Limitations, dangers and complications of cell transplantation

Regenerative-plastic medicine is based on the realization in the clinic of the totality and pluripotent properties of embryonic and progenitor stem cells, which allow in vitro and in vivo to create predetermined cell lines repopulating damaged tissues and organs of a sick person.

The real possibility of using the stem cells of the embryo and stem cells of definitive tissues (the so-called adult stem cells) of a human being for therapeutic purposes is no longer in doubt. However, experts at the National and Medical Academies of the United States (USA) recommend that the properties of stem cells in experiments be studied in more detail on adequate biological models and objectively evaluate all the consequences of transplantation, and only then use stem cells in the clinic.

It is established that stem cells are part of the tissue derivatives of all three embryonic leaflets. Stem cells are found in the retina of the eye, the cornea, the epidermis of the skin, the bone marrow and in the peripheral blood, in the vessels, the tooth pulp, the kidney, the epithelium of the digestive tract, the pancreas and the liver. With the help of modern methods it is proved that stem neural cells are localized in the adult brain and spinal cord. These sensational data attracted special attention of scientists and the media, since the neurons of the brain served as a classic example of a static cell population that does not recover. Both in the early and late periods of ontogenesis, neurons, astrocytes and oligodendrocytes are formed in the brain of animals and humans due to neuronal stem cells (Stem cells: scientific progress and future research directions., Nat Inst, of Health USA).

However, under normal conditions, the plasticity of stem cells of definitive tissues does not appear. To realize the plastic potential of stem cells of definitive tissues, they must be isolated and then cultured in media with cytokines (LIF, EGF, FGF). Moreover, stem cell derivatives successfully survive only when transplanted into the body of an animal with a depressed immune system (γ-irradiation, cytostatics, busulfan, etc.). To date, there has been no convincing evidence of stem cell plasticity in animals that have not been irradiated or otherwise exposed to deep immunosuppression.

In such conditions, dangerous potencies of ESCs are manifested, first of all, when they are transplanted into ectopic areas - with subcutaneous injection of ESC into immunodeficient mice teratocarcinomas are formed at the site of administration. In addition, in the development of the human embryo, the frequency of chromosomal abnormalities is higher than in embryogenesis in animals. At the stage of blastocyst, only 20-25% of human embryos consist of cells with normal karyotype, and in the predominant majority of early human embryos obtained after in vitro fertilization, chaotic chromosomal mosaicism is found, and numerical and structural aberrations are very common.

[1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]

Beneficial effect of stem cells

Preliminary results of clinical trials confirm the beneficial effect of stem cells on the patient, but so far there is no information on the long-term consequences of cell transplantation. In the literature, at first, reports of positive results of transplantation of brain fragments of embryos in Parkinson's disease predominated, but then data began to appear that denied the effective therapeutic effect of embryonic or fetal nervous tissue transplanted into the brain of patients.

In the middle of the 20th century, restoration of hematopoiesis in lethally irradiated animals after intravenous transfusion of bone marrow cells was first discovered, and in 1969 the American researcher D. Thomas performed the first bone marrow transplantation to a human. The lack of knowledge about the mechanisms of immunological incompatibility of the bone marrow cells of the donor and recipient at that time caused high lethality due to frequent non-grafting and development of the "graft versus host" reaction. The discovery of the main histocompatibility complex, which includes human leukocyte antigens (HbA), and the improvement of their typing methods, significantly increased the survival rate after bone marrow transplantation, which led to a wide spread of this method of treatment in oncohematology. After a decade, the first transplantation of hematopoietic stem cells (HSC) obtained from peripheral blood with the help of leukapheresis was performed. In 1988, in France, for the treatment of a child with Fanconi anemia, umbilical cord blood was used as the source of HSC for the first time, and since the end of 2000, reports began to print about the ability of HSC to differentiate into cells of various types of tissues, potentially expanding the area of their clinical application. However, it turned out that the material for transplantation, along with GSK, contains a significant number of non-hemopoietic cells, which are diverse in nature and properties. In connection with this, methods for purifying the graft and criteria for assessing its cellular purity are being developed. In particular, positive immunosection of CD34 + cells is used, which makes it possible to isolate HSC with the help of monoclonal antibodies.

