Search

Posttraumatic spinal cord cysts are difficult to treat with medication and surgery. Gene- cell therapy is a promising area of treatment for such patients. However, optimal gene-cell construct for this therapy has not been developed. We investigated the therapeutic efficiency of human olfactory ensheathing cells (OECs) transduced by adenoviral vector encoding the mature form of brain - derived neurotrophic factor (mBDNF) in spinal cord cysts. The adenoviral vectors Ad5/35-CAG-mBDNF and Ad5/35-CAG-Fluc were constructed. Spinal cysts were modeled in female Wistar rats. We selected animals at the early and the intermediate stages of recovery with scores to 13 according to the Basso, Beattie and Bresnahan (BBB) scale. The efficiency of therapy was evaluated by BBB tests. No cytotoxicity was detected using the Resazurin/AlamarBlue assay for both vectors at multiplicity of infection (MOIs) of 1, 5, and 25. There was an increase in the proliferation of cells treated with Ad5/35-CAG-mBDNF at MOIs of 5 and 25. The hind limb mobility after the transplantation of Ad5/35-CAG-mBDNF and Ad5/35-CAG-Fluc-transduced human OECs and non-transduced OECs had approximately the same tendency to improve. Cyst reduction was observed with the transplantation of all the samples. Although Ad5/35-CAG-mBDNF -transduced OECs had high BDNF expression levels in vitro, these cells lacked positive effect in vivo because they did not exhibit significant effect concerning functional test when comparing the groups that received the same numbers of OECs. The therapeutic efficiency of transduced OECs appears to be due to the cell component. The autological and tissue-specific human OECs are promising for the personalized cell therapy. It is extremely important to test new gene-cell constructs based on these cells for further clinical use.

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.

Enriched cultures of olfactory ensheathing cells and neural stem/progenitor cells were obtained according to our developed protocols from the olfactory mucosa of rat and human. It has been shown that only transplantation of human and rat olfactory ensheathing cells leads to a significant decrease in the size of cysts, as well as their complete disappearance in some animals.

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 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.

The experimental post-traumatic spinal cord cysts were modeled. It has been shown that NT-3 enhances the effectiveness of the use of human olfactory ensheathing cells in the treatment of experimental spinal cord cysts.

The pathological processes developing after spinal cord injuries often lead to formation of cysts. Existing surgical and medical methods are insufficient for treatment of post-traumatic spinal cord cysts. One of the emerging tools is cell therapy. Olfactory ensheathing cells (OECs) are perspective cells for cell therapy. In this study, we demonstrated that human OEC transplantation is effective in experimental spinal cysts. For our experiments, we selected animals only at the intermediate stage of recovery with scores from 8 to 13 according to the Basso, Beattie, and Bresnahan (BBB) scale. Cells were transplanted in different quantities (0.75 and 1.5 million) into the fully formed cysts and in the areas of injury without cysts. Improvement of limb mobility after human OEC transplantation into post-traumatic cysts was shown. In the group of rats with cysts, time-dependent increase in the BBB score was observed in subgroups treated with 0.75 and 1.5 million OECs with no statistically significant time-dependent dynamics of BBB values in the control group. When all three subgroups (control and two OEC doses) were compared, the Kruskal-Wallis test showed the presence of differences between subgroups after 1, 3, and 4 weeks of treatment with evidence of divergence increase. There was no statistically significant difference between the two doses of OEC treatment. The human OECs in the experiments without cysts were not effective. It was also shown that PKH26-labeled human OECs survive throughout the experiment and migrate to nearby areas of the cyst. Therefore, it was found that it is effective to transplant human OECs into fully formed cysts. In the future, autologous OECs can be used to personalize the treatment of patients with spinal cysts.

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 have shown that Y-27632, an inhibitor of Rho-associated kinase (ROCK), delays sarcomere assembly in cultured rat neonatal cardiomyocytes pretreated with angiotensin II. Y-27632 affects the beat rate of cardiomyocytes, but this effect is only observed at high cell density and, therefore, seems to be related to impairment of cell-cell interactions. Consistent with this suggestion, ROCK2 is localized in myofibrillar Z-disks of human myocardium as well as in intercalated disks. We propose that ROCK participates in maturation of the myocardial contractile system through phosphorylation of its molecular targets in Z-disks and in intercalated disks.

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility involving actin and myosin II. MLCK is widely present in vertebrate tissues including the myocardium. However, the role of MLCK in cardiomyocyte function is not known. Previous attempts to gain insight into possible roles and identify potential molecular partners were disappointing and equivocal due to cross reactivity of early antibodies with striated muscle MLCK, which has a different genetic locus and a divergent amino acid sequence from the above mentioned enzyme. Using an immunofluorescence approach and a panel of antibodies directed against MLCK, cytoskeletal, and sarcomeric proteins, we localized MLCK to myofibril precursors and Z-lines of sarcomeres in embryonic and adult cardiomyocytes. The same structures contained nonmuscle myosin IIB implicating this protein as a possible target of MLCK. Our results suggest a role for MLCK in cardiomyocyte differentiation and contraction through regulation of nonmuscle myosin IIB.