Mesenchymal stem cells

Among the regional stem cells, mesenchymal stem cells (MSCs) occupy a special place, the derivatives of which constitute the stromal matrix of all organs and tissues of the human body. Priority in the research of MSC belongs to representatives of Russian biological science.

In the middle of the last century, a homogeneous culture of multipotent stromal bone marrow stem cells was first isolated in the laboratory of A. Friedenshtein. Mesenchymal stem cells attached to the substrate retained a high intensity of proliferation for a long time, and in cultures with a low seeding density, clones of fibroblast-like cells lacking phagocytic activity were formed on the substrate after fixation. The stop of MSC proliferation was terminated by their spontaneous in vitro differentiation into bone, fat, cartilage, muscle or connective tissue cells. Further studies have made it possible to establish the osteogenic potential of fibroblast-like cells of the bone marrow stroma of various mammalian species, as well as their colony-forming activity. In vivo experiments, it was shown that both hetero- and orthotopic transplantation of colony-forming fibroblast-like cells is completed by the formation of bone, cartilaginous, fibrous and adipose tissue. Since bone marrow stem cells have a high capacity for self-renewal and a variety of differentiation within a single cell line, they have been termed multipotent mesenchymal progenitor cells.

It should be noted that for 45 years of fundamental research of mesenchymal stem cells, real conditions have been created for the use of their derivatives in clinical practice.

Today, there is no doubt that all the tissues of the human body are formed from the stem cells of different cell lines as a result of processes of proliferation, migration, differentiation and maturation. However, more recently, it was believed that stem cells in the adult body are tissue-specific, that is, capable of producing specialized cell lines only of the tissues in which they are located. This conceptual situation was refuted by the facts of transformation of hematopoietic stem cells not only into cellular elements of peripheral blood, but also into oval liver cells. In addition, and neural stem cells were able to give rise to both neurons and glial elements, as well as early committed lines of hematopoietic progenitor cells. In turn, mesenchymal stem cells, usually producing cellular elements of bone, cartilage and adipose tissue, are able to transform into neural stem cells. It is assumed that in the process of growth, physiological and reparative tissue regeneration, uncommitted progenitor cells are generated from tissue-specific stem reserves. For example, muscle tissue repair can be realized by means of mesenchymal stem cells that migrate from the bone marrow to skeletal muscles.

Although not all researchers recognize this cross-substitutability of stem cells, the possibility of clinical use of mesenchymal stem cells as a source for cell transplantation and the cellular vector of genetic information is not disputed by anyone, just like the multipotency of bone marrow stromal stem cells, which can be relatively easily isolated and multiplied in culture in vitro. At the same time, in the scientific literature, there continue to be reports of potential pluripotency in stem cells of bone marrow stroma. As evidence, research protocols are given in which, under the influence of specific inducers, transdifferentiation of MSCs is converted into nerve cells, cardiomyocytes and hepatocytes. However, in some scientists the possibility of re-activation and expression of genes early embryogenesis is highly questionable. At the same time, everyone understands that if conditions are found to expand the multipotency of mesenchymal stem cells to the pluripotency of ESC, in the regenerative and plastic medicine many ethical, moral, religious and legal problems are automatically resolved. In addition, since in this case the autologous stromal cells of the patient become the source of the regenerative stem potential, the problem of immune rejection of the cell transplant is solved. How real these prospects are, the near future will show.

[1], [2]

Use of mesenchymal stem cells in medicine

In the clinic, the use of mesenchymal stem cell derivatives is associated, first of all, with the restoration of tissue defects arising from extensive and profound thermal lesions of the skin. At the preclinical stage, an experimental assessment was made of the advisability of using allogeneic fibroblast-like mesenchymal stem cells for the treatment of deep burns. It is shown that fibroblast-like mesenchymal bone marrow stem cells form a monolayer in culture, which makes it possible to transplant them in order to optimize the processes of regeneration of deep burn wounds. The authors note that embryonic fibroblasts possess a similar property, but the clinical application of the latter is limited by existing ethical and legal problems. A deep thermal burn with damage to all layers of the skin was modeled on Wistar rats. The area of the burn was 18-20% of the total surface of the skin. The first experimental group included rats with deep thermal burn and transplantation of allogeneic fibroblast-like mesenchymal stem cells. The second group consisted of animals with a deep thermal burn and trans plantation of allogeneic embryonic fibroblasts. The third group was represented by control rats with a deep thermal burn, which did not carry out cellular therapy. A suspension of fibroblast-like mesenchymal stem cells and embryonic fibroblasts was applied to the surface of the burn wound with a pipette in an amount of 2 x 10 ⁴ cells on the 2nd day after modeling the burn and excising the necrotic scab. After cell transplantation, the burn surface was covered with a gauze cloth moistened with an isotonic sodium chloride solution with gentamycin. The collection of bone marrow cells for the production of MSCs followed by their induction into a line of fibroblast-like mesenchymal stem cells was performed in adult Wistar rats from the femur. Embryonic fibroblasts were obtained from the lungs of 14-17 day old embryos. Embryonic fibroblasts and bone marrow cells for the preparation of MSC were pre-cultured in Petri dishes at 37 ° C in a CO2 incubator in an atmosphere with 5% CO2 at 95% humidity. Embryonic fibroblasts were cultured for 4-6 days, whereas for the formation of a monolayer of MSC it took from 14 to 17 days. Subsequently, MSCs were preserved by cryopreservation method as a starting material for fibroblast-like mesenchymal stem cells, which were obtained by defrosting and culturing MSCs for 4 days. The number of formed fibroblast-like mesenchymal stem cells was more than 3 times greater than the number of embryonic fibroblasts that appeared during the same period of cultivation. To identify the transcultivated cells in burn wounds during the cultivation stage, their genome was labeled using a viral shuttle vector based on recombinant type V adenovirus bearing the 1ac-2 gene encoding ß-galactosidase of E. Coli. Living cells at different times after transplantation were detected immunohistochemically in cryosections with the addition of X-Gal substrate, which gives a characteristic blue-green coloration. As a result of dynamic visual, planimetric and histological evaluation of the burn wound condition, it was found that on the third day after cell transplantation, significant differences in the course of the wound process appear in the isolated groups. Especially distinct, this difference became on the 7th day after cell transplantation. In the animals of the first group, who had been transplanted with fibroblast-like mesenchymal stem cells, the wound acquired a uniformly pink intense color, the granulation tissue expanded throughout its area to the level of the epidermis, and the burned surface was significantly reduced in size. The collagen film formed on the surface of the wound was somewhat thinning, but it continued to cover the entire burn area. In animals of the second group, embryonic fibroblasts were transplanted, the granulation tissue was raised to the level of the epidermis of the wound edges, but only in places, while the plasmorrhoea from the wound was more intense than in the 1 st group, and the initially formed collagen film practically disappeared. In animals that did not receive cell therapy, on the 7th day, the burn wound was a pale, dug, necrotic tissue covered with fibrin. Plasmorrhea was noted throughout the burn surface. Histologically, in animals of the 1st and 2nd groups there was a decrease in cellular infiltration and development of the vasculature, and these signs of the beginning regenerative process were more pronounced in the rats of the 1 st group. In the control group there were signs of cellular infiltration of the wound, the histological pattern of the newly formed vessels was absent. On the 15th to 30th day of observation in animals of the 1st group, the burn surface area was significantly smaller than in the rats of the remaining groups, and the granulating surface was more developed. In the animals of the 2nd group, the area of the burn surface also decreased in comparison with the size of burn wounds in the rats of the control group, which was due to marginal epithelialization. In the control group, the burning surface in places remained pale with rare granulations, vascular asterisks appeared on it, there were islets of fibrinous plaque, moderate plasmorrhoea continued over the entire burn surface, and in some cases a hard-to-separate scab remained. In general, the animals of the third group also reduced the size of the wound, but the edges of the wound remained undercut.

Thus, during the comparative study of wound healing speed with the use of fibroblast-like mesenchymal stem cells and embryonic fibroblasts, and without the use of cell therapy, acceleration of the healing rate of the burn surface as a result of transplantation of fibroblast-like mesenchymal stem cells and embryonic fibroblasts was noted. However, in the case of allogeneic fibroblast-like mesenchymal stem cells, the rate of wound healing was higher than that of embryonic fibroblast transplantation. This was expressed in accelerating the phase change of the regenerative process - the terms of cellular infiltration were shortened, the rate of growth of the vasculature increased, as well as the formation of granulation tissue.

The results of dynamic planimetry indicate that the rate of spontaneous healing of the burn wound (without the use of cell therapy) was the lowest. On the 15th and 30th days after the transplantation of allogeneic fibroblast-like mesenchymal stem cells, the rate of wound healing was higher than in transplantation of embryonic fibroblasts. The histochemical method for the detection of beta-galactosidase showed that after transplantation of fibroblast-like mesenchymal stem cells and embryonic fibroblasts, during the whole period of observation at the surface and in the depth of the regenerating wounds, the transplanted cells remain viable. The authors believe that a higher rate of regeneration of burn wounds when using fibroblast-like mesenchymal stem cells is due to the release of biologically active growth-stimulating factors by these cells during maturation.

Transplantation of auto- or allogeneic keratinocytes and allogeneic fibroblasts for the treatment of burn wounds was also applied in the clinic. It should be noted that surgical treatment of children with extensive deep burns is the most difficult task due to high traumatic and multiple surgical interventions, significant blood loss, various reactions to infusion media used. The main difficulties in performing skin and plastic operations with extensive deep burns, over an area exceeding 40% of the body surface, are due to the severity of the condition of the victims and the lack of donor skin resources. The use of mesh grafts with a large perforation factor does not solve the problem, since the cells formed after perforation are epithelialized very slowly, and the skin flaps themselves are often lysed or dried. Such coatings of burn wounds as xenotic, cadaveric allografts, synthetic film coatings are not always effective enough, therefore new methods of closing burn surfaces with layers of cultured keratinocytes and fibroblasts are being developed. In particular, a method for closing burn surfaces with the help of cultured allofibroblasts, which have a pronounced stimulating effect on the proliferation of epidermal cells preserved in the wound with border burns, as well as keratinocytes in the ligaments of mesh grafts, is proposed. In the work of L. Budkevich and co-authors (2000) the results of the application of this method for the treatment of burns in children are presented. 31 children with thermal trauma aged between 1 and 14 years were under observation. In three children, the total area of burn wounds IIIA-B - IV degree was 40%, 25 - 50-70%, and in three - 71-85% of the body surface. Early surgical necrosectomy was combined with transplantation of cultured allof fibroblasts and autodermoplasty. At the first stage of the treatment, excision of necrotic tissues was carried out, on the second stage - transplantation of cultured allof fibroblasts on carrier films, at the third (48 hours after transplantation of cultured allof fibroblasts) - removal of the matrix and autodermoplasty with skin grafts with a perforation ratio of 1: 4. Three patients admitted to the clinic with a severe burn disease, cultured allof fibroblasts were transplanted to granulating wounds. Transplantation of cultured allo-fibroblasts was performed once in 18 children, twice in 11, and three times in two patients. The area of the wound surface covered by the cell culture was from 30 to 3500 cm2. The effectiveness of the application of cultured allo-fibroblasts was assessed by the total percentage of engraftment of skin flaps, the timing of healing of burns and the number of deaths of severe thermal trauma. The engraftment of transplants was complete in 86% of patients. A partial non-appearance of skin flaps was noted in 14% of cases. Despite ongoing treatment, six (19.3%) children died. The total skin lesion area in them was from 40 to 70% of the body surface. Transplantation of cultured allo-fibroblasts was not related to the fatal outcome of a burn injury in any patient.

