The analysis of the phenotypic features of embryonic stem cells
Of particular interest is the interspecific comparative analysis of phenotypic features of ESC. It has been established that human colonies of human ESC are dense accumulations of flattened, epithelioid-like cells, whereas embryoid bodies of mice consist of a loose conglomerate of rounded cells. In human ESC, the index of the nuclear-plasma ratio is lower than in mouse ESK. Embryonic stem cells of monkeys form more flat cell colonies with uneven edges. In early clones of ESC primates, single cells are easily seen. The proliferating ESC of all the animal species studied does not express MHC molecules of the first and second classes. At the same time, human ESCs respond positively to antibodies TERA 1-60 and GCTM-2, which indicates the presence of keratin / chondroitin sulfate proteoglycans characteristic for embryo (terato-carcinoma) stem cells on their surface. Expression in the ESC of all animal species of the oct4 gene suggests that despite phenotypic differences, the same set of genes responsible for the preservation of pluripotency appears to be activated in human and mouse ESOs (Peru, 2001). In addition, the ESC lines isolated from the embryos of rats, pigs, rabbits, primates and cattle have similar morphological characteristics, a similar set of molecular identification markers, and an almost identical molecular mechanism for implementing embryogenesis programs, which allows us to take a fresh look at the problem of xenotransplantation .
Unlike normal embryogenesis in vivo, the proliferation of ESC in vitro is not accompanied by the formation of embryonic sheets and proceeds against the background of the blocking of the homeotic Nogens, that is, without organogenesis. Because segmentation genes do not function, embryonic development, such as embryo insertion, embryo segmentation, yolk sac formation, allantoic and other provisional organs and tissues, can not be reproduced in ESC culture. The culture ESCs were frozen at the beginning of the formation of 350 restriction lines of specialized cells. Thus, the clone of daughter progenitor cells and centrally localized ESC are only a model of the embryo, in the course of development of which different lines of specialized cells, which nevertheless originate from common precursors, simultaneously form in different tissue regions. Despite the minimal level of receptors on the surface of the ESC, they retain the ability to perform primitive formative processes, imitating the voluminous structures of the early embryo: the suspension of ESCO in culture aggregates and forms structures resembling blastocysts or even later embryos (egg cylinders). Such suspension aggregates were appropriately named simple and complex embryoid bodies.
With mixed differentiation in different cells of one embryoid body, the early genes of the ectoderm (oct3, fgf-5, nodal), endoderm (gata-4), mesoderm (brachyury), cardiogenic mesoderm (pxx-2.5), neural tube (msx3 ) and hematopoiesis (elkf). With the help of various combinations of growth factors and cytokines, in some cases it was possible to obtain embryoid bodies in which the ectoderm or mesoderm genes were preferably expressed, which opens the way to modeling gastrulation and the initial phases of organogenesis.
Clonal growth of ESC is evidence of asymmetric cell division, in which only one of the ESCs in the center of the clone retains the unlimited reproductive potential, while the second daughter cell generates a generation of progenitor cells that are already in differentiation. Therefore, the rate of multiplication of the clone at the periphery of the embryoid body is higher than in the center. The marginal cells of the growing clone undergo spontaneous disordered differentiation, migrate or die by the mechanisms of apoptosis. These events determine the fate of the clone: if the proliferation rate exceeds the rate of migration and apoptotic cell death, the size of the clone continues to increase, stabilization occurs when the rate of apoptosis and the rate of formation of new cells are equal, regression with the inverse relationship of these processes. Progenitor cells divide symmetrically, that is, both daughter cells are further differentiated into mature specialized cell lines. The ratio of ESC / progenitor cells varies, but always the amount of ESC is only a fraction of a percent of the progenitor cell population. Therefore, only careful pipetting and timely disaggregation of clones can increase the number of ESCs in culture. To obtain the maximum yield of ESC, the most effective was the disaggregation of the clones at the stage of 10-12 cells. The direction and degree of differentiation of cells in the embryoid body depends on their location. External cells of the embryoid body do not express the oct4 gene and are differentiated into the cells of the primary endoderm, from which epithelio-like cells of the parietal and visceral extra-germinal endoderm are subsequently formed. The internal cells of the embryoid body express the oct4 gene and retain pluripotency for 48 hours of culture. However, then a morphological rearrangement of the culture takes place in the epithelial monolayer and expression of the genes controlling the development of the primary ectoderm begins. Then begins the process of total disordered cytodifferentiation with the appearance of various cell types that are the derivatives of all three germinal sheets. In the process of spontaneous differentiation of cells of the embryoid body, aggregates with endoderm markers in the form of fragments (cysts) of the yolk sac appear first. Further, angioblasts and endothelial cells of growing capillaries appear in these structures. At the final stages of spontaneous differentiation from the internal cells of the embryoid body, various terminal differentiated cells develop, including neurons, glial elements, cardiomyocytes, macrophages and erythrocytes. In a certain approximation (taking into account the spatial inversion of the formation of germ tissue sheets), the embryoid bodies can in vitro study morphogenetic processes and analyze the molecular mechanisms of the initial periods of embryonic cytodifferentiation, and also establish the role of specific genes in the realization of these processes.
Thus, within the clone are cells in which different genetic development programs are discovered - ESCs, early progenitors and differentiating progenitor populations. The cultivation of ESC by the methods of a drooping drop or mass culture without a feeder layer and without the addition of LIF in the medium inevitably leads to the formation of embryoid bodies. The morphology of the cells of the outer and inner layers of the embryoid bodies is different. The outer layer consists of large, process cells. Their surface, facing the environment, is covered with numerous microvilli. The outer layer of cells is separated from the internal basal membrane resembling the Reichert membrane, while the cells of the inner layer of the embryoid bodies are a cylindrical epithelium. Morphologically, the inner layer, although containing many dividing cells, is more reminiscent of undifferentiated ESC colonies.