[13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23]

Complications of stem cell therapy

Complications of bone marrow transplantation are most often hematological and are associated with a prolonged period of iatrogenic pancytopenia. The most common infections are anemia, anemia and hemorrhages. In this regard, it is extremely important to select the optimal mode of sampling, processing and storing the bone marrow for maximum preservation of stem cells, which will ensure rapid and stable recovery of hematopoiesis. When characterizing a transplant, it is currently accepted to evaluate the following parameters: the number of mononuclear and / or nucleated cells, colony-forming units and the content of SB34-positive cells. Unfortunately, these indicators only provide an indirect estimate of the real hematopoietic ability of the stem cell transplant population. For today, there are absolutely precise parameters for determining the sufficiency of the transplant for the long-term recovery of hemopoiesis in patients even in autologous bone marrow transplantation. The development of common criteria is extremely difficult due to the lack of rigid processing standards, cryopreservation and graft testing. In addition, it is necessary to take into account the variety of factors that influence the parameters of the successful recovery of hematopoiesis in each particular patient. In autologous bone marrow transplantation, the most important of them are the number of previous courses of chemotherapy, the peculiarities of the conditioning regime, the period of the disease in which bone marrow sampling is performed, and the regimens for applying colony-stimulating factors in the posttransplant period. In addition, it should not be forgotten that chemotherapy preceded by fetal grafting can have a negative effect on bone marrow stem cells.

The incidence of severe toxic complications increases significantly with allogeneic bone marrow transplantation. In this connection, statistical data on the transplantation of allogeneic bone marrow in thalassemia are of interest. In the reports of the European Bone Marrow Transplantation Group, about 800 bone marrow transplantations were recorded in patients with large thalassemia. Allogeneic transplantation in thalassemia in the vast majority of cases is performed from HLA-identical siblings, which is associated with severe complications and high mortality in transplantation of stem cell material from partially compatible related or compatible unrelated donors. To minimize the risk of fatal infectious complications, patients are placed in isolated aseptic boxes with a laminar flow of air, receive a low- or abacterial diet. For bacterial decontamination of the intestine per os prescribe non-resorptive forms of antibiotics, antifungal drugs. For the prevention of intravenous amphotericin B is administered. Prevention of systemic infections is fixed with amikacin and ceftazidime, which are prescribed the day before transplantation, continuing treatment until discharge of patients. All blood preparations before irradiation are irradiated at a dose of 30 Gy. Parenteral nutrition during transplantation is a prerequisite and begins immediately by limiting the intake of food naturally.

A number of complications are associated with high toxicity of immunosuppressive drugs, which often cause nausea, vomiting and mucositis, kidney damage and interstitial pneumonia. One of the most severe complications of chemotherapy is veno-occlusive disease of the liver, leading to death in the early post-transplant period. Among the risk factors for venous thrombosis in the portal system of the liver, the age of patients, the presence of hepatitis and liver fibrosis, and immunosuppressive therapy after bone marrow transplantation should be noted. Veno-occlusive disease is especially dangerous in thalassemia, which is accompanied by hemosiderosis of the liver, hepatitis and fibrosis - frequent satellites of transfusion therapy. Thrombosis of the veins of the portal system of the liver develops 1-2 weeks after transplantation and is characterized by a rapid increase in the content of bilirubin in blood and transaminase activity, the progression of hepatomegaly, ascites, encephalopathy and pain in the upper abdomen. Histologically, in the autopsy material, endothelium damages, subendothelial hemorrhages, damage to centrolobular hepatocytes, thrombotic venous obstruction and central veins of the liver are determined. In patients with thalassemia, cases of fatal cardiac arrest associated with toxic effects of cytostatics have been described.

In preparation for transplantation, cyclophosphamide and busulfan often cause toxic hemorrhagic cystitis with pathological changes in uroepithelial cells. The use of cyclosporine A in bone marrow transplantation is often accompanied by effects of nephro- and neurotoxicity, hypertension syndrome, fluid retention in the body, and cytolysis of hepatocytes. Violation of sexual and reproductive function is more often observed in women. In young children after transplantation pubertal development usually does not suffer, but in older children the pathology of the development of the genital sphere can be very serious - up to sterility. Complications directly related to the transplant itself include rejection of allogeneic bone marrow cells, incompatibility in the ABO system, acute and chronic forms of the "graft versus host" reaction.