Analyzing the results of the treatment, the authors note that earlier deep burns incompatible with life included a deep thermal damage to the skin with an area of 35-40% of the body surface (for children under 3 years old - critical burns with an area of 30%, for older children age - over 40% of the body surface). When performing surgical necrectomy with transplantation of cultured allof fibroblasts and subsequent autodermoplasty with skin flaps with a high perforation coefficient, IIIB - IV degrees burns remain critical, but now there are prospects in many cases to save life even for such victims. Surgical necrosectomy in combination with transplantation of cultured allof fibroblasts and autodermoplasty in children with deep burns proved to be especially effective in patients with widespread skin lesions with a deficiency of donor sites. Active surgical tactics and transplantation of cultured allo-fibroblasts contribute to the rapid stabilization of the general condition of such patients, a reduction in the number of infectious complications of burn disease, the creation of favorable conditions for transplant engraftment, a reduction in the recovery time of lost skin and the duration of inpatient treatment, and a reduction in the death rate among victims with extensive burns. Thus, transplantation of cultured allo-fibroblasts followed by autodermoplasty with skin flaps allows recovery in children with severe burns, which were previously considered doomed.

It is widely recognized that the primary objective of treating a burn disease is to maximize the full and rapid recovery of damaged skin to prevent the resulting toxic effects, infectious complications and dehydration of the body. The results of the application of cultured cells largely depend on the preparedness for transplantation of the burn wound itself. In cases of transplantation of cultured keratinocytes, 55% (by area) of the transplanted cells survive on the wound surface after surgical necrectomy, whereas in engrafting wounds the engraftment frequency is reduced to 15%. Therefore, successful treatment of extensive deep skin burns requires, in the first place, active surgical tactics. In the presence of burn wounds IIIB-IV degree, the burn surface is immediately released from necrotic tissues in order to reduce the effects of intoxication and reduce the number of complications of burn disease. The use of such tactics is the key to reducing the time from the moment of getting a burn to closing the wounds and the length of stay of patients with extensive burns in the hospital, and also significantly reducing the number of deaths.

The first reports on the successful use of cultured keratinocytes to cover the burn surface appeared in the early eighties of the last century. Subsequently, this manipulation was carried out with the help of layers of cultured keratinocytes, obtained most often from autostructures, much less often from allokeratinocytes. However, the technology of autokeratinocytoplasty does not allow the creation of a cell bank, while the time required to produce a sufficient graft from keratinocytes is large and amount to 3-4 weeks. During this period, the risk of developing infectious and other complications of burn disease increases sharply, which significantly prolongs the total length of stay of patients in the hospital. In addition, autokeratinocytes practically do not survive during transplantation into granulating burn wounds, and the high cost of special growth media and biologically active keratinocyte growth stimulants significantly limits their clinical application. Other biotechnological methods, such as collagenoplasty, transplantation of cryopreserved xenoids, and the use of various biopolymer coatings increase the effectiveness of treatment of extensive surface but not deep burns. The method of coating the wound surface with cultured fibroblasts is fundamentally different in that the main component of the cultured cell pool is not keratinocytes but fibroblasts.

The prerequisite for the development of the method was the data that pericytes surrounding small vessels are progenitor mesenchymal cells that can be transformed into fibroblasts that produce many growth factors and provide wound healing due to a strong stimulating effect on the proliferation and adhesion of keratinocytes. The use of cultured fibroblasts to close the wound surfaces immediately revealed a number of significant advantages of this method compared with the use of cultured keratinocytes. In particular, the production of fibroblasts in culture does not require the use of special nutrient media and growth stimulants, which reduces the cost of the transplant by more than 10 times the cost of obtaining keratinocytes. Fibroblasts are easily passaged, during which they partially lose the surface antigens of histocompatibility, which in turn opens the possibility of using allogeneic cells for the production of grafts and creating their banks. The time is short for obtaining grafts ready for use in the clinic, from 3 weeks (for keratinocytes) to 1-2 days (for fibroblasts). The primary culture of fibroblasts can be obtained by culturing skin fragments taken from autodermoplasty from cells, and the density of cell culture in the production of subcultures of human fibroblasts is only 20 x 10 ³ per 1 cm ².

To study the effect of fibroblasts and their regulatory proteins on the proliferation and differentiation of keratinocytes, a comparative analysis of the morphology and proliferation of keratinocytes on substrates from collagen I and III types as well as fibronectin in a co-culture with human fibroblasts was carried out. Human keratinocytes were isolated from fragments of the skin of patients with burns taken during the operation of autodermoplasty. The density of keratinocytes was 50 x 103 cells per cm2. The clinical efficacy of transplantation of cultured fibroblasts was evaluated in 517 patients. All patients were divided into two groups: 1st - adults affected with burns IIA, B - IV degree; 2nd - children with deep burns of IIIB - IV degree. Evaluation of the dynamics of the structural and functional organization of monoclonal fibroblasts with allowance for the role of glycosaminoglycans, fibronectin and collagen in the regeneration processes allowed the authors to determine the 3rd day as the most favorable period for the use of fibroblast cultures for the manufacture of grafts. Investigation of the influence of fibroblasts on the proliferation and differentiation of keratinocytes showed that in vitro fibroblasts exert a stimulating effect, primarily on the adhesion of keratinocytes, increasing the number of attached cells and their fixation rate more than 2-fold. Stimulation of adhesion processes is accompanied by an increase in the intensity of DNA synthesis and the level of proliferation of keratinocytes. In addition, it was found that the presence of fibroblasts and the extracellular matrix formed by them is a prerequisite for the formation of the tonofibrillar apparatus of keratinocytes, intercellular bonds and, ultimately, for the differentiation of keratinocytes and the formation of the basal membrane. In the treatment of children with deep burns, a high clinical efficacy of allo-fibroblast cell transplantation has been established, especially in the group of patients with extensive skin lesions in conditions of donor sites deficiency. Complex morphofunctional study showed that graft fibroblasts are characterized by active synthesis of DNA, as well as collagen, fibronectin and glycosaminoglycans that are part of the cell-formed extracellular matrix. The authors indicate a high percentage of engraftment of transplanted fibroblasts (up to 96%), a sharp reduction in the timing of their production (within 24-48 hours instead of 2-3 weeks in the case of keratinocytes), significant acceleration of the epithelialization of the burn surface, and a significant reduction in cost times) of the technology of growing the graft from fibroblasts in comparison with keratinocyte transplantation. The application of transplantation of cultured allo-fibroblasts makes it possible to save the lives of children with critical burns - a thermal lesion of more than 50% of the body surface, which was previously considered incompatible with life. It should be noted that during transplantation of allogeneic embryonic fibroblasts, it is also convincingly proved not only faster wound regeneration and reconvalescence of patients with different degree and area of burn, but also a significant reduction in their mortality.

Autologous fibroblasts are also used in such a complex area of plastic surgery as restorative correction of vocal cord injuries. Usually, for this purpose, bovine collagen is used, the duration of which is limited by its immunogenicity. Being a foreign protein, bovine collagen is sensitive to the recipient's collagenase and can induce immune reactions, in order to reduce the risk of which, technologies have been developed for obtaining collagen preparations cross-linked with glutaraldehyde. Their advantage lies in greater stability and less immunogenicity, which has found practical application in the elimination of defects and atrophy of the vocal cords. Injections of autologous collagen were first used in 1995. The technique ensured the preservation of the primary structure of autologous collagen fibers, including intramolecular enzymatically catalyzed crosslinks. The fact is that natural collagen fibers are more resistant to protease destruction than reconstituted collagen, in which telopeptides are cut. The integrity of telopeptides is important for the quaternary structure of collagen fibers and the formation of cross-links between adjacent collagen molecules. Unlike bovine collagen preparations, autologous collagen does not cause immune responses in the recipient, however, it is not effective enough as a replenishment agent. Persistent correction can be achieved by local production of collagen by autologous fibroblast transplantation. However, studies of the efficacy of autologous fibroblast transplantation in the clinic revealed certain difficulties. In the early period after fibroblast transplantation, the clinical effect was weaker compared to that after the administration of bovine collagen. In the cultivation of autologous fibroblasts, the possibility of transformation of normal fibroblasts into pathological so-called myofibroblasts responsible for the development of fibrosis and scar formation is possible, as evidenced by a reduction in the collagen gel caused by the specific interaction of fibroblasts and collagen fibrils. In addition, after serial passaging in vitro, fibroblasts lose the ability to synthesize extracellular matrix proteins.

Nevertheless, at present the technique of culturing autologous human fibroblasts has been experimentally eliminated, eliminating the above disadvantages and not leading to oncogenic transformation of normal fibroblasts. Autologous fibroblasts obtained by this method are used to fill the defects of the soft tissues of the face. In a study by H. Keller and co-authors (2000), 20 patients aged 37 to 61 years with wrinkles and atrophic scars received treatment. Skin biopsies (4 mm) of the BTE were transported to the laboratory in sterile tubes containing 10 ml of culture medium (Eagle's medium with antibiotic, myco-septic, pyruvate and fetal calf serum). The material was placed inside 3-5 culture dishes with a diameter of 60 mm and incubated in a thermostat with an atmosphere containing 5% CO2. After 1 week, the cells were removed from the dishes by trypsinization and placed in 25 cm2 vials. Cells were administered to patients in an amount of 4 x 107. Significant and persistent clinical effect was observed in patients with correction of nasolabial folds, as well as in patients with scars at 7 and 12 months after the third transplantation of autologous fibroblasts. According to flow cytometry, cultured fibroblasts produced a large amount of Type I collagen. In vitro studies, the normal contractility of injectable fibroblasts is shown. Two months after subcutaneous administration of cultured fibroblasts in a dose of 4 x 107 cells, nude mice were not detected. Injectable fibroblasts did not cause scar formation and diffuse fibrosis in patients. According to the author, the implanted autologous fibroblasts are able to constantly produce collagen, which will give cosmetic rejuvenation effect. In this case, since the life span of differentiated cells is limited, fibroblasts taken from a young patient are more effective than those obtained in elderly people. In the future, the possibility of cryopreservation of a fibroblast culture taken from a young donor is assumed to later transplant to an elderly patient his own young cells. In conclusion, it is not entirely correct conclusion that autologous fibroblasts under the condition of their functional preservation are the ideal means of correction of defects in the soft tissues of the face. At the same time, the author himself notes that in the process of research some problematic situations connected with the use of an autologous fibroblast-collagen system also arose. The clinical effect was often weaker than with the use of bovine collagen, which caused disappointment in patients.

