Hematopoietic stem cells from umbilical cord blood

Umbilical cord blood is a good source of hematopoietic stem cells in terms of proliferative potential and repopulation capabilities of hematopoietic cells. It has been repeatedly shown that by the time of birth, umbilical cord blood contains a sufficiently large number of weakly committed hematopoietic progenitor cells. Some authors believe that the advantage of cord blood hematopoietic stem cell transplantation is the lack of need to search for a donor compatible with HLA antigens. In their opinion, the immaturity of the newborn's immune system causes reduced functional activity of immunocompetent cells and, accordingly, a lower incidence of severe graft-versus-host disease than with bone marrow transplantation. At the same time, the survival rate of a cord blood cell transplant is not lower than that of bone marrow cells, even in the case of using a smaller number of HSCs administered per 1 kg of the patient's body weight. However, in our opinion, the issues of the optimal number of transplanted cord blood cells required for effective engraftment in the recipient’s body, their immunological compatibility, and a number of other aspects of the problem of transplantation of cord blood hematopoietic stem cells require more serious analysis.

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Obtaining hematopoietic stem cells from umbilical cord blood

The procedure for obtaining hematopoietic stem cells from umbilical cord blood requires its collection immediately after the birth of the child and its separation from the placenta when the placenta is in utero or ex utero, as well as during a cesarean section, but also ex utero. It has been shown that if the time from the moment of birth to the separation of the newborn from the placenta is reduced to 30 seconds, the volume of umbilical cord blood obtained increases by an average of 25-40 ml. If this procedure is performed later, the same amount of blood is lost. It has been established that early separation of the child from the placenta does not entail any negative consequences for the newborn.

The Russian Research Institute of Hematology and Transfusiology has developed effective and low-cost technologies for obtaining umbilical cord blood both during normal birth ((70.2+25.8) ml) and cesarean section ((73.4+25.1) ml). A method for separating umbilical cord blood with a sufficiently high yield of nucleated and mononuclear cells has been proposed - (83.1+9.6) and (83.4+14.1)%, respectively. A method for cryopreserving umbilical cord blood has been improved, which ensures high preservation of mononuclear cells and CFU-GM - (96.8+5.7) and (89.6+22.6)%, respectively. The efficiency of the drainage method for collecting umbilical cord blood using the Kompoplast-300 container (Russia) has been determined. The authors collected umbilical cord blood immediately after the birth of the child and its separation from the placenta, in conditions of the placenta placement in utero or ex utero. Before the puncture of the umbilical vein, the umbilical cord was treated once with 5% iodine tincture, and then twice with 70% ethyl alcohol. The blood flowed spontaneously through the connecting tubes into the container. The collection procedure took no more than 10 minutes. The average volume of 66 cord blood samples collected by drainage was (72+28) ml, and the number of leukocytes in the average total sample volume was (1.1+0.6) x 107. When analyzing cord blood for sterility (bacterial contamination, HIV-1, hepatitis B and C viruses, syphilis and cytomegalovirus infection), IgG antibodies to the hepatitis C virus were detected in only one sample. In another study, the placenta was placed fetal surface down on a special frame immediately after birth, the umbilical cord was treated with 5% iodine solution and 75% ethyl alcohol. The umbilical vein was drained using a needle from a transfusion system (G16). Blood flowed into the container spontaneously. The average volume of blood collected in this way was (55+25) ml. In the work of G. Kogler et al. (1996), umbilical cord blood was collected using a closed method and large volumes of blood were obtained - on average (79+26) ml. The authors note that among 574 umbilical cord blood samples, about 7% contained less than 40 ml of blood, which does not allow them to be used for transplantation. K. Isoyama et al. (1996), collecting cord blood by active exfusion using syringes, obtained an average of 69.1 ml of blood (the volume of umbilical cord blood varied from 15 to 135 ml). Finally, A. Abdel-Mageed PI et al. (1997) managed to obtain an average of 94 ml of umbilical cord blood (from 56 to 143 ml) through catheterization of the umbilical vein.

To reduce the risk of iatrogenic infection and contamination with maternal secretions, a closed blood collection system has been developed based on the widely used transfusion system of Baxter Healthcare Corp., Deerfield, IL (USA), containing 62.5 ml of CPDA (citrate-phosphate-dextrose with adenine) as an anticoagulant. The technology for obtaining the material is of primary importance for preparing a high-quality sample in terms of volume, content, and purity of the cell suspension. Of the existing methods for collecting umbilical cord blood, which are conventionally classified into closed, semi-open, and open systems, preference should be given to the first, since the closed system significantly reduces the risk of microbial contamination of the material, as well as contamination of the cell suspension with maternal cells.

