The first session of the workshop focused on mammalian embryo research and pluripotent stem cells with the aim of exploring the characteristics of mammalian embryo model systems and the potential benefits and limitations to using these models for studying human embryonic development. The session was moderated by Renee Reijo Pera, the vice president of Research and Economic Development at California Polytechnic State University, and featured presentations on the molecular mechanisms of lineage specification in human embryos, human and synthetic models of human development after implantation, and the clinical implications of studying pre-implantation human development.
MOLECULAR MECHANISMS OF LINEAGE SPECIFICATION IN HUMAN EMBRYOS
Kathy Niakan, a group leader at the Francis Crick Institute, a biomedical research institute in London, spoke about molecular mechanisms of lineage specification in human embryos. Little is known about how the first cell types that emerge in a human embryo become specialized in fate and function, so her laboratory uses transcriptome analysis to understand early human development and to refine methods for understanding gene function in human embryos. Currently, this knowledge
is used to rationally design and optimize culture conditions for human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). The members of Niakan’s lab hope to use the knowledge to identify biomarkers of successful embryo development with the aim of improving in vitro fertilization (IVF) culture conditions. In her presentation, Niakan highlighted distinct expression dynamics in human versus mouse embryos, explained why human pluripotent stem cell culture conditions need to be improved, and described how genome editing can contribute to the understanding of early human development. She also provided an overview of the regulatory framework for human embryonic research in the United Kingdom for the purposes of understanding the environment in which the work was conducted (see Box 2-1).
Rationale for Studying Human Pre-Implantation Development
Niakan’s laboratory focuses on understanding the mechanisms of early lineage specification in humans during the first 7 days of development, which encompasses the time immediately after the egg is fertilized until it forms a ball of approximately 200 cells called the blastocyst. The human blastocyst is composed predominantly of placental progenitor cells collectively called the trophectoderm, Niakan said. The human blastocyst also contains endoderm cells, which are the precursors of the yolk sac. Only a small subset of cells in the developing human pre-implantation
blastocyst—about 5 percent—have the unique potential to give rise to the entire embryo proper or the fetus. These are called the epiblast progenitor cells, and they express a key transcription factor called octamer-binding transcription factor 4 (OCT4).
In addition to fundamentally informing our understanding of human biology, studying early lineage specification and human pre-implantation development also has clinical implications, Niakan said. Most human IVF embryos will fail to develop to form a blastocyst, fail to implant, or fail to develop beyond 3 months. Across all IVF clinics in the United Kingdom, the aggregated ongoing pregnancy rate, irrespective of age, is only about 11 percent (Koot et al., 2012). Studying these early stages of development may lead to a better understanding of the conditions that human embryos need to continue development and help identify more accurate predictors of success in embryos used for transfer in treatment. Studying these early stages of development is also important for stem cell biology, because hESCs can be established from epiblast progenitor cells in the blastocyst. These cells have tremendous regenerative potential, Niakan said, because they can self-renew and could theoretically be directed to differentiate into any cell type; hence, the importance of understanding how pluripotency is established and maintained.
Distinct Expression Dynamics in Human Versus Mouse Embryos
To better understand the mechanisms of lineage specification, Niakan’s laboratory performed single-cell RNA sequencing to catalog which genes are expressed throughout the various pre-implantation stages of human development and then compared this list of genes with the corresponding one for the mouse, which remains the most important model organism for comparative analysis. A comparison of these transcriptome analyses for the two species revealed striking differences in the temporal gene expression patterns between the homologous or potentially orthologous genes in the two species (Blakeley et al., 2015). Differences in cell-type-specific expression were also found among other genes that may have more conserved expression patterns, including POU class 5 homeobox 1 (POU5F1)—which encodes the key transcription factor OCT4—as well as Krüppel-like factor 4 (KLF4). The expression of both of those genes is detectable much earlier within mouse development than within human development, she said.
