Hematopoietic stem cells of the yolk sac

Obviously, various proliferative and differentiating potentials of hematopoietic stem cells are determined by the peculiarities of their ontogenetic development, since even the localization of the main areas of hematopoiesis changes in humans during ontogenesis. Hematopoietic progenitor cells of the fetal yolk sac are committed to the formation of an exclusively erythropoietic cell line. After the migration of primary HSCs to the liver and spleen, the spectrum of commitment lines expands in the microenvironment of these organs. In particular, hematopoietic stem cells acquire the ability to generate lymphoid lineage cells. In the prenatal period, hematopoietic progenitor cells reach the zone of final localization and populate the bone marrow. During intrauterine development, the fetal blood contains a significant number of hematopoietic stem cells. For example, in the 13th week of pregnancy, the HSC level reaches 18% of the total number of mononuclear blood cells. Subsequently, a progressive decrease in their content is observed, but even before birth, the amount of HSCs in the umbilical cord blood differs little from their amount in the bone marrow.

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

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Questions about the origin of hematopoietic stem cells

There is no doubt that embryonic formation of erythrocytes originates in the blood islands of the yolk sac. However, the differentiation potential in vitro of yolk sac hematopoietic cells is very limited (they differentiate mainly into erythrocytes). It should be noted that transplantation of yolk sac hematopoietic stem cells is not able to restore hematopoiesis for a long time. It turned out that these cells are not the precursors of adult HSCs. True HSCs appear earlier, on the 3rd-5th week of intrauterine development, in the zone of formation of stomach tissue and endothelium of blood vessels (paraaortic splanchnopleura, P-SP), as well as in the place of the aorta, gonads and primary kidneys - in the mesonephros or so-called AGM region. It has been shown that AGM region cells are a source of not only HSCs, but also endothelial cells of blood vessels, as well as osteoclasts involved in bone tissue formation processes. At the 6th week of gestation, early hematopoietic progenitor cells from the AGM region move to the liver, which remains the main hematopoietic organ of the fetus until birth.

Since this point is extremely important from the point of view of cell transplantation, the problem of the origin of HSCs in the process of human embryogenesis deserves a more detailed presentation. The classical ideas that the hematopoietic stem cells of mammals and birds originate from an extraembryonic source are based on the studies of Metcalf and Moore, who were the first to use methods of cloning HSCs and their descendants isolated from the yolk sac. The results of their work served as the basis for the migration theory, according to which HSCs, having first appeared in the yolk sac, sequentially populate the transitory and definitive hematopoietic organs as the corresponding microenvironment is formed in them. This is how the point of view was established that the generation of HSCs, initially localized in the yolk sac, serves as the cellular basis for definitive hematopoiesis.

Yolk sac hematopoietic progenitor cells belong to the category of the earliest hematopoietic progenitor cells. Their phenotype is described by the formula AA4.1+CD34+c-kit+. Unlike mature bone marrow HSCs, they do not express Sca-1 antigens and MHC molecules. It would seem that the appearance of marker antigens on the surface membranes of yolk sac HSCs during cultivation corresponds to their differentiation during embryonic development with the formation of committed hematopoietic lines: the level of CD34 and Thy-1 antigen expression decreases, CD38 and CD45RA expression increases, and HLA-DR molecules appear. With subsequent specialization in vitro induced by cytokines and growth factors, expression of antigens specific for hematopoietic progenitor cells of a certain cell line begins. However, the results of the study of embryonic hematopoiesis in representatives of three classes of vertebrates (amphibians, birds and mammals) and, in particular, the analysis of the origin of HSCs responsible for definitive hematopoiesis in postnatal ontogenesis, contradict classical concepts. It has been established that in representatives of all the classes considered, two independent regions in which HSCs arise are formed during embryogenesis. The extraembryonic “classical” region is represented by the yolk sac or its analogues, while the recently identified intraembryonic zone of HSC localization includes the paraaortic mesenchyme and the AGM region. Today, it can be argued that in amphibians and birds, definitive HSCs originate from intraembryonic sources, while in mammals and humans, the participation of yolk sac HSCs in definitive hematopoiesis cannot yet be completely excluded.

