Hematopoietic stem cells

Hematopoietic stem cells (HSCs), like mesenchymal progenitor cells, are characterized by multipotency and give rise to cell lines, the final elements of which form the formed elements of the blood, as well as a number of specialized tissue cells of the immune system.

The hypothesis of the existence of a common precursor of all blood cells, as well as the term “stem cell” itself, belongs to A. Maksimov (1909). The potential for the formation of cellular mass in HSC is enormous - bone marrow stem cells daily produce 10 cells that make up the formed elements of peripheral blood. The very fact of the existence of hematopoietic stem cells was established in 1961 in experiments on the restoration of hematopoiesis in mice that received a lethal dose of radioactive irradiation that destroys bone marrow stem cells. After transplantation of syngeneic bone marrow cells to such lethally irradiated animals, discrete foci of hematopoiesis were found in the spleen of the recipients, the source of which were single clonogenic precursor cells.

Then the ability of hematopoietic stem cells to self-maintenance, providing the function of hematopoiesis in the process of ontogenesis, was proven. In the process of embryonic development, HSCs are distinguished by high migration activity, necessary for their movement to the zones of the formation of hematopoietic organs. This property of HSCs is also preserved in ontogenesis - due to their constant migration, a permanent renewal of the pool of immunocompetent cells occurs. The ability of HSCs to migrate, penetrate through histohematic barriers, implant in tissues and clonogenic growth served as the basis for transplantation of bone marrow cells in a number of diseases associated with pathology of the hematopoietic system.

Like all stem cell resources, hematopoietic stem cells are present in their niche (bone marrow) in very small quantities, which causes certain difficulties in their isolation. Immunophenotypically, human HSCs are characterized as CD34+NK cells capable of migrating into the bloodstream and populating the organs of the immune system or repopulating the bone marrow stroma. It should be clearly understood that HSCs are not the most immature cells of the bone marrow, but originate from precursors, which include dormant fibroblast-like CD34-negative cells. It has been established that cells with the CD34 phenotype are capable of entering the general bloodstream, where they change their phenotype to CD34+, but upon reverse migration into the bone marrow, under the influence of the microenvironment, they again become CD34-negative stem cell elements. In the resting state, CD34~ cells do not respond to paracrine regulatory signals of the stroma (growth factors, cytokines). However, in situations requiring increased intensity of hematopoiesis, stem cells with the CD34 phenotype respond to differentiation signals by forming both hematopoietic and mesenchymal progenitor cells. Hematopoiesis occurs through direct contact of HSCs with cellular elements of the bone marrow stroma, represented by a complex network of macrophages, reticular endothelial cells, osteoblasts, stromal fibroblasts and extracellular matrix. The stromal basis of the bone marrow is not just a matrix or “skeleton” for hematopoietic tissue; it carries out fine regulation of hematopoiesis due to paracrine regulatory signals of growth factors, cytokines and chemokines, and also provides adhesive interactions necessary for the formation of blood cells.

Thus, the constantly renewing system of hematopoiesis is based on a polypotent (from the point of view of hematopoiesis) hematopoietic stem cell capable of long-term self-maintenance. In the process of commitment, HSCs undergo primary differentiation and form clones of cells that differ in cytomorphological and immunophenotypic characteristics. The sequential formation of primitive and committed progenitor cells ends with the formation of morphologically identifiable progenitor cells of various hematopoietic lines. The result of subsequent stages of the complex multi-stage process of hematopoiesis is the maturation of cells and the release of mature formed elements into the peripheral blood - erythrocytes, leukocytes, lymphocytes and thrombocytes.

