Hematopoietic stem cells of the yolk sac

Obviously, the various proliferative and differentiating potencies of hematopoietic stem cells are due to the peculiarities of their ontogenetic development, since in the process of ontogenesis, even the localization of the main regions of hematopoiesis is changing in man. Hematopoietic progenitor cells of the yolk sac of the fetus are committed to the formation of an exclusively erythropoietic cell line. After migration of primary GSK to the liver and spleen in the microenvironment of these organs, the spectrum of the lines of commision is expanding. In particular, hematopoietic stem cells acquire the ability to generate lymphoid lineages. In the prenatal period, hematopoietic precursor cells reach the zone of final localization and colonize the bone marrow. In the process of fetal development in the blood of the fetus contains a significant number of stem hemopoietic cells. For example, at the 13th week of pregnancy, the HSC level reaches 18% of the total number of mononuclear blood cells. In the future there is a progressive decrease in their content, but even before the birth, the amount of HSC in the umbilical cord blood differs little from their number in the bone marrow.

According to classical ideas, the natural change of hematopoiesis localization in the process of embryonic development of mammals is carried out by migration and introduction into the new microenvironment of polypotent hematopoietic stem cells - from the yolk sac to the liver, spleen and bone marrow. Since in the early stages of embryonic development, the hematopoietic tissue contains a large number of stem cells, which decreases as the fetus matures, hematopoietic tissue of the embryonic liver, isolated from abortion on 5-8 weeks gestation, is considered the most promising for obtaining hematopoietic stem cells.

[1], [2], [3], [4], [5], [6]

The origin of hematopoietic stem cells

The fact that embryonic formation of erythrocytes originates in the blood islands of the yolk sac, is beyond doubt. However, the in vitro differentiation potential of the hematopoietic cells of the yolk sac is very limited (they differ primarily in erythrocytes). It should be noted that transplantation of hematopoietic stem cells of the yolk sac is not able to restore hemopoiesis for a long time. It turned out that these cells are not the precursors of the GSK of an adult organism. True GSK appear earlier, in the 3-5th week of intrauterine development, in the zone of formation of the tissues of the stomach and endothelium of the blood vessels (paraaortic splanchnopleura, P-SP), as well as in the place of the aorta, gonad and primary kidney - in the mesonephros or so called AGM-area. It is shown that AGM region cells are a source not only of HSC, but also endothelial cells of blood vessels, as well as osteoclasts involved in the processes of bone tissue formation. At the 6th week of gestation, early hematopoietic progenitor cells from the AGM region are transferred to the liver, which remains the main hematopoietic organ of the fetus until birth.

Since this moment is extremely important from the point of view of cell transplantation, the problem of the origin of HSC in the process of human embryogenesis deserves a more detailed exposition. The classic notion that mammalian and bird hematopoietic stem cells originate from an extraembryonic source is based on the studies of Metcalf and Moore, who first used cloning techniques for HSC and their descendants isolated from the yolk sac. The results of their work served as the basis for the migration theory, according to which GSK, first arising in the yolk sac, consistently populate the transitory and definitive hemopoietic organs as they form the corresponding microenvironment. This is how the view was established that the generation of GSK, initially localized in the yolk sac, serves as the cellular basis for definitive hematopoiesis.

The hematopoietic ancestral cells of the yolk sac belong to the category of the earliest precursor cells of hemopoiesis. Their phenotype is described by the formula AA4.1 + CD34 + c-kit +. Unlike GCS of mature bone marrow, they do not express Sca-1 antigens and MHC molecules. It would seem that the appearance of marker antigens on the surface membranes of the GSK yolk sac during culturing corresponds to their differentiation during the embryonic development with the formation of committed hemopoiesis lines: the expression level of CD34 and Thy-1 antigen decreases, the expression of CD38 and CD45RA increases, and HLA-DR molecules appear. In the subsequent, cytokine-induced and growth factors, in vitro specialization, the expression of antigens specific for hematopoietic progenitor cells of a particular cell line begins. However, the results of the study of embryonic hematopoiesis in representatives of three classes of vertebrate animals (amphibians, birds and mammals) and, in particular, analysis of the origin of HSCs responsible for definitive hematopoiesis in postnatal ontogenesis, contradict classical concepts. It is established that in representatives of all the classes examined in embryogenesis two independent regions are formed in which GSKs arise. The extraembryonic "classical" region is represented by the yolk sac or its analogues, whereas the recently identified intraembryonic zone of HSC localization includes the para-aortic mesenchyme and the AGM region. Today it can be argued that in amphibians and birds, definitive HSCs originate from intraembryonic sources, whereas in mammals and humans, the involvement of GSK of the yolk sac in definitive hematopoiesis can not be completely ruled out.

