Hemopoietic stem cells of umbilical cord blood

Umbilical cord blood acts as a source of hematopoietic stem cells on the proliferative potential and repopulation capabilities of hematopoietic cells. It has been repeatedly shown that, at the time of delivery, the umbilical cord blood contains a sufficiently large number of poorly committed hematopoietic progenitor cells. Some authors believe that the advantage of transplanting hematopoietic stem cells of cord blood is that there is no need to search for a donor compatible with HLA antigens. In their opinion, immaturity of the newborn's immune system causes a decreased functional activity of immunocompetent cells and, accordingly, the rate of development of the severe "graft versus host" reaction, as compared with bone marrow transplantation. At the same time, the survival rate of the umbilical cord blood transplant is not lower than that of the bone marrow cells, even if a smaller number of HSCs is administered, based on 1 kg of the body weight of the patient. However, in our opinion, the questions of the optimal number of umbilical cord transplant cells necessary for effective engraftment in the recipient organism, their immunological compatibility, and a number of aspects of the problem of transplantation of cord blood stem cells require more serious analysis.

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

Obtaining hematopoietic stem cells of umbilical cord blood

The procedure for obtaining hematopoietic stem cells of umbilical blood requires its collection immediately after the birth of the child and separating it from the afterbirth, when the placenta is in utero or ex utero, as well as in the cesarean section, but also ex utero. It is shown that in the case of a reduction in the time from birth to separation of the newborn from the placenta to 30 seconds, the volume of cord blood obtained increases on average by 25-40 ml. With a later procedure, the same amount of blood is lost. It is established that the early separation of the child from the afterburn does not entail any negative consequences for the newborn.

In the Russian Research Institute of Hematology and Transfusiology, effective and low-cost technologies for obtaining umbilical cord blood have been developed both in physiological births ((70.2 + 25.8) ml) and in cesarean section (73.4 + 25.1) ml. The technique of umbilical cord separation with a sufficiently high yield of nucleated and mononuclear cells (83.1 + 9.6) and (83.4 + 14.1)%, respectively, is proposed. The method of cryopreservation of umbilical cord blood has been improved, which ensures high safety of mononuclear cells and CFU-GM - (96.8 + 5.7) and (89.6 + 22.6)%, respectively. The efficiency of the drainage method of cord blood sampling using the "Kompoplast-300" container (Russia) was determined. Cord blood sampling was carried out immediately after the birth of the child and separation from the afterbirth, in conditions of placement of the placenta in utero or ex utero. Before the cord vein puncture, the umbilical cord was treated once with 5% tincture of iodine, and then twice with 70% ethyl alcohol. The blood flowed through the connecting tubes to the container spontaneously. The duration of the fence took no more than 10 minutes. The volume of 66 samples collected by drainage method of umbilical cord blood samples averaged (72 + 28) ml, and the number of leukocytes in the average total sample volume - (1,1 + 0,6) х х 107. When analyzing umbilical cord blood for sterility (bacterial contamination, HIV-1, hepatitis B and C viruses, syphilis and cytomegalovirus infection), IgG antibodies to hepatitis C virus were detected in only one sample. In another study, the placenta was placed immediately downstream of the fetal surface by a special frame, the umbilical cord was treated with a 5% solution iodine and 75% ethyl th alcohol. The veins of the umbilical cord were drained with a needle from the transfusion system (G16). The blood drained into the container spontaneously. The volume of blood taken in this way averaged (55 + 25) ml. In G. Kogler et al. (1996), umbilical cord blood was withdrawn by a closed method and large volumes of blood were obtained - on average (79 + 26) ml. The authors note that among the 574 umbilical cord blood samples, about 7% contain less than 40 ml of blood, which makes it impossible to use them for transplantation. K. Isoyama and co-authors (1996), taking cord blood by active exfusion using syringes, received an average of 69.1 ml of blood (cord blood volume varied from 15 to 135 ml). Finally, A. Abdel-Mageed PI co-authors (1997) managed to obtain on average 94 ml of umbilical cord blood (from 56 to 143 ml) by catheterization of the vulva.

To reduce the risk of iatrogenic infection and contamination with maternal secretions, a closed blood sampling system was developed based on the widely used transfusion system Baxter Healthcare Corp., Deerfield, IL (USA) containing 62.5 ml of CPDA (citrate-phosphate-dextrose with adenine) as anticoagulant. The technology of obtaining the material is of primary importance for the preparation of a qualitative sample with respect to the volume, content and purity of the cell suspension. Of the existing methods of cord blood sampling, which are classified into closed, half-open and open systems, the first should be followed, since the risk of microbial contamination of the material and the contamination of the cell suspension by mother cells are significantly reduced in a closed system.

A. Nagler and co-authors (1998) conducted a comparative analysis of the efficacy of all three cord blood sampling systems. In the first variant, the procedure was performed in a closed system by exfusion of blood directly into the container. In the second variant, cord blood was obtained by the method of active blood exfusion of syringes1 with further washing of the veins of the placenta and simultaneous drainage of blood into the container (open method). In the third variant, the blood was withdrawn in a semi-open system by actively extracting it with syringes and washing through the artery of the umbilical cord with simultaneous exfusion into the container. In the first version, the authors received umbilical cord blood in the volume (76.4 + 32.1) ml with a leukocyte count (10.5 + 3.6) x 10 ⁶ per 1 ml of blood. In the second variant, the corresponding indices were (174.4 + 42.8) ml and (8.8 + 3.4) x 10 ⁶ / ml; in the third - (173.7 + 41.3) ml and (9.3 + 3.8) x 10 ⁶ / ml. The most frequent infection of umbilical cord blood samples was noted when using an open system. A direct correlation between the placenta mass and the volume of extracted blood is established - with increasing placenta mass, the amount of blood collected increases.