In the body of patients after ADO-incompatible bone marrow transplantation, the "host versus ABO donor" isoagglutinins are produced within 330-605 days after transplantation, which can lead to prolonged hemolysis and sharply increase the need for blood transfusions. This complication is prevented by transfusion of erythrocytes only in group 0. After transplantation, autoimmune neutropenia, thrombocytopenia or pancytopenia are noted in a number of patients, for the correction of which splenectomy is necessary.

In 35-40% of recipients, the acute "graft versus host" reaction develops within 100 days after the transplantation of the allogeneic HbA-identical bone marrow. The degree of damage to the skin, liver and intestine varies from rash, diarrhea and moderate hyperbilirubinemia to skin desquamation, intestinal obstruction and acute liver failure. In patients with thalassemia, the incidence of an acute "graft-versus-host" reaction of the first degree after bone marrow transplantation is 75%, grade II and above - 11-53%. The chronic "graft versus host" reaction as a systemic multi-organ syndrome usually develops within 100-500 days after allogennial bone marrow transplantation in 30-50% of patients. The skin, mouth, liver, eyes, esophagus and upper respiratory tract are affected. There is a limited form of chronic "graft versus host" reaction when the skin and / or liver are affected, and common, when generalized skin damage is combined with chronic aggressive hepatitis, eye damage, salivary glands or any other organ. The cause of death is often the infectious complications that result from severe immunodeficiency. In thalassemia, the mild form of the chronic "graft versus host" reaction occurs in 12%, moderate in 3% and severe in 0.9% of allogeneic HLA-compatible bone marrow recipients. A serious complication in bone marrow transplantation is transplant rejection, which develops 50-130 days after the operation. The rejection frequency depends on the conditioning mode. In particular, in thalassemia patients receiving only methotrexate during the preparation period, bone marrow transplant rejection is observed in 26% of cases, with a combination of methotrexate and cyclosporin A - in 9%, and in the appointment of only cyclosporin A - in 8% of cases (Gaziev et al. ., 1995).

Infectious complications after bone marrow transplantation cause viruses, bacteria and fungi. Their development is associated with deep neutropenia, which chemotherapy drugs induce during conditioning, cytostatic damage to mucous membranes and the "graft-versus-host" reaction. Depending on the time of development, three phases of infectious complications are distinguished. In the first phase (developing in the first month after transplantation), mucosal barrier and neutropenia lesions predominate, often accompanied by viral infections (herpes, Epstein-Barr virus, cytomegalovirus, Varicella zoster), as well as infections caused by gram-positive and gram-negative bacteria, Candida fungi , aspergillomas. In the early posttransplant period (the second and third months after transplantation), the most serious infection is cytomegalovirus, which often leads to the death of patients in the second phase of infectious complications. In thalassemia, cytomegalovirus infection after bone marrow transplantation develops in 1.7-4.4% of recipients. The third phase is observed in the late post-transplant period (three months after the operation) and is characterized by severe combined immunodeficiency. In this period, there are usually infections caused by Varicella zoster, streptococcus, Carini pneumoniae, Neisseria meningitidis, Haemophilus influenzae, as well as hepatotropic viruses. In thalassemia, the mortality of patients after bone marrow transplantation is associated with bacterial and fungal sepsis, idiopathic interstitial and cytomegalovirus pneumonia, acute respiratory distress syndrome, acute heart failure, cardiac tamponade, cerebral hemorrhages, veno-occlusive disease of the liver and acute graft-versus-host reaction.

At present, certain progress has been made in developing methods for isolating from the bone marrow a pure population of stem hemopoietic cells. The technique of obtaining fetal blood from the umbilical cord has been improved and methods have been developed for isolating blood-forming cells from cord blood. In the scientific press there are reports that when cultured in media with cytokines, hematopoietic stem cells are capable of multiplication. When using specially designed bioreactors for the expansion of HSC, the biomass of stem hemopoietic cells isolated from bone marrow, peripheral or cord blood is significantly increased. The possibility of HSC expansion is an important step in the clinical development of cell transplantation.