In general, the literature data on the prospects of clinical use of mesenchymal stem cells look quite optimistic. Attempts are made to use autologous bone marrow multipotent mesenchymal progenitor cells for the treatment of degenerative joint lesions. The first clinical trials of cultured mesenchymal progenitor cells in the treatment of complex fractures of bone are conducted. Autologous and allogenic mesenchymal cells of bone marrow stroma are used to create cartilaginous tissue for the purpose of transplantation when correcting articular cartilage defects due to trauma or autoimmune lesions. Methods for the clinical use of multipotent mesenchymal progenitor cells for elimination of bone defects in children with severe forms of incomplete osteogenesis caused by mutations of the type I gene of collagen are being developed. After mieloablation, the bone marrow from HLA-compatible healthy donors is transplanted to recipient children, as unfractionated bone marrow can contain a sufficient number of mesenchymal stem cells to replenish a severe bone defect. After transplantation of allogeneic bone marrow, such children had positive histological changes in trabecular bones, an increase in the rate of growth and a decrease in the incidence of bone fractures. In some cases, a positive clinical result is achieved by transplanting closely related allogeneic bone marrow and osteoblasts. For the treatment of congenital fragility of bones due to imbalance of osteoblasts and osteoclasts in bone tissue, MSK transplantation is also used. Restoration of bone formation in this case is achieved due to chimerization of the pool of stem and progenitor stromal cells in the bone tissue of patients.

The methods of genetic modification of donor mesenchymal stem cells are being improved to correct genetic defects of stromal tissues. It is expected that in the near future mesenchymal progenitor cells will be used in neurology for directed chimerization of brain cells and creation of a healthy pool of cells capable of generating a deficient enzyme or a factor responsible for clinical manifestations of the disease. Transplantation of mesenchymal stem cells can be used to restore bone marrow stroma in cancer patients after radio and chemotherapy, and in combination with bone marrow cells - to restore hemopoiesis. The development of substitution therapy aimed at eliminating defects in the musculoskeletal system with the help of MSCs is promoted by engineering developments in the field of designing matrix biomaterials or biomimetics that form the skeletons populated by offspring of mesenchymal stem cells.

Sources of mesenchymal stem cells

The main source of mesenchymal stem cells is the bone marrow, whose hematopoietic stem cells in the mammalian organism are constantly differentiated into blood cells and the immune system, whereas mesenchymal stem cells are represented by a small population of fibroblast-like bone marrow stromal cells and contribute to the preservation of the undifferentiated state of hematopoietic stem cells. Under certain conditions, mesenchymal stem cells differentiate into cells of cartilaginous and bone tissue. When planted on a culture medium in conditions of low planting density, mononuclear bone marrow stromal cells form colonies of adhesive cells, which, in fact, are fibroblast-like multipotent mesenchymal progenitor cells. Some authors believe that uncommitted mesenchymal stem cells are deposited in the bone marrow, which, thanks to the ability to self renewal and high differentiation potential, provide all body tissues with mesenchymal precursors of stromal elements throughout the life of the mammalian organism.

In the bone marrow, stromal cell elements form a network that fills the space between sinusoids and bone tissue. The content of dormant MSC in the bone marrow of an adult is comparable to the number of hematopoietic stem cells and does not exceed 0.01-0.001%. Mesenchymal stem cells isolated from bone marrow and not subjected to cultivation are devoid of adhesive molecules. Such MSCs do not express CD34, ICAM, VCAM, type I and III collagen, CD44 and CD29. Therefore, in vitro, not mesenchymal stem cells are fixed on the culture substrate, but more advanced progenitor derivatives of mesenchymal stem cells that have already formed components of the cytoskeleton and receptor apparatus of cell adhesion molecules. Stromal cells with the phenotype CD34 are found even in peripheral blood, although they are much less in the bone marrow than CD34-positive mononuclear cells. The CD34 cells isolated from the blood and transferred into the culture attach to the substrate and form colonies of fibroblast-like cells.

It is known that in the embryonic period the stromal base of all organs and tissues of mammals and humans arises from a common pool of mesenchymal stem cells before and at the stage of organogenesis. Therefore, it is believed that in a mature body, most of the mesenchymal stem cells should be in connective and bone tissue. It has been established that the majority of cellular elements of the stroma of loose connective and bone tissue are represented by committed progenitor cells, which, however, retain the ability to proliferate and form clones in vitro. With the introduction of such cells into the total blood flow, more than 20% of the mesenchymal progenitor cells are implanted among the stromal elements of the hematopoietic tissue and the parenchymal organs.

Potential source of mesenchymal stem cells is adipose tissue, among the stem cells of which the adipocyte precursors commited to different degrees have been identified. The least mature progenitor elements of adipose tissue are stromal-vascular cells, which, like multipotent mesenchymal precursor cells of the bone marrow, are able to differentiate into adipocytes under the influence of glucocorticoids, insulin-like growth factor and insulin. In culture, stromal-vascular cells differentiate into adipocytes and chondrocytes, and in adipose tissue of bone marrow origin there are cells that form adipocytes and osteoblasts.

In the muscles, stromal stem sources were also found. In the primary culture of cells isolated from human skeletal muscles, cells of stellate form and multinucleated myotubes are detected. In the presence of horse serum, the stellate cells proliferate in vitro without signs of cytodifferentiation, and after the addition of dexamethasone to the nutrient medium, their differentiation is characterized by the appearance of cellular elements with a phenotype of skeletal and smooth muscle cells, bone, cartilaginous and adipose tissue. Consequently, both committed and uncommitted multipotent mesenchymal progenitor cells are present in the human muscle tissue. It was shown that the population of progenitor cells represented in skeletal muscle originates from uncompensated multipotent mesenchymal bone marrow precursors and differs from myogenic satellite cells.

In the myocardium of newborn rats, adhesive star cells, corresponding to the multipotent mesenchymal progenitor cells, are also found in the myocardium of the newborn rats, because they are differentiated into adipocytes, osteoblasts, chondrocytes, smooth muscle cells, myotubes of skeletal muscles and cardiomyocytes under the influence of dexamethasone. It has been shown that vascular smooth muscle cells (pericytes) are derived from undifferentiated perivascular multipotent mesenchymal progenitor cells. In culture, perivascular mesenchymal stem cells express smooth muscle a-actin and a platelet-derived growth factor receptor and are able to differentiate at least in smooth muscle cells.

A special place, in terms of stem reserves, is the cartilaginous tissue, whose extremely low reparative potential is believed to be due to a deficiency of multipotent mesenchymal progenitor cells or differentiation and growth factors. It is assumed that the multipotent mesenchymal progenitor cells pre-donated to the chondro- and osteogenesis enter the cartilaginous tissue from other tissue sources.

Tissue origin and conditions for the commission of mesenchymal progenitor cells in the tendons are also not established. Expert observations indicate that in the early postnatal period the cells of the Achilles tendon of rabbits in primary cultures and at the first passage retain expression of type I collagen and decorina, but with further cultivation they lose the differentiating markers of the tenocytes.

It should be noted that the answer to the question whether the multipotent mesenchymal progenitor cells localized in different tissues are always present in their stroma or whether the tissue pool of mesenchymal stem cells is compensated for by migration of bone marrow stromal stem cells is still not received.

In addition to the bone marrow and other mesenchymal tissue zones of an adult organism, another source of MSC can be cord blood. It was shown that cord blood veins contain cells that have similar morphological and antigenic characteristics with multipotent mesenchymal progenitor cells, are capable of adhesion and are not inferior to multipotent mesenchymal progenitors of bone marrow origin by differentiation potential. In cultures of mesenchymal stem cells of umbilical cord blood, 5 to 10% of uncommitted multipotent mesenchymal progenitor cells were detected. It turned out that their quantity in the cord blood is inversely proportional to the duration of gestation, which indirectly indicates the migration of multipotent mesenchymal progenitor cells to various tissues in the process of fetal development. The first information about the clinical use of mesenchymal stem cells derived from umbilical cord blood, as well as derived from embryonic biomaterial, was established, which is based on the known ability of fetal stem cells to integrate, to survive and function in the organs and tissues of adult recipients.

The search for new sources of mesenchymal stem cells

The use of mesenchymal stem cells of embryonic origin, like other fetal cells, creates a number of ethical, legal, legal and legislative problems. Therefore, the search for extraembryonic cellular donor material continues. The attempt of clinical application of human skin fibroblasts was unsuccessful, which was predetermined not only by the high financial capacity of the technology, but also by the rapid differentiation of fibroblasts into fibroblasts, which have significantly less proliferation potential and produce a limited number of growth factors. Further progress in studying the biology of MSK and multipotent mesenchymal bone marrow precursors allowed the development of a strategy for the clinical use of autologous mesenchymal stem cells. The technology of their isolation, cultivation, ex vivo reproduction and directed differentiation required, first of all, the study of the molecular marker spectrum of MSCs. Their analysis showed that in the primary cultures of human bone tissue there are several types of multipotent mesenchymal progenitor cells. The pro-osteoblast phenotype was found in cells expressing a marker of stromal precursor cells STRO-1, but not carrying an osteoblast marker - alkaline phosphatase. Such cells are characterized by a low ability to form a mineralized bone matrix, as well as the absence of expression of osteopontin and the parathyroid hormone receptor. Derivatives of STRO-1-positive cells that do not express alkaline phosphatase are represented by intermediate and completely differentiated osteoblasts. It is established that the cellular elements of cloned lines of STRO-1-positive cells of human trabecular bones are able to differentiate into mature osteocytes and adipocytes. The direction of differentiation of these cells depends on the effect of polyunsaturated fatty acids, proinflammatory cytokines - IL-1b and tumor necrosis factor a (TNF-a), as well as anti-inflammatory and immunosuppressive TGF-b.

Subsequently, it turned out that the multipotent mesenchymal progenitor cells lack a specific phenotype alone, but they express a complex of markers characteristic of mesenchymal, endothelial, epithelial and muscle cells in the absence of expression of immunophenotypic antigens of hemopoietic cells - CD45, CD34 and CD14. In addition, mesenchymal stem cells constitutively and inducibly produce hematopoietic and nonhematopoietic growth factors, interleukins and chemokines, and on multipotent mesenchymal progenitor cells, receptors for some cytokines and growth factors are expressed. Among the cells of the stromal base of the human body, dormant or resting cells with an immunophenotype almost identical to the antigenic profile of 5-fluorouracil untreated multipotent mesenchymal progenitor cells have been found-both of these cells express CD117 labeling "adult" stem cells.

Thus, a cell marker unique to mesenchymal stem cells has not yet been established. It is assumed that resting cells represent a population of uncommitted multipotent mesenchymal progenitor cells, since they do not express markers of cells commited to osteo- (Cbfa-1) or adipogenesis (PPAR-y-2). The prolonged exposure of slowly proliferating resting cells to fetal calf serum leads to the formation of terminally differentiated precursors, characterized by rapid growth. The clonal growth of such stem mesenchymal cells is supported by FGF2. It seems that the genome of stromal stem cells is sufficiently tightly "closed." There are reports of the absence of spontaneous differentiation in MSK - without special conditions for committing they are not transformed even into cells of the mesenchymal series.