Features of human embryonic stem cells
The absence of parenchymal-mesenchymal signaling interactions against the background of the blocking of the homeostasis leads to a disordered growth of ESC in the culture, since the bookmarking and the formation of the infrastructure of the provisional organs are disrupted. Unorganized growth and spontaneous spontaneous differentiation of ESCs in culture are caused by the absence of mesenchymal marking of the stromal framework of the future organs: it is quite possible to form millions of hepatocytes in vitro, but not a single lobule of the liver including such structural-functional elements as sinuses, Disse spaces and Kupffer cells.
It is believed that the pluripotency of ESC is realized exclusively in embryogenesis with the formation of tissues and organs of the embryo, while the placenta and umbilical cord are derived from the trophoblast. Prisoners in the trophectodermal envelope of the ESK consistently generate clones of provisional cells that realize the development program by combining the mRNA with the volume topographic matrix of Nokhteiov, which predetermine the spatial arrangement, shape and size, the number of cells of the provisional and definitive organs, and the assembly of the parenchyma into structural-functional units. In this case, ESCs remain the only type of cells in which the molecular mechanism for the realization of their potencies is completely disconnected from the genetic development program, and the ESCs themselves are unable to interact with other cells due to the blocking of both receptor perceptions and trans-signaling systems. However, adequate activation of ESC leads to a phased deployment of the program of embryogenesis, resulting in the birth of a fully formed and ready-to-fetal life of an organism consisting of billions of cells. In this short but time-consuming process in the cell space of the path, errors inevitably arise both in the molecular mechanisms that support the vital activity of cells and in programs controlling their proliferation, differentiation and specialization. Therefore, in modern pharmacogenomics, we separately consider the diseases of the molecular structure and the disease of cell programming. And the effect of most new drugs is aimed at correcting the programs of differentiation, proliferation and organogenesis, as well as regeneration of organs and tissues. In an adult organism, it is possible to control the behavior of stem / progenitor cells transplanted into the brain, liver, spleen, bone marrow and other human organs in an adult organism to restore the injured parenchyma of the recipient organs due to differentiation and specialization of donor cells on the preserved mesenchymal matrix. Essentially, the totipotency program begins to realize at the genome level the oocyte, zygote and blastomer, but these cells can not yet be cloned and passaged in quantities necessary for the needs of experimental and practical medicine. Therefore, ESC remains a unique source of genetic information containing codes for a three-dimensional embryo map and linear restriction codes for specialized cell lines during gastrulation.
Virtually unlimited regenerative capabilities of ESC are due to the fact that their genome, unlike the genetic apparatus of differentiated somatic cells, preserves pluripotency. One of the manifestations of the dormant state of the genetic information embedded in the ESC is the so-called minimal phenotype - a limited number of receptors is expressed on the surface of the ESC, and, accordingly, very few trans-logistic programs are deployed for the interaction of the nuclear apparatus of the cell with its microenvironment. Against the backdrop of the hibernation of genes responsible for the restriction of specialized cell lines and cell differentiation, only about 30 of 500 genes are activated, the products of which ensure the cell's connection with the surrounding microenvironment. Using the method of serial analysis of gene expression, it was shown that in the generality of the work of the main functional boxes of the genome regulating energy and metabolism in somatic cells and ESC, the latter determines a very low level of mRNA receptors, G-proteins, secondary messengers, transcriptases, expression cofactors and repression , that is, the entire system of transmembrane transfer of the regulatory signal into the cell. This is due to the lack or very low expression of transsanalization genes. In the period of induced differentiation, 18 functioning genes synchronously stop functioning on the background of activation of 61 gene of trans-signaling controlling the synthesis of cell adhesion receptors, extracellular matrix components, restriction transcriptases and messenger elements of the signal transmission system to the nuclear apparatus from the receptors of the plasma membrane of cells. At the same time, the expression of genes responsible for the synthesis of the silencer proteins, as well as gene expression co-inhibitors, which ensure the totipotency of the ESC genome, is blocked.
Genetic markers were found for the cells of all three embryonic leaflets. Identification of the ectodermal cell layer is carried out by the expression of the genes nodal, oct3 and fgf-5, mesoderm cells - brachyury, zeta-globin, endoderm - by expression of the gata-4 gene. In normal embryogenesis during the gastrulation period, immature populations of stem and progenitor cells are locally active, localizing the areas of development of the bones of the facial part of the skull, certain parts of the brain, peripheral nervous system, conduction system of the heart and thymus, the tissues of which are formed from clones of migrating cells. The marking of cells on the early genes of the embryonic sheets facilitates the task of topographical analysis of migration processes of progenitor cells in the developing embryo. It was found, in particular, that in the expression of the embryocarcinoma cells P19, the expression of the first brachyury mesoderm gene begins at a time when the expression of the tissue activator's genes for plasminogen, a-fetoprotein, keratin 8 and keratin 19, which are markers of the first migratory mesoderm populations. Consequently, the formation of mesodermal origin tissues begins only after the completion of the process of point migration and migration of mesodermal progenitor cells.
With extremely limited phenotypic signs and the absence of most trans-signaling units, ESCs still express some receptor molecules that can be used to identify them. It is noteworthy that antigens-markers of ESCs in humans and primates were found to be common. Most often, labeled antibodies to labeled antigens SSEA-3, SSEA-4 (unique lipid antigens representing the complex of glycolipid GL7 with sialic acid) and also high polymeric glycoproteins TRA-1-81, TRA-1-60 are used most often for the labeling of ESCs. In addition, ESCs express specific embryonic SSEA-1 antigen and endogenous alkaline phosphatase, as well as a specific Oct4 transcription factor. The latter is necessary to maintain the mechanisms of ESC proliferation - the specific transcription factor Oct4 activates the expression of the fibroblast growth factor 4 gene and stabilizes the expression of the box of genes responsible for unlimited DNA reduplication in immature cells. The most important intracellular marker proteins are Oct3, Oct4, Tcf, and Groucho, which are related to chromatin silencer proteins.
