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expert reaction to two preprints on stem cell-derived human embryo models

Two preprints, unpublished non-peer reviewed studies, look at the generation of stem cell- derived human embryo models.


Prof Alfonso Martinez Arias, ICREA Senior Research Professor, Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), said:

“The two manuscripts are very different.

“The aim is to reproduce human post implantation structures that combine extraembryonic and embryonic tissues.  Both are derived from the first cell, the zygote, but only the embryonic ones will contribute to the fetus, the others will form a complex of membranes that feeds the embryo (the yolk sac) as well as protects it and attaches it to the mother through the placenta.  When in these early stages people talk about three lineages they talk about the embryo, the yolk sac and the precursors of the placenta, the trophoblast.  At day 14 from conception, the embryo becomes organized and starts gastrulation with the localized expression of the gene Brachyury and the development of germ cells.

“With this in mind, we can look at the two manuscripts, briefly.

“The Zernicka-Goetz preprint reports aggregates of cells of some, but not all, the lineages without a clear organization.  The structures that form are difficult to interpret and only with the guidance of the authors can one gauge what might be going on.  The cells are heavily genetically manipulated and exposed to high doses of various signals which might explain why they are so disorganized.  From what I can see there is no hint of gastrulation and it is very difficult to ascertain the age of the structures relative to embryos.  Much of the identification of the cell types relies on a currently trendy technique that reveals the genes expressed by cells but, again, this needs interpretation and biases can be introduced in the analysis.  What we should be looking at is a structure with spatially patterned gene expression; we don’t see much that resembles the structures in vivo.  The manuscript does not contain much detail of how and why things happen and in my view would not on its own support an interpretation that embryo-like structures have definitely been produced in this instance.  In addition the manuscript doesn’t mention other work that has produced related structures with better accuracy and reproducibility.  Essentially, in my view this isn’t particularly new or noteworthy.

“The second preprint from Jacob Hanna seems to be a very sound and rigorous piece of work that recapitulates much of the development between day 7 and day 14.  The structures are recognizable and can be related to the embryo.  They form at a low frequency but some of them are remarkable copies of the natural structures.  Two features are worth highlighting amidst many.  The first one is that the authors managed to coax the cells to make those structures without genetic modifications, purely chemically.  The second is that they form so well even though they are not implanted.  One might have thought that the involved organization of the structures might be influenced by the attachment but they don’t.  Moreover they also report the initial stages of gastrulation.  Perhaps this is not so surprising as many mammalian embryos e.g. horses, implant after gastrulation.  The work is well described with much detail which will help reproducibility which, nonetheless, will not be easy.  A great deal of the work is focused on defining the conditions for the experiment to work.  All in all this is a useful piece of work and an important advanced in the field.  (I should also add that J Hanna presented this work at a meeting in Japan on March earlier this year).

“The first preprint does not have much to say to the ethical discussions.  However, the second one does raise some ethical issues that, I am confident are being discussed within the scientific community.  I do worry that we need to make sure the public discussion on these issues is fully informed and is at a time when we can be clear about and ready to discuss things such as the definition of an embryo from the perspective of these and other models.  This is a necessary discussion.  Nonetheless, there is little question of the value of these models to access important stages of human development that are nearly impossible to study.  However, ethical overseeing needs to go apace with the technical advances.”


Comment sent out by our colleagues at SMC Germany:

 Dr Jesse Veenvliet, Head of the Stembryogenesis Research Group, Max Planck Institute of Molecular Cell Biology and Genetics (CBG), Dresden, said:

On the paper by Jacob Hanna’s group:

“The similarity to the natural embryo is remarkable, almost uncanny. Coaxing the human embryonic stem cells into the embryo-like structures required extensive optimisation of stem cell culture protocols to achieve cellular states that most closely resemble the progenitors of the earliest embryonic and extra-embryonic lineages. Achieving this was the most critical advancement, together with very careful dissection of the exact culture conditions compatible with continuous growth and development of the embryo-like structures. It’s beautiful to see how more than a decade of very basic research focused on extremely careful dissection of human pluripotent stem cell states lead up to this.”

Does the group of Jacob Hanna also use the “ETiX”-Embryos” or do they use another approach?
“No, they are not. A unique feat of the Hanna work (also compared to the Zernicka-Goetz preprint) is that they achieved the formation of the embryo-like-structures without having to rely on transgenes (as was previously done in the ETiX). In fact, in their hands, it appears that bringing the pluripotent stem cells into the right state using chemical cocktails functions (much) better than using transgenes, which are a bit more of a sledgehammer to enforce the “right” states. It is fascinating how, once the human embryonic stem cells have been coaxed into states closely resembling the earliest embryonic and extra-embryonic cell states, they obtain the capacity to self-organise into structures that mimic the embryo – without any need for additional chemical modulation.”

“That said, it should also be emphasised that the protocol to coax these stem cell ensembles into embryo-like-structures is very much based on previous protocols to develop models of mouse embryo development in the labs of Magdalena Zernicka-Goetz & Nicolas Rivron, both pioneers in the modelling of pre-, peri-implantation, and early post-implantation development. In a way, it is beautiful to see how the work by the Hanna lab is a perfect synergy of more than a decade of very careful work on basic stem cell biology, and pioneering work on modelling the mammalian embryo in vitro (Stembryogenesis, as we call it in the lab [1].”

