Antos Shakhbazau

Morphological Evidence for a Transport of Ribosomes from Schwann Cells to Regenerating Axons

Recently, we showed that Schwann cells transfer ribosomes to injured axons. Here, we demonstrate that Schwann cells transfer ribosomes to regenerating axons in vivo. For this, we used lentiviral vector‐mediated expression of ribosomal protein L4 and eGFP to label ribosomes in Schwann cells. Two approaches were followed. First, we transduced Schwann cells in vivo in the distal trunk of the sciatic nerve after a nerve crush. Seven days after the crush, 12% of regenerating axons contained fluorescent ribosomes. Second, we transduced Schwann cells in vitro that were subsequently injected into an acellular nerve graft that was inserted into the sciatic nerve. Fluorescent ribosomes were detected in regenerating axons up to 8 weeks after graft insertion. Together, these data indicate that regenerating axons receive ribosomes from Schwann cells and, furthermore, that Schwann cells may support local axonal protein synthesis by transferring protein synthetic machinery and mRNAs to these axons.

Transfection efficiencies of PAMAM dendrimers correlate inversely with their hydrophobicity

Dendriplexes were characterized by ethidium bromide intercalation assay and their transfection efficiency was studied using HEK 293 cells and human mesenchymal stem cells. PAMAM G4 showed a higher transfection efficiency than PAMAM G3-G6, G4-OH, G4-25% or G4-50% dendrimers. Substitution of OH groups for the NH(2) surface groups rendered the dendrimer unable to form dendriplexes and to transfect cells. Partial (25%) substitution of CH(3) groups for the NH(2) groups markedly impaired transfection; 50% substitution decreased the ability of PAMAM G4 to transfect threefold. It was concluded that increased hydrophobicity decreased the ability of dendrimers to transfect. PAMAM G4-50% is highly hydrophobic and forms micelles in solution, which can transfect pGFP. The results of ethidium bromide intercalation assays, ANS fluorescence studies and transfection efficiencies of PAMAM dendrimers were correlated. Subsequently, we constructed a neurotrophin-encoding plasmid and studied its delivery to mesenchymal stem cells using PAMAM G4 dendrimer and Lipofectamine 2000. Lipofectamine 2000 was a more effective carrier (18.5%) than PAMAM G4 dendrimer (1.2%).

Poly(amidoamine) dendrimer complexes as a platform for gene delivery

Introduction: Gene therapy is one of the most effective ways to treat major infectious diseases, cancer and genetic disorders. It is based on several viral and non-viral systems for nucleic acid delivery. The number of clinical trials based on application of non-viral drug and gene delivery systems is rapidly increasing. Areas covered: This review discusses and summarizes recent advances in poly(amidoamine) dendrimers as effective gene carriers in vitro and in vivo, and their advantages and disadvantages relative to viral vectors and other non-viral systems (liposomes, linear polymers) are considered. Expert opinion: In this regard, dendrimers are non-immunogenic and have the highest efficiency of transfection among other non-viral systems, and none of the drawbacks characteristic for viral systems. The toxicity of dendrimers both in vitro and in vivo is an important question that has been addressed on many occasions. Several non-toxic and efficient multifunctional dendrimer-based conjugates for gene delivery, along with modifications to improve transfection efficiency while decreasing cytotoxicity, are discussed. Twelve paradigms that affected the development of dendrimer-based gene delivery are described. The conclusion is that dendrimers are promising candidates for gene delivery, but this is just the beginning and further studies are required before using them in human gene therapy.

Skin derived precursor Schwann cells improve behavioral recovery for acute and delayed nerve repair.

Previous work has shown that infusion of skin-derived precursors pre-differentiated into Schwann cells (SKP-SCs) can remyelinate injured and regenerating axons, and improve indices of axonal regeneration and electrophysiological parameters in rodents. We hypothesized that SKP-SC therapy would improve behavioral outcomes following nerve injury repair and tested this in a pre-clinical trial in 90 rats. A model of sciatic nerve injury and acellular graft repair was used to compare injected SKP-SCs to nerve-derived Schwann cells or media, and each was compared to the gold standard nerve isograft repair. In a second experiment, rats underwent right tibial nerve transection and received either acute or delayed direct nerve repair, with injections of either 1) SKP-SCs distal to the repair site, 2) carrier medium alone, or 3) dead SKP-SCs, and were followed for 4, 8 or 17weeks. For delayed repairs, both transected nerve ends were capped and repaired 11weeks later, along with injections of cells or media as above, and followed for 9 additional weeks (total of 20weeks). Rats were serially tested for skilled locomotion and a slip ratio was calculated for the horizontal ladder-rung and tapered beam tasks. Immediately after nerve injury and with chronic denervation, slip ratios were dramatically elevated. In the GRAFT repair study, the SKP-SC treated rats showed statistically significant improvement in ladder rung as compared to all other groups, and exhibited the greatest similarity to the sham controls on the tapered beam by study termination. In the ACUTE repair arm, the SKP-SC group showed marked improvement in ladder rung slip ratio as early as 5weeks after surgery, which was sustained for the duration of the experiment. Groups that received media and dead SKP-SCs improved with significantly slower progression. In the DELAYED repair arm, the SKP-SC group became significantly better than other groups 7weeks after the repair, while the media and the dead SKP-SCs showed no significant improvement in slip ratios. On histomorphometrical analysis, SKP-SC group showed significantly increased mean axon counts while the percent myelin debris was significantly lower at both 4 and 8weeks, suggesting that a less inhibitory micro-environment may have contributed to accelerated axonal regeneration. For delayed repair, mean axon counts were significantly higher in the SKP-SC group. Compound action potential amplitudes and muscle weights were also improved by cell therapy. In conclusion, SKP-SC therapy improves behavioral recovery after acute, chronic and nerve graft repair beyond the current standard of microsurgical nerve repair.

