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 blood cells, as well as a number of specialized tissue cells of the immune system.

The hypothesis about the presence of a common precursor of all blood cells, as well as the term stem cell, belongs to A. Maksimov (1909) .The potential for the formation of the cell mass in HSC is enormous - bone marrow stem cells daily produce 10 cells that make up the uniform elements of peripheral blood. The fact of the existence of hematopoietic stem cells was established in 1961 in experiments on the restoration of hemopoiesis in mice receiving a lethal dose of radioactive irradiation destroying the stem cells of the bone marrow. Ie transplantation of syngeneic bone marrow cells so lethally irradiated animals discrete foci of hematopoiesis were detected in the spleen of recipients whose source - single clonogenic progenitor cells.

Then, the ability of hematopoietic stem cells to self-support was demonstrated, which provides the function of hematopoiesis during ontogenesis. In the process of embryonic development, HSCs are characterized by high migration activity, which is necessary for their migration to the sites of the hematopoietic organs. This property of HSCs is retained also in ontogenesis - due to their constant migration, a permanent renewal of the pool of immunocompetent cells takes place. The ability of GSK to migrate, penetrate through histohematological barriers, implantation in tissues and clonogenic growth served as the basis for bone marrow transplantation in a number of diseases associated with the pathology of the hematopoiesis 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 allocation. Immunophenotypically, human HSCs are characterized as CD34 + NK cells capable of migrating to the bloodstream and populating the organs of the immune system or repopulating the stroma of the bone marrow. It is necessary to clearly realize that HSCs are not the most immature cells of the bone marrow, but come from predecessors, which include the dormant fibroblast-like SB34-negative cells. It has been established that cells with the CD34 phenotype are capable of reaching the general bloodstream, where they change their phenotype on CD34 +, but when they return to the bone marrow under the influence of the microenvironment, they again become CD34-negative stem cell elements. At rest, CD34-cells do not respond to paracrine regulatory stromal signals (growth factors, cytokines). However, in situations that require intensification of hematopoiesis, stem cells with the CD34 phenotype respond to differentiation signals by the formation of both hematopoietic and mesenchymal progenitor cells. Hooding is carried out by direct contact of HSC with cellular elements of bone marrow stroma, represented by a complex network of macrophages, reticular endothelial cells, osteoblasts, stromal fibroblasts and extracellular matrix. The bone marrow stromal stem is not just a matrix or a "skeleton" for hemopoietic tissue, it performs 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 basis of the constantly updated hemopoiesis system is the hemopoietic stem cell, which is capable of prolonged self-maintenance, is polypotent (from the point of view of hemopoiesis). During the process of committing, HSCs undergo primary differentiation and form clones of cells that differ in their cytomorphological and immunophenotypic characteristics. The consecutive formation of primitive and committed progenitor cells is completed by the formation of morphologically identifiable ancestor cells of various hematopoietic lines. The result of the subsequent stages of a complex multi-stage hematopoiesis process is the maturation of cells and the release into the peripheral blood of mature elements - erythrocytes, leukocytes, lymphocytes and platelets.

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

Sources of hematopoietic stem cells

Hematopoietic stem cells are considered the most studied stem source, which is largely due to their use in the clinic for bone marrow transplantation. At first glance, a lot is known about these cells. To some extent this is true, because intermediate and mature descendants of HSC are the most accessible cellular elements, each of which (red blood cells, leukocytes, lymphocytes, monocytes / macrophages and platelets) has been thoroughly studied at all levels - from light to electron microscopy, from biochemical and immunophenotypic characteristics before identification by PCR analysis. However, monitoring of morphological, ultrastructural, biochemical, immunophenotypic, biophysical and genomic parameters of HSC has not allowed to get answers to many problematic issues, the solution of which is necessary for the development of cell transplantology. Mechanisms for stabilizing HSC in the dopant state, their activation, reaching the stage of symmetrical or asymmetric division, and, most importantly, committing to the formation of so functionally different blood elements as erythrocytes, leukocytes, lymphocytes and platelets have not been established so far.