Embryonic hematopoiesis in the yolk sac is, in fact, primary erythropoiesis, which is characterized by the preservation of the nucleus at all stages of erythrocyte maturation and the synthesis of fetal-type hemoglobin. According to the latest data, the wave of primary erythropoiesis ends 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. At the next stage of embryogenesis, definitive erythroid progenitor cells - CFU-E, as well as (!) mast cells and CFU-GM are already formed. This is the basis for the point of view that definitive progenitor cells arise in the yolk sac, migrate with the bloodstream, settle in the liver and quickly initiate the first phase of intraembryonic hematopoiesis. According to these concepts, the yolk sac can be considered, on the one hand, as the site of primary erythropoiesis, and on the other, as the first source of definitive hematopoietic progenitor cells in embryonic development.

It has been shown that colony-forming cells with high proliferative potential can be isolated from the yolk sac as early as the 8th day of gestation, i.e., long before the closure of the vascular system of the embryo and the yolk sac. Moreover, the cells with high proliferative potential obtained from the yolk sac in vitro form colonies whose size and cellular composition do not differ from the corresponding parameters of the cultural growth of bone marrow stem cells. At the same time, when retransplanting colony-forming cells of the yolk sac with high proliferative potential, significantly more daughter colony-forming cells and multipotent progenitor cells are formed than when using bone marrow progenitor cells of hematopoiesis.

A final conclusion on the role of yolk sac hematopoietic stem cells in definitive hematopoiesis could be provided by the results of the work in which the authors obtained a line of yolk sac endothelial cells (G166), which effectively supported the proliferation of its cells with the phenotypic and functional characteristics of HSCs (AA4.1+WGA+, low density and weak adhesive properties). The content of the latter increased more than 100-fold when cultivated on a feeder layer of C166 cells for 8 days. Macrophages, granulocytes, megakaryocytes, blast cells and monocytes, as well as B- and T-lymphocyte precursor cells were identified in mixed colonies grown on a sublayer of C166 cells. Yolk sac cells growing on a sublayer of endothelial cells had the ability to self-reproduce and withstood up to three passages in the authors' experiments. Restoration of hematopoiesis with their help in 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 in their studies used yolk sac cells of a 10-day-old embryo, in which the extra- and intraembryonic vascular systems are already closed, which does not allow us to exclude the presence of intraembryonic HSCs among the yolk sac cells.

At the same time, the analysis of the differentiation potential of hematopoietic cells of early stages of development, isolated before the unification of the vascular systems of the yolk sac and embryo (8-8.5 days of gestation), revealed the presence of precursors of T- and B-cells 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 differentiated into pre-T- and mature T-lymphocytes. Under the same cultivation conditions, but on a monolayer of stromal cells of the liver and bone marrow, mononuclear cells of the yolk sac differentiated into pre-B-cells and mature IglVT-B-lymphocytes.

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

Other authors have also shown that the yolk sac contains cells with potential for lymphoid differentiation, and the resulting lymphocytes do not differ in antigenic characteristics from those in sexually mature animals. It has been established that the yolk sac cells of an 8-9-day-old embryo are capable of restoring lymphopoiesis in the athymocyte thymus with the emergence of mature CD3+CD4+- and CD3+CD8+-lymphocytes possessing a formed repertoire of T-cell receptors. Thus, the thymus can be populated by cells of extraembryonic origin, but it is impossible to exclude the probable migration of early T-lymphocyte precursor cells from intraembryonic sources of lymphopoiesis into the thymus.

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

It should be noted that the experiments used yolk sac hematopoietic cells with marker antigens of hematopoietic stem cells (c-kit+ and/or CD34+ and CD38+), which were injected directly into the liver or abdominal vein of the offspring of female mice that received an injection of busulfan on the 18th day of pregnancy. In such newborn animals, their own myelopoiesis was sharply suppressed due to the elimination of hematopoietic stem cells caused by busulfan. After transplantation of yolk sac hematopoietic stem cells, formed elements containing the donor marker - glycerophosphate dehydrogenase - were detected in the peripheral blood of recipients for 11 months. It was found that yolk sac HSCs restore the content of lymphoid, myeloid and erythroid lineage cells in the blood, thymus, spleen and bone marrow, and the level of chimerism was higher in the case of intrahepatic rather than intravenous administration of yolk sac cells. The authors believe that yolk sac HSCs of early-stage embryos (up to 10 days) require preliminary interaction with the hematopoietic microenvironment of the liver to successfully populate the hematopoietic organs of adult recipients. It is possible that there is a unique stage of development in embryogenesis, when yolk sac cells, initially migrating to the liver, then acquire the ability to populate the stroma of the hematopoietic organs of mature recipients.