Early Lineage Specification in Mice and Humans
In order to delve further into what is known about lineage specification in humans, Niakan first described what is known about how lineages
are specified in mice. In the mouse, the first lineage decision occurs at the morula stage, when outer cells initiate a program that ultimately gives rise to trophectoderm cells and inner cells subsequently segregate to form the primitive endoderm or the pluripotent epiblast. The mechanisms that underlie this first lineage decision in the mouse are thought to be shaped by differences in polarization state that influence cell fate (Nishioka et al., 2009). An anterior polarity complex comprising atypical protein kinase C and PAR proteins1 sequesters angiomotin to the cell membrane. This prevents the activation of Hippo kinases and allows the yes-associated protein 1 (YAP1) transcription factor to translocate into the nucleus, where it accumulates and activates trophectoderm genes. This process does not occur in the inner cells; instead, the Hippo kinases phosphorylate YAP and prevent it from translocating into the nucleus to drive trophectoderm cell initiation. Niakan’s laboratory is currently investigating whether these components are also expressed in human pre-implantation embryos.
Niakan explained that research on the first lineage segregation event serves to benchmark what is likely the next segregation event (i.e., segregation of the pluripotent epiblast from the primitive endoderm). In the mouse model it is well established that the pluripotent epiblast secretes a ligand fibroblast growth factor (FGF) that binds to an FGF receptor enriched on primitive endoderm-fated cells. Blocking either that receptor or the downstream mitogen-activated protein kinase/extracellular-signal-regulated kinase (MEK/ERK) pathway prevents the initiation of genes associated with primitive endoderm differentiation (Yamanaka et al., 2010). This finding has been used to improve mouse embryonic stem cell culture conditions in the naïve pluripotent stem cell state (Ying et al., 2008). For reasons not yet understood, hESCs seem to require FGF for their self-renewal (Thomson et al., 1998).
Rationale for Improving Human Pluripotent Stem Cell Culture
Decades of research using hESCs have been transformative for our understanding of human biology, Niakan said. However, there are important justifications for continued research into how pluripotent epiblast cells form and how to refine hESC culture conditions using actual human embryos, she continued. Decades of work with iPSCs and ESCs have been valuable, but pluripotent stem cell culture conditions need to be improved to more closely approximate the processes of normal embryonic development. Some pluripotent stem cells—especially human naïve cells—that are established in cultures have epigenetic abnormalities, such as abnormalities in genomic
___________________
1 PAR proteins are a highly conserved group of proteins across the metazoan that control cell polarity during development.
imprinting, which may preclude their use in understanding certain aspects of human biology. Improving these culture methods might also decrease known line-to-line variabilities between human ESCs and iPSCs as well as facilitating the generation of mature cell types with greater phenotypic fidelity to in vivo counterparts. On a pragmatic level, improving human pluripotent stem cell culture conditions may also be more cost permissive, especially for large-scale experiments such as three-dimensional (3D) cell-suspension cultures, she added.
Nodal Signaling and Insulin Growth Factor in Pre-Implantation Development
Niakan’s laboratory returned to its transcriptome datasets to investigate signaling pathways that could be modulated to rationally design human pluripotent culture conditions that more closely recapitulate the niche. They identified that a number of components of the Nodal Growth Differentiation Factor (Nodal) signaling pathway are transcribed specifically in the embryonic epiblast, including Nodal and the co-factors growth differentiation factor 3 (GDF3) and teratocarcinoma-derived growth factor 1 (TDGF1, which encodes Cripto). Next, they posited that blocking the Activin-receptor-like kinases involved in the Nodal signaling pathway would have a consequence for downstream NANOG2 expression, which has been demonstrated to be affected in hESCs following inhibition of this pathway. Indeed, treating human embryos with inhibitors of Nodal signaling led to a loss of downstream NANOG expression; the same effect was not observed in mouse embryos treated with the same inhibitors, suggesting potential differences. Thus, it appears that Nodal signaling may be necessary for the maintenance of NANOG in human epithelial cells (Blakeley et al., 2015). The signaling pathway for insulin growth factor (IGF1)—active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling—was also found to be active in human embryos (Wamaitha et al., 2020). By stimulating the Nodal signaling pathway with IGF1 in novel culture conditions, they were able to derive hESCs directly from embryos and reprogram fibroblasts into iPSCs. These cells express key pluripotency-associated transcription factors and cell-surface antigens and can be directed to differentiate into a variety of different lineages. Having established the novel culture conditions, Niakan and colleagues then assessed the transcriptional similarities and differences and used the novel culture conditions to make strides toward moving the cells closer to the embryonic epiblast. They found that irrespective of the culture conditions, established hESCs are somewhat transcrip-
___________________
2 NANOG is a transcription factor that helps embryonic stem cells maintain pluripotency by suppressing cell determination factors.
tionally different from the pluripotent epiblast, despite some similarities to the embryonic epiblast (Wamaitha et al., 2020). Niakan added that even naïve hESCs are still somewhat transcriptionally different, even though they have upregulated many genes that are uniquely expressed in the embryonic epiblast. This suggests that further refinements of human pluripotent stem cell culture conditions are needed and that a basic understanding of how the embryonic epiblast develops in human embryos may provide key insights into how to make further improvements.
