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In current in vitro study we have shown the impact of deuterium content in growth medium on proliferation rate of human cultured adipose-derived stem cells (ADSC). ADSCs have also demonstrated morphological changes when cultured in deuterated growth medium: the cell cultures did not reach confluence but acquired polygonal morphology with pronounced stress fibers. At high deuterium concentrations the ADSCs population doubling time increased which indicated the cell cycle retardation and decrease of cell proliferation rate. The deuterated and deuterium-depleted growth media demonstrated acute and chronic cytotoxicity, respectively. The minimal migration ability was observed in deuterated medium whereas the highest migration activity was observed in the medium with the deuterium content close to natural. The cells in deuterated growth medium demonstrated decrease in metabolic activity after three days in culture. In contrast, in deuterium-depleted medium there was an increase in ADSC metabolic activity.

Introduction. The adult neural crest-derived stem cells (NCSCs) have significant perspectives for use in regenerative medicine. The most attractive sources for adult NCSC isolation are the hair follicles (HF) and skin dermis (SD) because of easy access and minimally invasive biopsy. The aim of this study was to compare the biological properties of HF- and SD-derived NCSCs after their large-scale expansion. Methods. The conventional explant method was used to obtain HF NCSCs. For the isolation of SD NCSCs, a new combined technique consisting of preplating and subsequent culturing in 3D blood plasma-derived fibrin hydrogel was applied. The studied cells were characterized by flow cytometry, ICC, qPCR, Bio-Plex multiplex assay, and directed multilineage differentiation assays. Results. We have obtained both adult SD and HF NCSCs from each skin sample (n=5). Adult SD and HF NCSCs were positive for key neural crest markers: SOX10, P75 (CD271), NESTIN, SOX2, and CD349. SD NCSCs showed a higher growth rate during the large-scale expansion compared to HF NCSCs (p<0.01). Final population of SD NCSCs also contained more clonogenic cells (p<0.01) and SOX10+, CD271+, CD105+, CD140a+, CD146+, CD349+ cells (p<0.01). Both HF and SD NCSCs had similar gene expression profiling and produced growth factors, but some quantitative differences were detected. Adult HF and SD NCSCs were able to undergo directed differentiation into neurons, Schwann cells, adipocytes, and osteoblasts. Conclusion. The HF and SD are suitable sources for large-scale manufacturing of adult NCSCs with similar biological properties. We demonstrated that the NCSC population from SD was homogenous and displayed significantly higher growth rate than HF NCSCs. Moreover, SD NCSC isolation is cheaper, easier, and minimally time-consuming method.

The application of chitosan (Ch) as a promising biopolymer with hemostatic properties and high biocompatibility is limited due to its prolonged degradation time, which, in turn, slows the repair process. In the present research, we aimed to develop new technologies to reduce the biodegradation time of Ch-based materials for hemostatic application. This study was undertaken to assess the biocompatibility and hemostatic and tissue-regeneration performance of Ch-PEO-copolymer prepared by electrospinning technique. Chitosan electrospinning membranes (ChEsM) were made from Ch and polyethylene oxide (PEO) powders for rich high-porous material with sufficient hemostatic parameters. The structure, porosity, density, antibacterial properties, in vitro degradation and biocompatibility of ChEsM were evaluated and compared to the conventional Ch sponge (ChSp). In addition, the hemostatic and bioactive performance of both materials were examined in vivo, using the liver-bleeding model in rats. A penetrating punch biopsy of the left liver lobe was performed to simulate bleeding from a non-compressible irregular wound. Appropriately shaped ChSp or ChEsM were applied to tissue lesions. Electrospinning allows us to produce high-porous membranes with relevant ChSp degradation and swelling properties. Both materials demonstrated high biocompatibility and hemostatic effectiveness in vitro. However, the antibacterial properties of ChEsM were not as good when compared to the ChSp. In vivo studies confirmed superior ChEsM biocompatibility and sufficient hemostatic performance, with tight interplay with host cells and tissues. The in vivo model showed a higher biodegradation rate of ChEsM and advanced liver repair.

Aim: The purpose of this work was to obtain, multiply and characterize the adult neural crest-derived multipotent stem cells from human hair follicle for their further clinical use. Materials and Methods: Adult neural crest-derived multipotent stem cells were obtained from human hair follicle by explant method and were expanded at large-scale up to a clinically significant number. The resulted cell cultures were examined by flow cytometry and immunocytochemical analysis. Their clonogenic potential, ability to self-renewal and directed multilineage differentiation were also investigated. Results: Cell cultures were obtained from explants of adult human hair follicles. Resulted cells according to morphological, phenotypic and functional criteria satisfied the definition of neural crest-derived multipotent stem cells. They had the phenotype Sox2+Sox10+Nestin+CD73+CD90+CD105+CD140a+CD 140b+CD146+CD166+CD271+CD349+ CD34-CD45-CD56-HLA-DR-, showed high clonogenic potential, ability to self-renewal and directed differentiation into the main derivatives of the neural crest: neurons, Schwann cells, adipocytes and osteoblasts. Conclusion: The possibility of a large-scale expansion of adult neural crest-derived multipotent stem cells up to 40–200·106 cells from minimal number of hair follicles with retention of their phenotype and functional properties are the significant step towards their translation into the clinical practice.