Almost immediately after long-term attempts to cultivate ESKs outside the body were successful and the first cultures of stem cells isolated from mouse blastocysts and the culture of primary germ cells were obtained, the stage of studies of the pluripotent potential of ESCs was started when they were introduced into embryos at early stages of development. It was shown that in the stage of morula and blastocysts, ESCs are able to form chimeric embryos in which descendants of donor ESCs are detected in all somatic tissues and even in gametes. Thus, a "bridge" between experimental studies in vivo and in vitro was established in developmental biology with the help of ESCs, which greatly expanded the possibilities of studying the processes of laying primary tissues and organs, their differentiation and embryonic organogenesis.
It is clearly established that in vivo in the process of embryogenesis, ESCs are integrated into the early germ cell mass, and their derivatives are found in all organs and tissues. ESCs colonize in the chimeric embryo a line of sex cells, the descendants of which form full-fledged ovules and spermatozoa. Embryonic stem cells are clonogenic - a single ESC is able to create a genetically identical colony of cells with molecular markers, which include the expression of the oct4 and alkaline phosphatase gene, high telomerase activity, and the expression of certain embryonic antigens.
To study the mechanisms of embryogenesis with the help of ESC, a method of chimerization of morula was developed by creating a biological structure, outside of which a layer of tetraploid blastomeres of the recipient is located, and donor ESCs are inserted inside. Thus, the trophoblast is formed from descendants of tetraploid blastomeres of the recipient, which provides implantation and placentation, and donor ESCs act as the internal cell mass from which the body of the viable embryo and the line of primary progenitor germ cells are formed. The research value of ESC is not only that when manipulating in vitro with their genome, pluripotency is preserved, but also that the ability of ESCs to participate in the formation of primary sex cells of the chimeric fetus is preserved. It is shown that the descendants of just one genetically modified ESC are populating all the primary rudiments and emerging tissues of a chimeric embryo obtained by aggregation or co-cultivation of this cell with an 8-cell embryo. When transplanted into the morula of ESC mice transfected with the green fluorescent protein gene, the fluorescent descendants of this cell were found in all the test tissues of the developing embryo (Shimada, 1999). Transplantation of ESC into the morula allows the creation of viable mice, the body of which consists only of the descendants of the donor ESC, which opens prospects for various options for therapeutic cloning. Now this methodical approach is successfully used to study the problems of developmental biology, in particular, it analyzes the mechanisms of genetic inactivation of the X chromosome or the epigenetic instability of ESC. Transplantation of ESC into the early embryo is also used in biotechnology in agriculture, as well as in gene therapy experiments.
Transplants of genetically modified ESCs are used to test the target cells of mutant genes. In vitro cultured ESCs are used in biotechnology to create knockout mice. To do this, by homologous recombination, the gene to be examined is removed from the ESC, and the cells lacking this gene are isolated on selective media. Then knockout ESCs are injected into the blastocyst or aggregated with blastomeres of morula. The chimeric early embryos thus obtained transplanted to female recipients, and in the newborn mice, individuals with gametes, Nullizygotic for the gene, were selected. With this technology, many lines of knockout mice have been created, which are widely used in experimental biology and experimental medicine. On such biological models, the importance of certain genes in embryonic development is studied, as well as their role in the mechanisms of diseases and pathological conditions of man. In addition, the lines of knockout animals are used in the preclinical testing phase of new methods of gene therapy. For example, by transfection into the ESC genome of a normal allele of a mutant gene, it is possible to effectively correct a mutation that damages the hematopoiesis system. Introduction of alien genes into ESC allows the creation of lines of homozygous transgenic laboratory animals at an accelerated rate. However, it should be noted that the technique of directed recombination deletion of genes has been reliably worked out for the time being only with respect to the ESC of mice. Using mouse double-knockout ESCs, the functional role of the gene cluster on the 7th chromosome (copy of the genomic region on the 19th human chromosome), as well as the proximal portion of the 11th chromosome (a copy of the human 5d chromosome), was deletion of these genes in ESK mice allowed to evaluate the function of their analogues in humans.
The possibilities of studying the function of human embryogenesis genes, transfection of which into the ESC genome of laboratory animals, allowed, in particular, to clarify the role of the crypto gene in laying and formation of cardiogenic mesoderm, the gene-6 in the embryogenesis of the eye. The first maps of gene expression in immature proliferating ESK teratocarcinomas and blastocysts of mice are made, suppressive repression in ESC of transsignalization genes is confirmed. A combination of 60-80 mutant ESKs and 20-30 cells of normal pre-implantation mouse embryos leads to the development of chimeric embryos in which the organ buds consist of donor and recipient cells, which makes it possible to clarify the role of unknown genes in gastrulation and organogenesis. A functional map of the genes of developing mouse embryos was supplemented by information on the role of the sf-1 gene in the adrenal and genital buds, the wt-1 gene in the kidney bookmark, the myoD family genes in the skeletal muscle insertion, the genes of the gata-1-4 family in restriction maturation rudiments of erythro- and lymphopoiesis.
Directional deactivation of the maternal and paternal alleles of genes in the ESC by means of vector recombinases made it possible to clarify the functions of various genes in the early period of embryogenesis, and the technology of directional transfer of unknown human genes to mouse ESK promotes the discovery of new mutant genes responsible for the development of severe hereditary pathology. Using the knockout method, the obligate value of some genes for embryonic tissue laying was determined: gata-4 for myocardium, gata-1 for erythroid germ of hematopoietic tissue, myoD for skeletal muscles, brachyury for mesoderm, restriction transcripts hnf3 and hnf4 for stem cells of the liver, rag-2 - for the laying of clones of T- and B-lymphocytes (Repin, 2001). The double deletion of genes in the ESC has opened access to the study of the functional role of the germplasm genes, segmentation and homeostasis, and transplantation of ESC has made it possible to obtain viable interspecific embryos-hybrids. Using the improved technique of transplantation of a single donor ESC into the 8-cell embryo, the fact of chimerization at the cellular level of many organs of the embryo-recipient has been proved. Note that cell germs of human tissue are found in the organs of recipient mice and after the introduction of human hematopoietic stem cells into the blastocyst. It was established that pluripotent ESCs circulate in murine embryos during the period of organ formation in the blood. It is possible that their biological function is in the embryonic organization of the future immune system. With the help of ESC in the laboratory, adequate models of human genetic pathology are reproduced: a double knockout of the dystrophin gene models in mice Duchenne muscular dystrophy, deactivation of the atm gene (control of the synthesis of chromatin signal kinase) - ataxia-telangectasia. In this fatal hereditary disease in children due to defects in DNA repair, degeneration of Purkinje cells in the cerebellum develops, which is accompanied by an involution of the thymus due to the death of proliferating cells. The clinic, pathophysiology and pathomorphology of ataxia-telangiectasia, reproduced by the introduction of pathological genetic information in the ESC, in chimera mice correspond to those in humans. In addition to ataxia-telangiectasia using ESC and knockout mice, experimental models of some hereditary homozygous human diseases associated with the pathology of carbohydrate and lipid metabolism, catabolism of amino acids, excretion of copper and bilirubin have been developed, which greatly expanded the possibilities of experimental medicine at the stage of preclinical trials of new ways of treating relevant diseases rights.
