What is regenerative-plastic medicine?
Evolution has identified two basic options for the completion of cell life - necrosis and apoptosis, which at the tissue level are associated with proliferation and regeneration. Proliferation can be considered as a kind of sacrifice, when the filling of a defect in damaged tissue occurs due to its replacement by connective tissue elements: retaining structural integrity, the organism partially loses the function of the affected organ, which determines the subsequent development of compensatory reactions with hypertrophy or hyperplasia of structural and functional elements that have not been damaged. The length 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, sharp deterioration in quality and shortening of a person's life span begins. Physiological regeneration provides the processes of remodeling, that is, the replacement of the aging and dying by the mechanisms of natural cell death (apoptosis) of cells into new cells, stemming from the stem cell reserves of the human body. In the processes of reparative regeneration, the cellular resources of stem spaces are also involved, which, however, are mobilized in pathological conditions associated with disease or damage to tissues that initiate 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 the embryonic stem cell (ESC) is primarily due to the high potential possibilities of cellular or, as we call it, regenerative-plastic therapy. The basis for the development of methods for treating severe human diseases (degenerative pathology of the central nervous system, spinal cord and brain injury, Alzheimer's and Parkinson's diseases, multiple sclerosis, myocardial infarction, arterial hypertension, diabetes mellitus, autoimmune diseases and leukemia, burn disease and neoplastic processes not a complete list of them) are laid unique properties of stem cells, which allow the creation of new tissues in return, as previously thought, irreversibly damaged tissue zones n sick body.
The progress of theoretical studies of stem cell biology over the past 10 years has been realized by spontaneously emerging trends in the emerging regenerative and plastic medicine, the methodology of which is not only fully systematized, but also needs it. The first and most rapidly developing field of practical use of the regenerative potential of stem cells has become replacement regenerative-plastic therapy. Its path is easily traced in the scientific literature - from experiments on animals with myocardial necrosis to recent work aimed at restoring the postinfarction deficit of cardiomyocytes or to replenishing the losses of β cells of the pancreas and dopaminergic neurons of the central nervous system.
The basis of replacement regenerative-plastic medicine is cell transplantation. The latter should be defined as a complex of medical measures in which, for a short or long time, the patient's organism has direct contact with viable cells of auto-, allo-, iso- or xenogeneic origin. The means of cell transplantation is a suspension of stem cells or their derivatives, standardized by the number of transplantation units. The transplantation unit is the ratio of the number of colony forming units in culture to the total number of transplanted cells. Methods of carrying out cellular transplantation: intravenous, intraperitoneal, subcutaneous injection of a suspension of stem cells or their derivatives; injection of a suspension of stem cells or their derivatives into the ventricles of the brain, lymphatic vessels or cerebrospinal fluid.
In allo- and autologous cell transplantation, two fundamentally different methodological approaches to the realization of the pluri-, multi- or pluripotential potential of stem cells - in vivo or in vitro - are used. In the first case, the introduction of stem cells into a diseased organism is carried out without their preliminary differentiation, in the second - after multiplication in culture, directed differentiation and purification from undifferentiated elements. Among the numerous methods of substitutive cell therapy, three groups of methods are quite clearly distinguished: replacement of bone marrow and blood cells, replacement of cells of organs and soft tissues, replacement of rigid and solid elements of the body (cartilage, bone, tendons, heart valves and vessels of capacitive type). The latter direction should be defined as reconstructive-regenerative medicine, since the potential of stem cell differentiation is realized on a matrix - a biologically inert or absorbable structure that has the form of a replaceable part of the body.
Another way to increase the intensity of regeneration and plastic processes in the affected tissues is the mobilization of 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 yield of hematopoietic stem cells into the total bloodstream, which in the tissue damage zone provides regeneration processes due to their inherent plasticity.
Thus, the methods of regenerative medicine are aimed at stimulating the processes of restoring the lost function - either through the mobilization of its own stem reserves of the diseased organism, or by introducing allogenic cellular material.
