Limitations, dangers and complications of cell transplantation

Regenerative plastic medicine is based on the clinical implementation of the toti- and pluripotent properties of embryonic and progenitor stem cells, which allow in vitro and in vivo the creation of specified cell lines that repopulate damaged tissues and organs of a sick person.

The real possibility of using embryonic stem cells and stem cells of definitive tissues (the so-called “adult” stem cells) of humans for therapeutic purposes is no longer in doubt. However, experts from the National and Medical Academies of the USA (Stem cells and the future regenerative medicine National Academy Press) and the National Institute of Health of the USA (Stem cells and the future research directions. Nat. Inst, of Health USA) recommend a more detailed study of the properties of stem cells in experiments on adequate biological models and an objective assessment of all the consequences of transplantation, and only then use stem cells in the clinic.

It has been established that stem cells are part of the tissue derivatives of all three germ layers. Stem cells are found in the retina, cornea, skin epidermis, bone marrow and peripheral blood, in blood vessels, dental pulp, kidney, gastrointestinal epithelium, pancreas and liver. Using modern methods, it has been proven that neural stem cells are localized in the brain and spinal cord of an adult. These sensational data attracted special attention from scientists and the media, since neurons in the brain served as a classic example of a static cell population that is not restored. Both in the early and late periods of ontogenesis, neurons, astrocytes and oligodendrocytes are formed in the brain of animals and humans due to neural 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 manifest itself. To realize the plastic potential of stem cells of definitive tissues, they must be isolated and then cultivated in media with cytokines (LIF, EGF, FGF). Moreover, stem cell derivatives successfully engraft only when transplanted into the body of an animal with a depressed immune system (γ-irradiation, cytostatics, busulfan, etc.). To date, no convincing evidence has been obtained of the implementation of stem cell plasticity in animals that have not been exposed to radiation or other effects that cause deep immunosuppression.

In such conditions, the dangerous potential of ESCs manifests itself, first of all, during their transplantation into ectopic areas - during subcutaneous injection of ESCs into immunodeficient mice, teratocarcinomas are formed at the injection site. In addition, during the development of the human embryo, the frequency of chromosomal abnormalities is higher than in embryogenesis in animals. At the blastocyst stage, only 20-25% of human embryos consist of cells with a normal karyotype, and the overwhelming majority of early human embryos obtained after in vitro fertilization exhibit chaotic chromosomal mosaicism and very often encounter numerical and structural aberrations.

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Beneficial effects of stem cells

Preliminary results of clinical trials confirm the beneficial effect of stem cells on the patient, but there is still no information about the long-term effects of cell transplantation. The literature initially was dominated by reports of positive results of transplantation of embryonic brain fragments in Parkinson's disease, but then data began to appear denying the effective therapeutic effect of embryonic or fetal nerve tissue transplanted into the brain of patients.

In the mid-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 transplant in humans. The lack of knowledge about the mechanisms of immunological incompatibility of donor and recipient bone marrow cells at that time led to high mortality due to frequent transplant failure and the development of graft-versus-host reaction. The discovery of the major histocompatibility complex, which includes human leukocyte antigens (HbA), and the improvement of their typing methods made it possible to significantly increase survival after bone marrow transplantation, which led to the widespread use of this treatment method in oncohematology. A decade later, the first transplants of hematopoietic stem cells (HSCs) obtained from peripheral blood using leukapheresis were performed. In 1988, umbilical cord blood was first used as a source of HSCs in France to treat a child with Fanconi anemia, and since the end of 2000, reports have appeared in the press on the ability of HSCs to differentiate into cells of various tissue types, which potentially expands the scope of their clinical application. However, it turned out that the transplant material, along with HSCs, contains a significant number of non-hematopoietic cell impurities of various natures and properties. In this regard, methods for purifying the transplant and criteria for assessing its cellular purity are being developed. In particular, positive immunoselection of CD34+ cells is used, which allows for the isolation of HSCs using monoclonal antibodies.

