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Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop (2020)

Chapter: 5 Comparative Embryonic Development Across Species

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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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5

Comparative Embryonic Development Across Species

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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The fourth session of the workshop focused on comparative embryonic development across species. The session’s objectives were to understand the similarities and differences between nonhuman embryos, embryo models (e.g., chimeras), and human embryos and to identify scientific questions that may necessitate the study of human embryos. The session was moderated by Jianping Fu.

CROSS-SPECIES COMPARISON OF PRE-IMPLANTATION CHROMOSOMAL INSTABILITY

Shawn Chavez, an assistant professor in the Division of Reproductive and Developmental Sciences at Oregon Health & Science University, provided a comparison of pre-implantation chromosomal instability among different mammalian species, focusing on the prevalence of aneuploidy in pre-implantation embryos and mechanisms of mitotic mis-segregation and chromosome sequestering by micronuclei.

Aneuploidy in Pre-Implantation Embryos

Although in vitro fertilization (IVF) use has continued to increase each year, Chavez said, the success of IVF as measured by live births has not increased beyond 30–35 percent for decades. A leading cause of IVF failure and embryo loss is the presence of aneuploidy, which affects about

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

50–80 percent of cleavage-stage human embryos (Chavez et al., 2012; Chow et al., 2014; Huang et al., 2015; Johnson et al., 2010; McCoy, 2015; Vanneste et al., 2009a,b). Although an estimated 50 percent or more aneuploid embryos will arrest, they can still form blastocysts and may be morphologically indistinguishable from chromosomally normal (i.e., euploid) embryos. Both meiotic and mitotic errors contribute to aneuploidy, she noted. Until recently, errors involving chromosomal mis-segregation in oocytes during meiosis were considered the primary reason for aneuploidy, especially in cases of advanced maternal age. In maternal age-related aneuploidy, oocytes contribute approximately 90 percent of the meiotic errors. However, errors involving chromosomal mis-segregation during mitosis occur with equal frequency or perhaps even greater frequency than meiotic errors, irrespective of maternal age or fertility status.

Aneuploidies are diagnosed using pre-implantation genetic testing for aneuploidy (PGT-A), formerly known as pre-implantation genetic screening, Chavez said. PGT-A is associated with various risks and disadvantages based on the stage of development at which the biopsy occurs: polar body biopsy of a zygote, blastomere biopsy during cleavage stage, or trophectoderm biopsy of a blastocyst. At the zygote stage, PGT-A only detects meiotic errors. The biopsy of blastomeres at the cleavage-stage suffers from a high incidence of mosaicism,1 and there is evidence that the blastocyst stage is similarly mosaic. Biopsy performed during the blastocyst stage may also require extended culture. Regardless of the stage, all biopsies are considered invasive and may be detrimental to embryonic development.

Aneuploidy Frequency in Early Cleavage-Stage Embryos Across Mammals

Chavez outlined what is known about the range of aneuploidy frequency at the early cleavage stage across different mammalian species. As previously stated, aneuploidy occurs in about 50–80 percent of human embryos and in around 74 percent of nonhuman primates, specifically rhesus macaque embryos (Daughtry et al., 2019). In the cow, aneuploidy rates range from 32 to 85 percent in early cleavage-stage embryos (Destouni et al., 2016; Hornak et al., 2016; Tšuiko et al., 2017). This is a large range, Chavez said, but the majority of the studies were done using in vitro matured eggs, and in vitro maturation is known to increase aneuploidy due to both meiotic and mitotic errors. In the horse, the aneuploidy frequency is not yet known, although studies are ongoing using in vitro matured eggs. In a natural setting (i.e., no chemical induction), the mouse exhibits the least

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1 Mosaicism is the presence of more than one karyotypically distinct cell lineage in a single embryo (McCoy, 2017).

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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amount of aneuploidy (1 to 4 percent) (Bolton et al., 2016; Lightfoot et al., 2006; Macaulay et al., 2015; Treff et al., 2016), but this increases with maternal age and in vitro maturation.

Research Questions Regarding Aneuploidy in Pre-Implantation Embryos

Further research on aneuploidy in pre-implantation embryos is needed, Chavez said. One area to explore is the underlying mechanisms that may help explain why there is such a high incidence of aneuploidy in preimplantation embryos. Another question to investigate is whether live cell imaging—with or without labeled markers—can be used to dissect those mechanisms and to assess embryo developmental potential. Her laboratory focuses on the nuclear structure where the DNA is located, but she is also looking at cytoskeletal structure, given the cross-communication between those components. Determining whether there are corrective natural or therapeutic means to overcome chromosomal instability and aneuploidy generated during pre-implantation development should also be explored, Chavez said, adding that this area of research on pre-implantation development has the greatest potential to help the IVF community.

Mechanisms of Mitotic Mis-Segregation and Sequestering by Micronuclei

Current knowledge about four mechanisms of aneuploidy has been derived primarily from cancer cells and, more recently, from mouse embryos, Chavez said. Abnormal centrosome numbers—either too few or too many centrosomes—can have consequences such as multipolar divisions. Defective spindle attachments involve some kind of non-attachment or abnormal attachment. Compromised cell cycle checkpoints suggest that abnormal events such as an anaphase lagging chromosome go unrecognized and that the embryo divides before it has a chance to line up on the spindle; this is an area in which much of the mouse embryo work has been done. Loss of or prolonged chromosome adhesion are mechanisms by which chromosomes come apart too soon or stick together too much.