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

Hematopoietic cells in mammals are first detected by morphological methods on the 7th day of embryonic development and are represented by hematopoietic islands inside the vessels of the yolk sac. However, natural hematopoietic differentiation in the yolk sac is limited to primary erythrocytes that retain nuclei and synthesize fetal hemoglobin. Nevertheless, it was traditionally believed that the yolk sac serves as the only source of HSCs migrating to the hematopoietic organs of the developing embryo and providing definitive hematopoiesis in adult animals, since the appearance of HSCs 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 data that yolk sac cells, when cloned in vitro, give rise to granulocytes and macrophages, and in vivo - to splenic colonies. Then, in the course of transplantation experiments, it was established that the hematopoietic cells of the yolk sac, which in the yolk sac itself are capable of differentiating only into primary erythrocytes, in the microenvironment of the liver of newborn and adult SCID mice, the depleted thymus or stromal feeder acquire the ability to repopulate hematopoietic organs with the restoration of all hematopoietic lines even in adult recipient animals. In principle, this allows us to classify them as true HSCs - 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 HSCs for definitive hematopoiesis in mammals, but their contribution to the development of the hematopoietic system is still unclear. The biological meaning of the existence of two hematopoietic organs with similar functions in early mammalian embryogenesis is also unclear.

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 that restore lymphopoiesis in sublethally irradiated SCID mice with a pronounced deficiency of T- and B-lymphocytes. Yolk sac hematopoietic cells were injected both intraperitoneally and directly into the spleen and liver tissue. After 16 weeks, TCR/CD34 CD4+ and CD8+ T-lymphocytes and B-220+IgM+ B-lymphocytes labeled with donor MHC antrxgenes were detected in the recipients. At the same time, the authors did not find stem cells capable of such restoration of the immune system in the body of 8-8.5-day-old embryos.

Yolk sac hematopoietic cells have a high proliferative potential and are capable of prolonged self-reproduction in vitro. Some authors identify these cells as HSCs based on the prolonged (almost 7 months) generation of erythroid progenitor cells, which differ from bone marrow progenitors of the erythroid lineage by a longer passaging period, larger colony sizes, increased sensitivity to growth factors, and longer proliferation. In addition, under appropriate conditions of yolk sac cell cultivation in vitro, lymphoid progenitor cells are also formed.

The presented data generally allow us to consider the yolk sac as a source of HSCs, less committed and therefore possessing a greater proliferative potential than bone marrow stem cells. However, despite the fact that the yolk sac contains pluripotent hematopoietic progenitor cells that maintain various lines of hematopoietic differentiation in vitro for a long time, the only criterion for the completeness of HSCs is their ability to long-term repopulate the recipient's hematopoietic organs, whose hematopoietic cells are destroyed or genetically defective. Thus, the key question is whether pluripotent hematopoietic cells of the yolk sac can migrate and populate hematopoietic organs and whether it is advisable to revise the known works that demonstrate their ability to repopulate the hematopoietic organs of mature animals with the formation of the main hematopoietic lines. Intraembryonic sources of definitive GSCs were identified in bird embryos back in the 1970s, which already then cast doubt on the established ideas about the extraembryonic origin of GSCs, including in representatives of other classes of vertebrates. In the last few years, publications have appeared on the presence of similar intraembryonic areas containing GSCs in mammals and humans.

It should be noted once again that fundamental knowledge in this area is extremely important for practical cell transplantology, since it will help not only to determine the preferred source of HSCs, but also to establish the features of the interaction of primary hematopoietic cells with a genetically foreign organism. It is known that the introduction of hematopoietic stem cells of human fetal liver into a sheep embryo 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 HSCs do not change their karyotype, maintaining a high proliferation rate and the ability to differentiate. In addition, transplanted xenogeneic HSCs do not conflict with the immune system and phagocytes of the host organism and do not transform into tumor cells, which formed the basis for the intensive development of methods for intrauterine correction of hereditary genetic pathology using HSCs or ESCs transfected with deficient genes.

