Effect of TNTs-mediated mitochondrial transfer between mesenchymal stem cells and endothelial cells on vascularization in vitro

Abstract

Objective To explore the effect of tunneling nanotubes (TNTs) within mesenchymal stem cells (MSC)/human umbilical vein endothelial cells (HUVEC) co-culture cell spheroids on endothelial cell angiogenesis. Methods Pre-vascular network formation assay was used to compare the angiogenic ability among the MSC/HUVEC cell spheroid co-culture group, adherent co-culture group, HUVEC 3D culture group and MSC 3D culture group. The expression of β-catenin protein was detected by Western blotting and immunofluorescence staining. The cell spheroids were treated with microfilament depolymerization reagent (CytoD) as a control group, and the above parameters were tested again in the untreated and treated groups. The structure of TNTs was verified by fluorescent labeling and field emission scanning electron microscopy, mitochondrial communication was observed by adenovirus infection, and the mitochondrial status was assessed by mitochondrial membrane potential assay. Results Compared with the MSC/HUVEC cell spheroid co-culture group, the numbers of junctions and meshes were significantly lower in the adherent co-culture group, HUVEC 3D culture group and MSC 3D culture group (P < 0.05). Immunofluorescence staining for β-catenin protein suggested that the spheroid co-culture group had stronger expression than the adherent co-culture group. The results of Western blotting also showed that the intracellular β-catenin expression in the spheroid co-culture group was significantly higher than that in the adherent co-culture group (P < 0.05). The presence of TNT (F-actin) between MSC and HUVEC in the co-culture cellular spheroids was shown by phalloidine staining. When the cells were co-cultured as spheroids, the mitochondria derived from HUVEC were observed intracellularly in MSC, and the mitochondria derived from MSC were also observed intracellularly in HUVEC, and the above mitochondrial exchange was diminished in the CytoD-treated group. The mitochondrial membrane potential of the CytoD-treated spheroids was significantly lower than that of the untreated spheroids (P < 0.05), and the proliferation rate and migration range of HUVEC were lower and smaller than those of the untreated spheroids. Compared with the untreated cell spheroid group, the numbers of junctions and meshes were significantly lower in the CytoD-treated cell spheroid group (P < 0.05). The total amount of β-catenin protein was significantly higher in the untreated cell spheroid group compared to the CytoD-treated group (P < 0.05). Conclusion Within the co-culture cell spheroids, there are TNTs and their mediated bidirectional mitochondrial transfer between MSC and HUVEC, and they affect the angiogenic ability of HUVEC within these cellular spheroids probably through β-catenin related pathways.

Keywords

• cell spheroids
• tunneling nanotubes
• mitochondria
• vascularization
• β-catenin