Early regenerative effects of NGF-transduced Schwann cells in peripheral nerve repair.

Peripheral nerve injury leads to a rapid and robust increase in the synthesis of neurotrophins which guide and support regenerating axons. To further optimize neurotrophin supply at the earliest stages of regeneration, we over-expressed NGF in Schwann cells (SCs) by transducing these cells with a lentiviral vector encoding NGF (NGF-SCs). Transplantation of NGF-SCs in a rat sciatic nerve transection/repair model led to significant increase of NGF levels 2weeks after injury and correspondingly to substantial improvement in axonal regeneration. Numbers of NF200, ChAT and CGRP-positive axon profiles, as well as the gastrocnemius muscle weights, were significantly higher in the NGF-Schwann cell group compared to the animals that received control SCs transduced with a lentiviral vector encoding GFP (GFP-SCs). Comparison with other models of NGF application signifies the important role of this neurotrophin during the early stages of regeneration, and supports the importance of developing combined gene and cell therapy for peripheral nerve repair.

Use of polyamidoamine dendrimers to engineer BDNF-producing human mesenchymal stem cells

We report the use of polyamidoamine (PAMAM-NH2) dendrimers along with other non-viral vehicles for the in vitro transfection of human bone marrow mesenchymal stem cells (hMSCs) and for engineering MSCs to secrete brain-derived neurotrophic factor (BDNF). Different generations of cationic polyamidoamine dendrimers (generations 3–6) were tested on HEK 293T cells. hMSCs were then transfected with PAMAM-NH2 G4 dendrimers and Lipofectamine 2000, which elicited the expression of GFP reporter in around 6 and 20% of the cells, respectively. Both vehicles were then shown to elicit the expression of BDNF in MSCs from a bicistronic cassette. Non-virally induced neurotrophin expression may be a safe and easy method for adapting autologous stem cells for therapeutic treatment of diseases and neural system injuries.

Fourth generation phosphorus-containing dendrimers: prospective drug and gene delivery carrier.

Research concerning new targeting delivery systems for pharmacologically active molecules and genetic material is of great importance. The aim of the present study was to investigate the potential of fourth generation (P4) cationic phosphorus-containing dendrimers to bind fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS), anti-neoplastic drug cisplatin, anti-HIV siRNA siP24 and its capability to deliver green fluorescent protein gene (pGFP) into cells. The interaction between P4 and ANS (as the model drug) was investigated. The binding constant and the number of binding centers per one molecule of P4 were determined. In addition, the dendriplex between P4 and anti-HIV siRNA siP24 was characterized using circular dichroism, fluorescence polarization and zeta-potential methods; the average hydrodynamic diameter of the dendriplex was calculated using zeta-size measurements. The efficiency of transfection of pGFP using P4 was determined in HEK293 cells and human mesenchymal stem cells, and the cytotoxicity of the P4-pGFP dendriplex was studied. Furthermore, enhancement of the toxic action of the anti-neoplastic drug cisplatin by P4 dendrimers was estimated. Based on the results, the fourth generation cationic phosphorus-containing dendrimers seem to be a good drug and gene delivery carrier candidate.

Evidence for a systemic regulation of neurotrophin synthesis in response to peripheral nerve injury

J. Neurochem. (2012) 122, 501–511.

Non-Viral Engineering of Skin Precursor-Derived Schwann Cells for Enhanced NT-3 Production in Adherent and Microcarrier Culture

Genetic engineering of stem cells and their derivatives has the potential to enhance their regenerative capabilities. Here, dendrimer- and lipofection-based approaches were used for non-viral neurotrophin-3 (NT-3) over-expression in Schwann cells differentiated from skin precursors (SKP-SCs). A variety of dendrimers were first tested for transfection efficiency on HEK 293T cells, with PAMAMNH2 G4 found most effective and used subsequently for SKP-SCs transfection. Plasmid-based expression resulted in increased NT-3 release from SKP-SCs in both adherent and microcarrier-based culture. In a proof-of-concept study, the microcarrier/SKP-SCs were implanted into the injured nerve, and transfected cells were shown to detach, integrate into the nerve tissue and associate with regenerating axons. Virus-free systems for transient neurotrophin expression are a feasible and biologically safe option to increase the therapeutic value of stem cells and stem cell-derived cells in nerve repair strategies. Further work to develop bioprocesses for expansion of SKP-SCs on microcarriers in bioreactors is still needed.

Neurons and Stromal Stem Cells as Targets for Polycation-Mediated Transfection

Expression of transgenes in neurons and stromal/mesenchymal stem cells (MSC) can greatly enhance their therapeutic potential. In transfection experiments, we studied properties of linear and branched (dendrimers) polycations as transgene delivery vehicles. Linear polyethyleneimine transfected neurons, but was ineffective in MSC. Polyamidoamine dendrimers showed greater transfection efficiency and mean GFP fluorescence intensity compared to phosphorus dendrimers of the same (4th) generation. Expression of neurotrophic factor BDNF in MSC transfected with polyamidoamine dendrimers was also by more than 10 times higher.