Monkey Embryos Cultured to 20 Days

Five to six days after fertilization, human embryos form a blastocyst containing three different types of cells: epiblast, hypoblast, and trophectoderm, which are fated to become the embryo proper, yolk sac, and placenta, respectively [1]. Seven days after fertilization, developing human embryos implant into the uterus [1]. Several days later, implanted human embryos begin to gastrulate—that is, form the germ layers and the body plan [1].

A clearer understanding of the steps of development after implantation would shed light into major causes of early pregnancy loss. However, owed to the lack of access to primary human tissue for study and experimental manipulation, the processes that distinguish this phase of human development have remained mysterious for decades. Although some advances have been made in culturing human embryos in a dish, such experiments cannot be extended past 14 days because of legal and ethical constraints [2,3]. The study of nonhuman primate embryos presents a tantalizing alternative platform to model the early stages of primate development, with high relevance for human biology [4].

The two new studies in Science demonstrate that, as observed with both mouse and human embryos, monkey embryos have the potential to self-organize and develop up to 20 days postfertilization (d.p.f.) without the need for maternal tissues [5,6]. By altering the growth conditions in which embryos are grown, the two studies achieved the development of monkey embryos in a dish with key landmarks, including segregation of the epiblast and hypoblast and generation of the amniotic and yolk sac cavities [5,6]. The authors of both studies also identified hallmarks of gastrulation, as reflected in the manifestation of gastrulating cells and epithelial-to-mesenchymal of cells undergoing gastrulation [5,6]. In addition, the growth rates of the in vitro cultured embryos resembled that of in vivo embryos at analogous stages [5]. Both Ma et al. and Niu et al. observed the early stages of germ cell differentiation [5,6]. Moreover, Ma et al. demonstrated the establishment of an anterior–posterior axis, suggesting that the monkey embryos likely progressed beyond the initial stages of gastrulation [5]. Indeed, Ma et al. identified neural groove-like structures at d.p.f. 19, reminiscent of human embryo development at E17–19 (Carnegie stage 8–9) [5,7].

The most striking observation in the two studies is the similarity of in vitro cultured embryo cells to in vivo cells, at least at the gene expression level [5,6]. Single-cell RNA-seq analyses showed that cells from the embryonic and extraembryonic lineages of in vitro cultured embryos resembled those of in vivo-derived embryos at a transcriptomic level [5,6]. For instance, in vitro cultured embryos possessed different gastrulation-associated cell types manifest in in vivo embryos, including both early and late gastrulating cells [4 –6,8]. These data suggest that the embryonic development observed in the dish might actually correspond to development occurring in vivo [5,6].

A next step would be to revisit the gene expression data sets from Ma et al., Niu et al., and two other studies reporting gene expression data of in vivo-derived embryos [4 –7,9]. Such meta-analyses could provide a more comprehensive snapshot of the cellular states manifest in in vitro-cultured embryos, including cells of unreported fates [9].

What technical advances enabled the development of these embryo culture systems? Both studies adapted previously reported culture methods used to grow human embryos for 12–13 days [2,3]. The authors of both new studies identified specialized culture conditions that allowed for longer-term culture of monkey embryos [5,6]. Previous attempts at growing primate embryos for extended periods of time resulted in the collapse of embryo growth after 2 weeks [2,3]. In one study, the authors grew monkey embryos in different serum-containing media to facilitate the growth of monkey embryos to 3 weeks [5]. In the second study, the authors used alternative basal medium and the provision of rho-associated protein kinase (ROCK) inhibitor Y-27632 to facilitate embryo growth to 3 weeks [6].

The two studies further investigated cellular changes correlated with the development of monkey embryos in the dish. Their data suggested a key role for cellular polarization in epiblast development and cavity and lumen generation, observations that are consistent with previous studies in mice and humans [2,3,10]. While awaiting further validation, the concept that polarization is involved in the development of the epiblast lineage is reminiscent of the postulate that inhibiting differentiation and polarization stabilizes human pluripotent stem (PS) cells with features suggestive of a more primitive epigenetic state [11,12]. However, the primate embryo studies could not fully differentiate correlation from causation. To more robustly demonstrate the role of cellular polarization on development of in vitro cultured embryos, it will be of future interest to ascertain the functional consequences of inhibiting cellular polarization in in vitro cultured monkey embryos using small molecules.

In this vein, the two Science articles have the potential to improve our understanding of pluripotency in the embryo and in culture. For more than a decade, the notion that PS cells may be considered as existing in two distinct attractor states—naive and primed—has dominated our understanding of in vitro pluripotency [13]. An emerging concept in the field of PS cells is the idea that in vitro pluripotency comprises a continuum of different cellular states that spans the naive and primed polar extremes [14 –16]. Adding further nuance to this conception, the data in the two studies suggest complexity in the primed pluripotent epiblast of primates [5,6]. Another idea is that naive epiblast cells must transition through a “formative” phase, a stage when competency for differentiation is acquired, before pluripotent epiblast cells become primed for differentiation [15]. In this regard, the nature of gastrulating cells, their changing molecular features during in vitro development, and relatedness to conventional primed human PS cells are of interest [5,6]. Future experimental manipulation of in vitro cultured monkey embryos is likely to yield additional insights. For example, by modulating signaling pathway activities through small molecules provision or CRISPR/Cas9-mediated genome editing, it might become possible to identify key pathways and genes mediating embryo development [6]. The monkey embryo culture system presents new opportunities to dissect transitions between different pluripotent states, the molecular underpinnings underlying competence for differentiation, and mechanisms of embryo development in primates, with likely relevance for humans.