The presence in the bone marrow of cells with the CD34 phenotype, which are the ancestors of both mesenchymal and hematopoietic stem cells, raised the question of the existence of the earliest, cellular-differentiated precursors of CD4-negative cells into the stromal and hemopoietic lines. By the method of long-term culture, a so-called long-term culture-initiating cell (LTC-IC) was obtained. The lifetime of such progenitor cells with colony-forming activity on the bone marrow stromal base for a certain combination of growth factors is more than 5 weeks, whereas the viability of the committed colony-forming units (CFU) in culture is only 3 weeks. Currently, LTC-IC is considered to be a functional analog of HSC, since at a high repopulation potential about 20% of LTC-IC are characterized by the phenotype CD34 + CD38- and show a high ability for self-renewal. Such cells are found in the bone marrow of a person with a frequency of 1: 50,000. However, the closest to HSC is the recognition of myeloid lymphoidinitating cells, which are obtained under conditions of long-term (15 weeks) cultivation. Such cells, designated as LTC, are found 10 times less frequently in human bone marrow cells than LTC-IC, and form the cell lines of both myeloid and lymphoid hematopoietic growths.

Although marking of hematopoietic stem cells with monoclonal antibodies followed by immunophenotypic identification is the main method of recognition and selective sorting of hematopoietic cells with stem potential, the clinical application of the HSCs isolated in this way is limited. Blocking with antibodies of the CD34 receptor or other marker antigens in the process of immunopositive sorting inevitably changes the properties of the cell isolated with it. More preferable is the immuno-negative secretion of HSC on magnetic columns. However, in this case, as a rule, monoclonal antibodies fixed on a metallic carrier are used for sorting. In addition, importantly, both methods of isolating HSC are based on phenotypic, and not on functional characteristics. Therefore, many researchers prefer to use the analysis of clonogenic parameters of HSC, which allows the size and composition of colonies to determine the degree of maturity and the direction of differentiation of progenitor cells. It is known that in the process of committing the number of cells and the number of their types in the colony decreases. The hematopoietic stem cell and its early daughter cell, called "granulocyte-erythrocyte-monocyte-megakaryocytic colonization unit" (CFU-GEMM), create large multilinear colonies in culture containing respectively granulocytes, erythrocytes, monocytes and megakaryocytes. The granulocyte-monocyte-colony forming unit (CFU-GM) below forms the colony of granulocytes and macrophages, and the granulocyte colony-forming unit (CFU-G) is only a small colony of mature granulocytes. Early erythrocyte precursor - burst-forming unit of erythrocytes (CFU-E) - is the source of large, and more mature colony-forming unit of erythrocytes (CFU-E) - small erythrocyte colonies. In the general population, with the growth of cells on semi-solid media, it is possible to identify cells forming 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 the separation of HSC contains a significant number of concomitant cells. In this connection, a preliminary purification of the transplant, first of all, of the active cells of the immune system of the donor is necessary. Usually, immunosection based on lymphocyte expression of specific antigens is used for this, which makes it possible to isolate and remove them using monoclonal antibodies. In addition, the immunophoretic technique of T-lymphocytic depletion of the bone marrow transplant has been developed, which is based on the formation of complexes of CD4 + lymphocytes and specific monoclonal antibodies that are effectively removed by apheresis. This technique provides a purified cell material with 40-60% hematopoietic stem cells.

An increase in the number of precursor cells by removing mature blood cells 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 consistent application of these two techniques ensures a complete cleansing of the transplant from platelets, 89% from erythrocytes and 91% from white blood cells. Due to a significant reduction in HSC losses, the level of CD34 + cells in the total cell mass can be increased to 50%.

For the functional characteristics of isolated hematopoietic stem cells, their ability to create colonies of mature blood elements in culture is used. Analysis of the formed colonies makes it possible to identify and quantify the types of progenitor cells, the degree of their confor- mation, and also to establish the direction of their differentiation. Clonogenic activity is determined in semi-solid media on methylcellulose, agar, plasma or fibrin gel, which reduces the migration activity of cells, which prevents their attachment to the surface of glass or plastic. Under optimal culture conditions, clones from a single cell develop within 7-18 days. If there are less than 50 cells in the clone, it is identified as a single cluster if the number of cells exceeds 50 - as a colony. The number of cells capable of forming a colony (colony forming units - CFU or colony-forming cells - COCs) is taken into account. It should be noted that the parameters of CFU and COC do not correspond to the amount of HSC in the cell suspension, although it correlates with it, which again underscores the need to determine the functional (colony-forming) activity of HSC in vitro.

Among the cells of the bone marrow, hematopoietic stem cells have the highest proliferative potential, due to which the largest colonies are formed in culture. By the number of such colonies, it is proposed to indirectly determine the number of stem cells. After in vitro colony formation exceeding 0.5 mm in diameter and with a cell number of more than 1000, the authors tested these cells for resistance to sublethal doses of 5-fluorouracil and studied their ability to repopulate the bone marrow of deadly irradiated animals. According to these parameters, the isolated cells did not differ much from HSC and received the abbreviation symbol HPP-CFC - colony-forming cells with a high proliferative potential.