Embryonic hematopoiesis in the yolk sac is, in fact, the primary erythropoiesis, which is characterized by the preservation of the nucleus at all stages of erythrocyte maturation and the synthesis of hemoglobin of the fetal type. According to the latest data, the wave of primary erythropoiesis terminates in the yolk sac on the 8th day of embryonic development. It is followed by a period of accumulation of definitive erythroid progenitor cells - BFU-E, which are formed exclusively in the yolk sac and first appear on the 9th day of gestation. The next stage of embryogenesis is already forming the definitive erythroid progenitor cells - CFU-E, as well as (!) Mast cells and CFU-GM. This is the basis for the existence of a point of view according to which definitive progenitor cells appear in the yolk sac, migrate with blood, settle in the liver and quickly initiate the first phase of intraembryonic hematopoiesis. According to such ideas, the yolk sac can be considered, on the one hand, as the place of primary erythropoiesis, and on the other hand as the first source of definitive hematopoietic progenitor cells in embryonic development.

It has been shown that colony-forming cells with a high proliferative potential can be isolated from the yolk sac already on the 8th day of gestation, that is, long before the closure of the vascular system of the embryo and yolk sac. And cells from the yolk sac with a high proliferative potential in vitro form colonies whose size and cellular composition do not differ from the corresponding parameters of the culture growth of bone marrow stem cells. At the same time, when re-implanting colony-forming cells of the yolk sac with a high proliferative potential, much more daughter colony-forming cells and multipotent progenitor cells are formed than with the use of bone marrow progenitors of hemopoiesis.

The final conclusion on the role of the hematopoietic stem cells of the yolk sac in the definitive hematopoies could be obtained from the work in which the authors obtained a line of endothelial cells of the yolk sac (G166), which effectively supported the proliferation of its cells with the phenotypic and functional characteristics of GSK (AA4.1 + WGA + low density and weak adhesive properties). The content of the latter during cultivation on the feeder layer of C166 cells increased by more than 100 times within 8 days. In mixed colonies grown on a sublayer of C166 cells, macrophages, granulocytes, megakaryocytes, blast cells and monocytes, as well as progenitor cells of B and T lymphocytes, were identified. Yolk sac cells, growing on an underlayer of endothelial cells, had the ability to self-reproduce and survived in experiments of the authors up to three passages. Recovery with their help of hemopoiesis in sexually mature mice with severe combined immunodeficiency (SCID) was accompanied by the formation of all types of leukocytes, as well as T and B lymphocytes. However, the authors used yolk sac cells of the 10-day embryo in their studies, in which the extra- and intraembryonic vascular systems are already closed, which does not allow to exclude the presence of intracellular origin among the cells of the yolk sac.

At the same time, the analysis of the differentiation potential of the hematopoietic cells of the early stages of development, prior to the unification of the vascular systems of the yolk sac and embryo (8-8.5 days of gestation), revealed the presence of T and B cell precursors in the yolk sac, but not in the body of the embryo . In the in vitro system, by the method of two-stage cultivation on a monolayer of epithelial and subepithelial cells of the thymus, mononuclear cells of the yolk sac were differentiated into pre-T and mature T-lymphocytes. Under the same culture conditions, but on a monolayer of stromal cells of the liver and bone marrow, the yolk sac mononuclears differentiated into pre-B cells and mature IglVT-B lymphocytes.