However, before the reproduction of HSC in vitro, it is necessary to isolate a homogeneous population of hematopoietic stem cells. This is usually achieved using markers that allow selective marking of HSCs with monoclonal antibodies covalently bound to a fluorescent or magnetic label and isolating them using an appropriate cell sorter. At the same time, the issue of phenotypic characteristics of hematopoietic stem cells has not been finally solved. A. Petrenko., V. Grishchenko (2003), cells on the surface of which CD34, AC133 and Thyl antigens are present and CD38, HLA-DR and other markers of differentiation (cells with the CD34 + Liir phenotype) are considered as candidates for HSC. Linear differentiation markers (lineage, Lin) include glycophorin A (GPA), CD3, CD4, CD8, CD10, CD14, CD16, CD19, CD20 (Muench, 2001). Perspective for transplantation are cells with the phenotype CD34 + CD45RalüW CD71low, as well as CD34 + Thyl + CD38low / c-kit / low.

The problem of the number of HSCs that is sufficient for effective transplantation remains a problem. Currently, the source of stem blood-forming cells are the bone marrow, peripheral and cord blood, as well as the embryonic liver. Expansion of stem hemopoietic cells is achieved by culturing them in the presence of endotheliocytes and hematopoietic growth factors. In various protocols, myeloproteins, SCF, erythropoietin, insulin-like growth factors, corticosteroids and estrogens are used to induce HSC proliferation. When combinations of cytokines are used in vitro, a significant increase in HSC pool can be achieved with a peak of their release at the end of the second week of cultivation.

Traditionally, HSC cord blood is used mainly in hemoblastoses. However, the minimum dose of hematopoietic cells necessary for successful transplantation of cord blood cells is 3.7 x 10 ⁷ nucleated cells per 1 kg of body weight of the recipient. Using a smaller amount of HSC significantly increases the risk of graft failure and relapse of the disease. Therefore, transplantation of blood-forming cells of umbilical cord blood is mainly used in the treatment of hemoblastosis in children.

Unfortunately, there are still no procurement standards, as well as standardized protocols for the clinical use of cord blood hemopoietic cells. Accordingly, cord blood stem cells themselves are not a legally recognized source of hematopoietic cells for transplantation. In addition, there are neither ethical nor legal norms regulating the activity and organization of umbilical cord blood banks, which are available abroad. Meanwhile, for safe transplantation, all samples of umbilical cord blood should be carefully monitored. Before a blood sample is collected from a pregnant woman, her consent must be obtained. Each pregnant woman should be examined for carriage of HBsAg, the presence of antibodies to hepatitis C viruses, HIV infection and syphilis. Each cord blood sample should be routinely tested for the number of nucleated cells, CD34 + and colony-forming ability. In addition, HLA typing, determination of the blood group according to the ABO and its belonging to the Rhesus factor are carried out. The necessary testing procedures are bacteriological culture for sterility, serological testing for HIV-1 and HIV-2 infection, HBsAg, viral hepatitis C, cytomegalovirus infection, HTLY-1 and HTLY-II, syphilis and toxoplasmosis. In addition, a polymerase chain reaction is performed to detect cytomegalovirus and HIV infection. It seems advisable to supplement the test protocols with cord blood analysis for the detection of genetic diseases such as a-thalassemia, sickle cell anemia, adenosine deaminase deficiency, Bruton's agammaglobulinemia, Harler's and Ponter's disease.

At the next stage of preparation for transplantation, the question arises of the preservation of GSK. The most dangerous for the viability of cells when preparing them are freezing and thawing procedures. When freezing the hemopoietic cells, a significant part of them can be destroyed due to crystal formation. To reduce the percentage of cell death, special substances are used - cryoprotectants. Most often, as a cryoprotectant, DMSO is used at a final concentration of 10%. However, for DMSO, this concentration is characterized by a direct cytotoxic effect, which manifests itself even under conditions of minimal exposure. Reduction of the cytotoxic effect is achieved by strict maintenance of the zero temperature of the exposure mode, as well as by observance of the procedure for processing the material in the process and after defrosting (speed of all manipulations, application of reusable washing procedures). Do not apply a DMSO concentration of less than 5%, since in this case, the mass death of hematopoietic cells occurs during the freezing period.