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The use of stem cell cytohybrid
Hybrid cells obtained by fusion of ESCs with somatic cells are an adequate and promising model for studying the pluripotency of stem cells and reprogramming chromosomes of differentiated cells. Cytohybrids obtained by the fusion of ESCs with differentiated cells of an adult animal make it possible to study the relationship of genomes of different "ages": a unique situation develops when homologous chromosomes originating from cells of different stages of differentiation and different degrees of maturity are in the same nucleus where they can easily to exchange transient regulatory signals.It is difficult to predict how the cisregulatory epigenetic systems of homologous chromosomes that have developed during the course of the in vivo in hybrid cells, segregation of parental chromosomes occurs, which makes it possible to study the interaction of genomes at the level of individual chromosomes, that is, potentially to identify the involvement of specific chromosomes in maintaining pluripotency or, on the contrary, an output in differentiation.
As the first experimental model for studying the interaction of genomes with different "history of development", cytohybrids obtained by the fusion of pluripotent teratocarcinoma and differentiated somatic cells were used. In some cases, such hybrid cells retained pluripotent properties at a sufficiently high level. In particular, in vivo teratocarcinoma-somatic hybrid cells induced the development of true teratomas containing the derivatives of all three germinal sheets, and in vitro embryoid bodies were formed in the suspension cultures. Even in interspecific cytohybrids of this type, embryonic antigens were noted in cases where somatic partners in the fusion with teratocarcinoma cells had lymphocytes or thymocytes. It is noteworthy that the cyto-hybrids created by the fusion of teratocarcinoma cells with fibroblasts corresponded to fibroblasts according to the phenotype.
The most important established fact is that in teratocarcinoma-somatic hybrid cells there appeared signs of reprogramming the genome of differentiated cells, which was characterized by the reactivation of either individual genes or an inactive X chromosome of the somatic partner. Thus, the results of studies on cytohybridids such as teratocarcinoma-somatic cells indicate that hybrid cells often retain pluripotency and there are signs of reprogramming the genome of the somatic partner.
In the experiments on obtaining intraspecific embryonic hybrid cells by fusion of mouse ESK with splenocytes of an adult animal, the characteristics of such cytohybridids were studied, the segregation of parental chromosomes was analyzed, and the pluripotency of the hybrid genome was estimated. For intraspecific hybrid cells obtained from the fusion of teratocarcinoma cells with somatic cells, a low level of segregation of chromosomes with a tetraploid or near-tetraploid karyotype is typical. A similar chromosomal composition was observed in the cytohybrid by the fusion of primary sex cells with lymphocytes. At the same time, interspecific hybrid cells obtained as a result of the fusion of mouse teratocarcinoma cells with mink lymphocytes, marked intensive chromosome segregation of the somatic partner.
A qualitatively new stage in the study of segregation of parental chromosomes in intraspecific hybrids came after the development of a method for analyzing microsatellites using a polymerase chain reaction, through which several hundred markers were found for each chromosome of the mouse, which reliably discriminated any pair of homologous chromosomes in hybrid cells.
By fusion of ESC (HM-1 cells, deficient in hypoxanthine phosphoribosyltransferase activity, 2n = 40, XY, isolated from blastocysts of 129 / 01a mice) with splenocytes from mice of the congenial DD / c line, it was possible to obtain a set of hybrid clones morphologically similar to ESC. All clones were isolated on a selective medium in which only cells with active hypoxanthine phosphoribosyltransferase can grow. Electrophoretic analysis showed the presence in all clones of an allelic variant of hypoxanthine phosphoribosyltransferase, characteristic for DD / c mice. Using cytogenetic analysis, it was found that of the four hybrid clones, three had a near-diploid set of chromosomes. One near-tetraploid clone contained two populations of hybrid cells, one of which was tetraploid, and the second, the smaller, diploid.
Analysis of microsatellites, which allows discriminating any pair of homologous chromosomes of 129 / 01a and DD / c mice, in hybrid clones with a near-diploid set showed that in two clones there was a clear preferential elimination of the somatic partner's autosomes. Most autosomes in clones HESS2 and HESS3 had markers of the line 129 / 01a, that is, a pluripotent partner. The only exceptions were chromosomes 1 and I: clones of HESS2 and HESS3, along with markers of HM-1 cells, contained a small number of markers of the somatic partner. Such results may reflect the incompleteness of segregation of chromosomes 1 and 11 of the somatic partner and are consistent with cytogenetic data that trisomy on these chromosomes is observed in 30-40% of cells of clones HESS2 and HESS3. The clone of HESS4 was significantly different in chromosome composition: many autosomes in this clone originated from the genome of ESC (chromosomes 2, 3, 4, 5, 6, 7, 10, 13, 14 and 17), but chromosomes 1, 9, 11, 12, 15, 16, 18 and 19 were represented by homologues of both parents. The quantitative ratio of microsatellites labeling these homologous chromosomes approximately corresponded to 1: 1. This allowed the authors to assume that one homologue is derived from the genome of the ESC, and the other from the differentiated cells. In some subclones of the clone HESS4, only chromosomal markers 18 and 19 of the somatic partner were present. The results show that in the cells of the clone HESS4, in addition to chromosome segregation of the somatic partner, one or both of the homologues of the above-mentioned chromosomes of the pluripotent genome were eliminated, that is, bilateral segregation of chromosomes of both parents occurred - a very unusual phenomenon, since cytohybrids are characterized by chromosome segregation only one of the parents.