Based on this work, Niakan said she believes that FGF and Nodal signaling may differ in how they function in mouse and human preimplantation development. Her team found that IGF1 promotes proliferation of the pluripotent epiblast in ES cells and that Nodal (Activin) together with IGF1 supports the derivation and maintenance of hESCs and iPSCs. IGF1 likely functions as a proliferative factor and is not involved in lineage segregation, she added. It is not yet clear how the epiblast and the primitive endoderm diverge in humans, and this is likely a distinct mechanism compared to the mouse.
Use of Genome Editing to Understand Early Human Development
Genome editing approaches are another way to better understand early human development, Niakan said. Her laboratory has targeted OCT4 in human embryos using CRISPR-Cas9 genome editing as a proof-of-principle in order to determine if these methods are useful in understanding human biology (Fogarty et al., 2017). She said that they screened various methods in hESCs and in mouse embryos to optimize their techniques before they targeted human embryos. It appears that human embryos need OCT4 to correctly form a blastocyst (Fogarty et al., 2017). Human embryos in which OCT4 was targeted were significantly less likely to develop to the blastocyst stage—despite embryos initiating blastocyst formation, the blastocysts cannot be maintained. Genotyping and transcriptome analysis revealed that the OCT4-targeted cells are transcriptionally distinct from unedited cells with respect to NANOG expression. OCT4-targeted human embryos lacked detectable NANOG expression, while in mouse embryos, OCT4-null mutant mouse embryos retained NANOG expression, Niakan said. This suggests that NANOG is differentially regulated between the species. In addition to the retention of NANOG expression in OCT4null mutant mouse embryos, the expression of placental markers (e.g., CDX2) is retained (Frum et al., 2013). In fact, OCT4-null mutant mouse embryos plated in mouse embryonic culture conditions preferentially gave rise to trophoblast or placental outgrowths. In contrast, trophectoderm development is compromised in OCT4-targeted human embryos (Fogarty et al., 2017). This suggests that OCT4 is a negative regulator of the placental
program in the mouse and a positive regulator of the program in the human. Altogether, this suggests that while some developmental programs may be conserved across species, there may be important distinctions in early embryogenesis, underscoring the importance of studying human embryos directly, Niakan said.
BUILDING EMBRYO MODELS TO STUDY HUMAN DEVELOPMENT
Magdalena Zernicka-Goetz, a professor of mammalian development and stem cell biology at the University of Cambridge and the Bren Professor of Biology and Biological Engineering at the California Institute of Technology, described her laboratory’s work on early post-implantation human embryo development that led to the development of synthetic 3D stem cell models. She cited two reasons for studying early human development: (1) because it is the initiation of human life and (2) because of basic interest in understanding how human development is different than other models, particularly in the mouse. Research into early human development also provides a new frontier of opportunity for understanding why only about 30 percent of human pregnancies are successful, she said. During the preclinical stage, an estimated 30 percent of human pregnancies fail prior to the implantation phase of development and another 30 percent of pregnancies fail around the time of implantation or soon after; yet another 10 percent of pregnancies are lost to miscarriage (Macklon et al., 2002). The embryos themselves—rather than the endometrium—are typically the cause of the pregnancy loss. Around 85 percent of recovered embryos have chromosomal abnormalities.
Early Post-Implantation Human Embryo Development: Research Aims
Zernicka-Goetz outlined four research aims that her laboratory has pursued in the realm of early post-implantation human embryo development. The first aim was to develop the conditions that would allow human embryos to be cultured in vitro beyond implantation, in order to understand the morphogenesis of the human embryo at the time of implantation and soon after. In addition to uncovering the sequence of morphogenetic steps that an embryo undergoes, another aim of developing this model system was to create a molecular map of transitions that occur in the human embryo during those early stages using single-cell transcriptomic analysis. A third aim was to study the development of aneuploid and mosaic human embryos by investigating the developmental consequences of specific aneuploidies during post-implantation development. Mosaic embryos were of particular
interest because they contain both normal and abnormal cells, giving rise to questions about what happens to those abnormal cells when they are surrounded by normal cells in early development—for example, whether their development is normal and whether those cells can be manipulated by normal cells in ways that might lead to cell death, as Zernicka-Goetz’s laboratory recently demonstrated in the mouse. Using the knowledge gained from achieving the first three research aims, the lab has developed four different 3D model systems to mimic the early stages of development and explore the mechanisms of embryo self-organization.