The presence of red blood cell impurities in the suspension mixture GSK creates the danger of developing an incompatibility reaction for erythrocyte antigens. At the same time, with the removal of erythrocytes, the loss of hematopoietic cells significantly increases. In this connection, a method for unfractionated separation of GCS was proposed. In this case, 10% DMSO solution and cooling at a constant rate (HS / min) to -80 ° C are used to protect the nucleated cells from the damaging effect of low temperatures, after which the cell suspension is frozen in liquid nitrogen. It is believed that with this cryopreservation technique, a partial lysis of erythrocytes takes place, therefore blood samples do not require fractionation. Before transplantation, the cell suspension is thawed, washed free of hemoglobin and DMSO in a solution of human albumin or serum. Preservation of hematopoietic progenitors using this method is indeed higher than after fractionation of umbilical cord blood, but the danger of transfusion complications due to transfusion of ABO-incompatible erythrocytes persists.

The establishment of a system of banks for the storage of HSC tested and HSC samples could solve the above problems. However, for this it is necessary to develop ethical and legal norms, which are still only being discussed. Before the creation of a banking network, it is necessary to adopt a number of provisions and documents on the standardization of procedures for sampling, fractionation, testing and typing, and cryoconservation of GCW. An obligatory condition for the effective operation of GSK banks is the organization of a computer base for interrelating with the registries of the World Donor Medullary Association (WMDA) and the United States National Donor Medullary Program (NMDP).

In addition, it is necessary to optimize and standardize the methods of HSC expansion in vitro, primarily hematopoietic cord blood cells. Reproduction of HSC cord blood is necessary to increase the number of potential recipients compatible with the HLA system. Because of small volumes of cord blood, the amount of HSC contained in it is, as a rule, not able to provide bone marrow repopulation in adult patients. At the same time, for conducting unrelated transplantations, it is necessary to have access to a sufficient number of typical GSK samples (from 10,000 to 1,500,000 per 1 recipient).

Transplantation of stem hemopoietic cells does not eliminate complications accompanying bone marrow transplantation. The analysis shows that in the transplantation of umbilical cord blood stem cells, severe forms of the acute "graft-versus-host" reaction develop in 23%, chronic in 25% of recipients. In oncohematological patients recurrences of acute leukemia within the first year after transplantation of HSC cord blood are observed in 26% of cases.

In recent years, the methods of transplantation of peripheral hematopoietic stem cells have been developing intensively. The content of HSC in the peripheral blood is so small (there are 1 GSK per 100,000 blood cells), that their isolation without special preparation does not make sense. Therefore, the donor is previously given a course of drug stimulation of the release of hematopoietic bone marrow cells into the blood. To this end, such far-harmless drugs as cyclophosphamide and granulocyte colony-stimulating factor are used. But even after the procedure for mobilizing HSC in peripheral blood, the content of CD34 + cells in it does not exceed 1.6%.

To mobilize HSC in the clinic, C-CEC is more commonly used, which is characterized by relatively good tolerability, with the exception of the almost regular appearance of pain in the bones. It should be noted that the use of modern blood separators allows us to efficiently isolate stem progenitors of hematopoiesis. However, under conditions of normal hematopoiesis, at least 6 procedures must be performed to obtain a sufficient number of hematopoietic stem cells, comparable to the repopulative capacity of the bone marrow slurry. With each such procedure, the separator processes 10-12 liters of blood, which can cause thrombocytopenia and leukopenia. The separation procedure involves the administration of an anticoagulant (sodium citrate) to the donor, which does not exclude, however, contact activation of platelets during extracorporeal centrifugation. These factors create conditions for the development of infectious and hemorrhagic complications. Another drawback of the method lies in the considerable variability in the mobilization response, which requires monitoring of the content of HSC in peripheral blood donors, necessary to determine their maximum level.

Autologous transplantation of HSC, in contrast to allogeneic, completely excludes the development of the rejection reaction. Nevertheless, a significant disadvantage of autotransplantation of stem hemopoietic cells, which limits the spectrum of indications to its conduct, is a high probability of reinfusion of leukemic clone cells with a graft. In addition, the lack of an immuno-mediated "graft-versus-tumor" effect significantly increases the frequency of recurrences of malignant blood disease. Therefore, the only radical way to eliminate neoplastic clonal hemopoiesis and restore normal polyclonal hematopoiesis with myelodysplastic syndromes remains intensive polychemotherapy with transplantation of allogeneic HSCs.