After the sampling of umbilical cord blood, the separation step follows-isolation of mononuclear cells and purification of the cell suspension from erythrocytes. Under the experimental conditions, the nucleated cells are isolated by the method of their sedimentation with methylcellulose during the lysis of ammonium erythrocytes by chloride. However, for clinical purposes, methylcellulose should not be used, since the loss of hematopoietic stem cells reaches 50-90%. Lysis of erythrocytes in connection with large volumes of working solution in the clinic is also almost not carried out, although the percentage of isolation in this way of nucleated cells with the phenotype CD34 +, as well as precursor cells with CFU-GM and CFU-GEMM functions is much higher. A new agent for the isolation of mononuclear cells in the density gradient buyant density solution (BDS72) has been reported. This substance has the following physiological parameters: pH - 7.4, osmolality - 280 mosm / kg, density - 1.0720 g / ml. According to the authors, with its help it is possible to isolate up to 100% of CD34-positive cells and remove 98% of red blood cells. However, the clinic does not yet apply BDS72.

In proven methods for isolating nucleated cells from cord blood, a 10% solution of hydroxyethyl starch or a 3% gelatin solution is usually used. Efficiency of precipitation of erythrocytes and isolation of nucleated cells in both cases is approximately equal. However, when gelatin is used as a sedimenting agent, it is possible to obtain a somewhat larger amount of CFU-GM than with hydroxyethyl starch. It is assumed that the differences in the efficiency of the release of CFU-GM are due to the unequal precipitation rate of individual core cell fractions, or the ability of hydroxyethyl starch molecules to be absorbed on the surface of the receptors of hemopoietic cells and thereby block their sensitivity to colony-stimulating factors that are used in CFU-GM cultivation in vitro. Nevertheless, both sedimenters may well be suitable for isolating nucleated cells when creating large-scale umbilical cord blood banks.

Methods of separation and cryopreservation of umbilical cord blood in principle do not differ from those used in the work with hematopoietic stem cells of peripheral blood and bone marrow of adult donors. But when preparing a large number of umbilical cord blood samples for its banks, the methods of separation must be, first of all, low-cost. Therefore, now, unfortunately, for clinical needs already used routine methods of isolation and cryopreservation of umbilical cord blood cells are used, and more effective but financially effective methods remain the lot of experimenters.

In general, the criteria for estimating the number of hematopoietic cells and the requirements for the study of umbilical cord blood samples were established in order to identify infectious agents. In order to secure transplantation of hematopoietic cord blood cells, all blood samples must be examined primarily for hematogenous infections and genetic diseases. A number of authors recommend additional special methods for the investigation of umbilical cord blood in order to diagnose genetic diseases such as a-thalassemia, sickle cell anemia, adenosine deaminase deficiency, Bruton agammaglobulinemia, Harler and Ponter disease.

According to the recommendations of L. Ticheli and co-authors (1998), in each cord blood sample it is necessary to determine the number of nucleated cells, SB34-positive cells and CFU-GM, conduct HLA-typing, determine the blood group by ABO and its Rh-accessory. In addition, bacteriological inoculation, serological testing for HIV and cytomegalovirus infection, HBsAg, viral hepatitis C, HTLY-I and HTLV-II (human T-cell leukemia), syphilis and toxoplasmosis are carried out. A polymerase chain reaction to cytomegalovirus and HIV infection is mandatory.

The very procedure for obtaining cord blood should be carried out in strict accordance with the principles of medical bioethics. Before the start of blood collection it is necessary to obtain the consent of the pregnant woman for its implementation. Preliminary interview with a pregnant woman for obtaining informed consent to perform all manipulations, from blood exfusion to the completion of documentation, is carried out only by medical personnel. In no case is it acceptable to perform any of these procedures by personnel who have a biological, chemical, pharmaceutical and other non-medical education, in view of the violation of established norms of bioethics and human rights. With positive tests for carriage of HBsAg, the presence of antibodies to hepatitis C pathogens, HIV infection and syphilis, cord blood is not taken away, and samples of already collected blood are discarded and destroyed. It should be noted that the carriage of latent infections in newborns is much less common than in adults, therefore, the probability of hematogenous transfer and development of infectious complications in infusions of hematopoietic blood cells is significantly lower than in the case of the use of adult donors for bone marrow transplantation.

An important point in the application of cord blood in the clinic is the evaluation of the transplant, which is based on the determination of the number of hematopoietic stem cells in the cord blood sample and the cell doses required for transplantation. Standards for the optimal number of umbilical cord blood cells required for transplantation have not yet been developed. There is no generally accepted point of view even on such routine parameters as the number of CD34-positive cells and CFU-GM. Some authors evaluate the potential of hematopoietic cells by analyzing long-term cultures with the determination of the concentration of colony-forming units common to granulocytes, erythrocytes, monocytes and megakaryocytes - CFU-GEMM.

However, in clinical settings, the standard evaluation of cord blood transplantation usually includes only the determination of the number of nucleated or mononuclear cells.