A. Nagler et al. (1998) conducted a comparative analysis of the efficiency of all three systems for collecting umbilical cord blood. In the first variant, the procedure was carried out in a closed system by exfoliating blood directly into a container. In the second variant, cord blood was obtained by active exfusion of blood with an MP1 syringe followed by flushing of the placental veins and simultaneous drainage of blood into a container (open method). In the third variant, blood was collected in a semi-open system by actively extracting it with syringes and flushing it through the umbilical artery with simultaneous exfusion into a container. In the first variant, the authors obtained umbilical cord blood in a volume of (76.4+32.1) ml with a leukocyte content of (10.5+3.6) x 10 ⁶ in 1 ml of blood. In the second variant, the corresponding indicators 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 observed when using an open system. A direct correlation was established between the mass of the placenta and the volume of blood extracted - with an increase in the mass of the placenta, the amount of blood collected increases.

After cord blood collection, the separation stage follows - isolation of mononuclear cells and purification of the cell suspension from erythrocytes. In experimental conditions, nucleated cells are isolated by sedimentation with methylcellulose during lysis of erythrocytes with ammonium chloride. However, methylcellulose should not be used for clinical purposes, since losses of hematopoietic stem cells on it reach 50-90%. Lysis of erythrocytes is also almost never performed in the clinic due to the large volumes of the working solution, although the percentage of isolation of nucleated cells with the CD34+ phenotype, as well as progenitor cells with the CFU-GM and CFU-GEMM functions in this way is significantly higher. The emergence of a new means for isolating mononuclear cells in a density gradient, buyant density solution (BDS72), is reported. This substance has the following physiological parameters: pH - 7.4, osmolality - 280 mOsm/kg, density - 1.0720 g/ml. According to the authors, it can be used to isolate up to 100% of CD34-positive cells and remove 98% of erythrocytes. However, BDS72 is not yet used in the clinic.

In the approved methods of isolating nucleated cells from umbilical cord blood, a 10% hydroxyethyl starch solution or a 3% gelatin solution are usually used. The efficiency of sedimentation of erythrocytes and isolation of nucleated cells in both cases is approximately equal. However, when gelatin is used as a sedimentation agent, it is possible to obtain a slightly larger amount of CFU-GM than when using hydroxyethyl starch. It is assumed that the differences in the efficiency of CFU-GM isolation are due to different sedimentation rates of individual fractions of nucleated cells or the ability of hydroxyethyl starch molecules to be absorbed on the surface of hematopoietic cell receptors and thereby block their sensitivity to colony-stimulating factors used in culturing CFU-GM in vitro. Nevertheless, both sedimentators may well be suitable for isolating nucleated cells when creating large-scale cord blood banks.

Methods of cord blood separation and cryopreservation are basically no different from those used in work with hematopoietic stem cells of peripheral blood and bone marrow of adult donors. But when preparing a large number of cord blood samples for its banks, the methods of separation must, first of all, be low-cost. Therefore, unfortunately, at present, for clinical needs, already tested routine methods of isolating and cryopreserving cord blood cells are used, and more effective, but costly methods remain the lot of experimenters.

In general, criteria for assessing the number of hematopoietic cells and requirements for examining cord blood samples to identify infectious agents have been approved. In order to ensure the safety of cord blood hematopoietic cell transplantation, all blood samples must be examined primarily for hematogenously transmitted infections and genetic diseases. A number of authors recommend additional special methods for examining cord blood to diagnose genetic diseases such as a-thalassemia, sickle cell anemia, adenosine deaminase deficiency, Bruton's agammaglobulinemia, Hurler's and Ponter's diseases.

According to the recommendations of L. Ticheli and co-authors (1998), each cord blood sample must be tested for nucleated cells, CD34-positive cells, and CFU-GM, HLA typing must be performed, and the blood group must be determined according to ABO and its Rh factor. In addition, bacteriological culture, serological testing for HIV and cytomegalovirus infection, HBsAg, viral hepatitis C, HTLY-I and HTLV-II (human T-cell leukemia), syphilis, and toxoplasmosis must be performed. Polymerase chain reaction for cytomegalovirus and HIV infection is mandatory.

The procedure for obtaining cord blood must be carried out in strict accordance with the principles of medical bioethics. Before blood collection, it is necessary to obtain the consent of the pregnant woman to carry it out. A preliminary conversation with the pregnant woman to obtain informed consent for all manipulations, from blood exfusion to filling out the documentation, is carried out only by medical workers. In no case is it permissible for any of these procedures to be carried out by personnel with biological, chemical, pharmaceutical or other non-medical education, due to the violation of established norms of bioethics and human rights. In case of positive tests for HBsAg carriage, the presence of antibodies to the pathogens of hepatitis C, HIV infection and syphilis, cord blood is not collected, and samples of already collected blood are rejected and destroyed. It should be noted that carriage of latent infections in newborns is much less common than in adults, therefore, the probability of hematogenous transfer and development of infectious complications during infusions of cord blood hematopoietic cells is significantly lower than in the case of using adult donor bone marrow for transplantation.