How comparable are those synthetic embryos presented to embryos created through fertilization?
“The similarity in terms of cellular composition, architecture and organisation is incredible. Many key landmarks and milestones of early human post-implantation development are recapitulated. It is however noteworthy that owing to the nature of the protocol / formation process, the embryo-like-structures do not proceed through the earliest developmental stages. In particular, they do not transit through a blastocyst stage, the stage when the natural embryo implants into the uterus. It remains unclear how much this affects the developmental potential of the embryo-like-structures (the capacity to give rise to a healthy, fully formed fetus), but this would be hard to test given the current ethical, legal and – possibly – technical constraints. Another important thing to mention is that the remarkable resemblance to the natural embryo is only achieved in ~ 3% of the structures, and even within this 3% there is much more variation compared to the embryo. To realise the full potential of these embryo-like-structures, it will be critical to improve reproducibility and efficiency. This is especially the case if one wants to use the embryo-like structures for e.g. embryo toxicity testing. It should also be noted that the authors very much focused on the similarities, and did not report on any differences between the embryo and its model in detail.”

“The authors did not culture the embryo-like-structures beyond the equivalent of a 14-day old human embryo (which is ~day 8 in their protocol). However, it is not clear if this was not technically feasible or if the authors purposefully stopped their experiments for ethical considerations. Given the status of the ~3% of successful structures, I do not see a reason why these could not develop further. It should however be stressed that this is a model. For example, the structures cannot be implanted into a womb, since they do “skip” the implantation (blastocyst) stage. How far the structures could properly develop in the absence the maternal environment remains important to assess, since only that will inform us about the developmental potential, and hence about how much the moral status of the embryo-like structure should be considered similar to the embryo. This can only be addressed by direct comparison with the human embryo in vivo, which would require extension of the 14-day rule. I think this is justifiable on a case-by-case basis; Currently, what is happening beyond day 14, when the embryo starts to set up the Anlagen for the future organs, remains a black box, and it is pivotal to get a window into this stage to e.g. understand why many pregnancies fail during these stages. Embryo-like structures like the ones developed by Hanna lab offer a unique possibility for this, but it is essential to understand both the similarities AND differences between the embryo and its model.”

[1] Veenvliet JV (2021): Sculpting with stem cells: how models of embryo development take shape. Development. DOI: 10.1242/dev.192914.


Comment sent out by our colleagues at SMC Spain:

Prof Gemma Marfany, Professor of Genetics, Universitat de Barcelona (UB) and Head of Group at CIBERER, said:

Human embryonic development is largely unknown from implantation and placenta formation, which occurs around day 7.5 post-fertilisation. However, these early days of embryo formation are crucial in explaining fertility problems and failure of apparently healthy embryos to implant. So far, our knowledge has been derived from studies in embryos of other mammals, but not everything is directly extrapolatable. Regulations in many countries prohibit the in vitro study of human embryos beyond 14 days when embryo gastrulation and many organ formation processes begin. One way to overcome the lack of knowledge about human embryos is to generate “embryos-like” or embryoids from cultured embryonic cells. In these sets of cells, through genetic manipulation, it is possible to differentiate into different embryonic tissues and to study how this occurs and whether it recapitulates embryonic development.

“Both Jacob Hanna’s group (Israel) and Magdalena Zernicka-Goetz’s group (UK and USA) have been investigating early embryonic development for some time. They use different methods of aggregating embryonic stem cells with different differentiation profiles (Zernicka-Goetz does this by joining cells transgenic for different differentiation factors, while Hanna uses unmodified pluripotent cells, mixed with others that are genetically modified). The result is human embryoids, or rather, synthetic models of human embryos that recapitulate features of early embryo differentiation.

“Both refer to the generation of the amniotic sac (the extra-embryonic tissues that are biologically derived from the zygote but are not part of the embryo). The results, in preprint format, seem to support that the stem cell-derived embryonic models of Hanna’s group are structurally more embryo-like, while those of Zernicka-Goetz’s group would be less structured, but gene expression in these embryoids would support that they are differentiating into organ precursors.

“At the moment, they have not gone beyond the stage of a 14-day-old embryo, but they are synthetic embryonic models, created by joining different stem cells, and are not a viable human embryo. There is still a lot of research to be done and the main question at the moment is how these models are categorised, i.e. whether they are considered human embryos or not. At the moment they are not, because they are not viable and do not manifest the full potential of a human embryo. These stem cell-derived embryonic models answer questions about the early stages of embryos, they look very much like embryos, but they are not human embryos. The problem is that they are in legal limbo in many countries, and it is not known which regulations apply to them.

“We have to consider that the day when cellular and genetic manipulation techniques allow it, these models may have potential and viability, so it will be necessary to define – both from a bioethical-legal and scientific point of view – what they are and to determine what rules apply to them, how their generation is controlled and up to what point of development they can be investigated”.


Prof Daniel Brison, Honorary Professor of Clinical Embryology and Stem Cell Biology; Scientific Director of the Department of Reproductive Medicine, University of Manchester, said:

“Complex data like this does take time to review and has currently, in preprint stage, not undergone any independent expert assessment. While we can make some quick judgements about the work, a full peer review will take many hours of work and we should still wait to see the full published journal article before making too many conclusions. Ethical and regulatory issues raised by this work can also be sensibly discussed in time.”



‘Transgene directed induction of a stem cell-derived human embryo model’ by Bailey A. T. Weatherbee et al. was uploaded onto the preprint server bioRxiv on 15th June. It has not been through peer review. 

‘Transgene-Free Ex Utero Derivation of A Human Post-Implantation Embryo Model Solely from Genetically Unmodified Naive PSCs’ by Bernardo Oldak et al. was uploaded onto the preprint server bioRxiv on 15th June. It has not been through peer review.



Declared interests

Prof Alfonso Martinez Arias: “Have two patents on gastruloids, stem cell based embryo of mammalian development.”

Prof Gemma Marfany: The author has declared they have no conflicts of interest

Prof Daniel Brison: “No conflicts of interest to declare.”

For all other experts, no reply to our request for DOIs was received.

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