Effects of Tunneling Nanotubes on the Mitochondrial Regulation of the Amount and Subcellular Localization of Β-Catenin During Osteogenesis of MSC/HUVEC Spheroids

Abstract

Tunneling nanotubular expressways (TNTs), which allow direct cell-to-cell transfer of intracellular organelles, have been widely identified in various cell types. However, the precise functions of TNTs in intercellular communication and their practical application in tissue regeneration is still uncertain. Mesenchymal stem cells (MSCs) are commonly employed as seed cells in tissue engineering. The differentiation of MSCs requires sufficient energy, which can be regulated by the fusion and fission of mitochondria. The phenomenon of mitochondrial shuttle between cells has been observed, and has led to the hypothesis that applying TNTs to deliver mitochondria into MSCs might be a promising approach to stimulate osteogenic differentiation. In proliferating endothelial cells (ECs), cellular dynamics including the fusion and fission of mitochondria is increased, and thus ECs are considered as ideal candidates for mitochondria donors. In order to exploit the application of TNT-mediated mitochondria transfer, we employed mesenchymal stem cell/ human umbilical vein endothelial cell (MSC/HUVEC) spheroids as a research model, and investigated the transfer among them, as well as the underlying mechanism. Fluorescence staining showed that directional transfer of mitochondria between MSC-HUVEC pairs, especially from HUVEC to MSC. Through TNT-mediated mitochondrial transfer, osteogenesis markers were up-regulated, accompanied by an increased amount of β-catenin in MSCs. Moreover, the improved generation of pre-vascular network has also been observed in the spheroids, as a result of β-catenin translocation to the periphery of HUVECs. However, all of these effects are abolished by the destruction of TNTs. Collectively, our results indicate that the TNT strategy can be applied widely to various aspects of biological research, such as but not limited to tissue regeneration and targeted drug delivery.