To study the population structure of mesenchymal stem cell derivatives, a search for marker differentiation proteins on stromal cell lines and in primary cultures is carried out. In clonal analysis of colony-forming bone marrow cells in vitro, it was found that when exposed to primary cultures, EGF increases the average colony size and reduces clonal expression of alkaline phosphatase, while the addition of hydrocortisone activates the expression of alkaline phosphatase, which is a marker of the osteogenic orientation of MSK differentiation. Monoclonal antibodies to STRO-1 made it possible to separate and study the population of STRO-1-positive adhesive cells in a heterogeneous system of Dexter cultures. A spectrum of cytokines that regulate not only proliferation and differentiation of hematopoietic and lymphoid cells but also involved in the formation, formation and resorption of skeletal tissues through para-, auto- and endocrine mechanisms has been determined. Receptor-mediated release of such secondary messengers as cAMP, diacylglycerol, inositol triphosphate and Ca2 +, is also used for marker analysis of various categories of stromal tissue cells expressing the corresponding receptors. The use of monoclonal antibodies as markers made it possible to establish the belonging of the reticular cells of the stroma of the lymphoid organs to the T- and B-dependent zones.

For some time scientific disputes continued around the question of the possibility of the origin of MSC from the hematopoietic stem cell. Indeed, with the explantation of the suspension of bone marrow cells into monolayer cultures, discrete colonies of fibroblasts grow in them. However, it was shown that the presence of precursors of fibroblast colonies and various germs of hematopoietic tissue differentiation in the bone marrow is not a proof of their common origin from the stem cell. With the help of discriminant analysis of bone marrow stem cells, it is established that the microenvironment during heterotopic bone marrow transplantation is not transferred by hemopoietic cells, which proves the existence in the bone marrow of the MSC population histogenetically independent of the hematopoietic cells.

In addition, the selective cloning method made it possible to reveal a new category of stromal progenitor cells in monolayer cultures of bone marrow cells, to determine their number, to study their properties, proliferative and differentiating potencies. It turned out that stromal fibroblast-like cells in vitro proliferate and form diploid colonies, which, with the return of transplantation into the body, provide the formation of new hematopoietic organs. The results of the study of individual clones indicate that among the stromal progenitor cells there is a population of cells, in its proliferative and differentiating potential, capable of claiming the role of stem cells of stromal tissue histogenetically independent of stem hemopoietic cells. Cells of this population are characterized by self-supporting growth and differentiate into progenitor cells of bone, cartilage and reticular bone marrow tissue.

Of great interest are the results of the studies of R. Chailakhian and co-authors (1997-2001) who cultured bone marrow stromal progenitor cells of rabbits, guinea pigs and mice on a-MEM nutrient medium supplemented with fetal bovine serum. The authors carried out the explantation with an initial density of 2-4 x 103 bone marrow cells per 1 cm2. As a feeder, homologous or heterologous radiation-inactivated bone marrow cells were used in a dose retaining the feeder action but completely blocking their proliferation. Two-week primary discrete colonies of fibroblasts were trypsinized to produce monoclonal strains. Evidence of the clonal origin of the colonies was obtained using a chromosome marker in mixed cultures of bone marrow of the male and female guinea pigs, a time-lapse photography of live cultures, as well as in mixed cultures of syngeneic bone marrow of CBA and CBAT6T6 mice. Transplantation of a suspension of freshly isolated bone marrow cells or in vitro-grown stromal fibroblasts under the kidney capsule was carried out in isoval or gelatinous porous scaffolds, as well as in an inactivated rabbit bone matrix matrix. For the transplantation of clones in the bone cover, the guinea pig's femur was cleared of soft tissues and periosteum, the epiphyses were cut off and the bone marrow was thoroughly washed. The bone was cut into fragments (3-5 mm), dried and irradiated at a dose of 60 Gy. In the bony covers, individual fibroblast colonies were placed and implanted intramuscularly. For intraperitoneal transplantation of stromal fibroblasts grown in vitro, diffusion chambers of types A (V = 0.015 cm3, h = 0, l mm) and O (V = 0.15 cm3, h = 2 mm) were used.

In studying the growth dynamics of clonal strains, R. Chailakhian and co-authors (2001) found that individual cells forming fibroblast colonies, as well as their descendants, have a huge proliferative potential. By the 10th passage, the number of fibroblasts in some strains was 1.2-7.2 x 10 ⁹ cells. In the process of their development, they performed up to 31-34 cellular duplications. In this case, heterotopic transplantation of bone marrow strains, formed by stromal precursors of several dozen clones, led to the transfer of the bone marrow microenvironment and the formation of a new hematopoietic organ in the transplantation zone. The authors raised the question of whether individual clones are capable of transferring the bone marrow microenvironment of stromal cells or does it require the cooperation of several different clonal-derived stromal progenitors? And if individual clones are capable of transferring the microenvironment, will it be complete for all three germs, or do different clones provide a microenvironment for different hematopoietic sprouts? To address these issues, a technology was developed for culturing stromal progenitor cells on a collagen gel, allowing to remove from the surface grown colonies of fibroblasts for subsequent heterotopic transplantation. Individual clones of stromal fibroblasts grown from bone marrow cells of CBA mice and guinea pigs were excised together with a gel cover fragment and were transplanted heterotopically under a capsule of kidney syngenous mice or into the abdominal muscle of autologous guinea pigs. When transplantation into the muscle, the colonies on the gel were placed in the bony covers.

The authors found that in 50-90 days after transplantation of colonies of bone marrow fibroblasts, development of bone or bone and hemopoietic tissue was observed in the transplant zone in 20% of cases. In 5% of recipient animals, the foci of bone tissue formed contained a cavity filled with bone marrow. Inside the osseous cylinders, such foci had a rounded shape and a capsule, constructed from bone tissue with osteocytes and a well developed osteoblastic layer. Bone cavity contained a reticular tissue with myeloid and erythroid cells, the proportional ratio of which did not differ from that in the usual bone marrow. In the kidney, the graft was a typical bone marrow organ that formed when a native bone marrow transplantation, and the bone capsule covered the medullary cavity only from the side of the renal capsule. Hooded tissue included myeloid, erythroid and megakaryocytic elements. The stroma of the medullary cavity had a well developed sine system and contained typical fat cells. At the same time, bone tissue with no signs of hematopoiesis was found in the transplantation zone of some colonies under the kidney capsule. The study of the proliferative and differentiating potentials of individual clones was continued on monoclonal bone marrow strains of rabbits, the cells of which were resuspended in a nutrient medium and in a separate Ivalon sponge weighing 1-2 mg were inserted under the kidney capsule of a bone marrow donor. Such autotransplantation was subjected to the cells of 21 monoclonal strains. The results were taken into account in 2-3 months. The authors found that in 14% of the cases the transplanted monoclonal strains formed a bone marrow organ consisting of bone tissue and a bone marrow cavity filled with hematopoietic cells. In 33% of cases, the transplanted strains formed a compact bone of different sizes with osteocytes immured in the cavities and developed osteoblastic layer. In some cases, in the sponges with transplanted clones, the reticular tissue developed without bone or hemopoietic elements. Occasionally, a reticular stroma was formed with a well-developed network of sinusoids, but not populated with hematopoietic cells. Thus, the results obtained were similar to those obtained in the transplantation of clones on a collagen gel. However, if transplantation of clones grown on a substrate led to the formation of bone marrow tissue in 5% of cases, bone - in 15% and reticular tissue - in 80% of cases, when transplanting monoclonal strains, formation of bone marrow elements was observed in 14% of cases, in 53% and reticular - in 53% of cases. In the opinion of the authors, this indicates that the conditions for the realization of proliferative and differentiating potencies of stromal fibroblasts during transplantation on porous scaffolds proved to be more optimal than when they were transplanted in bone casings and on a collagen substrate. It is possible that the use of more advanced methods of cultivation and reverse transplantation of clones can improve the conditions for the clones to realize their differentiating potencies and change these relationships. One way or another, but the main significance of the studies is that some clones of stromal cells are able to form bone tissue and simultaneously provide a stromal hematopoietic microenvironment for three growths of bone marrow hematopoiesis: erythroid, myeloid and megakaryocytic, while creating fairly large bridgeheads and some bone mass.

Further, the authors solved the problem of the ability for these types of cell differentiation of individual clonal stromal progenitor cells in conditions of a closed system of diffusion chambers. In addition, it was necessary to find out whether individual clones possess polypotency, or for the manifestation of a differentiation potential, it is necessary to co-operative interaction of several clones with a fixed feature of cytodifferentiation, the different ratio of which determines the preferential formation of bone, reticular or cartilaginous tissue. Combining two methodological approaches - obtaining monoclonal strains of bone marrow stromal progenitor cells and transplanting them into diffusion chambers, R. Chaylakhyan and co-authors (2001) obtained results that made it possible to approach the understanding of the structural organization of the marrow stroma. Transplantation of monoclonal strains of stromal progenitor cells in O-type chambers resulted in the formation of both bone and cartilaginous tissue, which indicates the ability of descendants of a single stromal colony-forming cell to simultaneously form bone and cartilaginous tissue. The assumption that bone and cartilaginous tissue originates from the common stromal progenitor cell has been repeatedly expressed repeatedly. However, this hypothesis did not have a correct experimental confirmation. The formation of bone and cartilage in diffusion chambers was a necessary proof of the existence amongst stem cells of the bone marrow stroma of a common precursor cell for these two types of tissue.

Then, 29 clonal strains of the second and third passages obtained from the primary cultures of the rabbit's marrow were placed in diffusion chambers and implanted intraperitoneally with homologous animals. Studies have shown that 45% of bone marrow monoclonal strains have osteogenic potencies. Exceptionally, the reticular tissue contained 9 chambers, but together with bone and cartilaginous tissue it was present in 13 more chambers, which accounted for 76% of all strains. In chambers of type O, where differentiation was possible for both bone and cartilaginous tissue, 16 strains were studied. In four chambers (25%), both bone and cartilaginous tissue was formed. It should be noted again that in the studies of R. Chailakhian and co-authors (2001), individual progenitor cells underwent 31 to 34 doublings in the cell strain, and their progeny was 0.9-2.0 x 10 ⁹ cells. The number of mitoses to which the precursor cells of the polyclonal strains were exposed was practically the same as that of the cells of the monoclonal strains. At the same time, the rate of development of polyclonal strains, especially in the first phase of their formation, depended to a considerable extent on the number of colonies used for the initiation of strains. Diploid strains of human embryonic fibroblasts (WI-38) when re-formed at 12-15 levels of duplication also formed colonies that differ in diameter and in the content of cells in them. Large colonies containing more than 103 cells were only 5-10%. With the increase in the number of divisions, the percentage of large colonies decreased. Mono- and polyclonal strains of stromal bone marrow fibroblasts retained a diploid set of chromosomes after 20 or more doublings, and the trend of their development was comparable with the dynamics of development of diploid strains of embryonic fibroblasts. Analysis of the differentiation potential of individual bone marrow stromal progenitor cells, carried out by transplanting monoclonal strains into diffusion chambers, showed that half of them are osteogenic. Large colonies accounted for 10% of their total number. Consequently, the number of osteogenic colony-forming cells corresponded to approximately 5% of their total population. In the total mass of osteogenic progenitor cells identified by the authors, there were cells capable of forming bone and cartilaginous tissue simultaneously. It was first established that for these two types of tissue in the adult body there is a common precursor cell: 25% of the tested clones were created by similar cells, and their number among the general population of progenitor cells was at least 2.5%.

