Miniature organs
Maxim Rousseau, Polit.roo
The technology of growing miniature human organs from stem cells began to develop actively only in the last decade. However, scientists have already been able to obtain analogs of the heart, kidney, brain, stomach, lungs, retina, large and small intestines and so on in laboratory conditions. They have groups of differentiated cells similar to those found in full-sized organs.
To obtain an organoid, stem cells are placed in an environment that allows them to form a three-dimensional structure. There they self-organize and differentiate into cells of various types, repeating with some degree of accuracy the structure and even the functions of a real organ. Such organoids already serve for drug testing, but their role is no less important for basic research, since with their help it is possible to establish the genetic mechanisms of the formation of real organs during the development of the embryo.
The development of any organ is determined by a complex algorithm that provides for the inclusion and deactivation of specific genes at the right moments. Scientists are just beginning to learn the details of this program. A new technology allows us to do this – RNA sequencing from a single cell (single-cell RNA sequencing). Reading RNA molecules makes it possible to determine which genes are working at the moment, since it is with the help of so-called informational or matrix RNAs that the information encoded in the genes is transmitted to the ribosomes, where protein synthesis takes place. RNA is a short–lived molecule, so a specific matrix RNA can be found only during the operation of the associated gene, not earlier and not later.
Therefore, scientists grow an organoid from stem cells placed in a volumetric medium and in the process of its development determine, sequencing the RNA of individual cells, which genes and how active they are at the moment. Developmental biologist Jason Spence from the University of Michigan says that sequencing single cells is a great way to describe these processes with a sufficient degree of rigor.
The use of organoids also makes it much easier than, for example, studies on laboratory animals to apply various methods of influencing the genetic activity of cells. You can delete or insert individual genes using specially designed viruses, or use the CRISPR/Cas9 method of point editing of the genome. And then see what effect these changes have caused. Biologists have even learned to infect organoids with various bacterial or viral infections in order to determine the molecular mechanism of the disease. Now, for example, the effects of Zika virus fever on the brain are being studied in this way. In addition, systems have been developed for the joint cultivation of several organoids, reproducing the structure of parts of the body, including a network of neurons and cells of the immune system.
Last week, the most detailed study of the formation of a miniature liver from stem cells was published in the journal Nature. One of its authors, Takanori Takebe, who works at the universities of Yokohama and Cincinnati, was interested in whether artificially grown liver tissue could be used for transplantation to patients. He learned how to successfully grow mini-organs in his laboratory with a size of only a few millimeters from pluripotent stem cells that differentiated into hepatocyte progenitor cells, mesenchymal and endothelial cells.
But he understood that the liver from a Petri dish may differ from an organ of natural origin. Takebe's attention was attracted by the work of Barbara Treutlein from the Institute of Molecular Cell Biology and Genetics of the Max Planck Society. Barbara runs a laboratory that specializes in single cell RNA sequencing. In the work that Takebe drew attention to, she investigated the activity of genes in the formation of lungs in bat embryos. Takanori Takebe invited her to jointly study the genetic mechanisms of the growth of mini-liver from stem cells. Scientists were most interested in the interaction of different types of cells during the formation of an organ, because sometimes a protein secreted by a neighboring cell of another type serves as a signal to start a gene in a cell. Among the leading authors of the work were also Keisuke Sekine (Keisuke Sekine) from Yokohama and J. J. Gray Camp from the Department of Evolutionary Genetics at the Max Planck Society Institute for Evolutionary Anthropology.
Miniature livers were grown using Takanori Takebe's method, and at different stages of their development, researchers took cells and sequenced all the RNA molecules encoding proteins from them, determining the activity of genes. Each time they received a complete set of active transcription factors (proteins that control the work of other genes), signaling proteins and receptors involved at that particular moment.
For comparison, the activity of genes was also studied in human embryo cells and in adult liver cells. According to the data obtained, the patterns of gene activity in organoids are very similar to the processes in the natural embryonic liver, but differ from the liver of an adult.
A liver organoid grown from human pluripotent stem cells.
Hepatocytes are colored green, blood vessel cells are colored red.
In particular, for the first time in history, the authors were able to identify proteins that provide communication between different types of cells in a developing organoid. To test their results, the researchers created many new small livers, but during their development, inhibitors blocking the action of signaling proteins were added to the environment. This allowed scientists to turn off or turn on the processes of cellular differentiation and organ formation at will.
They also managed to establish the role of hypoxia – lack of oxygen – in the process of organoid growth. When the accumulation of cells becomes too large, those cells that are inside begin to experience oxygen deficiency. This causes the cells that are supposed to give rise to blood vessels to start producing the proteins responsible for this process. If an organoid is then transplanted into the liver of a laboratory mouse, it will be able to connect its forming vessels to its circulatory system.
"The possibility of creating a bioengineered transplantable liver or liver tissue will be very useful for people suffering from liver diseases, whose lives need innovative treatment methods to save," Takanori Teikbe commented on the results obtained. "Our data provide a new, detailed understanding of intercellular communication between developing liver cells and show that we can create fragments of the human liver that are very surprisingly close to the formations of embryonic cells that appear during natural human development."
In May of this year, the journal Nature Cell Biology published another paper that tested the possibility of using miniature lungs grown in the laboratory for the study of viral respiratory studies and cystic fibrosis. The team of researchers from Columbia University was led by Professor Hans-Willem Snoeck. Scientists have grown model organoids from pluripotent stem cells, ensuring that they have analogues of branching branches of the bronchi ending in alveoli. Then the organoids were exposed to the virus or, by editing the cell genome, reproduced the mutation responsible for cystic fibrosis. In both cases, they observed the effects characteristic of this disease, which means that such minilights can be used in the search for effective treatment methods.
Also this year, a group of scientists from the United States began using mini-organs in the treatment of prostate cancer. Doctors led by Hatem Sabaawy from the Rutgers Cancer Institute of New Jersey decided to grow model tumors from cells taken from patients and expose them to drugs proposed for the treatment of these patients. If the drug shows its effectiveness, it will be given to the patient.
Tumor cell cultures for testing various therapies have been grown for a long time, but researchers believe that flat tumor tissue in a Petri dish does not sufficiently reflect the complexity of the tumor and poorly predicts how patients will respond to treatment. Therefore, they decided to build three-dimensional analogues of the tumor-affected organ. The researchers also intend to sequence the DNA of tumor tissue to create a bank of genetic profiles that can be used to treat other patients.
Professor Hans Clevers from the Hubrecht Institute of the Royal Netherlands Academy of Sciences is currently leading a similar project that examines colon tumors. He says that although the study is at an early stage, the results obtained with the first patients look promising. According to Klevers, laboratory studies allow you to choose the most effective drug for a particular patient and avoid using those drugs to which the cells of this tumor are resistant. By the end of the year, two more organoid cancer research projects will be launched in the Netherlands, one will be dedicated to colorectal cancer, the other to breast cancer.
Jatin Roper, head of the Center for Hereditary Gastrointestinal Cancer Research at Tufts Medical Center in Boston, combines the use of organoids with research on laboratory animals. Mini-organs that simulate colon tissue with a tumor are grown in the laboratory and then implanted into the intestines of a mouse. There, tumor cells interact with other intestinal cells, which allows researchers to observe cancer in a more natural environment, while various genetic variants are reproduced using CRISPR/Cas9 technology.
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19.06.2017