Features of Remyelination after Transplantation of Olfactory Ensheathing Cells with Neurotrophic Factors into Spinal Cord Cysts.

This paper shows for the first time that co-transplantation of human olfactory ensheathing cells with neurotrophin-3 into spinal cord cysts is more effective for activation of remyelination than transplantation of cells with brain-derived neurotrophic factor and a combination of these two factors. The studied neurotrophic factors do not affect proliferation and migration of ensheathing cells in vitro. It can be concluded that the maximum improvement of motor function in rats receiving ensheathing cells with neurotrophin-3 is largely determined by activation of remyelination.

Modeling of Alzheimer's Disease to Study the Efficacy of Cell Therapy (Review).

We analyzed the main approaches to the modeling of Alzheimer's disease for studying the effectiveness of cell therapy. Recent advances in regenerative medicine in the field of neuroscience create prospects for the use of various cell preparations for the treatment of Alzheimer's disease. Experimental data on the use of neural stem/progenitor cells, mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells in various models of Alzheimer's disease are presented. Of particular importance is the standardization of protocols. The use of a standardized protocol in modeling of Alzheimer's disease will allow a comparative analysis of the effectiveness and safety of treatment to identify the optimal cell preparation. The data obtained on experimental animals can form the basis for further preclinical and clinical studies of cell therapy for Alzheimer's disease.

Application of Behavioral Tests for Evaluation of an Experimental Model of Alzheimer's Disease in Female Rats.

Alzheimer's disease was modeled in female Wistar rats aged 4 months by stereotaxic bilateral injection of a synthetic peptide ß-amyloid (Aß1-42) into the hippocampus. Behavioral tests (open field, Y-maze, passive avoidance, and Morris water maze) revealed significant impairment of memory and spatial navigation 8 weeks after ß-amyloid administration. At this term, the cognitive impairments typical of Alzheimer's disease are reproduced. The experimental model of Alzheimer's disease proposed by us can be used in preclinical studies of drugs for the treatment of this pathology.

Obtaining a New Gene-Cell Construct Based on Transduced Olfactory Ensheathing Cells for the Treatment of Spinal Cord Injuries.

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.

Neurotrophin-3 Enhances the Effectiveness of Cell Therapy in Chronic Spinal Cord Injuries.

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.

Application of a New Gene-Cell Construct Based on the Olfactory Mucosa Escheating Cells Transduced with an Adenoviral Vector Encoding Mature BDNF in the Therapy of Spinal Cord Cysts.

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.

The Effect of Transplantation of Olfactory Ensheathing Cells on the Size of Posttraumatic Spinal Cord Cysts.

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.

Preparation of Adhesion Culture of Neural Stem/Progenitor Cells of the Olfactory Mucosa for the Treatment of Spinal Cord Injuries.

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 ßIII-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.

Combined Preparation of Human Olfactory Ensheathing Cells in the Therapy of Post-Traumatic Cysts of the Spinal Cord.

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.

Energy Metabolism in Cellular Regenerative Processes: Focus on PPARα.

Reduced expression of the key regulator of cardiac metabolism, transcription factor PPARα, in surgical samples of the auricles from patients with coronary heart disease and heart failure was detected by real-time quantitative PCR. These changes indicate reduced activity of this factor and a shift of energy metabolism from oxidative phosphorylation to glycolysis typical of dedifferentiated cells. Electron microscopy revealed dedifferentiated cardiomyocytes with disassembled contractile apparatus and disorganized sarcomeres. In the examined specimens from patients with heart failure, severe myocardial fibrosis was revealed.

Neural Stem/Progenitor Cells of Human Olfactory Mucosa for the Treatment of Chronic 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).

Comparison of the Efficiency of Transplantation of Rat and Human Olfactory Ensheathing Cells in Posttraumatic Cysts of the Spinal Cord.

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.

Survival and Migration of Rat Olfactory Ensheathing Cells after Transplantation into Posttraumatic Cysts in the Spinal Cord.

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.