Modeling Human Embryo Development After Implantation
Zernicka-Goetz explained that until recently human post-implantation development could not be studied directly. Knowledge of this stage was limited to the analysis of a small number of embryos that had developed in vivo and were recovered after hysterectomy (Hertig et al., 1956). Her laboratory considered two alternative paths for exploring this phase of development: (1) to develop a system to culture human embryos beyond day 7 in vitro that is as similar as possible to human development in vivo and (2) to develop a system to model human development with stem cells. They had already developed a system to culture mouse blastocysts in vitro through the implantation stages into the post-implantation gastrulation stage, which initiates anterior-posterior polarity (Bedzhov and Zernicka-Goetz, 2014; Bedzhov et al., 2014; Morris et al., 2012), and they found that the same system could be used to culture human embryos from the blastocyst stage onwards (Deglincerti et al., 2016; Shahbazi et al., 2016). Today, many groups are using this stem cell–based system to identify the sequential morphogenic steps involved in early post-implantation human embryo development (Shahbazi and Zernicka-Goetz, 2018; Zhou et al., 2019). Zernicka-Goetz added that, in addition to morphogenesis, this stem cell culture system can also reveal mechanisms that underlie the sequential stages of post-implantation development and drive different tissues to develop the unique characteristics that enable future development of the fetus, placenta, and yolk sac.
Molecular Mapping of Human Embryos
Having the stem cell–based developmental system in place, Zernicka-Goetz’s laboratory looked at the genes that are expressed at different stages in various tissues to understand the molecular fate of those tissues. Focusing first on day 9 and day 11, they isolated cells and identified epiblast, hypoblast, cytotrophoblast, and syncytiotrophoblast tissues based on markers of gene expression. Zernicka-Goetz described this study as
important in providing the molecular fingerprint for morphogenic events observed in early embryonic development. Her lab will now be using this approach to understand signaling interactions between the tissues.
Developmental Consequences of Specific Aneuploidies
The culture conditions developed to study normal human embryo development can also be used to look at aneuploid embryos and identify the developmental consequences of specific aneuploidies, Zernicka-Goetz said. She maintained that this stem cell–based system has great potential to further our understanding of how genes on specific chromosomes contribute to the phenotype and behavior of trophectoderm, epiblast, and hypoblast. Using this method, she said, her lab will also be able to identify embryos that are misdiagnosed as aneuploid when they are actually mosaic.
Embryo Growth in the Stem Cell Culture System
Zernicka-Goetz explained how her lab determines whether the embryos are behaving normally in this model system. One method is to rely on established markers of in vivo developing embryos, but a clear indication that the embryos are behaving normally is that embryo growth is observed in the individual culture systems. In fact, embryo development is very simple prior to implantation—the embryo cleaves into smaller and smaller cells, albeit with a complex cell rate specification. Embryo growth does not begin until implantation, at which point all the tissues begin to proliferate, and major remodeling occurs. At days 7 and 8 the cells segregate into the epiblast and hypoblast lineages, marked by OCT4 and GATA6, respectively. At days 8 and 9 a group of epiblast cells forms the pre-amniotic cavity. Zernicka-Goetz and colleagues were able to mimic the formation of this cavity by culturing human stem cells in Matrigel, a 3D extracellular matrix (Bedzhov and Zernicka-Goetz, 2014; Shahbazi et al., 2017). They surrounded human ESCs with Matrigel, which allows the cells to become polarized. Upon polarization, the amniotic cavity is formed, she said. In this way, studying human stem cells elucidates the morphological transition for both the mouse and the human at this very early stage of development. Extraembryonic tissues begin to differentiate at days 10 and 11. Hypoblast tissue3 forms the prospective yolk sac, and trophoblast tissue differentiates into cytotrophoblast and syncytiotrophoblast. Zernicka-Goetz described this study as a breakthrough in showing that human embryos can self-organize in vitro outside the body of the mother and said that this model system can be used to mimic natural human development in vitro, at least to a certain extent.
