Embryonic stem cells

The discovery of embryonic stem cells did not arise by chance, but appeared on the prepared ground of scientific research in the field of developmental biology. The term “stem cell” was introduced into medicine in 1908 at the congress of the hematological society in Berlin by Alexander Maximov in relation to hematopoietic cells. Long before the isolation and production of stable lines of pluripotent embryonic stem cells, stem terato-(embryo-carcinoma) cells were used in studies of early development processes, with the help of which unknown mechanisms of embryogenesis were studied, including the sequence of expression of early genes and protein products of their activity.

But is the totipotency of the human genome irretrievably lost in the process of evolution? No, and embryogenesis is proof of this. If this is so, then when, in principle, will the second path of evolutionary development be realized? Probably, when man enters space, where environmental conditions will be relatively constant for a sufficiently long time. The loss of bone tissue (demineralization of bones in a state of weightlessness), which is very slowly subject to remodeling and regeneration, can be considered the first step in the process of adaptation of man, as a species, to existence in space conditions. However, the price for the second path of evolutionary development will be different - the price for the return of totipotency and absolute plasticity to all cells will be sterility. So, in this world of “evolutionary chameleons”, we will have to reproduce without meiosis, by budding. But we will live a long time. Telomerase immortality is the immortality of an amoeba. In a multicellular organism, stem cells are the substrate of quantitative and qualitative longevity.

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Sources of embryonic stem cells

Today, the sources of embryonic stem cells for laboratory research are mouse teratocarcinoma lines (129/sv, F19, F8, Zin 40, CGR 86, Rl, CCE, JM-1, E14TG2a, CGRSb) and human teratocarcinoma (NTERA-2, TERA-2, H-9 clone), as well as Trauneon ESC lines. However, the availability of a detailed cell passport indicating the immune phenotype, results of chromosomal analysis, mRNA expression profiles, exposed receptors and intracellular signaling proteins does not compensate for the significant shortcomings of teratocarcinoma ESC lines - rapid loss of totipotency and the impossibility of using them in clinical trials, while mixed differentiation in culture makes it very difficult to isolate a pure specialized line from a heterogeneous cell population. Therefore, the source of ESC lines created for clinical purposes is usually the inner cell mass of the blastocyst, individual blastomeres of 8-cell stage embryos, morula cells of later stages, as well as primordial germ cells.

It should be noted that teratocarcinoma cells, although they have the property of pluripotency, are characterized by a significantly lower pluripotent potential compared to ESCs. Their integration with embryonic cells rarely leads to the formation of chimeras, which, moreover, never form gametes with the genotype of teratocarcinoma cells. It is believed that this is due to the frequent occurrence of chromosomal abnormalities during the cultivation of teratocarcinoma cells: loss of the Y chromosome, various trisomies, deletions or translocations.

Attempts to isolate a human ESC line have been made repeatedly, but this task has not been solved, since normal human blastocysts are difficult to access. In addition, the frequency of chromosomal abnormalities in humans is higher than in animal embryogenesis. The overwhelming majority of early human embryos obtained after in vitro fertilization exhibit chaotic chromosomal mosaicism and often have numerical and structural aberrations. Even later, at the blastocyst stage, only 20-25% of human embryos consist of cells with a normal karyotype. It was virtually impossible to use such embryos to create ESCs, since zygotes were usually cultured to the two- or four-blastomere stage and then transplanted into the uterus. Only relatively recently has a reliable technique for culturing fertilized human eggs to the blastocyst stage been developed. The introduction of this technique into the practice of in vitro fertilization has not only increased the frequency of successful implantation outcomes, but has also made normal blastocysts a more accessible object.

Another pluripotent stem cell source is the primordial germ cells, which, unlike the more advanced progenitor populations of the germinal epithelium, do not have beta-integrin on their surface, but express high activity of alkaline phosphatase. It should be noted that populations of stem cells that formed from primordial germ cells have been studied experimentally since the 1980s. At that time, a technique for isolating primordial germ cells from the rudiment of the mouse embryo's gonad was developed. The first unsuccessful results of culturing primordial germ cells in vitro suggested the futility of these attempts, since the cells, although they survived, did not proliferate and died within the first day. It was later established that mouse primordial germ cells reproduce in vitro only in the presence of soluble and membrane-bound specific polypeptide growth factors in the culture medium. The results of numerous studies have shown that for the survival and proliferation of primary germ cells, the presence of not only LIF but also membrane-bound and soluble Steel factors (SIF) in the culture medium is necessary. These peptides are produced by somatic cells of embryos homozygous for the Steel mutation, and one of them is a ligand of the cKit proto-oncogene.

Primary germ cells of mammals and humans have an extragonadal origin and are the source of clonal development of the germ cell line. The origin of the primordial germ cell line, as well as all embryonic tissues and the extraembryonic mesoderm, is the epiblast (primary ectoderm) of early embryos, which has a mosaic structural organization. Using the method of microsurgical removal of various parts of the early embryo, a localization zone in the epiblast of the clone of committed precursors of primordial germ cells was established. Using rhodamine dextran, which was used as a cell marker, it was established that the precursors of primordial germ cells are localized in the proximal region of the epiblast, near the extraembryonic ectoderm. The primordial germ cell line arises from a 45-cell clone, the allocation of which occurs at the very beginning of gastrulation. Then the clone segregates, and during gastrulation the primary germ cells enter the extraembryonic mesoderm and are found at the base of the allantois rudiment, behind the primary streak. From there the primary germ cells migrate towards the ventral part of the hindgut endoderm and then actively move along the mesentery, populating the genital ridges at the end of migration. During migration, as well as in the first 2-3 days of localization in the gonad rudiment, the primary germ cells actively proliferate and undergo eight replicative cycles. If at the beginning of migration there are about 50 primary germ cells, then in the genital ridges of mouse embryos of twelve days of development the number of primary germ cells exceeds 25,000.

The functional similarity of ESCs and primordial germ cells is evidenced by the complete integration of the latter into the blastocyst with the replacement of the internal cell mass and subsequent full-fledged development of the embryo, the tissues of which consist only of the descendants of the primordial germ cells. In other properties, mouse primordial germ cells also turned out to be identical to ESCs, demonstrating the ability to differentiate in a variety of directions, to form embryoid bodies in vitro, and to form teratomas in vivo when administered subcutaneously to immunodeficient mice, resembling spontaneous testicular teratomas in 129/ter mice.

It was found that when LIF, membrane-bound and soluble SIF are added to the medium, isolated primary germ cells of 8-day-old mouse embryos survive and reproduce in the culture for 4 days, but then die. Moreover, the period when death of primary germ cells is observed in the culture coincides with the stage of development of mouse embryos (12.5-13.5 days) when female primary germ cells enter meiosis in the rudiments of the gonads, and mitotic divisions are blocked in male primary germ cells. However, if not only the growth factors LIF and SIF, but also FGF2 are added to the medium, the primary germ cells continue to proliferate, and colonies of cells capable of multiplying even after the removal of growth factors (SIF and FGF) from the medium are formed in the subcultures. Such cells can be cultured for a long time on a substrate of embryonic fibroblasts without adding the soluble growth factor LIF. It was proposed to call these stable cell lines obtained from primordial germ cells embryonic germ cells. This term is not at all successful, since it is impossible to obtain embryonic germ cells capable of performing subsequent stages of oogenesis or spermatogenesis when culturing EG cells. This is due to the fact that EG cell lines, although they originate from primordial germ cells, but, acquiring the properties of embryonic pluripotent stem cells in culture, lose the ability to commit to germ lineages. In other words, primordial germ cells, when cultivated, lose the properties of gamete precursors and are transformed into ESC-like pluripotent cells.

It has been noted that teratomas do not arise when EG cells are introduced into immunodeficient mice. It is assumed that the loss of the ability of human EG cells to give rise to teratomas is due to the fact that these lines were not created directly from cultured primary germ cells, but were obtained from cells isolated from embryoid bodies. Therefore, it is possible that they are descendants of pluripotent, but already committed cells.

It should be noted that there are fundamental differences between EG cells and primordial germ cells. The latter do not allow obtaining chimeric mouse embryos, which indicates the lack of ability of primordial germ cells to integrate into the inner cell mass or trophectoderm. The characteristics of the primordial germ cell population are more similar to committed lines of somatic cells of later embryos, the introduction of which into the blastocyst also does not lead to the formation of chimeric embryos.

Modification of the technique of culturing embryoid bodies obtained by aggregation of EG cells made it possible to obtain another population of pluripotent cells, called "embryoid body derived cells" (EBD cells), using selection on selective media. The ability of EBD cells to proliferate in culture for a long time made it possible to create stable cell lines of committed cells. Clones of cells expressing a wide range of mRNA and protein markers of specialized cells were obtained. This approach ultimately proved that human primary germ cells are pluripotent and differentiate in vitro into different cell types: neurons, neuroglia, vascular endothelium, hematopoietic cells, muscle and endodermal cells.

Alternative sources of embryonic stem cells

An alternative source of human ESC lines may be hybrid cells. Implantation into the uterus of pseudo-pregnant cows of a heterogeneous construct obtained by fusion by electroporation of somatic cells of the human fetus with a cow's egg from which the pronucleus is previously removed makes it possible to obtain an internal cell mass from an artificial embryo of pre-implantation stages of development. For this purpose, at the first stage a blastocyst is obtained from a cow's egg with a transplanted human cell nucleus.

