Mice are not enough in embryology, stem cells are the future

Mice as a test model are an invaluable tool in medical research. However, they are not ideal for all types of experiments and, at the same time, current trends are encouraging efforts to find alternative testing methods that reduce the use of animals. MUDr. Volodymyr Porokh, from the Department of Histology and Embryology at the FM MU, is working on one such method.

As part of the Proof of Concept project MUNI-BLAST, funded by the TA CR GAMA 2 programme, the team of the main researcher Zuzana Holubcova, PharmD, is developing an experimental model from human stem cells for pharmacological and toxicological testing. And while he admits that research will not be done without mice for a long time, his work could help answer numerous questions in the field of prenatal development in the future, in addition to streamlining embryotoxicity testing.

5 May 2022

What do we mean by embryotoxicity?

It is the reaction of the embryo to stimuli from the external environment. Whether it is the effect of light, heat, or a chemical, if the effect on the embryo is negative and damaging, we speak of embryotoxicity.

The purpose of the MUNI-BLAST project is to create a functional model to facilitate the pharmaceutical testing of new drugs and their effects on embryos, which is what mouse embryos are used for today. Can you explain what was the impulse for your project?

As part of its development, every drug must undergo a safety check. This testing is carried out, among other things, on human volunteers who have been instructed on the possible effects. But if you read the package leaflet of some medicine, you may find that it is not recommended for use by pregnant women. This is because it is not possible to do a test study on pregnant women, as the ethics committee would not allow it even if they wanted to. Therefore, mice are used to test the effects of the drug on the pregnant woman and the developing embryo. However, the consequence of this is that we know the effect on the animal but not on the human being for drugs tested in this way. Therefore, for most drugs, it is recommended that their use by a pregnant woman be restricted. There are very few drugs that are known from experience to be completely safe for pregnant women. Yet many women take drugs that they cannot stop taking, such as epilepsy drugs, so they take them with the risk that they may be embryotoxic. The main impulse of our project is therefore to provide a new test system to verify the effect of existing and new drugs on human embryonic cells.

What are the advantages and disadvantages of embryotoxicity testing in rodents?

Mouse or rat models are mainly a convenient and inexpensive solution. However, when it comes to reproduction, rodents are completely different from humans and have different physiology. Mice live only a short time and in order to preserve their species, they must produce many offspring, as a result of which their embryos are very resistant and very efficient in reproduction. In humans, on the other hand, reproductive physiology is evolutionarily disadvantageous, even though we have plenty of time to reproduce. The human egg also has a different mechanism for the distribution of genetic information in the form of chromosomes. Human eggs and early embryos are thus slightly more sensitive than their mouse counterparts. Therefore, although we can perform embryotoxicity tests on rodents in large numbers, such testing is not ideal.

Your work is based on relatively recent findings in synthetic embryology. Can you elaborate on what it is?

The current trend in reproductive and developmental biology is to try to replicate the basic biological processes underlying the development of an individual in vitro (in a test tube). A few years ago, it was discovered that mouse stem cells from a developing embryo are able, under the right conditions, to form 3D structures resembling the early embryo from which these cells were isolated. Essentially, you break a mouse embryo into its component parts, one of which is stem cells, and reassemble them into something that looks and molecularly resembles an actual naturally occurring embryo. This discovery has given rise to synthetic embryology, and in the last year, it has become clear that we can use human stem cells to create an artificial in vitro model. There are a number of such models resembling different stages of embryogenesis. We work with so-called blastoids showing the stage of development around the first week after pregnancy when the woman usually has no idea that she might be pregnant. This is a sensitive period and we generally don't know much about it, so we don't know what can affect it.

4. den vývoje myšího embrya

The Czech Centre for Phenogenomics is a leading facility for experiments on mice, where they can genetically modify and prepare mice for specific types of experiments. Will your blastoids be similarly adaptable?

