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

Abstract

Abstract. 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.

Quantification of the asymmetric migration of the lipophilic dyes, DiO and DiD, in homotypic co-cultures of chondrosarcoma SW-1353 cells

“…Therefore, DiO-type lipophilic dyes may be transferred and incorporated by unlabelled cells. Our previous studies revealed a similar phenomenon of DiO and DiD transfer between MSCs and NPCs in heterotypic co-culture (13). Using a fluorescent microscope, rapid movements of DiO and DiD particles in SW-1353 cells were detected (data not shown; video recording available on request from the corresponding author).…”

“…In our previous study, a predominance of DiD-stained cells over DiO-stained cells was observed by flow cytometry, which we termed ‘Q1-Q4 asymmetry’ (13). In the present study, homotypic co-cultures of SW-1353 cells were performed to precisely quantify the Q1-Q4 asymmetry.…”

“…For NPCs, the non-degenerate intervertebral disc tissue was dissected primarily from Th12 to L3 to separate the nucleus pulposus from the annulus fibrosus tissue, as described in our previous study (13). Briefly, the nucleus pulposus was enzymatically digested overnight at 37°C with 0.02% collagenase type II (Sigma-Aldrich; Merck Millipore) in serum-free Dulbecco's modified Eagle's medium/Nutrient F-12 Ham (DMEM/F-12; Sigma-Aldrich; Merck Millipore) containing 100 U/ml penicillin, 100 µg/ml streptomycin and 25 ng/ml amphotericin B (ABAM; Sigma-Aldrich; Merck Millipore).…”

“…The random subpopulation of MSCs obtained was subcultured in a chondrocyte selective cell culture medium (Human Mesenchymal Stem Cell Functional Identification kit, R&D Systems, Inc, Minneapolis, MN, USA). The chondrocyte phenotype was confirmed by quantitative polymerase chain reaction, as described in our previous study (13). MSCs destined for co-culture with NPCs were stained with lipophilic dyes and were subsequently cultured in an insert system or in direct co-culture with NPCs.…”

“…It is recommended that various factors be considered when staining with lipophilic dyes, including dye concentration, duration of staining and temperature (12). Our previous study demonstrated the asymmetry of DiO and DiD distribution in a heterotypic cell co-culture (13). Data concerning the transfer of DiO or DiD between cells are contradictory; certain authors suggest that lipophilic dyes undergo very low intercellular transfer, whereas others report very high transfer (14–19).…”