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Introduction and Overview
Early human embryo development is studied for a variety of reasons, including to better understand our fundamental origins, with the goal of using that information to provide insights into infertility, implantation, and placental development and to apply that knowledge to improve assisted reproductive technologies such as in vitro fertilization (IVF). The use of embryo models to study human embryogenesis can contribute new knowledge about an essential stage of human development that is otherwise inaccessible (see the section on the Current State of Regulation of Research on Human Embryo Models in Hyun et al., 2020). These embryo models can provide important information to guide infertility treatments, prevent disease, and create organs that could be used for pharmacologic screening or possibly even for transplantation.
A host of technical breakthroughs in modeling early embryonic development has been achieved in the past few years. A new technique of geometrical confinement has enabled the self-organization of human stem cells to be modeled in two dimensions (2D) using micropatterns (Warmflash et al., 2014). Three-dimensional (3D) culturing systems use hydrogels that modify the physical environment of embryonic development, allowing for the use of mouse stem cells to create 3D models of features of the embryo prior to implantation to illuminate the process of placental formation and elucidate the reasons for the high rates of early human embryo loss and human placental anomalies (Rivron et al., 2018b). Human pluripotent stem cells grown in microfluidic systems offer opportunities to study early post-implantation biology with even greater control and reproducibility than 3D modeling (Zheng et al., 2019). CRISPR-Cas9 techniques have
revolutionized gene editing in human cells and can be used to study the genetics underlying early developmental defects. Similarly, the advent of single-cell omics has been pivotal in understanding the genetic features of early embryos, while developmental genetics has generated a wealth of new information about developmentally important genes.
OVERVIEW OF THE WORKSHOP
Because of the recent advances in embryo modeling techniques, and at the request of the Office of Science Policy in the Office of the Director at the National Institutes of Health (NIH), the National Academies of Sciences, Engineering, and Medicine appointed a planning committee to develop and host a 1-day public workshop1 that would explore the state of the science of mammalian embryo model systems. The workshop, which took place on January 17, 2020, featured a combination of presentations, panels, and general discussions, during which panelists and participants offered a broad range of perspectives. The workshop began with a survey of the developments that have laid the groundwork for the field, then focused on opportunities and challenges for future work with embryo model systems. Presentations and discussions covered topics such as the characteristics of advanced mammalian embryo model systems, the differences between various mammalian embryo model systems and bona fide mammalian embryos, and the differences between mammalian embryo model systems and mammalian “embryoid bodies,” which arise via aggregation of stem cells but do not recapitulate regular organization. Participants considered whether embryo model systems—especially those that use nonhuman primate cells—can be used to predict the function of systems made with human cells. They also considered the functionality and organismal potential of the model systems—that is, whether embryo model systems have organismal potential if they lack trophoblast cells or other extraembryonic cell types. Presentations provided an overview of the current state of the science of in vitro development of human trophoblast cells. As requested by NIH, the discussions at the workshop focused on the state of the science, not on policy or ethical implications.
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1 This workshop was organized by an independent planning committee whose role was limited to identification of the topics and speakers. This Proceedings of a Workshop was prepared by the rapporteurs as a factual summary of the presentations and discussion that took place at the workshop. Statements, recommendations, and opinions expressed are those of individual presenters and participants and are not endorsed or verified by the National Academies of Sciences, Engineering, and Medicine, and they should not be construed as reflecting any group consensus. The workshop agenda, speaker biographies, planning committee Statement of Task, and a list of attendees can be found in Appendixes A, B, C, and D, respectively.
OPPORTUNITIES AND CHALLENGES WITH STEM CELL–BASED EMBRYO MODELS
Janet Rossant, a senior scientist in the Program in Developmental and Stem Cell Biology at The Hospital for Sick Children, University of Toronto, delivered an opening keynote that focused on the unique aspects of human embryology and opportunities and challenges with stem cell–based embryo models. Opportunities already exist to generate partial or more complete stem cell–derived human embryo models. However, she said, embarking on this human model pathway will require careful consideration of the similarities and differences between mouse and human systems in order to select the appropriate cell type for developing human models. Furthermore, it will require aligning directly with events in the human embryo itself, either via culture in vitro or in comparison with nonhuman primate models.
