Induced pluripotent stem cells on the way to the clinic
Childhood was returned to the cage
Galina Kostina, "Expert" No. 7-2011
The technology of reprogramming adult human cells into stem cells has been named by Nature magazine as one of the most significant breakthroughs in science of the last decade. Judging by the enchanting speed of scientific achievements in this field, such cells will be used for treatment and rejuvenation in the near future.
We all get out of one cell, which begins to divide, forming at first a kind of featureless ball, and then more and more similar to a small human body shape. At the first stages of division, all cells are the same. They are called embryonic stem cells (ESCs), and also — omnipotent, or pluripotent, because each of them can turn into a blood or skin cell, bone or brain, into any of the 220 types of cells in our body. They are in such an omnipotent state for only three to five days, then their path of differentiation into cells of specific tissues and organs begins. Scientists have long been attracted by the unique capabilities of embryonic stem cells, which can be an excellent material for the treatment of various diseases associated with cell defects. Researchers have already achieved a lot in this area, having learned how to isolate ESCs, cultivate them, grow various tissues based on them or use them in separate clinical trials. In the last decade, scientists have literally bombarded the media with such statements. However, the use of embryonic stem cells encountered a lot of difficulties of both an ethical and technical nature, in connection with which a search was conducted for the possibility of obtaining stem cells from adult organisms. This also turned out to be a difficult task. Firstly, in an adult body, stem cells no longer have total universality; secondly, they need to be extracted, as a rule, in a traumatic way.
At some point, a crazy thought appeared that the cell of an adult organism could be tried to return to its omnipotent state. Why not? Each cell has the same genome, but different programs are included: one set of genes works in embryonic stem cells, another in blood cells, a third in skin cells, etc. If you learn how to manipulate these programs, you can achieve the conversion of one cell into another. And scientists have succeeded. They are just a step away from creating a safe and effective cell reprogramming technology.
Mouse Tail CocktailAttempts to obtain omnipotent cells for therapy began immediately after the isolation of human embryonic stem cells by James Thomson from the University of Wisconsin in 1998.
In the course of research, such a technology for obtaining and storing ESCs was developed, in which cells could retain their universal potencies for a very long time. At the right time, they could be differentiated into different tissue cells. At the same time, scientists tried to solve the problem of cell compatibility, because embryonic cells are alien, and it is very difficult to find such donor ESCs that would fully fit a particular patient. Therefore, cloning technology was born: the nucleus of a somatic, or adult, cell is transferred to an egg, after which an organism identical to the one from whom the nucleus of a somatic cell was taken may appear. So the famous Dolly lamb appeared. In this way, mice, fish and other small animals were cloned in single copies, there were even scandalous reports about human cloning, but so far there is no official data on the successful receipt of human embryonic stem cells in this way. Since research in the field of ESCs is limited, and in some countries it is still not allowed (human embryos cannot be used), scientists have been looking for more ethical ways to obtain omnipotent cells similar in their properties to ESCs.
In November 2006, a sensational report by Shinya Yamanaki, a researcher from Kyoto University in Japan, appeared in the journal Cell, where it was said that he and his colleagues managed to obtain cells with the properties of embryonic stem cells from adult skin cells, that is, reprogram adult cells to a young state. The cells were called iPS cells — induced pluripotent stem cells (iPSCs).
This achievement was based on the tremendous work of geneticists around the world, done in the late twentieth — early twenty-first century. Thanks to the Human Genome project, all the genes working in the human body have become known. It was found out, in particular, which genes work, and not "silent" in embryonic stem cells. Among them, the genes of the so-called transcription factors have been discovered, which scientists have respectfully called master genes. These lord genes are not responsible for the routine activity of the cell, they activate other genes through the proteins encoded by them, which maintain the cellular omnipotent state. At the same time, gene manipulation technologies were rapidly developing. It became possible to cut them out of pieces of DNA, insert them into various structures and make them work by producing certain proteins.
