Background

Soon after the mouse egg is fertilized, successive cell divisions will increase the number of cells exponentially to form a compact mass of cells. Cells in the outer part of this compact mass will become the trophectoderm, while the inner cells will acquire two alternative fates: either primitive endoderm or epiblast. These distinctive specializations will differentially contribute to the development of the future embryo: while the epiblast will be responsible for the generation of the embryo proper, the extra-embryonic structures will be formed by both the trophectoderm (e.g., placenta) and the primitive endoderm (e.g., yolk sac). Around embryonic day 6.5 (E6.5) in the mouse, a process of finely orchestrated rearrangements of epiblast cells, known as gastrulation, begins which results in the formation of the three primary germ layers: ectoderm, mesoderm, and endoderm (Shahbazi and Zernicka-Goetz, 2018). As the basic body plan of the animal is established, specialized tissues and organs next develop from these germ layers. For example, the neural tube is formed through a process called neurulation, where a portion of the ectoderm (the neuroectoderm) folds into a tube at the midline of the embryo, eventually differentiating into specific regions such as the forebrain at the most anterior part or the spinal cord more posteriorly. The endoderm gives rise to the gastrointestinal and respiratory tract, whereas the mesoderm will form the urogenital system and the somites.

In mammals, embryos develop inside the mother’s body, making the study of early embryogenesis a very difficult task. To solve this problem and identify the key mechanisms required for building an embryo, scientists have been trying to develop in vitro systems that can recapitulate the early developmental stages of mammalian embryogenesis. Several stem cell models of the mouse embryo have been generated in the past decade (Harrison et al., 2017; Rivron et al., 2018; Sozen et al., 2018), suggesting a remarkable self-organizing capacity of the early mammalian embryo (Zhu and Zernicka-goetz, 2020). Scientists were also able to generate human blastocysts in vitro from induced pluripotent stem cells (Liu et al., 2021; Sozen et al., 2021). Additionally, gastruloids and trunk-like structures with a neural tube and somites were generated using mouse embryonic stem cells (Beccari et al., 2018; van den Brink et al., 2020; Sozen et al., 2021; Turner et al., 2017; Veenvliet et al., 2020), but these models are unable to recapitulate post-implantation embryogenesis since patterning signals originating form extraembryonic tissues are absent (Shahbazi and Zernicka-Goetz, 2018).

The Zernicka-Goetz Lab has experimented with different recipes to generate a synthetic mouse embryo and has now found two protocols that support the development of a synthetic embryo through post-implantation stages (Amadei et al., 2022a; Lau et al., 2022). The work led by Amadei (Amadei et al., 2022a) has now been published in Nature (Amadei et al., 2022b). In these two preprints, they describe how they were able to generate in vitro mouse embryoids, using two different approaches, that resemble E8.5 natural mouse embryos. These post-implantation-like embryos went through neurulation, developing forebrain and midbrain, produced pairs of somites, developed a beating heart, and initiated gut development. All this is remarkably similar to the development of a natural mouse embryo (Table 1,2).

Main findings

The ETiX-embryoids

In the first preprint from the Zernicka-Goetz Lab (Amadei et al., 2022a), synthetic embryos were generated by aggregating (1) embryonic stem cells (ESCs) from the epiblast, with (2) trophoblast stem cells (TSC; derived from the extraembryonic ectoderm) and (3) extraembryonic endodermal cells. These last ones were differentiated from ESCs by transiently inducing the expression of the extraembryonic visceral endoderm master regulator Gata4. These ETiX embryos developed structures that are remarkably similar to the ones observed in natural mouse embryos from E7.5 to E8.5 (Table 1).

Table 1. Summary table with the main features of the ETiX embryo

The EiTiX-embryoids

In a second preprint, the members of the Zernicka-Goetz Lab aimed to reconstruct mouse early development by solely using embryonic stem cells and transcription factor-mediated reprogramming (Lau et al., 2022). Synthetic mouse embryos, here named EiTiX-embryoids, were generated aggregating: (1) mESCs for epiblast formation; (2) mESCs with inducible Gata4 expression for extraembryonic endoderm formation; and (3) mESCs with inducible Cdx2 expression for trophectoderm formation. This new culture system using mouse embryonic stem cells simplifies the culture conditions since the three cell types use the same culture media.

