Stem cells and regenerative and plastic medicine

Today, there are few practicing doctors who do not know about the development of a new direction in the treatment of the most severe diseases, previously incurable by traditional and alternative medicine. We are talking about regenerative-plastic medicine, based on the use of the regenerative potential of stem cells. An unprecedented scientific discussion and pseudo-scientific hype have arisen around the developing direction, largely created thanks to the information hyperboles of the World Wide Web. In a very short time, laboratory studies of the therapeutic capabilities of stem cells have gone beyond the experiment and have begun to be actively introduced into practical medicine, which has given rise to a host of problems of a scientific, ethical, religious, legal and legislative nature. State and public institutions have clearly turned out to be unprepared for the speed of the transition of stem cells from Petri dishes to systems for intravenous administration, which is not beneficial to either society as a whole or a specific suffering person. It is not easy to understand the unimaginable amount of information about the capabilities of stem cells, both in quantity and quality, even for specialists (of which there are none, since everyone is trying to master the new scientific trend on their own), not to mention doctors who are not directly involved in regenerative plastic medicine.

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Why are such experiments needed and are they needed at all?

At first glance, the creation of cellular interspecies chimeras is the fruit of the unbridled fantasy of a fanatical scientist who has forgotten about bioethics. However, it is this approach that has significantly expanded our fundamental knowledge of embryogenesis, since it has made it possible to calculate the number of cells necessary for organogenesis (the formation of the liver, brain, skin, and organs of the immune system). In addition (perhaps this is the main thing in ESC biology), geneticists have received a unique tool at their disposal, with the help of which the functional purpose of genes can be established during the chimerization of embryos. First, a special double knockout technique is used to “switch off” the studied pair of genes in ESCs. Then such ESCs are introduced into a blastocyst and the changes that occur in the body of the developing chimeric embryo are monitored. In this way, the functions of the genes sf-1 (development of the adrenal gland and genital organs), urt-l (kidney anlage), muoD (skeletal muscle development), gata-l-4 (anlage of erythropoiesis and lymphopoiesis) were established. In addition, human genes that have not yet been studied can be introduced (transfected) into the ESCs of laboratory animals to determine their function using a chimeric embryo.

But, as a rule, justifying an experiment by obtaining new fundamental knowledge does not find support from a wide audience. Let us give an example of the applied significance of chimerization using ESCs. First of all, this is xenotransplantation, that is, transplantation of animal organs to humans. Theoretically, the creation of human-pig cell chimeras allows us to obtain an animal that is much closer in antigenic characteristics to the ESC donor, which in various clinical situations (diabetes mellitus, liver cirrhosis) can save the life of a sick person. True, for this we must first learn how to return the property of totipotency to the genome of a mature somatic cell, after which it can be introduced into a developing pig embryo.

Today, the ability of ESCs to divide almost infinitely under special cultivation conditions is used to produce totipotent cell mass with its subsequent differentiation into specialized cells, such as dopaminergic neurons, which are then transplanted into a patient with Parkinson's disease. In this case, the transplantation is necessarily preceded by targeted differentiation of the obtained cell mass into specialized cells needed for treatment and purification of the latter from undifferentiated cellular elements.

As it turned out later, the threat of carcinogenesis was far from the only obstacle to cell transplantation. Spontaneously, ESCs in embryoid bodies differentiate heterogeneously, that is, they form derivatives of a wide variety of cell lines (neurons, keratinocytes, fibroblasts, endotheliocytes). In the field of view of the microscope in this case, cardiomyocytes stand out among the cells of various phenotypes, each of which contracts in its own rhythm. However, to treat a patient, it is necessary to have pure cell populations: neurons - in case of stroke, cardiomyocytes - in case of myocardial infarction, β-cells of the pancreas - in case of diabetes mellitus, keratinocytes - in case of burns, etc.