Comparison of Human and Mouse Development
Rossant explained why mouse models are not sufficient for understanding early human development. Although human and mouse development have many similarities, the processes are morphologically and molecularly distinct (Rossant and Tam, 2017). To illustrate, she compared the timelines of human and mouse development. In both species the blastocyst is the first major differentiated state of the embryo, containing three cell types—trophectoderm, epiblast, and hypoblast. Many of the genes that are involved in specifying the three cell types in the mouse are also involved in the human blastocyst. However, there are differences in the time of activation of the genes that specify cell fate, which likely relates to a difference in the timing of zygote genome activation. Lineage-specific gene expression cannot begin until zygotic genome activation has occurred, which happens at a later developmental timepoint in the human than the mouse. Much larger morphological differences between the two species occur post-implantation. Regenerative medicine will be one of the areas that will benefit from a better understanding of the similarities and differences between human and mouse embryos and their derived stem cells, she added.
Timing of Blastocyst Lineage Commitment in Mouse and Human
The timing of blastocyst lineage commitment is different in mouse and human development, Rossant said. Studies of the timing of the commitment of inner cell mass (ICM) in trophectoderm in the blastocyst have established that mouse lineage commitment occurs in trophectoderm before the ICM, although both cell lineages are committed by the blastocyst stage (Posfai
et al., 2017; Wigger et al., 2017). That is, if the cells are taken apart and reaggregated, they cannot regenerate a blastocyst. She described some of the relevant gene expression and signaling pathways involved in this process. In addition to early cell biases, the Hippo/YAP and fibroblast growth factor (FGF) signaling pathways are critical to final cell fate specification in the mouse blastocyst. Less information is available on blastocyst lineage commitment in humans, although some studies suggest that human early blastocyst cells remain uncommitted to lineage (De Paepe et al., 2013).
Timing of Gene Expression
The timing of the expression of key transcription factors differs between mouse and human, Rossant said, and those factors may also differ in function between the species. For instance, single-cell RNA sequencing shows that many key genes in the mouse early embryo are conserved in humans, but there are also divergent lineage-specific genes (Blakeley et al., 2015; Petropoulos et al., 2016). For example, caudal type homeobox 2 (CDX2), which is presumed to be important for trophectoderm, does not begin expression in the human until after blastocyst formation, which gives rise to questions about how it can drive cell fate specification (Niakan and Eggan, 2013). Furthermore, knock-out gene editing studies suggest that OCT4 may play a much different role in human development than in the mouse (Fogarty et al., 2017). It has been established that the morphogenesis of the human blastocyst precedes lineage specification, but it is still unknown whether CDX2 is important to trophectoderm or whether Hippo signaling is required. Rossant explained that these differences are important because they may relate to the derivation of different types of pluripotent stem cells from the blastocyst.
Differences in Post-Implantation Morphogenesis
Additional morphological distinctions emerge post-implantation in human and mouse development, Rossant said. The major difference between the mouse and the human post-implantation is the formation of the extraembryonic ectoderm from the polar trophectoderm, which proliferates and pushes the ICM into the blastocoel to form a cup-shaped epiblast. The polar trophectoderm does not proliferate in this way in humans; instead of a cup shape, the epiblast is shaped like a flat sheet. In vitro outgrowths of human embryos across the implantation period (i.e., less than 14 days) have demonstrated morphogenesis of the post-implantation embryos (Deglincerti et al., 2016; Shahbazi et al., 2016), although the human embryos were still largely disorganized. Two laboratories have recently developed techniques to extend in vitro cultures of cynomolgus monkey embryos for longer than
14 days in the hope of achieving better morphology, she added (Ma et al., 2019; Niu et al., 2019).