Thus, heterotopic transplantation of individual clones of bone marrow fibroblasts has opened new aspects of the structural organization of the population of mesenchymal progenitor cells. Stromal progenitor cells were found capable of transferring a specific microenvironment immediately for all hematopoietic germs, the number of which among the large clones studied in different models is 5-15% (0.5-1.5% of the total number of precursor cells detected). Along with the clones that carry the full bone marrow microenvironment, there are progenitor cells that are determined only to osteogenesis, forming bone tissue in the open system that does not support the development of hemopoiesis. Their number from the total number of progenitor cells is 1.5-3%. Some of these cells can form bone tissue with a limited period of self-maintenance. Consequently, the population of stromal progenitor cells is heterogeneous in its differentiation potential. Among them there is a category of cells claiming the role of stem stromal cells that can differentiate in all three directions peculiar to bone marrow stromal tissue, thus forming bone, cartilaginous and reticular tissue. These data allow us to hope that with the help of various cell markers it will be possible to determine the contribution of each type of stromal cells to the organization of a specific microenvironment and the support of hematopoiesis in Dexterian cultures.

Features of mesenchymal stem cells

In recent years, it has been established that in stationary bone marrow cultures, multipotent mesenchymal progenitor cells are represented by a limited population of small agranular cells (RS-1 cells), characterized by low colony formation and the absence of Ki-67 antigen expression specific for proliferating cells. The antigenic parameters of the dormant RS-1 cells differ from the antigen spectrum of rapidly proliferating commited stromal progenitor cells. It was found that a high rate of proliferation of committed progenitor cells was observed only in the presence of RS-1 cells. In turn, RS-1 cells increase the growth rate under the influence of factors secreted by the most mature derivatives of multipotent mesenchymal progenitor cells. It seems that RS-1-cells are a subclass of uncommitted MSCs that are capable of recycling. In vitro, 5-fluorouracil-resistant stromal bone marrow precursors have a low RNA content and a high level of expression of the ornithine decarboxylase gene, a marker of non-proliferating cells.

Intensive reproduction of stromal progenitor cells begins after fixation on the substrate. In this case, the marker profile of weakly differentiated cells is expressed: SH2 (receptor TGF- (3), SH3 (domain of signal protein), collagen I and III types, fibronectin, adhesion receptors VCAM-1 (CD106) and ICAM (CD54), cadherin-11 , CD44, CD71 (transferrin receptor), CD90, CD120a and CD124, but without expression of characteristic markers of hematopoietic stem cells (CD34, CD14, CD45). Clonal growth allows multiple passages of mesenchymal stem cells to form numerous genetically homogeneous stromal progenitor pluripotent cells. In the culture of sufficient density, after stopping proliferation, stromal progenitor cells, unlike hematopoietic fibroblasts, differentiate into adipocytes, myocytes, cartilage cells and bone tissue. A combination of three regulatory differentiation signals , including 1-methyl-isobutylxanthine (inducer of intracellular cAMP formation), dexamethasone (inhibitor of phospholipases A and C), and indomethacin (a cyclooxygenase inhibitor that reduces and activity of thromboxane synthase), is converted into adipocytes up to 95% of the progenitor mesenchymal cells. The formation of adipocytes from immature stromal elements is confirmed by the expression of the lipoprotein lipase gene, histochemical detection of apolipoproteins and peroxisomal receptors. The cells of the same clone under the influence of TGF-b in a serum-free medium create a homogeneous chondrocyte population. The multilayer cell culture of this cartilaginous tissue is characterized by a developed intercellular matrix consisting of proteoglycan and type II collagen. In a nutrient medium with 10% fetal serum, the effect of a complex of differentiation signals consisting of b-glycerophosphate (an inorganic phosphate donor), ascorbic acid and dexamethasone, in the same culture of stromal progenitor progenitor cells leads to the formation of cellular aggregates. In such cells, there is a progressive increase in the activity of alkaline phosphatase and osteopontin, which indicates the formation of bone tissue, the mineralization of cells is confirmed by a progressive increase in intracellular calcium content.

According to some data, the ability of mesenchymal stem cells to unrestricted division and reproduction of various cell types of the mesenchymal line of differentiation is combined with a high degree of plasticity. When inserted into the ventricles or white matter of the brain, the mesenchymal stem cells migrate to the parenchyma of the neural tissue and differentiate into derivatives of the glial or neuronal cell line. In addition, there is evidence of transdifferentiation of MSCs into stem hemopoietic cells both in vitro and in vivo. With more in-depth analysis, the exceptional plasticity of MSC is determined in individual studies, which is manifested in their ability to differentiate into astrocytes, oligodendrocytes, neurons, cardiomyocytes, smooth muscle cells and skeletal muscle cells. In a number of studies on the transdifferentiation potential of MSC in vitro and in vivo, it has been established that multipotent mesenchymal precursor cells of bone marrow origin terminally differentiate into the cellular lines forming bone, cartilage, muscle, nerve and adipose tissue, as well as the tendon and stroma supporting hematopoiesis .

However, in other studies, no evidence of restriction of pluripotency of the genome of mesenchymal stem cells and progenitor populations of stromal cells was found, although more than 200 clones of MSC isolated from one primary culture were examined to test for possible pluripotency of stromal cells. The vast majority of clones in vitro retained the ability to differentiate in the osteogenic, chondrogenic and adipogenic directions. With the exclusion of the probability of migration of recipient cells by transplantation of mesenchymal stem cells under the capsule of the kidney or in diffusion chambers, it turned out that stromal precursor cells in situ retain a heterogeneous phenotype, indicating either the absence of restriction factors in the transplantation zone or the absence of pluripotency of MSCs as such. At the same time, the existence of a rare type of somatic pluripotent stem cells, which are common precursors of all adult stem cells, is allowed.

The multi- but not the pluripotency of true mesenchymal stem cells, which constitute a very small fraction of bone marrow cells and capable of proliferating in vitro under certain conditions without differentiation, is indicated by their induced commision into bone, cartilaginous, fat, muscle tissue cells , as well as in the tenocytes and stromal elements supporting hematopoiesis. As a rule, prolonged exposure in a culture medium with fetal calf serum provokes the output of MSCs into the commited stromal progenitor cells, the offspring of which undergo spontaneous final differentiation. In vitro, it is possible to achieve directed formation of osteoblasts by adding dexamethasone, ß-glycerophosphate and ascorbic acid to the conditioning medium, whereas the combination of dexamethasone and insulin differentiation signals induces the formation of adipocytes.

It was established that, before reaching the stage of terminal differentiation of MSC of the bone marrow, when creating certain cultivation conditions, initially differentiate into fibroblast-like mesenchymal stem cells. Derivatives of these cells in vivo are involved in the formation of bones, cartilage, tendons, fat and muscle tissue, as well as stroma supporting hematopoiesis. Many authors understand the concept of "multipotent mesenchymal progenitor cells" as the actual MSC, as well as the committed stromal progenitors of bone marrow and mesenchymal tissues. Clonal analysis of multipotent mesenchymal precursor cells of bone marrow origin showed that slightly more than one third of all clones differentiate into osteochondrial and adipocytes, whereas the remaining clone cells have only an osteogenic potential and form only chondro- and osteocytes. This clone of multipotent mesenchymal progenitor cells, like BMC-9, under different microenvironment conditions, is differentiated into cells with the phenotype and functional characteristics of not only osteoblasts, chondrocytes and adipocytes but also stromal cells supporting hematopoiesis. Isolated from rat fetal bone marrow, the clone of RCJ3.1 cells differentiates into mesenchymal cells of various phenotypes. In the complex effect of ascorbic acid, b-glycerophosphate and dexamethasone, the cellular elements of this clone first form multinuclear myocytes, and then, sequentially, adipocytes, chondrocytes and islets of mineralized bone tissue. The population of granular cells from the rat fetal period corresponds to uncommitted multipotent mesenchymal progenitor cells, because it has a low proliferation rate, does not express differentiation markers, and under cultivation conditions differentiates to form chondro, osteo-and adipocytes, and also smooth muscle cells.

Thus, it should be recognized that the question of the pluripotent or multipotentiality of the genome of mesenchymal stem cells remains open, which, accordingly, affects the concept of the differentiation potential of stromal progenitor cells, which is also not definitively established.

The experimentally proven and important characteristic of mesenchymal stem cells is their ability to leave the tissue niche and circulate in the general bloodstream. To activate the genetic program of differentiation, such circulating stem cells should fall into the appropriate microenvironment. It has been shown that when the MSC is systematically introduced into the bloodstream of the recipient animals, immature cells are implanted into different organs and tissues, then differentiating into blood cells, myocytes, adipocytes, chondrocytes and fibroblasts. Consequently, in the local tissue zones, there is a signal-regulatory interaction between non-credited and committed stromal progenitor cells, and also between them and surrounding mature cells. It is assumed that the induction of differentiation is effected by paracrine regulatory factors of mesenchymal and non-mesenchymal origin (growth factors, eicosanoids, extracellular matrix molecules) that provide spatial and temporal links in the microenvironment of multipotent mesenchymal progenitor cells. Therefore, local damage to the mesenchymal tissue should lead to the formation of microenvironment zones of multipotent mesenchymal progenitor cells that are qualitatively different from the complex of regulatory signals of intact tissues in which the processes of physiological and not reparative regeneration occur. This difference is extremely important in terms of the specialization of the cell phenotype in a normal and damage-induced microenvironment.

According to the ideas, it is here that the mechanisms of the fundamental difference of two known processes - physiological regeneration and inflammatory proliferation - are laid. The first of them ends with the restoration of specialized cellular tissue composition and its function, whereas the result of the proliferation process is the formation of mature connective tissue elements and loss of function of the damaged tissue zone. Thus, for the development of optimal programs for the use of multipotent mesenchymal progenitor cells in regenerative-plastic medicine, a careful study of the characteristics of the influence of microenvironment factors on the differentiation of MSCs is necessary.

Dependence of the structure of the compartment of stem cells on cellular para- and autocrine regulators, the expression of which is modulated by external signals, no one doubts. Among the functions of regulatory factors, the most important are the control of asymmetric division of MSCs and the expression of genes determining the stages of commision and the number of cell divisions. External signals, on which the further development of MSC depends, are provided by their microenvironment. In the immature state, MSCs proliferate for a long time, while retaining the ability to differentiate in the adipocyte line, myofibroblasts, hematogenous stroma, cartilage and bone cells. It has been established that a limited population of SB34-negative stromal cell elements circulating in the blood from the total blood flow returns to the stroma of the bone marrow tissue, where it is transformed in the line of CD34-positive hematopoietic stem cells. These observations suggest that recirculation of progenitor mesenchymal cells in the bloodstream provides support for tissue balance of stromal stem cells in different organs by mobilizing a common pool of immature bone marrow stromal elements. The differentiation of MSCs into cells with multiple mesenchymal phenotypes and their involvement in the regeneration or repair of bone, cartilaginous, adipose tissue and tendons in vivo has been demonstrated with the help of models of adoptive transfer in experimental animals. According to other authors, distant migration of MSK along the vascular bed is combined with a short-distance or local displacement of multipotent mesenchymal progenitor cells within the tissue during cartilage repair, muscle regeneration and other restorative processes.