In addition, after the 20th passage, all clones of hybrid cells contained exclusively X-chromosome markers of the somatic partner, that is, in the clones, the X chromosome of ESC was replaced with the X chromosome of the somatic partner. This is confirmed by in situ hybridization data using a FITC-labeled probe specific for the mouse X-chromosome: a positive signal was detected only on one chromosome. It should be noted that at earlier stages of cultivation (up to the 15th passage), according to cytogenetic data, in many cells there were two X-chromosomes. Consequently, the use of selective media makes it possible to manipulate the chromosomal composition of hybrid cells and selectively target clones carrying single chromosomes of the somatic partner against the background of the ESC genome.
Since the unique feature of the cytohybrid genome is the localization of parental genomes in one nucleus, naturally, the question arises of preserving the pluripotent properties of the embryonic genome in ESC-somatic cell hybrids under conditions of its close contact with the genome of the differentiated cell. Morphologically the cytohybrid of ESC and somatic cells resembled the parental line of the ESC. The evaluation of pluripotency showed that all clones with a near-diploid set of chromosomes were able to form embryoid bodies in suspension cultures in which derivatives of three germinal sheets were present.
Most hybrid cells contained the ECMA-7 antigen, a marker characteristic of early mouse embryos, and also had a high activity of alkaline phosphatase. The most convincing data on the high pluripotent properties of hybrid cells were obtained in experiments to obtain a series of injection chimeras involving hybrid cells of the clone HESS2. An analysis of biochemical markers showed that descendants of donor hybrid cells were found in most chimera tissues. Therefore, hybrid cells obtained by fusion of ESC and somatic differentiated cells retain pluripotency at a high level, including the ability to form chimeras when inserted into the blastocyst cavity.
Clones HESS2 and HESS4 differed significantly in the composition of the parent chromosomes, but they had similar pluripotent properties. One could believe that pluripotency in the hybrid genome manifests itself as a dominant feature, but it is possible that not all chromosomes of the embryonic genome are involved in the process of maintaining pluripotency. If this assumption is correct, one can expect that the elimination of some chromosomes of the pluripotent partner from the hybridoma genome will not be accompanied by a change in their pluripotent status. In this case, an analysis of the segregation of parental chromosomes in embryonic hybrid cells would allow a close approach to the identification of chromosomes responsible for controlling the pluripotency of embryonic cells.
O. Serov and co-authors (2001) did not find among 50 descendants obtained by crossing chimeras with normal mice, such as would have the genotype of mice 129 / 01a and carrying the X chromosome of DD mice. The authors see the reason for this in the reduction of pluripotency in hybrid cells under the influence of the somatic genome. An alternative explanation may be the negative effect of trisomy on some autosomes and imbalances in sex chromosomes (XXY were observed in cells up to the 15th passage) in hybrid cells when they passed meiosis. It is known that cells of XXY can not pass through meiosis and form gametes. Trisomy is also able to cause a decrease in proliferative activity of hybrid cells, as a result of which the selective advantage in the development of chimeras can belong to the cells of the recipient embryo. It follows that to adequately evaluate the pluripotent potential of hybrid cells, it is necessary to obtain hybrid clones with a normal diploid set of chromosomes.
In the experiments of O. Serov and co-authors (2001), the possibility of reprogramming the X chromosome of a somatic cell in the genome of hybrid cells was demonstrated for the first time. This conclusion follows from an analysis of the expression of hprt gene (X chromosome marker) in chimeras: the presence of allelic variant of hdt mice of DD / c was detected in all analyzed chimeric tissues. It is pertinent to emphasize that after the introduction of hybrid cells into the blastocyst cavity, the cytohybrids fall into non-selective conditions and the preservation of the X chromosome in the genome of the hybrid cells means that it has become its obligate component and the gene does not discriminate it from the U chromosome of the pluripotent partner.
Summarizing the results of the analysis of the interaction of somatic and pluripotent genomes in hybrid embryonic cells, the authors conclude that in some cytohybrids pluripotency manifests itself as a dominant trait. A hybrid genome is able to reprogram individual chromosomes of differentiated cells, which, however, does not exclude the possibility of the reverse effect of the somatic genome on the pluripotency of the embryonic genome. In the cultivation of hybrid cells, induction of differentiation occurs much more often than in the original parent line of the ESC NM-1. A similar effect is observed in the formation of primary colonies: many primary colonies of embryonic hybrid cells differentiate in the early stages of formation with large losses of clones during their selection and multiplication.
Thus, cytohybrides created by the fusion of ESCs with somatic cells, despite close contact with the genome of differentiated cells, preserve pluripotency as a unique property of the embryonic genome. Moreover, in such hybrid cells, it is possible to reprogram the individual chromosomes originating from the diffused cells. It remains unclear how fully the pluripotent properties of the embryonic genome in hybrid cells persist, in particular, their ability to participate in the formation of the embryonic pathway in chimeras. For this, it is necessary to obtain embryonic hybrid cells with a normal karyotype. In any case, pluripotent embryonic hybrid cells can become a real genetic model for the identification of chromosomes involved in maintaining pluripotency or its controlling, since two-sided segregation of parental chromosomes potentially provides such an opportunity.
No less attractive is the study of the phenomenon, which O. Serov and co-authors (2001) define as "chromosomal memory". In a hybrid genome, homologous chromosomes are in two alternative configurations: the homologues of the somatic partner have once been differentiated, whereas in the pluripotent partner homologues, this process is just beginning. Consequently, the persistence of high pluripotent properties by hybrid cells suggests that the "pluripotent" configuration of ESC homologues is fairly stable in the hybrid genome, despite the influence of the transmitting factors originating from the somatic partner. The above-described signs of reprogramming homologous chromosomes of a differentiated genome in the development of chimeras do not exclude the fact that in the first stages of the formation and cultivation of cytohybrids in vitro they retain their status acquired during in vivo differentiation. According to recently obtained data, when embryonic hybrid cells are transferred to a nonselective medium, intensive elimination of chromosomes from only the somatic partner is observed in them, that is, the hybrid cell genome easily discriminates homologues after in vitro culture for 10-15 passages. Thus, embryonic hybrid cells represent a promising experimental model for studying not only such a fundamental property of the embryonic genome as pluripotency, but also its alternatives - embryonic differentiation.