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Complications of stem cell therapy

Complications in bone marrow transplantation are most often hematological and associated with a long period of iatrogenic pancytopenia. Infectious complications, anemia and hemorrhages develop most often. In this regard, it is extremely important to select the optimal mode of bone marrow collection, processing and storage for maximum preservation of stem cells, which will ensure rapid and stable restoration of hematopoiesis. When characterizing a transplant, the following parameters are currently commonly assessed: the number of mononuclear and/or nucleated cells, colony-forming units and the content of CD34-positive cells. Unfortunately, these indicators provide only an indirect assessment of the real hematopoietic capacity of the stem cell population of the transplant. Today, there are no absolutely accurate parameters for determining the sufficiency of a transplant for long-term restoration of hematopoiesis in patients, even with autologous bone marrow transplantation. The development of general criteria is extremely difficult due to the lack of strict standards for processing, cryopreservation and testing of the transplant. In addition, it is necessary to take into account the whole variety of factors influencing the parameters of successful restoration of hematopoiesis in each individual patient. In autologous bone marrow transplantation, the most important of them are the number of previous chemotherapy courses, the characteristics of the conditioning regimen, the period of the disease in which the bone marrow was collected, and the schemes for using colony-stimulating factors in the post-transplant period. In addition, it should not be forgotten that chemotherapy preceding the transplant collection can have a negative effect on bone marrow stem cells.

The incidence of severe toxic complications increases significantly during allogeneic bone marrow transplantation. In this regard, statistical data on allogeneic bone marrow transplantation in thalassemia are of interest. The reports of the European Bone Marrow Transplantation Group have registered about 800 bone marrow transplantations to patients with thalassemia major. Allogeneic transplantation in thalassemia is performed in the vast majority of cases from HLA-identical siblings, which is associated with severe complications and high mortality during 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 laminar air flow and receive a low- or abacterial diet. For bacterial decontamination of the intestine, non-resorbable forms of antibiotics and antifungal drugs are prescribed per os. For prophylaxis, amphotericin B is administered intravenously. Prevention of systemic infections is reinforced with amikacin and ceftazidime, which are prescribed the day before transplantation, continuing treatment until the patient is discharged. All blood products are irradiated at a dose of 30 Gy before transfusion. Parenteral nutrition during transplantation is a necessary condition and begins immediately after restriction of food intake in a natural way.

A number of complications are associated with the 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. Risk factors for thrombosis of the veins of the portal system of the liver include the age of patients, the presence of hepatitis and liver fibrosis, as well as immunosuppressive therapy after bone marrow transplantation. Veno-occlusive disease is especially dangerous in thalassemia, which is accompanied by hemosiderosis of the liver, hepatitis and fibrosis - frequent companions 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 and transaminase activity in the blood, progression of hepatomegaly, ascites, encephalopathy and pain in the upper abdomen. Histologically, autopsy material reveals endothelial damage, subendothelial hemorrhages, damage to centrilobular hepatocytes, thrombotic obstruction of venules and central veins of the liver. Cases of fatal cardiac arrest associated with the toxic effects of cytostatics have been described in patients with thalassemia.

During the pre-transplant period, 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 nephro- and neurotoxicity, hypertension syndrome, fluid retention in the body, and hepatocyte cytolysis. Sexual and reproductive dysfunction is more often observed in women. In young children, pubertal development is usually not affected after transplantation, but in older children, pathology of the development of the sexual sphere can be very serious - up to sterility. Complications directly related to the transplant itself include rejection of allogeneic bone marrow cells, ABO incompatibility, acute and chronic forms of graft-versus-host disease.

In patients with ABO-incompatible bone marrow transplantation, host-versus-ABO donor isoagglutinins are produced for 330-605 days after transplantation, which can lead to prolonged hemolysis and dramatically increase the need for blood transfusions. This complication is prevented by transfusing only type 0 red blood cells. After transplantation, some patients experience autoimmune neutropenia, thrombocytopenia, or pancytopenia, which require splenectomy to correct.