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Sources of hematopoietic stem cells

Hematopoietic stem cells are considered to be the most studied stem cell source, which is largely due to their clinical use in bone marrow transplantation. At first glance, quite a lot is known about these cells. To some extent, this is true, since intermediate and mature descendants of HSCs are the most accessible cellular elements, each of which (erythrocytes, leukocytes, lymphocytes, monocytes/macrophages and platelets) has been carefully studied at all levels - from light to electron microscopy, from biochemical and immunophenotypic characteristics to identification by PCR analysis methods. However, the monitoring of morphological, ultrastructural, biochemical, immunophenotypic, biophysical and genomic parameters of HSCs has not provided answers to many problematic issues, the solution of which is necessary for the development of cell transplantology. The mechanisms of stabilization of hematopoietic stem cells in a dormant state, their activation, entry into the stage of symmetrical or asymmetrical division, and most importantly, commitment to the formation of such functionally different formed elements of the blood as erythrocytes, leukocytes, lymphocytes and platelets have not yet been established.

The presence in the bone marrow of cells with the CD34 phenotype, which are the progenitors of both mesenchymal and hematopoietic stem cells, raised the question of the existence of the earliest precursors of cellular differentiation into stromal and hematopoietic lineages, close to CD34-negative cells. The so-called long-term culture-initiating cell (LTC-IC) was obtained using the long-term cultivation method. The lifespan of such precursor cells with colony-forming activity on the stromal basis of bone marrow with a certain combination of growth factors exceeds 5 weeks, while the viability of committed colony-forming units (CFU) in culture is only 3 weeks. Currently, LTC-IC is considered to be a functional analogue of HSCs, since with a high repopulation potential, about 20% of LTC-IC are characterized by the CD34+CD38- phenotype and exhibit a high capacity for self-renewal. Such cells are found in human bone marrow with a frequency of 1:50,000. However, myeloid-lymphoid-initiating cells, which are obtained under long-term (15 weeks) cultivation conditions, should be recognized as the closest to HSCs. Such cells, designated as LTC, are among the cells of the bone marrow human brain are found 10 times less frequently than LTC-IC and form cell lines of both myeloid and lymphoid hematopoietic lineages.

Although labeling of hematopoietic stem cells with monoclonal antibodies followed by immunophenotypic identification is the main method for recognition and selective sorting of hematopoietic cells with stem potential, the clinical application of the thus isolated HSCs is limited. Blocking of the CD34 receptor or other marker antigens with antibodies during immunopositive sorting inevitably changes the properties of the cell isolated with its help. Immunonegative isolation of HSCs on magnetic columns is considered more preferable. However, in this case, monoclonal antibodies fixed on a metal carrier are usually used for sorting. In addition, which is important, both methods of HSC isolation are based on phenotypic rather than functional characteristics. Therefore, many researchers prefer to use the analysis of clonogenic parameters of HSCs, which allows the degree of maturity and the direction of differentiation of progenitor cells to be determined by the size and composition of colonies. It is known that during the process of commitment the number of cells and their types in the colony decreases. The hematopoietic stem cell and its early daughter cell, called the “granulocyte-erythrocyte-monocyte-megakaryocyte colony-forming unit” (CFU-GEMM), create large multilineage colonies in culture containing granulocytes, erythrocytes, monocytes and megakaryocytes, respectively. The granulocyte-monocyte colony-forming unit (CFU-GM), located downstream along the commitment line, forms colonies of granulocytes and macrophages, and the granulocyte colony-forming unit (CFU-G) forms only a small colony of mature granulocytes. The early erythrocyte precursor, the burst-forming unit of erythrocytes (CFU-E), is the source of large erythrocyte colonies, and the more mature colony-forming unit of erythrocytes (CFU-E) is the source of small erythrocyte colonies. In general, when cells grow on semi-solid media, cells can be identified that form six types of myeloid colonies: CFU-GEMM, CFU-GM, CFU-G, CFU-M, BFU-E, and CFU-E).

However, in addition to hematopoietic derivatives, any source material for isolating HSCs contains a significant number of accompanying cells. In this regard, preliminary purification of the transplant is necessary, first of all, from active cells of the donor's immune system. Usually, immunoselection is used for this purpose, based on the expression of specific antigens by lymphocytes, which makes it possible to isolate and remove them using monoclonal antibodies. In addition, an immunorosette method of T-lymphocyte depletion of bone marrow transplant has been developed, which is based on the formation of complexes of CD4+ lymphocytes and specific monoclonal antibodies, effectively removed using apheresis. This method ensures the production of purified cellular material with 40-60% content of hematopoietic stem cells.

