Chemical Reprogramming of Somatic Cells in Neural Direction: Myth or Reality?

In in vitro experiments on cultures of human multipotent stem cells from the human bonearrow and dental pulp, we studied direct reprogramming towards neuro-glial lineage cells using a cocktail of small molecules. Reprogramming by the previously published protocol (with a cocktail containing ß-mercaptoethanol, LIF, VPA, CHIR99021, and RepSox) and by the optimized protocol (VPA, RG108, А83-01, dorsomorphin, thiazovivin, CHIR99021, forskolin, and Isx9) allows obtaining cells with immunophenotypic and genetic signs of neural stem cells. However, neither the former, nor the optimized protocols allowed preparing neural progenitors capable of adequate terminal differentiation from both bone marrow-derived mesenchymal stem cells and nestin-positive neural crest-derived mesenchymal stem cells. Real-time PCR demonstrated the expression of some neurogenesis markers, but neural stem cell-specific expression pattern was not observed. The findings lead us to a conclusion that reprogramming with small molecules without additional factors modifying gene expression does not allow reproducible production of human neural stem cell-like progenitors that can be used as the source of neural tissue for the regenerative therapy.

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.

Site-directed delivery of VEGF-targeted liposomes into intracranial C6 glioma.

The efficiency of conventional chemotherapy for aggressive tumors in the CNS remains low and new strategies for the targeted delivery of anti-tumor substances are now actively developed. Pegylated liposomes covalently conjugated with monoclonal antibodies to VEGF synthesized by us are nanoparticle characterized by narrow size distribution and high dispersion stability. Immunochemical activity of antibodies after conjugation was 70% of initial level. The anti-VEGF liposomes developed by us were highly specific for VEGF(+) tumor cells (in vitro and in vivo). Intravenous injection of VEGF-liposomes to rats with intracranial C6 glioma was followed by their specific accumulation in the malignant tissues and engulfment by glioma cells, which attested to target delivery and selective accumulation of anti-VEGF-liposomes in the brain tumor. Thus, the use of targeting molecules can significantly increase the distribution and efficiency of delivery of nanocontainers to a tumor characterized by hyperexpression of the target proteins.

Targeted delivery of cisplatin by сonnexin 43 vector nanogels to the focus of experimental glioma C6.

The aim of this study was to create a nanocontainer conjugated with monoclonal antibodies to connexin 43 (Cx43) that is actively expressed at the periphery of C6 glioma and in the astroglia roll zone. Stable vector nanogels with high (up to 35%) cisplatin load were synthesized. The antitumor effects of Cx43-modified cisplatin-loaded nanogels, free cisplatin, and nonspecific drugs were carried out on C6 glioma model. Vector nanogels reduced systemic toxicity of cisplatin, effectively inhibited tumor growth, and significantly prolonged the lifespan of animals with experimental tumors.

Tissue Engineered Neural Constructs Composed of Neural Precursor Cells, Recombinant Spidroin and PRP for Neural Tissue Regeneration.

We have designed a novel two-component matrix (SPRPix) for the encapsulation of directly reprogrammed human neural precursor cells (drNPC). The matrix is comprised of 1) a solid anisotropic complex scaffold prepared by electrospinning a mixture of recombinant analogues of the spider dragline silk proteins - spidroin 1 (rS1/9) and spidroin 2 (rS2/12) - and polycaprolactone (PCL) (rSS-PCL), and 2) a "liquid matrix" based on platelet-rich plasma (PRP). The combination of PRP and spidroin promoted drNPC proliferation with the formation of neural tissue organoids and dramatically activated neurogenesis. Differentiation of drNPCs generated large numbers of ßIII-tubulin and MAP2 positive neurons as well as some GFAP-positive astrocytes, which likely had a neuronal supporting function. Interestingly the SPRPix microfibrils appeared to provide strong guidance cues as the differentiating neurons oriented their processes parallel to them. Implantation of the SPRPix matrix containing human drNPC into the brain and spinal cord of two healthy Rhesus macaque monkeys showed good biocompatibility: no astroglial and microglial reaction was present around the implanted construct. Importantly, the human drNPCs survived for the 3 month study period and differentiated into MAP2 positive neurons. Tissue engineered constructs based on SPRPix exhibits important attributes that warrant further examination in spinal cord injury treatment.