At the second stage, an embryoblast is isolated from the blastocyst, and from it, ESCs are isolated using the Thomson method. It is noteworthy that the best results in isolating ESC lines using this method were obtained using nuclei of follicular cells or primary germ cells that remain in the human body in a state of hibernation. This is due to the fact that the nuclei of human cells transplanted into a cow's egg must have non-shortened telomeres and high telomease activity, which helps avoid premature aging of ESC clones obtained from a hybrid egg (Repin, 2001). It is known that the most important intracellular marker proteins of ESCs are Oct3, Oct4, Tcf, Groucho, which belong to the so-called chromatin silencer proteins. Silencers provide a particularly compact package of heterochromatin, which prevents the formation of euchromatin loops. Chromatin packaging mediated by these proteins correlates with the totipotency of the ESC genome. It has been established to date that mature bovine and human oocytes are the only type of specialized cells that contain high concentrations of silencer proteins in the cytoplasm. On this basis, a method was developed for obtaining hybrid ESCs by transferring somatic cell nuclei into enucleated bovine oocytes. Preliminary in vitro studies have shown that the cytoplasm of bovine oocytes restores the totipotency of the human somatic cell nuclei genome after 12-24 hours of cultivation.

Of particular interest are the data on the peculiarities of preimplantation development of human embryos, indicating a later replacement of totipotent cells by a population of pluripotent cells than in mice. A study of cellular transformations showed that trophoblast cells also arise from the cells of the inner cell mass of human blastocysts, in addition to ESCs, which indicates their total potency.

It is known that at the blastocyst stage, two differently committed cell populations arise. One of them forms the outer layer of the blastocyst - the trophectoderm, the derivatives of which are the trophoblast cells and other embryonic components of the placenta. The second population of cells is grouped into a dense mass contacting the inner surface of the trophectoderm. The derivatives of the population of cells of the inner cell mass are all tissues and rudiments of the organs of the embryo. At the stage of the late blastocyst, the extraembryonic endoderm is formed from the inner cell mass and the epiblast (primary ectoderm) is formed. In this case, the epiblast cells retain pluripotency, while the ability to differentiate cells of the extraembryonic endoderm is limited.

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Obtaining human embryonic stem cells

Until recently, it was believed that it was impossible to obtain ESCs from trophoblast. However, a line of diploid trophectoderm stem cells isolated from a blastocyst proliferates and transforms into stem cells in a medium containing FGF2 and heparin instead of LIF. If FGF2 is removed from the medium, trophectoderm cells stop multiplying, chromosome endoreduplication begins in them, and trophectoderm cellular elements gradually transform into giant trophoblast cells. Probably, LIF does not stimulate trophectoderm cell proliferation due to the fact that FGF2 triggers a different trans-signaling mechanism, since FGF2, binding to the plasma receptor (FGFR2), activates MAP kinases in the cytoplasm - ERK1 and ERK2. Consequently, when one signaling pathway (LIF - gpl30 - JAK kinase - STAT3) is switched on in blastocyst cells, the cells of the inner cell mass are transformed into pluripotent ESCs, and when the second mechanism of transmembrane signal transduction (FGF2 - FGFR2 - MAP kinase ERK1/ERK2) is activated, trophectoderm stem cells are formed in the blastocyst. The choice of the signaling pathway, in turn, depends on the activity of the oct4 gene. This gene, which belongs to the POU domain, is located in the t locus of autosome 17 and is expressed during oogenesis, during the cleavage period, as well as in the cells of the inner cell mass of the blastocyst and in the primary germ cells. The functional role of the oct4 gene is to encode a transcription factor necessary for the emergence of pluripotent cells, their differentiation and dedifferentiation.

Expression of the oct4 gene in ESCs varies depending on the interaction of this transcription factor with cofactors. Directed regulation of oct4 expression in blastocysts showed that when its activity is reduced, half of the cells form trophectoderm, whereas when induced expression of oct4 increases, predominantly ESCs arise.

In the experiment, ESCs cannot be transferred into a line during the cultivation of totipotent blastomeres at the cleavage stage, as well as at the gastrulation stage and later stages of embryonic development. Mouse ESCs are usually isolated on the 3.5-4.5th day of pregnancy, which corresponds to the sixth (single-layer blastocyst) and seventh stages (two-layer blastocyst - early egg cylinder) of normal embryogenesis. Obviously, only in the preimplantation period do mouse embryos contain cell populations capable of transforming into ESCs. Consequently, the isolation of ESC lines is possible only at certain stages of embryogenesis. The zygote and blastomeres arising during cleavage are totipotent, from the point of view of the possibility of developing a viable embryo with embryonic membranes and placenta. The loss of total potency of germ cells begins at the late morula stage, when further commitment of blastomeres depends on their location. Early morula blastomeres retain totipotency, since experimental manipulations with changes in their localization, such as inversion of their location, do not prevent the development of a full-fledged embryo.

It has been established that the efficiency of isolating ESCs into a line is affected by the condition of blastocysts at the time of their explantation. The use of blastocysts after modeling a seven-day diapause in the reproductive tract of mice ovariectomized on the 3.5th day of pregnancy and given progesterone facilitates more successful isolation of embryonic stem cell lines. It is assumed that under such conditions the number of blastomeres forming the inner cell mass increases. It is also possible that the cell cycle is lengthened and most blastomeres enter the G0 phase.

In addition, the creation of stable pluripotent ESC lines depends on the genotype of the embryos: ESCs are fairly easily isolated from blastocysts of the 129 mouse line, they are much more difficult to obtain using CS7BL/6 mice, and it is virtually impossible to isolate an ESC line from blastocysts of CBA/Ca mice. Obviously, early embryos have some genetic features that affect the development of a pluripotent ESC line. Nevertheless, when culturing isolated epiblasts, as well as by selective selection of differentiating cells, ESC lines were nevertheless isolated from early embryos of CBA/Ca mice.

A proven standard technique for obtaining ESC lines from blastocysts is given in laboratory manuals on the technique of experiments with early embryos. Experimental ESC lines can also be obtained by culturing isolated epiblast (primary ectoderm) of 4.5-day-old mouse embryos using a rather complex microsurgical technique and modified culturing conditions. The labor intensity of this procedure is justified, since the frequency of ESC line formation in this case turned out to be significantly higher than in works with the inner cell mass of the blastocyst.

To isolate ESC lines, each clone is transferred to a microwell, an aggregate of 40-60 cells is grown, and then dispersed again. Multiple repetitions of this procedure allow us to obtain an immortalized ESC line with the maximum proliferation rate of normokaryotypic cells attached to plastic, which retain totipotency and high telomerase activity after 50-100 passages. In the process of maintaining ESC lines, the greatest danger is contamination of the medium or serum with bacterial endotoxins - even trace concentrations of endotoxin in the culture medium cause mass death of immature germ cells. With careful monitoring of linear growth and timely dispersion, ESCs in culture are capable of symmetrical division, in which both daughter cells remain pluripotent and capable of performing an unlimited number of cell cycles, maintaining a diploid karyotype and total potency.

Selection of a pure population of human ESCs can be performed after transfection of their genome with recombinant DNA molecules containing the gene encoding the synthesis of green fluorescent protein (GFP). GFP expression increases when ESCs are grown under conditions that support their proliferation, whereas with the onset of differentiation the expression level of this gene decreases, which allows selection of pure stable pluripotent cell lines on a selective medium. When culturing ESCs isolated using GFP selection, the frequency of colony formation increases many times over, since the powerful antiproliferative effect of differentiated cells is eliminated under the conditions of selection cultures.

The translation of human embryonic stem cells into a line is carried out using the method of their isolation from preimplantation embryos (at the stage of 80-120 cells), which remain after the in vitro fertilization procedure. For this purpose, artificially obtained “excess” embryos are mechanically dispersed in the Delbecco-Eagle medium. After labeling the cells with selective monoclonal antibodies with a fluorescent label, the embryoblast cells are isolated. The embryoblast is dispersed into individual cells using a dispase-collagenase mixture. The dissociated cells are grown in a special medium (80% Delbecco's medium + 20% fetal calf serum in the presence of 500 μg/ml IL-6, LIF and SCF) over a feeder monolayer of embryonic fibroblasts of the first 3 passages. In this case, the survival and proliferation of stem and progenitor cells is maintained due to the effect of IL-6, LIF and SCF. In such a medium, ESCs grow as suspension clones of unattached balled cells, which must be dissociated by soft, repeated pipetting. New clones appear in the suspended culture on the 5th-7th day. The maximum growth rate of ESCs is achieved by repeated dissociation of clones at the stage of 10-15 cells. Then, each clone is transferred to a microwell and grown to an aggregate of 40-50 cells. The procedure is repeated many times in passages, increasing the volume of the culture to a density of 5-10 million cells per 6-cm dish. Using such passaging, Thomson isolated 10 immortal clones of human ESCs, which after 100 passages retained high telomerase activity, the ability to proliferate vigorously, minimal phenotypic characteristics, and total potency with the ability to differentiate into any of 350 specialized cell lines derived from the ecto-, meso-, and endoderm. Differentiation of human ESCs began (upon medium change, addition of serum, and elimination of LIF) with cell attachment to the substrate, indicating the development of the cytoskeleton and expression of adhesion receptors. Importantly, with unlimited proliferation, human ESCs retained a normal karyotype.

