The thesis presents the results of scientific research on morphological and functional properties of adult neural crest-derived multipotent stem cells (NCSCs) from mouse whiskers and human hair follicles as well as some biotechnological aspects of their use in regenerative medicine. New markers of postnatal NCSCs from the hair follicle transcription factor Sox2 and membrane receptor CD349 (Frizzled-9) were identified in the study. For the first time, the hierarchical organization of the NCSC culture is shown, which is justified by the presence of different types of CFUs and a subpopulation of ALDHbright cells. Also, the data provided show that the uses of a modified by author the cell growth medium without chicken embryonic extract (CEE) allows obtaining a culture of postnatal mouse NCSCs, but is not able to maintain them in stem cell state during expansion. Thus further expansion of mouse postnatal NCSCs up to P3 results in loss of phenotypic and functional signs of stem cells (Nestin, high activity of ALDH, CFU type III), which may indicate a spontaneous differentiation of NCSCs. Thus, a growth medium without of the CEE needs further optimization, that was done by selecting the optimal cell density, use of low oxygen content in the gas phase and use of recombinant growth factors. So, initial low cell seeding density positively influenced the rate of mouse NCSCs proliferation, increasing the population doubling number and reducing the average cell population doubling time. The positive impact of various factors (bFGF, EGF, low plating density, low oxygen content in the gas phase) on the proliferation of adult NCSCs was detected. It was established that bFGF and EGF have a dose-dependent mitogenic effect, which is more pronounced for bFGF. It was found that low plating density and low concentration of oxygen (5 % and 10 %) are preferred for the culturing of adult NCSCs. It was shown that adult NCSCs have the ability to self-renewal and directed multilineage differentiation into neurons, glia (Schwann cells), adipocytes, osteoblasts and chondrocytes. The viability and proliferative activity of adult NCSCs during cultivation in fibrin and collagen 3D hydrogels was investigated. A method for creating tissue-engineered biomedicinal products based on adult cultured NCSCs, fibrin hydrogel and osteoplastic materials was developed. The therapeutic potential of adult NCSCs was evaluated in models of the calvarial bone critical defects, peripheral nerve injury and short-term oxygen-glucose deprivation of the hippocampus organotypic culture. It was shown that adult NCSCs stimulated reparative regeneration processes of the skull bones and peripheral nerve, and also promoted the neurons survival in the hippocampus CA1 zone. The study shows the stimulating effect of mice postnatal cultured NCSCs transplantation on reparative regeneration of the sciatic nerve. Thus, during the morphometric method used for the peripheral segment of the damaged nerve measurement, it was found that the total number of nerve fibers in mice subjected to NCSCs transplantation was higher than that of control animals (10522.8 ± 1044.0 vs 8409.5 ± 739.5, p < 0.05). The transplanted postnatal mice NCSCs survived on organotypic slices of the hippocampus during the entire observation period (14 days), but they did not differentiate into cells that are characteristic of the CNS. However, transplantation of NCSCs resulted in the survival of the oxygen/glucose deprivation-damaged neurons in the hippocampal CA1 region, which may be due to the paracrine (trophic) effect reflected by cell production of neurotrophins and growth factors with anti-apoptotic action. A method for large-scale expansion of human adult NCSCs with a preservation of their morphology and functional properties was also developed in order to create biomedicinal cell- and tissue-engineered products for regenerative medicine.