Storage of hematopoietic stem cells of umbilical cord blood

Some problems exist in the technology of storing hematopoietic cord blood cells. In the cryopreservation of hemopoietic stem cells, in order to achieve the optimal mode of their freezing, it is necessary to minimize the amount of cord blood as much as possible, and also to remove red blood cells beforehand in order to avoid hemolysis and the danger of developing an incompatibility reaction with red blood cell antigens (ABO, Rh). For these purposes, various methods for isolating nucleated cells are suitable. In the early 90s of the last century, the most widely used method was the isolation of nucleated cells in a density gradient based on a ficoll with a density of 1.077 g / ml or percola with a density of 1,080 g / ml. Separation of cord blood in the density gradient allows isolating predominantly mononuclear cells, but leads to significant losses of hematopoietic progenitor cells - up to 30-50%.

The sedimentation efficiency of hydroxyethyl starch in the process of isolation of umbilical cord blood cells is estimated in different ways. Some authors indicate a low quality of separation using this method, while other researchers, on the contrary, among all possible methods prefer to allocate HSC cord blood precisely using a 6% solution of hydroxyethyl starch. This highlights the high efficiency of sedimentation of hemopoietic cells, which, according to some data, reaches from 84% to 90%.

Supporters of another point of view believe that practically all methods of fractionation involve large losses of nucleated cells and suggest separation by centrifugation, dividing the umbilical cord blood into 3 fractions: erythrocyte, leukocyte ring and plasma. By isolating the cells in this way, the authors found that the content of mononuclear cells, early hematopoietic progenitor cells, and cells with the immunophenotype CD34 + ultimately amounted to 90, 88, and 100% of the original level, respectively. Similar values of the growth of umbilical cord blood cells purified by this method were obtained by other researchers: 92% of nucleated, 98% - mononuclear, 96% - CD34-positive cells and 106% of colony-forming units were isolated after sedimentation.

In the late 1990s, gelatin was widely used as a sedimenting agent. In clinical practice, with the help of gelatin, hematopoietic stem cells from umbilical cord blood have been isolated since 1994. When using a 3% solution of gelatin, the efficiency of isolation of nucleated cells reaches 88-94%. The widespread use of gelatin in the development of the cord blood bank has confirmed its advantages over other sedimentation agents. A comparative analysis of the efficacy of all the above methods of isolating nucleated cells in the conditions of their sequential use on each of the tested umbilical cord samples proved that the optimal sedimentator for the yield of mononuclear cells with the CD34 + / CD45 + phenotype, as well as the number of CFU-GM and CFU-GEMM is 3% gelatin solution. Significantly less effective were methods using a gradient of ficoll density, as well as the use of hydroxyethyl starch and methyl cellulose, in which the loss of hemopoietic cells reached 60%.

The expansion of the volume of stem cell transplantation of umbilical cord blood is associated not only with the development of methods for their production, but also storage. There are many problems directly related to the preparation of cord blood for long-term storage and the choice of the optimal cryopreservation technology for its samples. Among them are the issues of the expediency of performing separation procedures, the use of various cryopreserving media, and the use of methods for preparing thawed cells for transplantation. Transportation of native samples of cord blood is often carried out from regions remote from hematological centers. In connection with this, the problem arises of the permissible periods for storage of cord blood from the moment of its receipt to the beginning of cryopreservation, which is of particular importance in the development of cord blood banks.

Investigation of the functional activity of hematopoietic cord blood cells after prolonged storage (up to 12 years) in liquid nitrogen allowed us to establish that about 95% of the hematopoietic cells do not lose their high proliferative capacity during this time. S. Yurasov and co-authors (1997) have shown that storage of umbilical cord blood at room temperature (22 ° C) or at 4 ° C for 24 and 48 hours does not significantly reduce the viability of hematopoietic cells, which is 92 and 88%. However, if the storage time is extended to three days, the number of viable nucleated cells in the cord blood is significantly reduced. At the same time, during other studies, it was found that when stored for 2-3 days at a temperature of 22 or 4 ° C, the viability of mature granulocytes, rather than hematopoietic cells, primarily affects.

The viability of hematopoietic stem cells of cord blood can be adversely affected by system components for its collection. Analysis of the effect of various anticoagulants, whose mechanism of action is due to the binding of calcium ions (ACD, EDTA, XAPD-1), to hematopoietic progenitor cells under cord blood storage conditions from 24 to 72 hours revealed their adverse effect on the viability of nucleated cells. In this regard, the authors recommend using PBS (phosphate buffer solution) with the addition of native heparin without a preservative at a concentration of 20 U / ml, which, in their opinion, allows prolonging the storage time of unfractionated cord blood to 72 hours and preserves the functional activity of the colony-forming units. However, when studying the safety of CFU-GM and CFU-G, it is shown that the cord blood storage time prior to cryopreservation should not exceed nine hours. Obviously, in this case, the principle should operate, according to which, in the presence of conflicting data, the minimum recommended shelf life of cord blood should be used and proceed to programmable freezing of the isolated cells as soon as possible.

When freezing hematopoietic stem cells of umbilical cord blood, a 10% solution of DMSO is usually used as a cryoprotectant. However, in addition to the expressed cryoprotective effect, dimethylsulfoxide in this concentration has a direct cytotoxic effect, even under the condition of minimal exposure to the blood-forming cells of the umbilical cord blood. To reduce the cytotoxic effect of DMSO, a zero exposure temperature, an increase in the speed of all manipulations and repeated washing after thawing of umbilical cord samples are applied.

The Institute of Hematology and Transfusiology of the Academy of Medical Sciences of Ukraine has been developing a scientific direction since 1995, whose goal is to study the cord blood as an alternative source of stem hemopoietic cells. In particular, new technologies for low-temperature cryopreservation of hemopoietic cells of unfractionated and fractionated cord blood have been developed. As a cryoprotectant, low molecular weight medical polyvinylpyrolidone is used. The method of cryopreservation of unfractionated cord blood is based on the original technology of pre-preparation of cells for freezing and the technique of special treatment of cell suspension immediately before transplantation.