Local stem reserves of the stromal tissue base play the role of the source of cells in the processes of physiological tissue regeneration and are replenished by the distant transport of MSCs as the stromal-tissue stem resources are used up. However, in the context of the need for emergency mobilization of the reparative cell potential, for example, in polytrauma, the whole echelon of MSC takes part in the processes of reparative regeneration, and mesenchymal bone marrow precursors are recruited to the periphery through the general bloodstream.

Transplantation of mesenchymal stem cells

There are certain parallels between the processes of physiological regeneration of tissues and their formation during the period of intrauterine development. In the embryogenesis of humans and mammals, the formation of various types of specialized cells occurs from the ecto-, meso- and endodermal pool of embryonic leaves, but with the obligatory participation of the mesenchyme. The loose cellular network of embryonic mesenchymal tissue performs numerous regulatory, metabolic, skeletal and morphogenetic functions. The tabulation of provisional organs is carried out only after the condensation of the mesenchyme due to the clonogenic growth of the progenitor cells, which generate the primary morphogenetic signals of organogenesis. The stromal derivatives of the embryonic mesenchyme create a cellular framework of provisional organs and form the basis for their future energy supply by growing primary blood and lymphatic vessels. In other words, the stromal elements of the microcirculatory unit of the fetal organs appear before the formation of their structural-functional units. In addition, active migration of mesenchymal cells during organogenesis provides spatial orientation of developing organs due to the marking of their volume boundaries by restriction of homeotic Noch-Teps. On the stromal scaffold there is also an assembly of structural and functional units of parenchymatous organs, which often contain morphogenetically and functionally completely different cells. Consequently, in embryogenesis, mesenchymal functions are primary and are realized by generating regulatory signals that activate regional proliferation and differentiation of progenitorial epithelial cells. Embryonic mesenchyme cells produce growth factors such as LEG, HGF-b, CSF, for which parenchymal progenitor cells have corresponding receptors. In the mature mature tissue of the adult organism, the stromal cell network also generates signals to maintain the viability and proliferation of progenitor cells of non-mesenchymal origin. However, the spectrum of stromal regulatory signals in postnatal ontogenesis is different (SCF, HGF, IL-6, IL-1, IL-8, IL-11, IL-12, IL-14, IL-15, GM-CSF, flt-3, LIF, etc.) and is aimed at providing physiological regeneration or repair of damaged tissue zones. Moreover, the spectral characteristics of stromal regulatory factors in each kind of tissue and even within the same organ are different. In particular, hematopoiesis and lymphopoiesis with the multiplication and differentiation of hematopoietic and immunocompetent cells occurs only in certain organs within the boundaries of which a stromal microenvironment operates providing conditions for the maturation of hematopoietic and lymphoid cells. It is from the regulatory factors of the microenvironment that the ability of the hematopoietic and lymphoid cells to repopulate this organ, proliferate and mature in its microstructural niches depends.

Among the components of the extracellular matrix that produce multipotent mesenchymal progenitor cells, mention should be made of fibronectin, laminin, collagen and proteoglycans, as well as CD44 (the hyaluronan and osteopontin receptor), which are involved in the organization of intercellular interaction and the formation of extracellular matrix in the bone marrow and bone tissue . It has been proved that bone marrow multipotent mesenchymal cells-predecessors create a stromal microenvironment providing inductive and regulatory signals not only for MSC, but also hemopoietic progenitors and other non-mesenchymal stem cells of the bone marrow. It is known that the involvement of MSC in the hematopoiesis is determined by their ability to differentiate into stromal cells supporting hematopoiesis, and this instruction signal MSC is obtained directly from the hematopoietic stem cells. That is why in culture the network of stromal progenitor cells serves as a feeder base for the development of all clones of hemopoietic cells.

In a mature organism, the intensity of hemo- and lymphopoiesis is in a state of dynamic equilibrium with the "expenditure" of mature blood cells and cells of the immune system at the periphery. Since the stromal cells of the bone marrow and lymphoid organs are updated extremely rarely, significant rearrangement of the stromal structures does not occur in them. The system can be removed from the dynamic equilibrium by mechanical damage of any of the organs of hemo- or lymphopoiesis, which leads to the same type of consistent changes that affect not only and not so much the hemopoietic or lymphoid elements as the stromal structures of the injured organ. In the process of reparative regeneration, the stromal base is first formed, which is then repopulated by hematopoietic or immunocompetent cells. This long-known fact makes post-traumatic regeneration a convenient model for studying the stromal microenvironment of the hematopoietic organs. In particular, for the study of reparative bone marrow regeneration, mechanical emptying of the medullary cavity of tubular bones is used - curettage, which allows the hematopoietic tissue to be quickly and efficiently removed from the state of dynamic equilibrium. When studying the processes of reparative regeneration of the hematopoietic and stromal components of the bone marrow after mechanical depopulation of the medullary cavity of the tibia of guinea pigs, it is established that there is no direct correlation between the parameters of regeneration of hematopoietic and stromal cells (number of hematopoietic cells, concentration and number of stromal progenitor cells). In addition, it turned out that an increase in the population of stromal progenitor cells occurs at an earlier time after curettage, and the stromal fibroblasts themselves become phosphatase-positive, which is characteristic of osteogenic tissue. It is also established that the curettage of 3-5 tubular bones leads to an increase in this population of cells in the bone marrow of the unoperated bones and even in the spleen, which in guinea pigs is exclusively a lymphopoietic organ.

The morphological picture of reparative processes in the bone marrow of cortical tibiae of guinea pigs as a whole corresponds to the data described in the literature obtained in experiments with animals of other species, the dynamics of changes occurring after removal of the hematopoietic tissue is the same for all species of animals, and the difference concerns only temporal parameters . The morphological phase order of hematopoiesis recovery in the emptied medullary cavity consists in successive processes of blood clot organization, formation of coarse-fibrous bone tissue, its resorption, development of sinusoids and formation of the reticular stroma, which is subsequently repopulated by hematopoietic elements. At the same time, the number of progenitor hematopoietic cells in the process of regeneration of bone marrow tissue increases in parallel with an increase in the content of stem hemopoietic cells.

Y. Gerasimov and co-authors (2001) compared changes in the number of hematopoietic cells and the number of stromal progenitor cells in individual phases of the regeneration process. It turned out that the quantitative changes in bone marrow cells in the cured bone correspond to the dynamics of the morphological characteristics of regeneration. Decrease during the first three days of cellular content in the regenerate is attributed to the death of the hematopoietic cells due to the adverse effects of the microenvironment, which creates a proliferating reticular tissue in the preserved bone marrow in the epiphysis area, as well as the formation of osteoid tissue in the last foci and vascular damage in curettage. On the 7th-12th day, the increase in the level of nucleated cells coincides with the appearance of separate foci of myeloid hematopoiesis in the zones of proliferation of stromal elements. On the 20th day there are significant areas of regenerated bone marrow and well-developed sinuses, which is accompanied by a significant increase in the total number of cells. However, the number of hematopoietic elements in this period is 68% of the control level. This is consistent with previously published data that the number of hematopoietic cells after curettage reaches the norm only on the 35-40th day after the operation.

In the early post-traumatic period, the main cellular source for the restoration of hemopoiesis is the cellular elements preserved in the curettage. In later terms, the main source of regeneration of the bone marrow hematopoietic tissue are stem cells, repopulating the free stromal zones. As for certain categories of stromal cells (endothelial, reticular and osteogenic), the sources providing their formation during the reconstruction of the medullary cavity remain unexplained. The results of Yu.V. Gerasimov and co-authors (2001) testify that in the bone marrow preserved after curettage the concentration of cells forming fibroblast colonies is significantly higher than in the normal bone marrow. The authors believe that with curettage there is a more intensive selective leaching of the hematopoietic cells in comparison with the colony-forming stromal cells that participate in the formation of the stroma and are more strongly associated with its basic substance than the hematopoietic cells.

The dynamics of the number of cells forming the colony of fibroblasts correlates with the intensity of the processes of osteogenesis, subsequent resorption of bone trabeculae and the formation of the reticular stroma, which is populated by the hematopoietic cells. Most of the stromal progenitor cells form a coarse-fibrous bone tissue and a reticular stroma at the indicated regeneration times. At fractures of the femurs in the conditions of prolonged osteosynthesis on the 5th day in the regeneration zone, the concentration and number of cells forming the colony of fibroblasts increases, and in the period of intensive bone formation their number increases 6-fold. It is known that bone marrow cells forming fibroblast colonies possess osteogenic properties. The number of stromal progenitor cells increases before colonization of the area of the cortexed bone marrow by hematopoietic cells. This is in good agreement with the evidence that stromal cells provide the formation of a hematopoietic microenvironment. Obviously, the creation of a hematopoietic microenvironment corresponds to a certain level of regeneration of the stromal tissue, and the number of hematopoietic cells increases with the expansion of the stromal bridgehead suitable for hematopoiesis.

Of greatest interest are the authors' data that immediately after curettage the number of stromal progenitor cells in the remote parts of the skeleton increases. From the sixth hour through the twentieth day inclusive, in the contralateral tibia there is more than a twofold increase in both the concentration and number of cells forming the colony of fibroblasts. The mechanism of this phenomenon is probably connected with the fact that a massive trauma of the bone marrow leads to the formation of a large number of blood clots with the simultaneous destruction of a significant number of platelets and the release of platelet-derived growth factor (RBSK) into the blood, which is known to cause the proliferation of colonizing cells fibroblasts located in the body outside the proliferative pool. In experiments on rabbits, the local administration of MSC promotes the restoration of the cartilaginous tissue of a surgically damaged knee, which can be related to the formation of chondrocytes originating from injected MSCs. However, reparative regeneration of bone defects in laboratory rats is greatly enhanced by the use of mesenchymal stem cells encased in a ceramic framework. Therefore, it can be assumed that, if not for PBOK, then some other factor originating from the damaged stromal cells has a distant stimulating effect on the proliferation of mesenchymal progenitor cells in intact bone marrow regions and stimulates their migration to the area of the defect of the bone marrow tissue. In turn, this contradicts the literature data of past years, indicating that the stromal cells responsible for the microenvironment, unlike the hematopoietic cells, are not capable of migration and come from local sources.

Nevertheless, the results of a study by Yu. Gerasimov and co-authors (2001) indicate that the application of a mechanical trauma causes not only a sharp rearrangement of stromal tissue in the cured bone, but also significant changes in the stroma in distant intact bones, that is, there is a systemic response stromal tissue for local trauma. And when applying polytrauma - multiple curettage - this reaction is amplified and observed not only in the operated bone and the remote parts of the skeleton, but also in lymphoid organs, in particular in the spleen. The mechanism of such a systemic response of bone marrow stromal tissue and spleen to local trauma and polytrauma remains unknown. It is assumed that this process is associated with the effect of the humoral factor released by the mesenchymal stroma of the medullary cavity of the bone marrow. The possibility of producing bone marrow and spleen by the stromal cells of an organonesspecific humoral factor responsible for the proliferation of cells forming fibroblast colonies is evidenced by data on their colony-stimulating activity in monolayer bone marrow cultures.

