[Comparative transcriptome pairwise analysis of spontaneously transformed multipotent stromal cells from human adipose tissue].

Potential markers and the mechanism of the spontaneous transformation of multipotent stromal cells (MSC) with perivascular immunophenotype have been determined. A transcriptome comparative study was performed involving six paired specimens of normal and spontaneously transformed MSC with perivascular immunophenotype, obtained in the first passages following the isolation of adipose tissue of six healthy donors. According to the results obtained using the microarray Illumina HT-12 v4 with the Significance Analysis of Microarrays software, differentially expressed transcripts were revealed and a statistical analysis using Gene Ontology and Molecular Signatures Databases was conducted. The association of the spontaneous transformation of isolated MSC with perivascular immunophenotype with previously identified oncogenic cell transformation pathways (E2F, ATR/ATM, RAS, and RHOA) is suggested and further aims for more detailed study are set. Potential transformation markers, including largely unknown genes and those previously not associated with cancer genes (HSPB6, PLAC9, FEZ1, DTWD1, APH1A, and ATP5L) are described.

Tissue engineering construct on the basis of multipotent stromal adipose tissue cells and Osteomatrix for regeneration of the bone tissue.

We developed a new method of creation of tissue engineering constructs for regeneration of the bone tissue based on the principle of free distribution of cells in a fibrin clot within a scaffold. The tissue engineering construct includes multipotent stromal adipose tissue cells committed in osteogenic lineage, platelet-rich plasma, and resorbed material on the basis of xenogeneic bone collagen. The culture of bone progenitor cells was characterized by the main markers of osteoblastic differon. The material meets all requirements for materials intended for tissue engineering. An innovative high-technological tissue engineering product for clinical application is prepared.

Isolation of insulin-producing cells from different populations of multipotent stromal cells of the umbilical cord and human adipose tissue.

Stromal cells of adipose tissue and human umbilical cord were isolated by the original method from general populations of multipotent subpopulation of multipotent stromal cells exhibiting perivascular phenotype (CD146+, CD31-). Effective directed differentiation of these cells into insulin-producing cells by transient transfection of the gene Pdx1 was demonstrated. Transfection multipotent stromal cells CD146-, CD31- derived from adipose tissue and umbilical cord and isolated by the standard method, did not result in activation of insulin gene transcription. It was shown that the expression of nestin was not necessary for effective pancreatic cell differentiation.

Preparation and characterization of culture of CD146+ cells from human adipose tissue.

A method for isolation of homogenous culture of cells expressing CD146 marker from adipose tissue lipoaspirate was developed. The resultant clonogenic cultures retained high proliferative activity, immunophenotype, and morphology after numerous passages. The presence of insulin in the medium served as the selective factor for maintenance of the population phenotype. These cultures effectively differentiate into CD31(+)endothelial cells and can be used in regenerative medicine.

Angiogenesis after transplantation of auto- and allogenic cells.

Neoangiogenesis after transplantation of auto- and allogenic mononuclears and multipotent stromal cells from the bone marrow was studied on the model of inflammatory angiogenesis. Transplanted auto- and allogenic cells stimulate the formation of new blood vessels in the granulation tissue, this manifesting in an increase in the quantity and volume density of blood vessels. The most pronounced angiogenesis was observed after transplantation of allogenic mononuclears and multipotent stromal cells. It was associated with intense inflammatory infiltration, with less numerous and mature collagen fibers in the granulation tissue. Injection of allogenic cells led to stimulation and chronization of inflammation, infiltration with inflammatory and poorly differentiated cells, and more pronounced and lasting angiogenesis. However, neither auto-, nor allogenic transplanted labeled cells were detected in the walls of new blood vessels. Hence, it seems that bone marrow mononuclears and multipotent stromal cells stimulated angiogenesis mainly at the expense of production of angiogenic factors, and after transplantation of allogenic cells also by stimulating the inflammation.

Regeneration of skull bones in adult rabbits after implantation of commercial osteoinductive materials and transplantation of a tissue-engineering construct.

We performed a comparative study of reparative osteogenesis in rabbits with experimental critical defects of the parietal bones after implantation of commercial osteoinductive materials "Biomatrix", "Osteomatrix", "BioOss" in combination with platelet-rich plasma and transplantation of a tissue-engineering construct on the basis of autogenic multipotent stromal cells from the adipose tissue predifferentiated in osteogenic direction. It was found that experimental reparative osteogenesis is insufficiently stimulated by implantation materials and full-thickness trepanation holes were not completely closed. After transplantation of the studied tissue-engineering construct, the defect was filled with full-length bone regenerate (in the center of the regenerate and from the maternal bone) in contrast to control and reference groups, where the bone tissue was formed only on the side of the maternal bone. On day 120 after transplantation of the tissue-engineering construct, the percent of newly-formed bone tissue in the regenerate was 24% (the total percent of bone tissue in the regenerate was 39%), which attested to active incomplete regenerative process in contrast to control and reference groups. Thus, the study demonstrated effective regeneration of the critical defects of the parietal bones in rabbits 120 days after transplantation of the tissue-engineering construct in contrast to commercial osteoplastic materials for directed bone regeneration.