An important practical result of the discovery of embryonic stem cells is therapeutic cloning, based on the understanding of the triggering mechanisms of embryogenesis. If the initial signal for the onset of embryogenesis is the pre-mRNA complex located in the cytoplasm of the oocyte, 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 take part in the implementation of the embryogenesis program. What happens to them afterwards, after birth, during periods of growth, maturity and aging? The answer to this question was given by Dolly the sheep: they are preserved. Using the most modern methods of research, it has been proved that the nuclei of adult cells store all the codes necessary for the formation of embryonic stem cells, embryonic leaflets, organogenesis and restriction maturation (differentiation and specialization) of mesenchymal, ecto-, endo- and mesodermal . Therapeutic cloning as a direction was formed already at the earliest stages of the development of cellular transplantology and provides for the return of totipotency to the patient's own somatic cells for obtaining a genetically identical transplant material.
The discovery of stem cells began "from the end", since the term introduced into biology and medicine by A. Maximov referred to bone marrow stem cells that 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 a differentiated predecessor. A common source for absolutely all somatic cells is the embryonic stem cell. It should be noted that the concepts of "embryonic stem cells" and "embryonic stem cells" are by no means identical. Embryonic stem cells were isolated by J. Thomson from the internal cell mass of the blastocyst and transferred to long-lived cell lines. Only these cells have the facsimile "ESC". Leroy Stevens, who discovered embryonic stem cells in experiments in mice, called them "embryonic pluripotent stem cells," meaning the ability of ESC to differentiate into the derivatives of all three embryonic leaflets (ecto-, meso-, and endoderm). But all the cells of the embryo of 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 separation according to their ability (potency) to give rise to cellular lines, which is defined as totality, pluri-, multi-, poly-, bi-and unipotency. Totipotency, that is, the ability to recreate a genetically programmed organism as a whole, has zygotic cells, blastomeres and embryonic stem cells (blastocyst inner mass cells). Another group of totipotent cells, which are formed at later terms of embryo development, is represented by primary hermetic cells of the embryonic sexual zone (genital tubercles). Pluripotency, under which the ability to differentiate into cells of any organ or tissue, is inherent in the embryonic cells of three germinal leaves - ecto-, meso-, and endoderm. It is believed that multipotency, that is, the ability to form any cells within a single specialized line, is characteristic of only two types of cells: the so-called mesenchymal stem cells that are formed in the neural crest and are the precursors of all cells of the connective tissue base of the organism, including neuroglia cells, as well as hematopoietic hematopoietic stem cells, which gives rise to all lines of blood cells. In addition, bi-and unipotent stem cells are isolated, in particular, the progenitor cells of the myeloid, lymphoid, monocytic and megakaryocytic hematopoietic sprouts. The existence of unipotent stem cells is clearly demonstrated on the example of liver cells - the loss of a significant part of the hepatic tissue is compensated for by the intensive division of differentiated polyploid hepatocytes.
In the process of development, all organs and tissues are formed as a result of the proliferation and differentiation of the internal cell mass of the blastocyst, the cells of which are in the strict sense totipotent embryonic stem cells. The first work on isolating embryonic stem cells was performed by Evans, who showed that blastocysts implanted in the brains of mice give rise to teratocarcinomas whose cells clone form lines of pluripotent embryonic stem cells (the initial name for these cells is embryonal carcinoma cells or in the abbreviation ECC in currently not applicable). 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 animals as well as humans.
In the literature of recent years, there are more and more reports on the plasticity of stem cells, which are 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, it is possible in principle to return the somatic differentiated cell to the stage of embryonic development with recapitulation (return) of pluripotency and its realization into a differentiation with the formation of cells of a different type. It is reported, in particular, that hematopoietic stem cells are capable of transdifferentiating with the formation of hepatocytes, cardiomyoblasts and endotheliocytes.
The scientific debate regarding the separation of stem cells through their plasticity continues, that is, the terminology and glossary of cell transplantation are in the process of becoming, which is of direct practical importance, since it is on the use of plastic properties and the ability of stem cells to differentiate into different cell lines that most methods of regenerative plastic medicine.