In this regard, it should be noted that when systemic introduction of multipotent mesenchymal progenitor cells, their derivatives are repopulated not only by the bone marrow, but also by other tissues, which is used, in particular, for gene therapy. It was shown that with intravenous administration of large amounts of MSC with wild type genome to mice with a mutation of the collagen I gene, donor cells replace up to 30% of cells in the bone and cartilage tissue of recipients, and transfected mesenchymal mouse stem cells secreting human IL-3 for 9 months effectively support hematopoiesis in case of their simultaneous administration with human hematopoietic stem cells to immunodeficient mice.

[3], [4], [5], [6], [7], [8], [9], [10]

Genetic modification of mesenchymal stem cells

Among the successes of the experimental genetic modification of MSC is the transfection of the factor IX gene in human MSC with the subsequent transfer of transfectant cells to immunodeficient mice, which leads to the appearance of antihemophilic factor B in the blood for 8 weeks after transplantation. In this experiment, posttranslational modification of factor IX with γ-glutamyl carboxylase was performed in transfected cells. The transduction of MSC by a retroviral vector encoding human factor IX was less successful - the subsequent administration of these cells to a dog with hemophilia B provided a therapeutic level of factor IX, which maintains the normal intensity of coagulation hemostasis, for only 12 days.

The transplantation of mesenchymal stem cells into the brain parenchyma of animals has shown that donor immature cells are transformed both in the population of neurons and glia. The engraftment of neuronal derivatives of healthy donor mesenchymal tissue theoretically makes it possible to correct genetic abnormalities of brain metabolism in patients with Gauchers disease and other disorders of lipid metabolism, gangliosides or carbohydrates.

The experimental search for conditions of transdifferentiation of stem cells of the bone marrow stroma into the progenitor cells of the nervous and hepatic tissues continues. The attention of researchers is focused on combinations of differentiation inducers and special conditioning media. In particular, for the isolation of the primary culture of stromal cells, the bone marrow cells washed and resuspended in DMEM / F12 (1/1) culture medium with 10% fetal calf serum are sown at a density of 200,000 / cm2. After 24 hours, non-adherent cells are removed, and fibroblast-like cells attached to the plastic are cultured for one week. For the differentiation of bone marrow stromal cells into the neuroblasts, a conditioning medium obtained by three-day culturing of the primary embryonic fibroblast culture of the mouse, as well as DMEM / F12 (1/1) medium with 2% fetal calf serum and adding 20 ng / ml LNP or 10-6M retinoic acid (neuroinductors, which are used for neural differentiation of mouse and human embryonic stem cells). Differentiation of bone marrow stromal cells into hepatocyte precursor cells is induced in a conditioned medium created by three-day culturing of the primary embryonic mouse liver culture culture in DMEM / F12 (1/1) medium supplemented with 10% fetal bovine serum.

Here it should be noted again that the colony-forming cells of bone marrow stroma are heteromorphic and can be divided into two types. The first type includes fibroblast-like cells that form filopodia cells with large nuclei and one or two nucleoli. The second type is represented by small cells of a spindle-shaped shape. In the cultivation of cells of both types in the conditioned medium obtained on the feeder layer of primary mouse embryonic fibroblasts, cells similar to neuroblasts appear on the third and fourth days in culture. At this stage they often have a spindle-shaped form with one or two long processes ending with filopodia. Pyramidal or stellate cells with short dendrites are less common. Dendrites of some neuroblasts have characteristic extensions (buds of growth) and branching in their distal part, others - distinct growth cones with filopodia, with which the growth of dendrites occurs. Similar morphological signs (kidneys and cones of growth with filopodia), inherent in neuroblasts, differentiating into neurons, are described in detail in works on neurogenesis. On this basis, some authors conclude that the cells they detect in culture are neuroblasts. In particular, E. Shchegelskaya and co-authors (2002), after culturing the primary culture of stromal cells for two weeks in a conditioned environment for every 3 to 4 days, established that a part of the cells proliferate, preserving the undifferentiated state. Outwardly, such cells looked like fibroblasts and were identified in culture along with differentiating neuroblasts. Most of the cells (about 80%) were at different stages of differentiation into cells of the nervous tissue, mainly into neurons. The dendritic processes of these cells closely contacted each other, so that gradually the cells formed on the substrate sections of the nerve network in the form of long multicellular strands. Dendritic processes of neuroblasts grew much longer, some of them 8-10 times higher than the length of the body of the neuron itself. Gradually the proportion of pyramidal and stellate cells increased. Dendrites of stellate cells branched. According to the authors, the later differentiation of pyramidal and stellate cells in comparison with spindle-shaped ones corresponds to the sequence of stages of normal neurogenesis in animals. As a result, the authors conclude that stem cells of the bone marrow stroma undergo induced neurogenesis, during which in vitro from neuroblasts all three main types of neurons are formed. Predecessors of nerve cells were also detected during the cultivation of bone marrow stroma cells for 3-4 days in medium with 2% fetal serum and 20 ng / ml LIF. But in this case the stem cells were divided very slowly, the differentiation of the neuroblasts occurred only in 30% of cases and they did not form neural networks. Using as an inducer the differentiation of nerve cells of retinoic acid, the authors obtained in culture up to 25-30% of nerve cells with a predominance of glial elements - astrocytes and oligodendrocytes. Neurons accounted for only a third of all nerve cells, although they were represented by all three types: fusiform, pyramidal, and stellate cells. On the 6th day of cultivation of stromal cells in a medium with retinoic acid, nerve cells became more differentiated, and axons were found in individual pyramidal neurons, which in normal neurotogenesis appear later than the formation of dendritic processes. According to the authors, despite the low yield of nerve cells, the induction method with retinoic acid has its advantages: oligodendrocytes and astrocytes perform myelinating and feeding functions during the growth of dendrites and axons and are necessary for the normal formation of nerve tissue. Therefore, to repair its damaged sites in vivo, it is better to use a suspension of neurons enriched with glial cells.

In the second series of experiments, the authors attempted to induce differentiation of bone marrow stromal cells into liver cells. After a three-day cultivation of bone marrow stromal stem cells in the conditioned medium obtained by incubating mouse embryonic hepatocytes, large, spherical-shaped cells, often double-nucleated, with cytoplasmic inclusions of different sizes were found. These cells were at different stages of differentiation and differed in size, number of nuclei and inclusions in the cytoplasm. In most of these cells, glycogen was detected, on the basis of which the authors identified them as progenitor cells of hepatocytes. Since no neuroblast-like cells were found in the culture, it was concluded that in the conditioned environment resulting from the cultivation of embryonic hepatocytes, there are no factors for nerve cell differentiation and, conversely, there are factors inducing differentiation of bone marrow stromal cells into progenitor cells of hepatocytes . In conclusion, the authors suggest the presence of pluripotency in bone marrow stromal cells, as they differentiate in vitro into cells of the nervous or hepatic tissue, depending on the specific media and inducers used.

In some works, the differentiation of bone marrow stroma cells into cardiomyocytes, cartilage, bone and nervous tissue cells is indeed correctly shown. There is information that among the cells of the bone marrow there are populations of stem cells that can differentiate into hepatocytes. In the light of these data, the results of the above experiments on mice can still be considered as another confirmation of the presence in the bone marrow of pluripotent mesenchymal stem cells that have the ability to differentiate into cells of different tissues of an adult organism.

Transplantation of mesenchymal stem cells

In clinical transplantology, human mesenchymal stem cells can be used to support the expansion of hematopoietic stem cells, as well as their early pre-mated offspring. In particular, the introduction of autologous hematopoietic stem cells and MSC to oncological patients after high-dose chemotherapy accelerates the recovery of the number of neutrophils and platelets in the peripheral blood. Allogeneic and autologous transplants of mesenchymal stem cells are used to treat multiple myeloma, aplastic anemia, and spontaneous thrombocytopenia, diseases associated with the primary stromal defect of the hematopoietic tissue. The effectiveness of cell therapy in oncohematological pathology is in many cases higher with the simultaneous introduction of stromal and hematopoietic stem cells, which is manifested by a reduction in the postoperative hematopoiesis recovery period, a reduction in the number of deaths due to nonselective destruction of regional and circulating cancer cells, in which the progenitor cells of the patient die. Prospective application of MSC and other multipotent mesenchymal progenitor cells in clinical practice is due to the relative ease of obtaining them from bone marrow aspirates, expansion in culture and transfection of therapeutic genes. In this case, local implantation of multipotent mesenchymal progenitor cells can be used to replenish local tissue defects, and in the case of systemic dysfunctions of mesenchymal tissue, their introduction into the total blood flow is not excluded.

More cautious in their reasoning are the authors of works in which the prospects of using MSC for local, systemic transplantation and gene therapy are analyzed from the point of view of the biology of stromal cells. Postnatal bone marrow is traditionally regarded as an organ consisting of two main systems of clearly expressed cell lines - the actual hematopoietic tissue and associated supporting stroma. Therefore, mesenchymal stem cells of the bone marrow were initially regarded solely as a source of stromal base for the production of regulatory factors in the hemopoietic microenvironment. Then the attention of the researchers switched to studying the role of MSC as a stem source of skeletal tissues. The latest data indicate an unexpected potential of differentiation of bone marrow stromal cells with the formation of a neural or muscle tissue. In other words, mesenchymal stem cells exhibit transgermal plasticity - the ability to differentiate into cellular types that are phenotypically unrelated to the cells of the original tissue. At the same time, some aspects of the biology of bone marrow stromal cells remain unclear and unresolved both in general biological terms and in individual details, including the identification, nature, origin, development and function of bone marrow stromal cells in vivo, as well as the permissible ex vivo differentiation potential and possibilities therapeutic use in vivo. The obtained data on the potential capabilities of MSCs, as well as the results of research on the regenerative potential of other stem cells, are in sharp contradiction with the dogmas established in biology.

When cultured under low density conditions, bone marrow stem stromal cells form distinct colonies, each of which is a derivative of a single precursor cell. The percentage of stromal progenitor cells in nucleated bone marrow cells, determined by the ability to form colonies, depends significantly both on the conditions of cultivation and on the species belonging to the MSC. For example, in rodents, the presence of bone marrow and serum in the culture of irradiated feeder cells is absolutely necessary for the production of the maximum number of stromal progenitor cells, whereas in man the colony-forming efficiency of mesenchymal stem cells does not depend on either the feeder or the nutrient medium. The number of known mitogenic factors stimulating the proliferation of stromal progenitor cells is limited. These include PDGF, EGF, FGF, TGF-b, and also IGF1. Under optimal culture conditions, the polyclonal lines of MSC survive in vitro more than 50 cell divisions, which makes it possible to obtain billions of bone marrow stromal cells from 1 ml of its aspirate.

However, the population of bone marrow stromal cells is heterogeneous, which manifests itself as a variability in the size of colonies, different rates of their formation, and a variety of cellular morphology that encompasses a range from fibroblast-like fusiform to large flat cells. With the development of such crops after 20 days, phenotypic heterogeneity is also noted. Some colonies are highly expressed by alkaline phosphatase, others do not express it at all, and the third type of colonies are phosphatase-positive in the central region and phosphatase-negative at the periphery. Separate colonies form nodules of bone tissue (the beginning of matrix mineralization is marked when stained with alizarin red or on calcium by Van-Koss). In other colonies, fat accumulation takes place, identified by G-staining with oil red. Less often the colonies of the mesenchymal stem cells form cartilages that are colored with alcyan blue).