The second method of isolating human ESC lines is based on the use of primary germ cells. Experimental studies have shown that E cells lines can be obtained from the genital folds of 12.5-day-old mouse embryos. However, in these cases the frequency of formation of progenitor cell lines was significantly lower than in experiments with earlier embryos. At the same time, primary germ cells from the gonads of mouse embryos of 13.5 days of gestational age are not capable of transforming into lines at all.

The first stable lines of pluripotent human EG cells were obtained from primary gonocytes isolated from the gonads of 5-9-week-old embryos. The isolated cells were cultured on a substrate of inactivated mouse embryonic fibroblasts in DMEM medium with fetal serum supplemented with mercaptoethanol, forskolin, and recombinant human growth factors (FGF and LIF). After 7-12 days, multicellular colonies appeared in the culture, corresponding to human EG cells by morphological features and molecular markers. After aggregation, these cells formed embryoid bodies, with further development of which specialized cells characteristic of derivatives of all three germ layers appeared. Over the course of 10-20 passages, EG cell lines retained a normal karyotype and did not lose pluripotency.

It has also been shown that the combined action of LIF, membrane-bound and soluble Steel factors, and TGF-b alters the developmental program of primordial germ cells. Instead of ceasing mitotic divisions and beginning to differentiate toward oogenesis or spermatogenesis, primordial germ cells continue to proliferate. After several additional mitotic cycles, they become similar to epiblast cells and, losing the properties of germ cell precursors, are transformed into pluripotent embryonic stem EG cells.

Thus, in 1998, immortalized lines of primordial germ cells were isolated for the first time from the genital rudiment of human fetal autopsy tissue. In human embryogenesis, primordial germ cells appear in the yolk sac in the 3rd week of development, and in the 4th-5th weeks, these cells migrate to the genital tubercle zone, where they form dormant populations of primary gonocytes. In an inactive state, primordial germ cells are preserved in the embryo until birth. Lines of primordial germ cells are isolated from the fetal genital tubercle of 5-9-week embryos, the extracted tissue of which is ex tempore treated with a mixture of collagenases types IV-V, hyaluronidase and DNase to increase the quantitative and qualitative yield of cells. Primordial germ cells in the tissue of the fetal genital tubercle are surrounded by stromal (mesenchymal) Sertoli cells. The functional purpose of Sertoli cells is to produce antiapoptotic factors (Fas ligand), mitogens, and immunosuppressants that protect germ cells from immune attack by the body. In addition, the stromal microenvironment of the genital tubercle plays an important role in the maturation of gametes. The isolated primary germ cells are planted in culture over a feeder stromal layer consisting of fetal fibroblasts of the first three passages. The most effective combination of mitogens is a complex consisting of LIF, FGF, and forskolin (a stimulator of cAMP formation). Proliferation of primary germ cells in vitro requires the addition of fetal serum, in the presence of which the reproduction of primary gonocytes in culture is accompanied by the formation of clones of spherical cells not attached to the substrate.

At the National Institutes of Health, USA, based on a summary of the existing data on methods for isolating human ESC lines from blastocysts, a preliminary conclusion was made that successful isolation of ESCs is most likely when culturing blastocysts with a well-formed inner cell mass (Stem cells: scientific progress and future research directions. Nat. Inst, of Health USA). From this point of view, the optimal source of ESCs for creating lines are human blastocysts on the 5th day of development, from which the trophectoderm should be carefully removed when isolating the inner cell mass. The isolated inner cell mass, consisting of 30-35 cells at this stage, should be cultured on a substrate of embryonic mouse fibroblasts, which is a decisive condition for the formation of ESC colonies in culture.

Analysis of phenotypic features of embryonic stem cells

Of particular interest is the interspecies comparative analysis of the phenotypic features of ESCs. It was found that human ESC colonies are dense clusters of flattened, epithelial-like cells, whereas mouse embryoid bodies consist of a loose conglomerate of rounded cells. In human ESCs, the nuclear-plasma ratio index is lower than in mouse ESCs. Embryonic stem cells of monkeys form flatter colonies of cells with uneven edges. Individual cells are easily visible in early clones of primate ESCs. Proliferating ESCs of all studied animal species do not express MHC class I and II molecules. At the same time, human ESCs give a positive reaction to TERA 1-60 and GCTM-2 antibodies, which indicates the presence of keratin/chondroitin sulfate proteoglycans on their surface, characteristic of embryo-(terato)-carcinoma stem cells. Expression of the oct4 gene in ESCs of all animal species suggests that, despite phenotypic differences, the same set of genes responsible for maintaining pluripotency is apparently activated in human and mouse ESCs (Peru, 2001). In addition, ESC lines isolated from rat, pig, rabbit, primate and cattle embryos 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 new look at the problem of xenotransplantation.

Unlike normal embryogenesis in vivo, proliferation of ESCs in vitro is not accompanied by the formation of germ layers and occurs against the background of blocking of homeotic Hoxgenes, i.e., without organogenesis. Since the segmentation genes do not function, it is impossible to reproduce such periods of embryogenesis as the laying of somites, embryo segmentation, formation of the yolk sac, allantois and other provisional organs and tissues in ESC culture. Cultured ESCs seem to have frozen at the beginning of the process of formation of 350 restriction lines of specialized cells. Thus, a clone of daughter progenitor cells and a centrally localized ESC represent only a model of an embryo, during the development of which different lines of specialized cells are simultaneously formed in different tissue regions, which, however, originate from common precursors. Despite the minimal level of receptors on the surface of ESCs, they retain the ability to carry out primitive morphogenetic processes, imitating the volumetric structures of an early embryo: a suspension of ESCs in culture aggregates and forms structures resembling blastocysts or even later embryos (egg cylinders). Such suspension aggregates are respectively called simple and complex embryoid bodies.

In mixed differentiation, early genes of the ectoderm (oct3, fgf-5, nodal), endoderm (gata-4), mesoderm (brachyury), cardiogenic mesoderm (pkh-2.5), neural tube (msx3) and hematopoiesis (elkf) are simultaneously expressed in different cells of one embryoid body. Using various combinations of growth factors and cytokines for targeted action on the formation of germ layer cells in vitro, it was possible in a number of cases to obtain embryoid bodies in which ectoderm or mesoderm genes were preferentially expressed, which opens the way to modeling gastrulation and the initial phases of organogenesis.

Clonal growth of ESCs is evidence of asymmetric cell division, in which only one ESC in the center of the clone retains unlimited proliferation potential, while the second daughter cell generates a generation of progenitor cells that are already undergoing differentiation. Therefore, the clone proliferation rate 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 apoptotic mechanisms. These events determine the fate of the clone: if the proliferation rate exceeds the rate of migration and apoptotic cell death, the clone continues to increase in size, stabilization occurs when the apoptosis rate and the rate of new cell formation are equal, and regression occurs when the ratio of these processes is inverse. Progenitor cells divide symmetrically, i.e., both daughter cells subsequently differentiate into mature specialized cell lines. The ratio of ESCs to progenitor cells varies, but the number of ESCs is always 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 the culture. Disaggregation of clones at the stage of 10-12 cells turned out to be most effective for obtaining the maximum yield of ESCs. The direction and degree of differentiation of cells in the embryoid body depend on their location. The outer cells of the embryoid body do not express the oct4 gene and undergo differentiation into cells of the primary endoderm, from which epithelial-like cells of the parietal and visceral extraembryonic endoderm are subsequently formed. The inner cells of the embryoid body express the oct4 gene and retain pluripotency for 48 hours of cultivation. However, the culture is then morphologically restructured into an epithelial monolayer and expression of genes controlling the development of the primary ectoderm begins. Next, the process of total disordered cytodifferentiation begins with the appearance of various cell types that are derivatives of all three germ layers. In the process of spontaneous differentiation of the cells of the embryoid body, aggregates with endoderm markers in the form of fragments (cysts) of the yolk sac are the first to appear. Then, angioblasts and endothelial cells of the growing capillaries appear in these structures. At the final stages of spontaneous differentiation, various terminally differentiated cells develop from the internal cells of the embryoid body, including neurons, glial elements, cardiomyocytes, macrophages, and erythrocytes. To a certain extent (taking into account the spatial inversion of the formation of the germinal tissue layers), using embryoid bodies, it is possible to study morphogenetic processes in vitro and analyze the molecular mechanisms of the initial periods of embryonic cytodifferentiation, as well as to establish the role of specific genes in the implementation of these processes.

Thus, within the clone there are cells in which different genetic development programs have been discovered - ESCs, early progenitors and differentiating progenitor populations. Cultivation of ESCs by the hanging drop or mass culture methods without a feeder layer and without adding LIF to the medium inevitably leads to the formation of embryoid bodies. The morphology of the cells of the outer and inner layers of embryoid bodies differs. The outer layer consists of large, branched cells. Their surface facing the environment is covered with numerous microvilli. The outer layer of cells is separated from the inner layer by a basal membrane resembling Reichert's membrane, while the cells of the inner layer of embryoid bodies are columnar epithelium. Morphologically, the inner layer, although it contains many dividing cells, more closely resembles undifferentiated colonies of ESCs.