The number of publications in the field of fundamental and applied problems of regenerative and plastic medicine is growing rapidly. The range of various methodological approaches aimed at the most optimal use of the regenerative-plastic potential of stem cells has already been outlined. Zones of their vital interests were defined by cardiologists and endocrinologists, neuropathologists and neurosurgeons, transplantologists and hematologists. In the plastic possibilities of stem cells, ophthalmologists, tuberculosis specialists, pulmonologists, nephrologists, oncologists, genetics, pediatricians, gastroenterologists, therapists and pediatricians, surgeons and obstetrician-gynecologists are looking for solutions to acute problems. All representatives of modern medicine hope to get a cure for the fatal diseases that have been considered so far.
Is cell transplantation another "panacea" from all ills?
This question arises quite rightly among all doctors and scientists who are thoughtful and analyzing the current state of medical science. The situation is complicated by the fact that on one side of the field of scientific confrontation there are "healthy conservatives", on the other - "sick fanatics" of cell transplantology. Obviously, the truth, as always, lies between them - on a "no-man's land". Without touching on issues of law, ethics, religion and morality, let us consider the pros and cons of the indicated areas of regenerative and plastic medicine. The "light breeze" of the first scientific reports on the therapeutic possibilities of ESCs already a year after their discovery turned into a "gust of wind", swirling in 2003 in the "information tornado." The first series of publications concerned the cultivation of embryonic stem cells, their multiplication and directed differentiation in vitro.
It turned out that for unlimited reproduction of embryonic stem cells in culture, a number of conditions must be strictly observed. Three factors must necessarily be present in the conditioned environment: interleukin-6 (IL-6), stem cell factor (SCF) and leukosinhibiting factor (LIF). In addition, embryonic stem cells should be grown on a substrate (a feeder layer of cells) from embryonic fibroblasts and in the presence of fetal calf serum. Under these conditions, ESCs in the culture grow clones and form embryoid bodies - aggregates of suspension clones of globular cells. The most important feature of the ESC clone is that in the culture the embryoid body ceases to grow when accumulated in the aggregate 50-60, a maximum of 100 cells. During this period, an equilibrium state sets in - the rate of cell division within the clone is equal to the rate of apoptosis (programmed cell death) at its periphery. After reaching such a dynamic equilibrium, the peripheral cells of the embryoid body undergo spontaneous differentiation (usually with the formation of endodermal yolk sac, angioblast and endotheliocyte fragments) with loss of totipotency. Therefore, to obtain a sufficient amount of totipotent cell mass, the embryonic body needs to be disaggregated weekly with a transplantation of individual embryonic stem cells into a new nutrient medium, a process that is laborious enough.
The discovery of embryonic stem cells did not give an answer to the question of what exactly and how it launches embryogenesis programs encoded in the zygote DNA. It remains unclear how the program of the genome unfolds in the process of human life. At the same time, the study of embryonic stem cells made it possible to develop a concept about the mechanisms for preserving the totality, pluri- and multipotency of stem cells in the process of their division. The main distinguishing feature of the stem cell is its ability to self-reproduction. This means that the stem cell, unlike the differentiated one, is divided asymmetrically: one of the daughter cells gives rise to a specialized cell line, and the second preserves the totality, pluri- or multipotency of the genome. It remained unclear why and how this process occurs at the earliest stages of embryogenesis, when the dividing internal cellular mass of blastocysts is all totipotent, and the ESC gene is in a dormant (dormant, inhibited) state. If the process of duplication necessarily precedes the activation and expression of a whole complex of genes when dividing an ordinary cell, then this does not happen when dividing the ESC. The answer to the question "why" was obtained after the discovery of pre-existing mRNA (pre-mRNA) in the ESC, some of which are formed in follicular cells and remain in the cytoplasm of the ovum and the zygote. The second discovery answered the question "how": special enzymes, called "editases", were found in the ESC. Edithases perform three major functions. First, they provide an alternative epigenetic (without the involvement of the genome) reading and duplication of pre-mRNA. Secondly, they realize the process of activation of pre-mRNA (splicing - excision of introns, that is, inactive regions of RNA, which 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 mRNA, which are the repressors of the mechanisms of gene expression, which preserves the dense packing of chromatin and the inactive state of the genes. Protein products synthesized on such secondary mRNAs and called protein-silencer or genomic guardians are present in human ovules.