In 35-40% of recipients, acute graft-versus-host disease develops within 100 days after allogeneic HLA-identical bone marrow transplantation. The degree of skin, liver, and intestinal involvement varies from rash, diarrhea, and moderate hyperbilirubinemia to skin desquamation, intestinal obstruction, and acute liver failure. In patients with thalassemia, the incidence of grade I acute graft-versus-host disease after bone marrow transplantation is 75%, and grade II and higher is 11-53%. Chronic graft-versus-host disease as a systemic multiple organ syndrome usually develops within 100-500 days after allogeneic bone marrow transplantation in 30-50% of patients. The skin, oral cavity, liver, eyes, esophagus, and upper respiratory tract are affected. A distinction is made between a limited form of chronic graft-versus-host disease, when the skin and/or liver are affected, and a widespread form, when generalized skin lesions are combined with chronic aggressive hepatitis, lesions of the eyes, salivary glands, or any other organ. Death is often caused by infectious complications resulting from severe immunodeficiency. In thalassemia, a mild form of chronic graft-versus-host disease occurs in 12%, a moderate form in 3%, and a severe form in 0.9% of recipients of allogeneic HLA-compatible bone marrow. A severe complication of bone marrow transplantation is graft rejection, which develops 50-130 days after surgery. The frequency of rejection depends on the conditioning regimen. In particular, in patients with thalassemia who received only methotrexate during the preparation period, rejection of the bone marrow transplant was observed in 26% of cases, with a combination of methotrexate with cyclosporine A - in 9% of cases, and with the administration of only cyclosporine A - in 8% of cases (Gaziev et al., 1995).

Infectious complications after bone marrow transplantation are caused by viruses, bacteria and fungi. Their development is associated with deep neutropenia induced by chemotherapy drugs during the conditioning period, damage to mucous barriers by cytostatics and the graft-versus-host reaction. Depending on the time of development, three phases of infectious complications are distinguished. In the first phase (develops in the first month after transplantation), damage to mucous barriers and neutropenia 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, aspergilli. In the early post-transplantation period (the second and third months after transplantation), the most severe 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. Infections caused by Varicella zoster, streptococcus, Pneumocystis carinii, Neisseria meningitidis, Haemophilus influenzae, and hepatotropic viruses are common during this period. In thalassemia, mortality in 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 hemorrhage, veno-occlusive liver disease, and acute graft-versus-host disease.

At present, certain successes have been achieved in the development of methods for isolating pure populations of hematopoietic stem cells from bone marrow. The technique for obtaining fetal blood from the umbilical cord has been improved, and methods for isolating hematopoietic cells from cord blood have been created. There are reports in the scientific press that hematopoietic stem cells are capable of multiplying when cultivated in media with cytokines. When using specially designed bioreactors for the expansion of hematopoietic stem cells, the biomass of hematopoietic stem cells isolated from bone marrow, peripheral or umbilical cord blood increases significantly. The possibility of expanding hematopoietic stem cells is an important step towards the clinical development of cell transplantation.

However, before in vitro propagation of hematopoietic stem cells, it is necessary to isolate a homogeneous population of hematopoietic stem cells. This is usually achieved using markers that allow selective labeling of hematopoietic stem cells with monoclonal antibodies covalently linked to a fluorescent or magnetic label and their isolation using an appropriate cell sorter. At the same time, the issue of phenotypic characteristics of hematopoietic stem cells has not been finally resolved. A. Petrenko, V. Grishchenko (2003) consider cells with CD34, AC133, and Thyl antigens on their surface and no CD38, HLA-DR, or other differentiation markers (cells with the CD34+Liir phenotype) as candidates for hematopoietic stem cells. Lineage (Lin) markers include glycophorin A (GPA), CD3, CD4, CD8, CD10, CD14, CD16, CD19, CD20 (Muench, 2001). Cells with the CD34+CD45RalüW CD71low phenotype, as well as CD34+Thyl+CD38low/c-kit/low phenotype, are considered promising for transplantation.

The issue of the number of hematopoietic stem cells sufficient for effective transplantation remains problematic. Currently, the sources of hematopoietic stem cells are bone marrow, peripheral and cord blood, and embryonic liver. Expansion of hematopoietic stem cells is achieved by culturing them in the presence of endothelial cells and hematopoietic growth factors. In various protocols, myeloproteins, SCF, erythropoietin, insulin-like growth factors, corticosteroids, and estrogens are used to induce HSC proliferation. When using combinations of cytokines in vitro, it is possible to achieve a significant increase in the HSC pool with a peak in their output at the end of the second week of cultivation.