Isolation of Rat Olfactory Ensheathing Cells and Their Use in the Therapy of Posttraumatic Cysts of the Spinal Cord.

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.

Pathological Remodeling of the Myocardium in Chronic Heart Failure: Role of PGC-1α.

Pathological remodeling of the myocardium in chronic heart failure includes the development of pathological cardiac hypertrophy, reactivation of the fetal genetic program, and disorders in cardiac energy metabolism. Coactivator-1α of receptor γ activated by peroxisome proliferator (PGC-1α), a transcription coactivator of nuclear receptors and metabolism master regulator, plays an important role in cardiac metabolism regulation. Studies on the animals models of chronic heart failure have demonstrated the development of pathological cardiac hypertrophy, metabolic disorders, and reactivation of the fetal genetic program; these processes are mutually related. An important role in regulation of these processes belongs to PGC-1α; its low expression indicates low activity and down-regulation of this coactivator. Pathological cardiac hypertrophy, decrease of PGC-1α activity, and reactivation of the fetal genetic program in chronic heart failure are demonstrated.

Preparation of Human Olfactory Ensheathing Cells for the Therapy of Spinal Cord Injuries.

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 Cell Therapy in Traumatic Spinal Cord Injury].

The opportunities and the most promising ways of using cellular technology in traumatic spinal cord injury are considered in this review. A large number of experimental and clinical studies with the use of different types of cells: embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, Schwann cells, olfactory mucosa cells, and others ­ was conducted. The use of these types of cells in traumatic spinal cord injury treatment often demonstrated a positive therapeutic effect: the motor and sensory function recovery of the spinal cord. However, some types of cell preparations involve some methodological and ethical problems; some types of cell therapies are ineffective or give rise to side effects. These factors complicate the selection of optimal cell therapy for the traumatic spinal cord injury treatment. The most promising cells seem to be the cells of the olfactory mucosa. Getting the olfactory mucosa is considered to be a feasible and safe procedure for patients. The clinical application of the cells of the olfactory mucosa is effective in motor function recovery due to remyelination and axonal regeneration after spinal cord injury. These cells are tissue-specific and autologous since they can be obtained from a patient with spinal cord injury, and after cultivation, expansion, and directed differentiation they can be transplanted to the same patient. The presented benefits of olfactory mucosa cells open up the possibility for its clinical application in the cell therapy.

Myosin-activating protein kinases are possible regulators of nonmuscle myosin in developing human heart.

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.

[Rho-associated protein kinase is involved in establishing the contractile phenotype of cardiomyocytes].

It has been shown that Y-27632, an inhibitor of Rho-associated kinase, delays sarcomere assembly in rat neonatal cardiomyocytes pretreated with angiotensin II. Y-27632 affects the beat rate of cardiomyocytes; however, this effect is only observed at high cell density and, therefore, seems to be related to the formation of gap junctions between adjacent cardiomyocytes. Consistent with this suggestion, we established that Rho-associated kinase is localized in myofibrillar Z-discs of human myocardium and intercalated discs, the structures enriched in gap junctions. We propose that Rho-associated kinase participates in the maturation of the myocardial contractile system through phosphorylation of its molecular targets in Z-discs and intercalated discs.

[Penetrating peptide inhibitor of the myosin light chain kinase suppresses hyperpermeability of vascular endothelium].

Nonapeptide H-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 corresponding to a modified sequence of autoinhibitory region of myosin light chain kinase (MLCK) was synthesized from L-amino acids and from D-amino acids. Using nuclear magnetic resonance spectroscopy it has been demonstrated that D-peptide is significantly more stable in human blood plasma than its L-enantiomer. D-peptide accumulated in cultured human umbilical vein endothelial cells suppressed development of hyperpermeability in endothelial monolayer induced by thrombin addition. Following intravenous administration D-peptide decreased the extent of lung oedema in rats induced by infusion of oleic acid in bloodstream. Thus, the peptide molecules based on an autoinhibitory peptide of MLCK may serve as a prototype for development of a novel antioedematous drugs that directly affect the MLCK-dependent motile processes in vascular endothelium.