The results of these studies indicate the possibility of developing cells of the immune system from the extraembryonic tissue of the yolk sac, and the formation of primary T and B cell lines depends on the factors of the stromal microenvironment of embryonic hematopoietic organs.

In the works of other authors it was also shown that the yolk sac contains cells with potencies to lymphoid differentiation, and the resulting lymphocytes do not differ in antigenic characteristics from those of sexually mature animals. It was found that cells of the yolk sac of the 8-9-day-old embryo are able to restore lymphopoiesis in the atimocyte thymus with the appearance of mature CD3 + CD4 + and CDZ + CD8 + lymphocytes possessing a formalized repertoire of T-cell receptors. Thus, the thymus can be populated with cells of extraembryonic origin, but it is impossible to exclude the probable migration to the thymus of early T-lymphocyte precursor cells from the intraembryonic sources of lymphopoiesis.

At the same time, the transplantation of the hematopoietic cells of the yolk sac to adult irradiated recipients does not always result in a long repopulation of the devastated zones of hematopoietic tissue localization, and in vitro the cells of the yolk sac form significantly less spleen colonies than the cells of the AGM region. In some cases, using the cells of the yolk sac of the 9-day embryo, it is still possible to achieve a prolonged (up to 6 months) repopulation of the hematopoietic tissue of irradiated recipients. The authors believe that the cells of the yolk sac with the CD34 + c-kit + phenotype, in terms of their ability to repopulate devastated hemopoietic organs, not only do not differ from those of the AGM region, but also more efficiently restore hemopoiesis, since they contain in the yolk sac almost 37 times more .

It should be noted that in the experiments we used hematopoietic cells of the yolk sac with marker antigens GSK (c-kit + and / or CD34 + and CD38 +) that were injected directly into the liver or into the ventral vein of the offspring of female mice receiving busulfan injection on the 18th day of pregnancy. In such newborn animals, myelopoiesis was sharply depressed due to the elimination of stem hemopoietic cells caused by busulfan. After transplantation of HSC of the yolk sac, for 11 months in the peripheral blood of the recipients, the uniform elements containing the donor marker, glycerophosphate dehydrogenase, were detected. It was established that GSK from the yolk sac reconstituted the contents of the cells of the lymphoid, myeloid and erythroid lines of differentiation in the blood, thymus, spleen and bone marrow, the level of chimerism was higher in the case of intrahepatic, than intravenous administration of the cells of the yolk sac. The authors believe that GSK yolk sac embryos early stages of development (up to 10 days) for successful colonization of the hematopoietic organs of adult recipients need a preliminary interaction with the hemopoietic microenvironment of the liver. It is possible that in embryogenesis there is a unique stage of development, when cells of the yolk sac, migrating initially to the liver, then acquire the ability to colonize the stroma of the hematopoietic organs of sexually mature recipients.

In this regard, it should be noted that the chimerism of cells of the immune system is often observed after bone marrow transplantation to irradiated sexually mature recipients - in the blood of the latter, the cells of the donor phenotype are found in a fairly large number among the B, T cells and granulocytes of the recipient, which continues at least 6 months.

By morphological methods, hematopoietic cells in mammals are first detected on the 7th day of development of the embryo and are represented by hematopoietic islets within the vessels of the yolk sac. However, natural hematopoietic differentiation in the yolk sac is limited to primary red blood cells, which retain nuclei and synthesize fetal hemoglobin. Nevertheless, traditionally it was believed that the yolk sac is the only source of GSK migrating to the hematopoietic organs of the developing embryo and providing definitive hematopoiesis in adult animals, since the appearance of GSK in the body of the embryo coincides with the closure of the vascular systems of the yolk sac and embryo. This point of view is supported by the evidence that yolk sac cells in cloning in vitro give rise to granulocytes and macrophages, and in vivo to splenic colonies. Then during transplantation experiments it was found that the hematopoietic cells, which in the yolk sac can differentiate only into primary red blood cells, in the microenvironment of the liver of newborns and adults of SCID mice, devastated thymus or stromal feeder, acquire the ability to repopulate the hematopoietic organs with restoration of all lines of hemopoiesis even in adult recipient animals. In principle, this makes it possible to classify them as true GSKs - as cells that function in the postnatal period. It is assumed that the yolk sac, along with the AGM region, serves as a source of HSC for definitive hematopoiesis in mammals, but their contribution to the development of the hematopoietic system is still unclear. The biological sense of the existence in the early embryogenesis of mammals of two hemopoietic organs with similar functions is also not understandable.