Table 2. Summary table with the main features of the EiTiX embryo

 

Of note, a similar approach has been developed by Tarazi and colleagues which was recently published in the journal Cell (Tarazi et al., 2022). Similar to the EiTiX system, their synthetic embryos (sEmbryos) were formed by aggregating non-transduced ESCs with Cdx2-ESCs for the formation of the trophectoderm lineage and Gata4-ESCs to promote primitive endoderm lineage formation. These sEmbryos were grown on an adapted electronic platform which the researchers had previously deployed to grow ex utero mouse embryos for prolonged periods of time (Aguilera-Castrejon et al., 2021).

All these three recent works – ETiX (Amadei et al., 2022a), EiTiX (Lau et al., 2022), as well as the sEmbryos model (Tarazi et al., 2022) – reveal that embryoids recapitulate the development of both embryonic and extraembryonic lineages of the natural mouse embryo, thanks to a detailed analysis of morphology, marker gene expression, and single-cell RNA-seq analyses. Of note, the EiTiX-embryoid model presents some limitations, such as depletion of cell types from the ectoplacental cone lineage, frequently enlarged heart structures, or asynchronicity in cell differentiation. Similarly, sEmbryos presented some differences and/or abnormalities when their development was carefully compared to natural mouse embryos (Tarazi et al., 2022). Nevertheless, these three recipes, even with their current limitations, show that synthetic embryos can adequately complete gastrulation, neurulation, and organogenesis of embryonic and extraembryonic tissues; thus, highlighting the intrinsic capability of naïve pluripotent stem cells to self-organize and give rise to the whole organism in a dish.

Things we like

We are amazed by the striking resemblance of synthetic embryos to natural ones. If one had to blindly assign images to synthetic or natural embryos, it would be a very hard game to play. This resemblance was carefully evaluated by the authors, comparing the embryoids to the natural mouse embryos by immunofluorescence, as well as with a detailed molecular characterization using single-cell RNA-seq. Lastly, the fact that the authors could rescue, to some extent, neural tube defects in the embryoids reveals the great potential of these technologies to model neurodevelopmental disorders and find appropriate treatments. 

When the two preprints, along with the Cell paper from the laboratory of Jacob H. Hanna, became available online we immediately thought that they would have a huge impact on the scientific community and beyond. We were right! After just a few weeks, one of the preprints was published in Nature (Amadei et al., 2022b), and the great resemblance of the synthetic embryos to natural ones was quickly picked up by the press (example: https://www.bbc.com/news/health-62679322). Of course, Twitter has also been buzzing with comments and great discussion points.

The peer-reviewed version (Amadei et al., 2022b) of the preprint (Amadei et al., 2022a) that we’ve highlighted here includes some exciting and noteworthy changes. Some that we particularly liked are: the catchier new title, implementations of the histological characterization, more in-depth analysis of the single-cell RNA-sequencing used to compare cell identities in EiTX-embryoids to natural embryos, new experiments evaluating the consequences of Pax6 deletion, and the more elaborate discussion section. We are now looking forward to seeing the second preprint (Lau et al., 2022) appear in a peer-reviewed journal!

Questions for the authors

• What is the proportion of aggregates that successfully become ETiX or EiTiX? Do you have thoughts on how the efficiency could be improved?
• Did you check whether patterning mechanisms (e.g., organizers, signaling centers, the somitogenesis clock) are correctly in place within the ETiX/EiTiX-embryoids?
• Can you speculate on the requirements for longer culture of ETiX/EiTiX-embryoids? How far do you think the system can be pushed?
• Do you think a similar approach could be used for building human synthetic embryoids? What are the ethical restrictions on synthetic embryoids currently in place?

References

 Aguilera-Castrejon, A., Oldak, B., Shani, T., Ghanem, N., Itzkovich, C., Slomovich, S., Tarazi, S., Bayerl, J., Chugaeva, V., Ayyash, M., et al. (2021). Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis. Nature 593, 119–124.