Characteristics of human embryonic stem cells

The absence of parenchymatous-mesenchymal signaling interactions against the background of homeosis gene blocking leads to disordered growth of ESCs in culture, since this disrupts the formation and development of the infrastructure of provisional organs. Disorganized growth and disordered spontaneous differentiation of ESCs in culture are due to the absence of mesenchymal marking of the stromal framework of future organs: in vitro, it is quite possible to form millions of hepatocytes, but it is impossible to obtain a single liver lobule that includes such structural and functional elements as sinuses, Disse spaces, and Kupffer cells.

It is believed that the pluripotency of ESCs is realized exclusively in embryogenesis with the formation of tissues and organs of the embryo, while the placenta and umbilical cord are derivatives of the trophoblast. ESCs enclosed in the trophectodermal membrane sequentially generate clones of provisional cells that implement the development program through the combinatorial mRNA of the volumetric topographic matrix of Nokhteyov, which predetermine the spatial arrangement, shape and size, the number of cells of provisional and definitive organs, as well as the assembly of the parenchyma into structural and functional units. At the same time, ESCs remain the only type of cells in which the molecular mechanism for the implementation of their potencies is completely disconnected from the genetic development program, and the ESCs themselves are deprived of the ability to interact with other cells due to the blocking of both receptor perceptions and transsignaling systems. However, adequate activation of ESCs leads to a step-by-step development of the embryogenesis program, ending with the birth of a fully formed organism, consisting of billions of cells, ready for extrauterine life. On this short-term but unimaginably long-term path in cellular space, errors inevitably arise both in the molecular mechanisms that ensure the vital activity of cells and in the programs that control their proliferation, differentiation and specialization. Therefore, in modern pharmacogenomics, diseases of the molecular structure and diseases of cell programming are considered separately. Moreover, the action of most new drugs is aimed at correcting the programs of differentiation, proliferation and organogenesis, as well as the regeneration of organs and tissues. In an adult organism, ESCs make it possible to control the behavior of stem/progenitor cells transplanted into the brain, liver, spleen, bone marrow, and other human organs to restore damaged parenchyma of the recipient's organs by differentiating and specializing donor cells on the preserved mesenchymal matrix. In essence, the totipotency program begins to be realized at the level of the oocyte, zygote, and blastomere genomes; however, these cells have not yet been cloned and passaged in quantities necessary for the needs of experimental and practical medicine. Therefore, ESCs remain a unique source of genetic information containing codes for a three-dimensional map of the embryo and codes for linear restriction of specialized cell lines during gastrulation.

The virtually limitless regenerative potential of ESCs is due to the fact that their genome, unlike the genetic apparatus of differentiated somatic cells, retains pluripotency. One of the manifestations of the dormant state of the genetic information embedded in ESCs is the so-called minimal phenotype - a limited number of receptors are expressed on the surface of ESCs, and, accordingly, very few trans-signaling programs are deployed for the interaction of the cell's nuclear apparatus with its microenvironment. Against the background of 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 connection of the cell with the surrounding microenvironment. Using the method of serial analysis of gene expression, it was shown that with the common work of the main functional boxes of the genome regulating energetics and metabolism in somatic cells and ESCs, the latter have a very low level of mRNA of receptors, G proteins, secondary messengers, transcriptases, expression and repression cofactors, i.e., the entire system of transmembrane transmission of the regulatory signal to the cell. This is due to the absence or very low expression of transsignaling genes. During the period of induced differentiation in the ESC genome, 18 functioning genes synchronously stop working against the background of activation of 61 transsignaling genes controlling the synthesis of cell adhesion receptors, components of the extracellular matrix, restriction transcriptases and messenger elements of the signal transmission system to the nuclear apparatus from the receptors of the cell plasma membrane. At the same time, the expression of genes responsible for the synthesis of silencer proteins is blocked, as well as co-inhibitors of gene expression that ensure the totipotency of the ESC genome.

Gene markers have been found for cells of all three germ layers. Identification of the ectodermal cell layer is carried out by the expression of the nodal, oct3 and fgf-5 genes, mesoderm cells - by the brachyury, zeta-globin genes, endoderm - by the expression of the gata-4 gene. In normal embryogenesis during the gastrulation period, active migration of immature populations of stem and progenitor cells is observed, locally marking the development zones of the facial bones of the skull, some parts of the brain, peripheral nervous system, cardiac conduction system and thymus, the tissues of which are formed from clones of migrant cells. Marking of cells by early genes of the germ layers facilitates the task of topographic analysis of the processes of migration of precursor cells in the developing embryo. It has been established, in particular, that in aggregates of P19 embryocarcinoma cells, the expression of the first mesoderm gene brachyury begins during the period of decreased expression of the genes of tissue plasminogen activator, a-fetoprotein, keratin 8 and keratin 19, which are markers of the first migrating mesoderm populations. Consequently, the formation of tissues of mesodermal origin begins only after the completion of the process of point migration and dispersal of mesodermal progenitor cells.

Despite extremely limited phenotypic features and the absence of most trans-signaling units, ESCs still express some receptor molecules that can be used for their identification. It is noteworthy that ESC marker antigens in humans and primates turned out to be common. Most often, labeled antibodies to membrane-bound antigens SSEA-3, SSEA-4 (unique lipid antigens representing a complex of glycolipid GL7 with sialic acid), as well as high-polymer glycoproteins TRA-1-81, TRA-1-60 are used for ESC labeling. In addition, ESCs express the specific embryonic antigen SSEA-1 and endogenous alkaline phosphatase, as well as the specific transcription factor Oct4. The latter is necessary for maintaining 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 replication 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 many years of attempts to cultivate ESCs outside the body were successful and the first cultures of stem cells isolated from mouse blastocysts and cultures of primary germ cells were obtained, a stage of research on the pluripotent potential of ESCs began when they were introduced into embryos at early stages of development. It was shown that at the morula and blastocyst stages, ESCs are capable of forming chimeric embryos in which the descendants of donor ESCs are detected in all somatic tissues and even in gametes. Thus, in developmental biology, using ESCs, a “bridge” was established between experimental studies in vivo and in vitro, which significantly expanded the possibilities for studying the processes of laying down primary tissues and organs, their differentiation and embryonic organogenesis.

It is clearly established that in vivo during embryogenesis ESCs are integrated into the cell mass of the early embryo, and their derivatives are found in all organs and tissues. ESCs colonize a line of germ cells in the chimeric embryo, the descendants of which form full-fledged eggs and sperm. Embryonic stem cells are clonogenic - a single ESC is capable of creating a genetically identical colony of cells with molecular markers, which include expression of the oct4 gene and alkaline phosphatase, high telomerase activity, and expression of certain embryonic antigens.

To study the mechanisms of embryogenesis using ESCs, a method of morula chimerization has been developed by creating a biological construct, outside of which there is a layer of recipient tetraploid blastomeres, and donor ESCs are introduced inside. Thus, the trophoblast is formed from the descendants of the recipient's tetraploid blastomeres, which ensures implantation and placentation, and donor ESCs act as an internal cell mass from which the body of a viable embryo and a line of primary progenitor germ cells are formed. The research value of ESCs lies not only in the fact that pluripotency is preserved during in vitro manipulations with their genome, but also in the fact that the ability of ESCs to participate in the formation of primary germ cells of a chimeric embryo is preserved. It has been shown that the descendants of just one genetically modified ESC populate all primary rudiments and developing tissues of a chimeric embryo obtained by aggregation or cocultivation of this cell with an 8-cell embryo. When ESCs transfected with the green fluorescent protein gene were transplanted into the morula of mice, fluorescent descendants of this cell were found in all studied tissues of the developing embryo (Shimada, 1999). Transplantation of ESCs into the morula allows the creation of viable mice whose organism consists only of the descendants of the donor ESC, which opens up prospects for various options for therapeutic cloning. This methodological approach is now successfully used to study problems of developmental biology, in particular, it is used to analyze the mechanisms of genetic inactivation of the X chromosome or epigenetic instability of ESCs. Transplantation of ESCs into an early embryo is also used in agricultural biotechnology, as well as in gene therapy experiments.

Transplantation of genetically modified ESCs is used to test target cells of mutant genes. In vitro cultured ESCs are used in biotechnology to create knockout mice. For this purpose, the gene to be studied is removed from the ESCs by homologous recombination (knockout) and cells lacking this gene are isolated on selective media. Knockout ESCs are then introduced into the blastocyst or aggregated with morula blastomeres. The chimeric early embryos obtained in this way are transplanted into recipient females, and individuals with gametes nullizygous for a given gene are selected from among the newborn mice. This technology has been used to create many lines of knockout mice, which are widely used in experimental biology and experimental medicine. Such biological models are used to study the significance of certain genes in embryonic development, as well as their role in the mechanisms of human diseases and pathological conditions. In addition, knockout animal lines are used in the preclinical testing of new gene therapy methods. For example, by transfecting the normal allele of a mutant gene into the ESC genome, it is possible to effectively correct a mutation affecting the hematopoietic system. The introduction of foreign genes into ESCs allows for the rapid creation of lines of homozygous transgenic laboratory animals. However, it should be noted that the technique of targeted recombination gene deletion has so far only been reliably developed in relation to mouse ESCs. Using double knockout mouse ESCs, the functional role of the gene cluster region on chromosome 7 (a copy of the genomic region on human chromosome 19) and the proximal region of chromosome 11 (a copy of human chromosome 5g) was established - deletion of these genes in mouse ESCs made it possible to evaluate the function of their analogs in humans.