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

In the human embryo, an intraembryonic region containing HSCs homologous to the AGM region of animals has also been identified. 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 also present in the yolk sac. A detailed analysis of their localization showed that hundreds of such cells are collected in 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 is also traced at the immunochemical level - both the cells of the hematopoietic clusters and the endothelial cells express the vascular endothelial growth factor, Flt-3 ligand, their receptors FLK-1 and STK-1, as well as the transcription factor of leukemia stem cells. In the AGM region, mesenchymal derivatives are represented by a dense strand of rounded cells located along the entire dorsal aorta and expressing tenascin C - a glycoprotein of the ground substance actively involved in the processes of intercellular interaction and migration.

Multipotent stem cells of the AGM region after transplantation quickly restore hematopoiesis in mature irradiated mice and provide effective hematopoiesis for a long time (up to 8 months). The authors did not find cells with such properties in the yolk sac. The results of this study are confirmed by the data of another work, which showed 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 mature irradiated recipients.

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

Extended immunophenotyping of the cellular composition of the AGM region showed that it contains not only multipotent hematopoietic cells, but also cells committed to myeloid and lymphoid (T- and B-lymphocytes) differentiation. However, molecular analysis of individual CD34+c-kit+ cells from the AGM region using polymerase chain reaction revealed activation of only beta-globin and myeloperoxidase genes, but not lymphoid genes encoding the synthesis of CD34, Thy-1, and 15. Partial activation of lineage-specific genes is characteristic of early ontogenetic stages of the generation of HSCs and progenitor cells. Considering 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, hematopoiesis in the AGM region is just beginning, whereas in the main hematopoietic organ of the fetus during this period, the hematopoietic lines have already developed.

Indeed, unlike earlier (9-11 days) hematopoietic stem cells of the yolk sac and AGM region, which repopulate the hematopoietic microenvironment of the newborn, but not the adult organism, hematopoietic progenitor cells of the 12-17-day embryonic liver no longer require an early postnatal microenvironment and populate the hematopoietic organs of an adult animal no worse than a newborn. After transplantation of embryonic liver HSCs, hematopoiesis in irradiated adult recipient mice had a polyclonal character. In addition, using labeled colonies, it was shown that the functioning of the engrafted clones is completely subject to the clonal succession revealed in the adult bone marrow. Consequently, embryonic liver HSCs, labeled under the most gentle conditions, without prestimulation with exogenous cytokines, already possess the main attributes of adult HSCs: they do not require an early postembryonic microenvironment, enter a state of deep dormancy after transplantation, and are mobilized into clonal formation sequentially in accordance with the clonal succession model.

Obviously, it is necessary to dwell on the phenomenon of clonal succession in some more detail. Erythropoiesis is carried out by hematopoietic stem cells that have a high proliferative potential and the ability to differentiate into all lines of committed precursor cells of blood cells. At normal intensity of hematopoiesis, most hematopoietic stem cells are in a dormant state and are mobilized for proliferation and differentiation, sequentially forming clones that replace each other. This process is called clonal succession. Experimental evidence of clonal succession in the hematopoietic system was obtained in studies with HSCs marked by retroviral gene transfer. In adult animals, hematopoiesis is maintained by many simultaneously functioning hematopoietic clones, derivatives of HSCs. Based on the phenomenon of clonal succession, a repopulation approach to the identification of HSCs has been developed. According to this principle, a distinction is made between long-term haematopoietic stem cell (LT-HSC), which is capable of restoring the hematopoietic system throughout life, and short-term HSC, which performs this function for a limited period of time.