An increase in the number of progenitor cells due to the removal of mature formed elements of the blood from the leukapheresis product is achieved by countercurrent centrifugation followed by filtration (in the presence of a chelator - trisodium citrate) through columns containing nylon fibers coated with human immunoglobulin. The sequential use of these two methods ensures complete purification of the transplant from platelets, 89% from erythrocytes and 91% from leukocytes. Due to a significant decrease in the loss of HSCs, the level of CD34+ cells in the total cell mass can be increased to 50%.

The ability of the isolated hematopoietic stem cells to form colonies of mature blood cells in culture is used for the functional characterization of the cells. Analysis of the formed colonies allows identifying and quantifying the types of progenitor cells, the degree of their commitment, and establishing the direction of their differentiation. Clonogenic activity is determined in semi-solid media on methylcellulose, agar, plasma or fibrin gel, which reduce the migration activity of cells, preventing their attachment to the surface of glass or plastic. Under optimal cultivation conditions, clones develop from a single cell in 7-18 days. If a clone contains fewer than 50 cells, it is identified as a single cluster; if the number of cells exceeds 50, it is identified as a colony. The number of cells capable of forming a colony is taken into account (colony-forming units - CFU or colony-forming cells - COC). It should be noted that the parameters of CFU and COC do not correspond to the number of HSCs in the cell suspension, although they correlate with it, which once again emphasizes the need to determine the functional (colony-forming) activity of HSCs in vitro.

Among bone marrow cells, hematopoietic stem cells have the highest proliferative potential, due to which they form the largest colonies in culture. The number of such colonies is proposed to indirectly determine the number of stem cells. After the formation of colonies in vitro exceeding 0.5 mm in diameter and with a cell count of more than 1000, the authors tested such cells for resistance to sublethal doses of 5-fluorouracil and studied their ability to repopulate the bone marrow of lethally irradiated animals. According to the specified parameters, the isolated cells were almost indistinguishable from HSCs and received the abbreviation symbol HPP-CFC - colony-forming cells with high proliferative potential.

The search for better quality isolation of hematopoietic stem cells continues. However, hematopoietic stem cells are morphologically similar to lymphocytes and represent a relatively homogeneous set of cells with almost round nuclei, finely dispersed chromatin and a small amount of weakly basophilic cytoplasm. Their exact number is also difficult to determine. It is assumed that HSCs in human bone marrow occur with a frequency of 1 per 106 nucleated cells.

Identification of hematopoietic stem cells

To improve the quality of identification of hematopoietic stem cells, a sequential or simultaneous (on a multichannel sorter) study of the spectrum of membrane-bound antigens is carried out, and in HSCs the CD34+CD38 phenotype should be combined with the absence of linear differentiation markers, especially antigens of immunocompetent cells, such as CD4, surface immunoglobulins and glycophorin.

Almost all hematopoietic stem cell phenotyping schemes include determination of the CD34 antigen. This glycoprotein with a molecular weight of about 110 kDa, carrying several glycosylation sites, is expressed on the plasma cell membrane after activation of the corresponding gene localized on chromosome 1. The function of the CD34 molecule is associated with L-selectin-mediated interaction of early hematopoietic progenitor cells with the stromal basis of the bone marrow. However, it should be remembered that the presence of the CD34 antigen on the cell surface allows only a preliminary assessment of the HSC content in the cell suspension, since it is also expressed by other hematopoietic progenitor cells, as well as bone marrow stromal cells and endothelial cells.

During the differentiation of hematopoietic progenitor cells, CD34 expression is permanently reduced. Erythrocyte, granulocyte, and monocytic committed progenitor cells either weakly express CD34 antigen or do not express it on their surface at all (CD34 phenotype). CD34 antigen is not detected on the surface membrane of differentiated bone marrow cells and mature blood cells.