Therapeutic efficacy of embryonic stem cell transplantation
Before analyzing the therapeutic efficacy of ESC transplantation and their derivatives, we summarize the above material. The potential of ESC in terms of the full implementation of in vitro embryogenesis is inadequate, since developmental defects in this case are due to the absence of mesenchymal stem cells, which in the body arise autonomously and independently of ESC. The genetic potency of ESC is less than the genetic potential of the zygote, therefore it is not directly used for the cloning of embryos. The unique biological potential of ESC as the only cells in which development programs are deployed in full sequential implementation, finds application in studies on the function of genes. With the help of the ESC, the first combinations of signals that activate the expression of early and late genes encoding the development of three embryonic sheets are deciphered. Preservation of the pluripotency of the ESC genome in vitro makes them a unique tool for reparative regeneration, capable of automatically replacing cellular losses in damage to organs and tissues. In an ideal hypothetical variant, it can be assumed that "... In the transplantation of donor ESCs, compactly packaged programs are transferred to the recipient organism which, under favorable conditions, are realized in the construction of a new tissue'7 capable of" ... Effectively integrating into the recipient organism as a morphological, both functional and functional. "
Naturally, following the development of methods for monodifferentiation of ESC, the in vivo study of the functional activity of cells obtained in vitro from a single specialized clone began. The proliferating ESO clone generates populations of migrating progenitor cells that are really capable of actively integrating into the tissue damage zones of the recipient, which is used in regenerative-plastic medicine. It has been established that transplantation of Dopa-neurons in substantia nigra reduces clinical manifestations in experimental hemiparkinsonianism. Regional transplants of donor neural stem cells reduce the degree of motor disorders caused by trauma or contusion of the spinal cord and brain. Received and the first positive results of stem cell transplantation in demyelinating diseases. It would seem that the regenerative-plastic potencies of ESCs open up unlimited possibilities for using cellular transplantation in practical medicine. However, when transplanting into ectopic zones, ESCs inevitably transform into tumors. When subcutaneous injection of ESC in immunodeficient mice teratomas are formed. When the ESK suspension is transplanted under the capsule of the testis in syngeneic mice, a teratoma is also formed, consisting of different tissues, the cells of which are derived from all three embryonic leaflets. In such teratomas, the processes of reduced organogenesis are extremely rare.
A number of works provide information on the positive results of transplantation of early derivatives of ESCOs to animals with an ex-perimental pathology. Cell neurotransplantation using ESC derivatives is further developed in the experiment and the first clinical trials to correct functional disorders in cerebral and spinal injuries, treatment of syringomyelia and multiple sclerosis (Repin, 2001). With the advent of the technique of neuronogenesis from ESK in vitro, instead of using embryonic brain tissue, methods of transplantation of derivatives of neurospheres derived from embryonic nerve tissue cultures are being developed. Such transplantation suspensions are much more homogeneous and contain committed neuronal and neuroglia precursors.
With the regular addition of retinoic acid to the culture medium at a dose of 10 μg / ml for 6 weeks, more than 80% of postmitotic neurons are formed in the embryo-(terato-carcinoma) line of human NTERA-2. The complete homogeneity of the neuronal population is achieved through the flow sorting of immunophenotypic markers marked by mature neurons, which allows you to get rid of the remains of teratocarcinoma and immature cells. After transplantation into various regions of the brain of experimental animals, such neurons not only survive, but also are built into regional neural networks. In animals with experimental models of local CNS defects, neurotransplantation reduces the clinical manifestations of such a human pathology as the consequences of cerebral trauma, stroke, demyelinating diseases, hereditary defects of the cerebellum, diseases of lipid deposition and polysaccharides.
To optimize the regeneration processes in degenerative diseases of the central nervous system, technologies for the preparation of myelin-producing oligodendrocytes from ESK are being developed. The first stage traditionally involves the proliferation of ESCs with the multiplication of the number of cells necessary for transplantation. In the second stage, directed differentiation of cells into a population of myelin-producing oligodendrocyte precursors is carried out, which is controlled by selective marker antigens.
Certain perspectives are opened for the use of ESK derivatives in order to develop methods for correcting immunodeficiencies caused by genetic defects in thymus maturation. In studies on knockout (mouse) mice with an induced gene defect, a violation of the V (D) J recombination mechanism of TCR gene loci, leading to a loss of T-lymphocyte function, transplantation of early ESK derivatives into the thymus of animals restores the maturation of normal populations of immune clones responsible for cellular immunity. Clinical trials of transplantation of preformed in vitro ESK for the treatment of fatal hereditary anemia in children are conducted.
Objections to the rapid introduction of stem cell transplantation into the clinic are justified by a limited number of stable lines of human embryonic stem cells and the need for their standardization. To increase the purity of standardized ESC lines, as well as adult stem cells, a method of line selection based on molecular genetic analysis of short tandem repeats of DNA is suggested. It is also necessary to test the ESC lines for the presence of small chromosomal rearrangements and genetic mutations, the potential possibility of their occurrence under conditions of cell cultivation is sufficiently high. The thesis about compulsory testing of properties of all types of ESC and regional pluripotent stem cells is advanced, since their reproduction in vitro can lead to the appearance of new characteristics not inherent in stem cells of the embryo or definitive tissues. In particular, it is permissible that prolonged cultivation in cytokine media approximates ESK lines to tumor cells, since similar changes in the ways of regulation of cell cycles take place with the acquisition of the ability to perform an unlimited number of cell divisions. Some authors, on the basis of the potential for the development of tumors, consider human transplantation of early derivatives of embryonic stem cells as recklessness. In their opinion, it is much safer to use the committed descendants of the ESC, that is, the lines of the ancestors of differentiated cells. However, a reliable technique for obtaining stable human cell lines that differentiate in the right direction has not yet been developed.