The search for the possibility of a more qualitative selection of hematopoietic stem cells continues. However, stem hematopoietic 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. The exact number is also difficult to determine. It is assumed that GSK in the bone marrow of a person occurs with a frequency of 1 per 106 nucleus-containing cells.

Identification of hematopoietic stem cells

To improve the quality of hematopoietic stem cell identification, sequential or simultaneous (multi-channel assay) studies of the spectrum of membrane-bound antigens are carried out. In the HSC, the CD34 + CD38 phenotype must be combined with the absence of linear differentiation markers, especially antigens of immunocompetent cells such as CD4, surface immunoglobulins and glycophorin.

Practically all schemes of phenotyping of hematopoietic stem cells include the determination of CD34 antigen. This glycoprotein with a molecular mass of about 110 kDa, carrying several glycosylation sites, is expressed on the plasma cell membrane after activation of the corresponding gene located on the 1 st chromosome. The function of the CD34 molecule is associated with the L-selectin-mediated interaction of early hematopoietic precursor cells with the bone marrow stromal base. However, it should be remembered that the presence of CD34 antigen on the cell surface allows only a preliminary assessment of the content of HSC in the cell suspension, since it is expressed by other precursor cells of hemopoiesis, 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 monocyte commited progenitor cells either weakly express CD34 antigen, or it is absent on their surface at all (phenotype CD34). On the surface membrane of differentiated cells of the bone marrow and mature blood cells, CD34 antigen is not detected.

It should be noted that the dynamics of differentiation of hematopoietic progenitor cells not only reduces the expression level of CD34, but also the expression of CD38 antigene, an integral membrane glycoprotein with a molecular mass of 46 kDa, which has NAD-glycohydrolase and ADP-ribosyl cyclase activity, progressively increases, which implies its participation in transport and synthesis of ADP-ribose. Thus, there is the possibility of a dual control of the degree of committing the hematopoietic progenitor cells. The population of cells with the CD34 + CD38 + phenotype, comprising 90 to 99% of CD34-positive bone marrow cells, contains progenitor cells with limited proliferative and differentiation potential, whereas cells with the CD34 + CD38 phenotype may claim the role of GSK.

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

Relatively recently, CD34-positive cells express two more markers - AC133 and CD90 (Thy-1), which are also used to identify hematopoietic stem cells. Thy-1 antigen is co-expressed with the CD117 receptor (c-kit) on CD34 + cells of bone marrow, umbilical cord and peripheral blood. This is a surface phosphatidylinositol binding glycoprotein with a molecular weight of 25-35 kDa, which takes part in cell adhesion processes. Some authors believe that the Thy-1 antigen is the marker of the most immature CD34-positive cells. Self-reproducing cells with the phenotype CD34 + Thy-1 + give rise to long-cultivated lines with the formation of daughter cells. It is suggested that the Thy-1 antigen blocks the regulatory signals that cause the cell division to stop. Despite the fact that CD34 + Thy1 + cells are capable of self-reproduction and the creation of long-cultivated lines, their phenotype can not be attributed exclusively to HSC, since the Thy-1 + content in the total mass of CD34-positive cell elements is about 50% the number of hematopoietic cells.

More promising for the identification of hematopoietic stem cells is 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 GSK maturation - it is possible that even earlier than the antigen CD34. In the studies of A. Petrenko and V. Grishchenko (2003), it was found that AC133 expresses up to 30% of CD34-positive cells of the embryonic liver.

Thus, the ideal phenotypic profile of hematopoietic stem cells, according to today's ideas, is composed of a cellular outline in which contours of antigens CD34, AC133 and Thy-1 should be present, but there is no place for molecular projections of CD38, HLA-DR and linear differentiation markers GPA , CD3, CD4, CD8, CD10, CD14, CD16, CD19, CD20.

The variation of the phenotypic portrait of HSC can be a combination of CD34 + CD45RalowCD71low, since the properties of the cells described by this formula do not differ from the functional parameters of cells with the phenotype CD34 + CD38. In addition, human GSK can be identified by the phenotypic signs of CD34 + Thy-1 + CD38Iow / 'c-kit / low - only 30 of these cells completely restore hematopoiesis in lethally irradiated mice.