One of the most important factors affecting the level of functional activity of cryopreserved hemopoietic stem cells is the cooling rate of cell suspension, especially during the crystallization phase. A software approach to solving the problem of speed and time of freezing provides great opportunities for creating simple and highly effective methods of cryopreservation, without washing the cell suspension from cryoprotectants before transplantation.

The stages of immediate freezing and thawing are most dangerous for the viability of cells during their preparation. When freezing the hemopoietic cells, a significant part of them can be destroyed at the moment of transition of the intercellular medium from the liquid to the solid phase - crystallization. To reduce the percentage of cell death, cryoprotectants are used, the mechanisms of action and cryoprotective effectiveness of which have been adequately covered in the scientific literature.

A promising direction in optimizing the methods of cryopreservation of the bone marrow and cord blood cells is the combination in one solution of low concentrations of several cryoprotectants with different mechanisms of action, for example, acting at the intracellular level of DMSO and hydroxyethyl starch or albumin, which possess an extracellular barrier effect.

For the cryopreservation of umbilical cord blood cells, a 20% solution of DMSO is traditionally used which, while mechanically stirring constantly in an ice bath, is slowly poured into the cell suspension until an equal (1: 1) ratio of cryoprotector and cell suspension is achieved. The final concentration of dimethyl sulphoxide is 10%. The cell suspension is then cooled on a software cryostat with a speed of HS / min to -40 ° C, after which the cooling rate is increased to 10 ° C / min. After reaching -100 ° C, the container with the cell suspension is placed in liquid nitrogen (-196 ° C). With this method of cryopreservation, the safety of functionally active mononuclear cells after thawing reaches 85% of the initial level.

Modifications of cryopreservation methods are aimed at reducing the concentration of DMSO by adding hydroxyethyl starch (final concentrations of dimethyl sulfoxide and hydroxyethyl starch are 5% and 6%, respectively). The high efficiency of this combination of cryoprotectants is observed when the suspension of myeloid cells is frozen, with no less cytoprotection than with a single 10% solution of dimethylsulfoxide. The number of viable nucleated cells reached 96.7% of the baseline level, and their functional activity, estimated by the number of CFU-GM, was 81.8%.

Using a solution of dimethylsulfoxide in concentrations from 5 to 10% in combination with 4% hydroxyethyl starch (final concentration) it was established that the safety of CD34-positive cells in such ranges of dimethyl sulfoxide practically does not change. At the same time, under conditions of decreasing dimethyl sulfoxide concentration from 5 to 2.5%, mass loss of umbilical cord blood cells is observed - the number of viable cell units decreases from 85.4 to 12.2%. Other authors also came to the conclusion that exactly 5 and 10% solutions of dimethylsulfoxide (in the author's version - in combination with autologous serum) provide cytoprotection with cryopreservation of HSC cord blood with maximum efficiency. In addition, high preservation of successively frozen and thawed cells is noted in the case of a combination of 5 or 10% dimethylsulfoxide with 4% hydroxyethyl starch solution, especially at a controlled cooling rate of HS / min. In another work, a cryoprotection solution consisting of three ingredients - DMSO, purified human albumin and RPMI medium in a ratio of 1: 4: 5, was added, which was added to the cell suspension to an equal volume ratio (final concentration of dimethylsulfoxide was 5%). After defrosting in a water bath at a temperature of + 4 ° C, the safety of the CFU-GM exceeded 94%.

Some authors suggest using unfractionated cord blood for cryopreservation, since significant amounts of hematopoietic cells are lost during the removal of red blood cells. In this embodiment, a 10% solution of dimethylsulfoxide is used to protect mononuclear cells from the damaging effects of cryocrystallization. Freezing is carried out at a constant cooling rate of HS / min to -80 ° C, after which the suspension of umbilical cord blood cells is immersed in liquid nitrogen. With this method of freezing, a partial lysis of the erythrocytes takes place, therefore blood samples do not require fractionation. After thawing, the cell suspension is washed from free hemoglobin and dimethylsulfoxide in a solution of human albumin or in the autologous serum of the patient and used for transplantation.

Preservation of hematopoietic progenitor cells after unfractionated unfractionated cord blood is indeed higher than fractionated blood, but due to the cryostability of the part of red blood cells, serious posttransfusion problems can arise due to transfusion of ABO-incompatible erythrocytes. In addition, the volume of stored unfractionated blood significantly increases. From a clinical point of view, it is still preferable to cryopreserve pre-isolated and purified from other cell fractions of hematopoietic cord blood cells.

In particular, a method for cryopreservation of cells of fractionated cord blood has been developed, which makes it possible to remove erythrocytes at the stage of preparation for freezing, in which a 6% solution of hydroxyethyl starch is used as part of the plasma-substituting solution "Stabisol". After thawing, the cell suspension thus obtained is ready for clinical use without additional manipulation.

Thus, at present, there are many quite effective ways of cryopreservation of umbilical cord blood. The principal difference is that blood samples are frozen unfractionated or subjected to separation into cell fractions during the preparation stage and harvested nucleated cells without an admixture of erythrocytes.