If we consider hematopoietic stem cells from the point of view of the repopulation approach, then the peculiarity of hematopoietic cells of the embryonic liver is their ability to create colonies that are significantly larger in size than those in the growth of cord blood or bone marrow HSCs, and this applies to all types of colonies. This fact alone indicates a higher proliferative potential of hematopoietic cells of the embryonic liver. A unique property of hematopoietic progenitor cells of the embryonic liver is a shorter cell cycle compared to other sources, which is of great importance from the point of view of the effectiveness of hematopoietic organ repopulation during transplantation. Analysis of the cellular composition of the hematopoietic suspension obtained from sources of a mature organism indicates that at 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 hematopoietic tissue. In particular, suspensions of mononuclear cells of bone marrow and cord blood consist of more than 50% mature cells of the lymphoid series, while the hematopoietic tissue of the embryonic liver contains less than 10% lymphocytes. In addition, the cells of the myeloid lineage in the embryonic and fetal liver are represented mainly by the erythroid series, while in cord blood and bone marrow, granulocyte-macrophage elements prevail.

It is also important that the embryonic liver contains a complete set of the earliest hematopoietic precursors. Among the latter, erythroid, granulopoietic, megakaryopoietic and multilineage colony-forming cells should be noted. Their more primitive precursors - LTC-IC - are capable of proliferating and differentiating in vitro for 5 weeks or more, and also retain functional activity after engraftment in the recipient's body during allogeneic and even xenogeneic transplantation to immunodeficient animals.

The biological expediency of the predominance of erythroid cells in the embryonic liver (up to 90% of the total number of hematopoietic elements) is due to the need to provide the rapidly increasing blood volume of the developing fetus with erythrocyte mass. In the embryonic liver, erythropoiesis is represented by nuclear erythroid precursors of varying degrees of maturity containing fetal hemoglobin (a2u7), which, due to its 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 for the realization of the hematopoietic potential of hematopoietic cells in the embryonic liver, whereas a combination of cytokines and growth factors consisting of EPO, SCF, GM-CSF and IL-3 is required for the commitment of bone marrow and cord blood HSCs to erythropoiesis. At the same time, early hematopoietic progenitor cells isolated from the embryonic liver, which do not have receptors for EPO, do not respond to exogenous erythropoietin. For the induction of erythropoiesis in a suspension of mononuclear cells of the embryonic liver, the presence of more advanced erythropoietin-sensitive cells with the CD34+CD38+ phenotype, which express the EPO receptor, is necessary.

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

It should be noted once again that the embryonic liver, compared to other sources, is characterized by the highest content of HSCs. Approximately 30% of CD344 cells of the embryonic liver have the CD38 phenotype. At the same time, the number of lymphoid progenitor cells (CD45+) in the early stages of hematopoiesis in the liver is no more than 4%. It has been established 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 HSCs permanently decreases.

The functional activity of hematopoietic stem cells also depends on the period of embryonic development of their source. The study of the colony-forming activity of liver cells of human embryos at 6-8 and 9-12 weeks of gestation during cultivation 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 is 1.5 times higher when seeding HSCs of embryonic liver at early stages of development. At the same time, the number of myelopoiesis progenitor cells such as CFU-GEMM in the liver at 6-8 weeks of embryogenesis is more than three times higher than their number at 9-12 weeks of gestation. In general, the colony-forming activity of hematopoietic liver cells of embryos in the first trimester of gestation was significantly higher than that of fetal liver cells in the second trimester of pregnancy.

The above data indicate that the embryonic liver at the beginning of embryogenesis is distinguished not only by an increased content of early hematopoietic progenitor cells, but its hematopoietic cells are characterized by a wider spectrum of differentiation into various cell lines. These features of the functional activity of hematopoietic stem cells of the embryonic liver may have a certain clinical significance, since their qualitative characteristics allow us to expect a pronounced therapeutic effect when transplanting even a small number of cells obtained at early stages of gestation.

Nevertheless, the problem of the quantity 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 when stimulated by cytokines and growth factors. With constant perfusion of early embryonic liver HSCs in a bioreactor, after 2-3 days, it is possible to obtain a quantity of hematopoietic stem cells at the output that is 15 times higher than their initial level. For comparison, it should be noted that at least two weeks are required to achieve a 20-fold increase in the output of human cord blood HSCs under the same conditions.

Thus, the embryonic liver differs from other sources of hematopoietic stem cells by a higher content of both committed and early hematopoietic progenitor cells. In 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 bone marrow HSCs. The most pronounced differences in the specified sources are in the content of early hematopoietic progenitor cells that form mixed colonies - the amount of CFU-GEMM in the embryonic liver exceeds that in cord blood and bone marrow by 60 and 250 times, respectively.

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