Traditionally, cord blood hematopoietic stem cell transplantation is used mainly for hemoblastoses. However, the minimum dose of hematopoietic cells required for successful cord blood cell transplantation is 3.7 x 10 ⁷ nucleated cells per 1 kg of recipient body weight. Using a smaller number of cord blood hematopoietic stem cells significantly increases the risk of graft failure and disease relapse. Therefore, cord blood hematopoietic stem cell transplantation is mainly used to treat hemoblastoses in children.

Unfortunately, there are still no standards for the procurement or standardized protocols for the clinical use of cord blood hematopoietic cells. Accordingly, cord blood stem cells themselves are not a legally recognized source of hematopoietic cells for transplantation. In addition, there are no ethical or legal norms governing the activities and organization of cord blood banksb, which exist abroad. Meanwhile, for safe transplantation, all cord blood samples must be carefully monitored. Before collecting blood from a pregnant woman, her consent must be obtained. Each pregnant woman must be examined for HBsAg carriage, the presence of antibodies to hepatitis C, HIV and syphilis viruses. Each cord blood sample must be tested as standard for the number of nucleated cells, CD34+ and colony-forming capacity. In addition, HbA typing, determination of the blood group by ABO and its belonging by the Rh factor are carried out. The necessary testing procedures are bacteriological culture for sterility, serological testing for HIV-1 and HIV-2 infections, HBsAg, viral hepatitis C, cytomegalovirus infection, HTLY-1 and HTLY-II, syphilis and toxoplasmosis. In addition, polymerase chain reaction is performed to detect cytomegalovirus and HIV infections. It seems advisable to supplement the testing protocols with an analysis of cord blood GSC to detect such genetic diseases as a-thalassemia, sickle cell anemia, adenosine deaminase deficiency, Bruton's agammaglobulinemia, Hurler's and Ponter's diseases.

The next stage of preparation for transplantation is the question of preserving the hematopoietic stem cells. The most dangerous procedures for the viability of the cells during their preparation are freezing and thawing. When freezing hematopoietic cells, a significant part of them can be destroyed due to crystal formation. Special substances - cryoprotectors - are used to reduce the percentage of cell death. Most often, DMSO is used as a cryoprotector in a final concentration of 10%. However, DMSO in such a concentration is characterized by a direct cytotoxic effect, which manifests itself even under conditions of minimal exposure. A decrease in the cytotoxic effect is achieved by strict maintenance of zero temperature of the exposure mode, as well as compliance with the regulations for processing the material during and after defrosting (speed of all manipulations, use of multiple washing procedures). DMSO concentrations of less than 5% should not be used, since this causes massive death of hematopoietic cells during the freezing period.

The presence of erythrocyte impurities in the suspension mixture of hematopoietic stem cells creates a risk of developing an incompatibility reaction for erythrocyte antigens. At the same time, when erythrocytes are removed, the loss of hematopoietic cells increases significantly. In this regard, a method of unfractionated isolation of hematopoietic stem cells has been proposed. In this case, a 10% DMSO solution and constant-rate cooling (GS/min) to -80°C are used to protect nucleated cells from the damaging effects of low temperatures, after which the cell suspension is frozen in liquid nitrogen. It is believed that this cryopreservation method results in partial lysis of erythrocytes, so blood samples do not require fractionation. Before transplantation, the cell suspension is defrosted, washed from free hemoglobin and DMSO in a solution of human albumin or in blood serum. The preservation of hematopoietic precursors using this method is indeed higher than after fractionation of umbilical cord blood, but the risk of transfusion complications due to transfusion of ABO-incompatible erythrocytes remains.

The establishment of a banking system for storing HLA-tested and typed HSC samples could solve the above problems. However, this requires the development of ethical and legal norms, which are currently only being discussed. Before creating a banking network, it is necessary to adopt a number of regulations and documents on standardization of procedures for collection, fractionation, testing and typing, as well as cryopreservation of HSC. A mandatory condition for the effective operation of HSC banks is the organization of a computer base for interaction with the registries of the World Marrow Donor Association (WMDA) and the National Marrow Donor Program of the United States (NMDP).