The possibilities of studying the function of human embryogenesis genes have expanded, transfection of which into the genome of ESCs of laboratory animals has made it possible, in particular, to clarify the role of the crypto gene in the formation and development of the cardiogenic mesoderm, the pax-6 gene - in eye embryogenesis. The first maps of gene expression in immature proliferating ESCs of teratocarcinoma and blastocysts of mice are being compiled, and suppressive repression of transsignaling genes in ESCs has been confirmed. A combination of 60-80 mutant ESCs and 20-30 cells of normal preimplantation mouse embryos leads to the development of chimeric embryos in which organ primordia consist of donor and recipient cells, which makes it possible to clarify the role of unknown genes in gastrulation and organogenesis. The functional map of genes of developing mouse embryos was supplemented by information on the role of the sf-1 gene in the formation of the adrenal gland and gonads, the wt-1 gene in the formation of the kidney, genes of the myoD family in the formation of skeletal muscle, and genes of the gata-1-4 family in the restriction maturation of the erythropoiesis and lymphopoiesis rudiments.

Directed switching off of maternal and paternal alleles of genes in ESCs using vector recombinases allowed to clarify the functions of various genes in the early period of embryogenesis, and the technology of directed transfer of unknown human genes into mouse ESCs contributes to the discovery of new mutant genes responsible for the development of severe hereditary pathology. Using the knockout method, the obligatory significance of some genes for the formation of embryonic tissues was determined: gata-4 - for the myocardium, gata-1 - for the erythroid lineage of hematopoietic tissue, myoD - for skeletal muscles, brachyury - for the mesoderm, restriction transcriptases hnf3 and hnf4 - for liver stem cells, rag-2 - for the formation of T- and B-lymphocyte clones (Repin, 2001). Double deletion of genes in ESCs has opened access to the study of the functional role of germ layer genes, segmentation and homeosis, and ESC transplantation has made it possible to obtain viable interspecies hybrid embryos. Using an improved technique for transplanting a single donor ESC into an 8-cell embryo, the fact of chimerization at the cellular level of many organs of the recipient embryo has been proven. It should be noted that cell sprouts of human tissue have been found in the organs of recipient mice after the introduction of human hematopoietic stem cells into the blastocyst. It has been established that pluripotent ESCs circulate in the blood of mouse embryos during the period of organ formation. It is possible that their biological function lies in the embryonic organization of the future immune system. With the help of ESCs, adequate models of human genetic pathology have been reproduced in laboratory conditions: double knockout of the dystrophin gene models Duchenne muscular dystrophy in mice, and the shutdown of the atm gene (control of chromatin signal kinase synthesis) - ataxia-telangectasia. In this fatal hereditary disease in children, degeneration of Purkinje cells in the cerebellum develops due to defects in DNA reparation, which is accompanied by involution of the thymus due to the death of proliferating cells. The clinical picture, pathophysiology and pathomorphology of ataxia-telangectasia, reproduced by introducing pathological genetic information into ESCs, in chimera mice correspond to those in humans. In addition to ataxia-telangectasia, using ESCs and knockout mice, experimental models of some hereditary homozygous human diseases associated with pathology of carbohydrate and lipid metabolism, amino acid catabolism, and copper and bilirubin excretion have been developed, which has significantly expanded the capabilities of experimental medicine at the stage of preclinical testing of new methods for treating the corresponding human diseases.

[ 12 ], [ 13 ], [ 14 ], [ 15 ]

Use of stem cell cytohybrids

Hybrid cells obtained by fusing ESCs with somatic cells are an adequate and promising model for studying the pluripotency of stem cells and reprogramming the chromosomes of differentiated cells. Cytohybrids obtained by fusing ESCs with differentiated cells of an adult animal make it possible to study the relationships between genomes of different “ages”: a unique situation arises when homologous chromosomes originating from cells at different stages of differentiation and different degrees of maturity are located in the same nucleus, where they can easily exchange trans-acting regulatory signals. It is difficult to predict how the cisregulatory epigenetic systems of homologous chromosomes, formed during individual development, will react to the influence of trans-acting signals emanating from embryonic related genomes. In addition, segregation of parental chromosomes occurs in hybrid cells, which allows us to study the interaction of genomes at the level of individual chromosomes, that is, to potentially identify the participation of specific chromosomes in maintaining pluripotency or, conversely, the exit to differentiation.

Cytohybrids obtained by fusing pluripotent teratocarcinoma and differentiated somatic cells were used as the first experimental model for studying the interaction of genomes with different “developmental histories”. In some cases, such hybrid cells retained pluripotent properties at a fairly high level. In particular, in vivo teratocarcinoma-somatic hybrid cells induced the development of true teratomas containing derivatives of all three germ layers, and in vitro in suspension cultures they formed embryoid bodies. Even in interspecific cytohybrids of this type, the presence of embryonic antigens was noted in cases where the somatic partners in the fusion with teratocarcinoma cells were lymphocytes or thymocytes. It is noteworthy that cytohybrids created by fusing teratocarcinoma cells with fibroblasts corresponded to fibroblasts in phenotype.

The most important established fact is that in teratocarcinoma-somatic hybrid cells, signs of reprogramming of the differentiated cell genome appeared, which was characterized by reactivation of either individual genes or the inactive X chromosome of the somatic partner. Thus, the results of studies on cytohybrids of the teratocarcinoma-somatic cell type indicate that pluripotency is often preserved in hybrid cells and there are signs of reprogramming of the somatic partner genome.

In experiments on obtaining intraspecific embryonic hybrid cells by fusing mouse ESCs with adult splenocytes, the characteristics of such cytohybrids were studied, segregation of parental chromosomes was analyzed, and the pluripotency of the hybrid genome was assessed. Intraspecific hybrid cells obtained by fusing teratocarcinoma cells with somatic cells are usually characterized by a low level of chromosome segregation with a tetraploid or near-tetraploid karyotype. A similar chromosomal composition was observed in cytohybrids during the fusing of primary germ cells with lymphocytes. At the same time, interspecific hybrid cells obtained by fusing mouse teratocarcinoma cells with mink lymphocytes showed intense segregation of the chromosomes of the somatic partner.

A qualitatively new stage in the study of segregation of parental chromosomes in intraspecific hybrids began after the development of a method for analyzing microsatellites using the polymerase chain reaction, thanks to which several hundred markers were found for each mouse chromosome, allowing reliable discrimination of any pair of homologous chromosomes in hybrid cells.

By fusing ESCs (HM-1 cells deficient in hypoxanthine phosphoribosyltransferase activity, 2n = 40, XY, isolated from blastocysts of 129/01a mice) with splenocytes of the congenic DD/c mice, it was possible to obtain a set of hybrid clones morphologically similar to ESCs. All clones were isolated on a selective medium in which only cells with active hypoxanthine phosphoribosyltransferase can grow. Electrophoretic analysis showed that all clones had an allelic variant of hypoxanthine phosphoribosyltransferase characteristic of DD/c mice. Cytogenetic analysis showed that three of the four hybrid clones 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, smaller one, was diploid.

Microsatellite analysis, which allows discrimination of any pair of homologous chromosomes of mice 129/01a and DD/c, in hybrid clones with a near-diploid set showed that in two clones there was a clear preferential elimination of autosomes of the somatic partner. Most autosomes in clones HESS2 and HESS3 had markers of the 129/01a line, i.e., the pluripotent partner. The exception were chromosomes 1 and I: in clones HESS2 and HESS3, along with markers of HM-1 cells, markers of the somatic partner were present in small quantities. Such results may reflect incomplete segregation of chromosomes 1 and I of the somatic partner and are consistent with cytogenetic data that trisomy for these chromosomes is observed in 30-40% of cells of clones HESS2 and HESS3. Clone HESS4 differed significantly in its chromosomal composition: many autosomes in this clone originated from the ESC genome (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 homologs of both parents. The quantitative ratio of microsatellites marking these homologous chromosomes was approximately 1:1. This allowed the authors to assume that one homolog originated from the ESC genome, and the other from differentiated cells. In some subclones of clone HESS4, only markers of chromosomes 18 and 19 of the somatic partner were present. The obtained results indicate that in the cells of the HESS4 clone, in addition to the segregation of the chromosomes of the somatic partner, the elimination of one or both homologues of the above-listed chromosomes of the pluripotent genome occurred, that is, there was a bilateral segregation of the chromosomes of both parents - a very unusual phenomenon, since segregation of the chromosomes of only one of the parents is characteristic of cytohybrids.