Study of the safety of transplantation of cultured autologous human chondroblasts.

Study of tumorigenic activity of cultured human chondroblasts intended for autologous transplantations showed that subcutaneous injection of human chondroblast suspension in a dose of 10(7)cells/0.2 ml did not cause the development of tumors in C.B-17 SCID immunodeficient mice over 12 weeks. Subcutaneous formations of compact cartilaginous consistency persisted at the site of chondroblast transplantation in all animals; these formations gradually shrank. Subcutaneous injection of tumor cell suspension (A204 human embryonic sarcoma) to C.B-17 SCID mice under the same conditions caused 100% development of tumors (poorly differentiated sarcomas) by day 14.

Effect of dexamethasone on differentiation of multipotent stromal cells from human adipose tissue.

Effect of dexamethasone on differentiation of multipotent stromal cells from human adipose tissue was evaluated. Addition of dexamethasone to growth medium resulted in active adipogenesis. Addition of dexamethasone to the osteogenic medium (containing active vitamin D3 form as the main inductor) led to simultaneous realization of the adipogenic and osteogenic potencies of multipotent stromal cells of the adipose tissue. Hence, the quality of the transplant on the basis of predifferentiated multipotent stromal cells from the adipose tissue for bone tissue repair can be deteriorated by dexamethasone directing some cells to adipogenic development.

Application of multipotent mesenchymal stromal cells from human adipose tissue for compensation of neurological deficiency induced by 3-nitropropionic Acid in rats.

We evaluated possible therapeutic effect of multipotent mesenchymal stromal cells from human adipose tissue differentiated to neuronal phenotype with retinoic acid on Wistar rats subjected to toxic effect of 3-nitropropionic acid. Transplantation of mesenchymal stromal cells from human adipose tissue considerably decreased neurological symptoms, normalized exploratory activity (open field test) and long-term memory (Morris test), which correlated with normalization of pathomorphological manifestations in the brain. Destructive changes in the caudate nucleus caused by treatment with 3-nitropropionic acid (reduced size of neurons, changes in their shape, and cell edema) tended to decrease under the effect of multipotent mesenchymal stromal cells: the area of neurons increased 2-fold, the cells acquired typical round shape, cell edema decreased.

Injection form of tissue-engineering construct on the basis of autogenous chondroblasts for regeneration of the cartilaginous tissue.

We present an injection form of tissue-engineering construct on the basis of autogenous human chondroblasts for regeneration of the cartilaginous tissue. A material meeting the requirements for the creation of the injection form of an ITC construct (injectable chondrocyte transplantation) was used as the matrix carrier. The developed construct on the basis of autogenous human chondroblasts and homogenized hemostatic gelatin sponge is now at the stage of complex experimental testing.

Clinical study of the efficiency of combined cell transplant on the basis of multipotent mesenchymal stromal adipose tissue cells in patients with pronounced deficit of the maxillary and mandibulary bone tissue.

The use of synthetic osteoplastic materials not always provides the required amount of the bone tissue. Transplantation of tissue-engineering constructs containing osteogenic precursor cells can be an alternative high-technology implantation method. Here we present the results of a pilot clinical study demonstrating safety of this method, accelerated healing of the operation wound, formation of young bone tissue after transplantation, and the possibility of mounting implants after 3 months in case of sufficient amount of the bone for primary fixation.

Aneuploidy of stem cells isolated from human adipose tissue.

The incidence of autosomes 6 and 8 aneuploidy in stem cell cultures derived from adipose tissue was evaluated at different stages of culturing. Monosomy was more incident than trisomy during the early passages. Distribution of cultures by the incidence of aneuploidy in different chromosomes was virtually the same. Clones with chromosome 6 monosomy were detected in two cultures during late passages.

Chromosome variability of human multipotent mesenchymal stromal cells.

We elaborated a method of preparing cytogenetic preparations of cultured multipotent mesenchymal stromal cells from the adipose tissue. It was found that karyotypic changes (monosomy, translocations) appear in some samples during culturing. Clones with changed karyotype were detected in 11-14-passage cultures from 2 of 7 individuals. The percent of aberrant cells in cultures from different individuals varied from 1.5 to 5.95 per 100 cells, which attested to karyotype instability. These data substantiate the need for cytogenetic control of cells before their transplantation into donor organism and further investigation of chromosome variability in stem cells.

[Prospects for using combined three-dimensional injection of gel transplants with autologous and allogenic cell cultures in reproductive medicine].

A combined cellular transplant for injection has been designed, by using lowly differentiated stromal fat tissue cells obtained from Wistar rats and it preserved biological and physical properties after passing through a small-diameter injection needle (an insulin syringe). A morphological study of the results of its transplantation into paraurethral tissues in 28 female rats showed that the transplant cells, autologous ones in particular, activated the proliferation of local connective tissue cell populations and retained their viability and proliferative and synthetic activities up 20 days or more. At the end of the experiment (on days 27-35), marked focal connective tissue enlargement at the site of injection of both allo- and autografts results in the narrowing of the urethral lumen. The injection implantation of combined allo- and autografts opens up new avenues for the treatment of pathological processes of the reproductive and urinary tracts.