An important aspect of cord blood clinical use is transplant evaluation, which is based on determining the amount of hematopoietic stem cells in a cord blood sample and the doses of cells required for transplantation. At present, standards for the optimal amount of 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 determination of the content of colony-forming units common to granulocytes, erythrocytes, monocytes and megakaryocytes - CFU-GEMM.

However, in a clinical setting, standard evaluation of a cord blood transplant typically involves only determination of the number of nucleated or mononuclear cells.

Storage of cord blood hematopoietic stem cells

There are also some problems in the technology of storing hematopoietic cells of umbilical cord blood. When cryopreserving hematopoietic stem cells, in order to achieve the optimal freezing mode, it is necessary to reduce the volume of umbilical cord blood as much as possible, and also to remove erythrocytes in advance to avoid hemolysis and the risk of developing an incompatibility reaction for erythrocyte antigens (ABO, Rh). Various methods of isolating nucleated cells are suitable for these purposes. In the early 90s of the last century, the most widely used method was isolating nucleated cells in a density gradient based on Ficoll with a density of 1.077 g / ml or Percoll with a density of 1.080 g / ml. Separation of umbilical cord blood in a density gradient allows for the isolation of 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 isolating cord blood hematopoietic cells is assessed differently. Some authors point to the low quality of separation using this method, while other researchers, on the contrary, among all possible methods, give preference to isolating cord blood HSC using a 6% hydroxyethyl starch solution. At the same time, the high efficiency of hematopoietic cell sedimentation is emphasized, which, according to some data, reaches from 84% to 90%.

Proponents of a different point of view believe that virtually all fractionation methods are associated with large losses of nucleated cells and propose to perform separation by centrifugation, dividing the umbilical cord blood into 3 fractions: erythrocytes, leukocyte ring and plasma. By isolating cells in this way, the authors found that the content of mononuclear cells, early hematopoietic progenitor cells and cells with the CD34+ immunophenotype ultimately amounted to 90, 88 and 100% of the initial level, respectively. Similar values for the increase in cord blood cells purified by this method were also obtained by other researchers: after sedimentation, 92% of nucleated cells, 98% of mononuclear cells, 96% of CD34-positive cells and 106% of colony-forming units were isolated.

In the late 1990s, gelatin was widely used as a sedimentation agent. In clinical practice, gelatin has been used to isolate hematopoietic stem cells from umbilical cord blood since 1994. When using a 3% gelatin solution, the efficiency of isolating nucleated cells reaches 88-94%. The large-scale use of gelatin in creating a cord blood bank has confirmed its advantages over other sedimentation agents. A comparative analysis of the efficiency of all the above methods for isolating nucleated cells under the conditions of their sequential use on each of the tested umbilical cord blood samples has proven that a 3% gelatin solution is the optimal sedimentation agent in terms of the yield of mononuclear cells with the CD34+/CD45+ phenotype, as well as in terms of the number of CFU-GM and CFU-GEMM. Methods using a Ficoll density gradient, as well as the use of hydroxyethyl starch and methylcellulose, were significantly less effective, with losses of hematopoietic cells reaching 60%.

The expansion of cord blood stem cell transplantation volumes is associated not only with the development of methods for their acquisition, 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 technology for cryopreservation of its samples. Among them are the issues of the feasibility of performing separation procedures, using various cryopreservation media and applying methods for preparing defrosted cells for transplantation. Transportation of native cord blood samples is often carried out from regions remote from hematological centers. In this regard, the problem of acceptable storage periods for cord blood from the moment of its acquisition to the beginning of cryopreservation arises, which is of particular importance when creating cord blood banks.

A study of the functional activity of hematopoietic cells in umbilical cord blood after long-term storage (up to 12 years) in liquid nitrogen has shown that about 95% of hematopoietic cells do not lose their high proliferative capacity during this period. In the work of S. Yurasov and co-authors (1997), it was proven that storing 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% of the initial level, respectively. However, if the storage period is extended to three days, the number of viable nucleated cells in the umbilical cord blood decreases significantly. At the same time, other studies have shown that when stored for 2-3 days at 22 or 4°C, the viability of mature granulocytes, rather than hematopoietic cells, suffers first and foremost.