___________________
3 Hypoblast tissue is the equivalent of primitive endoderm in the mouse.
Four 3D Stem Cell Models of Early Development
Zernicka-Goetz described how her laboratory has developed four 3D stem cell embryo model systems to generate embryo-like structures. To do so they applied the knowledge gleaned from observing the development of normal and abnormal embryos on morphological and molecular levels using their initial culture system. Their research aim was to inform the mechanistic understanding of development by reconstructing mammalian embryos using the building blocks of stem cells for three distinct tissues: ESCs from epiblast, extraembryonic endoderm (XEN) cells from primitive endoderm, and trophoblast stem cells (TSCs) from trophectoderm.
Pre-Implantation Blastocyst Model
In the first mouse model, Zernicka-Goetz and colleagues sought to mimic the formation of the pre-implantation blastocyst using expanded-potential ESCs, which can form both epiblast and primitive endoderm. TSCs were added to make a chimera of the two types of stem cells. The cells self-organized to form blastocyst-like structures (i.e., “blastoids”) with all three constituent cell types (Li et al., 2019; Rivron et al., 2018b; Sozen et al., 2019). The advantage of this model is that it provides insight into the blastocyst stage, she said. The drawback is that the model does not develop very well to post-implantation stages, because it induces an implantation-like reaction but does not progress to post-implantation stages.
Peri-Implantation Blastocyst Model
The second 3D model developed by Zernicka-Goetz’s laboratory was designed to look at the peri-implantation phase. Mouse or human ESCs are embedded in Matrigel and allowed to undergo polarization and lumenogenesis (Bedzhov and Zernicka-Goetz, 2014; Shahbaz et al., 2017). This model offers insights in the cellular and molecular mechanisms underlying mouse and human lumenogenesis, which opens up an amniotic cavity as well as the interrelationships between different pluripotent states and tissue architectures.
Post-Implantation Polarizing ETS Model4
Zernicka-Goetz noted that in normal embryonic development, ESCs are surrounded by extraembryonic cells to induce polarization. Their next
___________________
4 The ETS model refers to an in vitro model that combines embryonic and extraembryonic stem cells on a 3D matrix. More details about the ETS model can be found in Harrison et al. (2017).
model was designed to mimic this phenomenon (Harrison et al., 2017). Mouse ESCs were combined with TSCs, leading to the formation of “ET embryos” composed of embryonic and extraembryonic compartments. These mouse ET embryos grow and appear similar to normal embryos. She remarked that this model provided important insights (e.g., that the processes of symmetry breaking, mesoderm specification, and primordial germ cell formation do not require the presence of the anterior signaling center that is normally involved in those processes).
Post-Implantation Embryo Model
The fourth model was designed to study the mechanisms that enable cells to communicate and self-assemble to create the embryo structure with all three compartments and therefore a model that can induce the formation of not only posterior but also anterior structures. To create this model system, Zernicka-Goetz’s group combined mouse ESCs, TSCs, and XEN cells to build post-implantation-stage embryo models that look extremely similar to natural embryos (Sozen et al., 2018). This model makes it possible to study the principles directing self-organization leading up to initiation of gastrulation.
CLINICAL PERSPECTIVE ON PRE-IMPLANTATION HUMAN EMBRYO DEVELOPMENT
Heidi Cook-Andersen, an assistant professor of reproductive medicine at the University of California, San Diego, offered a clinical perspective on how studying pre-implantation human embryo development can elucidate inefficiencies in human reproduction in order to improve treatments for infertility and assisted reproductive technology. Her laboratory is exploring developmental benchmarks in embryos that can be reproduced and studied in model systems to improve IVF and reproductive treatments.