Replacing Human Embryos with Self-Organizing Stem Cell Cultures
The use of human or nonhuman primate embryos as model systems will not be sufficient, Rossant said. The use of human embryos can be considered ethically challenging in many regions, while nonhuman primate embryos are expensive and only available in limited research centers. These challenges have led researchers to replace human embryos with self-organizing stem cell model systems in order to study development without the use of embryos. The use of self-organizing embryo stem cell cultures began and is most advanced with the mouse system, she explained. Fortunately, the mouse system has three lineage-specific stem cell lines that represent the three lineages of the blastocyst: trophectoderm, epiblast, and primitive endoderm. This provides a starting population of cells that can be combined to model some of the events of blastocyst formation and early post-implantation development. Embryonic stem cells (ESCs) come from the ICM of the blastocyst, Rossant said, and express the pluripotency genes. When placed back in the chimera, they contribute to the fetus but usually not to the placenta or the yolk sac. Trophoblast stem cells (TSCs) arise from the trophectoderm and have a different growth factor requirement which enables them to grow indefinitely in culture; they make placental tissues later in development. Extraembryonic endoderm (XEN) cells can also grow indefinitely in culture and contribute only to yolk sac endoderm. One laboratory has already been able to take these three cell types from the blastocyst and recombine them to form 3D “artificial” mouse embryo models which look like early post-implantation embryos, and to model early interactions between these cell lineages which are critical in later development (Sozen et al., 2018). Nicolas Rivron’s laboratory has been able to create mini blastoids that resemble the mouse blastocyst by combining ESCs and TSCs and allowing them to reaggregate such that TSCs form an outer trophectoderm layer (Rivron et al., 2018b). These efforts demonstrate that the use of stem cells in mouse systems has the potential to mimic pre-implantation and early post-implantation development by aggregate systems, she explained.
Use of Mouse Stem Cells for Model Generation
Rossant provided updates on progress in the use of mouse stem cells for model generation, with the caveat that the three original ES, TS, and XEN cell types are not necessarily completely equivalent to the three cell types
of the blastocyst and that more work is needed to mimic the three lineages of the mouse blastocyst. Nicolas Rivron’s laboratory has shown that TSCs that express higher levels of CDX2 are more like the polar trophectoderm (TE) and are thus more likely to accurately represent the cells that would reconstitute a blastoid rather than post-implantation trophoblast (Aldeguer et al., 2019). Josh Brickman’s laboratory has demonstrated that culture conditions from ESCs can make naïve extraembryonic endoderm (nEND) cells that are closer to the primitive endoderm than XEN cells, suggesting that they may provide a better system for embryo models (Anderson et al., 2017). Several laboratories have derived extended-potential stem cells that are purported to be able to generate extraembryonic tissues themselves; because they have an extended potential beyond pluripotent cells, they can generate epiblast and primitive endoderm when combined with TSCs to make a blastoid (Sozen et al., 2019). Extended-potential stem cells may also have the potential to make trophoblast-like cells as well, meaning that the extended-potential cells could make blastoids (Li et al., 2019).
Potential to Use Human Stem Cells for Embryo Models
To offer more context on the shift from mouse to human stem cells for embryo models,2 Rossant provided a brief overview of the literature to date about what can be modeled with stem cells (Hyun et al., 2020). Mouse systems have been used for several years to model blastocyst formation and post-implantation interactions between the trophoblast, endoderm, and epiblast; ESCs have also been used to create gastrula-like structures. On the human side, there have been no reports of blastoid formation published to date, although studies have used micropattern cultures to model some of the events of gastrulation in 2D and 3D. Microfluidic systems have also been used to create small amniotic sac structures. Only ESCs have been used thus far in human models, Rossant said. She discussed whether naïve ES, TS, and XEN-type cells are available for use in human stem cell models.
Similarities Between Mouse and Human Stem Cells
Human naïve ESCs do exist and are the closest cell type to the epiblast or the blastocyst; thus, they may be good candidates for use in an embryo model, Rossant said. It has been suggested that the naïve state of ESCs in the human may be different than in the mouse. However, like mouse naïve ESCs, human naïve ESCs can grow in 2i LIF+FGFR inhibitor (Anderson et al., 2017). Human TSCs cannot be derived from blastocysts in humans
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2 Rossant noted that related ethical and regulatory issues were beyond the scope of the workshop, but a review is available in Hyun et al. (2020).
using mouse conditions, which involves activating FGF signaling. Because the early trophoblast in the human does not proliferate, it does not respond to FGF signaling in the embryo itself. However, good-quality TSCs have been generated from human blastocyst and early villus structures that express and have many of the properties of early placental cells (Okae et al., 2018). The lack of dependence on FGF in human TSCs indicates that a different set of signaling pathways is important in humans. The question remains as to whether there is a blastocyst-type TE cell to be found in humans or whether they do not exist, in which case human TSCs may be the best tools to monitor the state of development, Rossant said. It is possible to overexpress SOX7 (a member of the SRY-related HMG-box family of genes) (Seguin et al., 2008) as well as expressing other genes that would drive human ESCs toward primitive endoderm. Brickman’s laboratory has been able to culture human nEND cells using the same conditions to make mouse nEND cells, which are posited to mimic the primitive endoderm and can be cultured indefinitely (Linneberg-Agerholm et al., 2019). The expanded-potential cells that have been reported in the mouse to produce trophoblast and primitive endoderm have also been reported in the human, using culture conditions similar to the mouse (Gao et al., 2019). Because these cells have similar properties to the mouse cells, there is great interest in whether they can produce blastoids as well.