But even in this case, treatment for most hemoblastoses is aimed only at increasing the survival time of patients and improving their quality of life. According to several large studies, prolonged disease-free survival after HSC allotransplantation is achieved in 40% of oncohematological patients. When using stem cells of HbA-compatible sibling, the best results are observed in young patients with a short history of the disease, the number of blast cells up to 10%, and favorable cytogenetics. Unfortunately, the mortality associated with the procedure for HSC allotransplantation in patients with myelodysplastic diseases remains high (in most reports - about 40%). The results of the 10-year work of the National Bone Marrow Donor Program (510 patients, median age - 38 years) indicate that disease-free survival for two years is 29% with a relatively low probability of recurrence (14%). However, the mortality caused by the procedure of GSC allotransplantation from an unrelated donor is extremely high and reaches 54% over a two-year period. Similar results were obtained in the European study (118 patients, median age 24 years, 2-year relapse-free survival 28%, recurrence 35%, mortality 58%).

When carrying out intensive courses of chemotherapy with the subsequent restoration of hematopoiesis by allogeneic hemopoietic cells, immunohematological and transfusion complications often arise. In many ways, they are related to the fact that blood groups in humans are inherited independently of MHC molecules. Therefore, even if the donor and recipient are compatible with the main HLA antigens, their erythrocytes may have a different phenotype. There is a "big" incompatibility, when the recipient pre-existent antibodies to the antigens of the donor's erythrocytes, and "small" when the donor has antibodies to the antigens of the erythrocytes of the recipient. There are cases of a combination of "big" and "small" incompatibilities.

The results of a comparative analysis of the clinical efficacy of bone marrow allotransplantation and stem hemopoietic cord blood cells in hemoblastoses indicate that in children after allografting of HSC cord blood, the risk of graft-versus-host reaction is significantly reduced, but a longer recovery of neutrophil and platelet counts is observed a higher frequency of a 100-day post-transplant mortality.

The study of the causes of early lethality made it possible to clarify the contraindications to the allogeneic transplantation of GSK, among which the most important are:

• presence in the recipient or donor of positive tests for cytomegalovirus infection (without carrying out preventive treatment);
• acute radiation sickness;
• presence or even suspicion of the presence of a mycotic infection in the patient (without systemic early prophylaxis with fungicidal drugs);
• hemoblastoses, in which patients received prolonged treatment with cytostatics (because of the high probability of sudden cardiac arrest and multiple organ failure);
• transplantation from HLA-non-identical donors (without prevention of the acute "transplant against host" reaction by cyclosporin A);
• chronic viral hepatitis C (due to a high risk of developing veno-occlusive disease of the liver).

Thus, GSK transplantation can cause serious complications, which often lead to death. In the early period (up to 100 days after transplantation), infectious complications, acute graft-versus-host reaction, transplant rejection reaction (non-pretreatment of the donor GCS), veno-occlusive disease of the liver, as well as toxicity-related tissue conditioning conditions, characterized by high the speed of remodeling (skin, vascular endothelium, intestinal epithelium). Complications of the late post-transplant period include the chronic "graft-versus-host" reaction, relapses of the underlying disease, growth retardation in children, impaired reproductive system and thyroid function, eye damage.

Recently, in connection with the appearance of publications on the plasticity of bone marrow cells, the idea of using GSK for the treatment of heart attacks and other diseases has arisen. Although some experiments on animals also support this possibility, the conclusions about the plasticity of bone marrow cells need to be confirmed. This circumstance should be taken into account by those researchers who believe that the transplanted cells of the human bone marrow are easily transformed into cells of skeletal muscle, myocardium or CNS. The hypothesis that GSKs are a natural cellular source of regeneration of these organs requires serious evidence.

In particular, the first results of an open, randomized trial by V. Belenkov (2003) were published, the aim of which was to study the effect of C-SCC (ie, mobilization of autologous HSCs) on the clinical, hemodynamic and neurohumoral status of patients with moderate and severe chronic heart failure, as well as an assessment of its safety against standard therapy (angiotensin-converting enzyme inhibitors, beta-blockers, diuretics, cardiac glycosides). In the first publication of the results of the study, the authors of the program note that the only argument in favor of O-CP is the results of treatment of one patient, who, against the background of therapy with this drug, established an undeniable improvement in all clinical and hemodynamic parameters. However, the theory of GSK mobilization in the bloodstream followed by myocardial regeneration in the postinfarction zone was not confirmed - even in a patient with a positive clinical dynamics, stress echocardiography with dobutamine did not reveal the appearance of zones of viable myocardium in the scar area.