The search for answers to these questions continues. In vivo, it was possible to prove the presence in the yolk sac of 8-8.5 day old embryos of cells restoring lymphopoiesis in sublethally irradiated SCID mice with a pronounced deficiency of T and B lymphocytes. The hematopoietic cells of the yolk sac were injected both intraperitoneally and directly into the tissue of the spleen and liver. After 16 weeks, TCR / CD34 \ CD4 + and CD8 + T-lymphocytes and B-220 + IgM + B-lymphocytes, labeled with MHC donor antrhogens, were detected in the recipients. In the body, 8-8.5-day embryos of stem cells, capable of such restoration of the immune system, the authors did not find.

The hematopoietic cells of the yolk sac have a high proliferative potential and are capable of prolonged self-reproduction in vitro. Some authors identify these cells as HSC on the basis of a prolonged (almost 7 months) generation of erythroid progenitor cells that differ from bone marrow forefathers of the erythroid line with a longer duration of passage, larger colonies, increased sensitivity to growth factors, and longer proliferation. In addition, under the appropriate conditions for the cultivation of cells of the yolk sac in vitro, lymphoid precursor cells are also formed.

The data given in general make it possible to treat the yolk sac as a source of HSC, and less committed and therefore have a greater proliferative potential than bone marrow stem cells. However, in spite of the fact that the yolk sac contains polypotent hematopoietic progenitor cells that support different hematopoietic differentiation lines for a long time in vitro, the only criterion for the usefulness of HSC is their ability to prolong repopulation of the hematopoiesis of the recipient whose hematopoietic cells are destroyed or genetically defective. Thus, the key question is whether the polypotent hematopoietic cells of the yolk sac can migrate and colonize the hematopoietic organs and whether it is advisable to revise known works in which their potential for repopulation of the hematopoietic organs of sexually mature animals with the formation of the main hematopoietic lines is demonstrated. In embryos of birds, as early as the 1970s, intraembryonic sources of definitive HSCs were identified, which even then challenged the established ideas of the extraembryonic origin of HSC, including those of representatives of other classes of vertebrates. In the last few years, there have been publications about the presence of similar intraembryonic sites containing HSC in mammals and humans.

Once again, it should be noted that fundamental knowledge in this field is extremely important for practical cell transplantology, since it will help not only to determine the preferred source of HSC, but also to establish the features of the interaction of primary hematopoietic cells with a genetically alien organism. It is known that the introduction of stem hematopoietic cells of human fetal liver into the embryo of the sheep at the stage of organogenesis leads to the birth of chimera animals, in the blood and bone marrow of which 3 to 5% of human hematopoietic cells are stably determined. At the same time, human GCSs do not change their karyotype, retaining a high rate of proliferation and the ability to differentiate. In addition, transplanted xenogeneic HSCs do not conflict with the immune system and phagocytes of the host organism and are not transformed into tumor cells, which formed the basis for intensive development of methods for intrauterine correction of hereditary genetic pathology with the help of GSK or ESC transfected with deficient genes.