The viability of cord blood hematopoietic stem cells may be negatively affected by components of cord blood collection systems. An analysis of the effect of various anticoagulants whose mechanism of action is due to calcium ion binding (ACD, EDTA, XAPD-1) on hematopoietic progenitor cells under conditions of cord blood storage for 24 to 72 hours revealed their negative 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 increasing the storage period of unfractionated cord blood to 72 hours and preserves the functional activity of colony-forming units. However, a study of the safety of CFU-GM and CFU-G showed that the storage time of cord blood before cryopreservation should not exceed nine hours. Obviously, the principle that should apply in this case is that in the presence of conflicting data, the minimum recommended storage period for cord blood should be used and programmed freezing of the isolated cells should be initiated as soon as possible.

When freezing cord blood hematopoietic stem cells, a 10% DMSO solution is usually used as a cryoprotectant. However, in addition to the pronounced cryoprotective effect, dimethyl sulfoxide in such a concentration also has a direct cytotoxic effect, even with minimal exposure to cord blood hematopoietic cells. To reduce the cytotoxic effect of DMSO, zero exposure temperature is used, the speed of all manipulations is increased, and multiple washings are performed after thawing of cord blood samples.

Since 1995, the Institute of Hematology and Transfusiology of the Academy of Medical Sciences of Ukraine has been developing a scientific direction aimed at a comprehensive study of umbilical cord blood as an alternative source of hematopoietic stem cells. In particular, new technologies for low-temperature cryopreservation of hematopoietic cells of unfractionated and fractionated umbilical cord blood have been developed. Low-molecular medical polyvinylpyrrolidone is used as a cryoprotectant. The method of cryopreservation of unfractionated umbilical cord blood is based on an original technology for pre-preparation of cells for freezing and a method for special processing of cell suspension immediately before transplantation.

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

The most dangerous stages for the viability of cells during their preparation are the stages of direct freezing and thawing. When freezing hematopoietic 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, cryoprotectors are used, the mechanisms of action and cryoprotective efficiency of which are sufficiently covered in scientific literature.

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

For cryopreservation of umbilical cord blood cells, a 20% DMSO solution is traditionally used, which is slowly poured into the cell suspension with constant mechanical stirring in an ice bath until an equal (1:1) ratio of the cryoprotectant and cell suspension volumes is achieved. The final concentration of dimethyl sulfoxide is 10%. The cell suspension is then cooled in a programmed cryogenic unit at a rate of GS/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 cryopreservation technique, the preservation of functionally active mononuclear cells after defrosting reaches 85% of the original level.

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

When using a dimethyl sulfoxide solution in concentrations from 5 to 10% in combination with 4% hydroxyethyl starch (final concentration), it was found that the safety of CD34-positive cells in such ranges of dimethyl sulfoxide remains virtually unchanged. At the same time, when the concentration of dimethyl sulfoxide decreases from 5 to 2.5%, massive death 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 it is 5 and 10% dimethyl sulfoxide solutions (in the author's version - in combination with autologous serum) that provide cytoprotection with maximum efficiency during cryopreservation of umbilical cord blood HSCs. In addition, high preservation of successively frozen and thawed cells is noted in the case of a combination of 5 or 10% dimethyl sulfoxide with a 4% hydroxyethyl starch solution, especially at a controlled cooling rate of GS/min. In another study, a cryoprotective solution was used consisting of three ingredients - DMSO, purified human albumin and RPMI medium in a ratio of 1:4:5, which was added to the cell suspension to an equal volume ratio (the final concentration of dimethyl sulfoxide was 5%). After defrosting in a water bath at a temperature of +4 GS, the preservation of CFU-GM exceeded 94%.

Some authors suggest using unfractionated cord blood for cryopreservation, since significant amounts of hematopoietic cells are lost during the process of removing red blood cells. In this variant, a 10% solution of dimethyl sulfoxide is used to protect mononuclear cells from the damaging effects of cryocrystallization. Freezing is carried out at a constant cooling rate of GS/min to -80°C, after which the cord blood cell suspension is lowered into liquid nitrogen. This freezing method results in partial lysis of red blood cells, so blood samples do not require fractionation. After defrosting, the cell suspension is washed from free hemoglobin and dimethyl sulfoxide in a solution of human albumin or in the patient's autologous blood serum and used for transplantation.

The preservation of hematopoietic progenitor cells after thawing of unfractionated cord blood is indeed higher than that of fractionated cord blood, however, due to the cryostability of some erythrocytes, serious post-transfusion problems may arise due to transfusion of ABO-incompatible erythrocytes. In addition, the volume of stored unfractionated blood increases significantly. From a clinical point of view, cryopreservation of previously isolated and purified from other cell fractions cord blood hematopoietic cells is still preferable.

In particular, a method for cryopreservation of fractionated umbilical cord blood cells has been developed, allowing the removal of 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 "Stabizol". After defrosting, the cell suspension obtained in this way is ready for clinical use without additional manipulations.