Regardless of the mechanism of aneuploidy, Chavez said, the embryo knows that a chromosome has been mis-segregated. This has been demonstrated by staining cleavage-stage embryos with DAPI for DNA as well as by using the nuclear envelope marker LMNB1 to show that mis-segregated chromosomes have been encapsulated in micronuclei during the next mitotic division (Chavez et al., 2012). These micronuclei have also been observed in nonhuman primates (Daughtry et al., 2019), the horse (Brooks et al., 2019), and the cow; however, micronuclei formation

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

is rarely observed in the early cleavage stages of the mouse (Chavez et al., 2014). Knowledge about the potential fate of these chromosome-containing micronuclei has been gleaned from cancer cells as well as from bovine and mouse embryos (Brooks et al., 2019; Chavez et al., 2012; Crasta et al., 2012; Daughtry et al., 2019; Liu et al., 2018; Vazquez-Diez et al., 2016; Zhang et al., 2015). One fate is unilateral inheritance, whereby the micronucleus that has formed goes on to replicate and divide just like the primary nucleus, so it is propagated with development. This has been demonstrated in bovine embryos at the early cleavage stage, in mouse embryos at the morula stage, and in cancer cells. Another fate is nuclear fusion, in which the nuclear envelope breaks down and the micronucleus fuses back with the primary nucleus. Work on cancer cells indicates that this is highly detrimental, because these micronuclei have undergone severe mutations and chromosome chaos.

Aneuploidy and Chromosome Loss Due to Cellular Fragmentation

Research on human and rhesus embryos has shown that one phenotype highly associated with micronuclei formation is a process called cellular fragmentation, Chavez said. DAPI, LMNBI, and CENP-A staining techniques have been used to show that a highly fragmented embryo can contain DNA not just in micronuclei, but also within cellular fragments. In fact, aneuploidy is often, but not always, associated with cellular fragmentation (Alikani et al., 1999; Antczak and Van Blerkom, 1999; Buster et al., 1985; Chavez et al., 2012; Dozortsev et al., 1998; Hardy et al., 2001; Pereda and Croxatto, 1978; Rambags et al., 2005). This process is observed in about 50 percent or more primate and equine embryos and about 15–20 percent of bovine embryos, although it is rarely seen in mouse embryos unless induced experimentally. Cellular fragmentation is distinct from the DNA fragmentation that can occur following cell death late in pre-implantation development and evidence suggests that it occurs in vivo for multiple species, including humans. Fragmented embryos often exhibit chromosome loss from blastomeres, Chavez said, which complicates aneuploidy assessment.

Chavez highlighted a study that she and her colleagues conducted of aneuploidy and chromosome loss via cellular fragmentation in rhesus embryos using single-cell, next-generation sequencing of 254 blastomeres, 42 polar bodies, and 175 cellular fragments isolated from 50 cleavage-stage embryos (Daughtry et al., 2019). The majority of the polar bodies, whose identity was confirmed by single-nucleotide polymorphism genotyping, were chromosomally normal. However, only about one-quarter of the blastomeres were completely normal; high degrees of aneuploidy and mosaicism were observed in the blastomeres. The researchers also found

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

that the missing chromosomes that had been lost from blastomeres were encapsulated within cellular fragments. There did not appear to be any preferential sequestering of specific chromosomes, and the pieces of chromosomes observed were quite large (6–85 megabases). Multipolar divisions were the most frequent phenotype that resulted in the formation of chromosome-containing cellular fragments, and all of the associated blastomeres exhibited chaotic aneuploidy. However, because not every single embryo exhibited this phenotype, Chavez’s group is investigating other mechanisms, such as chromatin or anaphase bridging.

DNA Damage in Chromosome-Containing Cellular Fragments and Micronuclei

Eighteen percent of the rhesus embryos in the study had chromosome-containing fragments, Chavez said, but only 6 percent of all the fragments had DNA detectable by sequencing. Daughtry et al. (2019) surmised that once the cellular fragments encapsulate the DNA, they become highly damaged because they no longer contain a nuclear envelope. Micronuclei can form as early as the zygote stage, but they can also be retained in the blastocyst stage—either as previous or new events—and may have substantial DNA damage. Ongoing work has shown that there are micronuclei in the trophectoderm as well as in the inner cell mass (ICM) of blastocysts; Chavez’s team is currently analyzing blastocysts at the single-cell level for aneuploidy and to establish whether DNA damage has occurred. Fluorescent live-cell imaging is being used to identify in real time which cleavage-stage embryos have cellular fragments that contain DNA so that they can be isolated before they start to degrade. The aim is to identify which blastomeres contain DNA so that they can be carried forward for sequencing. The fluorescent labels do not interfere with DNA sequencing, Chavez said.

Chromosomally Mosaic Embryos Can Lead to the Birth of Healthy Offspring

Several years ago, Chavez said, the IVF community was rocked by the finding that in about 50 percent of cases, chromosomally mosaic embryos can result in the live birth of healthy offspring (Greco et al., 2015). Some of these studies have since explored a potential post-implantation mechanism for this phenomenon (Bolton et al., 2016; Fragouli et al., 2017; Gleicher et al., 2016). However, to help the IVF community specifically will require a better understanding of the mechanisms that occur during preimplantation. To explore whether there are selective growth advantages for euploid cells or mechanisms for the elimination of aneuploid cells during

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

pre-implantation development, Chavez and colleagues cultured 92 rhesus embryos with time-lapse imaging, with a blastocyst formation rate of 54.3 percent. At the cleavage stage, 26.5 percent of the embryos were euploid and 32.6 percent were mosaic, suggesting that some of those blastocysts would be chromosomally abnormal to a certain degree.

By returning to the time-lapse imaging, they found that some of these rhesus embryos exhibited cellular fragmentation that was excluded from the rest of the embryo and appeared not to participate in embryo development. About 16 percent of the rhesus blastocysts exhibited blastomere exclusion—that is, upon blastocyst transition one or more of the embryos’ blastomeres fails to divide from the 2- to 4-cell stage and is sequestered to the blastocoel. DAPI staining was used to reveal the exclusion of DNA-containing cellular fragments upon embryo hatching. The researchers also found that excluded blastomeres were multi-nucleated, had substantial DNA damage, and were highly chromosomally abnormal. This suggests that blastomere exclusion is at least one of the mechanisms that serves to overcome aneuploidy during pre-implantation development, Chavez said. This mechanism also appears to be conserved across species, because it has also been observed in horse and cow embryos.

