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 double-stained. 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.

Fig. 1A–B

The flowcharts illustrate our experimental method. (A) Cell growth, telomerase activity, and cell phenotype of MSCs, NPCs, and cocultured cells were examined. (B) Quantitative reverse transcriptase-polymerase chain reaction (Q-PCR), FACS, and fluorescence microscopy were examined.

Fig. 2

A graph shows the results of the cell growth assay after coculture for 14 days. The number of cells increased gradually in all the cultured cell groups. The cell growth rate was greater in NPCs cocultured with MSCs than in NPCs cultured alone (MSCs versus NPCs, p = 1.14 × 10⁻⁵; MSCs versus cocultured cells, p = 0.24; NPCs versus cocultured cells, p = 3.2 × 10⁻⁴; n = 6).

Fig. 3A–B

The graphs show telomerase activity after coculture for 14 days. (A) After normalization to the internal control, MSCs cultured alone exhibited the highest telomerase activity levels. The telomerase activity level was greater in cocultured cells than in NPCs cultured alone (MSCs versus NPCs, p = 1.2 × 10⁻¹²; MSCs versus cocultured cells, p = 3.5 × 10⁻¹¹; NPCs versus cocultured cells, p = 0.001; n = 6). (B) Using NPCs and MSCs alone in alginate beads as a control for calculation, the ratios of cocultured cells to MSCs and NPCs were 35.8% ± 2.8% (p = 7.5 × 10⁻¹⁴, n = 6) and 191.7% ± 20.2% (p = 5.9 × 10⁻⁷, n = 6).

Fig. 4

A graph shows relative gene expression levels in NPCs and MSCs after coculture for 14 days. As compared with NPCs and MSCs cultured alone, cocultured cells exhibited elevated levels for the Type II collagen gene (4.2-fold greater than the levels in NPCs, p = 0.002, n = 4; 38.3-fold greater than the levels in MSCs, p = 1.5 × 10⁻⁵, n = 4) and aggrecan gene (4.4-fold greater than the levels in NPCs, p = 0.002, n = 4; 16.4-fold greater than the levels in MSCs, p = 3.2 × 10⁻⁵, n = 4).

Fig. 5A–C

The photomicrographs illustrate the results of immunohistochemical analysis for Type II collagen. When compared with (A) MSCs, immunohistochemical analysis revealed strong staining for Type II collagen (brown) in (B) NPCs and (C) cocultured cells (Stain, immunostain; original magnification, ×200; size bar, 1 unit = 50 μm).

Fig. 6A–C

The photomicrographs illustrate the results of immunohistochemical analysis for KS. When compared with (A) MSCs, immunohistochemical analysis revealed strong staining for KS (brown) in (B) NPCs and (C) cocultured cells (Stain, immunostain; original magnification, ×200; size bar, 1 unit = 50 μm).

Fig. 7A–B

Histologic observation revealed cell fusion had occurred among cocultured cells. Two different types of nuclei were seen in the fused cells (arrows). The detection of cells with (A) two nuclei or (B) hybrid nuclei among the cocultured cells raised the possibility of cell fusion (Stain, safranin O; original magnification, ×400; size bar, 1 unit = 50 μm).

Fig. 8A–D

The photomicrographs illustrate in situ cell labeling with PKH to trace cell fusion. (A) PKH26-stained cells (red) and (B) PKH67-stained cells (green) were observed. (C) Some cocultured cells expressed both PKH dyes and were detected at the same position on the slide, with the same focus (yellow) (Original magnification, ×200). (D) A scatterplot shows the detection of PKH-stained cells by cell sorting. FACS analysis revealed approximately 42% of the cocultured cells took up both stains.