Thus, at present there are many quite effective methods of cryopreservation of umbilical cord blood. Their fundamental difference is that blood samples are frozen unfractionated or are subjected to separation into cell fractions at the preparation stage and nucleated cells without admixture of erythrocytes are prepared.

Cord blood hematopoietic stem cell transplantation

In the late 1980s and early 1990s, it was established that umbilical cord blood, which provides the fetus with life support during pregnancy, has a high content of hematopoietic stem cells. The relative simplicity of obtaining umbilical cord blood cells and the absence of obvious ethical problems contributed to the use of umbilical cord blood stem cells in practical medicine. The first successful cord blood transplant to a child with Fanconi anemia served as a starting point for expanding the volume of cord blood stem cell transplants and creating a system for its banking. In the world system of cord blood banks, the largest is the New York Placental Blood Center, which is on the balance sheet of the US National Institute of Health. The number of stored umbilical cord blood samples in this bank is approaching 20,000. The number of recipients (mostly children) who have undergone a successful transplant is also growing. According to the US Department of Health, the relapse-free period of post-transplant life of cord blood transplant recipients already exceeds 10 years.

This is not surprising, since numerous studies of the hematopoietic potential of umbilical cord blood have shown that in terms of the quantity and quality of the earliest stem cells, it is not only not inferior to the bone marrow of an adult, but also exceeds it in some respects. The higher proliferative potential of cord blood stem cells is due to the ontogenetic features of cellular signaling, the presence of receptors for specific growth factors on the HSC, the ability of cord blood cells to autocrine production of growth factors, and the large size and length of telomeres.

Thus, the genomic and phenotypic characteristics of cord blood hematopoietic stem cells predetermine high-quality engraftment of the transplant with a high potential for restoration of donor hematopoiesis in the recipient’s body.

Benefits of cord blood hematopoietic stem cells

Among the real advantages of using cord blood hematopoietic stem cells for transplantation over other sources of hematopoietic cells, it is worth noting the virtually zero risk to the donor's health (if we do not consider the placenta as such) and the absence of the need for general anesthesia. The use of cord blood expands the possibilities of cell transplantation due to partially HLA-compatible transplants (incompatibility from one to three antigens). A method for long-term storage of cord blood hematopoietic cells in a frozen state has been developed, which increases the likelihood of obtaining rare HLA types and reduces the time to search for an HLA-compatible transplant for allogeneic transplantation. At the same time, the risk of developing certain latent infections transmitted by transmissible means is significantly reduced. In addition, an inexpensive form of biological life insurance arises due to the possibility of using cord blood cells for autologous transplantation.

However, due to the small volumes of blood that can be collected from the placenta (on average no more than 100 ml), the problem of obtaining the maximum possible amount of blood from the umbilical cord vein comes to the fore while strictly observing the condition of minimal risk of bacterial contamination of the obtained umbilical cord blood samples.

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

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

As already mentioned, significant differences between bone marrow and cord blood are in the amount of hematopoietic cells used for transplantation obtained during one collection procedure. If during bone marrow transplantation the loss of cell mass during separation, cryopreservation, thawing and testing is acceptable within 40-50%, then for cord blood such cell losses are very significant, since if an insufficient amount of HSC is used, the transplant may prove to be ineffective. According to G. Kogler et al. (1998), for cell transplantation with a recipient's body weight of 10 kg, all cord blood samples can be potential transplants (the total number of collected cord blood samples is 2098), with a body weight of 35 kg - 67%, and only 25% of samples can provide effective transplantation in patients with a body weight of 50-70 kg. This clinical situation indicates the need to optimize and improve the efficiency of existing methods of collecting, reproducing and storing umbilical cord blood cells. Therefore, the literature currently widely discusses the issues of standardizing methods of collecting, testing, separating and cryopreserving umbilical cord blood to create blood banks, its use in the clinic, and also stipulates the conditions and terms of storing hematopoietic stem cells of umbilical cord blood.

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Use of cord blood hematopoietic stem cells in medicine

^{Usually, it is possible to isolate up to 10 6} hematopoietic stem cells from umbilical cord blood, rarely more. In this regard, the question of whether such a quantity of hematopoietic cells from umbilical cord blood is sufficient to restore hematopoiesis in an adult recipient remains open to this day. Opinions on this matter are divided. Some researchers believe that such a quantity is quite sufficient for transplantation to children, but too little for transplantation to an adult, for whom the optimal amount is the introduction of (7-10) x 10 ⁶ CD34-positive cells per 1 kg of body weight - an average of 7 x 10 ⁸ per transplant. From these calculations it follows that one sample of umbilical cord blood contains 700 times fewer 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 bone marrow cells and does not take into account the ontogenetic features of hematopoiesis at all.