In closing, Chavez reiterated that rhesus pre-implantation embryos have the same incidence of micronucleation, aneuploidy, and cellular fragmentation as human embryos. However, models for aneuploidy related to maternal age are still needed, she said, because the monkeys used are relatively young. The mouse model could still be useful for maternal age-related aneuploidy and post-implantation, she said, while the monkey would be useful for modeling pre-implantation development.

TROPHOBLAST DIFFERENTIATION FROM PRIMATE PLURIPOTENT CELLS

Ted Golos, a professor and the chair of the Department of Comparative Biosciences at the University of Wisconsin, considered whether trophoblast differentiation can occur with primate pluripotent cells. He described the formation of embryoid bodies from human embryonic stem cells and the use of rhesus embryoid bodies to model trophoblast differentiation. He also examined the predictive capacity of nonhuman primate embryo model systems.

Breakthroughs in Modeling Trophoblast Differentiation with hESCs

In the mid-1990s, Golos said, James Thomson at the Wisconsin National Primate Research Center modified methods previously used with mouse embryonic stem cells to produce multiple embryonic stem cell (ESC) lines

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

using blastocysts from the rhesus monkey produced in vivo (Thomson et al., 1995). It was also observed that ESCs from the rhesus monkey and the common marmoset can spontaneously give rise to gene expression associated with trophoblasts, because those spontaneously differentiating ESCs initiate the expression of chorionic gonadotropin (CG) and its alpha and beta subunits. Thomson was later able to derive human ESCs (hESCs) based on the spontaneous differentiation and secretion of CG (Thomson et al., 1998). This established that trophoblast formation is also a hallmark of hESCs, which is not, Golos noted, the case for mouse ESC models. This work gave rise to the question of whether primate and human ESCs can provide a system for modeling trophoblast differentiation and placental morphogenesis. Spontaneous differentiation of hESCs to the trophoblast lineage in vitro or in teratomas is of low efficiency; a breakthrough occurred when Xu and colleagues discovered that bone morphogenetic protein 4 (BMP4) or other related ligands can be used to produce a morphologically homogeneous population of cells (Xu et al., 2002). Although it may not have been a homogeneous trophoblast population, this new model clearly had great potential.

Embryoid Body Formation from hESCs

While this work was ongoing with BMP and other ligands, Golos and colleagues decided to use the embryoid body (EB) as an approximation of a spherical embryoid structure, by lifting the hESC colonies and maintaining them in suspension in two-dimensional (2D) culture (Gerami-Naini et al., 2004). They also developed a method for three-dimensional (3D) culture of EBs from hESCs in extracellular matrix by physically inserting EBs into droplets of Matrigel and maintaining them in suspension. Golos explained that EBs in suspension culture initiate the secretion of the “holy trinity” of protein and steroid hormones characteristic of primate placental function: CG, progesterone, and estrogen (the latter is secreted if given an androgenic precursor; see Gerami-Naini et al., 2004). Furthermore, EBs growing in a 3D Matrigel scaffold initiate the sustained secretion of CG as well as of progesterone and estradiol. This established that both 2D and 3D Matrigel environments were able to support placenta trophoblast secretions.

Adaptation of hESC Paradigms to In Vivo Implantation

Next, Golos and colleagues sought to adapt these paradigms to a system that might eventually mimic implantation. Although they had seen outgrowths into Matrigel in the previous work with embryoid bodies, they had not seen villous morphogenesis. Because the morphogenesis of chorionic villi and implantation are similar—but not identical—between humans and macaques, they used rhesus ESCs for the BMP4 differentiation

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

paradigm. They also plated EBs on 2D Matrigel and inserted EBs into a 3D Matrigel environment. However, neither BMP4 nor the 2D/3D Matrigel culture stimulated the secretion of monkey CG or progesterone, nor did they observe any expression of endocrine function when using hormone secretion as the readout for whether mESCs were equivalent to hESCs. To explore whether a non-endocrine differentiation marker would be more productive, they used flow cytometry for the rhesus monkey homolog of HLA-G (i.e., Mamu-AG). In the primary trophoblast cultures, there was clear expression of placenta-specific Mamu-AG with differentiation in the 2D EB culture paradigm.2 However, one of the rhesus ESC lines did occasionally have outgrowths in which some of the cells expressed Mamu-AG, so the approach was not fully consistent. Coupled with the lack of endocrine differentiation, this called into question the use of CG as a readout and suggested that perhaps the macaque placental endocrine profile may not be appropriate for these approaches.

Use of Rhesus Embryoid Bodies to Model Trophoblast Differentiation

It was established in 1974 that CG is rapidly secreted 10–11 days after implantation in rhesus monkeys. Although CG is somewhat truncated, it is clearly a marker and a signaling mechanism for corpus luteum rescue (Hodgen et al., 1974). Therefore, Golos and colleagues returned to the use of embryos produced in vitro and inserted hatched blastocysts into the 3D Matrigel environment that they had used with EBs (Chang et al., 2018). They observed outgrowths from those blastocysts into the Matrigel as well as the initiation of the secretion of CG, progesterone, and estradiol. This work also provided a good visual depiction of the embryonic structure in the Matrigel. Wispy areas can be observed where the trophoblasts are growing out into the Matrigel as well as digestion degradation of the Matrigel environment, which is consistent with the metalloproteinase expression and secretion that is also seen with the interstitial migration of extravillous trophoblast (EVT) into the maternal decidua. This work suggests that the actual rhesus embryo might be a more appropriate model of trophoblast differentiation than pluripotent cells, Golos said. Recently, Okae et al. (2018) have reported the derivation of human trophoblast stem cells (TSCs). Using the Okae approach, Golos and his colleagues have generated eight macaque (rhesus and cynomolgus) TSC lines and, under the appropriate paradigms, they have been able to use TSC differentiated to syncytiotrophoblast (STB) to initiate substantial monkey CG secretion into the culture medium.

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2 Mark Garthwaite and Svetlana Dambaeva, unpublished data.