Yes, that is one of their advantages. In some countries, such as Belgium, England, or Finland, human embryos can actually be used for research, so in theory embryotoxicity could be studied directly on human embryos. These are embryos that are unused in assisted reproduction and whose donors agree that they can be used for research instead of being discarded after a period of time. However, there are not many of these embryos, so they cannot be used for extensive testing, and they are each a combination of different eggs with different sperm, so they are completely different. In general, embryos produced naturally (whether mouse or human) are characterized by a great deal of genetic variability. Thus, differences in the development of individual embryos make it difficult to interpret the results of testing. Whereas when we take blastoids, they all come from the same stem cell line, from which we are able to produce hundreds of thousands of exactly the same blastoids, and so we can compare the effects of external stimuli - and drugs - on a large scale. So these are identical models which, yes, we can modify according to specific needs.

As this is the second time we have mentioned the ethical aspect of the project, where did you get the input material for it in the form of human stem cells?

It is derived from human embryos, which is allowed by Czech legislation. We are part of the Department of Histology and Embryology, where the Head of the Department, Aleš Hampl, together with the then Head of the Department of Biology, Petr Dvořák, were the first in the Czech Republic and the third in the world to successfully derive these cells from human embryos. Since 2003, there has been a bank of cell lines or a set of registered and well-characterised cell lines that we can use for our research. And the more lines we use for testing, the more statistically significant the results are.

Switzerland recently held a referendum in which the people voted in favour of continuing animal experimentation. What is your view on the issue? Because if you develop your model successfully, there will be no need to experiment on mice, right?

Not really, mice will probably be needed for a long time. After all, in drug safety testing they are used to test effects on other organ systems, so overall they are a good model. For example, to test the effects of toxicity on the liver or the brain. There is no better alternative at the moment and probably won't be for some time. While there are in vitro models for each organ system, these will never replace tissue that has developed naturally. For example, digestion in rodents is similar to humans, but reproduction, which is what I am concerned with, is where animals are completely different from humans, for example because they do not have a menstrual cycle. That is why it would be so useful to have a non-animal model for these purposes. Mind you, this is not to say that any reproductive science found in mice is useless, but most of what we find in mice needs to be verified in another model that is closer to the human model.

Can it be assumed that, in addition to the higher accuracy of your model, its benefit will be economic, i.e. that it will lead to lower testing costs?

The higher accuracy is due to the fact that we can produce a large number of identical blastoids, so that the results are then more valuable from a statistical point of view. In terms of cost, here the comparison is more debatable. These are two different ways of testing drug safety with different starting material and thus different implementation requirements. But when I compare the number of blastoids I will be able to produce in one week with the number of embryos I am able to get in the same amount of time from mice, then yes, in this respect our method seems to be more efficient.

Your research may find applications in the future, for example in pharmacology or assisted reproduction. At what stage would you ideally see it in, say, ten years' time? It would probably be a nice feeling to know that once you open the package insert and it states that the drug is not suitable for pregnant women, that it has been verified by your method...

That's a very nice thought, if in ten years the testing and classification of embryotoxicity had changed. Today it is stated that either they don't know what effect the drug has or they know it is bad. But then there are also many drugs that are stated to be known or predicted to have a negative effect on animals. That does not necessarily mean that they are bad for humans, so we use them de facto at our peril. Or rather, in such cases, the doctor must assess the level of potential risk on an individual basis. I would like to see our practice become repeatable and universal. However, this is a long run, where, in addition to the biological challenges, we will also face a lot of administrative and legislative hurdles.

More about the Masaryk University project, which was supported by the TA ČR GAMA 2 programme

The university-wide project under the TA CR GAMA 2 programme entitled "Strengthening the system of commercialisation of R&D results at Masaryk University" is administered by the Centre for Technology Transfer of Masaryk University. The aim of the Proof of Concept projects is to evaluate and verify the application potential of the achieved results of research and development at MU by means of so-called sub-projects, which are selected for support on the basis of internal evaluation and implemented at individual MU departments. The awarding of projects is decided by the Council for TT and Commercialisation, which is an advisory body to the Rector and whose members are also experts from the application sphere.

For more information see this link.

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