The next stage in the development of cell transplantology was associated with the development of technologies for obtaining a sufficient number (millions of cells) of such pure cell populations. The search for factors causing the directed differentiation of ESCs was empirical in nature, since the sequence of their synthesis during embryogenesis remained unknown. At first, it was established that the formation of the yolk sac is induced by adding cAMP and retinoic acid to the ESC culture. Hematopoietic cell lines were formed in the presence of 1L-3, SCF, fibroblast growth factor (FGH), insulin-like growth factor (IGF-1), 1L-6, and granulocyte colony-stimulating factor (G-СSF) in the culture medium. Nervous system cells were formed from ESCs after the removal of LIF and the fibroblast layer, which served as a feeder. After treatment with retinoic acid in the presence of fetal serum, ESCs began to differentiate into neurons, and cardiomyocytes were obtained by adding dimethyl sulfoxide (DMSO), which provides targeted delivery of hydrophobic signaling molecules to the cell nucleus. In this case, the accumulation of active oxygen species in the culture medium, as well as electrical stimulation, contributed to the formation of mature contractile cardiomyocytes.

Enormous efforts and resources were spent on finding conditions for differentiation of ESCs into insulin-producing cells of the pancreas. However, it soon became clear that a number of specialized cell lines (pancreatic β-cells, immune and endocrine cells, adipocytes) do not arise from ESCs when stimulated according to the principle of “one stimulating factor - one cell line”. This principle turned out to be valid only for a limited number of cell lines. In particular, the formation of neurons can be induced by retinoic acid, muscle cell line - by transforming growth factor-β (TCP-β), erythroid lines - 1L-6, monocytic-myeloid line - 1L-3. Moreover, the effects of these factors on the differentiation of ESCs turned out to be strictly dose-dependent.

The stage of searching for combinations of growth factors that would advance ESCs to later stages of embryogenesis with the formation of mesoderm (the source of cardiomyocytes, skeletal muscles, renal tubule epithelium, myeloerythropoiesis and smooth muscle cells), ectoderm (epidermis, neurons, retina) and endoderm (epithelium of the small intestine and secretory glands, pneumocytes) began. Nature seemed to force researchers to move forward along the path of embryogenesis, repeating its stages in a Petri dish, not giving the opportunity to immediately and easily obtain the desired result. And such combinations of growth factors were found. Activin A in combination with TGF-β turned out to be a powerful stimulator of the formation of mesodermal cells from ESCs, while blocking the development of the endoderm and ectoderm. Retinoic acid and a combination of bone marrow morphogenetic protein (BMP-4) and epidermal growth factor (EGF) signals activate the formation of ecto- and mesoderm cells, stopping the development of the endoderm. Intensive cell growth of all three germ layers is observed with the simultaneous effect of two factors on ESCs - hepatocyte growth factor (HGF) and nerve cell growth factor.

Thus, to obtain the necessary cell lines, it is necessary to first transfer embryonic stem cells to the stage of formation of cells of some germ layer, and then select a new combination of growth factors capable of inducing the directed differentiation of ecto-, meso- and endoderm into specialized cells necessary for transplantation to the patient. The number of combinations of growth factors today is in the thousands, most of them are patented, some are not disclosed at all by biotech companies.

It was time to purify the obtained cells from undifferentiated cellular impurities. The cells differentiated in the culture were labeled with markers of mature cell lines and passed through a high-speed laser immunophenotypic sorter. The laser beam found them in the general cellular flow and directed them along a separate path. Laboratory animals were the first to receive the obtained purified cellular material. It was time to evaluate the effectiveness of using ESC derivatives on models of diseases and pathological processes. One of such models was experimental Parkinson's disease, which is well reproduced in animals using chemical compounds that destroy dopaminergic neurons. Since the disease in humans is based on an acquired deficiency of dopaminergic neurons, the use of replacement cell therapy in this case was pathogenetically justified. In animals with experimental hemiparkinsonism, about half of the dopaminergic neurons obtained from ESCs and introduced into the brain structures took root. This was sufficient to significantly reduce the clinical manifestations of the disease. Attempts to restore the function of damaged CNS structures in experimental strokes, injuries, and even spinal cord ruptures have proven quite successful.

However, it should be noted that almost all cases of successful use of differentiated ESC derivatives for correction of experimental pathology were undertaken in the acute period of the simulated pathological situation. Remote treatment results were not so comforting: after 8-16 months, the positive effect of cell transplantation disappeared or decreased sharply. The reasons for this are quite clear. Differentiation of transplanted cells in vitro or in loco morbi inevitably leads to the expression of cellular markers of genetic foreignness, which provokes an immune attack from the recipient's body. To resolve the problem of immunological incompatibility, traditional immunosuppression was used, in parallel with which clinical trials began to realize the potential of transdifferentiation and genetic correction of autologous hematopoietic and mesenchymal stem cells that do not cause an immune conflict.