In particular, the fact of a higher proliferative potential of cord blood hematopoietic stem cells compared to bone marrow hematopoietic progenitor cells is ignored. The results of in vitro colony-forming potential studies suggest that one dose of cord blood is capable of providing reconstitution of adult recipient hematopoiesis. On the other hand, it should not be forgotten that the number of cord blood stem cells decreases even during embryonic development: the content of CD34-positive cells in cord blood decreases linearly by 5 times in the period from 20 weeks (blood for the study was obtained during premature termination of pregnancy) to 40 weeks of gestation (the period of physiological labor), which is accompanied by a parallel, permanently increasing expression of linear cytodifferentiation markers.

Due to the lack of a standardized approach to the quantitative determination of progenitor cells in cord blood samples, debates about the optimal dose of cord blood hematopoietic stem cells continue. Some researchers believe that the number of nucleated cells and mononuclear cells recalculated for the recipient's body weight, i.e., their dose, can be used as criteria for selecting cord blood samples. Some authors believe that the minimum quantitative threshold of CD34+ cells even for autotransplantation of HSCs is 2 x 10 ⁶ /kg. At the same time, an increase in the dose of hematopoietic cells to 5 x 10 ⁶ cells/kg (only 2.5 times) already ensures a more favorable course of the early post-transplant period, reduces the incidence of infectious complications and shortens the duration of preventive antibiotic therapy.

According to E. Gluckman et al. (1998), in oncohematology the condition for successful cord blood cell transplantation is the introduction of at least 3.7 x 10 ⁷ nucleated cells per 1 kg of recipient body weight. When the dose of hematopoietic stem cells is reduced to 1 x 10 ⁷ or less nucleated cells per 1 kg of patient body weight, the risk of transplant failure and relapse of blood cancer increases sharply. It should be recognized that the minimum number of progenitor cells necessary for rapid restoration of hematopoiesis after allotransplantation of HSCs is still unknown. Theoretically, this can be achieved using one cell, but in clinical practice of bone marrow transplantation, rapid and stable engraftment is guaranteed by transfusing at least (1-3) x 10 ⁸ nucleated cells per 1 kg of patient body weight.

A recent detailed study to determine the optimal number of HSCs in oncohematology included observation of patients in three groups, allocated depending on the content of CD34-positive cells in the transplanted material. Patients of the first group were administered (3-5) x 10 ⁶ cells/kg. The HSC dose in patients of the second group was (5-10) x 10 ⁶ cells/kg, and patients of the third group were transplanted with more than 10 x 10 ⁶ CD34+ cells/kg. The best results were observed in the group of recipients who received 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 x 10 ⁶ /kg, statistically significant advantages were not revealed. In this case, a very high content of HSCs in the transplant (> 10 x 10 ⁶ /kg) is associated with reinfusion of a significant number of residual tumor cells, which leads to relapse of the disease. A direct relationship between the number of transplanted allogeneic progenitor cells and the development of the graft-versus-host reaction has not been established.

The accumulated world experience of cord blood transplantation confirms their high repopulation potential. The rate of engraftment of the cord blood transplant correlates with the number of introduced nucleated cells. The best results are observed with transplantation of 3 x 10 ⁷ /kg, while for bone marrow this dose is 2 x 10 ⁸ /kg. According to the data of coordinating centers, at the end of 2000, 1200 cord blood cell transplantations were performed worldwide, mainly from related donors (83%). It is obvious that cord blood should be considered as an alternative to bone marrow for transplantation to patients with hemoblastoses.

At the same time, the neonatal nature of the cord source of hematopoietic tissue inspires optimism due to the presence of functional features of its HSC. At the same time, only clinical experience can answer the question of the sufficiency of one cord blood sample to restore hematopoiesis in an adult recipient with hematopoietic aplasia. Transplantation of umbilical cord blood cells is used in treatment programs for many tumor and non-tumor diseases: leukemia and myelodysplastic syndromes, non-Hodgkin's lymphoma and neuroblastoma, aplastic anemia, congenital Fanconi and Diamond-Blackfan anemias, leukocyte adhesion deficiency, Barr syndrome, Gunther's disease, Hurler syndrome, thalassemia.

Immunological aspects of cord blood hematopoietic cell transplantation deserve close attention and a separate study. It has been shown that in the case of cord blood stem cell transplantation from donors with incomplete HLA compatibility, the transplantation results are quite satisfactory, which, according to the authors, indicates a lower immunoreactivity of cord blood cells than bone marrow.