Inefficiencies in Human Reproduction
Compared with reproduction in other animals, human reproduction is markedly inefficient, Cook-Andersen said. The mouse cyclic fecundity rate is around 80 percent, and the nonhuman primate rate is reported to be as high as 70 percent, yet the human rate is just 20–30 percent (Silver et al., 1995; Stevens, 1997; Wilcox et al., 1988; Zinaman et al., 1996). Understanding the molecular defects in human embryo development that lead to these inefficiencies is critical to improving the diagnosis and treatment of infertility, she said. Four decades after the first IVF baby was born, observations from IVF and embryo culture in IVF clinics can provide
critical insights about the most likely sources of inefficiencies in human reproduction (Steptoe and Edwards, 1978). What is learned from IVF can help to improve embryo model systems and, in turn, the embryo model systems will make it possible to understand the causes leading to these inefficiencies in our reproduction, she said. One of the major questions is precisely how IVF and embryo culture mimic what is happening in vivo. Although this question may never be fully addressed, IVF protocols provide a unique and important opportunity to observe the earliest stages of human development, Cook-Andersen said.
In humans, reproductive inefficiencies tend to occur in three different stages of embryo development, Cook-Andersen said. The first is high rates of developmental arrest: 40–50 percent of embryos arrest during the transition from the cleavage stage (day 3) to the blastocyst stage (day 5) (Hardy et al., 2001). Many of those embryos are aneuploid, but it is estimated that up to one-third of them could potentially be euploid (Maurer et al., 2015; Munné et al., 1994). For embryos that develop to the blastocyst stage, high rates of aneuploidy are a major clinical problem that is largely age-based, occurring in 25–60 percent of the embryos in women less than 40 years old and up to 90 percent of the embryos in women older than 40, she said. Even in the youngest women with the best prognoses, embryo aneuploidy rates are still as high as 20–25 percent (Franasiak et al., 2014). The third inefficiency is the high rate of implantation failure, which ranges from 30 to 70 percent per embryo transferred. Implantation failure is based partially on ploidy, but it is also related to morphology and other factors that are not yet well understood. Because these inefficiencies are more common in humans, pre-implantation developmental progression, aneuploidy and implantation are important benchmarks to study in human embryos and to reproduce in model embryo systems in order to enable the mechanistic studies required to understand the causes of these inefficiencies that cannot be performed in the embryos themselves, Cook-Andersen said.
Exploring the Molecular Basis for Successful Implantation of the Human Embryo
Cook-Andersen and her colleagues are investigating high rates of implantation failure in human embryos in order to understand ultimately what is required at the molecular level for successful implantation. Decades of studies in IVF clinics have strongly established that embryos with “good morphology” have much lower implantation failure rates and much higher live birth rates than embryos with poor morphologies (Irani et al., 2017; Nazem et al., 2019). However, the differences at the molecular level between embryos of good and poor morphology that may explain their difference in implantation potential are still unknown. Morphology is not a perfect
predictor—some embryos with good morphology will not implant, and some with poor morphology will implant, Cook-Andersen said. However, her research group posits that euploid blastocysts with good morphology are enriched for embryos with high implantation potential, so euploid blastocysts with good morphology should also be enriched for the factors required for successful implantation. In the absence of being able to do rigorous genetics in humans, her laboratory is working to uncover the differences between these two groups of embryos with disparate implantation potential at the molecular level. To do this, they are exploring the gene expression patterns within and cell–cell communication networks between cells of the two major compartments of the human blastocyst: the mural trophectoderm and the inner cell mass. By directly comparing their findings in blastocysts of good and poor morphology, this approach provides the opportunity to develop prioritized gene lists and identify the molecular pathways important for successful human embryo implantation and the defects in these pathways that might contribute to the high rates of implantation failure seen in humans. Early findings suggest primitive endoderm development in pre-implantation embryos may play an important role in the success or failure of implantation, at least in the IVF setting, she said. Cook-Andersen stated that this and other similar studies in human embryos will provide important focus for studies in human embryo model systems and help lead to much needed advances in our treatments for infertile couples and in our understanding of the earliest stages of our own development.
PANEL DISCUSSION
Interactions Between Cells During Early Human Development
Discussions about human development often focus on heterogeneity and stochasticity, R. Pera said, and she asked panelists to comment about the nature of interactions that occur among cells during development (e.g., whether they are coordinated from cell to cell or are cell independent). Zernicka-Goetz replied that one of her primary research interests is to understand how an embryo breaks its symmetry with respect to the development of the major body axes and also when and how the cells within the embryo begin to differentiate and for the very first time start to initiate their identity. It is not yet known when human embryos initiate this process, but in mouse models the cells initiate this process by the four-cell stage or perhaps even earlier. The cells still have enormous plasticity potential to derive all cell lineages at that stage, Zernicka-Goetz said, albeit with different respective propensities to do so, and the potency in early blastomeres is preserved. Niakan added that disaggregating blastomeres
and allowing them to develop following reaggregation has suggested that there is plasticity of cells in human embryos until even after a blastocyst is formed, but systematic lineage tracing studies to investigate this process have yet to be conducted in humans. When blastomeres are separated at the two-cell stage in the mouse, one blastomere has been recently shown to have a greater developmental potential than the other, Zernicka-Goetz said.