What is regenerative plastic medicine?

Evolution has determined two main options for the end of a cell's life - necrosis and apoptosis, which at the tissue level correspond to the processes of proliferation and regeneration. Proliferation can be considered as a kind of sacrifice, when the filling of the defect of damaged tissue occurs due to its replacement with connective tissue elements: while maintaining structural integrity, the body partially loses the function of the affected organ, which determines the subsequent development of compensatory reactions with hypertrophy or hyperplasia of the structural and functional elements that remain intact. The duration of the compensation period depends on the volume of structural lesions caused by the factors of primary and secondary alteration, after which, in the vast majority of cases, decompensation occurs, a sharp deterioration in the quality and reduction in the duration of human life. Physiological regeneration ensures remodeling processes, that is, the replacement of aging and dying cells by the mechanisms of natural cellular death (apoptosis) with new ones originating from the stem cell reserves of the human body. The processes of reparative regeneration also involve the cellular resources of the stem spaces, which, however, are mobilized under pathological conditions associated with disease or tissue damage, initiating cell death through necrosis mechanisms.

The close attention of scientists, doctors, the press, television and the public to the problem of studying the biology of embryonic stem cells (ESC) is due, first of all, to the high potential of cellular or, as we call it, regenerative-plastic therapy. The development of methods for treating the most severe human diseases (degenerative pathology of the central nervous system, spinal cord and brain injuries, Alzheimer's and Parkinson's diseases, multiple sclerosis, myocardial infarction, arterial hypertension, diabetes mellitus, autoimmune diseases and leukemia, burn disease and neoplastic processes make up a far from complete list) is based on the unique properties of stem cells, which allow the creation of new tissues to replace, as was previously believed, irreversibly damaged tissue areas of a diseased organism.

The progress of theoretical research into stem cell biology over the past 10 years has been realized by spontaneously emerging areas of the emerging regenerative-plastic medicine, the methodology of which is not only quite amenable to systematization, but also requires it. The first and most rapidly developing area of practical use of the regenerative potential of stem cells has become replacement regenerative-plastic therapy. Its path is quite easily traced in the scientific literature - from experiments on animals with myocardial necrosis to the works of recent years aimed at restoring the post-infarction deficiency of cardiomyocytes or replenishing the loss of β-cells of the pancreas and dopaminergic neurons of the central nervous system.

Cell transplantation

The basis of substitutive regenerative-plastic medicine is cell transplantation. The latter should be defined as a complex of medical measures, during which the patient's body has direct contact with viable cells of auto-, allo-, iso- or xenogenic origin for a short or long period of time. The means of cell transplantation is a suspension of stem cells or their derivatives, standardized by the number of transplantation units. A transplantation unit is the ratio of the number of colony-forming units in the culture to the total number of transplanted cells. Methods of cell transplantation: intravenous, intraperitoneal, subcutaneous administration of a suspension of stem cells or their derivatives; administration of a suspension of stem cells or their derivatives into the ventricles of the brain, lymphatic vessels or cerebrospinal fluid.

Allo- and autologous cell transplantation employ two fundamentally different methodological approaches to the implementation of the pluri-, multi- or polypotent potential of stem cells - in vivo or in vitro. In the first case, the introduction of stem cells into the patient's body is carried out without their preliminary differentiation, in the second - after reproduction in culture, targeted differentiation and purification from undifferentiated elements. Among the numerous methodological techniques of replacement cell therapy, three groups of methods are quite clearly distinguished: replacement of bone marrow and blood cells, replacement of organ and soft tissue cells, replacement of rigid and solid elements of the body (cartilage, bone, tendons, heart valves and capacitive vessels). The latter direction should be defined as reconstructive and regenerative medicine, since the differentiation potential of stem cells is realized on a matrix - a biologically inert or absorbable structure shaped like the replaced area of the body.

Another way to increase the intensity of regenerative-plastic processes in damaged tissues is to mobilize the patient's own stem resources by using exogenous growth factors, such as granulocyte and granulocyte-macrophage colony-stimulating factors. In this case, the rupture of stromal connections leads to an increase in the release of hematopoietic stem cells into the general bloodstream, which in the area of tissue damage provide regeneration processes due to their inherent plasticity.

Thus, the methods of regenerative medicine are aimed at stimulating the processes of restoration of lost function - either through the mobilization of the patient's own stem reserves, or by introducing allogeneic cellular material.

