Person for 4%

The proportion of human cells in the chimera with a mouse was raised to four percent

Polina Loseva, N+1

American biologists managed to create chimeric human and mouse embryos, in which the proportion of human cells was up to four percent. In previous experiments, human cells were poorly embedded in such embryos, and they could not always be detected. Progress was made possible thanks to special processing of human embryonic stem cells: researchers "slowed down" their development by synchronizing their state with mouse embryonic cells. As a result, human cells have penetrated into a variety of organs, from the liver to the retina. The work was published in the journal Science Advances (Hu et al., Transient inhibition of mTOR in human pluripotent stem cells enables robust formation of mouse-human chimeric embryos).

Chimeric embryos from different mammalian species are not only a tool for studying embryonic development, but also a promising biotechnology. Scientists have repeatedly suggested using animals as incubators for growing human organs. Such chimeric embryos have already been created not only for laboratory organisms – for example, mice and rats – but also with the participation of human cells.

So, a few years ago, the pig-man chimeras were obtained, and in the summer of 2019, Spanish scientists announced that they had created a monkey-man chimera in China. However, all these embryos are destroyed at the early stages of development in order to avoid ethical disagreements. And only in the summer of 2019, Japanese scientists received permission not only to inject human cells into a mouse embryo, but also to plant a chimera in the uterus of pregnant mice.

The problem is this: at the stage of development when it is possible to create a chimera, human and mouse cells are in different states. The mouse embryo at this moment consists of naive embryonic stem cells (ESCs) – they are able to give rise to all germinal, as well as some extra-germinal tissues. Human cells at the same stage are called primed ESCs – extra-germ tissues will not turn out of them, since they have already begun differentiation.

Now there are several ways to transfer human ESCs from a primed state to a naive one – these are, for example, chemical inhibitors or the expression of certain genes (we also talked about one mechanical method). But in all these cases, the effectiveness of embedding in the post-implantation embryo is low: in the human-pig chimeras, only individual human cells could be seen, and in the human-mouse chimeras, they practically cannot be seen.

Jiang Feng from New York State University in Buffalo and his colleagues have proposed another way to solve the problem. The fact is that the naivety of mouse ESCs may be associated with diapause, a condition in which the development of embryos is inhibited for a while while the mother is carrying or feeding the previous generation of cubs. It is also known that diapause in mouse embryos can be caused if the mTOR protein complex is blocked, which spurs cellular metabolism. Therefore, Feng and his colleagues suggested that it is possible to treat human ESCs with mTOR blockers and thereby synchronize them with cells in the mouse embryo.

To begin with, the researchers checked that their chosen method works. They developed a protocol involving a common mTOR blocker, rapamycin, as well as an alternative blocker, Torin1. The resulting colonies of naive ESCs expressed all the necessary markers and formed tumors (teratomas) when injected into the mouse body, that is, they met the criteria of embryonic cells. At the same time, their functional properties have changed: for example, they have become better at sharing. While the original culture of primed ESCs increased 10 times in 6 days, the culture of naive ESCs increased almost 1000 times. The changes also affected gene expression and the level of DNA methylation.

Then the authors of the work introduced naive human ESCs expressing green fluorescent protein into mouse morules – embryos of the first days of development, which are simply a group of identical cells. From chimeric morules, blastocysts developed – cellular balls with a loose mass inside – and human cells shone both in the outer layer and in the inner. Then these blastocysts were planted in mice and the resulting embryos were taken for analysis up to the 17th day of development (counting from the moment of fertilization). The researchers were interested in which tissues human cells could integrate into. As a result, they were found both in the liver (a derivative of the inner germ leaf, endoderm), and in the retina (a derivative of the outer leaf, ectoderm), and even in the heart and bone marrow (derivatives of the middle leaf, mesoderm).

A section of a chimeric embryo with different colors. Green – human cells, blue arrows mark the places of integration of these cells into the organs of the embryo. 1 – the heart, 2 – the retina of the eye. Figure from the article by Hu et al.

By calculating the proportion of total human DNA in the chimeric embryo, the authors found that from 0.14 to 4.06 percent of the cells (depending on the specific embryo) were human. Compared with previous studies, in which these cells were hardly detected, here we can talk about the stable embedding of human cells into the mouse embryo after implantation.

To avoid ethical disputes, the researchers checked: human cells did not get into the rudiments of the genitals of mice (this is one of the strict restrictions on the creation of such chimeras). As for the nervous system, the authors of the work note: although individual cells were found there, in such a modest amount and at such early stages they could not make a significant contribution to the formation of the mouse brain. Nevertheless, their research demonstrates that the fundamental difficulties in creating chimeric embryos are not so much ethical as technical, but, unlike ethical ones, they are gradually being overcome.

Portal "Eternal youth" http://vechnayamolodost.ru