Transplantation of hemopoietic stem cells of umbilical cord blood

In the late 80's - early 90's of the last century it was found that the umbilical cord blood supplying the fetus during pregnancy is characterized by a high content of hematopoietic stem cells. The relative simplicity of obtaining umbilical cord blood cells and the absence of obvious ethical problems promoted the use of cord blood stem cells in practical medicine. The first successful transplantation of cord blood to a child with Fanconi anemia served as a starting point for expanding the volumes of cord blood stem cell transplantation and creating a system for its bank provision. In the global system of cord blood banks, the largest is the New York Center for Placental Blood, which is on the balance sheet of the National Institutes of Health. The number of stored cord blood samples in this bank is approaching 20 LLC. The number of recipients (mainly children) is also growing, and successful transplantation has been carried out. According to the US Department of Health, the relapse-free period of post-transplant life of recipients of HSC cord blood has already exceeded 10 years.

This is not surprising, since numerous studies of the hematopoietic potential of cord blood showed that the quantity and quality of the earliest stem cells is not only inferior to the bone marrow of an adult person, but it exceeds it by some indicators. The higher proliferative potential of cord blood stem cells is due to the ontogenetic features of cellular signaling, the presence of specific growth factors on HSC receptors, the ability of cord blood cells to autocrine growth factor production, large size and telomere length.

Thus, genomic and phenotypic features of stem blood-forming umbilical cord blood cells predetermine the qualitative engraftment of a transplant with a high potential for the restoration of donor hemopoiesis in the recipient organism.

Advantages of hematopoietic stem cells of umbilical cord blood

Among the real benefits of using hematopoietic stem cells of umbilical cord blood for the purpose of transplantation in front of other sources of hematopoietic cells, one should note the almost zero risk to the health of the donor (if not count as a placenta) and the absence of the need for general anesthesia. The use of umbilical cord blood extends the possibilities of cell transplantation due to partially compatible transplantation in the HLA system (incompatibility from one to three antigens). A technique for prolonged storage of cord blood hemopoietic cells in the frozen state has been developed, which increases the probability of obtaining rare HLA types and shortens the search time of an HLA-compatible transplant for allogeneic transplantation. At the same time, the risk of developing some latent infections transmitted by the transmission route is significantly reduced. In addition, there is an inexpensive form of biological life insurance in connection with the possibility of using cord blood cells for autologous transplantation.

However, due to the small amounts of blood that can be collected from the placenta (an average of no more than 100 ml), the problem of obtaining the greatest possible amount of blood from the vein of the umbilical cord, with strict adherence to the minimum risk of bacterial contamination of the obtained umbilical cord blood samples, comes to the fore.

Primitive hematopoietic cord blood cells are usually identified by the presence on their surface of glycosophosphoprotein CD34, and also on the basis of their functional properties by studying clonogenicity or colony formation in vitro. Comparative analysis showed that in the cord blood and bone marrow the maximum content of CD34-positive cells in the fraction of mononuclear cells is 1.6 and 5.0%, the maximum level of colony-forming units in the CD34 + cell subpopulation is 80 and 25%, the total cloning efficiency of CD34 + cells - 88 and 58%, the maximum content of colony-forming cells with a high proliferation potential (HPP-CFC in the CD34 + population) is 50 and 6.5%. To this, it should be added that the efficiency of cloning of CD34 + CD38 cells and the ability to respond to cytokine stimulation is also higher in hematopoietic stem cells of umbilical cord blood.

The combination of the phenotypic antigens Thy-1, CD34 and CD45RA confirms the high proliferative potential of the hematopoietic blood-forming cells, and the expression of these three antigens on the surface of the umbilical cord blood cells indicates their belonging to stem cells. In addition, it has been established that cord blood contains cells with a CD34 + phenotype that do not have linear differentiation markers. The level in the umbilical cord blood of cell subpopulations with the phenotypic profile of CD34 + / Lin is about 1% of the total number of CD34-positive cells. Hematopoietic cord blood precursor cells give rise to both the lymphoid cell line and the polypotent myeloid series of linear cell differentiation, which also indicates their belonging to stem cells.

As already mentioned, the essential differences between bone marrow and cord blood are the amount of hematopoietic cells used for transplantation, obtained with one procedure. If during bone marrow transplantation, cell mass loss during separation, cryopreservation, defrosting and testing is permissible within 40-50%, then for cord blood, such cell losses are very significant, since using an insufficient number of GSK graft may prove to be untenable. According to G. Kogler and co-authors (1998), for all cell transplantations with a recipient body weight of 10 kg, all cord blood samples can be potential grafts (total number of cord blood samples collected - 2098), with a body weight of 35 kg - 67%, and only 25% of the samples will be able to provide effective transplantation in patients with a body weight of 50-70 kg. This clinical situation indicates the need to optimize and improve the effectiveness of existing methods of sampling, reproduction and storage of umbilical cord blood cells. Therefore, at present in the literature there are widely discussed the issues of standardization of methods for sampling, testing, separation and cryopreservation of umbilical cord blood for the creation of blood banks, its application in the clinic, as well as conditions and terms of storing hematopoietic stem cells of cord blood.

[7], [8], [9],

The use of hematopoietic stem cells of umbilical cord blood in medicine

Usually, up to 10 ⁶ hematopoietic stem cells can be isolated from umbilical cord blood , rarely more. In connection with this, until today, the question remains of the sufficiency of so many hematopoietic cord blood cells to restore the hematopoiesis of the adult recipient. Opinions on this issue were divided. Some researchers believe that this amount is sufficient for transplantation to children, but too little for a transplant to an adult, for which the optimal administration (7-10) x 10 ⁶ CD34-positive cells per 1 kg of body weight is on average 7 x 10 ⁸ per transplant. From these calculations it follows that one umbilical cord sample contains 700 times less hematopoietic stem cells than is required for one transplantation to an adult patient. However, such a quantitative assessment is made by analogy with the number of transfused cells in the bone marrow and completely ignores the ontogenetic features of hematopoiesis.