In addition, it is necessary to optimize and standardize the methods of in vitro HSC expansion, primarily cord blood hematopoietic cells. Expansion of cord blood HSC is necessary to increase the number of potential recipients compatible with the HLA system. Due to the small volumes of cord blood, the number of HSCs contained in it is usually not able to ensure bone marrow repopulation in adult patients. At the same time, to perform unrelated transplants, it is necessary to have access to a sufficient number of typed HSC samples (from 10,000 to 1,500,000 per recipient).

Transplantation of hematopoietic stem cells does not eliminate the complications that accompany bone marrow transplantation. Analysis shows that with cord blood stem cell transplantation, severe forms of acute graft-versus-host disease develop in 23% of recipients, and chronic forms in 25% of recipients. In oncohematological patients, relapses of acute leukemia during the first year after cord blood stem cell transplantation are observed in 26% of cases.

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

For the mobilization of hematopoietic stem cells in the clinic, S-SEC is most often used, which is characterized by relatively good tolerance, with the exception of the almost natural occurrence of bone pain. It should be noted that the use of modern blood separators allows for the effective isolation of hematopoietic stem cells. However, under normal hematopoiesis conditions, at least 6 procedures must be performed to obtain a sufficient number of hematopoietic stem cells comparable in repopulation capacity to bone marrow suspension. Each such procedure requires 10-12 liters of blood to be processed on the separator, which can cause thrombocytopenia and leukopenia. The separation procedure involves the introduction 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 disadvantage of the method is the significant variability of the mobilization response, which requires monitoring the content of HSCs in the peripheral blood of donors, which is necessary to determine their maximum level.

Autogenous transplantation of hematopoietic stem cells, unlike allogeneic transplantation, completely eliminates the development of rejection reaction. However, a significant disadvantage of autotransplantation of hematopoietic stem cells, which limits the range of indications for its implementation, is the high probability of reinfusion of leukemic clone cells with the transplant. In addition, the absence of the immune-mediated “graft versus tumor” effect significantly increases the frequency of relapses of malignant blood disease. Therefore, the only radical method of eliminating neoplastic clonal hematopoiesis and restoring normal polyclonal hematopoiesis in myelodysplastic syndromes remains intensive polychemotherapy with transplantation of allogeneic hematopoiesis.

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, long-term relapse-free survival after allotransplantation of HSCs is achieved in 40% of oncohematological patients. When using stem cells of an HLA-compatible sibling, the best results are observed in young patients with a short history of the disease, a blast cell count of up to 10% and favorable cytogenetics. Unfortunately, mortality associated with the procedure of allotransplantation of HSCs in patients with myelodysplastic diseases remains high (in most reports - about 40%). The results of 10-year work of the National Bone Marrow Donor Program in the USA (510 patients, median age - 38 years) indicate that the relapse-free survival for two years is 29% with a relatively low probability of relapse (14%). However, mortality associated with the procedure of allotransplantation of HSCs from an unrelated donor is extremely high and reaches 54% over a two-year period. Similar results were obtained in a European study (118 patients, median age - 24 years, two-year relapse-free survival - 28%, relapse - 35%, mortality - 58%).

During intensive chemotherapy courses with subsequent restoration of hematopoiesis with allogeneic hematopoietic cells, immunohematological and transfusion complications often occur. They are largely due to the fact that human blood groups are inherited independently of MHC molecules. Therefore, even if the donor and recipient are compatible for the main HLA antigens, their erythrocytes may have different phenotypes. A distinction is made between “major” incompatibility, when the recipient has pre-existing antibodies to the donor’s erythrocyte antigens, and “minor” incompatibility, when the donor has antibodies to the recipient’s erythrocyte antigens. Cases of a combination of “major” and “minor” incompatibility are possible.