An important practical result of the discovery of embryonic stem cells is therapeutic cloning based on understanding the triggers of embryogenesis. If the initial signal for the onset of embryogenesis is the pre-mRNA complex located in the oocyte cytoplasm, then the introduction of the nucleus of any somatic cell into the enucleated egg should trigger the embryo development program. Today we already know that about 15,000 genes participate in the implementation of the embryogenesis program. What happens to them later, after birth, during the periods of growth, maturity and aging? The answer to this question was given by Dolly the sheep: they are preserved. Using the most modern research methods, it has been proven that the nuclei of adult cells retain all the codes necessary for the formation of embryonic stem cells, germ layers, organogenesis and restriction maturation (exit to differentiation and specialization) of cell lines of mesenchymal, ecto-, endo- and mesodermal origin. Therapeutic cloning as a direction was formed already at the earliest stages of the development of cell transplantology and provides for the return of totipotency to the patient's own somatic cells to obtain genetically identical transplant material.

The discovery of stem cells began “from the end”, since the term introduced into biology and medicine by A. Maksimov referred to bone marrow stem cells, which give rise to all mature cellular elements of peripheral blood. However, hematopoietic stem cells, like cells of all tissues of an adult organism, also have their own, less differentiated predecessor. The common source for absolutely all somatic cells is the embryonic stem cell. It should be noted that the concepts of “embryonic stem cells” and “embryo stem cells” are by no means identical. Embryonic stem cells were isolated by J. Thomson from the inner cell mass of the blastocyst and transferred to long-lived cell lines. Only these cells have a facsimile of “ESC”. Leroy Stevens, who discovered embryonic stem cells in experiments on mice, called them “embryonic pluripotent stem cells,” referring to the ability of ESCs to differentiate into derivatives of all three germ layers (ecto-, meso-, and endoderm). However, all cells of the embryo at later stages of development are also stem cells, since they give rise to a huge number of cells that form the body of an adult. To define them, we propose the term “embryonic pluripotent progenitor cells.”

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Types of stem cells

The modern classification of stem cells is based on the principle of their division by their ability (potency) to give rise to cell lines, which is defined as toti-, pluri-, multi-, poly-, bi- and unipotency. Totipotency, that is, the ability to recreate a genetically programmed organism as a whole, is possessed by zygote cells, blastomeres and embryonic stem cells (cells of the inner mass of the blastocyst). Another group of totipotent cells, which are formed at later stages of embryonic development, is represented by primary germinal cells of the embryonic genital zone (genital tubercles). Pluripotency, which is the ability to differentiate into cells of any organ or tissue, is inherent in embryonic cells of the three germ layers - ecto-, meso- and endoderm. It is believed that multipotency, i.e. the ability to form any cells within one specialized line, is characteristic of only two types of cells: the so-called mesenchymal stem cells, which are formed in the neural crest and are the precursors of all cells of the connective tissue base of the body, including neuroglia cells, as well as hematopoietic hematopoietic stem cells, which give rise to all blood cell lines. In addition, bi- and unipotent stem cells are distinguished, in particular, the precursor cells of the myeloid, lymphoid, monocytic and megakaryocytic hematopoietic sprouts. The existence of unipotent stem cells has been clearly proven using the example of liver cells - the loss of a significant part of liver tissue is compensated for by the intensive division of differentiated polyploid hepatocytes.

During development, all organs and tissues are formed as a result of the proliferation and differentiation of the inner cell mass of the blastocyst, the cells of which are, in the strict sense, totipotent embryonic stem cells. The first work on the isolation of embryonic stem cells was carried out by Evans, who showed that blastocysts implanted in the brain of mice give rise to teratocarcinomas, the cells of which, when cloned, form lines of pluripotent embryonic stem cells (the original name of these cells - embryonal carcinoma cells or in the abbreviation ECС - is currently not used). These data were confirmed in a number of other studies in which embryonic stem cells were obtained by culturing blastocyst cells of mice and other animal species, as well as humans.

In recent years, the literature has increasingly reported on the plasticity of stem cells, which is considered not only as the ability of the latter to differentiate into different types of cells at different stages of development, but also to undergo dedifferentiation (transdifferentiation, retrodifferentiation). That is, the fundamental possibility of returning a somatic differentiated cell to the stage of embryonic development with recapitulation (return) of pluripotency and its implementation in repeated differentiation with the formation of cells of a different type is admitted. In particular, it is reported that hematopoietic stem cells are capable of transdifferentiation with the formation of hepatocytes, cardiomyoblasts and endotheliocytes.