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Predictive Capacity of Nonhuman Primate Embryo Model Systems

To the question of whether nonhuman primate embryo model systems can predict the function of systems made with human cells, Golos said that currently rhesus pluripotent cells are not transparently equivalent to human cells for trophoblast differentiation. In addition to the approaches that Golos described, there are other potential avenues for discovering the differences between rhesus and human pluripotent cells, including differentiation paradigms, receptor complement, feeder cells, trophoblast differentiation niche, maternal factors, genetic background, and organoid aggregates with placental stroma. On the other hand, it is well established that rhesus IVF blastocysts implant in vitro, secrete stereotypical placental hormones, and have invasive trophoblasts that invade and degrade a Matrigel environment. Thus, rhesus embryos might be a tractable system with which to study some of these important processes in early pregnancy. Furthermore, given the limited availability of monkey embryos, rhesus TSCs also could be a high-throughput model for the differentiation of in situ trophoblasts and putative EVT. In addition to forming monkey CG–secreting multinuclear STB and putative EVT, rhesus TSCs express placenta-specific major histocompatibility complex class I molecules; RNA sequencing shows that they appear to be authentic trophoblasts.

Answering Scientific Questions by Studying the Human Embryo

There are a number of scientific questions that could be answered by studying the human embryo, Golos said. Within a few weeks of fertilization, the human embryo has invasive primitive STB that becomes buried in the decidua (West et al., 2019). That invasion process may not be easy to model in nonhuman primates and may require human systems, he said; however, useful information about the peri-implantation period may potentially be gathered from other species. A strength of the nonhuman primate model, he said, is that it provides opportunities to interrogate through pre-implantation embryo manipulation different types of embryonic events that are critical for in vivo placentation, such as villous morphogenesis, spiral artery endovascular migration of EVT, and vessel remodeling. These are experiments that cannot be conducted at the requisite stage of human pregnancy, so toggling back and forth between different kinds of models may be needed in order to answer precise questions about the components that underlie pregnancy success and adverse pregnancy outcomes.

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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EARLY NEURAL CREST FORMATION: FROM BIRDS TO HUMANS

Martín García-Castro, an associate professor of biomedical sciences at the University of California, Riverside, made a case for pursuing early studies of neural crest cells, both in human and in alternative model organisms. He explained the ontogeny and differentiation potential of the neural crest and described how human neural crest formation can be modeled using pluripotent stem cells.

Neural Crest and Neural Crest Derivatives

In 1868 Wilhelm His used the chicken embryo to pinpoint the cells between the prospective epidermis and the neural ectoderm, which he predicted would also be found at different stages in the elevated neural folds, inside the neural tube, outside the neural tube, and migrating to colonize cranial ganglia (His, 1868). His initially referred to these cells as the intermediate cord, but they were later renamed the neural crest (Marshall, 1879). Subsequent work has shown that these cells arise along the anterior–posterior axis of the embryo at the edge of the neural plate and are marked by the transcription factors Pax7/Pax3 among others. After these cells reach the neural folds, they undergo an epithelial-to-mesenchymal transition to delaminate from the neural tube and migrate extensively through stereotypic pathways in very precise routes, generating a series of neural crest derivatives. Throughout the body, they contribute to neurons and glia of the peripheral nervous system and generate the melanocytes in the skin that provide color and protect from ultraviolet rays. They also make specific derivatives in distinct territories, including cartilage, bone, and connective tissue in the cranial region and sympathoadrenal cells in the trunk. Neural crest derivatives are thought to be involved in one-third of all birth defects, including orofacial clefts and rare syndromes. They also contribute to cancers, including melanoma. Because neural crest cells are involved in so many pathologies, García-Castro said, these cells hold great promise for clinical studies.

Differentiation Potential of Neural Crest

García-Castro described his work, which focuses on the origin of the neural crest and, specifically, trying to understand how it is possible to generate so many different cell types (e.g., neurons, glia, melanocytes, adipocytes, odontoblasts, osteoblasts, chondroblasts, myocytes, oxygen-sensing cells of the carotid body, and thymus mesenchyme) given the putative origin of these cells. The standard story is that after fertilization, cells differentiate

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

to generate epiblast cells, which in turn undergo gastrulation that generates the three germ layers: the endoderm that makes the internal organs; the mesoderm that forms bone, cartilage, adipose tissue, and blood-forming cells; and the ectoderm that produces the central nervous system and the epidermis. Neural crest cells are purportedly derived from the ectoderm and, as such, they are progenitors of the neurons and glia of the peripheral nervous system as well as of the melanocytes in the skin. This story is somewhat puzzling, García-Castro said, given that the neural crest cells also make many other derivatives in the cranial region that are normally associated with mesoderm. He questioned how a cell that is derived from the ectoderm could simultaneously generate all of the neural crest derivatives. This phenomenon is discordant with the Waddington’s epigenetics and post gastrulation restrictions that suggest that mesoderm, ectoderm, and endoderm should only differentiate into derivatives of each lineage. “For an ectodermaly derived neural crest to generate a mesoderm derivative, it would have to bypass the ectoderm restriction, climb up a hill and go to the other valley,” he remarked.

Ontogeny of Neural Crest

García-Castro’s laboratory is using a variety of model organisms and model systems to revisit the ontogeny of neural crest cells and determine when those cells are specified.

Specification of Neural Crest Cell Fate

The perception that neural processes are specified in the ectoderm comes from a series of studies from the 1980s and 1990s, which showed that neural crest cells appear in between the neural and non-neural ectoderm. This suggests that those two tissues interact with mesoderm that lies underneath them, inducing the appearance of neural crest cells in a classical fashion. This is thought to be mediated by the BMP, fibroblast growth factors (FGFs), and Wnt pathways, among others, which lead to a cascade of transcription factors and other markers that are expressed at the neural plate border and are responsible for the development of neural crest cells. However, the work of García-Castro’s laboratory suggests that the specification of these neural crest cells can actually be traced much earlier to the epiblast, before the definitive mesoderm and neural tissues appear. The researchers in García-Castro’s laboratory have identified a specific region of the pre-gastrula epiblast with cells that are poised to make neural crest cells, although they do not express known markers. To frame his discussion, García-Castro defined the concept of specification in this context. Specification is the initiation of a program—in this case, the initiation of the neural

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

crest cell fate program. The specified cell does not display known neural crest markers yet, but it is able to do so under “undisruptive” conditions. The researchers use the markers that are seen later on in neural crest cells to assess cell fate. This does not suggest that these cells are committed to form neural crest cells, García-Castro said; they are still plastic and can be persuaded to do otherwise in the embryo. In researching early avian neural crest development, García-Castro and colleagues identified the expression of Pax7 in a restricted region between the prospective neural plate and epidermis at the early stages of development. No other marker of the neural process is expressed in such a restricted fashion at this early stage, and it precedes the expression of later markers such as Snail2, SOX9, and Ets1. Inhibiting Pax7 blocked the expression of several of those markers, demonstrating that it was required for neural crest cells and further indicating that some cells are already specified in the embryo before they express Pax7 (Basch et al., 2006).

