Evaluation of the Optimal Number of Implanted Mesenchymal Stem Cells for the Treatment of Post-Traumatic Syrinx and Recovery of Motor Activity after Chronic Spinal Cord Injury.

The present work aims at determining the most effective dose (number) of mesenchymal stem cells (MSC) for its transplantation in order to treat chronic spinal cord injury (SCI) in mature Sprague-Dawley rats (n=24). MSC were obtained from bone marrow of 4-6-month-old Sprague-Dawley rats. Four weeks after SCI, MSC suspension (4 µl) was injected to experimental animals into the injured area in doses of 4×105, 8×105, or 106. Using MRI, diffusion tensor imaging (DTI), diffusion tensor tractography (DTT), immunohistochemistry, histological staining, and behavioral tests, we studied the effect of transplantation of MSC in different doses on the following parameters in rats with SCI: the size of lesion cavity and post-traumatic syrinx (PTS), glial scar formation, neuronal fibers remodeling, axonal regeneration and sprouting, vascularization, expression of neuronal factors, and motor functions. MSC administration improved motor function in rats after SCI due to stimulation of regeneration and sprouting of the axons, enhanced recovery of locomotor functions, reduction of PTS and the glial scar, and stimulation of vascularization and expression of the neurotrophic factors. The effects of MSC were dose-dependent; the most effective dose was 106 cells.

Therapeutic Efficacy and Migration of Mesenchymal Stem Cells after Intracerebral Transplantation in Rats with Experimental Ischemic Stroke.

We studied therapeutic efficacy and migration characteristics of mesenchymal stem cells isolated from the human placenta after their intracerebral (stereotactic) administration to rats with the experimental ischemic stroke. It was shown that cell therapy significantly improved animal survival rate and reduced the severity of neurological deficit. New data on the migration pathways of transplanted cells in the brain were obtained.

Expression of Epithelial Cell Adhesion Molecule (EpCAM) in Tumor Spheroids of Human Colorectal Adenocarcinoma Cells.

We studied the formation of spheroids by Caco-2, SW480, and HCT116 human colorectal adenocarcinoma cell lines under low-adhesion culturing conditions. Of these three cell lines, only HCT116 formed stable tumor spheroids. Flow cytometry analysis of 19 surface markers in monolayer HCT116 culture and spheroids formed by these cells revealed considerable similarity of the expression profiles in these two culturing modes. The only exception was EpCAM molecule: its expression in spheroids was 3-fold higher than in the monolayer culture. Scanning confocal laser microscopy showed equal EpCAM distribution in the inner mass of the spheroids.

Distribution and Migration of Human Placental Mesenchymal Stromal Cells in the Brain of Healthy Rats after Stereotaxic or Intra-Arterial Transplantation.

Human placenta mesenchymal stromal cells were injected to healthy rats either stereotaxically into the striatum or intra-arterially through the internal carotid artery. Some cells injected into the brain migrated along the corpus callosum both medially and laterally or concentrated around small blood vessels. A small fraction of MSC injected intra-arterially adhered to the endothelium and stayed inside blood vessels for up to 48 hours mostly in the basin of the middle cerebral artery. Neither stereotaxic, nor intra-arterial transplantation of mesenchymal stromal cells modulated the proliferation of neural stem cells in the subventricular zone of the brain, but stereotaxic transplantation suppressed activation of their proliferation in response to traumatization with the needle.

Internalization of Vectorized Liposomes Loaded with Plasmid DNA in C6 Glioma Cells.

We studied internalization of vector nanocarriers loaded with plasmid DNA into C6 glioma cells. For improving selectivity of plasmid delivery, the liposomes were conjugated with monoclonal antibodies to VEGF and its receptor VEGFR2. Flow cytofluorometry and laser scanning confocal microscopy showed more intensive (more than 2-fold) internalization and accumulation of antibody-vectorized liposomes in C6 glioma cells in comparison with the control (liposomes conjugated with non-specific antibodies and non-vectorized liposomes). Using quantitative analysis of fluorescent signal, we showed that cationic immunoliposomes significantly more effective delivered pCop-Green-N plasmid DNA and ensured effective transfection of C6 glioma cells.

Internalization of Vectored Liposomes in a Culture of Poorly Differentiated Tumor Cells.