In addition, after the 20th passage, all clones of hybrid cells contained only markers of the somatic partner's X chromosome, i.e., the ESC X chromosome was replaced by the somatic partner's X chromosome in the clones. This important fact is confirmed by in situ hybridization data using an 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 (before the 15th passage), according to cytogenetic data, many cells contained two X chromosomes. Therefore, the use of selective media allows manipulating the chromosomal composition of hybrid cells and selectively selecting clones carrying single chromosomes of the somatic partner against the background of the ESC genome.

Since a unique feature of the cytohybrid genome is the localization of the parental genomes in one nucleus, the question naturally arises about the preservation of the pluripotent properties of the embryonic genome in ESC-somatic cell hybrids under conditions of its close contact with the genome of a differentiated cell. Morphologically, the cytohybrids of ESCs and somatic cells were similar to the parental ESC line. Evaluation of pluripotency showed that all clones with a near-diploid set of chromosomes were capable of forming embryoid bodies in suspension cultures, in which derivatives of three germ layers were present.

Most hybrid cells contained the ECMA-7 antigen, a marker characteristic of early mouse embryos, and also had high alkaline phosphatase activity. The most convincing data on the high pluripotent properties of hybrid cells were obtained in experiments on obtaining a series of injection chimeras involving hybrid cells of the HESS2 clone. Analysis of biochemical markers showed that the descendants of the donor hybrid cells were present in most tissues of the chimeras. Therefore, hybrid cells obtained by fusing ESCs and somatic differentiated cells retain pluripotency at a high level, including the ability to form chimeras when injected into the blastocyst cavity.

Clones HESS2 and HESS4 differed significantly in the composition of parental chromosomes, but had similar pluripotent properties. It could be assumed that pluripotency in the hybrid genome manifests itself as a dominant trait, 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, then it can be expected that the elimination of some chromosomes of the pluripotent partner from the genome of hybrid cells will not be accompanied by a change in their pluripotent status. In this case, the analysis of segregation of parental chromosomes in embryonic hybrid cells would allow us to closely approach the identification of chromosomes responsible for the control of pluripotency of embryonic cells.

O. Serov et al. (2001) did not find any offspring among 50 offspring obtained by crossing chimeras with normal mice that had the 129/01a mouse genotype and carried the X chromosome of DD mice. The authors believe that this is due to a decrease in 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 imbalance in sex chromosomes (XXY were observed in cells up to the 15th passage) in hybrid cells during meiosis. It is known that XXY cells are unable to undergo meiosis and form gametes. Trisomy can also cause a decrease in the proliferative activity of hybrid cells, as a result of which the selective advantage in the development of chimeras may belong to the cells of the recipient embryo. It follows that for an adequate assessment of 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 of the authors follows from the analysis of the expression of the hprt gene (an X chromosome marker) in chimeras: the presence of the allelic variant of hprt of DD/c mice was detected in all analyzed chimeric tissues. It is appropriate 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 hybrid cells means that it has become its obligate component and the genome does not discriminate it from the Y chromosome of the pluripotent partner.

Summarizing the results of the analysis of the interaction of the somatic and pluripotent genomes in hybrid embryonic cells, the authors conclude that in some cytohybrids pluripotency is manifested as a dominant trait. The hybrid genome is capable of reprogramming individual chromosomes of differentiated cells, which, however, does not exclude the possibility of a reverse effect of the somatic genome on the pluripotency of the embryonic genome. When culturing hybrid cells, induction of differentiation occurs significantly more often than in the original parental line of ESCs HM-1. A similar effect is observed during the formation of primary colonies: many primary colonies of embryonic hybrid cells differentiate at early stages of formation with large losses of clones during their selection and reproduction.

Thus, cytohybrids created by the fusion of ESCs with somatic cells, despite close contact with the genome of differentiated cells, retain pluripotency as a unique property of the embryonic genome. Moreover, in such hybrid cells, reprogramming of individual chromosomes originating from differentiated cells is possible. It remains unclear to what extent the pluripotent properties of the embryonic genome are retained in hybrid cells, in particular, their ability to participate in the formation of the germ line in chimeras. This requires obtaining embryonic hybrid cells with a normal karyotype. In any case, pluripotent embryonic hybrid cells can become a real genetic model for identifying chromosomes involved in maintaining pluripotency or controlling it, since bilateral segregation of parental chromosomes potentially provides such an opportunity.

No less attractive is the study of the phenomenon that O. Serov et al. (2001) define as “chromosomal memory”. In the hybrid genome, homologous chromosomes are in two alternative configurations: homologs of the somatic partner have once undergone differentiation, whereas in homologs of the pluripotent partner this process is just beginning. Consequently, the preservation of high pluripotent properties by hybrid cells indicates that the “pluripotent” configuration of ESC homologs is sufficiently stable in the hybrid genome, despite the influence of transacting factors emanating from the somatic partner. The above-described signs of reprogramming of homologous chromosomes of the differentiated genome during the development of chimeras do not exclude the possibility that at the first stages of formation and cultivation of cytohybrids in vitro they retain their status acquired during differentiation in vivo. According to recently obtained data, when embryonic hybrid cells are transferred to a non-selective environment, they show intensive elimination of chromosomes of only the somatic partner, i.e., the genome of hybrid cells easily discriminates homologues after in vitro cultivation 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 alternative - embryonic differentiation.

Therapeutic efficacy of embryonic stem cell transplantation

Before analyzing the therapeutic efficacy of transplantation of ESCs and their derivatives, we summarize the above material. The capabilities of ESCs in terms of full implementation of embryogenesis in vitro are insufficient, since developmental defects in this case are due to the absence of mesenchymal stem cells, which arise in the body autonomously and independently of ESCs. The genetic potential of ESCs is less than the genetic potential of the zygote, therefore ESCs are not used directly for cloning embryos. The unique biological potential of ESCs as the only cells in which development programs are fully deployed in a consistent implementation is used in studies of gene function. With the help of ESCs, the first combinations of signals activating the expression of early and late genes encoding the development of three germ layers are decoded. Preservation of the pluripotency of the ESC genome in vitro makes them a unique tool for reparative regeneration, capable of automatically replenishing cellular losses in case of damage to organs and tissues. In an ideal hypothetical scenario, it can be assumed that “... when transplanting donor ESCs, compactly packed programs are transferred into the recipient’s body, which, under favorable conditions, are realized in the construction of new tissue’7, capable of “... being effectively integrated into the recipient’s body at both the morphological and functional levels.”

Naturally, following the development of methods for monodifferentiation of ESCs, the in vivo study of the functional activity of cells obtained in vitro from one specialized clone began. A proliferating ESC clone generates populations of migrating progenitor cells that are truly capable of actively integrating into areas of recipient tissue damage, which is used in regenerative-plastic medicine. It has been established that transplantation of DOPA neurons into the substantia nigra reduces clinical manifestations in experimental hemiparkinsonism. Regional transplants of donor neural stem cells reduce the degree of motor disorders caused by trauma or contusion of the spinal cord and brain. The first positive results of stem cell transplantation in demyelinating diseases have also been obtained. It would seem that the regenerative-plastic potential of ESCs opens up unlimited possibilities for the use of cell transplantation in practical medicine. However, when transplanted into ectopic zones, ESCs inevitably transform into tumors. Teratomas are formed when ESCs are injected subcutaneously into immunodeficient mice. When ESC suspensions are transplanted under the capsule of the testicle in syngeneic mice, teratomas are also formed, consisting of different tissues, the cells of which are derivatives of all three germ layers. In such teratomas, processes of reduced organogenesis are extremely rare.

A number of studies provide information on the positive results of transplanting early ESC derivatives to animals with experimental pathology. Cellular neurotransplantation using ESC derivatives is being further developed in experiments and the first clinical trials to correct functional disorders in brain and spinal injuries, and to treat syringomyelia and multiple sclerosis (Repin, 2001). With the advent of the technique of in vitro neurogenesis from ESCs, instead of using embryonic brain tissue, methods are being developed for transplanting neurosphere derivatives obtained from embryonic nervous tissue cultures. Such transplant suspensions are significantly more homogeneous and contain committed precursors of neurons and neuroglia.

With regular addition of retinoic acid at a dose of 10 μg/ml to the culture medium for 6 weeks, more than 80% of postmitotic neurons are formed in the human embryo-(terato)-carcinoma line NTERA-2. Complete homogeneity of the neuronal population is achieved by flow sorting of mature neurons labeled with immunophenotypic markers, which allows getting 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 integrate into regional neural networks. In animals with experimental models of local CNS defects, neurotransplantation reduces the clinical manifestations of such human pathologies as the consequences of traumatic brain injury, stroke, demyelinating diseases, hereditary defects in the development of the cerebellum, diseases of lipid and polysaccharide deposition.

To optimize regeneration processes in degenerative diseases of the central nervous system, technologies are being developed for obtaining myelin-producing oligodendrocytes from ESCs. The first stage traditionally includes the proliferation of ESCs with the reproduction of the number of cells required for transplantation. At the second stage, targeted differentiation of cells into a population of myelin-producing oligodendrocyte precursors is carried out, which is controlled by selective marker antigens.

Certain prospects are opening up for the use of ESC derivatives for the development of methods for correcting immunodeficiencies caused by genetic defects in thymus maturation. In studies on knockout (rag 1) mice with an induced gene defect - a disruption of the recombination mechanism of V(D)J loci of TCR genes, leading to the loss of T-lymphocyte function, transplantation of early ESC derivatives into the thymus of animals restores the maturation of normal populations of immune clones responsible for cellular immunity. Clinical trials of transplantation of in vitro preformed ESCs for the treatment of fatal hereditary anemias in children are underway.