It should be noted that in the dynamics of differentiation of hematopoietic progenitor cells not only the level of CD34 expression decreases, but also the expression of the CD38 antigen, an integral membrane glycoprotein with a molecular weight of 46 kDa, which has NAD-glycohydrolase and ADP-ribosyl cyclase activity, progressively increases, which suggests its participation in the transport and synthesis of ADP-ribose. Thus, the possibility of double control of the degree of commitment of hematopoietic progenitor cells appears. The population of cells with the CD34+CD38+ phenotype, which constitutes from 90 to 99% of CD34-positive bone marrow cells, contains progenitor cells with limited proliferative and differentiating potential, whereas cells with the CD34+CD38 phenotype can claim the role of HSC.

Indeed, the bone marrow cell population described by the formula CD34+CD38- contains a relatively large number of primitive stem cells capable of differentiating in the myeloid and lymphoid directions. Under conditions of long-term cultivation of cells with the CD34+CD38- phenotype, it is possible to obtain all mature formed elements of the blood: neutrophils, eosinophils, basophils, monocytes, megakaryocytes, erythrocytes and lymphocytes.

It has been established relatively recently that CD34-positive cells express two more markers, AC133 and CD90 (Thy-1), which are also used to identify hematopoietic stem cells. The Thy-1 antigen is coexpressed with the CD117 receptor (c-kit) on CD34+ cells of the bone marrow, umbilical cord, and peripheral blood. It is a surface phosphatidylinositol-binding glycoprotein with a molecular weight of 25-35 kDa, which participates in cell adhesion processes. Some authors believe that the Thy-1 antigen is a marker of the most immature CD34-positive cells. Self-reproducing cells with the CD34+Thy-1+ phenotype give rise to long-term cultured lines with the formation of daughter cells. It is assumed that the Thy-1 antigen blocks regulatory signals that cause cell division arrest. Despite the fact that CD34+Thy1+ cells are capable of self-reproduction and the creation of long-term cultured lines, their phenotype cannot be attributed exclusively to HSCs, since the content of Thy-1+ in the total mass of CD34-positive cellular elements is about 50%, which significantly exceeds the number of hematopoietic cells.

More promising for the identification of hematopoietic stem cells should be recognized as AC133 - an antigen marker of hematopoietic progenitor cells, the expression of which was first detected on embryonic liver cells. AC133 is a transmembrane glycoprotein that appears on the surface of the cell membrane at the earliest stages of HSC maturation - it is possible that even earlier than the CD34 antigen. In the studies of A. Petrenko, V. Grishchenko (2003) it was established that AC133 is expressed by up to 30% of CD34-positive embryonic liver cells.

Thus, the ideal phenotypic profile of hematopoietic stem cells, according to current concepts, consists of a cellular outline, the contours of which should include configurations of the CD34, AC133 and Thy-1 antigens, but there is no room for the molecular projections of CD38, HLA-DR and the linear differentiation markers GPA, CD3, CD4, CD8, CD10, CD14, CD16, CD19, CD20.

A variation of the phenotypic portrait of HSCs may be the combination CD34+CD45RalowCD71low, since the properties of the cells described by this formula do not differ from the functional parameters of cells with the CD34+CD38 phenotype. In addition, human HSCs can be identified by the phenotypic features CD34+Thy-l+CD38Iow/'c-kit /low - only 30 such cells completely restore hematopoiesis in lethally irradiated mice.

The 40-year period of intensive research into HSCs, which are capable of both self-reproduction and differentiation into other cellular elements, began with the analysis of the general phenotypic characteristics of bone marrow cells, which made it possible to justify the use of bone marrow transplantation for the treatment of various pathologies of the hematopoietic system. New types of stem cells discovered later have not yet been widely used in clinical practice. At the same time, stem cells of umbilical cord blood and embryonic liver are capable of significantly expanding the scale of cell transplantation not only in hematology, but also in other areas of medicine, since they differ from bone marrow HSCs in both quantitative characteristics and qualitative features.