Scientific debates regarding the division of stem cells according to their plasticity continue, that is, the terminology and glossary of cell transplantation are in the process of formation, which has direct practical significance, since most methods of regenerative plastic medicine are based on the use of plastic properties and the ability of stem cells to differentiate into various cell lines.

The number of publications in the field of fundamental and applied problems of regenerative-plastic medicine is rapidly increasing. A range of different methodological approaches aimed at the most optimal use of the regenerative-plastic potential of stem cells has already been outlined. Cardiologists and endocrinologists, neurologists and neurosurgeons, transplantologists and hematologists have identified their areas of pressing interest. Ophthalmologists, phthisiologists, pulmonologists, nephrologists, oncologists, geneticists, pediatricians, gastroenterologists, therapists and pediatricians, surgeons and obstetrician-gynecologists are looking for a solution to pressing problems in the plastic capabilities of stem cells - all representatives of modern medicine hope to get the opportunity to cure diseases that were previously considered fatal.

Is cell transplantation the next “cure-all”?

This question quite rightly arises in all thoughtful doctors and scientists who analyze the current state of medical science. The situation is complicated by the fact that on one side of the field of scientific confrontation are “healthy conservatives”, on the other – “sick fanatics” of cell transplantology. Obviously, the truth, as always, is between them – in the “no man’s land”. Without touching on the issues of law, ethics, religion and morality, let us consider the pros and cons of the designated areas of regenerative-plastic medicine. The “light breeze” of the first scientific reports on the therapeutic possibilities of ESCs turned into a “squally wind” a year after their discovery, which swirled into an “information tornado” in 2003. The first series of publications concerned the issues of culturing embryonic stem cells, their reproduction and directed differentiation in vitro.

It turned out that for unlimited reproduction of embryonic stem cells in culture it is necessary to strictly observe a number of conditions. Three factors must be present in the conditioned medium: interleukin-6 (IL-6), stem cell factor (SCF) and leukase inhibitory factor (LIF). In addition, embryonic stem cells must be grown on a substrate (feeder layer of cells) of embryonic fibroblasts and in the presence of fetal calf serum. If these conditions are met, ESCs in culture grow as clones and form embryoid bodies - aggregates of suspension clones of spherical cells. The most important feature of the ESC clone is that in culture the embryoid body stops growing when 50-60, maximum 100 cells accumulate in the aggregate. During this period, an equilibrium state occurs - the rate of cell division inside the clone is equal to the rate of apoptosis (programmed cell death) on its periphery. After achieving such a dynamic equilibrium, the peripheral cells of the embryoid body undergo spontaneous differentiation (usually with the formation of endodermal fragments of the yolk sac, angioblasts and endotheliocytes) with the loss of totipotency. Therefore, to obtain a sufficient amount of totipotent cell mass, the embryoid body must be disaggregated weekly with the transplantation of individual embryonic stem cells to a new nutrient medium - a rather labor-intensive process.

The discovery of embryonic stem cells did not answer the question of what exactly and how triggers the embryogenesis programs encrypted in the zygote DNA. It remains unclear how the genome program unfolds during human life. At the same time, the study of embryonic stem cells made it possible to develop a concept of the mechanisms for maintaining the toti-, pluri- and multipotency of stem cells during their division. The main distinguishing feature of a stem cell is its ability to self-reproduce. This means that a stem cell, unlike a differentiated cell, divides asymmetrically: one of the daughter cells gives rise to a specialized cell line, and the second retains the toti-, pluri- or multipotency of the genome. It remained unclear why and how this process occurs at the earliest stages of embryogenesis, when the dividing inner cell mass of the blastocyst is entirely totipotent, and the ESC genome is in a dormant (sleeping, inhibited) state. If during division of an ordinary cell the process of duplication is necessarily preceded by activation and expression of a whole complex of genes, then during division of ESC this does not happen. The answer to the question “why” was obtained after the discovery of pre-existing mRNA (pre-mRNA) in ESCs, some of which are formed in follicular cells and are stored in the cytoplasm of the egg and zygote. The second discovery answered the question “how”: special enzymes called “editases” were found in ESCs. Editases perform three important functions. Firstly, they provide alternative epigenetic (without participation of the genome) reading and duplication of pre-mRNA. Secondly, they implement the process of pre-mRNA activation (splicing - cutting out introns, that is, inactive sections of RNA that inhibit the process of protein synthesis on mRNA), after which the assembly of protein molecules begins in the cell. Thirdly, editases promote the formation of secondary mRNAs, which are repressors of gene expression mechanisms, which maintains the dense packing of chromatin and the inactive state of genes. Protein products synthesized on such secondary mRNAs and called silencer proteins or genome guardians are present in human egg cells.