Blastula Studies of Neural Crest Cell Specification

García-Castro provided an overview of his laboratory’s recent work on the specification of neural crest cells in chicks during the blastula stage. In the first phase, his team monitored for the expression of the Pax7 marker after dissecting epiblast tissues, explanting them into collagen gels, and culturing them in isolation under non-inductive conditions. A clear spatiotemporal restriction was observed between explants that can and cannot express Pax7, with Pax7 only appearing in the intermediate, not the center or the most lateral territories. After the explants had been incubated for a longer period, other neural crest markers emerged, and eventually the definitive markers of migratory neural crest cells were evident in the restricted intermediate territories. García-Castro and his colleagues followed up with a series of experiments to determine whether neural crest markers are induced in culture. First, they cut the explants and disassociated the cultures that were generating a low density of prospective neural crest cells—that is, the cells were not isolated, but they were far apart from each other. Then the researchers compared the explant from the central (control) territory with the explants from the lateral territory, which were cultured under the same conditions. At the time the explants were cut, the cells in the intermediate territory were already poised to express Pax7. After 25 hours, the researchers could identify cells that were clearly far away from each other in the large-volume culture, which makes it unlikely that the cells were responding to contact mediated signals from neighbors and induced in the classical way. These cells that were disassociated long before gastrulation at stage XII of embryonic development and cultured at low density went on to express Pax7 and SOX9, while those cultured from the central territory

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

did not. A subsequent set of experiments looked at epiblast fate mapping by labeling cells in the epiblast with fluorescent dyes (DiI and DiO) and found that intermediate territories contributed to cells that go on to express Pax7 in the lateral territory that abuts the neural folds; the researchers also observed the label in the neural folds and migratory neural crest cells. Based on these findings, García-Castro and colleagues have built a map that shows the lateral region as containing prospective neural crest cells at these early stages of development. This is aligned with previous work suggesting that neural cells are specified in the center of the epiblast (Wilson et al., 2001).

Modeling Early Human Neural Crest Development with Pluripotent Stem Cells

Considering the question of whether human neural crest also specified before gastrulation and whether the knowledge gained from neural crest in chicks can be translated to humans, García-Castro said that knowledge of neural crest cell development in human embryos is still limited to histological descriptions of neural crest cells under migratory routes, with minimal molecular information available. To address this knowledge gap, García-Castro and colleagues obtained a collection of embryos and characterized a battery of markers expressed by human neural crest cells in pre-migratory or migratory states (Betters et al., 2010). However, this was not sufficient to determine whether specification was occurring at the very early stage, and those types of embryos are difficult to access. Thus, the researchers turned to modeling the development of human neural crest with PSCs, following work initiated by Pomp et al. (2005) and carried out in various other labs, and informed by the García-Castro lab’s own work on neural crest cell development in the embryo. The researchers developed a system to rapidly derive human neural crest from hESCs via Wnt in defined media (Leung et al., 2016). Using low-density cultures of cells that display characteristics of stem cells but no neural crest cell markers, they observed robust expression of Pax7 and SOX10 in just 5 days. Ultimately, the model was used to show the expression of all neural crest cell markers of interest as well as to demonstrate the capacity to generate all of the neural crest cell derivatives that are expected from neural crest cells. This demonstrates that the Wnt-induced cells are bona fide neural crest cells, García-Castro said.

Advantages of the Model

García-Castro’s model follows many others that have induced neural crest from hPSC (Chambers et al., 2013; Fukuta et al., 2014; Lee et al., 2007a; Menendez et al., 2011; Mica et al., 2013; Pomp et al., 2005). However, he said, his model is unique in several ways. It is the most rapid,

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

and it is the only one that does not use either fading of serum replacement or TGF-beta inhibition. His team has used the model to characterize the signaling parameters of neural crest cell development, including the window of time of exposure for Wnt and the concentration of Wnt. Using the model, it is possible to modulate the identity of the neural crest cells and make neural crest derivatives according to the desired axial region—that is, higher levels of Wnt can make posterior neural crest cells and low levels of Wnt can make anterior neural crest cells (Gomez et al., 2019a,b). The protocol has also been modified to generate a robust platform that has high efficiency across multiple types of cells, including ESCs or induced pluripotent stem cells (Hackland et al., 2019). Furthermore, the model addresses the ontogeny of the neural crest cells by demonstrating—contrary to the prevailing model—that neither neural ectoderm nor mesoderm is required to form neural crest cells. Instead, the model holds that there is a very early lineage derived from the epiblast, which García-Castro calls “pre-border,” that expresses specific markers that lead to neural crest cell formation. This holds specifically for early anterior neural crest cells, he noted. To further analyze the ontogeny of human neural crest cells, García-Castro’s laboratory has looked at different characteristics of early induction in the epiblast using epigenetic, transcriptomic, and functional assays, including a comparison of the epigenetic regulation and expression of ESC and neural crest cell markers after Wnt induction. He and his colleagues have found that neural crest proteins are upregulated in prospective neural crest cells soon after Wnt activation, as indicated by the expression of neural crest markers (Leung et al., 2019). Additionally, functional assays suggest that 6 hours after exposure to Wnt, the cells have initiated the path to neural crest cell formation.