The volume of hematopoietic stem cell mass required for transplantation is usually obtained from bone marrow, peripheral and cord blood, and embryonic liver. In addition, hematopoietic progenitor cells can be obtained in vitro by multiplying ESCs with their subsequent directed differentiation into hematopoietic cellular elements. A. Petrenko, V. Grishchenko (2003) rightly note significant differences in the immunological properties and ability to restore hematopoiesis of HSCs of different origins, which is due to the unequal ratio of early pluripotent and late committed progenitor cells contained in their sources. In addition, hematopoietic stem cells obtained from different stem sources are characterized by quantitatively and qualitatively completely different associations of non-hematopoietic cells.

Bone marrow has already become a traditional source of hematopoietic stem cells. Bone marrow cell suspension is obtained from the ilium or sternum by washing under local anesthesia. The suspension obtained in this way is heterogeneous and contains a mixture of HSCs, stromal cell elements, committed progenitor cells of myeloid and lymphoid lines, as well as mature formed elements of the blood. The number of cells with the CD34+ and CD34+CD38 phenotypes among bone marrow mononuclear cells is 0.5-3.6 and 0-0.5%, respectively. Peripheral blood after G-CSF-induced mobilization of HSCs contains 0.4-1.6% CD34+ and 0-0.4% CD34+CD38.

The percentage of cells with the immunophenotypes CD34+CD38 and CD34+ is higher in umbilical cord blood - 0-0.6 and 1-2.6%, and their maximum number is detected among the hematopoietic cells of the embryonic liver - 0.2-12.5 and 2.3-35.8%, respectively.

However, the quality of the transplanted material depends not only on the number of CD34+ cells it contains, but also on their functional activity, which can be assessed by the level of colony formation in vivo (bone marrow repopulation in lethally irradiated animals) and in vitro - by colony growth on semi-liquid media. It turned out that the colony-forming and proliferative activity of hematopoietic progenitor cells with the CD34+CD38 HLA-DR phenotype isolated from the embryonic liver, fetal bone marrow and cord blood significantly exceeds the proliferative and colony-forming potential of hematopoietic cells of the bone marrow and peripheral blood of an adult. Quantitative and qualitative analysis of HSCs of various origins revealed significant differences in both their relative content in the cell suspension and functional capabilities. The maximum number of CD34+ cells (24.6%) was found in the transplanted material obtained from fetal bone marrow. The bone marrow of an adult contains 2.1% of CD34-positive cellular elements. Among the mononuclear cells of the peripheral blood of an adult, only 0.5% have the CD34+ phenotype, while in cord blood their number reaches 2%. At the same time, the colony-forming capacity of CD34+ cells of fetal bone marrow is 2.7 times higher than the clonal growth capacity of bone marrow hematopoietic cells of an adult, and umbilical cord blood cells form significantly more colonies than hematopoietic elements isolated from the peripheral blood of adults: 65.5 and 40.8 colonies/105 cells, respectively.

Differences in proliferative activity and colony-forming capacity of hematopoietic stem cells are associated not only with different degrees of their maturity, but also with their natural microenvironment. It is known that the intensity of proliferation and the rate of differentiation of stem cells are determined by the integral regulatory effect of a multicomponent system of growth factors and cytokines that are produced both by the stem cells themselves and by the cellular elements of their matrix-stromal microenvironment. The use of purified cell populations and serum-free media for cell culturing made it possible to characterize growth factors that have a stimulating and inhibitory effect on stem cells of various levels, progenitor cells, and cells committed in one or another linear direction. The results of the studies convincingly indicate that HSCs obtained from sources with different levels of ontogenetic development differ both phenotypically and functionally. HSCs at earlier stages of ontogenesis are characterized by high self-reproduction potential and high proliferative activity. Such cells are distinguished by longer telomeres and undergo commitment to form all hematopoietic cell lines. The immune system response to HSCs of embryonic origin is delayed, since such cells weakly express HLA molecules. There is a clear gradation of the relative content of HSCs, their self-renewal capacity and the number of types of commitment lines they form: CD34+ cells of embryonic liver > CD34+ cells of cord blood > CD34+ cells of bone marrow. It is important that such differences are inherent not only to the intra-, neo- and early postanatal periods of human development, but also to the entire ontogenesis - the proliferative and colony-forming activity of HSCs obtained from the bone marrow or peripheral blood of an adult is inversely proportional to the age of the donor.