Differences Between Mouse and Human Stem Cells
Rossant highlighted several differences between mouse and human stem cells. Although it appears that human naïve ESCs can be cultured in conditions similar to those used for mouse cells, the lineage status of the human cells is less clear. Naïve ESCs in the mouse are restricted to the epiblast lineage, so they do not normally make the other blastocyst cell types—many other factors need to be added to generate extended-potential cells. However, evidence suggests that human naïve ESCs may have some intrinsic extended potential; their expression profile shows similarity to some stages in early pre-implantation development, as well as to the epiblast (Stirparo et al., 2018). Transposon expression and other properties of these cells have also been reported to be similar to cleavage stages (Theunissen and Jaenisch, 2017). Rossant noted that in humans, even the blastocyst stage shows late plasticity, with no lineage commitment. She surmised that this may explain why these naïve cells seem to have an extended potential: they are behaving like blastocyst cells. Data also suggest that human expanded-potential stem cells (Gao et al., 2019)—and naïve cells (Dong et al., 2020)—may be more readily able to generate TSCs in the appropriate culture conditions than mouse expanded-potential stem cells.
NEW MODELS MAY OFFER A NEW FRONTIER OF DATA
There has been enormous progress in the past 5 years in using cell culture systems to model mammalian embryonic development, said Martin Pera, a professor at The Jackson Laboratory in Bar Harbor, Maine, and member of the workshop planning committee. Technologies to replicate cell specification and differentiation events have been available for much longer, but novel systems have opened up a new frontier in providing the opportunity to model cell patterning and morphogenetic events in space and time. With these advances comes the potential to better understand the earliest stages of human development, he said, and the advances may offer the means to explore and ultimately intervene to address disorders that may have their origin in early embryonic development. At the level of basic science, these systems can not only shed light on fundamental questions about our human origins, but elucidate similarities and differences in embryonic development across species. These systems can also contribute to a better understanding of the origin of germ cells in order to address issues of infertility.
New data are also emerging from studies characterizing the molecular attributes of embryos from the mouse, human, and nonhuman primate. Moving the field of mammalian embryology forward with these transformative new systems and techniques will involve venturing down new avenues of research to explore how embryo research interacts with stem cell models to harness the vast potential of pluripotent cells. It will also require conducting comparative cross-species studies of early embryonic development and investigating the nature of the broad range of cell types that can now be generated and manipulated, M. Pera said.
ORGANIZATION OF THE PROCEEDINGS OF A WORKSHOP
Following this introductory chapter, Chapter 2 explores the current state of mammalian embryo model systems, the use of pluripotent stem cells to generate those models, and the potential benefits and limitations of using these models for studying human embryonic development. Speakers discussed the molecular mechanisms of lineage specification in human embryos, described the development of embryo models, and considered the clinical implications of modeling pre-implantation embryo development. Chapter 3 examines the development of extraembryonic lineages and the impact of those lineages on human embryo model systems, featuring presentations on modeling trophoblast differentiation using pluripotent stem cells and molecular innovation in the human trophoblast lineage. Chapter 4 provides an overview of cutting-edge novel systems that have
been developed to model human embryos from stem cells. Speakers described techniques for modeling attachment and self-organization in human embryonic development using 3D models and 2D micropatterns, systems for modeling the pre-implantation embryo in 3D, and the use of microfluidic systems to model the peri-implantation embryo. Chapter 5 summarizes the workshop session on comparative embryonic development across species. Speakers explored mechanisms underlying pre-implantation chromosomal instability, systems to differentiate trophoblast from primate pluripotent cells, models of early neural crest formation in humans, and models of pre-implantation development generated from pluripotent stem cells with expanded potential. Finally, Chapter 6 summarizes the workshop’s final panel discussion and closing keynote, which focused on future opportunities and challenges of mammalian embryo model systems.
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