A paper published in Nature look at the cultivation of synthetic mouse embryo-like structures from stem cells.
This Roundup accompanied an SMC Briefing.
Prof Robin Lovell-Badge FRS FMedSci, Group Leader, Francis Crick Institute, said:
“The paper by Amadei and colleagues from Magda Zernika-Goetz’s lab as well as one published a few weeks ago from Jacob Hanna’s lab (Tarazi et al, 2 August 2022, Cell) both show that it is possible to derived quite complex structures that closely resemble day 8.5 mouse embryos from embryonic stem cells entirely in culture. Both groups also compared gene activity within cells of their embryo models and normal embryos of an equivalent stage (as judged by morphology) and find that the two give very similar profiles and very similar spatial patterns of specific gene markers. The protocols used are similar, relatively straightforward, and it is reassuring that the results in the two labs are comparable, with reproducibility being very important. However, this includes the rather low efficiency to go from the starting stem cell types all the way through to the embryo-like structures, that the latter are not identical to normal embryos in either case, and there is no data (at least yet) to indicate that they could be implanted into a uterus and develop further, let alone to term. It is important, therefore, to avoid thinking of these embryo-like models as being the real thing – even if they are getting close to the latter. And if these had been derived from human stem cells, and it is accepted that these should never be transplanted into a uterus, we will never know if they are equivalent.
These are “integrated stem cell-based embryo models” with extraembryonic tissues as well as an embryonic part that has formed early-stage organs with tissues that seem to be patterned correctly. The published work is also using mouse ES cells with the advantage that the embryo-like models can be readily compared with normal embryos. It will be interesting to see if similar protocols can be made to work with human ES cells or iPS cells and I would not be surprised if both labs are working on this. It would be very valuable to have accurate stem cell-based models of early human embryos, especially to be able to study events during and just after gastrulation, when the basic body plan is established as well as tissues such as those of the central nervous system, heart, gut and germ cells, about which we know relatively little and where there may well be differences with the mouse and other animal embryos. However, there will be a need to validate any human stem cell-based embryo model against normal human embryos and in the UK as well as many other countries this would necessitate a change in law or commonly accepted guidelines where there is currently a 14-day limit for embryo culture, which is just at the point where gastrulation may begin. Given the similarity with real embryos, it follows that consideration also needs to be given as to whether and how such integrated stem cell-based embryo models should be regulated. They probably do not need the same level of scrutiny, partly because they can be generated in the many thousands and this would be an unnecessary regulatory burden, especially if there is no proof that they are identical to normal embryos. But the public might well wish some degree of regulation and oversight and scientists appreciate having boundaries for their research, so that they know it is not crossing into something that might be unacceptable. This is something that the HFEA needs to consider as they currently have no say in what happens to ES cells once they are derived and they do not regulate iPS cell derivation and use at all.
I personally do not like the term ‘synthetic embryos’, which might give the impression that they are entirely artificial when they come from ES cells derived from an embryo. If induced pluripotent stem cells were to be used instead (and there is no reason why these would be any different) they would come from an adult. In either case the donors would be expected to give consent for the research and these individuals deserve respect.”
Some added background:
“Mammalian embryos develop within an opaque environment, the uterus and in the mother, where it is challenging to observe them or to carry out interventions. Ways to model aspects of embryo development of mammals in vitro are therefore proving to be very important to gain greater understanding of both normal processes, such as how cell fate is determined or organs begin to form, and to understand what goes wrong with deleterious genetic variants or with external influences, including chemicals, viruses, etc. This is particularly the case for human embryo development about which we know far less than for animals commonly used in research, notably the mouse, and it is increasingly becoming clear that while general principles can be the same, many details are not. Models derived from human stem cells will be very important to understand what goes wrong with specific gene mutations and help to find ways to avoid the problems or to test potential treatments for a wide range of disorders. However, if this is to be done with any degree of confidence in the results, the methods have to be far more efficient that reported by either Amadei et al or by Tarazi et al, where the vast majority of embryo-like structures show abnormalities or fail completely.
While many stem-cell derived models focus on deriving specific cell types or organ-like structures, there has been progress in mimicking complex dynamic aspects of post-implantation development using so called gastruloids that fall into the category defined by the ISSCR (the International Society of Stem Cell Research) of “non-integrated stem cell-based embryo models”, because they lack the extraembryonic tissues that are needed for an embryo to implant and develop in a uterus. Several groups have also been trying to derive “integrated stem cell-based embryo models”, which incorporate cells able to give both extraembryonic tissues (notably trophectoderm that gives rise to the placenta and extraembryonic endoderm that gives rise to the yolk sac and to some cells in the gut) and to cells of the embryo proper which develops from the ‘epiblast’ layer of an early post-implantation embryo. In addition to using embryonic stem cells (ESCs) that are derived from early preimplantation stage embryos (blastocysts), it is also possible to use induced pluripotent stem cells (iPSCs), which can have their origin with adult body cells that are reprogrammed into an ESC-like state. Critically these can be derived from any individual including those suffering from a genetic disorder. However, with both ESCs and iPSCs, in order to get extraembryonic cell types and the stem cells that give rise to these, they have to be cultured in conditions that will make them “naïve” or factors that promote their differentiation have to be added.”