Early Neural Crest Cell Development in Mammals

Reflecting on how to translate gains from chick and model human neural crest studies to mammalian embryos in order to enable more robust comparisons, García-Castro said that much progress has been made using mouse models to study neural crest cells in the later stages of development, but little is known about how the neural crest cells are formed or specified (Murdoch et al., 2010, 2012). A challenge lies in rodent-specific pre-gastrula development. A chick embryo has a large territory that allows for high spatiotemporal resolution; the mouse embryo is very small in comparison, which makes it difficult to dissect effectively. Another issue is that the mouse embryo adopts a cup-like shape rather than lying flat, and the murine germ layers are inverted in early stages. This makes the mouse a less appropriate model for modeling neural crest cell development in a way that is informative for other mammals, including humans. Consequently,

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

García-Castro’s laboratory is working to integrate the rabbit as a mammalian alternative model of neural crest cell development. The rabbit is larger than the mouse, allowing for better spatiotemporal resolution, and it has a flat blastodisk like the chick and human embryos. By characterizing neural crest development in very early stages in the rabbit, he and his colleagues have found the expression of mesoderm, neural, and neural crest markers. They identified Pax7 as the first restricted marker of neural crest cells as well as identifying several other NC markers, including SOX9 and SOX10, that have been shown in other model organisms. As shown in the mouse, they observed neural crest emigration before the tube closes. Most importantly, they found that rabbit neural crest cells are specified during gastrulation (Betters et al., 2018). For the first time in a mammal, they were able to dissect the epiblast of the gastrula embryo to generate and culture explants, demonstrating that although the intermediate territory is specified to form neural crest cells, it does not co-express mesoderm markers. Over time, they express crest markers, and they are subjected to signaling restrictions similar to those found in the chick.

BLASTOCYST-LIKE STRUCTURES GENERATED FROM PLURIPOTENT STEM CELLS WITH EXPANDED POTENTIAL

Jun Wu, an assistant professor of molecular biology at the University of Texas Southwestern Medical Center, explained how blastocyst-like structures (blastoids) can be generated from pluripotent stem cells systems in vitro in order to study early development. He described how the in vitro model mimics pre-implantation development in vivo and considered the development potential of these model structures in utero.

Building a Blastocyst-Like Structure Using Stem Cells

Describing the fundamentals of building a blastocyst-like structure using pluripotent stem cells, Wu explained that pluripotency is a continuum rather than a singular state of development (Graham and Zernicka-Goetz, 2016). To construct a blastocyst-like structure, the task is to select which type of pluripotent stem cells to use in order to adapt the in vivo continuum of pluripotency to an in vitro environment. Pluripotent stem cells can be generated by (1) isolating them from early embryos by taking out the ICM and then expanding them under different culture conditions or (2) inducing them from fibroblast and other somatic cells. Depending on when and what kind of cells are isolated, pluripotent cells have different properties. By developing different culture conditions, stem cells can be cultured in several different pluripotent states. The most well-known pluripotent states are naïve and primed. The naïve in vitro pluripotent state represents an earlier

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

type of pluripotent stem cell in vivo, which is very similar to the epiblast of an E4.5 embryo. The prime pluripotent state represents a peri-gastrulation stage of the epiblast when it is primed for differentiation.

In order to generate a blastocyst-like structure, the naïve pluripotent stem cell would seem to be the logical choice, Wu said; however, the blastocyst contains more than one cell type, so generating a blastocyst requires at least two to three different types of cells. Over the past several decades, three stem cell lines have been generated: trophoblast stem cells (TSCs) (Tanaka et al., 1998), extraembryonic endoderm (XEN) stem cells (Kunath et al., 2005), and ESCs (Evans and Kaufman, 1981; Martin, 1981). Rivron has demonstrated that aggregating TSCs and ESCs in culture can generate a blastoid structure, which looks like a blastocyst and expresses several blastocyst markers (Rivron et al., 2018b). Several years ago another type of pluripotent stem cell was derived from early embryos, called extended- or expanded-potential pluripotent stem cells (EPSCs) (Yang et al., 2017a,b). These cells can generate both embryonic and extraembryonic lineages, such as placenta and yolk sac. A single EPSC injected into an 8-cell stage embryo can contribute to both the trophectoderm and the ICM of a blastocyst.

Building a Blastocyst-Like Structure from Expanded-Potential Pluripotent Stem Cells

Wu and colleagues looked at whether EPSCs can self-organize to generate a blastocyst-like structure (blastoid) in culture (Li et al., 2019). Figure 5-1 depicts an overview of the process. After aggregating four to five EPSCs under the appropriate conditions for 4–6 days, the cells differentiated and self-organized into a blastoid-like structure, which Wu’s team named an “EPS-blastoid.” Initially, the cells formed a cluster without distinct morphology, but after a few days, a blastocyst-like cavity and an ICM-like structure began to form. Visual comparison revealed that the EPS-blastoid looked similar to a blastocyst isolated from an in vivo E3.5 stage embryo. Using two different cell lines, they found that about 15 percent of the structures looked like blastocysts, but the other structures were disorganized and showed markers in different locations.

First Cell Fate Determination in EPS-Blastoids: Trophectoderm and Inner Cell Mass

To further characterize the EPS-blastoid, Wu and colleagues looked first at the expression of two transcription factors that are characteristic of the trophectoderm (TE) and the ICM: CDX2 and SOX2. CDX2 was expressed in the outer layer of the cells of the blastoid, while SOX2 was localized to

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×
Image
FIGURE 5-1 Blastocyst-like structure generated in vitro using expanded- or extended-potential stem cells.
NOTE: EPS = expanded-potential stem; ESC = embryonic stem cell; TSC = trophoblast stem cell; XEN = extraembryonic endoderm.
SOURCES: Jun Wu workshop presentation, January 17, 2020.
Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

the ICM. This suggested that at least the first cell fate commitment in some of the structure had been made. In about 75 percent of the aggregates, the inside cells expressed SOX2, NANOG, or OCT4, and the outside cells expressed CDX2. However, other structures were generated that did not appear like blastocysts. To further investigate, the researchers counted the numbers of cells in the ICM and TE. At day 6, the numbers of both the CDX2-positive cells and the SOX2-positive cells were comparable to a blastocyst isolated in vivo.