Shinya Yamanaka has identified 24 genes that, in his opinion, play a major role in the embryonic stem cell. He took the fibroblast (the precursor of a skin cell) of a mouse tail and inserted all 24 genes into its nucleus using the so-called retroviral technology, when an artificial virus with genes sewn to it is introduced into a cell and embedded in its genome in the nucleus. The experiment was a success. Then the game of sorting out genes has already gone on. Yamanaka first divided 24 genes into two arbitrary halves and again conducted experiments with groups of 12 genes, then divided them in half, compared the results — so he looked for the most important genes for maintaining a state close to ESK, and the most effective ones. In the end, he settled on the four genes, which was later called the "Yamanaki magic cocktail". The cocktail includes Oct3/4, Sox2, c-Myc, Klf4 genes.
Following Yamanaka, James Thomson from the University of Wisconsin-Madison also announced successes in reprogramming cells. Initially, he identified 35 genes important for ESCs, and then also stopped at the four, where two were the same genes that Yamanaka used, Oct3/4 and Sox2, and two others — Nanog and Lin28. Thomson managed to use his four to convert first mouse and then human fibroblast into IPSC — induced pluripotent cell.
After these reports in influential scientific journals, many laboratories joined the research race. There was something to work on. Firstly, scientists recognized that the delivery of the necessary genes into the cell using viral vectors can only be used in vitro. It is dangerous to introduce such a structure into the body: the virus embeds it directly into the genome, after which unintended rearrangements can begin in the genome. Secondly, the Yamanaki cocktail contained a very strong proto-oncogene c-Myc, capable of activating the appearance of tumors in the body.
"Many genes associated with pluripotency are also associated with tumor formation," explains Professor Sergey Kiselyov, head of the Laboratory of Cell Technologies at the Vavilov Institute of General Genetics. — We analyzed and compared embryonic stem cells, induced pluripotent, adult and cancer cells. Embryonic, induced pluripotent and cancerous have a lot in common not only in the genetic program, but also in the epigenetic one. Epigenetic factors influence the work of the genome depending on external circumstances. And when we turn an adult cell into an induced pluripotent one, those genes that should work in pluripotent cells turn on, and those genes that worked in an adult somatic cell turn off. At the same time, her epigenetic profile also changes — from an adult to one that is characteristic of pluripotent cells."
Make the gentlemen workIn order to eliminate the risk of developing tumors and other genomic rearrangements, the same Yamanaka and other scientists tried to further narrow down the set of necessary genes by getting rid of the c-Myc proto-oncogene in the first place.
"It turned out that in some somatic cells, for example neuronal, two of the four genes of the Yamanaki cocktail work quite productively anyway," comments Sergey Kiselyov. "And it's enough to add only one gene there, which, as it turned out in a number of studies, is the most important — Oct3/4, so that cells begin to turn into pluripotent."
Scientists have been thinking about how else to make these "gentlemen" work. A group from the Scripps Research Institute in California first tried to reprogram adult mouse cells not with the help of the four genes themselves, but with the help of proteins produced by these genes. It succeeded. A little later, a group of the same biologists led by Shen Dean stated that they managed to create induced pluripotent cells from human skin cells in this way. A similar technology was repeated by the groups of Robert Lanza from Stem cell & Regenerative Medicine and Kwan Soo from Harvard.
"However, the technology of production of recombinant proteins used for this technology is quite complex and expensive,— says Sergey Kiselyov. — Therefore, the idea arose, and not to try to launch into the cell not proteins, but their matrix RNA (recall that in the body, to create the necessary protein, first information is read from the DNA gene to the matrix RNA, and then a protein is collected from this information to the RNA. — “Expert"). RNA is injected into the cell, produces proteins, which trigger the reprogramming process. This work was done by our group at the Institute of General Genetics. At first, we also repeated the work with viral vectors and Yamanaka genes, and then decided to use the RNAs synthesized by us from these genes. And we did it. We patented this method in 2009. Later, the results of similar work were published by groups from Israel and the USA."