In particular, the fact of a higher proliferative potential of hematopoietic stem cells of umbilical cord blood is ignored in comparison with hemopoietic precursor cells of the bone marrow. The results of studies of the colony-forming potential in vitro suggest that one dose of cord blood can provide a reconstitution of the hematopoiesis of the adult recipient. On the other hand, it should not be forgotten that the amount of HSC decreases even in the process of embryonic development: the content of CD34-positive cells in the umbilical cord is linearly reduced 5-fold in the time frame from 20 weeks (blood obtained for premature termination of pregnancy) to 40 weeks of gestation (the period of physiological births), which is accompanied by a parallel, permanent increase in the expression of linear cytodifferentiation markers.

Due to the lack of a standardized approach to the quantification of progenitor cells in umbilical cord blood samples, controversy over the optimal dose of hematopoietic cord blood stem cells continues. Some researchers believe that the number of nucleated cells and mononuclear cells, recalculated for the body weight of the recipient, that is, their dose, can be used as criteria for the selection of umbilical cord samples. Some authors believe that the minimum quantitative threshold of CD34 + cells, even for conducting autologous transplantation of HSC, is 2 x 10 ⁶ / kg. At the same time, increasing the dose of hemopoietic cells to 5 x 10 ⁶ cells / kg (only 2.5 times) already provides a more favorable course of the early post-transplant period, reduces the incidence of infectious complications and shortens the duration of the period of preventive antibiotic therapy.

According to E. Gluckman and co-authors (1998), in oncohematology, the condition for the successful transplantation of umbilical cord blood cells is the introduction of at least 3.7 x 10 ⁷ nucleated cells per 1 kg of body weight of the recipient. With a decrease in the dose of hematopoietic stem cells to 1 x 10 ⁷ and less nucleated cells per 1 kg of body weight, the risk of graft failure and recurrence of cancer is sharply increased. It should be recognized that the minimum number of progenitor cells required for the rapid recovery of hemopoiesis after GSG allotransplantation is not yet known. Theoretically, this can be achieved with a single cell, but in the clinical practice of bone marrow transplantation, rapid and stable engraftment is guaranteed by transfusion of at least (1-3) x 10 ⁸ nucleated cells per 1 kg of the patient's body weight.

A recent detailed study to determine the optimal amount of HSC in oncohematology included the observation of patients of three groups isolated depending on the content of CD34-positive cells in the transplant material. Patients of the first group received (3-5) x 10 ⁶ cells / kg. The dose of HSC in patients of the second group was (5-10) x 10 ⁶ cells / kg, and the patients of the third group received transplantation more than 10 x 10 ⁶ CD34 + cells / kg. The best results were observed in the group of recipients receiving a transplant with the number of CD34-positive cells equal to (3-5) x 10 ⁶ / kg. With an increase in the dose of transplanted cells above 5 × 10 ⁶ / kg, no statistically significant benefits were identified. At the same time, a very large HSC content in the transplant (> 10 x 10 ⁶ / kg) is associated with reinfusion of a significant number of residual tumor cells, which leads to a relapse of the disease. There was no direct relationship between the number of transplanted allogeneic progenitor cells and the development of the "graft versus host" reaction.

The accumulated world experience of transplantation of HSC cord blood confirms their high repopulation potential. The engraftment rate of the cord blood transplant correlates with the number of nucleated cells introduced. The best results are observed with a transplantation of 3 × 10 ⁷ / kg, while for the bone marrow this dose is 2 × 10 ⁸ / kg. According to the data of the coordination centers, in the end of 2000 1200 transplants of umbilical cord blood cells were made in the world, mainly from donor relatives (83%). Obviously, cord blood should be considered as an alternative to the bone marrow for transplantation to patients with hemoblastoses.

At the same time, the neonatal nature of the cord source of the hematopoietic tissue inspires optimism due to the presence of functional features of its GSK. In this case, only clinical experience can provide an answer to the question of the sufficiency of a single cord blood sample for restoring the hematopoiesis of an adult recipient with hematopoiesis aplasia. Transplantation of umbilical cord blood cells is used in the treatment programs of many diseases of tumor and non-tumorous nature: leukemias and myelodysplastic syndromes, non-Hodgkin's lymphoma and neuroblastoma, aplastic anemia, congenital anemia of Fanconi and Diamond-Blackfen, leukocyte adhesion deficit, Barr's syndrome, Gunther's disease, Harler's syndrome, thalassemia .

Close attention and separate research deserve the immunological aspects of transplantation of blood-forming cells of the umbilical cord blood. It is shown that in the case of transplantation of cord blood stem cells from donors with incomplete HLA-compatibility, the results of transplantation are quite satisfactory, which, according to the authors, indicates a lesser immunoreactivity of umbilical cord blood cells than bone marrow.

A detailed study of the cellular composition of the cord blood revealed features of both the phenotypic spectrum of the effector cells of the immune system and their functional activity, which made it possible to treat cord blood as a source of HSC with a relatively low risk of developing a "graft versus host" reaction. Among the signs of functional immaturity of immunocompetent umbilical cord blood cells, one should note an imbalance in cytokine production and a decrease in sensitivity to the cytokine regulation of the immune response. The resulting inhibition of cytotoxic lymphocyte activity is considered a factor contributing to the formation of immunological tolerance to the transplanted hemopoietic tissue. In the umbilical cord blood lymphocyte population, in contrast to peripheral blood and bone marrow of adult donors, inactive, immature lymphocytes and suppressor cells predominate. This indicates a decreased availability of cord blood T-lymphocytes to the immune response. An important feature of the monocyte population of umbilical cord blood cells is the low content of functionally complete and active antigen-presenting cells.