With the analysis of the general phenotypic characteristics of bone marrow cells, the 40-year period of intensive GSK research, which is simultaneously capable of both self-reproduction and differentiation into other cellular elements, has begun, which allowed to substantiate the application of bone marrow transplantation to treat various pathologies of the hematopoiesis system. Recently discovered new types of stem cells have not yet been widely used in clinical practice. At the same time, stem cells of umbilical cord blood and embryonic liver can significantly expand the scale of cell transplantation not only in hematology, but also in other fields of medicine, since they differ from the HSC of the bone marrow both by quantitative characteristics and qualitative characteristics.

The volume of stem hematopoietic cell mass required for transplantation is usually obtained from bone marrow, peripheral and cord blood, and also from the embryonic liver. In addition, hematopoiesis progenitor cells can be obtained in vitro by the propagation of ESCs followed by their directional differentiation into hematopoietic cell elements. A. Petrenko, V. Grishchenko (2003) rightly note significant differences in immunological properties and the ability to restore hematopoiesis of HSC of different origin, which is due to the unequal correlation of the early pluripotent and late committed precursor cells contained in their sources. In addition, hematopoietic stem cells derived from different stem sources have completely different associations of non-hemopoietic cells quantitatively and qualitatively.

A traditional source of hematopoietic stem cells is the bone marrow. A suspension of bone marrow cells is obtained from the abdominal bone or sternum by leaching under local anesthesia. The suspension thus obtained is heterogeneous and contains a mixture of HSC, stromal cell elements, committed progenitor cells of the myeloid and lymphoid lines, as well as mature blood elements. The number of cells with the phenotypes CD34 + and CD34 + CD38 among bone marrow mononuclear cells is 0.5 to 3.6 and 0 to 0.5%, respectively. Peripheral blood after G-CSF-induced mobilization of HSC contains 0.4-1.6% of CD34 + and 0-0.4% of CD34 + CD38.

The percentage of cells with immunophenotypes CD34 + CD38 and CD34 + in cord blood is higher - 0-0.6 and OD-2.6%, and their maximum number is detected among 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 contained in it, but also on their functional activity, which can be assessed in terms of the level of colony formation in vivo (bone marrow repopulation in lethally irradiated animals) and in vitro - the growth of colonies on semi-fluid media . It turned out that the colony-forming and proliferative activity of hematopoietic progenitor cells with the phenotype CD34 + CD38 HLA-DR isolated from the embryonic liver, fetal bone marrow and cord blood significantly exceeds the proliferative and colony-forming potential of the hematopoietic cells of the bone marrow and peripheral blood of an adult. Quantitative and qualitative analysis of HSC of various origin 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 transplantation material obtained from fetal bone marrow. The bone marrow of an adult human contains 2.1% of CD34-positive cell elements. Among the mononuclear cells of the peripheral blood of an adult human, only 0.5% have a CD34 + phenotype, whereas in cord blood, their number reaches 2%. At the same time, colony-forming ability of CD34 + cells of fetal bone marrow is 2.7 times higher than the possibility of clonal growth of bone marrow hematopoietic cells of an adult human, and umbilical cord blood cells form much more colonies than haematopoietic elements isolated from peripheral blood of adults: 65.5 and 40 , 8 colonies / 105 cells, respectively.

Differences in proliferative activity and colony-forming ability 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 stem cell differentiation is 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 the purpose of cell culture has made it possible to characterize growth factors that exert a stimulating and inhibitory effect on stem cells of different levels, progenitor cells, and cells committed in a particular linear direction. The results of the conducted studies convincingly testify that GSK, obtained from sources with different levels of ontogenetic development, differ both phenotypically and functionally. For GSK, staying at earlier stages of ontogeny, characterized by a high potential for self-reproduction and high proliferative activity. Such cells are distinguished by a longer telomere length and are subjected to committing with the formation of all hematopoietic cell lines. The reaction of the immune system to HSC embryonic origin is delayed, since such cells mildly express HLA molecules. There is a clear gradation in the relative content of HSCs, their ability to self-renew and the number of types of lines of commission they generate: CD34 + embryonic liver cells> CD34 + cord blood cells> CD34 + bone marrow cells. It is important that such differences are inherent not only in intra-, neo-and early post-natal periods of human development, but also in the ontogenesis-the proliferative and colony-forming activity of HSC obtained from the bone marrow or peripheral blood of an adult is inversely proportional to the age of the donor.