But scientists are not satisfied with this either. The same Sheng Ding, who used proteins of important genes, proposed a fundamentally new technology, which, in his opinion, can not only make iPSCs safer, but also cheaper. In 2008, he stated that he was able to replace two of the four genes with low-molecular-weight chemical compounds, and at the end of last year he found a way to replace three genes with "chemistry". According to Dean, these chemicals are well known and can not harm the work of the genome in any way, besides they do not require such costs as biological components. "We are just one step away from the ultimate goal," Dean said. "Our technology will revolutionize." Sheng Ding already calls his chemical cocktail the Holy Grail. However, whether this last component will be found remains the main intrigue, because the most important gene for omnipotent cells, Oct3/4, has remained unconquered.
It's up to the clinicSuch close attention to IPSC is understandable.
They can be used to treat diseases that are not yet curable or poorly curable, many of which are associated with genetic mutations, as well as for transplantation of the necessary tissues and organs.
The first confirmation that induced pluripotent cells are used to treat genetic diseases in mice was obtained in the laboratory of Whitehead Professor Rudolf Janisch. Model mice with human sickle cell anemia, a hereditary disease in which the protein hemoglobin is disrupted, were cured using gene therapy and induced cells. First, the genes with a breakdown were "corrected" in adult mouse skin cells. Then the cells were reprogrammed to return to a pluripotent state, and then grow normal blood cells from them, which are not enough in the sick body. These cells are transplanted into the blood of a sick mouse, and it begins to recover.
A similar confirmation of the working technology was demonstrated by scientists from the Center for Regenerative Medicine in Barcelona (Spain), but already on human cell culture. Scientists have tried to defeat such a rare genetic disease as Fanconi anemia, which leads to early death due to a number of disorders in the body, including tumor processes. The disease can be caused by a mutation in one of the 13 genes associated with Fanconi anemia. Scientists worked with fibroblasts of patients: first they corrected the "sick" genome, then they turned these corrected fibroblasts using viral vectors with sewn genes into induced pluripotent cells, and then directed their specialization towards hematopoietic cells — precursors of blood cells. The researchers have successfully passed all stages of preclinical trials. "We have not cured the person yet, but we have cured his cells," said the head of the group, Juan Carlos Belmonte. "Theoretically, we can transplant them to a patient and defeat the disease." Clinical trials were held back by the fact that the Spaniards used viral technologies fraught with the risk of developing tumors. But since there are other methods of reprogramming cells, these problems are, in principle, solvable.
"In fact, various methods can be used for reprogramming," Sergey Kiselyov believes, "depending on the tasks set. In particular, we use both viral vectors and RNA in our laboratory. Viral vectors can be used to create model systems, and methods using RNA or low molecular weight compounds, if such a cocktail is still created, can already be applied in therapy." Model systems can be useful for finding medicines. For example, in the laboratory of Sergey Kiselyov, approaches to the treatment of Parkinson's disease and Huntington's disease are being sought in this way. For model systems, neurons damaged by the disease are needed, but you can't dig them out of the patient's skull. Therefore, its fibroblasts are taken, induced pluripotent cells are made of them. In these cells, genetic mutations are not corrected, because scientists then screen chemicals on these cells turned into neurons. "Thus, we can choose a medicine not only for a specific disease, but also for a specific patient," explains Kiselyov. — By the way, the therapeutic use of induced pluripotent cells also, in my opinion, needs an individual approach. So far, scientists do not know how and when to inject induced pluripotent cells, for example, into the brain. So far, the clinical application for diseases related to blood cells is more understandable, because there they take root and work thanks to blood circulation." Perhaps very soon, induced pluripotent cells will be used for the treatment of complex hereditary diseases associated with blood, which will be "their own" for each patient. To treat other diseases, according to Kiselyov, there is still a long way to go: it is necessary to create technologies for the safe and effective introduction of cells in the right place and at the right time so that they are accepted by the body and work in the right direction. However, judging by the speed with which the field of cellular technologies is developing, the clinical application of induced pluripotent cells for the treatment of many diseases, transplantation of tissues and organs, as well as rejuvenation of the body is looming on the horizon.
Portal "Eternal youth" http://vechnayamolodost.ru21.02.2011