Thus, in the literature there is more and more data on the positive therapeutic effect of transplantation of human embryonic stem cell derivatives. However, many of these works are subject to revision and criticism. Some researchers believe that the results of early clinical trials are preliminary in nature and suggest only that stem cells can have a beneficial effect on the clinical course of a disease. Therefore, it is necessary to obtain data on the long-term results of cell transplantation. As an argument, the stages of development of clinical neurotransplantology are given. Indeed, in the literature, publications on the high efficiency of transplanting brain fragments of embryos in Parkinson's disease initially prevailed, but then reports began to appear that denied the therapeutic efficacy of embryonic or fetal nervous tissue transplanted into the brain of patients.
The first clinical trials were conducted to assess the safety of neuroblast transplantation - the NTERA-2 teratocarcinoma-derived ESK derivatives, the immature cells of which underwent proliferation in culture before the accumulation of 100 million cell masses. Some of the cells thus obtained were used to characterize the phenotype and to determine cell impurities, as well as to test for possible contamination by viruses and bacteria. LIF and the feed layer of stromal fetal cells were removed from the culture medium and conditions were created for the targeted differentiation of ESC into neuroblasts by a combination of cytokines and growth factors. Then, the neuroblasts were purified from immature teratocarcinoma cells on a flow cage sorter. After the secondary purification and characteristics of the phenotype of the transplanted cells, a suspension of neuroblasts (10-12 million) was injected into the basal nucleus of the brain of patients (with a special microcannula and syringe under the control of stereotaxis and computed tomography) (the seventh month after the hemorrhagic stroke). Post-transplantation one-year screening of the consequences of neuronal transplantation in the stroke zone showed no side effects and undesirable effects. Half of the patients experienced an improvement in motor function in the period from 6 to 12 months after transplantation. Positive clinical changes were accompanied by increased blood supply to the stroke zone after cell transplantation: the average increase in absorption of fluorescently-labeled 2-deoxyglucose, according to positron emission tomography, reached 18%, and in individual patients - 35%.
However, the US National Institute of Health conducted an independent study of the clinical efficacy of neurotransplantation in patients with Parkinsonism. Patients in the first group were transplanted with embryonic nerve tissue producing dopamine, while the second group of patients was undergoing a false operation. The results indicate a zero clinical efficacy of such neurotransplantation, despite the fact that dopamine-producing embryonic neurons survived in the brain of recipients. Moreover, 2 years after embryonic nerve tissue transplantation, 15% of patients developed persistent dyskinesia, which was absent in patients in the placebo group (Stem cells: scientific progress and future research directions., Nat. Inst., Of Health. USA). Observations of the further development of the disease in these patients continue.
Some authors link the inconsistency of the literature data on the evaluation of the clinical efficacy of neurotransplantation with a different approach to the selection of patient groups, inadequate choice of methods for objective evaluation of their condition, and, most importantly, different terms of embryonic nerve tissue development and different brain regions from which this tissue was obtained, transplant and methodical features of surgery.
It should be noted that attempts to directly transplant pluripotent stem embryonic cells into the striatum of rat brain with experimental hemiparkinsonism were accompanied by proliferation of ESC and their differentiation into dopaminergic neurons. It should be assumed that newly formed neurons were effectively built into neuronal networks, since after transplantation of ESC the correction of behavioral anomalies and motor asymmetry in the apomorphine test was observed. At the same time, some of the animals died due to transformation of transplanted ESK in the brain tumor.
Experts at the National and Medical Academies of the United States, experts at the National Institutes of Health of the United States believe that the clinical potential of ESCs deserves the most serious attention, but they insist on the need for a detailed study of their properties, the likelihood of complications and long-term consequences in experiments on adequate biological models of human diseases (Stem cells and the future regenerative medicine National Academy Press, Stem cells and the future research directions., Nat. Inst, of Health USA).
From this point of view, it is important that the comparative histological analysis of the experimental teratomas obtained during transplantation in the testis of the ESC suspension, with teratomas formed as a result of the early embryo transplantation, which also contains ESC, showed that ESCs, regardless of their source of origin or interaction with by those or other surrounding cells in the same way realize their tumorigenic potencies. It has been proved that such teratomas are of clonal origin, since tumors consisting of the derivatives of all three embryonic leaflets can arise from one ESC. (Peera, 2001). It is noteworthy that when transplanting immunodeficient mice cloned ESK with a normal karyotype, teratomas were also formed, consisting of different types of differentiated somatic cells. These experimental data are the perfect proof of the clonal origin of the teratom. From the point of view of developmental biology, they indicate that not multiple committed precursor cells, but a single pluripotent stem cell, acts as a source of differentiated derivatives of all three embryonic leaves constituting teratoma. However, on the path of practical cell transplantation, the results of these studies are, if not a prohibitory, then a warning sign of potential danger, since inoculation of ESC or primary germ cells into different tissues of adult immunodeficient mice inevitably causes the development of tumors from transplanted stem cells. The neoplastic degeneration of ectopically transplanted ESCs is accompanied by the emergence of satellite populations of differentiated cells - due to the partial differentiation of ESC and progenitor clones into specialized lines. Interestingly, when transplanting ESC into skeletal muscles next to teratocarcinoma cells, neurons are most often formed. However, the introduction of ESC into a crushed ovum or blastocyst is accompanied by the complete integration of cells into the embryo without the formation of neoplastic elements. In this case, ESCs are built into virtually all organs and tissues of the embryo, including the sexual rudiment. Such allophenic animals were first obtained by introducing the cells of teratocarcinoma 129 into early embryos in the stages of 8-100 cells. In allophenic mice, populations of heterogeneous cell-derived ESK donors are introduced into the tissues of the bone marrow, intestine, skin, liver and genital organs, which makes it possible to create even interspecific cellular chimeras in the experiment. The shorter the development of the early embryo, the higher the percentage of cellular chimerization, with the highest degree of chimerization observed in the hematopoietic system, skin, nervous system, liver and small intestine of the allophane embryo. In the adult organism of chimerization, tissues protected from the immune system of the recipient by histohematological barriers are susceptible: the transplantation of primary germ cells into the testicle parenchyma is accompanied by the insertion of donor stem cells into the hermetic layer of the recipient tissue. Nevertheless, with the transplantation of ESC into the blastocyst, the formation of chimeric rudiments of the genital organs with the generation of donor primary sexual cells does not occur. Pluripotency of ESC in creating special conditions can also be used for cloning: transplantation of ESK mice into an 8-16-cell murine embryo, cell mitoses in which are blocked by cytalocalin, facilitates the realization of normal embryogenesis with development of the embryo from donor ESC.