The extended monkey culture system could enable deeper insights into the nature of pluripotency. It has become increasingly clear that the relationships between naive pluripotency and extraembryonic lineages in mice, such as the inability of pluripotent cells to give rise to extraembryonic fates, differ in primates. The extraembryonic potential of different types of human PS cells, particularly naive PS cells, is currently receiving considerable attention in the PS cell field [16,17]. For example, human naive-like PS cells have been recently reported to exhibit a potential to form trophoblast stem cells [18,19]. Might their in vivo monkey counterparts, preimplantation epiblast, also possess trophoblastic potential? Conversely, might trophoblast cells in primate embryos be able to interconvert into preimplantation epiblast-like cells? Another peculiarity is the potential of the epiblast to form amniotic epithelial fates, a feature of early primate development [16,17]. Ma et al. and Niu et al. identified primate amnion cells and characterized their transcriptional features at different stages [5,6]. It remains unclear which PS cell type possesses the potential to form amniotic epithelial cells [16,17]. Molecular analyses from Ma et al. and Niu et al. provide a useful resource for benchmarking claims of amniotic epithelial cell fate in vitro [5,6]. The potential insights that could be gained from extended monkey culture systems could provide insights into regulatory networks that underlie the apparent extraembryonic plasticity of some human PS cell types [18 –21].

In addition, the data raise questions about the roles of amnion cells in gastrulation and germ cell specification that warrant further investigation [5,6,8]. The two studies observed the emergence of gastrulating cells from two sources: the dorsal amnion and between the epiblast and visceral endoderm [5]. These data possibly suggest that the gastrulating cells might have originated from the epiblast and/or the amnion, a divergence from the rodent paradigm [5,6]. In addition, the in vitro findings from Niu et al. seem to support a previous study of in vivo embryos demonstrating that primordial germ cells come from amniotic epithelial cells, thus further underscoring the importance of amniotic epithelial cellular biology [6,7].

In addition, Ma et al. and Niu et al. open up new opportunities to improve our understanding of errors in early primate development, with high relevance for gaining insights into early pregnancy failure in humans [5,6]. Altogether, spontaneous failures comprise 70% of all human embryos [22]. The possible insights into early human development that could be gained from studies of monkey embryos could aid clinicians in identifying when problems might emerge [9].

Finally, the extended monkey embryo culture could be repurposed to aid efforts to generate human–animal interspecies chimeras for producing human organs inside livestock species, including pigs and sheep. The author has previously suggested that injecting human PS cells into monkey embryos and culturing to early postimplantation studies would advance our ability to engineer interspecies chimeric compatibility between humans and more distant species such as pigs [23]. In this regard, the extended monkey embryo culture system possesses tremendous potential.

Whether considering the extent to which studies of monkey embryos can be extrapolated to humans or human–monkey embryonic chimeras, it is important to emphasize that monkey and human embryos still exhibit important differences. These include timing of implantation (humans—6–7 days; cynomolgus monkeys 10 days) and the likely existence of differences in the dynamics in the formation of different embryonic and extraembryonic lineages between monkey and human embryos [5,6]. For example, bone morphogenetic protein (BMP) signaling components LEFTY1, LEFTY2, and NODAL exhibited different expression patterns during pluripotent state transitions between humans and monkeys [24]. Differences between monkey and human embryos are perhaps not that surprising, given the differences in the timing of implantation, as well as the 30–40 million-year divergence that separates monkeys and humans. These differences highlight the importance of studying human embryos directly. However, there exist constraints for what types of experiments can be conducted with human embryos in the laboratory [25 –27]. Currently, human embryo culture experiments are stopped before primitive streak formation or 14 days, whichever comes first. The 14 days rule is an international rule that for decades has limited the development of human embryos in the dish [25 –27]. The author notes that in some countries, extended culture past 14 days is illegal [25 –27]. According to the 2016 International Society for Stem Cell Research (ISSCR) guidelines, in vitro culture of human embryos past 14 days is considered a prohibited research activity and should not be pursued at this time [28].

In addition, beyond the ethical and legal constraints, scientific methods have not yet been developed to culture human embryos past 14 days [2,3]. Indeed, past efforts that have cultured human embryos in vitro observed collapse of cultures within 14 days [2,3]. In light of the recent advances enabling monkey embryo culture to 20 days, it is highly likely it will be technically feasible to culture human embryos beyond 14 days and the initial stages of gastrulation, as well as early neural development [5,6]. Consequently, it is becoming increasingly crucial that stakeholders reappraise the 14 days rule and its advantages and disadvantages [25 –27].

In the author's view, there now exists a compelling rationale for eliminating or revising the 14 days rule to allow for studies of milestone developmental events during and after human gastrulation [25 –27]. If the 14 days rule is revisited, precisely if and when human embryo development in a dish should be halted would be the critical question. In this regard, it is important to consider researchers will eventually have to grapple with. What if future findings extend the in vitro culture period to 4 weeks, or if co-development with (artificial) placentae extends the period of ex vivo development for even longer? What would comprise an absolute limit that would prohibit the use of in vitro cultured embryos beyond such a limit for research purposes? These are important questions for future discussion.

In light of the new ethical questions raised, additional deliberation and a revisiting of guidelines is likely to occur in the very near future [27]. Although the insights gained from extended culture of human embryos are tremendous, it is important to move forward with the utmost caution [27]. Practitioners should minimize any chance that laws or guidelines can be violated [25 –27]. Until such policies and guidelines are revised in favor of culture beyond 14 days, the author emphasizes that, at this time, research should adhere to current international legal and ethical standards [25 –28].

Stem cell and embryology researchers need to educate the public about recent advances in extended embryo cultures and their relevance [27]. The scientific community should determine how to oversee this burgeoning field of research in light of these recent advances in monkey embryo research. It is necessary that scientists, regulators, and ethicists proceed together to avoid public backlash, which could harm progress in this provocative but potentially crucial area of research.