But at what stage of embryogenesis is it more appropriate to carry out such correction? For the first time, cells that are determined for hematopoiesis appear in mammals immediately after implantation (the 6th day of gestation), when morphological signs of hematopoietic differentiation and presumptive hematopoietic organs are still absent. At this stage, the dispersed cells of the mouse embryo are capable of repopulating the hematopoietic organs of irradiated recipients with the formation of erythrocytes and lymphocytes different from the host cells, respectively, by the type of hemoglobin or glycerophosphate isomerase, and also by an additional chromosomal marker (Tb) of donor cells. In mammals, as in birds, simultaneously with the yolk sac until the closure of the common vascular bed, hemopoietic cells appear directly in the body of the embryo in the para-aortic splanchnopleura. From the AGM-region, hematopoietic cells of phenotype AA4.1 +, characterized as multipotent hematopoietic cells, forming T- and B-lymphocytes, granulocytes, megakaryocytes and macrophages, were isolated. Phenotypically, these multipotent progenitor cells are very close to the HSC of the bone marrow of adult animals (CD34 + c-kit +). The number of multipotent AA4.1 + cells among all cells of the AGM-region is small - they make up no more than 1/12 of its part.

In the human embryo, a homologous AGM-region of animals, an intraembryonic region containing HSC, was also found. Moreover, in humans more than 80% of multipotent cells with high proliferative potential are contained in the body of the embryo, although such cells are present in the yolk sac. A detailed analysis of their localization showed that hundreds of such cells are assembled into compact groups that are located in close proximity to the endothelium of the ventral wall of the dorsal aorta. Phenotypically, they are CD34CD45 + Lin cells. On the contrary, in the yolk sac, as well as in other hematopoietic organs of the embryo (liver, bone marrow), such cells are single.

Consequently, in the human embryo, the AGM region contains clusters of hematopoietic cells closely associated with the ventral endothelium of the dorsal aorta. This contact can also be traced at the immunochemical level - and the cells of the hematopoietic clusters, and the endotheliocytes express the vascular endothelial growth factor, the Flt-3 ligand, their FLK-1 and STK-1 receptors, as well as the transcription factor of the leukemia stem cells. In the AGM region, the mesenchymal derivatives are represented by a tight cord of rounded cells located along the entire dorsal aorta and expressing tenascin C-glycoprotein of the basic substance, which actively participates in the processes of intercellular interaction and migration.

The multipotent stem cells of the AGM region after transplantation quickly restore hematopoiesis in sexually mature irradiated mice and provide effective hematopoiesis for a long time (up to 8 months). In the yolk sac of cells with such properties, the authors did not reveal. The results of this study are supported by data from another study showing that in embryos of early stages of development (10.5 days) the AGM region is the only source of cells that correspond to the definition of HSC, restoring myeloid and lymphoid hematopoiesis in sexually irradiated recipients.

AGM-S3 stromal line was isolated from the AGM-region, the cells of which support the generation of commited progenitor cells CFU-GM, BFU-E, CFU-E and colony-forming units of mixed type in the culture. The content of the latter during cultivation on the feeder sublayer of AGM-S3 cells increases from 10 to 80 times. Thus, in the microenvironment of the AGM region, stromal base cells are present that effectively support hematopoiesis, so the AGM region itself may well act as an embryonic hematopoietic organ - the source of definitive GSK, that is, GCK, forming the hematopoietic tissue of an adult animal.

Expanded immunophenotyping of the AGM-cell cellular structure has shown that it contains not only multipotent hematopoietic cells, but also cells that are committed to myeloid and lymphoid (T and B-lymphocytes) differentiation. However, in the molecular analysis of individual CD34 + c-kit + cells from the AGM region, activation of only beta-globin and myeloperoxidase but not lymphoid genes encoding the synthesis of CD34, Thy-1, and 15 was detected using a polymerase chain reaction. Partial activation of lineage-specific genes is characteristic of early ontogenetic stages of generation of HSC and progenitor cells. Given that the number of committed progenitor cells in the AGM region of a 10-day embryo is 2-3 orders of magnitude lower than in the liver, it can be argued that on the 10th day of embryogenesis, hemopoiesis in the AGM region is just beginning, whereas in the main the hematopoietic lines are already deployed during this period.

