Mesenchymal Stem Cell and Nucleus Pulposus Cell Coculture Modulates Cell Profile

Abstract

Spontaneous cell fusion can occur in cocultured stem cells. We examined whether telomerase activity change and cell fusion occurred in mesenchymal stem cell (MSC) and nucleus pulposus cell (NPC) coculture. MSCs and NPCs were labeled with PKH26 and PKH67 dyes and cocultured at a 50:50 ratio. An equal number of MSCs or NPCs were used as the control. After 14 days, cells were evaluated by cell growth, telomerase activity, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and histologic observation. Cell fusion was confirmed by microscopic observation and fluorescence-activated cell sorter (FACS) analysis. The results suggested cell growth rate and telomerase activity were higher in cocultured cells than in NPCs cultured alone. The mRNA expression levels of the Type II collagen and aggrecan were elevated in cocultured cells. Immunohistochemical analysis revealed positive staining for Type II collagen and keratan sulfate in NPCs cultured alone and in a proportion of cocultured cells. Histologic observation revealed binucleated cocultured cells expressed both PKH dyes in the same location and slide focus. The FACS analysis revealed 42% of cocultured cells were doublestained. Cocultured cells partially maintained the NPC phenotype. The partially maintained phenotype of the NPCs may be attributable to spontaneous cell fusion in association with increased telomerase activity.

Expression and significance of telomerase in the nucleus pulposus tissues of degenerative lumbar discs

“…Following transfection of the nucleus pulposus with the telomerase gene, the percentage of senescent cells to the total nucleus pulposus cells was 3-30% (following telomerase gene transfection) and 4-64% (control group). In addition, the generation of extracellular matrix products, such as collagen, were significantly increased within 120 days and the notable difference was identified at day 21 with a yield that was 7.2 times as high as the control group (14,15). In the present study, the expression levels of chondroitin sulfate and type II collagen in the nucleus pulposus were also significantly lower in the experimental group compared with those in the control group.…”

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

“…In rabbit culture, PKH26 (red) and PKH67 (green) lipophilic dyes were used. Subsequent to a two-week incubation period on alginate beads 42% of the cells were double-stained (4). A flow cytometric analysis revealed that the exchange of dyes between rabbit NPCs and MSCs is balanced and the cytometric diagram was less asymmetrical (in relation to a 45˚ axis) than our diagram, nevertheless, it is probable that the MSCs secreted more PKH26 due to fewer PHK67-stained NPCs observed (4).…”

“…Lipophilic dye exchange has been studied in rabbit and human NPCs and MSC co-culture systems (4,10). In rabbit culture, PKH26 (red) and PKH67 (green) lipophilic dyes were used.…”

“…Studies on the interaction between the IVD and multipotent mesenchymal stromal cells (MSCs) have been performed in human and animal culture systems (2)(3)(4). Direct co-culture between human MSCs and human AF cells was required to observe an increase in the glycosaminoglycan (GAG) content (5).…”

Advancing the cellular and molecular therapy for intervertebral disc disease

“…MSCs can activate NP cells by paracrine stimulation, cell-cell contact or exchange of membrane components [93,94]. Furthermore, anti-inflammatory and immunemodulating activity has been attributed to MSCs [95,96].…”