A detailed study of the cellular composition of umbilical cord blood revealed the features of both the phenotypic spectrum of effector cells of the immune system and their functional activity, which made it possible to consider cord blood as a source of HSCs with a relatively low risk of developing a 'graft versus host' reaction. Among the signs of functional immaturity of immunocompetent cells of umbilical cord blood, it is necessary to note the imbalance in the production of cytokines and a decrease in sensitivity to cytokine regulation of the immune response. The resulting inhibition of the activity of cytotoxic lymphocytes is considered a factor contributing to the formation of immunological tolerance to the transplanted hematopoietic tissue. In the population of umbilical cord blood lymphocytes, in contrast to the peripheral blood and bone marrow of adult donors, inactive, immature lymphocytes and suppressor cells predominate. This indicates a reduced readiness of umbilical cord blood T-lymphocytes for an immune response. An important feature of the monocytic population of umbilical cord blood cells is the low content of functionally full-fledged and active antigen-presenting cells.

On the one hand, the low maturity of the immune system effector cells in 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 cells of the donor and the recipient. But, on the other hand, it is known about the existence of 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 this regard, a study was conducted on the antitumor cytotoxicity of cord blood cells. The results obtained indicate that, despite the truly weakened response of immunocompetent cord blood cells to antigen stimulation, the lymphocytes that are primarily activated are natural killers and killer-like cells that take an active part in the mechanisms of implementation of antitumor cytotoxicity. In addition, subpopulations of lymphocytes with the CD16+CD56+ and CD16"TCRa/p+ phenotypes were found in cord blood. It is assumed that these cells in their activated form implement the “graft versus leukemia” reaction.

At the Institute of Oncology of the Academy of Medical Sciences of Ukraine, cryopreserved hematopoietic cells of umbilical cord blood were administered to cancer patients with persistent hematopoietic hypoplasia due to chemo- and radiotherapy. In such patients, transplantation of hematopoietic stem cells of umbilical cord blood quite effectively restored depressed hematopoiesis, as evidenced by a persistent increase in the content of mature formed elements in the peripheral blood, as well as an increase in the indicators characterizing the state of cellular and humoral immunity. The stability of the repopulation effect after transplantation of hematopoietic cells of umbilical cord blood allows continuing radiation and chemotherapy without interrupting the course of treatment. There is information on a higher efficiency of allotransplantation of umbilical cord blood stem cells to oncohematological patients: the annual risk of relapse of a tumor disease with their use was 25% versus 40% in patients with transplanted allogenic bone marrow.

The mechanism of action of cryopreserved cord blood stem cells should be considered the result of humoral stimulation of recipient hematopoiesis caused by the unique ability of neonatal cells to autocrine production of hematopoietic growth factors, as well as a consequence of temporary engraftment of donor cells (as evidenced by a reliable increase in the content of fetal hemoglobin in the peripheral blood of recipients on the 7-15th day after transfusion compared to the initial data). The absence of post-transfusion reactions in cord blood recipients is the result of the relative tolerance of its immunocompetent cells, as well as a confidence criterion for the biological adequacy of the cryopreserved material.

Cord blood T-lymphocyte killer progenitor cells are capable of activation under the influence of exogenous cytokine stimulation, which is used to develop new ex vivo and in vivo methods for inducing antitumor cytotoxicity of transplant lymphoid elements for subsequent immunotherapy. In addition, the “immaturity” of the genome of umbilical cord blood immunocompetent cells allows them to be used to enhance antitumor activity using molecular modeling methods.

Today, cord blood has found wide application primarily in pediatric hematology. In children with acute leukemia, allotransplantation of cord blood hematopoietic stem cells, compared with allotransplantation of bone marrow, significantly reduces the incidence of graft-versus-host disease. However, this is accompanied by a longer period of neutro- and thrombocytopenia and, unfortunately, a higher 100-day post-transplant mortality rate. A longer period of recovery of granulocyte and platelet levels in the peripheral blood may be due to insufficient differentiation of individual subpopulations of CD34-positive cord blood cells, as evidenced by the low level of absorption of radioactive rhodamine and 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 both a compatible unrelated bone marrow donor and the possibility of mobilizing autologous HSCs, showed a high one-year relapse-free survival in the group of patients under 30 years of age (73%). Expanding the age range of recipients (18-46 years) led to a decrease in survival to 53%.

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

Cord Blood Stem Cell Banks

For the proper development of a new area of practical medicine - cord blood stem cell transplantation, as well as for the implementation of bone marrow hematopoietic stem cell transplants, it is necessary to have an extensive network of blood banks, which have already been created in the USA and Europe. Domestic cord blood bank networks are united by the Netcord Bank Association. The expediency of creating an international association of cord blood banks is determined by the fact that a large number of typed cord blood samples are needed to perform unrelated transplants, which allows selecting an HLA-identical donor. Only the establishment of a system of banks with storage of blood samples of various HLA types can really solve the problem of finding the necessary donor. The organization of such a cord blood bank system requires preliminary development of ethical and legal norms, which are currently being discussed at the international level.