It should be noted that, by the present moment, data that allow to recommend substitutive cellular therapy for widespread introduction into everyday clinical practice is clearly not enough. We need well-designed and qualitatively performed clinical studies aimed at determining the effectiveness of various variants of regenerative cell therapy, developing indications and contraindications to it, as well as methodological recommendations for the combined use of regenerative-plastic therapy and traditional surgical or conservative treatment. There is still no answer to the question of which particular population of bone marrow cells (stem hemopoietic or stromal stem cells) can give rise to neurons and cardiomyocytes, and it is not clear which conditions contribute to this in vivo.

Work in these areas is carried out in many countries. In the summary of the Symposium on Acute Hepatic Insufficiency of the National Institutes of Health of the United States, along with liver transplantation, transplantation of xeno- or allogeneic hepatocytes and extracorporeal connection of bioreactors with liver cells were noted among promising methods of treatment. There is direct evidence that only foreign, functionally active hepatocytes can provide effective support for the liver of the recipient. For clinical use of isolated hepatocytes, it is necessary to create a cell bank, which will significantly reduce the time between the release of cells and their use. The most acceptable for creating a bank of isolated hepatocytes is the cryopreservation of liver cells in liquid nitrogen. When using such cells in the clinic in patients with acute and chronic hepatic insufficiency, a rather high therapeutic effect was revealed.

Despite the optimistic and encouraging results of the application of liver cell transplantation in the experiment and clinic, there are many problems still far from their solution. These include a limited number of suitable organs for the production of isolated hepatocytes, inadequate methods for their isolation, the lack of standardized methods for the preservation of liver cells, unclear ideas about the mechanisms of regulating the growth and proliferation of transplanted cells, the lack of adequate methods for assessing the engraftment or rejection of allogeneic hepatocytes. The presence of transplantation immunity in the use of allo- and xenogeneic cells, although less than in orthotopic liver transplantation, but requiring the use of immunosuppressors, encapsulation of isolated hepatocytes or their special treatment with enzymes, should also be cited here. Transplantation of hepatocytes often leads to an immune conflict between the recipient and the donor as a rejection reaction, which requires the use of cytostatics. One solution to this problem may be the use of polymeric microporous carriers to isolate the liver cells, which will improve their survival, since the capsule membrane effectively protects the hepatocytes, despite the immunization of the host.

However, with acute hepatic insufficiency such a transplantation of hepatocytes does not give an effect due to the sufficiently long time necessary for engraftment of liver cells in a new medium with reaching the stage of optimal functioning. Potential limitation is the secretion of bile in ectopic transplantation of isolated hepatocytes, and when bioreactors are used, a significant physiological barrier is the species discrepancy between human proteins and proteins that produce xenogenous hepatocytes.

There are reports in the literature that local transplantation of bone marrow stromal stem cells contributes to the effective correction of bone defects, with the restoration of bone tissue in this case taking place more intensively than with spontaneous reparative regeneration. Several preclinical studies in experimental models have convincingly demonstrated the possibility of using bone marrow stromal bone marrow transplants in orthopedics, although further work is needed to optimize these techniques, even in the simplest cases. In particular, the optimal conditions for the expansion of osteogenic stromal cells ex vivo have not yet been found, the structure and composition of their ideal carrier (matrix) remain unworked. The minimum number of cells required for bulk bone regeneration is not determined.

It is proved that mesenchymal stem cells exhibit transgermal plasticity - the ability to differentiate into cellular types that are phenotypically unrelated to the cells of the original line. Under optimal cultivation conditions, the polyclonal lines of stem cells of the bone marrow stroma survive in vitro more than 50 divisions, which makes it possible to obtain billions of stromal cells from 1 ml of bone marrow aspirate. However, the population of mesenchymal stem cells is heterogeneous, which manifests itself as a variability in the size of colonies, different rates of their formation, and morphological diversity of cellular types - from fibroblast-like fusiform to large flat cells. After 3 weeks of cultivation of stromal stem cells phenotypic heterogeneity is observed: some colonies form nodules of bone tissue, others - accumulations of adipocytes, others, more rare, form islands of cartilaginous tissue.