This is how the mechanism of formation of immortal cell lines of embryonic stem cells is presented today. Simply put, the signal to launch the embryogenesis program, the initial stages of which consist of the formation of totipotent cell mass, comes from the cytoplasm of the egg. If at this stage the internal cell mass of the blastocyst, i.e. ESC, is isolated from further regulatory signals, the process of self-reproduction of cells occurs in a closed cycle without the participation of the genes of the cell nucleus (epigenetically). If such a cell is provided with nutrient material and isolated from external signals that promote differentiation of the cell mass, it will divide and reproduce its own kind indefinitely.

The first results of experimental attempts to use totipotent cells for transplantation were quite impressive: the introduction of embryonic stem cells into the tissues of mice with an immune system weakened by immunosuppressants led to the development of tumors in 100% of cases. Among the cells of the neoplasm, the source of which were ESCs, there were differentiated derivatives of the totipotent exogenous cellular material, in particular neurons, but the growth of teratocarcinomas reduced the value of the obtained results to nothing. At the same time, in the works of L. Stevens, ESCs introduced into the abdominal cavity formed large aggregates in which embryonic muscles, heart, hair, skin, bones, muscles and nervous tissue were fragmentarily formed. (Surgeons who opened dermoid cysts should be familiar with this picture). Interestingly, suspended mouse embryoblast cells behave in exactly the same way: their introduction into the tissues of adult immunocompromised animals always causes the formation of teratocarcinomas. But if a pure line of ESCs is isolated from such a tumor and introduced into the abdominal cavity, then again specialized somatic derivatives of all three germ layers are formed without signs of carcinogenesis.

Thus, the next problem that had to be solved was to purify the cellular material from impurities of undifferentiated cells. However, even with a very high efficiency of targeted cellular differentiation, up to 20% of the cells in the culture retain their totipotent potential, which in vivo, unfortunately, is realized in tumor growth. Another "slingshot" of nature - on the scale of medical risk, the guarantee of recovery of the patient balances with the guarantee of his death.

The relationship between tumor cells and embryonic pluripotent progenitor cells (EPPCs), which are more advanced in development than ESCs, is quite ambiguous. The results of our studies have shown that the introduction of EPPCs into various transplantable tumors in rats can lead to the disintegration of tumor tissue (G), a rapid increase in tumor mass (D), its reduction (E-3), or does not affect the size of spontaneous central focal necrosis of neoplastic tissue (I, K). It is obvious that the result of the interaction of EPPCs and tumor cells is determined by the total set of cytokines and growth factors produced by them in vivo.

It is noteworthy that embryonic stem cells, responding with carcinogenesis to contact with adult tissues, are perfectly assimilated with the cellular mass of the embryo, integrating into all organs of the embryo. Such chimeras, consisting of the embryo's own cells and donor ESCs, are called allophene animals, although, in fact, they are not phenotypic chimeras. The hematopoietic system, skin, nervous tissue, liver and small intestine undergo maximum cellular chimerization when ESCs are introduced into an early embryo. Cases of chimerization of the genitals have been described. The only zone inviolable for ESCs are primary germ cells.

That is, the embryo retains the genetic information of its parents, which protects the purity and continuation of both the genus and the species.

Under conditions of blockade of cell division of the early embryo using cytoclazine, the introduction of embryonic stem cells into the blastocyst leads to the development of an embryo whose primary germ cells, like all others, were formed from donor embryonic stem cells. But in this case, the embryo itself is completely donor, genetically alien to the body of the surrogate mother. The mechanisms of such a natural block of the potential for mixing one's own and foreign hereditary information have not yet been clarified. It can be assumed that in this case, the apoptosis program is realized, the determinants of which are not yet known to us.