Objections to the rapid introduction of stem cell transplantation into the clinic are based on the 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 human stem cells, it is proposed to use a method of line selection based on molecular genetic analysis of short tandem DNA repeats. It is also necessary to test ESC lines for the presence of small chromosomal rearrangements and genetic mutations, the potential for which to occur under cell culture conditions is quite high. A thesis is put forward about the mandatory testing of the properties of all types of ESCs and regional pluripotent stem cells, since their reproduction in vitro can lead to the emergence of new characteristics that are not inherent in embryonic stem cells or definitive tissues. In particular, it is assumed that long-term cultivation in media with cytokines brings ESC lines closer to tumor cells, since they undergo similar changes in the pathways of cell cycle regulation with the acquisition of the ability to carry out an unlimited number of cell divisions. Some authors, based on the potential for tumor development, consider transplantation of early embryonic stem cell derivatives into humans to be reckless. In their opinion, it is much safer to use committed descendants of ESCs, i.e., lines of differentiated cell progenitors. However, at present, a reliable technique for obtaining stable human cell lines that differentiate in the desired direction has not yet been developed.

Thus, more and more data on the positive therapeutic effect of transplantation of human embryonic stem cell derivatives are appearing in the literature. However, many of these studies are being reviewed and criticized. Some researchers believe that the results of early clinical trials are preliminary in nature and only indicate that stem cells are capable of exerting a favorable effect on the clinical course of a particular disease. Therefore, it is necessary to obtain data on the remote results of cell transplantation. The stages of development of clinical neurotransplantology are cited as an argument. Indeed, at first, publications on the high efficiency of transplantation of embryonic brain fragments in Parkinson's disease prevailed in the literature, but then reports began to appear denying the therapeutic efficiency of embryonic or fetal nerve tissue transplanted into the brain of patients.

The first clinical trials were conducted to assess the safety of transplantation of neuroblasts derived from NTERA-2 teratocarcinoma ESCs, immature cells of which were subjected to proliferation in culture until accumulation of 100 million cell mass. Some of the cells obtained in this way were used to characterize the phenotype and determine cellular impurities, as well as to test for possible contamination with viruses and bacteria. LIF and the feeder layer of fetal stromal cells were removed from the culture medium, and conditions were created for targeted differentiation of ESCs into neuroblasts using a combination of cytokines and growth factors. Then the neuroblasts were purified from immature teratocarcinoma cells on a flow cell sorter. After secondary purification and phenotype characterization of transplanted cells, a suspension of neuroblasts (10-12 million) was injected into the basal nucleus of the patients' brains (in the seventh month after hemorrhagic stroke) using a special microcannula and syringe under stereotaxic and computed tomography control. Post-transplant one-year screening of the consequences of neuron transplantation into the stroke zone did not reveal any side or undesirable effects. Half of the patients showed 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 the absorption of fluorescently labeled 2-deoxyglucose, according to positron emission tomography, reached 18%, and in some patients - 35%.

However, the US National Institutes of Health conducted an independent study of the clinical effectiveness of neurotransplantation in patients with Parkinsonism. Patients in the first group were transplanted with sections of embryonic nerve tissue that produce dopamine, while the second group of patients underwent a sham operation. The results indicate zero clinical effectiveness of such neurotransplantation, despite the fact that dopamine-producing embryonic neurons survived in the recipients' brains. Moreover, 2 years after the transplantation of embryonic nerve tissue, 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 further development of the disease in these patients are ongoing.

Some authors associate the contradictory literature data on the assessment of the clinical effectiveness of neurotransplantation with different approaches to the selection of patient groups, inadequate choice of methods for objectively assessing their condition, and, most importantly, different periods of development of embryonic nervous tissue and different areas of the brain from which this tissue was obtained, different transplant sizes and methodological features of surgical intervention.

It should be noted that attempts at direct transplantation of pluripotent embryonic stem cells into the striatum region of the brain of rats with experimental hemiparkinsonism were accompanied by proliferation of ESCs and their differentiation into dopaminergic neurons. It should be assumed that newly formed neurons were effectively integrated into neural networks, since after ESC transplantation, correction of behavioral anomalies and motor asymmetry in the apomorphine test was observed. At the same time, some animals died due to the transformation of transplanted ESCs into brain tumors.

Experts from the National and Medical Academies of the USA, specialists from the National Institute of Health of the USA believe that the clinical potential of ESCs deserves the most serious attention, but 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 a comparative histological analysis of experimental teratomas obtained by transplantation of ESC suspension into the testis with teratomas formed as a result of transplantation of an early embryo, which also contains ESC, showed that ESC, regardless of their source of origin or interaction with certain surrounding cells, implement their tumorigenic potential in the same way. It has been proven that such teratomas have a clonal origin, since tumors consisting of derivatives of all three germ layers can arise from one ESC (Rega, 2001). It is noteworthy that when cloned ESCs with a normal karyotype were transplanted into immunodeficient mice, teratomas consisting of different types of differentiated somatic cells were also formed. These experimental data are impeccable proof of the clonal origin of teratomas. From the point of view of developmental biology, they indicate that not multiple committed progenitor cells, but a single pluripotent stem cell acts as a source of differentiated derivatives of all three germ layers that make up a teratoma. However, on the path to practical cell transplantation, the results of these studies are, if not a prohibitive, then a warning sign of potential danger, since inoculation of ESCs or primordial germ cells into various tissues of adult immunodeficient mice inevitably causes the development of tumors from transplanted stem cells. Neoplastic degeneration of ectopically transplanted ESCs is accompanied by the emergence of satellite populations of differentiated cells - due to partial differentiation of ESCs and progenitor clones into specialized lines. Interestingly, when ESCs are transplanted into skeletal muscles, neurons are most often formed next to teratocarcinoma cells. However, the introduction of ESCs into a dividing egg or blastocyst is accompanied by complete integration of the cells into the embryo without the formation of neoplastic elements. In this case, ESCs are integrated into virtually all organs and tissues of the embryo, including the genital primordium. Such allophene animals were first obtained by introducing teratocarcinoma 129 cells into early embryos at the 8-100 cell stages. In allophene mice, populations of heterogeneous cells derived from donor ESCs are integrated into the tissues of the bone marrow, intestine, skin, liver, and genitals, which makes it possible to create even interspecies cell chimeras in the experiment. The shorter the development period of the early embryo, the higher the percentage of cell chimerization, with the highest degree of chimerization observed in the hematopoietic system, skin, nervous system, liver, and small intestine of the allophene embryo. In an adult organism, tissues protected from the recipient's immune system by histohematic barriers are susceptible to chimerization: transplantation of primary germ cells into the testicular parenchyma is accompanied by the incorporation of donor stem cells into the recipient's germinal tissue layer. However,When transplanting ESCs into a blastocyst, the formation of chimeric rudiments of the sexual organs with the generation of donor primary germ cells does not occur. The pluripotency of ESCs, when special conditions are created, can also be used for cloning: transplanting mouse ESCs into an 8-16-cell mouse embryo, in which cellular mitoses are blocked by cytocalsin, promotes the implementation of normal embryogenesis with the development of an embryo from donor ESCs.

Therefore, an alternative to allogeneic ESC 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 lines of ESCs genetically identical to the donor of the somatic nucleus are then isolated. Technically, this idea is quite feasible, since the possibility of creating ESC lines from blastocysts obtained after transplantation of somatic nuclei into enucleated eggs has been repeatedly proven in experiments on laboratory animals (Nagy, 1990; Munsie, 2000). In particular, in mice homozygous for the rag2 gene mutation, fibroblasts obtained by culturing subepidermal tissue cells were used as donors of nuclei, which were transplanted into enucleated oocytes. After oocyte activation, the “zygote” was cultured until blastocyst formation, from the inner cell mass of which ESCs were isolated and transferred to a line of cells nullizygous for the mutant gene (rag2~/~). The mutation of one allelic gene was corrected in such ESCs by the method of homologous recombination. In the first series of experiments, embryoid bodies were obtained from ESCs with a recombinant restored gene, their cells were transfected with a recombinant retrovirus (HoxB4i/GFP) and, after reproduction, were injected into the vein of rag2~/~ mice. In the second series, tetraploid blastomeres were aggregated with genetically modified ESCs and transplanted into recipient females. The resulting immunocompetent mice served as bone marrow donors for transplantation into rag2~/~ mutant mice. In both series the result was positive: after 3-4 weeks mature normal myeloid and lymphoid cells capable of producing immunoglobulins were found in all mice. Thus, transplantation of somatic cell nuclei into oocytes 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 this direction of cell transplantation, in addition to bioethical problems, has its limitations. It is unclear how safe the transplantation of therapeutically cloned cells with a genotype identical to the genotype of a specific patient will be, since such cells can introduce mutations that create a predisposition to other diseases. Normal human eggs remain a difficult-to-access object, whereas even with the transplantation of somatic nuclei into enucleated animal eggs, only 15-25% of the constructed "zygotes" develop to the blastocyst stage. It is not yet determined how many blastocysts are required to obtain one line of pluripotent cloned ESCs. It is also worth noting the high level of financial costs associated with the complexity of the therapeutic cloning methodology.