The results of a comparative analysis of the clinical effectiveness of allotransplantation of bone marrow and cord blood hematopoietic stem cells in hemoblastoses indicate that in children after allotransplantation of cord blood hematopoietic stem cells, the risk of developing a graft-versus-host reaction is significantly reduced, but a longer period of recovery of the number of neutrophils and platelets is observed with a higher incidence of 100-day post-transplant mortality.

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

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

Thus, HSC transplantation can cause serious complications, which often lead to death. In the early (up to 100 days after transplantation) period, these include infectious complications, acute graft-versus-host disease, graft rejection (failure of donor HSCs), veno-occlusive liver disease, as well as tissue damage caused by the toxicity of the conditioning regimen, which is characterized by a high remodeling rate (skin, vascular endothelium, intestinal epithelium). Complications of the late post-transplantation period include chronic graft-versus-host disease, relapses of the underlying disease, growth retardation in children, dysfunction of the reproductive system and thyroid gland, and eye damage.

Recently, in connection with the publications on the plasticity of bone marrow cells, the idea of using HSCs to treat heart attacks and other diseases has arisen. Although some animal experiments 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 transplanted human bone marrow cells are easily transformed into skeletal muscle, myocardial or CNS cells. The hypothesis that HSCs are a natural cellular source of regeneration of these organs requires serious evidence.

In particular, the first results of an open randomized study by V. Belenkov (2003) have been published. Its purpose was to study the effect of C-SvK (i.e., mobilization of autologous HSCs into the blood) on the clinical, hemodynamic, and neurohumoral status of patients with moderate to severe chronic heart failure, as well as to evaluate its safety against the background of standard therapy (angiotensin-converting enzyme inhibitors, beta-blockers, diuretics, cardiac glycosides). In the first publication of the study results, the authors of the program note that the only argument in favor of O-SvK is the results of treating one patient, who showed an indisputable improvement in all clinical and hemodynamic parameters against the background of therapy with this drug. However, the theory of HSC mobilization into the bloodstream with subsequent myocardial regeneration in the post-infarction zone was not confirmed - even in a patient with 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 at present there is clearly insufficient data to recommend replacement cell therapy for widespread implementation in everyday clinical practice. Well-designed and high-quality clinical studies are needed to determine the effectiveness of various options for regenerative cell therapy, develop indications and contraindications for it, as well as guidelines 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 hematopoietic or stromal) can give rise to neurons and cardiomyocytes, and it is also unclear what conditions contribute to this in vivo.

Work in these areas is being carried out in many countries. In the summary of the symposium on acute liver failure of the National Institutes of Health of the USA, among promising methods of treatment, along with liver transplantation, the transplantation of xeno- or allogeneic hepatocytes and extracorporeal connection of bioreactors with liver cells are noted. There is direct evidence that only foreign functionally active hepatocytes are able to provide effective support to the recipient's liver. For the clinical use of isolated hepatocytes, it is necessary to create a cell bank, which will significantly reduce the time between the isolation of cells and their use. The most acceptable method for creating a bank of isolated hepatocytes is cryopreservation of liver cells in liquid nitrogen. When using such cells in the clinic in patients with acute and chronic liver failure, a fairly high therapeutic effect has been revealed.

Despite the optimistic and encouraging results of liver cell transplantation in experiments and clinical practice, there are still many problems that are far from being solved. These include a limited number of organs suitable for obtaining isolated hepatocytes, insufficiently effective methods for their isolation, the absence of standardized methods for preserving liver cells, unclear ideas about the mechanisms of growth and proliferation regulation of transplanted cells, the absence of adequate methods for assessing the engraftment or rejection of allogeneic hepatocytes. This also includes the presence of transplant immunity when using allogeneic and xenogeneic cells, although less than in orthotopic liver transplantation, but requiring the use of immunosuppressants, encapsulation of isolated hepatocytes or their special treatment with enzymes. Hepatocyte transplantation often leads to an immune conflict between the recipient and the donor in the form of a rejection reaction, which requires the use of cytostatics. One solution to this problem may be the use of polymeric microporous carriers to isolate liver cells, which will improve their survival, since the capsule membrane effectively protects hepatocytes despite host immunization.