On the one hand, the low degree of maturity of the effector cells of the immune system in the cord blood expands the indications for its use in the clinic, since these features provide a decrease in the intensity of the immune conflict between the donor and recipient cells. But, on the other hand, it is known that there is a correlation between the degree of development of the "graft versus host" reaction and the antitumor effect of transplantation, that is, the development of the "graft versus leukemia" effect. In connection with this, a study was conducted of the antitumoral cytotoxicity of umbilical cord blood cells. The obtained results show that, despite the really weakened response of immunocompetent cord blood cells to antigenic stimulation, primarily activated lymphocytes are natural killers and killer-like cells that actively participate in the mechanisms of antitumor cytotoxicity. In addition, subpopulations of lymphocytes with the phenotype CD16 + CD56 + and CD16 TCRa / p + have been found in the umbilical cord blood, it is these cells that are supposed to activate the "graft versus leukemia" reaction in an activated form.

At the Institute of Oncology of the Academy of Medical Sciences of Ukraine, cryopreserved hematopoietic cord blood cells were administered to an oncological patient with persistent hypoplasia of hematopoiesis due to chemo- and radiotherapy. In such patients, transplantation of hematopoietic stem cells of umbilical blood sufficiently effectively restored the oppressed hematopoiesis, as evidenced by a persistent increase in the content of mature elements in the peripheral blood, as well as an increase in the parameters characterizing the state of cellular and humoral immunity. Stability of the repopulation effect after transplantation of blood-forming cells of umbilical cord blood allows continuing radiation and chemotherapy without interrupting the course of treatment. There is evidence of a higher efficiency of stem cell allografts allergy to oncohematological patients: the annual risk of relapse of the tumor in their use was 25% versus 40% in patients with allogeneic bone marrow transplantation.

The mechanism of action of cryopreserved stem cells of umbilical cord blood should be considered as the result of humoral stimulation of hematopoiesis of recipients caused by the unique ability of neonatal cells to autocrine production of hematopoietic growth factors, as well as the result of temporary engraftment of donor cells (as confirmation - a significant increase in fetal hemoglobin of peripheral blood of recipients at 7-15 day after transfusion in comparison with baseline data). The absence of post-transfusion reactions in cord blood recipients is a result of the relative tolerance of its immunocompetent cells, as well as a confidence criterion for the biological usefulness of cryopreserved material.

The progenitor cells of cord blood umbilical T-lymphocytes are capable of activation under the influence of exogenous cytokine stimulation, which is used to develop new ex vivo and in vivo techniques for inducing the antitumoral cytotoxicity of lymphoid elements of the graft for subsequent immunotherapy. In addition, the "immaturity" of the genome of immunocompetent cord blood cells allows them to be used to enhance antitumor activity by molecular modeling methods.

Today, cord blood has found wide application primarily in pediatric hematology. In children with acute leukemia, the allotransplantation of hematopoietic stem cells in the umbilical cord, in comparison with bone marrow allotransplantation, significantly reduces the incidence of graft-versus-host disease. However, there is a longer period of neutropenia and thrombocytopenia, and, unfortunately, a higher level of 100-day post-transplant mortality. A longer period of recovery in peripheral blood of granulocytes and platelets may be due to the inadequate differentiation of individual subpopulations of CD34-positive cord blood cells, as indicated by the low level of absorption of radioactive rhodamine and the low expression of CD38 antigens on their surface.

At the same time, transplantation of hematopoietic stem cells of umbilical cord blood to adult patients, performed due to the absence of a compatible unrelated donor of the bone marrow, and the possibility of mobilizing autologous HSC, showed a high annual disease-free survival in the group of patients aged no more than 30 years (73%) . Expansion of the age range of recipients (18-46 years) resulted in a decrease in survival rate to 53%.

Quantitative analysis of cells with the CD34 + phenotype in the bone marrow and cord blood showed a higher (3.5 times) their content in the bone marrow, however, in the cord blood, a significant predominance of cells with the phenotypic profile of CD34 + HLA-DR is known. It is known that the cells depend on the DNA mammography markers CD34 + HLA-DR proliferates more actively than cells with immunophenotype CD34 + HLA-DR +, which is confirmed in experimental studies of the growth of long-term hematopoietic cell culture in vitro. Primitive cellular precursors with the CD34 + CD38 phenotype are found both in the cord blood and in the bone marrow, but cord blood cells with the CD34 + CD38 marker set have a higher clonogenic activity than the hematopoietic cells of the same phenotype isolated from the bone marrow of adult donors. In addition, cord blood cells with immunophenotype CD34 + CD38 proliferate faster in response to cytokine stimulation (IL-3, IL-6, G-CSF) and reproduce 7-fold more colonies in long-term cultures than bone marrow cells.

Banks of cord blood stem cells

For the proper development of a new field of practical medicine - the transplantation of cord blood stem cells, as well as for the transplantation of hematopoietic stem cells of the bone marrow, it is necessary to have a ramified network of blood banks that have already been established in the US and Europe. The intra-state networks of cord blood banks are united by the Association of Netcord Banks. The expediency of creating an international association of umbilical cord blood banks is determined by the fact that for performing unrelated transplantations a large number of typed umbilical cord samples is necessary, which allows selecting an HLA-identical donor. Only the establishment of a system of banks with the storage of blood samples of different HLA types in them can really solve the problem of finding the necessary donor. The organization of such a system of cord blood banks requires the preliminary development of ethical and legal norms, which are currently being discussed at the international level.