In this study, we performed an adipogenic differentiation of human adipose-derived stem cells (ADSCs) in vitro with different deuterium content (natural, low and high) in the culture medium during differentiation process with parallel analysis of the gene expression, metabolic activity and cell viability/ toxicity. After ADSCs differentiation into adipocytes we have done the analysis of differentiation process efficiency and determined a type of resulting adipocytes (by morphology, gene expression, UCP1 protein detection and adipokine production analysis). We have found that high (5 × 10 5 ppm) deuterium content significantly inhibit in vitro adipogenic differentiation of human ADSCs compared to the groups with natural (150 ppm) and low (30 ppm) deuterium content. Importantly, protocol of differentiation used in our study leads to white adipocytes development in groups with natural (control) and high deuterium content, whereas deuterium-depleted differentiation medium leads to brown-like (beige) adipocytes formation. We have also remarked the direct impact of deuterium on the cellular survival and metabolic activity. Interesting, in deuterium depleted-medium, the cells had normal survival rate and high metabolic activity, whereas the inhibitory effect of deuterated medium on ADSCs differentiation at least was partly associated with deuterium cytotoxicity and inhibitory effect on metabolic activity. The inhibitory effect of deuterium on metabolic activity and the subsequent decrease in the effectiveness of adipogenic differentiation is probably associated with mitochondrial dysfunction. Thus, deuterium could be considered as an element that affects the substance chirality. These findings may be the basis for the development of new approaches in the treatment of obesity, metabolic syndrome and diabetes through the regulation of adipose-derived stem cell differentiation and adipocyte functions. In the 21st century, non-communicable diseases (NCD) like obesity, metabolic syndrome and type 2 diabetes mellitus (T2DM) became the main medical problems of the humanity 1-4. These diseases started in the Western world, but in parallel with the improving of human life standards, technological progress and the spread of the Western lifestyle also around the world. So, these diseases have become a global epidemic 5. Currently, although there remains a correlation between the level of economic development and the frequency of these diseases, they have ceased to be a medical problem in high-income countries, but also have become an urgent item for the low-income and middle-income countries 6. A characteristic feature of obesity, metabolic syndrome and T2DM is the defection of glucose and lipids metabolism, which is manifested in insulin resistance, impaired fasting glucose, dyslipidemia, high blood sugar, high serum triglycerides, imbalance of different types of lipoproteins in blood serum 7,8 .

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Aim: Based on our preliminary positive clinical results with use of cultured bone marrow-derived multipotent mesenchymal stem/stromal cells in traumatology, our aim was to develop living three-dimensional tissue-engineered bone equivalent transplantation technology for restoration of critical sized bone defects caused by combat related high energy trauma. Materials and Methods: To fabricate bone equivalent we used devitalized allogeneic bone scaffolds (blocks and chips) seeded with cultured autologous cells: bone marrow-derived multipotent mesenchymal stem/stromal cells in mix with periosteal progenitor cells and endothelial progenitor cells. Quality/identity of cell cultures was assured by donor and cell culture infection screening (immunofluorescence assay, polymerase chain reaction), flow cytometry (cell phenotype), karyotyping (GTG banding), functional assays (colony forming units analysis, multilineage differentiation assay). Bone defect treatment with bone equivalent application was fully completed in 39 combat-injured with 42 defects. New bone formation was assessed by the radiographic examination. Results: Casualties were included in a treatment program an average of 10.1 months after injury, provided the ineffectiveness of conventional surgery methods. All cell type cultures had a normal karyotype and appropriate phenotype, differentiation potential and functional properties, ~30% colony forming units frequency and hadn’t any signs of cell senescence. The fluorescein diacetate/propidium iodide combined staining and histology analysis of graft samples before transplantation showed their regular seeding with viable cells. Pathomorphological analysis of bone equivalent specimens 3–6 months post-op revealed the active remodeling processes and immature bone tissue formation. Bone defect restoration was observed 5–6 months post-op. Conclusion: The developed biotechnology of living three-dimensional tissue-engineered bone equivalent transplantation with overall effectiveness 90.4% allows restoring the bone integrity, forming new bone tissue in a site of bone defect, and significantly reducing the rehabilitation period of a patient.