It should be noted that the embryogenesis of animals of different species is never coordinated: when implementing the donor program of organogenesis in the body of the recipient embryo of xenogeneic embryonic stem cells, the embryo dies in utero and is resorbed. Therefore, the existence of chimeras "rat-mouse", "pig-cow", "human-rat" should be understood as cellular, but not morphological mosaicism. In other words, when ESCs of one mammal species are introduced into the blastocyst of another species, offspring of the maternal species always develop, in which, among their own cells of almost all organs, inclusions are found, and sometimes clusters of structural and functional units consisting of genetically alien material of ESC derivatives. The term "humanized pig" cannot be perceived as a designation of some kind of monster endowed with intelligence or external characteristics of a human. This is just an animal, part of whose body cells originate from human ESCs introduced into the blastocyst of a pig.

Prospects for the use of stem cells

It has long been known that diseases associated with genopathology of hematopoietic and lymphoid lineage cells are often eliminated after allogeneic bone marrow transplantation. Replacement of one's own hematopoietic tissue with genetically normal cells from a related donor leads to partial and sometimes complete recovery of the patient. Among the genetic diseases that are treated with allogeneic bone marrow transplantation, it is worth noting combined immunodeficiency syndrome, X-linked agammaglobulinemia, chronic granulomatosis, Wiskott-Aldrich syndrome, Gaucher's and Hurler's diseases, adrenoleukodystrophy, metachromatic leukodystrophy, sickle cell anemia, thalassemia, Fanconi's anemia, and AIDS. The main problem in the use of allogeneic bone marrow transplantation in the treatment of these diseases is associated with the selection of an HbA-compatible related donor, for the successful search of which an average of 100,000 samples of typed donor hematopoietic tissue are required.

Gene therapy allows correcting a genetic defect directly in the patient's hematopoietic stem cells. Theoretically, gene therapy provides the same advantages in the treatment of genetic diseases of the hematopoietic system as allogeneic bone marrow transplantation, but without all the possible immunological complications. However, this requires a technique that allows for the effective transfer of a full-fledged gene into hematopoietic stem cells and maintaining the required level of its expression, which in certain types of hereditary pathology may not be very high. In this case, even a slight replenishment of the protein product of the deficient gene gives a positive clinical effect. In particular, in hemophilia B, 10-20% of the normal level of factor IX is quite sufficient to restore the internal mechanism of blood clotting. Genetic modification of autologous cellular material has proven successful in experimental hemiparkinsonism (unilateral destruction of dopaminergic neurons). Transfection of rat embryonic fibroblasts with a retroviral vector containing the tyrosine hydroxylase gene ensured the synthesis of dopamine in the central nervous system: intracerebral administration of transfected fibroblasts sharply reduced the intensity of clinical manifestations of an experimental model of Parkinson's disease in experimental animals.

The prospect of using stem cells for gene therapy of human diseases has posed many new challenges for clinicians and experimenters. The problematic aspects of gene therapy are associated with the development of a safe and effective system for gene transport into the target cell. At present, the efficiency of gene transfer into large mammalian cells is very low (1%). Methodically, this problem is solved in various ways. In vitro gene transfer involves transfection of genetic material into the patient's cells in culture, with their subsequent return to the patient's body. This approach should be recognized as optimal when using genes introduced into bone marrow stem cells, since the methods for transferring hematopoietic cells from the body to culture and back are well established. Retroviruses are most often used for gene transfer into hematopoietic cells in vitro. However, the bulk of hematopoietic stem cells are in a dormant state, which complicates the transport of genetic information using retroviruses and requires a search for new ways of effective gene transport into dormant stem cells. Currently, gene transfer methods such as transfection, direct microinjection of DNA into cells, lipofection, electroporation, “gene gun”, mechanical coupling using glass beads, transfection of hepatocytes with receptor-dependent DNA coupling to asialoglycoprotein, and aerosol introduction of the transgene into the cells of the alveolar epithelium of the lungs are used. The efficiency of DNA transfer by these methods is 10.0-0.01%. In other words, depending on the method of introducing genetic information, success can be expected in 10 patients out of 100 or in 1 patient out of 10,000 patients. It is obvious that an effective and at the same time safe method of transferring therapeutic genes has yet to be developed.