Dr Darius Widera, Associate Professor in Stem Cell Biology and Regenerative Medicine, University of Reading, said:
“This study is impressive and represents another milestone in the field of developmental biology. Despite significant progress in the field, complex models of embryonic development are still rare.
“The authors have generated one of the most advanced embryo models so far and this could help us understanding many aspects of early development. The structures created by Amadei and colleagues recapitulated developmental processes occurring in natural mouse embryos fairly accurately and contained structures such as early brain, beating heart-like aggregates and gut-like structures. Therefore, the mouse embryo models developed in this study could pave the way for better understanding of animal and human development and potentially birth defects.
“However, although the data is very promising, the efficacy of the protocol is still moderate. Moreover, natural mouse embryos of similar age contain a wider range of cell types. Lastly, the model mouse embryos were comparable to very early stage of development. Thus, further studies are needed to optimize the procedures and generate later stage embryonic structures in order to substantially reduce the need for experimental animals.
“It should also be noted that there are fundamental differences between mouse and human development.”
Lluís Montoliu, research professor at the National Biotechnology Centre (CNB-CSIC) and at the CIBERER-ISCIII, said:
“The fundamental challenge in biology remains to understand how a living organism as complex as any of us develops from a single cell. Understanding how hundreds of different cell types emerge during embryonic development from a single single cell embryo, which is the product of the fertilisation of an egg by a sperm. To answer these questions, we can witness the development of other animals, such as zebrafish, whose embryos develop externally, inside transparent eggs that allow us to see everything that is going on inside. However, for mammals, like us, it is much more difficult, since the embryo implants in the uterus of the female and develops inside anatomical structures that are difficult to see and access. Our ability to observe in detail, directly, what happens during the development of a mammalian embryo is limited to the early stages, prior to the implantation of the embryo in the uterus.
“This is the challenge that Magdalena Zernicka-Goetz’s lab set itself and whose results she now presents in her paper published in the journal Nature. She has managed to reproduce the initial stages of development of a mammalian embryo, from a mouse, in the laboratory, without needing the participation of a female to implant the embryo. And they have also achieved this without the need to resort to the fertilisation of an egg by a spermatozoon. Instead, these researchers have used different types of embryonic stem cells (stem cells) which, when mixed together, give rise to a new biological structure that looks very much like a natural embryo, without being one. These are synthetic embryos, developed entirely in the laboratory.
“For the extra-uterine development of these synthetic embryos, Zernicka’s laboratory at Caltech (California, USA) has used a device, an artificial incubator, which makes it possible to simulate the physiological conditions that exist in the female uterus. This ingenious technical solution was developed by the laboratory of Jacob Hanna of the Weizmann Institute in Israel, co-author of this study and who also reported similar experiments a few weeks ago, published in the journal Cell.
“The synthetic embryos reach a stage equivalent to that of natural embryos at 8-9 days gestation, almost half the pregnancy time in mice, which is 19-20 days. And they manage to develop very similar anatomical structures, such as the heart, with its beat, and the brain, with its different areas. These synthetic embryos are not embryos, but they are useful for research.
“Since these synthetic embryos are derived from embryonic stem cells in culture, they can also be generated from cells that contain a mutation in some gene, and thus investigate the effect that this mutation produces in the initial phases of development, observing directly in the laboratory what happens at each moment. This is a privilege that researchers did not have before with mammalian embryos.
“We are undoubtedly facing a new technological revolution, still very inefficient (it is very difficult to get stem cells to spontaneously generate a synthetic embryo), but with enormous potential. It is reminiscent of such spectacular scientific advances as the birth of Dolly the sheep, which we met in 1997, reconstructing an embryo with the nucleus of a somatic cell, or the inducible pluripotent embryonic stem cells, iPS cells, described by Yamanaka in 2006, which led him to win the Nobel Prize in Physiology or Medicine in 2012, shared with John Gurdon, pioneer of animal cloning in amphibians. A revolution that naturally also raises new ethical dilemmas, if we ever think of transferring these experiments to the human species for the generation of synthetic human embryos, perhaps with the aim of using them to obtain new tissues or organs to repair or replace those that are damaged, as Hanna has already proposed to explore, through a company he has created ad hoc“.
Dr Christophe Galichet, Senior Laboratory Research Scientist, Francis Crick Institute, said:
“The work from Amadei et al is extremely similar to the work published earlier this month from the Hanna’s lab with minute differences in the way synthetic embryos are cultivated.