In order to create cord blood banks in Ukraine, a whole series of regulations and documents must be worked out.

First of all, these are issues of standardization of methods of collection, fractionation and freezing of umbilical cord blood. It is necessary to regulate the rules of collection of umbilical cord blood in maternity hospitals in accordance with the requirements of medical ethics, to determine the minimum volume of umbilical 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 diagnostic methods for genetic and infectious diseases that can be transmitted during infusion of umbilical cord blood cells, to establish common criteria for the selection of healthy donors. It is also worth discussing the issues of creating separate storage facilities for serum, cells and DNA obtained from umbilical cord blood.

It is absolutely necessary to organize a computer network of cord blood data to link with bone marrow donor registries. For further development of cell transplantology, it is necessary to develop special protocols for comparing the results of cord blood and bone marrow transplantation from HLA-identical relatives and unrelated donors. Standardization of documentation, including informed consent of parents, as well as notification of the mother or relatives about genetic and/or infectious diseases detected in the child, can help solve the ethical and legal problems of the clinical use of cord blood cells.

The defining condition for the development of cell transplantology 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 Marrow Donor Association (WMDA), the US National Marrow Donor Program (NMDP) and other registries.

Summarizing the still short history of the development of cord blood hematopoietic stem cell transplantation, we note that the first assumptions about the possibility of using cord blood in a clinic, expressed back in the early 70s, were confirmed in the 80s by the results of experimental studies on animals, and in 1988 the world's first transplantation of cord blood hematopoietic cells to a human was performed, after which a global network of cord blood banks began to be created. In 10 years, the number of patients with transplanted cord blood hematopoietic cells approached 800. Among them were patients with various diseases of tumor (leukemia, lymphoma, solid tumors) and non-tumor (congenital immunodeficiencies, anemia, diseases associated with metabolic disorders) nature.

The content of early and committed cell precursors in umbilical cord blood is higher than in the peripheral blood of an adult. In terms of 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, greater proliferative activity and viability of umbilical cord blood cells than bone marrow cells have been noted. Critical moments in cord blood stem cell transplantation are the number and hematopoietic potential of nucleated cells, the presence of cytomegalovirus infection, HLA compatibility of the donor and recipient, body weight and age of the patient.

However, cord blood stem cell transplantation should be considered as an alternative to bone marrow transplantation for the treatment of severe blood diseases, primarily in children. Clinical problems of cord blood cell transplantation are gradually being resolved - there are already quite effective methods for collecting, separating and cryopreserving cord blood cells, conditions are being provided for the formation of cord blood banks, and methods for testing nucleated cells are being improved. 3% gelatin solution and 6% hydroxyethyl starch solution should be considered optimal for separation during large-scale procurement of cord blood hematopoietic stem cells when creating banks.

P. Perekhrestenko and co-authors (2001) rightly note that cord blood stem cell transplantation should take its rightful place in the complex of therapeutic measures to overcome hematopoietic depressions of various genesis, since cord blood stem cells have a number of significant advantages, among which the most important are the relative simplicity of their procurement, the absence of risk for the donor, low contamination of neonatal cells with viruses and the comparatively low cost of transplantation. Some authors point out that cord blood cell transplantation is less often accompanied by complications associated with the graft-versus-host reaction than bone marrow cell transplantation, which is due, in their opinion, to the weak expression of HLA-DR antigens on cord blood cells and their immaturity. However, the main population of nucleated cells in cord blood are T lymphocytes (CD3-positive cells), the content of which 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 influencing the survival rate in cord blood stem cell transplantation, it is worth noting the age of the patients (the best results are observed in recipients aged from one to five years), early diagnosis of the disease and the form of leukemia (the effectiveness is significantly higher in acute leukemia). Of great importance are the dose of nucleated cord blood cells, as well as their HLA compatibility with the recipient. It is no coincidence that the analysis of the clinical effectiveness of cord blood HSC transplantation in oncohematology shows the best treatment results when using related transplants: the one-year relapse-free survival in this case reaches 63%, while with unrelated transplantation - only 29%.

Thus, the presence of a large number of stem cells in cord blood and the high repopulation capacity of neonatal hematopoietic stem cells allow them to be used for allogeneic transplantation in oncohematological patients. However, it should be noted that the recapitulation of hematopoiesis after transplantation of cord blood hematopoietic cells is “stretched out in time”: restoration of the neutrophil content in the peripheral blood is usually observed by the end of the 6th week, and thrombocytopenia usually disappears after 6 months. In addition, the immaturity of cord blood hematopoietic cells does not exclude immunological conflicts: severe acute and chronic graft-versus-host disease is observed in 23 and 25% of recipients, respectively. Relapses of acute leukemia by the end of the first year after cord blood cell transplantation are noted in 26% of cases.

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