A fundamentally different solution to the problem of rejection of allogeneic cellular material in cell transplantology is the use of high doses of embryonic pluripotent progenitor cells to achieve the effect of reinstallation of the antigen homeostasis control system of an adult organism (the Kukharchuk-Radchenko-Sirman effect), the essence of which lies in the induction of immunological tolerance by creating a new base of immunocompetent cells with simultaneous reprogramming of the antigen homeostasis control system. After the introduction of high doses of EPPC, the latter are fixed in the tissues of the thymus and bone marrow. In the thymus, EPPC, under the influence of a specific microenvironment, differentiate into dendritic, interdigitate cells and epithelial-stromal elements. During the differentiation of EPPCs in the recipient's thymus, along with the recipient's own molecules of the major histocompatibility complex (MHC), MHC molecules that are genetically determined in donor cells are expressed, that is, a double standard of MHC molecules is established, according to which positive and negative selection of T-lymphocytes is realized.

Thus, the renewal of the effector link of the recipient's immune system occurs through the known mechanisms of positive and negative selection of T-lymphocytes, but through the double standard of MHC molecules - the recipient and donor EPPCs.

Reprogramming the immune system using EPPC not only allows cell transplantation without subsequent long-term use of immunosuppressants, but also opens up completely new prospects in the treatment of autoimmune diseases, and provides a foothold for the development of new ideas about the human aging process. To understand the mechanisms of aging, we have proposed a theory of depletion of the body's stem spaces. According to the main provision of this theory, aging is a permanent reduction in the size of the body's stem spaces, which is understood as a pool of regional ("adult") stem cells (mesenchymal, neuronal, hematopoietic stem cells, progenitor cells of the skin, digestive tract, endocrine epithelium, pigment cells of the ciliary folds, etc.), replenishing the cellular losses of the corresponding tissue in the process of body remodeling. Body remodeling is the renewal of the cellular composition of all tissues and organs due to stem space cells, which continues throughout the life of a multicellular organism. The number of cells in the stem spaces is determined genetically, which determines the limited size (proliferative potential) of each stem space. In turn, the size of the stem spaces determines the rate of aging of individual organs, tissues and body systems. After the depletion of the cellular reserves of the stem spaces, the intensity and rate of aging of a multicellular organism is determined by the mechanisms of aging of somatic differentiated cells within the Hayflick limit.

Therefore, at the stage of postnatal ontogenesis, the expansion of stem spaces can not only significantly increase the lifespan, but also improve the quality of life by restoring the body's remodeling potential. The expansion of stem spaces can be achieved by introducing large doses of allogeneic embryonic pluripotent progenitor cells, provided that the recipient's immune system is simultaneously reprogrammed, which significantly increases the lifespan of old mice in the experiment.

The theory of stem space depletion can change the existing ideas not only about the mechanisms of aging, but also about the disease, as well as the consequences of its drug-induced treatment. In particular, the disease can develop as a result of pathology of stem space cells (oncopathology). Depletion of the mesenchymal stem cell reserve disrupts the processes of connective tissue remodeling, which leads to the appearance of external signs of aging (wrinkles, flabbiness of the skin, cellulite). Depletion of the stem reserve of endothelial cells causes the development of arterial hypertension and atherosclerosis. The initially small size of the thymus stem space determines its early permanent age-related involution. Premature aging is a consequence of the initial pathological decrease in the size of all stem spaces of the body. Drug and non-drug stimulation of stem cell reserves improves the quality of life by reducing its duration, since it reduces the size of the stem spaces. The low efficiency of modern geroprotectors is due to their protective effect on aging differentiated somatic cells, and not on the stem spaces of the body.

In conclusion, we would like to note once again that regenerative-plastic medicine is a new direction in the treatment of human diseases based on the use of the regenerative-plastic potential of stem cells. In this case, plasticity is understood as the ability of exogenous or endogenous stem cells to be implanted and give rise to new specialized cell sprouts in damaged tissue areas of a diseased organism. The object of regenerative-plastic medicine is fatal human diseases that are currently incurable, hereditary pathology, diseases in which traditional medicine methods achieve only a symptomatic effect, as well as anatomical defects of the body, the restoration of which is the goal of reconstructive-plastic regenerative surgery. In our opinion, it is too early to consider the first attempts to recreate whole and functionally complete organs from stem cells as a separate area of practical medicine. The subject of regenerative-plastic medicine are stem cells, which, depending on the source of their receipt, have different regenerative-plastic potential. The methodology of regenerative plastic medicine is based on the transplantation of stem cells or their derivatives.