“By combining sets of stem cells together and by providing a proper environment, synthetic embryos develop, albeit inefficiently, past the point when natural embryos must implant into the mother’s womb to form organs such as brain, heart, and gut. The synthetic embryos resemble age-matched natural embryos in all aspects analysed. However, like the study published earlier this month, synthetic embryos fail to develop past day 8. This blockade is not understood and needs to be overcome if one desires to grow mouse synthetic embryos past day 8.
“Synthetic embryos could substitute natural embryos to study early development in both health and disease once the efficiency issue is addressed. To validate synthetic embryos as a replacement to natural embryos, the authors generated genetically altered synthetic embryos which were comparable to matched genetically altered natural embryos. The authors engineered embryonic stem cells lacking the gene Pax6, which is involved in neural tube, brain, and eye formation. Synthetic embryos generated from Pax6-null embryonic stem cells resemble Pax6-null natural embryos.
“While it is premature to talk about real world impacts because of the inefficiency of the system and the developmental blockade, it is now within scientist’s grasps to generate post-implantation human synthetic embryos with formation of organs such as the brain and beating heart. It is therefore crucial to discuss the ethical and regulatory implications of such methods to study human embryonic development.”
Prof Alfonso Martinez Arias, ICREA Senior Research Professor, Department of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF), said:
“This work follows the one from Jacob Hanna’s lab last week in Cell (https://bit.ly/3ccZQEA). Zernicka Goetz’s group has been pursuing this aim for a number of years, aggregating extraembryonic and embryonic cell types of the early stages of mouse development in the hope to produce a mouse embryo. They have achieved this now and the result seems to be due to some changes in the initial cell population and the use of a culture system developed by the Hanna group.
“The work shows the requirement for the interactions between the embryonic and extraembryonic tissue to lay down the mammalian body plan. Significantly it shows that this can happen outside of the uterus.
“As a proof of concept this work, as that from Hanna’s group, is fine and means some progress in our understanding of the early stages of mammalian development. However, if the system is to be used in any practical way, there are issues that will need to be addressed in the future. In particular, the frequency of these synthetic embryos is very low (about 1% of the starting cultures), the ones that make it collapse in a few days before maturing and all exhibit many defects in the organization of the tissues and organs. For the moment it is not clear how this system will substitute the animal that provides embryos more efficiently and robustly. The early stages are somewhat more robust and maybe they could be used to study early interactions between embryonic and extraembryonic tissues but, again, reproducibility and faithfulness will have to be improved.
“Perspective on this report is important since, without it, the headline that a mammalian embryo has been built in vitro can lead to the thought that the same can be done with humans soon. While this is likely to be the case in the future, it will take time and first will require sorting the issues of efficiency and fidelity raised above in the mouse system. In any case, the result does herald that, in the future, similar experiments will be done with human cells and that, at some point, will yield similar results. This should encourage considerations of the ethics and societal impact of these experiments before they happen.
“This should not be about a headline but about the Science and, on this, there is work to do. It is very important that journalists look at the small print, the Science, which sometimes is hidden. This is an advance but at a very early stage of development, a rare event which while superficially looking like an embryo, bears defects which should not be overlooked.”
Dr James Briscoe, Principal Group Leader – Assistant Research Director, Francis Crick Institute, said:
“Similar to the recent work reported by Hanna and colleagues (Weizmann Institute), this is a valuable proof of concept demonstration that a synthetic mouse embryo-like structure can be assembled from stem cells. It builds on previous advances in developmental biology that identified the molecular recipes for producing the set of stem cells necessary for initiating embryo formation. By combining these cells together, the study shows that it is possible to coax the development of something that resembles a mouse embryo at a stage when the main organs of the body are beginning to be established, including the nervous system, heart and gut. An important observation, however, is that the formation of these “synthetic embryos” was very inefficient. Moreover, even the successful synthetic embryos appeared not as well organised as natural embryos and failed to develop beyond the equivalent of embryonic day 8.5 (this is just under half way through normal mouse gestation). The reason for the block in further development is unclear but might relate to the defects in the formation of some of the placental cell types that the authors report. This emphasises how much we still have to learn about how embryos build themselves. The technique reported in this study is a promising approach to provide new insights into how mammalian embryos organise and construct organs. Nevertheless, the study has broad implications as, although the prospect of synthetic human embryos still requires further research (as human embryos are not identical to mouse embryos), now is a good time to engage in wider discussions about the legal and ethical implications of such research.”
‘Synthetic embryos complete gastrulation to neurulation and organogenesis’ by Prof Magdalena Zernicka-Goetz et al. was published in Nature at 16:00 UK time on Thursday 25th August 2022.
Prof Robin Lovell-Badge: “No competing financial interests.”
Dr Darius Widera: “No competing interests to disclose.”
Dr Lluís Montoliu: “No conflict of interest.”
Dr Christophe Galichet: “No conflicts of interest.”
Prof Alfonso Martinez Arias: “My group has developed alternative stem cell based models of animal development.”
Dr James Briscoe: “No conflicts of interest declared.”