Second Cell Fate Determination in EPS-Blastoids: Epiblast and Primitive Endoderm

Next, Wu and colleagues looked at whether EPS-blastoids could further develop into structures resembling a mature blastocyst, which contains three cell lineages instead of two. In some of the EPS-blastoids, they observed that some inside cells expressed GATA4, a marker of the primitive endoderm (PE) while other inside cells expressed NANOG. This suggested that the second cell fate determination had also occurred in some of the structures. However, the efficiency was low, with only 20 percent of the EPS-blastoids actually giving rise to a mature blastocyst-like structure containing all three cell lineages. The numbers of cells in the structures were comparable to those in a mature blastocyst at the E4.5 stage, with both NANOG-positive cells and GATA4-positive cells coming from the epiblast and primitive endoderm lineages.

Transcriptomic Features of EPS-Blastoids

A global transcriptome analysis was performed to identify transcriptomic features of EPS-blastoids, Wu said. First, they used bulk RNA sequencing to compare the EPS-blastoid with natural blastocysts and morulae. It appeared as though the blastoid they generated was closer to a blastocyst than a morula, suggesting that the EPS-blastoid was more like a 3.5-day blastocyst than a 2.5-day morula embryo. Single-cell RNA-sequencing analysis confirmed that all three cell lineages in the EPS-blastoid had been properly segregated. Of interest, the researchers observed that many cells that were intermediate between the epiblasts and trophectoderm, suggesting that the first cell commitment in some of the aggregates was not properly allocated.

EPS-Blastoid Formation Mimics Pre-Implantation Development

Wu and colleagues then looked at whether the system for EPS-blastoid formation could be used to model pre-implantation development (Ramanujam et al., 2017). Several morphological and molecular processes

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

occur during pre-implantation development, he said, including compaction, polarization, differential yes-associated protein (YAP) localization in the TE and ICM compartments, and X chromosome inactivation in the trophectoderm layer via preferential silencing of the X chromosome from the paternal side. He and his colleagues observed compaction occurring as early as day 1 as well as polarization occurring during blastoid formation. Looking at Hippo–YAP signaling in the outer layer cells of the trophectoderm, they saw most of YAP localized to the nucleus. However, in the ICM there was a diffused staining of YAP. Wu said that they used an inhibitor of YAP that was effective in stalling mouse embryo development, so the EPSC aggregates failed to form any blastoid-type structures in the presence of that inhibitor. He and his colleagues observed that in about 80 percent of the structures, the TE-like layer preferentially silenced the X chromosome from the paternal side. A functional test of the EPS-blastoid was whether it could be used to derive all three types of stem cells, Wu said. And, indeed, using the EPS-blastoid, they were able to derive all three types: (1) ESCs, which gave rise to adult chimeras, (2) TSCs, which could contribute to placenta tissue, and (3) XEN cells, which contributed to the yolk sac membrane after injection into the blastocyst.

Developmental Potential of EPS-Blastoids Post-Implantation and In Utero

The next step was to culture the EPS-blastoid in vitro to post-implantation-like structure, which they performed using the culture protocol developed by Zernicka-Goetz (Bedzhov et al., 2014). During this extended culture of the EPS-blastoid, they observed morphogenic events that modeled peri- and post-implantation development. In some of the structures the epiblast became polarized by day 3 and pro-amniotic cavities started to form by day 5. However, the efficiency decreased dramatically the longer they were cultured in vitro. To explore the in utero developmental potential of EPS-blastoids, they transferred them into the uterus and observed implantation. Some of the deciduae generated by the EPS-blastoid were similar in size to an in vivo decidua, and markers for EPI, TE, and PE were expressed in some of the structures’ cells. However, the structures were malformed, disorganized, and abnormal compared with the normal development of an embryo after transplantation into the uterus. He and his colleagues have also been able to derive and implant blastoids using iPS-cell-derived EPSCs, he added, which were generated with similar efficiency, as would be expected from EPSCs derived from embryos.

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

Use of Human Expanded-Potential Stem Cells

In addition to the mouse studies, two studies have shown that human EPSCs can be derived from the early embryo or induced from somatic cells (Gao et al., 2019; Yang et al., 2017b). Injecting a single human EPSC can contribute to the TE and ICM compartments in a mouse blastocyst, but it is inefficient for generating any type of blastocyst-like structure. Although some of the structures look similar to a natural human blastocyst, after staining the proper markers are not expressed in the right locations. “It’s safe to say that, using the mouse protocol, we haven’t been able to generate similar structures from human EPSCs that resemble a human blastocyst,” Wu said.

PANEL DISCUSSION

Use of Human Expanded-Potential Stem Cells in Mouse Models

Why do hEPSCs not appear to work as well as mouse EPSCs in the model produced by Wu’s team? one workshop participant asked. Wu offered several reasons why his team did not observe successful formation of the human blastoid from EPSCs: (1) EPS culture conditions and cell quality are different for human and mouse, and blastocyst-like structures could be generated from early but not late passage human EPSCs; (2) species differences between human and mouse during early development may also play a role, for example, if the two species differ in the timing of TE, EPI, and PE lineage specifications; and (3) the protocol used for generating blastoids from mouse EPSCs is not necessarily applicable to humans. The success in generating the mouse EPS-blastoid relied on an initial biased differentiation toward the TE lineage, Wu said. In the mouse, adding in factors like FGF4 or Wnt agonist can promote TE differentiation of the EPS cells. However, the signals that promote the human TE differentiation from human EPS cells remain unknown.

