Co‑culture of human nucleus pulposus cells with multipotent mesenchymal stromal cells from human bone marrow reveals formation of tunnelling nanotubes.

Degeneration of the intervertebral disc (IVD) is the main cause of age-related damage of spinal tissues. Using multipotent mesenchymal stromal cells (MSCs) regenerative medicine intends to restore the IVD components of annulus fibrosus (AF) and nucleus pulposus (NP). In the present study NP cells (NPCs) and MSCs obtained from adolescent patients suffering from scoliosis were used. IVDs and vertebrae were obtained during surgery and subsequently processed in order to establish cultures of NPCs and MSCs. The two cell types were co-cultured in 1-µm pore size insert system (indirect co-culture) or on one surface (direct co-culture). Prior to co-culture in these systems one of the cell types was stained by lipophilic fluorescent dye DiD (red). The results demonstrated that regardless of the cell type, the flow of DiD from stained to non-stained cells was more efficient in the direct co-culture in comparison with the insert system. Moreover, in the direct system the DiD flow was more efficient from MSCs towards NPCs compared with that in the opposite direction. These data indicated that the membrane interchange between the two cell types was asymmetric. To discriminate the subpopulation of cells that underwent membrane interchange, cells were double stained with DiD and DiO (green). In the first part of the experiment NPCs were stained by DiO and MSCs by DiD. In the second, NPCs were stained by DiD and MSCs by DiO. The cells were co-cultured in the direct system for 8 days and subsequently analyzed by flow cytometry and confocal microscopy. This analysis revealed that >50% of cells were stained by the DiO and DiD dyes. NPCs and MSCs formed structures similar to tunnelling nanotubes (TnT). In conclusion, the formation of TnT-like structures is able to promote, phenotypic changes during the direct co-culture of NPCs with MSCs.