Amadei, G., Handford, C.E., De Jonghe, J., Hollfelder, F., Glover, D., and Zernicka-Goetz, M. (2022a). Stem cell-derived mouse embryos develop within an extra-embryonic yolk sac to form anterior brain regions and a beating heart. BioRxiv 2022.08.01.502375.

Amadei, G., Handford, C.E., Qiu, C., De Jonghe, J., Greenfeld, H., Tran, M., Martin, B.K., Chen, D.-Y., Aguilera-castrejon, A., Hanna, J.H., et al. (2022b). Synthetic embryos complete gastrulation to neurulation and organogenesis. Nature 0–1.

Beccari, L., Moris, N., Girgin, M., Turner, D.A., Baillie-Johnson, P., Cossy, A.-C., Lutolf, M.P., Duboule, D., and Arias, A.M. (2018). Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids. Nature 562, 272–276.

van den Brink, S.C., Alemany, A., van Batenburg, V., Moris, N., Blotenburg, M., Vivié, J., Baillie-Johnson, P., Nichols, J., Sonnen, K.F., Martinez Arias, A., et al. (2020). Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids. Nature 582, 405–409.

Harrison, S.E., Sozen, B., Christodoulou, N., Kyprianou, C., and Zernicka-Goetz, M. (2017). Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro. Science (80-. ). 356, eaal1810.

Lau, K.Y.C., Rubinstein, H., Gantner, C.W., Amadei, G., Stelzer, Y., and Zernicka-Goetz, M. (2022). Mouse-embryo model derived exclusively from embryonic stem cells undergo neurulation and heart development. BioRxiv 2022.08.01.502371.

Liu, X., Tan, J.P., Schröder, J., Aberkane, A., Ouyang, J.F., Mohenska, M., Lim, S.M., Sun, Y.B.Y., Chen, J., Sun, G., et al. (2021). Modelling human blastocysts by reprogramming fibroblasts into iBlastoids. Nature 591, 627–632.

Rivron, N.C., Frias-Aldeguer, J., Vrij, E.J., Boisset, J.-C., Korving, J., Vivié, J., Truckenmüller, R.K., van Oudenaarden, A., van Blitterswijk, C.A., and Geijsen, N. (2018). Blastocyst-like structures generated solely from stem cells. Nature 557, 106–111.

Shahbazi, M.N., and Zernicka-Goetz, M. (2018). Deconstructing and reconstructing the mouse and human early embryo. Nat. Cell Biol. 22.

Sozen, B., Amadei, G., Cox, A., Wang, R., Na, E., Czukiewska, S., Chappell, L., Voet, T., Michel, G., Jing, N., et al. (2018). Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures. Nat. Cell Biol. 20, 979–989.

Sozen, B., Jorgensen, V., Zernicka-Goetz, M., and Chen, S. (2021). Reconstructing aspects of human embryogenesis with pluripotent stem cells. Nat. Commun. 1–13.

Tarazi, S., Aguilera-castrejon, A., Joubran, C., Ghanem, N., Roncato, F., Wildschutz, E., Haddad, M., Gomez-cesar, E., Livnat, N., Viukov, S., et al. (2022). Post-Gastrulation Synthetic Embryos Generated Ex Utero from Mouse Naïve ESCs. Cell.

Turner, D.A., Girgin, M., Alonso-crisostomo, L., Trivedi, V., Baillie-johnson, P., Glodowski, C.R., Hayward, P.C., Steventon, B., Lutolf, M.P., and Arias, A.M. (2017). Anteroposterior polarity and elongation in the absence of extra- embryonic tissues and of spatially localised signalling in gastruloids : mammalian embryonic organoids. Development 3894–3906.

Veenvliet, J. V, Bolondi, A., Kretzmer, H., Haut, L., Scholze-Wittler, M., Schifferl, D., Koch, F., Guignard, L., Kumar, A.S., Pustet, M., et al. (2020). Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites. Science (80-. ). 370, eaba4937.

Zhu, M., and Zernicka-goetz, M. (2020). ll Principles of Self-Organization of the Mammalian Embryo. Cell 183, 1467–1478.

Tags: stem cells, synthetic embryo

doi: https://doi.org/10.1242/prelights.32657

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