Impact of Chromosomal Disruption on Fertility

What is the impact of chromosomal disruption on human infertility, a workshop participant asked, and are there implications for pre-implantation diagnosis? The participant noted that cows are highly fertile, but the degree of chromosomal disruption in cows is comparable to that in humans. The use of nonhuman primate studies can help shed light on this issue, Chavez said, adding that when she and her colleagues investigated this in the time-mated breeding colony at their primate center, they found that 74 percent (the same percentage of rhesus cleavage-stage embryos that were aneuploid

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

to some degree) of the confirmed ovulation and mating cases did not result in a live birth. Thus, only about 30 percent of those resulted in a live birth, which is relatively similar to what is seen in humans. The impact of in vitro versus in vivo maturation of oocytes is a relevant consideration for cows, she added. However, a comparison between bovine embryos derived in vivo and in vitro revealed an aneuploidy rate of 18 percent even in the embryos derived in vivo.

Fragmentation in Aneuploid Embryos

In response to a question about whether all aneuploid embryos appear to fragment, Chavez replied that this question has persisted for decades. In fact, clinicians used to remove fragments because they thought it would improve the embryo. Her group’s working hypothesis is that fragmentation is an inefficient process that is a response to micronucleation. Fragmented embryos have yet to be observed in any species that does not also have micronuclei formation, which suggests that fragmentation will not occur without aneuploidy. These observations are based on recapitulation in the nonhuman primate, she said, and it may not be possible to study this in humans. It is challenging to study maternal-age-related aneuploidy in nonhuman primates, she added, because most of their monkeys are relatively young. Thus, they observe fewer meiotic errors than in a typical human IVF population. The invasive primitive in situ trophoblast does not seem to be an extensive population in monkeys, Golos said. Rather than invasion, there is more of an expansive proliferation of a trophoblastic cell. Although there are pockets of in situ trophoblasts, they do not intrude substantially into the decidua, as the human embryo does. This suggests that nonhuman primates may not provide the best model for understanding the earliest events contributing to the high rate of implantation failure in human embryos, he said. Much of this increased aneuploidy might be driven by recombination rates, which vary across species, a participant said.

Alternative Nonhuman Primate Models

Golos and Chavez were asked whether nonhuman primates that are closer to humans than macaques would be more useful systems for modeling in certain situations. Access to more closely related nonhuman primates would be challenging, Golos said, but iPS cells might offer an alternative. Marmosets are also an option, but marmoset embryology is even more different from humans than macaque embryology. Chavez replied that at her primate center they have cynomolgus macaques (cynos), rhesus macaques, baboons, and marmosets. Baboons are primarily used for contraceptive studies. Gibbons have an interesting genome for study, but

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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they are protected as an endangered species. Cynos have the advantage of being non-seasonal breeders, while rhesus have a decline in oocyte quality during the summer months.

Improvement of In Vitro Fertilization Culture Conditions

Chavez was asked about the percentage of aneuploid embryos in IVF that arise from meiosis versus mitosis and if cultures and protocols might be altered to either prevent aneuploidy from occurring or encourage its correction. Meiotic errors are more common in older women, Chavez said, but her group has intentionally tried to look at younger women to obtain a better assessment of error types and frequencies. She added that she is particularly interested in studying embryos that have a meiotic error and that then have a greater propensity to have a mitotic error. She said that she is often asked whether aneuploidy is a consequence of in vitro culture. Although this is difficult to study in humans, the phenomenon of cellular fragmentation is highly associated with aneuploidy and occurs in vivo in humans and many other species. Culture conditions could still probably be improved for humans and other mammals, she added.

Metabolic Stress and Culture Conditions

A participant commented that research comparing cortical organoids from humans to primary data have revealed gene differences related to metabolic stress across all of the published protocols, indicating that culture conditions somehow create metabolic stress. Correcting for metabolic stress eliminated the abnormal gene expression in the cortical organoids, and the fidelity of the model improved. The participant suggested that it might also hold true for the iPS of the stem cell–derived blastocyst and perhaps even in the IVF clinic. If so, it might be possible to correct for this and improve culture conditions. Wu remarked that when culturing pluripotent cells, the cells are removed from the early embryo and placed in a culture condition that is very different from the in vivo environment. In human naïve-like embryonic stem cells or iPS cells that have been generated, defects have been observed in imprinting status, and if cultured long enough, metabolic defects would likely also be observed. The cells also accumulate genomic instabilities and karyotypical abnormalities after long-term culture. Better culture conditions that more closely mimic the in vivo environment could enable cells to grow and differentiate more naturally. However, comparing transcriptomes from pluripotent stem cells cultured in vitro to in vivo embryos shows that in vitro cultured cells always cluster together and separate from in vivo counterparts, regardless of the culture conditions used. This suggests that there are some in vivo features that have not yet

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
×

been recapitulated in vitro. Although some features can be preserved in pluripotent stem cell cultures (e.g., mouse ESCs can generate chimera similar to the early epiblast) other features differ between in vivo and in vitro, such as metabolic and epigenetic features. Chavez added that an ongoing metabolomics analysis of monkey oocytes fertilized in culture from which the spent culture media was obtained indicates that stress may actually be set up even in the oocyte. The glucocorticoid pathway is one of the main signaling pathways observed in oocytes that go on to fertilize, cleave, and progress to the blastocyst stage. She noted that people often assume that stress experienced by a woman is the cause of oocyte difficulties. However, her group is finding that the systemic level of metabolites appears to be quite different than the levels in the follicular environment, underscoring the importance of good starting material. “If we do not have a good egg to begin with, we are probably not going to have a good embryo,” she said. Golos noted that extended culture experiments are typically performed in 20 percent oxygen, which is not the natural environment for an embryo or trophoblast. Chavez added that according to probe studies in the monkey, oxygen concentration is only about 1 percent even in the ovary, suggesting that culturing should be performed in low oxygen.

Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Suggested Citation:"5 Comparative Embryonic Development Across Species." National Academies of Sciences, Engineering, and Medicine. 2020. Examining the State of the Science of Mammalian Embryo Model Systems: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25779.
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Next: 6 Exploring Opportunities and Challenges with Mammalian Embryo Model Systems »
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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, the National Academies of Sciences, Engineering, hosted a 1-day public workshop 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. 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. Presentations provided an overview of the current state of the science of in vitro development of human trophoblast. This publication summarizes the presentation and discussion of the workshop.

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