In conclusion, it should be noted that in ESCs, the pluripotency of the genome with hypomethylated DNA is combined with high telomerase activity and a 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 ESCs in culture does not interfere with their differentiation into any specialized cell line of the organism when proliferation is stopped and appropriate regulatory signals are added. Restriction differentiation of ESCs into somatic cell lines in vitro is realized without the participation of the mesenchyme, bypassing the Nochteys, outside organogenesis and without embryo formation. Ectopic introduction of ESCs in vivo inevitably leads to the formation of teratocarcinomas. Transplantation of ESCs into a blastocyst or early embryo is accompanied by their integration with the embryonic tissues and stable chimerization of its organs.

Regenerative-plastic technologies based on cell transplantation are the point of intersection of 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 results of experimental studies prove the possibility of reprogramming stem cells with a targeted change in their properties, which opens up prospects for controlling the processes of cytodifferentiation using growth factors - for myocardial regeneration, restoration of CNS lesions and normalization of the function of the islet apparatus of the pancreas. However, for the widespread introduction of transplantation of ESC derivatives into practical medicine, it is necessary to study the properties of human stem cells in more detail and continue experiments with ESCs on experimental disease models.

Bioethical issues and the problem of rejection of an allogeneic cell transplant could be resolved by the discovered plasticity of the genome of regional stem cells of an adult organism. However, the initial information that when transplanting isolated and carefully characterized hematopoietic autologous cells into the liver, from which new hepatocytes are derived, integrating into the liver lobules, is 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 sprouts, and transplantation of brain neural stem cells into the bone marrow leads to the formation of hematopoietic sprouts with long-term donor myelo- and erythropoiesis. Consequently, pluripotent stem cells capable of reprogramming the genome to the potential of ESCs can persist in the organs of an adult organism.

The source of obtaining ESC for medical purposes remains the human embryo, which predetermines the inevitability of a new intersection of moral, ethical, legal and religious issues at the point of origin of human life. The discovery of ESC has given a powerful impetus to the resumption of tough discussions about where the line is between living cells and matter, being and personality. 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 adopt them. Each state, within the framework of its legislation, solves this problem independently. For their part, doctors around the world continue to try to take regenerative-plastic medicine beyond the scope of such discussions, primarily through the use of adult stem cell reserves rather than embryonic stem cells.

A little bit of history of the isolation of embryonic stem cells

Terato(embryo)carcinoma cells were isolated from spontaneously occurring testicular teratomas of 129/ter-Sv mice, spontaneous ovarian teratomas of Lt/Sv mice, and from teratomas derived from ectopically transplanted embryonic cells or tissues. Among the stable mouse terato(embryo)carcinoma cell lines obtained in this manner, some were pluripotent, others differentiated only into one specific cell type, and some were completely incapable of cytodifferentiation.

At one time, the focus was on studies whose results indicated the possibility of returning terato-(embryo)-carcinoma cells to a normal phenotype after their introduction into the tissues of a developing embryo, as well as work on the in vitro creation of genetically modified terato-(embryo)-carcinoma cells, with the help of which mutant mice were obtained for biological modeling of human hereditary pathology.

Conditioned suspension cultivation was used to isolate terato-(embryo)-carcinoma cell lines. In culture, terato-(embryo)-carcinoma cells, like ESCs, grow to form embryoid bodies and require mandatory dissociation for line transfer, maintaining pluripotency on a feeder layer of embryonic fibroblasts or during suspension cultivation in a conditioned medium. Cells of pluripotent terato-(embryo)-carcinoma lines are large, spherical, characterized by high alkaline phosphatase activity, form aggregates and are capable of multidirectional differentiation. When introduced into a blastocyst, they aggregate with the morula, which leads to the formation of chimeric embryos, in the composition of various organs and tissues of which derivatives of terato-(embryo)-carcinoma cells are found. However, the overwhelming majority of such chimeric embryos die in utero, and in the organs of surviving newborn chimeras, foreign cells are rarely detected and at low density. At the same time, the incidence of tumors (fibrosarcoma, rhabdomyosarcoma, other types of malignant tumors and pancreatic adenoma) increases sharply, and tumor degeneration often occurs during the period of intrauterine development of chimeric embryos.

Most terato-(embryo)-carcinoma cells in the microenvironment of normal embryonic cells almost naturally acquire malignant neoplastic characteristics. It is believed that irreversible malignancy is due to the activation of proto-oncogenes in the process of structural rearrangements. One of the exceptions are cells of the embryocarcinoma line SST3, obtained from mouse testicular teratomas (line 129/Sv-ter), which exhibit a high ability to integrate into the tissues and organs of the embryo without subsequent tumor formation in chimeric mice. Derivatives of terato-(embryo)-carcinoma cell lines in chimeric mice practically do not participate in the formation of primary gonocytes. Apparently, this is due to the high frequency of chromosomal aberrations characteristic of most terato-(embryo)-carcinoma lines, in the cells of which both aneuploidy and chromosomal abnormalities are observed.

Several stable lines of human terato-(embryo)-carcinoma cells characterized by pluripotency, high proliferative activity and the ability to differentiate during growth in cultures were obtained in laboratory conditions. In particular, the line of human terato-(embryo)-carcinoma cells NTERA-2 was used to study the mechanisms of neural cytodifferentiation. After transplantation of cells of this line into the subventricular region of the forebrain of newborn rats, their migration and neurogenesis were observed. Attempts were even made to transplant neurons obtained by culturing cells of the terato-(embryo)-carcinoma line NTERA-2 to patients with strokes, which, according to the authors, led to an improvement in the clinical course of the disease. At the same time, there were no cases of malignancy of transplanted cells of the terato-(embryo)-carcinoma line NTERA-2 in patients with stroke.

The first lines of undifferentiated pluripotent mouse embryonic stem cells were obtained in the early 1980s by Evans and Martin, who isolated them from the inner cell mass of the blastocyst - the embryoblast. The isolated ESC lines retained pluripotency and the ability to differentiate into various cell types under the influence of factors in a special culture medium for a long time.

The term “embryonic pluripotent stem cell” itself belongs to Leroy Stevens, who, while studying the effect of tobacco tar on the incidence of tumor development, drew attention to the spontaneous occurrence of testicular teratocarcinoma in linear (129/v) mice of the control group. The cells of testicular teratocarcinomas were characterized by a high proliferation rate, and in the presence of fluid from the abdominal cavity they spontaneously differentiated with the formation of neurons, keratinocytes, chondrocytes, cardiomyocytes, as well as hair and bone fragments, but without any signs of ordered cytoarchitecture of the corresponding tissue. When placed in culture, teratocarcinoma cells grew as pluripotent clones unattached to the substrate and formed embryoid bodies, after which they ceased dividing and underwent spontaneous disordered differentiation into neurons, glia, muscle cells, and cardiomyocytes. Stevens found that mouse teratocarcinoma 129/v contains less than 1% of cells capable of differentiating into a variety of specialized somatic lines, and the differentiation itself depends on the factors that affect them (the composition of the peritoneal fluid, products of mature cells or tissues added to the culture). Leroy Stevenson's hypothesis about the presence of embryonic progenitor cells of the germ line among teratocarcinoma cells was confirmed: a suspension of embryoblast cells from preimplantation embryos in the tissues of adult mice formed teratocarcinomas, and pure cell lines isolated from them after intraperitoneal administration to recipient animals differentiated into neurons, cardiomyocytes and other somatic cells derived from all three germ layers. In in vivo experiments, transplantation of ESCs (obtained from the embryoblast, but not the trophoblast) into mouse embryos of another line at the blastomere stages 8-32 resulted in the birth of chimeric animals (without the development of tumors), in whose organs sprouts of donor tissue were found. Chimerism was observed even in the germ cell line.

Primary progenitor germ cells isolated from the genital rudiment of a mouse embryo corresponded in morphology, immunological phenotype and functional characteristics to ESCs obtained by Stevenson from teratocarcinoma and embryoblast. In chimeras born after ESCs were introduced into a blastocyst, allophene morphogenesis of organs was characterized by a mosaic alternation of donor and recipient structural and functional units of the liver, lungs and kidneys. In some cases, the formation of intestinal crypts or liver lobules consisting of both recipient and donor cells was observed. However, morphogenesis was always realized according to the genetic program of the species to which the recipient belonged, and chimerism was limited exclusively to the cellular level.

It was then established that proliferation of ESCs without cytodifferentiation on a feeder layer of mesenchyme-derived cells (fetal fibroblasts) occurs with the obligatory presence of LIF in selective nutrient media, which selectively ensure the survival of only stem and progenitor cells, while the overwhelming majority of specialized cellular elements die. Using such methods, in 1998 James Thomson isolated five immortalized lines of embryonic stem cells from the inner cell mass of a human blastocyst. In the same year, John Gerhart developed a method for isolating immortal ESC lines from the genital tubercle of four- to five-week-old human embryos. Due to their unique properties, just two years later, embryonic stem cells and stem cells of definitive tissues began to be used in the practice of regenerative medicine and gene therapy.