Indeed, unlike the earlier (9-11th) hematopoietic stem cells of the yolk sac and the AGM region that repopulate the hematopoietic microenvironment of a newborn, but not an adult organism, the hematopoietic precursor cells of the 12-17-day-old embryonic liver no longer need early postnatal microenvironment and occupy the organs of hematopoiesis of an adult animal no worse than a newborn. After transplantation of HSC of the embryonic hematopoietic liver in irradiated adult recipient mice, it was polyclonal in nature. In addition, with the help of labeled colonies, it was shown that the functioning of clinging clones completely submits to the clonal succession revealed in the adult bone marrow. Consequently, HSCs of the embryonic liver, labeled under the most sparing conditions, without prestigeation by exogenous cytokines, already possess the basic attributes of adult GSK: they do not need an early postembryonic microenvironment, go into a state of deep rest after transplantation and are mobilized into cloning sequentially in accordance with the model of clonal succession.

Obviously, we should dwell in somewhat more detail on the phenomenon of clonal succession. Erythropoiesis carries stem hemopoietic cells that have a high proliferative potential and the ability to differentiate in all lines of committed precursor blood cells. With normal hematopoiesis, most of the hematopoietic stem cells remain in the dermatological state and are mobilized for proliferation and differentiation, successively forming successive clones. This process is called clonal succession. Experimental evidence of clonal succession in the hematopoietic system was obtained in studies with GSK marked with retroviral gene transfer. In adult animals, hematopoiesis is maintained by many simultaneously functioning hemopoietic clones derived from GSK. Based on the phenomenon of clonal succession, a repopulation approach to the identification of GCS was developed. This principle distinguishes long-term haematopoietic stem cell (LT-HSC), capable of restoring the hematopoietic system throughout life, and a short-term GSK that performs this function for a limited period of time.

If we consider hematopoietic stem cells from the point of view of the repopulation approach, the peculiarity of hematopoietic cells of the embryonic liver is their ability to create colonies that are much larger in size than those with the growth of HSC cord blood or bone marrow, and this applies to all types of colonies. This fact already indicates a higher proliferative potential of the hematopoietic cells of the embryonic liver. The unique property of hematopoietic precursor cells of the embryonic liver is a shorter cycle of cells compared to other sources, which is of great importance in terms of the effectiveness of repopulation of the hematopoiesis during transplantation. Analysis of the cellular composition of the hematopoietic suspension obtained from mature sources indicates that in all stages of ontogenesis, nuclear cells are predominantly represented by finally differentiated cells, the number and phenotype of which depend on the ontogenetic age of the donor of the hematopoietic tissue. In particular, bone marrow and cord blood mononuclear cell suspensions are more than 50% composed of mature lymphoid cells, whereas in hematopoietic embryonic liver tissue less than 10% of lymphocytes are contained. In addition, the cells of the myeloid line in the embryonic and fetal liver are represented mainly by the erythroid series, while in the cord blood and bone marrow predominate granulocyte-macrophagal elements.

Important is the fact that the embryonic liver contains a full set of the earliest predecessors of hemopoiesis. The latter include erythroid, granulopoietic, megakaryopoietic and multilinear colony-forming cells. Their more primitive precursors, LTC-IC, are capable of proliferating and differentiating in vitro for 5 or more weeks, and also retaining functional activity after engraftment in the recipient organism for allogenic and even xenogeneic transplantation to immunodeficient animals.

The biological expediency of the predominance in the embryonic liver of cells of the erythroid series (up to 90% of the total number of hematopoietic elements) is due to the need to provide the erythrocyte mass of the rapidly increasing volume of the developing fetus. In the embryonic liver, erythropoiesis is represented by nuclear erythroid precursors of varying degrees of maturity, containing fetal hemoglobin (a2y7), which, due to a higher affinity for oxygen, ensures effective absorption of the latter from maternal blood. Intensification of erythropoiesis in the embryonic liver is associated with a local increase in the synthesis of erythropoietin (EPO). It is noteworthy that the presence of erythropoietin alone is sufficient to realize the hematopoietic potential of the hematopoietic cells of the embryonic liver, whereas a combination of cytokines and growth factors consisting of EPO, SCF, GM-CSF, and IL-3 is required to be used for the compilation of bone marrow and cord blood erythropoiesis. In this case, early hematopoietic progenitor cells isolated from the embryonic liver that do not have EPO receptors do not respond to exogenous erythropoietin. The induction of erythropoiesis in the suspension of fetal mononuclear cells requires the presence of more advanced erythropoietin-sensitive cells with the CD34 + CD38 + phenotype that express the EPO receptor.