To create cord blood banks in Ukraine, it is necessary to work out a number of provisions and documents.

First of all, these are questions of standardizing the methods of sampling, fractionation and freezing of umbilical cord blood. It is necessary to regulate the rules for sampling cord blood in maternity hospitals in accordance with the requirements of medical ethics, to determine the minimum amount of cord blood that ensures successful transplantation. It is necessary to compare and standardize various criteria for assessing the quality and quantity of hematopoietic progenitor cells, as well as HLA typing methods and methods for diagnosing genetic and infectious diseases that can be transmitted by infusing cord blood cells, and establishing general criteria for selecting healthy donors. It is also worth discussing the creation of separate storage facilities for serum, cells and DNA derived from cord blood.

It is absolutely necessary to organize a computer network of data on cord blood for the implementation of the relationship with the registers of bone marrow donors. To further develop cell transplantology, special protocols for comparing the results of cord blood and bone marrow transplantation from HLA-identical relatives and unrelated donors should be developed. In the solution of ethical and legal problems of the clinical application of umbilical cord blood cells, the standardization of documentation, including informed consent of the parents, and notification of the mother or relatives about the child's genetic and / or infectious diseases, can help.

The decisive condition for the development of cellular transplantation in Ukraine will be the adoption of the National Stem Cell Donation Program and the development of international cooperation with other countries through the World Mediation Donor Association (WMDA), the National Donor Medullary Program (NMDP) and other registries.

Summarizing the still short history of the development of the transplantation of hematopoietic stem cells in the umbilical cord blood, we note that the first assumptions about the possible use of umbilical cord blood in the clinic, expressed in the early 1970s, were confirmed in the 1980s by the results of experimental animal studies, and in 1988 the first in the world transplantation of blood-forming cells of umbilical cord blood to a man was already conducted, after which the world network of umbilical cord blood banks was created. After 10 years, the number of patients with transplanted blood-forming cells of umbilical cord blood was close to 800. Among them were patients with various diseases of the tumor (leukemia, lymphomas, solid tumors) and non-tumorous (congenital immunodeficiencies, anemias, metabolic diseases) of nature.

In cord blood, the content of early and committed cell progenitors is higher than in the peripheral blood of an adult. By the number of granulocyte-macrophage colony-forming units and their proliferative potential, umbilical cord blood significantly exceeds the peripheral blood of adults even after the introduction of growth factors. In long-term cell cultures in vitro, there was a greater proliferative activity and viability of umbilical cord blood cells than bone marrow cells. Critical moments in the transplantation of cord blood stem cells are the number and haemopoietic potential of nucleated cells, the presence of cytomegalovirus infection, HLA-compatibility of the donor and recipient, body weight and age of the patient.

Nevertheless, stem cell transplantation of umbilical cord blood should be considered as an alternative to bone marrow transplantation in order to treat severe blood diseases, especially in children. Clinical problems of transplantation of umbilical cord blood cells are gradually resolved - there are already quite effective methods of sampling, separation and cryopreservation of umbilical cord blood cells, conditions for the formation of umbilical cord blood banks are provided, methods for testing nucleated cells are improved. Optimal for separation with a large-scale procurement of hematopoietic stem cells of umbilical cord blood when creating banks should be considered 3% gelatin solution and 6% solution of hydroxyethyl starch.

P. Perekhrestenko and co-authors (2001) rightly point out that transplantation of cord blood stem cells should take a proper place in the complex of therapeutic measures to overcome depressions of hemopoiesis of various genesis, since HSC cord blood differ in a number of significant advantages, among which the relative simplicity of their harvesting, no risk to the donor, low contamination of neonatal cells by viruses and a relatively low cost of transplantation. Some authors indicate that transplantation of umbilical cord blood cells is less common than bone marrow transplantation, accompanied by complications associated with the "graft versus host" reaction, which, in their opinion, is due to weak expression on the cord blood cells of HLA-DR antigens and their immaturity. Nevertheless, the main population of cord blood nuclei is T-lymphocytes (CCD-positive cells), whose content is about 50%, which is 20% less than in the peripheral blood of an adult, but the phenotypic differences in T-cell subpopulations from these sources are insignificant.

Among the factors directly affecting the survival rate of umbilical cord blood stem cell transplantation, the age of patients should be noted (the best results are observed in recipients aged between 1 and 5 years), early diagnosis of the disease and the form of leukemia (efficacy is much higher with acute leukemia). Of great importance are the dose of nucleated umbilical cord blood cells, as well as their HLA compatibility with the recipient. It is no coincidence that the analysis of the clinical efficacy of HSC cord blood transplantation in oncohematology testifies to the best results of treatment with related grafts: annual recurrence-free survival in this case reaches 63%, while in unrelated transplantation - only 29%.

Thus, the presence of a large number of stem cells in the umbilical cord blood and the high repopulation capacity of neonatal hematopoietic stem cells make it possible to use them for allogeneic transplantation in oncohematological patients. However, it should be noted that recapitulation of hemopoiesis after transplantation of blood-forming cells of cord blood is "stretched in time": recovery of peripheral blood neutrophils is usually observed at the end of the 6th week, and the phenomena of thrombocytopenia disappear, usually after 6 months. In addition, the immaturity of the hematopoietic cells in the umbilical cord blood does not exclude immunological conflicts: the severe course of the acute and chronic "graft-versus-host" reaction is observed in 23 and 25% of the recipients, respectively. Relapses of acute leukemia by the end of the first year after transplantation of umbilical cord blood cells are noted in 26% of cases.

[10], [11]