This is how the mechanism for the formation of immortal cell lines of embryonic stem cells is represented today. Simply put, the signal to start the embryogenesis program, whose initial stages consist in the formation of totipotent cell mass, comes from the cytoplasm of the egg cell. If at this stage the internal cellular mass of the blastocyst, that is, the ESC, is isolated from further regulatory signals, the process of cell self-reproduction takes place in a closed cycle without the involvement of the genes of the cell nucleus (epigenetically). If we provide such a cell with nutrient material and isolate it from external signals that promote the differentiation of the cell mass, it will divide and reproduce itself similar infinitely.
The first results of experimental attempts to use totipotent cells for transplantation were very impressive: the introduction of embryonic stem cells into tissues of mice with weakened immunosuppressors by the immune system in 100% of cases led to the development of tumors. Among the neoplasmic cells from which the ESC originated, differentiated derivatives of totipotent exogenous cell material, in particular neurons, were found, but the growth of teratocarcinoma reduced the value of the results obtained to naught. 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, this picture should be familiar). It is interesting that suspended mouse embryoblast cells behave in exactly the same way: their introduction into adult tissues of immunocompromised animals always causes the formation of teratocarcinomas. But if a clear line of ESC is isolated from this tumor and inserted into the abdominal cavity, then again, specialized somatic derivatives of all three embryonic sheets without signs of carcinogenesis are formed.
Thus, the next problem that needed to be solved was the purification of the cellular material from the impurities of undifferentiated cells. However, even with very high efficiency of directed cell 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 scales of the scales of medical risk the guarantee of recovery of the patient balances with the guarantee of his death.
The relationship between tumor cells and the more advanced in development than embryonic pluripotent progenitor cells (EECC) is very ambiguous. The results of our studies have shown that the administration of ESRP to various transplanted tumors in rats can lead to the disintegration of tumor tissue (D), a rapid increase in tumor mass (D), its reduction (E-3), or does not affect the size of spontaneous central focal necrosis Neoplastic tissue (I, K). It is obvious that the result of the interaction of EKPK and tumor cells is determined by the total set of cytokines and growth factors produced by them in vivo.
It is noteworthy that the embryonic stem cell, responding to carcinogenesis for contact with the tissues of an adult organism, is perfectly assimilated with the cell mass of the embryo, integrating into all the organs of the embryo. Such chimeras, consisting of intrinsic embryonic cells and donor ESCs, are called allophenic animals, although, in fact, they are not phenotypic chimeras. Maximum cell chimerization when introducing ESC into the early embryo undergoes a hematopoietic system, skin, nerve tissue, liver and small intestine. Cases of chimerization of genital organs are described. The only untouchable zone for the ESA was the primary sex cells.
That is, the embryo stores the genetic information of its parents, which preserves the purity and continuation of both the genus and the species.
In the conditions of blocking the division of cells of the early embryo with the help of cytoclasin, the introduction of embryonic stem cells into the blastocyst leads to the development of the embryo, in which 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 organism of the surrogate mother. The mechanisms of such a natural block of the potential possibility of mixing own and foreign hereditary information have not yet been clarified. It can be assumed that in this case an apoptosis program is implemented, the determinants of which are not yet known.
Attention should be paid to the fact that embryogenesis of animals of different species is never coordinated: when the donor organogenesis program is implemented in the body of the embryo-recipient of xenogeneic embryonic stem cells, the embryo dies in utero and resorbed. Therefore, the existence of rat-mouse, pig-cow, and rat-man chimeras must be understood as cellular, but not morphological mosaicism.In other words, when a single mammal is introduced into a blastocyst of another species, the progeny of the maternal species always develops, in which among the own cells of almost all organs there are inclusions and sometimes clusters of structural and functional units consisting of genetically alien material of ESK derivatives. "The term" humanized naya pig "as a designation of a certain monster endowed with reason or external signs of a person. This is just an animal, part of the cells of the body of which comes from the pigs of the human ESCs injected into the blastocyst.