Transplantologists are waiting for the continuation

Human muscle grown inside a pig embryo

Polina Loseva, N+1

American biologists have created chimeric human and pig embryos to grow human muscles in them. To do this, they needed a CRISPR/Cas9 genetic editing system: the genes responsible for muscle development were removed from pigs, and the gene associated with apoptosis in human cells. The researchers grew chimeric embryos up to 27 days and made sure that the muscle tissue in them develops from human cells, and the nervous tissue and genitals from pig cells. The work was published in the journal Nature Biomedical Engineering (Maeng et al., Humanized skeletal muscle in MYF5/MYOD/MYF6-null pig embryos).

Biologists have repeatedly proposed to solve the problem of the shortage of donor organs with the help of animals. There may be several solutions here. You can transplant your own animal organ, but the human immune system does not always (so in the text – VM) agree to this.

You can genetically modify an animal to make its cells more like human cells. We have already written about the birth of humanized pigs and the first skin transplant from such a pig to a human. However, it is still unclear how humanized organs are safe, for example, they can carry embedded viruses that are potentially contagious to humans.

Finally, you can use the animal as an incubator in which human organs will grow. To do this, it is necessary to create a chimeric embryo in which most of the cells will belong to the animal itself (for example, a pig), but some will be human and will be able to form the necessary organ.

This result can be achieved using the blastocyst complementation method: several human cells are injected into the pig embryo at the early stages of development. At the same time, if you take a modified pig that has the gene responsible for the development of an organ turned off, then it will have to grow this organ from human cells alone.

This is how the blastocyst complementation method works. Figures from the article by Maeng et al.

This technique has been repeatedly tested on mice, but experiments with human chimeras have so far been limited to the creation of chimeric embryos at the earliest stages. To move from them to targeted organ cultivation, three problems need to be solved: 1) find and disable the genes responsible for the development of organs in an incubator animal 2) teach human cells to take root inside a chimera 3) make sure that human cells do not penetrate into the genitals or the brain of an animal, because in this case the experiments may not receive the approval of ethical committees and regulators.

A group of researchers from the University of Minnesota, led by Daniel and Mary Garry, tried to solve these problems on pigs. They chose skeletal muscles as the target organ because this tissue is very difficult to obtain from a donor (after death, muscles are not transplanted, and during life they are not easy to remove). Using the CRISPR/Cas9 system, the researchers created modified fetuses of pigs deprived of the three genes MYF5, MYF6 and MYOD, which are key for muscle development. Such embryos developed at least until the 28th day, but their limbs were severely deformed.

Then the authors of the work checked whether it was possible to "save" the modified pigs with the help of blastocyst complementation. To do this, one cell of an ordinary pig with a built-in green fluorescent protein gene was added to each such embryo on the fourth day of development. Then these chimeric embryos were planted in the uterus of pigs, and chimeric animals were born. Their muscles were completely donated (fluoresced green), but the chimera piglets moved and behaved exactly the same as ordinary animals.

A chimera of two pigs (top) and an ordinary pig (bottom). Externally, animals and their muscles look the same.

After that, the researchers moved on to creating human chimeras. But they suspected that a single human cell might not be enough to form muscles in a pig's body. Therefore, they decided to remove from human cells some gene that could prevent them from taking root in the embryo of a pig. To do this, they compared gene expression in early pig and human embryos and found 257 differences in the work of genes associated with division and apoptosis. From these, the researchers selected the TP53 gene, which encodes the p53 protein, the main "engine" of apoptosis and removed it using CRISPR/Cas9, and also supplied the cells with a green fluorescent protein.

Finally, the authors introduced modified human embryonic cells into the embryos of modified pigs. Such cells really took root and divided better than ordinary ones (p<0.0001). Chimeric embryos were planted in the uterus of pigs and grew them up to 20 or 27 days of development. Outwardly, they looked completely normal.

After that, the researchers measured the content of human cells inside the embryos: it ranged from one per thousand to one per hundred thousand pig cells. At the same time, 99.2 percent of the muscle cells (judging by the expression of the MYOD marker) that were found inside the embryos glowed green, that is, the muscles inside the chimera turned out to be completely human.

Human cells as future muscles in a pig embryo: they glow green and express muscle markers.

In addition, the researchers were interested in whether human cells were embedded in other pig organs. However, they found no traces of human cells either in the heart muscle or in the nervous tissue (they obtained similar results for chimeras from two pigs).

The nerve tissue in the pig embryo (red) does not contain human cells (purple).

Thus, the authors of the work managed to obtain a humanized tissue inside the chimeric embryo. However, additional efforts will be needed to bring this method to practical application. Firstly, ethical restrictions on experiments in many countries do not allow growing embryos with human nervous tissue for longer than 14 days. And although in this particular experiment no human cells were found in the brain of developing embryos, researchers have yet to confirm this repeatedly and at the same time find out why this happens and how to predict it.

Secondly, hardly anyone will allow people to transplant cells without the TR53 gene, one of the main defenders against tumor transformation. In this particular experiment, the removal of this gene served only as proof that genetic modification can help cells survive inside the chimeric embryo. However, for practical application, you will probably have to pick up some other candidates for removal.

In 2019, we talked about the fact that in Japan they approved the creation of chimeric embryos from human and rodent cells, and in China has already created (and then destroyed) chimeric human and ape embryos. And in the fall of 2020, we asked our readers if they were ready for the appearance of human and pig chimeras. You can take this survey in the material "Pig Heart" and compare the results with the position of the Japanese and Americans.

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