Cell-permeable peptides (CPPs) promote the transduction of nonpermissive cells by recombinant adenovirus (rAd) to improve the therapeutic efficacy of rAd. In this study, branched oligomerization of CPPs significantly enhanced the transduction of human mesenchymal stem cells (MSCs) by rAd in a CPP type-independent manner. In particular, tetrameric CPPs increased transduction efficiency at 3000-5000-fold lower concentrations than did monomeric CPPs. Although branched oligomerization of CPPs also increases cytotoxicity, optimal concentrations of tetrameric CPPs required for maximum transduction are at least 300-1000-fold lower than those causing 50% cytotoxicity. Furthermore, although only B60% of MSCs were maximally transduced at 500 mM of monomeric CPPs, 495% of MSCs were transduced with 0.1 mM of tetrameric CPPs. Tetrameric CPPs also significantly increased the formation and net surface charge of CPP/rAd complexes, as well as the binding of rAd to cell membranes at a greater degree than did monomeric CPPs, followed by rapid internalization into MSCs. In a critical-size calvarial defect model, the inclusion of tetrameric CPPs in ex vivo transduction of rAd expressing bone morphogenetic protein 2 into MSCs promoted highly mineralized bone formation. In addition, MSCs that were transduced with rAd expressing brain-derived neurotrophic factor in the presence of tetrameric CPPs improved functional recovery in a spinal cord injury model. These results demonstrated the potential for tetrameric CPPs to provide an innovative tool for MSC-based gene therapy and for in vitro gene delivery to MSCs.

A novel actin-based bridge connecting cells has been recognized as a new pathway for the distant transport of cytoplasmic components, viruses, or pathogenic substances between cells. However, it is not yet known how such a fine structure extends over several hundred micrometres and remains robust for several hours. Using optical fluorescence imaging methods, we found that random contact promotes the formation of filopodial bridges through N-cadherin interactions between filopodia, which are slender actin-rich plasma membrane protrusions. These filopodial bridges eventually evolve into a single actin-based bridge (intercellular nanotube) that connects two cells via an intermediate state that involves a helical structure. Surprisingly, the twisting of two filopodia is likely to result from the rotational motion of actin filaments inside the filopodia by myosin V. The accumulated torsion of the filopodia triggers the release of one of the paired filopodia, whose end is attached to the other cell body by an N-cadherin cluster. The resulting retraction of the filopodium by retrograde F-actin flow leaves a single bridge. The N-cadherin/catenin cluster is likely to form a synapse between the intercellular nanotube and the cell body. This study sheds light on the formation mechanism of the filopodial bridge-based intercellular nanotubes for longdistance communication between cells.