Human liver in the mouse body: details
We recently published a popular summary of an article published on July 4 in the journal Nature by Takebe et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Here its contents are retold in much more detail.
The rudimentary liver grown in the laboratory turned into a mouse body
to a functioning body
Two days after the implantation of such a rudimentary liver into the body of the experimental animal, the graft grew to the blood vessels of the host and began to function and develop. When hepatitis was artificially started, the transplant helped the experimental animal survive by taking over the functions of its liver.
The production of artificial organs from induced pluripotent cells is an area of research that is rapidly progressing right before our eyes. Some time ago, the "Elements" talked about the creation of artificial bones (Bone grafts can be obtained from reprogrammed human cells); for this purpose, the precursor cells of bone tissue were grown on a special basis obtained from the calf bone after the removal of its cells. And recently, an article appeared in the journal Nature Communications about obtaining an artificial heart, in fact, by the same method – by cultivating human cells on a frame obtained from the mouse heart, from which all mouse cells were removed (the humanized mouse heart – VM).
In addition, an interesting alternative was found to the method of growing artificial organs outside the recipient's body: it turns out that in the laboratory it is enough to get only the rudiment of the future organ, which, when implanted into the body of the future host, will develop into a full-fledged organ with the required characteristics. The effectiveness of this approach was demonstrated by a group of Japanese researchers who received human liver transplants from small embryonic-like embryonic embryos grown in the laboratory.
Such rudiments are arranged incomparably simpler than mature organs, therefore, complex bioreactors and structural foundations of anatomical shape are not needed for their cultivation. To obtain the rudiment of the liver, it is necessary to have only several types of cells: vascular cells, mesenchymal stem cells, as well as liver endoderm cells corresponding to those cells of the intestine of the embryo that protrude from it, forming the rudiment of the future liver.
Fig. 1. Formation of a compact liver germ during joint cultivation of mesenchymal cells, vascular endothelial cells and liver endoderm cells on a Petri dish. Here and further – images from the discussed article in Nature.The latter cells were obtained from induced pluripotent human cells, turning them first into endoderm cells (cells of the primary intestine of the embryo), and then into liver endoderm cells, in which some genes characteristic of mature liver cells already work.
With the correct quantitative ratio of these types of cells, as well as when culturing them on a suitable substrate, conditions are created that resemble those in which the embryo's rudimentary liver is formed. At the same time, cells growing on an ordinary Petri dish form a compact structure that can be successfully transplanted into the recipient organism (see Fig. 1, Fig. 2, as well as the video).
Fig. 2. A compact rudiment of the liver is formed only when cells are grown on a suitable substrate (figure on the right). LAM – laminin, COL – collagen IV, ENT – entactin.The rudiments of the liver were implanted into the mesentery of the experimental animal or into its head.
The advantage of the latter, seemingly strange method is the ability to observe the development of the graft during the life of the mouse. In this case, a small window is cut through the animal's skull, into which the rudiment of the liver is implanted, and the hole is closed with thin glass from above. Thus, the developing rudiment of the liver is visible through the glass, and there is no need to slaughter the animal and extract the graft in order to study its development.
Two days after the operation, the vessels of the mouse, to which the rudimentary liver was transplanted, fused with the vessels of the transplant, and on the third day normal blood flow was established through them. At the same time, the cells of the implanted rudiment of the liver multiplied, and the network of its vessels expanded and became more complicated (Fig. 3).
Fig. 3. The development of the vascular network in the human liver germ transplanted to the experimental animal. Vascular cells are shown in green, mesenchymal stem cells are shown in red, from which pericytes are formed (cells necessary to maintain the stability of the vascular network).
To assess the maturity of graft cells in the blood of experimental animals, the level of human serum albumin was measured. This protein is synthesized by liver cells, and measuring its amount allows you to judge liver function. Human serum albumin appeared in the blood of experimental animals ten days after transplantation, and then its amount constantly increased, which indicated the maturation of the cells of the implanted rudiment of the human liver. Also, by analyzing the activity levels of various genes, it was revealed that two months after transplantation, the main part of the cells of the implanted rudiment of the liver turned into mature cells.
Interestingly, the liver implanted in the mouse, obtained from human cells, continued to function in a characteristic way for humans. In addition to the active production of the aforementioned human serum albumin, it also performed its other functions "humanly".
One of the key responsibilities of the liver is the neutralization of various substances foreign to the body by converting them into other compounds that are less toxic and easier to remove from the body. Medications, which are also substances foreign to the body, are also destroyed in the liver. The ways of such transformations of the same substances may differ in different organisms. In particular, certain drugs are known – ketoprofen and Debrisoquine – which are converted in different ways in the human liver and in the mouse liver.
Liver transplants obtained from human cells transformed ketoprofen and debrisoquin along a pathway peculiar to humans, but not peculiar to mice. Therefore, mice with such transplants can be used as models for studying human metabolism (Fig. 4).
Fig. 4. The scheme of the experiment. Liver endoderm cells (iPSC-HE, liver endoderm cells – precursors of mature liver cells) were obtained from human pluripotent cells (iPSC). These cells were cultured on a Petri dish together with mesenchymal cells (MSC) and vascular endothelial cells (HUVEC). After the formation of a compact rudiment of the liver, it was transplanted to immunodeficient mice and the functioning of the graft was investigated.
To check the quality of the liver transplants obtained by this method, a special line of mice was used, in which hepatitis could be caused when they were injected with a small dose of diphtheria toxin. A week after implantation of the human liver germ, the disease of the animal's own liver was triggered, and the survival rate of animals that underwent such an operation significantly exceeded the survival rate of animals that did not have a graft implanted. Thus, it was demonstrated that the implanted rudiment of the liver is able to perform its functions effectively enough a week after the transplant.
An important result of the work can be considered a demonstration of the key role of the cellular environment, in which the rudiment of the future organ is formed, for its subsequent functioning. Thus, liver cells can be grown separately from pluripotent cells and transplanted to an experimental animal, but such cells will work less efficiently than cells originally grown in the environment of mesenchymal and vascular cells and transplanted into the body already in the form of a rudiment of the future liver.
The authors of the work managed to artificially obtain functioning rudiments of the human liver, capable of continuing development when transplanted into the recipient's body. The rudiments were obtained by creating a cellular environment similar to the embryonic one for the liver endoderm cells, and it turned out that the signals sent by the cells to each other were enough to form a rudimentary liver of the correct shape, even if the cells were grown simply on a flat surface.
This means that when receiving artificial organs, it is extremely important to choose the initial composition of cells: there must be not only cells that will be the main working cells of the future organ, but also cells that will send them the signals necessary for proper development. The quantitative composition and the substrate on which the cells grow are also important for the formation of the rudiment of the organ. If the conditions for the formation of the embryo sufficiently resemble the natural conditions in which this organ is formed in a human embryo, in the future the resulting embryo is likely to develop optimally, and the organ can be formed definitively already in the recipient's body. When artificial organs are obtained by this method, there is no need for complex anatomical bioreactors, as well as in finding a way to organize the correct internal structure of the organ, since it will be formed mainly already in the recipient's body, adapting to it. The success of Japanese researchers allows us to hope for the effectiveness of this interesting approach to obtaining transplants.
Portal "Eternal youth" http://vechnayamolodost.ru29.08.2013