However, in acute liver failure, such hepatocyte transplantation is ineffective due to the relatively long time required for liver cells to engraft in a new environment and reach the stage of optimal functioning. A potential limitation is the secretion of bile during ectopic transplantation of isolated hepatocytes, and when using bioreactors, a significant physiological barrier is the species mismatch between human proteins and the proteins produced by xenogenic hepatocytes.

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

It has been proven that mesenchymal stem cells exhibit transgermal plasticity, i.e., the ability to differentiate into cell types phenotypically unrelated to the cells of the original line. Under optimal cultivation conditions, polyclonal bone marrow stromal stem cell lines can withstand more than 50 divisions in vitro, 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 is manifested by both variability in colony sizes, different rates of their formation, and morphological diversity of cell types, from fibroblast-like spindle-shaped to large flat cells. Phenotypic heterogeneity is observed after just 3 weeks of stromal stem cell cultivation: some colonies form nodules of bone tissue, others form clusters of adipocytes, and others, rarer, form islands of cartilage tissue.

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

In the clinic, careful studies of two groups of patients, in which neither the patients nor the doctors observing them knew (double-blind study), that one group of patients was transplanted with embryonic tissue with neurons producing dopamine, and the second group of patients underwent a sham operation, gave unexpected results. Patients who were transplanted with embryonic nerve tissue felt no better than patients in the control group. In addition, 5 of 33 patients developed persistent dyskinesia 2 years after transplantation of embryonic nerve tissue, which was not present in patients in the control group (Stem cells: scientific progress and future research directions. Nat. Inst, of Health. USA). One of the unsolved problems of clinical research of neural stem cells of the brain remains the analysis of the real prospects and limitations of transplantation of their derivatives for correction of CNS disorders. It is possible that neuronogenesis in the hippocampus induced by prolonged seizure activity, leading to its structural and functional reorganization, may be a factor in the progressive development of epilepsy. This conclusion deserves special attention, since it points to possible negative consequences of the generation of new neurons in the mature brain and the formation of aberrant synaptic connections by them.

It should not be forgotten that cultivation in media with cytokines (mitogens) brings the characteristics of stem cells closer to those of tumor cells, since similar changes in the regulation of cell cycles occur in them, determining the ability for unlimited division. It is reckless to transplant early derivatives of embryonic stem cells into a person, since in this case the threat of developing malignant neoplasms is very high. It is much safer to use their more committed progeny, that is, precursor cells of differentiated lines. However, at present, a reliable technique for obtaining stable lines of human cells that differentiate in the desired direction has not yet been developed.

The use of molecular biology technologies for the correction of hereditary pathology and human diseases by modifying stem cells is of great interest to practical medicine. The features of the stem cell genome make it possible to develop unique transplantation schemes to correct genetic diseases. However, there are a number of limitations in this area that need to be overcome before the practical application of stem cell genetic engineering. First of all, it is necessary to optimize the process of ex vivo stem cell genome modification. It is known that long-term (3-4 weeks) proliferation of stem cells reduces their transfection, so several transfection cycles are necessary to achieve a high level of their genetic modification. However, the main problem is associated with the duration of therapeutic gene expression. Until now, in no study has the period of effective expression after transplantation of modified cells exceeded four months. In 100% of cases, over time, the expression of transfected genes decreases due to inactivation of promoters and/or death of cells with a modified genome.

An important issue is the cost of using cell technologies in medicine. For example, the estimated annual need to finance only the medical expenses of a bone marrow transplant unit designed to perform 50 transplants per year is about US$900,000.

The development of cell technologies in clinical medicine is a complex and multi-stage process that involves constructive cooperation between multidisciplinary scientific and clinical centers and the international community. At the same time, issues of scientific organization of research in the field of cell therapy require special attention. The most important of them are the development of clinical research protocols, control over the reliability of clinical data, the formation of a national registry of studies, integration into international programs of multicenter clinical studies and the implementation of results in clinical practice.

In conclusion of the introduction to the problems of cell transplantology, I would like to express the hope that the unification of the efforts of leading Ukrainian specialists from various fields of science will ensure significant progress in experimental and clinical research and will make it possible in the coming years to find effective ways to provide assistance to seriously ill people in need of organ, tissue and cell transplantation.

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