For the treatment of degenerative diseases of the central nervous system, transplantation of embryonic nerve tissue was first used. In recent years, instead of the tissue of the embryonic brain, the cellular elements of neurospheres derived from neural stem cells have been transplanted (Poltavtseva, 2001). Neurospheres contain committed neural precursors and neuroglia - this gives hope for restoration of lost brain functions after their transplantation. After transplanting cells of dispersed neurospheres into the striatum of the rat brain, their proliferation and differentiation into dopaminergic neurons was noted, which eliminated motor asymmetry in rats with experimental hemiparkinsonism. However, in some cases, tumor cells developed from the cells of the neurosphere, which led to the death of animals (Bjorklund, 2002).

In the clinic, careful studies of two groups of patients in which neither the patients nor the physicians watching them knew (double-blind study) that one group of patients was transplanted with embryonic tissue with dopamine-producing neurons and the second group of patients were doing a false operation, gave unexpected results . Patients who were transplanted with embryonic nerve tissue felt no better than the patients of the control group. In addition, 5 out of 33 patients developed persistent dyskinesia 2 years after the transplantation of embryonic nerve tissue, which was not present in patients in the control group (Stem cells: scientific progress and future research trends, Nat. Inst., Of Health. USA). One of the unsolved problems of clinical investigation of brain neural stem cells remains an analysis of the real prospects and limitations of transplantation of their derivatives for the correction of CNS disorders. It is not excluded that the induced neuronal genesis in the hippocampus, resulting in its structural and functional rearrangements, may be a factor in the progressive development of epilepsy. Such a conclusion deserves special attention, since it indicates possible negative consequences of the generation of new neurons in the mature brain and the formation of aberrant synaptic connections.

It should not be forgotten that cultivation in environments with cytokines (mitogens) approximates the characteristics of stem cells to those of tumor cells, as close changes in the regulation of cell cycles occur that determine the ability to unrestricted division. It is foolish to transplant to humans early derivatives of embryonic stem cells, since in this case the threat of development of malignant neoplasms is very great. It is much safer to use their more committed offspring, that is, progenitor cells of differentiated lines. However, at present a reliable technique for obtaining stable human cell lines differentiating in the right direction has not yet been worked out.

The use of molecular biology technologies for the correction of hereditary pathology and human diseases with the help of stem cell modification is of great interest for practical medicine. Features of the genome of stem cells enable the development of unique transplantation schemes with the aim of correcting genetic diseases. But in this direction there are also a number of limitations that need to be overcome before the practical application of genetic engineering of stem cells begins. First of all, it is necessary to optimize the process of stem cell genome modification ex vivo. It is known that a prolonged (3-4 weeks) proliferation of stem cells reduces their transfection, so several cycles of transfection are necessary to achieve a high level of their genetic modification. However, the main problem is related to the duration of expression of the therapeutic gene. Until now, in none of the studies, the period of effective expression after transplantation of the modified cells did not exceed four months. In 100% of cases over time, the expression of transfected genes is reduced due to inactivation of promoters and / or death of cells with a modified genome.

An important problem is the cost of using cellular technologies in medicine. For example, the estimated annual funding requirement for only the medical expenses of a bone marrow transplant department, designed to perform 50 transplants per year, is about $ 900,000.

The development of cellular technologies in clinical medicine is a complex and multi-stage process involving constructive cooperation of multidisciplinary scientific and clinical centers and the international community. At the same time, special attention is paid to the scientific organization of research in the field of cell therapy. The most important of these are the development of protocols for clinical trials, monitoring the validity of clinical data, the formation of a national research register, integration into international programs of multicenter clinical trials, and the introduction of results into clinical practice.

Concluding the introduction to the problems of cellular transplantation, I would like to express the hope that the unification of the efforts of the leading Ukrainian specialists from different fields of science will ensure significant progress in experimental and clinical research and will allow in the coming years to find effective ways to provide assistance to seriously ill people who need organ transplantation , tissues and cells.

[24], [25], [26], [27], [28], [29]