After ectopic transplantation to experimental animals, the MGK polyclonal lines form an ectopic bone with a reticular stroma associated with myelopoiesis and adipocytes, and, more rarely, with cartilaginous tissue. When transplanting monoclonal stromal bone marrow lines, chimerism is observed in some cases in which the de novo bone formed consists of bone cells, contains stroma and adipocytes of donor origin, while the cells of the hematopoietic line and the vascular system are derived from the recipient.

The results of these studies confirm the stem nature of the stromal bone marrow precursor, from which the clonal line was obtained. They also simultaneously show that not all cloning in culture cells are indeed multipotent stem cells. Some researchers believe and we share their opinion that the most reliable information on the real potential of differentiation of individual clones can be obtained only in vivo after transplantation, and not by determining the phenotype of their derivatives in vitro. Expression in culture of phenotypic markers of osteo-, chondro- or adipogenesis (determined by mRNA or by histochemical technique) and even the production of a mineralized matrix does not reflect the degree of pluripotency of an individual clone in vivo. Therefore, the identification of stem cells in the stromal cell group is possible only posteriori, under the appropriate conditions of a biological transplantation test. In particular, chondrogenesis is very rarely observed in open transplantation systems, whereas cartilage formation is not uncommon in closed systems, such as diffusion chambers or in micromass cultures of stromal cells in vitro, where a local low oxygen tension is achieved that facilitates the formation of cartilaginous tissue. Therefore, even the technique of transplantation, as well as nonspecific in vitro cultivation conditions, significantly affect the range of MSC differentiation.

Experimental transplantation with observance of given experimental conditions is the golden standard for determining the potential for differentiation of bone marrow stromal cells and the key element of their proper identification. Historically, bone marrow stromal bone marrow transplantation studies are associated with a common bone marrow transplantation problem. It was established that hemopoietic microenvironment is created by transplantation of bone marrow stromal cells and provides ectopic development of hemopoietic tissue in the transplantation zone. The origin of the microenvironment from the donor, and hematopoietic tissue - from the host allows us to treat the ectopic bone as a true "inverted" bone marrow transplantation. Local transplantation of bone marrow stromal cells promotes effective correction of bone defects, more pronounced than in 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 the most thorough work and analysis 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 established, the structure and composition of the ideal carrier remain unused, as well as the number of cells necessary for bulk bone regeneration.

In addition to the use of ex vivo reproduced bone marrow stromal cells for the purpose of tissue regeneration of mesenchymal origin, the unorthodox plasticity of MSC opens up the potential for their use in regenerating neural cells or in the delivery of gene products to the central nervous system. In principle, this simplifies cellular therapy in the defeat of the nervous system, since there is no need to obtain autologous neural stem cells from humans. It is reported about the possibilities of using bone marrow cells for the generation of cardiomyocytes and myogenic precursor cells as a truly stromal and extrinsic origin.

Experiments are under way on systemic transplantation of bone marrow stromal cells for the treatment of common skeletal diseases. There is no doubt that bone marrow stromal cells represent a population responsible for genetic disorders in diseases of the skeleton, which is well illustrated by the vector transfer of these genetic information through these cells, which leads to the formation of pathological bone tissue in experimental animals. However, the ability of stromal cells to implant, grow, multiply and differentiate in the bones of the skeleton after introduction into the general blood stream has not yet been proven.

This is partly due to the fact that in the standard procedure of bone marrow transplantation the stroma is not transplanted together with the hematopoietic tissue, therefore, strict criteria for evaluating the successful engraftment of systemically administered stromal cells have yet to be developed. It should be remembered that the presence of marker genes in tissue extracts or the isolation in the culture of cells of donor origin indicates not about the engraftment of cells, but only their survival. Even intra-arterial injection of bone marrow stromal cells into the mouse's limb can lead to a virtually zero result of engraftment, despite the fact that cells of donor origin are found in large numbers within the microvascular network of the bone marrow. Unfortunately, such cells are usually described as "engrafted" only on the basis of the results of the determination of marker donor genes under ex vivo culture conditions. In addition, it is necessary to provide convincing evidence of long-term integration in the tissues of differentiated and functionally active cells of donor origin. In many published works, where it is reported about the engraftment of bone marrow stromal cells in the skeleton, the lack of clear data of this kind is striking. Nevertheless, it should be noted that in some correct experiments on animals, however, a limited but real engraftment of stromal progenitor cells after their systemic administration has been established.

These data are consistent with the results of the study of the possibility of delivering myogenic bone marrow precursor cells to the muscles through the vascular system. However, it should not be forgotten that both skeletal and muscular tissues are formed during development and growth on the basis of extravascular cell movements that use migration processes that do not involve circulation in the blood. If there really exists an independent circulatory pathway for the delivery of precursor cells to solid phase tissues, is it possible to allow the existence of physiologically circulating mesenchymal progenitor cells? What is the origin of these cells in both the developing and postnatal organism, and how do they penetrate the vascular wall? The solution of these issues is absolutely necessary and requires the most thorough preclinical analysis. Even after the answers to these questions are found, the problematic kinetic aspects associated with skeletal growth and remodeling of connective tissue remain unsolved. At the same time, treatment of osteogenesis disorders by replacing the entire population of mutated skeletal progenitor cells with healthy stromal cells appears to be a real clinical perspective. In this case, local zones of fracture or deformation due to pathological osteogenesis, as well as destructive changes in bone tissue, can be corrected by cultured in vitro stromal stem cells. Therefore, the direction of future research should be focused on the problems of transformation or genetic correction of autologous mutated osteogenic progenitor cells ex vivo.

The genetic engineering of cells, short-term or permanent, has become the basis of cellular and molecular biology, the source of many scientific discoveries concerning the role of individual proteins in cellular metabolism in vitro and in vivo. The use of molecular technologies to correct hereditary pathology and human diseases is very promising for practical medicine, since the properties of bone marrow stromal stem cells enable the development of unique transplantation schemes for the correction of genetic diseases of the skeleton. In this case, the mesenchymal progenitor cells can be easily obtained from the future recipient, they are amenable to genetic manipulation and are able to multiply in large numbers in a short period of time. The use of mesenchymal stem cells avoids the limitations and risks associated with the delivery of genetic information material directly to the patient through vtruvous vector structures. Such a strategy is applicable pi to embryonic stem cells, however autologous postnatal bone marrow stromal cells are the more preferred material, as their administration excludes possible immunological post-transplant complications. To achieve a short-term effect, for example, in order to accelerate bone regeneration, the most optimal method is the genetic modification of mesenchymal stem cells by means of electropores, chemical fusion, lipofection, plasmids and adenoviral constructs. In particular, viral transfection into bone marrow stromal BMP-2 cells was effective in accelerating the regeneration of bones in experimental polytrauma. Creation of adenoviral vector structures is preferable because of the absence of toxicity. However, the genetic modification of bone marrow stromal cells in this case is characterized by extremely low stability. In addition, normal transformed bone marrow stromal cells require the use of vector carriers of genetic information 10 times more infectious than other cell types, which significantly increases the percentage of death of transfected cells.

For the treatment of recessive diseases caused by low or zero biological activity of certain genes, a prolonged or permanent modification of mesenchymal stem cells is required, which requires the use of adeno-associated viruses, retroviruses, lentiviruses or adeno-retroviral chimeras. Transport sites of these viruses are capable of carrying large DNA transfects (up to 8 kb). In the scientific literature, information has already appeared on the exogenous biological activity of bone marrow stromal cells transfected with retroviral constructs coding for the synthesis of regulatory and marker molecules - IL-3, CD2, factor VIII, and enzymes involved in the synthesis of L-DOPA. But in these works the authors point out a number of limitations that must be overcome before the practical application of this technology begins. The first problem is to optimize the MCK ex vivo modification process. It is known that a prolonged (3-4 weeks) proliferation of bone marrow stromal cells in vitro reduces their transfection. At the same time, several cycles of transfusion are required to achieve a high level of genetic modification of MSCs. The second problem is related to the duration of expression of the therapeutic gene, which does not yet exceed four months. A natural decrease in effective gene expression is due to the inactivation of promoters and the death of modified cells. With the general promise of transferring genetic information through mesenchymal stem cells, the results of preliminary studies indicate the need for further optimization of ex vivo transfection methods, the selection of an appropriate promoter regulating biological activity in the desired direction and enhancing the ability of modified bone marrow stromal cells to self-sustain in vivo after transplantation. It should be noted that the use of retroviral designs to modify bone marrow stromal cells in the right direction does not always require their mandatory engraftment. Transfected mesenchymal stem cells can perform a corrective function against a background of stable residency and without mandatory active physical incorporation and functioning in connective tissue. In this case, they should be considered as a biological mini-pump, producing in vivo factor, the deficit of which determines the manifestation of genetic pathology.

The use of transformed bone marrow stromal cells to treat a dominant genetic pathology that is characterized by the expression of a gene with pathological or abnormal biological activity is much more problematic, since in this case it is necessary to block the transmission or implementation of distorted genetic information. One of the methods of genetic engineering is the homologous recombination of embryonic stem cells in order to create transgenic animals. However, an extremely low degree of homologous recombination combined with problems of identification, separation and expansion of such recombinants is unlikely to promote the wide application of this method in the near future, even if new technological methods are developed. The second approach in the gene therapy of the dominant pathology is based on automatic correction of damaged DNA, since genetic mutations can be corrected by introducing exogenous DNA with the desired sequence (short DNA oligonucleotides or chimeric RNA / DNA oligonucleotides) that binds to homologs in the damaged genome. The third option involves blocking the transmission of pathological information, which is achieved through the use of specifically designed oligonucleotides that bind to a specific gene to form a ternary spiral structure that excludes the possibility of transcription.

Despite the fact that the correction of genetic disease at the genome level remains the most optimal and preferred therapeutic method, mRNA is also a promising vector (perhaps even more accessible) for blocking the dominant negative gene. For the purpose of inhibiting translation and / or increasing degradation of mRNA, protein molecules with antisense oligonucleotide or complete sequences that block the binding of mRNA to the biosynthetic apparatus of the cell have long been used. In addition, double-stranded RNA induces rapid degradation of mRNA, the mechanism of which remains unclear. However, it is unlikely that the mere elimination of mRNA transcribed from a mutant allele with short or single mutations will facilitate the expression of the normal allele mRNA. An alternative is the use of hammerhead ribosins and hairpin, which are able to bind to highly specific regions of mRNA, followed by induction of their cleavage and inactivation during translation. Currently, the possibility of using this method in the treatment of pathological osteogenesis is being studied. Regardless of what exactly is the target - genomic or cytoplasmic elements, the success of new gene therapy technologies will be determined by the effectiveness of the inclusion of reagents in bone marrow ex vivo stromal cells, the optimal choice of a specific vector and the stable ability of mesenchymal stem cells to express the necessary factors in vivo.

Thus, the discovery of mesenchymal stem cells with their unexpected properties creates a new conceptual scheme for the development of cell lines. However, further interdisciplinary research is needed to understand the biological role of stromal stem cells, their nature, their ability to transdifferentiate or dedifferentiate, their physiological significance in the process of embryonic development, postnatal growth, maturation and aging, and in human diseases.