuva de Sousa Lopes S., Mertes H.

Abstract The current article provides an ethical reflection on the moral status of the human embryo, which is a crucial factor in determining permissible actions involving embryos and the extent of their protection. It advocates for the extension of the research period for embryos to 28-days post fertilization. It also states that integrated embryo-like structures (ELSs) should not currently be given the same moral status as natural embryos. However, if they pass the relevant tests, they should be subject to the same rules as natural embryos.

Martinez Arias A., Rivron N., Moris N., Tam P., Alev C., Fu J., Hadjantonakis A., Hanna J.H., Minchiotti G., Pourquie O., Sheng G., Solnica Krezel L., Veenvliet J.V., Warmflash A.

Pluripotent stem cells are being used to generate models of early embryogenesis that are promising for discovery and translational research. To be useful, these models require critical consideration of their level of efficiency and fidelity to natural embryos. Here we propose criteria with which to raise the standards of stem-cell-based embryo models of human embryogenesis.

Le Goff A., Jeffries Hein R., Hart A.N., Roberson I., Landecker H.L.

In vitro gametogenesis (IVG), the reconstitution of germ cell development in vitro, is an emerging stem cell-based technology with profound implications for reproductive science. Despite researchers' long-term goals for future clinical applications, little is currently known about the views of IVG held by the stakeholders potentially most affected by its introduction in humans. We conducted focus groups and interviews with 80 individuals with lived experience of infertility and/or LGBTQ+ family formation in the US, two intersecting groups of potential IVG users. Respondents expressed hope that IVG would lead to higher reproductive success than current assisted reproductive technology (ART), alleviate suffering associated with ART use, and promote greater social inclusion, while expressing concerns predominantly framed in terms of equity and safety. These findings underscore the importance of sustained engagement with stakeholders with relevant experience to anticipate the implications of IVG for research and clinical translation.

Pennings G.

Embryo-like structures (ELS) are intended for the study of embryonic development without the use of human supernumerary embryos. Scientists working in countries that do not allow research on embryos hope that these structures will replace natural embryos. The interest in ELS is largely based on two misconceptions: the belief that there is a shortage of research embryos and the belief that research on ELS will make research on natural embryos redundant. This paper argues that research efforts should be refocused on natural embryos.

Rossant J.

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.

Wu Y., Wang C., Fan X., Ma Y., Liu Z., Ye X., Shen C., Wu C.

It is widely acknowledged that we are currently facing a critical tipping point with regards to global extinction, with human activities driving us perilously close to the brink of a devastating sixth mass extinction. As a promising option for safeguarding endangered species, induced pluripotent stem cells (iPSCs) hold great potential to aid in the preservation of threatened animal populations. For endangered species, such as the northern white rhinoceros (Ceratotherium simum cottoni), supply of embryos is often limited. After the death of the last male in 2019, only two females remained in the world. IPSC technology offers novel approaches and techniques for obtaining pluripotent stem cells (PSCs) from rare and endangered animal species. Successful generation of iPSCs circumvents several bottlenecks that impede the development of PSCs, including the challenges associated with establishing embryonic stem cells, limited embryo sources and immune rejection following embryo transfer. To provide more opportunities and room for growth in our work on animal welfare, in this paper we will focus on the progress made with iPSC lines derived from endangered and extinct species, exploring their potential applications and limitations in animal welfare research.

Denker H.

Marx V.

Research with human embryos and embryo models, this year’s Method of the Year, can be fraught. In contrast, digital embryos could be studied, even perturbed, in computational what-happens-when experiments.

Haniffa M., Maartens A., Teichmann S.A.

In vivo developmental atlases provide a crucial reference for the new class of stem-cell-derived human embryo models, helping accelerate insights into the mechanisms of human development.

Denker H.

While research on stem cell-derived tissues and organoids is rapidly expanding, the technically related creation of complex embryoids has recently excited a vivid discussion since it raises ethical questions about individuation and the possible gain of viability. The present study focuses on the onset of organismic development and the proposed biological and legal definitions for the terms embryo, embryoid, and organoid. It is concluded that such considerations have become important for investigators’ choices of the appropriate in vitro model systems, allowing the formation of organoids vs. complex embryoids.

Rivron N.C., Martinez-Arias A., Sermon K., Mummery C., Schöler H.R., Wells J., Nichols J., Hadjantonakis A., Lancaster M.A., Morris N., Fu J., Sturmey R.G., Niakan K., Rossant J., Kato K.

Human embryology is flourishing thanks to an impetus provided by embryo models formed from stem cells. These scientific advances require meticulous experimental work and a refined ethical framework, but also sensible public communication. Securing public support is essential to achieve societal impact.