Expression of NMDA receptors in multipotent stromal cells of human adipose tissue under conditions of retinoic acid-induced differentiation.

The system of NMDA glutamate receptors in human adipose tissue multipotent stromal cells and SH-SY5Y human neuroblastoma cells was used as a model for studies of NMDA receptor expression during neurodifferntiation. Glutamate NMDA receptors were detected in multipotent stromal cells of human adipose tissue. The expression of NRI subunits of NMDA receptors increased significantly after 6-day incubation of multipotent stromal cells of human adipose tissue with 10 microM retinoic acid. Only NR1 subunits of NMDA receptors were expressed in SH-SY5Y neuroblastoma cells. Incubation with retinoic acid did not promote the appearance of mRNA of other subunits (NR2A-D, NR3). The results indicate that expression of NMDA receptors can serve as an indicator of neuronal differentiation of cells and as a marker of the efficiency of neuronal differentiation protocol.

Myogenesis in hemopoietic tissue mesenchymal stem cell culture.

The myogenic differentiation capacity of prenatal mesenchymal stem cells from the main sites of hemopoiesis (bone marrow, thymus, liver, and spleen) was studied. Myogenesis was observed in all studied cell cultures except splenic mesenchymal stem cells. Differentiating cells from the thymus, bone marrow, and liver were positively stained for skeletal muscle markers (myogenin and MyoD). Autonomously contracting structures positively stained for cardiotroponin I and slow muscle myosin, were detected in the same cultures. Our experiments revealed differences in differentiation of mesenchymal stem cells from hemopoietic organs depending on the source of cells.

Electron probe microanalysis of potassium and sodium in clonogenic culture of human neural stem cells.

The mean potassium and sodium concentrations and distribution of potassium in clonogenic culture of human neural stem cells (neurosphere) were estimated by means of electron probe microanalysis. High sodium concentration was typical of undifferentiated cells. Potassium was irregularly distributed in the test structure. Our results confirm published data on heterogeneous morphological structure of neurospheres.

Reparative osteogenesis during transplantation of mesenchymal stem cells.

Reparative osteogenesis was studied after xenotransplantation of suspension cell graft from human mesenchymal stem cells. A model of experimental damage to rat femoral diaphysis was developed. The state of animals was satisfactory and non-depressed in the early and late postoperation period. We revealed no local pathological reactions and complications. Administration of mesenchymal stem cells into the area of bone defect accelerated and improved regeneration. Unilateral transplantation of the cell graft stimulated regeneration in the contralateral limb due to acceleration of bone tissue maturation. On day 90 after treatment the bone regenerate was completely developed in the area of defect in animals of various groups. The newly formed bone tissue was well integrated into the bone organ.

Isolation and phenotypical characterization of mesenchymal stem cells from human fetal thymus.

Stem cells from human fetal thymus ectomesenchyma capable of forming colonies during in vitro culturing were isolated and characterized. Selection of culturing conditions showed that the growth and phenotypical characteristics of these cultures depended on seeding density and presence of basic fibroblast growth factor in the medium. After nonspecific inhibition of proliferation clonogenic cultures of thymic mesenchymal stem cells differentiated into myoblasts, formed characteristic myotubes, and expressed specific myogenesis markers. Colonies of thymic mesenchymal stem cells differentiated into chondrogenic, osteogenic, and adipogenic lines under conditions described for bone marrow mesenchymal stem cells. Cytofluorometric analysis of surface epitopes of thymic mesenchymal stem cells showed that the majority of cells expressed mesenchymal markers Thy-1, CD44, and CD105. Testing for CD34, CD38, CD45, and HLA-DR were negative in all cases. The main cell population (70-95%) did not express MHCl antigens during long-term culturing.

Stimulation of reparative osteogenesis after xenotransplantation of human prenatal mesenchymal stem cells and chondroblasts.

Regeneration of the bone tissue after unilateral xenogeneic transplantation of a suspension of human mesenchymal cells and chondroblasts was studied in rats with experimental damage to both femurs. The state of animals was satisfactory and non-depressed in the early and late postoperation period. No local pathological reactions and complications were seen. Administration of mesenchymal stem cells and chondroblasts into the area of bone defect accelerated regeneration on days 10 and 30 (compared to the control group). Implantation of mesenchymal stem cells more significantly stimulated reparative osteogenesis compared to treatment with chondroblasts. This was mainly associated with increased ratio of mature lamellar bone tissue. Unilateral transplantation of the cell suspension stimulated regeneration in the contralateral limb due to accelerated maturation of the bone tissue. The bone tissue formed after transplantation of mesenchymal stem cells and chondroblasts was integrated into bone organ and underwent complete remodeling. Xenotransplantation of prenatal mesenchymal stem cells and chondroblasts without immunosuppression was not followed by the development of early and delayed complications or local reactions of graft rejection.