Therefore, an alternative to allogeneic ESK transplantation is therapeutic cloning based on the transplantation of somatic cell nuclei into an enucleated egg to create a blastocyst, from the inner cell mass of which then lines of genetically identical to the donor of the somatic nucleus of ESC are isolated. Technically, this idea is quite feasible, since the possibility of creating ESK lines from blastocysts obtained after transplantation of somatic nuclei into enucleated eggs has been repeatedly proved in experiments on laboratory animals (Nagy, 1990, Munsie, 2000). In particular, in mice homozygous for the mutation of the rag2 gene, the fibroblasts obtained by culturing the cells of the subepidermal tissue were used as donors of nuclei that were transplanted into enucleated oocytes. After activation of the oocytes, the zygote was cultured to form a blastocyst, from which the inner cell mass was isolated by ESCs and transferred to the line of Nullizygotes by the mutant cell gene (rag2 ~ / ~). By homologous recombination in such ESCs, the mutation of one allelic gene was corrected. In the first series of experiments, embryoid bodies were obtained from the ESC with the recombinant reconstituted genome, their cells were transfected with recombinant retrovirus (HoxB4i / GFP), and after multiplication they were introduced into the vein of mice rag2 ~ / ~. In the second series, tetraploid blastomeres were aggregated with genetically modified ESCs and transplanted to their female recipients. The born immunocompetent mice served as bone marrow donors for transplantation to mutant mice rag2 ~ / ~. In both series, the result was positive: in 3-4 weeks all mature mice were found to have normal normal myeloid and lymphoid cells capable of producing immunoglobulins. Thus, the transplantation into the oocyte of somatic cell nuclei can be used not only to obtain ESC lines, but also for cytogenotherapy - correction of hereditary anomalies, using ESC as a vector for transport of corrective genetic information. But in this direction of cell transplantation, besides bioethical problems, there are limitations. It is not clear how safe the transplantation of therapeutically cloned cells with a genotype identical to the genotype of a particular patient will be, since such cells can introduce mutations that predispose to other diseases. Normal human ovules remain a hard-to-reach object, whereas even when somatic nuclei are transplanted into enucleated ovum animals, only 15-25% of the constructed "zygotes" develop to the blastocyst stage. It is not determined how much blastocyst is required to obtain a single line of pluripotent cloned ESCs. It should be noted and high level of financial costs associated with the complexity of the therapeutic cloning methodology.
In conclusion, we note that in the ESC, the pluripotency of the genome with hypomethylated DNA is combined with high telomerase activity and the short C ^ phase of the cell cycle, which ensures their intensive and potentially infinite reproduction, during which ESCs retain a diploid set of chromosomes and a "juvenile" set of phenotypic characteristics. Clonal growth of ESC in culture does not prevent their differentiation into any specialized cell line of the organism when the proliferation stops and the corresponding regulatory signals are added. Restrictive differentiation of ESC in the line of somatic cells in vitro is realized without participation of the mesenchyme, bypassing Nokhteiov, outside organogenesis and without the formation of an embryo. Ectopic administration of ESC in vivo inevitably leads to the formation of teratocarcinomas. Transplantation of ESC into the blastocyst or early embryo is accompanied by their integration with the tissues of the embryo and the stable chimerization of its organs.
Regenerative-plastic technologies based on cell transplantation are the point of intersection of the interests of representatives of cell biology, developmental biology, experimental genetics, immunology, neurology, cardiology, hematology and many other branches of experimental and practical medicine. The most important are the results of experimental studies proving the possibility of reprogramming stem cells with a directional change in their properties, which opens up prospects for managing the processes of cytodifferentiation with the help of growth factors-for myocardial regeneration, restoration of CNS lesions, and normalization of the function of the islet apparatus of the pancreas. However, for the wide introduction of transplantation of ESK derivatives into practical medicine, it is necessary to study in more detail the properties of human stem cells and to continue experiments with ESC in experimental models of diseases.
Bioethical issues and the problem of rejection of an allogeneic cell transplant could be solved by the revealed plasticity of the genome of regional stem cells of an adult organism. However, the initial information that when transplanted into the liver isolated and carefully characterized hematopoietic autogenous cells, from which new hepatocytes are built in to the liver lobules, are now being revised and criticized. Nevertheless, data have been published that transplantation of neural stem cells into the thymus causes the formation of new donor T- and B-lymphocyte germs, and transplantation of brain stem nerve cells into the bone marrow leads to the formation of hematopoietic germs with prolonged donor myelo- and erythropoiesis . Consequently, in the organs of an adult organism pluripotent stem cells capable of reprogramming the genome to the potential of an ESC can be preserved.
The human embryo remains the source of receiving the ESC for medical purposes, which predetermines the inevitability of a new intersection of moral, ethical, moral, legal and religious problems at the point of the birth of human life. The discovery of ESCs gave a powerful impetus to the resumption of harsh discussions about where the line between living cells and matter, substance and personality lies. At the same time, there are no universal norms, rules and laws regarding the use of ESC in medicine, despite repeated attempts to create and accept them. Each state within its legislation solves this problem on its own. For their part, physicians from all over the world continue to try to develop regenerative plastic medicine beyond such discussions, primarily through the use of non-embryonic stem cells, and the stem cell reserves of an adult organism.