In the literature there is still no consensus on the formation of hemopoiesis in the embryonic period. The functional significance of the existence of extra- and intraembryonic sources of hematopoietic precursor cells has not been established. However, there is no doubt that in human embryogenesis the liver is the central organ of hemopoiesis and at 6-12 weeks of gestation serves as the main source of hematopoietic stem cells that colonize the spleen, thymus and bone marrow, the GDRs perform the corresponding functions in the pre- and postnatal periods development.

It should be noted again that the embryonic liver in comparison with other sources is characterized by the highest content of HSC. Approximately 30% of CD344 cells of the embryonic liver have a phenotype of CD38. At the same time, the number of lymphoid precursor cells (CD45 +) at the early stages of hematopoiesis in the liver is no more than 4%. It was found that, as the fetus develops, from 7 to 17 weeks of gestation, the number of B lymphocytes progressively increases with a monthly "step" of 1.1%, while the level of GSC is permanently reduced.

The functional activity of hematopoietic stem cells also depends on the period of embryonic development of their source. A study of the colony-forming activity of human liver cells of the 6th-8th and 9th-12th weeks of gestation when cultured in a semi-liquid medium in the presence of SCF, GM-CSF, IL-3, IL-6 and EPO showed that the total number of colonies in 1 , 5 times higher when sowing HSV embryonic liver early in development. At the same time, the number in the liver of such myelopoiesis precursor cells, like CFU-GEMM, is more than three times as high as the 9th to 12th weeks of gestation at the 6th to 8th weeks of embryogenesis. In general, the colony-forming activity of hematopoietic liver cells of embryos of the first trimester of gestation was significantly higher than that of the second trimester of the fetal liver cells.

The data presented above indicate that the embryonic liver at the beginning of embryogenesis differs not only in the increased content of early hematopoiesis precursor cells, but its hemopoietic cells are characterized by a wider spectrum of differentiation into different cell lines. These features of the functional activity of stem hematopoietic cells of the embryonic liver may have a certain clinical significance, since their qualitative characteristics allow one to expect a pronounced therapeutic effect in transplantation even of a small number of cells obtained at early gestation.

Nevertheless, the problem of the number of hematopoietic stem cells required for effective transplantation remains open and relevant. Attempts are being made to solve it using the high potential of self-reproduction of hematopoietic cells of the embryonic liver in vitro with their stimulation by cytokines and growth factors. With constant perfusion in the bioreactor of early GSC of the embryonic liver, after 2-3 days at the output it is possible to obtain the number of stem hemopoietic cells 15 times higher than their baseline level. For comparison, it should be noted that to achieve a 20-fold increase in the yield of HSC cord blood in the same conditions, it takes at least two weeks.

Thus, the embryonic liver differs from other sources of hematopoietic stem cells with a higher content of both committed and early hematopoietic progenitor cells. In a culture with growth factors, embryonic liver cells with the CD34 + CD45Ra1 CD71l0W phenotype form 30 times more colonies than similar cord blood cells, and 90 times more than the HSC of the bone marrow. The differences in the content of early hematopoietic progenitor cells that form mixed colonies are most pronounced in these sources: the amount of CFU-GEMM in the embryonic liver exceeds that in the cord blood and bone marrow, respectively, by 60 and 250 times.

Important is the fact that up to the 18th week of embryonic development (the period of onset of hemopoiesis in the bone marrow) more than 60% of the liver cells are involved in the hematopoiesis function. Since the thymus and thymocytes do not exist in the human fetus until the 13th week of development, transplantation of hematopoietic cells of the embryonic liver of 6-12 weeks gestation significantly reduces the risk of graft-versus-host reaction and does not require the selection of a histocompatible donor, since it makes it relatively easy to achieve hemopoietic chimerism.