Internalization of liposomal nanocontainers conjugated with monoclonal antibodies to VEGF, VEGFR2 (KDR), and proteins overproduced in the tumor tissue was studied in vitro on cultures of poorly differentiated tumor cells. Comparative analysis of accumulation of vectored liposomes in the tumor cells was performed by evaluating co-localization of labeled containers and cell organelles by laser scanning confocal microscopy. We observed nearly 2 times more active penetration and accumulation of liposomes vectored with antibodies in the tumor cells in comparison with non-vectored liposomes. Selective clathrin-dependent penetration of vectored liposomes into tumor cells was demonstrated by using pharmacological agents inhibiting endocytosis.

Antitumor Activity of Rat Mesenchymal Stem Cells during Direct or Indirect Co-Culturing with C6 Glioma Cells.

The tumor-suppressive effect of rat mesenchymal stem cells against low-differentiated rat C6 glioma cells during their direct and indirect co-culturing and during culturing of C6 glioma cells in the medium conditioned by mesenchymal stem cells was studied in an in vitro experiment. The most pronounced antitumor activity of mesenchymal stem cells was observed during direct co-culturing with C6 glioma cells. The number of live C6 glioma cells during indirect co-culturing and during culturing in conditioned medium was slightly higher than during direct co-culturing, but significantly differed from the control (C6 glioma cells cultured in medium conditioned by C6 glioma cells). The cytotoxic effect of medium conditioned by mesenchymal stem cells was not related to medium depletion by glioma cells during their growth. The medium conditioned by other "non-stem" cells (rat astrocytes and fibroblasts) produced no tumor-suppressive effect. Rat mesenchymal stem cells, similar to rat C6 glioma cells express connexin 43, the main astroglial gap junction protein. During co-culturing, mesenchymal stem cells and glioma C6 cells formed functionally active gap junctions. Gap junction blockade with connexon inhibitor carbenoxolone attenuated the antitumor effect observed during direct co-culturing of C6 glioma cells and mesenchymal stem cells to the level produced by conditioned medium. Cell-cell signaling mediated by gap junctions can be a mechanism of the tumor-suppressive effect of mesenchymal stem cells against C6 glioma cells. This phenomenon can be used for the development of new methods of cell therapy for high-grade malignant gliomas.

[Hedgehog signaling in the pathogenesis of neuro-oncology diseases].

The review summarizes current knowledge on the Hedgehog signaling pathway, its role in normal embryogenesis and/or initiation and progression of neuro-oncological diseases, especially of high-grade gliomas, the most malignant neuroepithelial tumors. The main proteins forming the Hedgehog signaling pathway include Shh, PTCH1, SMO, HHIP, SUFU and GLI1 isoforms. Effects of other signaling pathways on the family of transcription factors GLI and other proteins are described. The review summarizes modern data about the impact of the Hedgehog signaling pathway on proliferation, migration activity and invasiveness, and also on tumor neoangiogenesis and tumor cell chemoresistance. The role of the Hedgehog signaling pathway in origin of cancer stem cells and epithelial-mesenchymal transition is also analyzed. Some prospects for new anticancer drugs acting on components of the Hedgehog signaling pathway inhibitors are demonstrated.

Functionally Active Gap Junctions between Connexin 43-Positive Mesenchymal Stem Cells and Glioma Cells.

The formation of functional gap junctions between mesenchymal stem cells and cells of low-grade rat glioma C6 cells was studied in in vitro experiments. Immunocytochemical analysis with antibodies to connexin 43 extracellular loop 2 showed that mesenchymal stem cells as well as C6 glioma cells express the main astroglial gap junction protein connexin 43. Analysis of migration activity showed that mesenchymal stem cells actively migrate towards C6 glioma cells. During co-culturing, mesenchymal stem cells and glioma C6 form functionally active gap junctions mediating the transport of cytoplasmic dye from glioma cells to mesenchymal stem cells in the opposite direction. Fluorometry showed that the intensity of transport of low-molecular substances through heterologous gap junctions between mesenchymal stem cells and glioma cells is similar to that through homologous gap junctions between glioma cells. This phenomenon can be used for the development of new methods of cell therapy of high-grade gliomas.