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In this work, an optimal protocol was developed for obtaining adhesion culture of neural stem/progenitor cells (NSPC) of rat olfactory mucosa. During the development of the protocol, the conditions for cell culturing on adhesion substrates fibronectin and laminin in DMEM/F-12 and neurobasal media with the same culture additives were compared. Cell proliferation was maximum during culturing on both substrates in the neurobasal medium. Using the immunofluorescence method, we found that culturing on fibronectin in the neurobasal medium ensured maximum (52.22%) content of nestin-positive cells in comparison with other culturing conditions. The highest percentage of βIII-tubulin-positive cells was detected in cultures growing on fibronectin in the neurobasal medium and in DMEM/F-12 (79.11 and 83.52%, respectively). Culturing in adhesion cultures in the neurobasal medium on fibronectin allowed obtaining cultures enriched with NSPC and neurons differentiating from them in a quantity sufficient for further transplantation. The developed protocol can be recommended for obtaining NPSC from human olfactory mucosa for the treatment of spinal cord injuries.

In experiments on rats, co-transplantation of olfactory ensheathing cells of the human olfactory mucosa and neural stem/progenitor cells from the same source into post-traumatic cysts of the spinal cord led to improvement of the motor activity of the hind limbs and reduced the size of the cysts in some animals by 4-12%. The transplantation of a combination of the olfactory mucosa cells is effective and can be used in preclinical trials for the treatment of spinal cord injuries.

We studied the efficiency of transplantation of neural stem/progenitor cells from human olfactory mucosa in chronic spinal cord injury. Neural stem/progenitor cells were obtained by a protocol modified by us and transplanted to rats with spinal post-traumatic cysts. It was shown that transplantation of neural stem/progenitor cells from human olfactory lining improved motor activity of hind limbs in the recipient rat with spinal post-traumatic cysts (according to BBB scale).

We developed a viral vector Ad5/35-CAG-mBDNF expressing the mature form of BDNF (mBDNF). On the basis of olfactory ensheathing cells transduced with this adenovector, a new gene-cell construct was obtained. In experiments in vitro, high viability of the transduced olfactory ensheathing cells and enhanced secretion of BDNF by these cells were observed. It is possible that a new gene-cell construct will significantly increase the regenerative effects of transplanted olfactory ensheathing cells.

A gene-cell construct based on rat olfactory mucosa ensheathing cells transduced with an adenoviral vector encoding a mature form of brain neurotrophic factor (mBDNF) was transplanted into post-traumatic cysts of rat spinal cord. Transplantation of the gene-cell construct improved motor activity of the hind limbs and reduced the size of cysts in some animals. However, comparison of the effects of transduced and non-transduced ensheathing cells revealed no significant differences. In parallel in vitro experiments, a decrease in the proliferation of transduced cells compared to non-transduced cells was observed. It is likely that mBDNF reduces proliferation of transduced cells, which can affect their efficiency. The therapeutic efficacy of the new gene-cell construct is most likely provided by the cellular component.

Neurotrophin-3 enhances the effectiveness of human olfactory ensheathing cells in improving hind limb mobility in rats with post-traumatic cysts of the spinal cord. Transplantation of olfactory ensheathing cells into spinal cord cysts reduced their size; neurotrophin-3 did not modulate this effect. Combined preparation of human olfactory ensheathing cells and neurotrophin- 3 can be used in neurosurgery for the treatment of patients with spinal cord injuries.

We studied the effect of transplantation of ensheathing cells obtained from the olfactory mucosa of rats and humans on the size of posttraumatic spinal cord cysts. MRI examination showed that transplantation of these cells into experimental posttraumatic cysts of the spinal cord led to a significant decrease in cyst volume and even their complete disappearance in two animals receiving transplantation of rat or human cells. These findings attested to regenerative processes developing as a result of ensheathing cell transplantation. Further studies in this field will be aimed at elucidation of the mechanisms underlying spinal cord regeneration in the area of posttraumatic cysts after transplantation of ensheathing cells.

We evaluated the efficacy of rat olfactory ensheathing cells in the therapy of experimental cysts of the spinal cord. Improvement of the motor function of the hind limbs after transplantation of the olfactory ensheathing cells into the posttraumatic spinal cord cysts rats was found. We also determined the required number of cells for transplantation and demonstrated a neuroprotective effect of this dosage. For further clinical studies, autologous tissue-specific cell preparation of olfactory ensheathing cells has to be created. Cell therapy in combination surgical and pharmacological treatment will substantially improve the quality of life of patients with posttraumatic spinal cord cysts.

We developed an optimal protocol for preparing and culturing of olfactory ensheathing cells from human olfactory mucosa. Using this protocol, we obtained a culture enriched with human olfactory ensheathing cells. Immunofluorescence analysis by simultaneous expression of GFAP and p75NTR markers showed that the content of ensheathing cells was maximum in passage 3 and 4 cultures (94 and 89.5%, respectively). The developed protocol can be recommended for obtaining autologous preparations of human ensheathing cells for cell therapy of spinal cord injuries.

Olfactory ensheathing cells showed significant effects on the regeneration of the spinal cord in experimental models and in clinical trials. However, the use of these cells in the therapy of posttraumatic cysts of the spinal cord has not been studied. Cultures of human and rat olfactory mucosa were obtained according to the protocols developed by us. Passage 3-4 cultures are most enriched with olfactory ensheathing cells and are preferable for transplantation. We performed transplantation of 750,000 olfactory ensheathing cells into the region of modeled cysts. The therapeutic effect of human cells was more pronounced. The positive dynamics of recovery of motor activity in the hind limbs of rats can reflect regenerative processes in the spinal cord after transplantation of olfactory ensheathing cells into the region of posttraumatic cysts.

We studied survival of rat ensheathing cells after transplantation into experimental posttraumatic cysts. These cells were prepared according to our original protocol, labeled with intravital membrane dye PKH26, and transplanted into posttraumatic cysts of the spinal cord. The presence of cysts was verified by magnetic resonance imaging. Olfactory ensheathing cells were detected in the spinal cord by the immunofluorescence method. It was shown that rat olfactory ensheathing cells survived in the spinal cord over 4 weeks and their migration was observed. High survival rate and the possibility of obtaining olfactory ensheathing cells from the olfactory mucosa of patients for creation of an autologous preparation allow considering them as very promising material for the treatment of patients with posttraumatic cysts of the spinal cord.

We studied the localization of myosin-activating protein kinases in cardiomyocytes obtained from fetal human heart at 8–9 weeks gestation. It was found that at this developmental stage, smooth muscle/nonmuscle myosin light chain kinase (MLCK, 108 kDa) and its high-molecular weight isoform (MLCK, 210 kDa), skeletal MLCK and death-associated protein kinase (DAPK) are co-localized with nonmuscle myosin IIB in the premyofibrils. The data obtained suggest that cardiac nonmuscle myosin at 8–9 weeks gestation may serve as the substrate of the studied myosin-activating protein kinases that are likely to cooperatively regulate the formation of myofibrils. We revealed high-molecular weight isoform of smooth muscle/nonmuscle kinase MLCK-210 in developing human heart and determined the ratios of MLCK-108 and MLCK-210 at different gestational stages. In this case, the approximate time period of changes in these isoforms ratio was revealed (between 8–9 and 13 weeks), that can be associated with functional changes in the developing myocardium.