Naïve and Expanded-Potential Stem Cells
Panelists were asked for clarification about the definitions of naïve and expanded-potential stem cells. Earlier studies showed that ESCs can contribute to extraembryonic mesoderm, but more recent studies indicate that there is variability between naïve and expanded-potential cells. Mouse naïve cells are equivalent to epiblast at roughly 4.5 days, said Rossant. In a chimera they make epiblast derivatives but typically do not make the other cell types. Extended-potential or expanded-potential cells seem to be able to make contributions to the trophoblast. But even if they contribute to the post-implantation trophoblast, most of those cells may not necessarily be making trophoblast, although they may have further potential to make XEN and TS cells in culture, she added. In humans, these cells appear to be able to make TSCs easily, suggesting that they may have more genuinely expanded potential.
Defects in Development of Primitive Endoderm
Thus far, it appears as though the absence of the primitive endoderm signal does not affect the epiblast in terms of gene expression, but it would be expected to have an effect at later stages, said M. Pera. He asked if a similar specific defect in the extended development of primitive endoderm has been observed in embryos grown through longer stages. No specific defect in the formation of the primitive endoderm has been observed in the embryos that her laboratory has grown to post-implantation stages, Zernicka-Goetz said. However, a defect they often observe is a reduced ability of the epiblast to proliferate in vitro. She noted that specification of the epiblast and primitive endoderm occurs later in humans than in the mouse. This tissue, called hypoblast in human embryos, is essential for later development. She said that chemical signaling between the tissues is critical to maintain development of both tissues: ligands and receptors for FGF are expressed in both extraembryonic tissue and embryonic tissue, while epiblast expresses ligand. Cook-Andersen added that they have observed that the factors for those pathways were higher in good embryos than in poor ones. Her group’s hypothesis is that different embryos with poor morphology might have different defects but that these different defects
might all manifest similarly downstream during the development of the primitive endoderm. Some differences in gene expression can be observed in the epiblast, but they tend to be more subtle than changes in the primitive endoderm. A workshop participant suggested that single-cell databases for nonhuman primates could be analyzed for evidence of evolutionary changes that might explain the role of primitive endoderm in implantation failure in humans.
Embryo-to-Embryo Variability in Modeling Human Development
The question of how to account for embryo-to-embryo genetic and epigenetic variability in humans when translating work done using mouse models to human models was raised by a workshop participant. This variability is an important consideration and can be a confounding effect in this research, Niakan said, noting that mouse lines of certain strains also have known differences. It is difficult to work with embryos because only a fraction of them make it to the developmental stages that can be studied, she continued. Another confounding effect is that the embryos they have access to are surplus to family building (i.e., ones that were not immediately selected for IVF), so they may not be the highest quality. Some jurisdictions, such as the United Kingdom, allow the creation of embryos for research. Another option is to use surplus eggs from egg banks, which tend to have a lower maternal age of donation and may be of better quality. Zernicka-Goetz highlighted two further potential confounding factors in developing a synchronized population of human embryos to study: (1) potential mosaicism in some of the embryos and (2) determination of exactly when human embryos were activated by fertilization. The oocytes that are obtained in IVF may also be at fundamentally different developmental stages as well, R. Pera added. Obtaining a sufficient number of cells from higher-quality embryos to draw meaningful conclusions is a substantial limitation of the system, Cook-Andersen said, and another issue to consider is underlying infertility in patients, although the extent to which infertility contributes to these inefficiencies in unclear. Additionally, mosaicism and different types of aneuploidy affect large proportions of these embryos, which is a barrier to computational analysis.
Genetic and Epigenetic Aberration in Blastocysts
Suboptimal culture conditions are thought to be largely responsible for the high rate of aberration observed in embryos cultured in vitro, one workshop participant said. However, it is unclear whether naïve conditions are better or worse than standard prime conditions, the participant added. The low rate of reproductive success in humans may be due to an inherent
low stability in the human system (e.g., pluripotent stem cells and certain early developmental states are transient in vivo). Potentially high rates of aberration in vivo are being “weeded out” by the biological system, but in vitro systems are being set up by selecting for cells that are compatible with culture conditions. Some data indicate that ESC lines are generally clonal, which suggests a very high selectivity for cells that may not be representative of the actual cells that contribute to reproductive success in vivo. The workshop participant went on to ask panelists if they knew of any evidence from either in vivo or in vitro cultured embryos that genetic or epigenetic aberrations may accumulate in the blastocyst itself, not in cultured cell lines where technical issues can cause aberrations. Niakan responded that more basic research is needed, but human ESC lines seem to have a high rate of acquired genomic abnormalities, such as aneuploidies or dysregulation of genes like p53, which may also underlie instability in the cells. The culture conditions for propagating human ESC and iPSC lines require optimization, Niakan said, and it would be helpful to inform approaches to optimize the culture methods by using insights from studying the embryonic compartment in the developing embryo. Due to selective pressure, embryos or cells with genomic abnormalities generally do not survive beyond a certain stage of development, Niakan said. In contrast, researchers seeking to capture these types of cells in culture in perpetuity may be selecting for a certain type of cell with slightly different properties. “This idea that you can capture exactly the equivalent stage of an embryonic transient epiblast in an in vitro culture is not necessarily an accurate way of thinking about it,” Niakan said. These are useful systems for disease modeling and drug discovery, she said, but they will not always recapitulate exactly what is happening in the transient embryonic epiblast.
Future Research Directions
Panelists were asked to reflect on the research questions that emerged from the first session. Rossant commented that using stem cells to make embryo models will require answering two fundamental questions: (1) how a normal embryo develops in a human and (2) what is being captured when different stem cell states are captured. Research should be aimed at better understanding what these cells states actually are, Rossant said, rather than focusing exclusively on getting stem cells to reflect exactly what happens in the embryo. Cook-Andersen called for studies to dissect what goes wrong at each of three steps of embryo development that appear to be particularly inefficient or prone to defects. This would be valuable from a biological perspective, assuming that similar failures happen in natural development, and from a clinical perspective, because intervening to address failures during those three steps would be most beneficial for helping people with
infertility. She suggested several areas of focus for future research: (1) the molecular mechanisms underpinning developmental progression on days 3–5 (which might largely reflect oocyte quality prior to fertilization); (2) the mechanisms of aneuploidy and the embryo’s capacity for self-correction (including whether aneuploidy is predetermined before oocyte stimulation or if alterations in IVF stimulation protocols might decrease aneuploidy rates); and (3) the molecular requirements for successful implantation (including methods to improve embryo selection prior to transfer).
Zernicka-Goetz said that in developing their synthetic 3D models, her group drew on knowledge about mouse and human development at the time of implantation to strategically select the types and numbers of cells that are known to be involved in the epiblast, primitive endoderm, and trophectoderm at the time of implantation. This basic knowledge came from years of observations of mouse and human development before, during, and after implantation. Although synthetic models have limitations, they are a powerful resource for identifying paths of development. It would be beneficial to focus research efforts on aneuploid embryos and especially embryos that have some aneuploid cells, but many euploid cells, the so-called mosaic embryos, Zernicka-Goetz said. In the mouse, those aneuploid cells are eliminated in a part of the embryo to make the fetus, but not in trophectoderm. The elimination process peaks after implantation when the p53 pathway is initiated. If the same process is found to occur in human embryos, then the underlying mechanism could—potentially, she stressed—be enhanced through culturing embryos at the time of implantation. Determining the relevance of cell heterogeneity is another important research question, she said. Unlike ESCs in culture, the cells in the embryo can be homogenous before developing heterogeneity and beginning to make different structures. She suggested that this heterogeneity might help to initiate and guide this process because the embryo has limited time before it implants and must generate the three tissue types in the correct spatial-temporal order.
Niakan predicted that the advent of novel, powerful omics technologies will facilitate important comparative embryology analysis between species and help to refine hypotheses about conserved or divergent molecular programs. Furthermore, she suggested that new genome editing technologies and methods for protein depletion and manipulating gene function will contribute to identifying molecular mechanisms across species and improving stem cell biology techniques. However, she cautioned against underestimating the value of mouse and nonhuman primate models.
