Differentiation of intervertebral notochordal cells through live automated cell imaging system in vitro

Study Design. We demonstrated the differentiation of notochordal cells by direct observation using a live automated cell imaging system. We also hypothesized that notochordal cells have characteristics of chondrocyte-like cells. Objective. To determine characteristics of notochordal cells by matrix protein expression and their differentiation using a live automated cell imager. Summary of Background Data. Although notochordal cells are critical to homeostasis of intervertebral disc, their fate has not been extensively studied and there is little evidence of notochordal cells as progenitors. Methods. Notochordal cells purified from rabbit nucleus pulposus were isolated after serial filtration. Notochordal cells in 3-dimensional culture were compared to chondrocyte-like cells by ³⁵S sulfate incorporation into proteoglycan and reverse transcription polymerase chain reaction for gene expression(collagen II and aggrecan). Notochordal cells in 2-D culture were used for immunocytochemical staining (collagen II, aggrecan, and SOX9) and time-lapsed cell tracking study. Results. Notochordal cells were capable of proteoglycan production at a rate comparable to chondrocyte-like cells (108% ± 22.6% to chondrocyte-like cells) and expressed collagen II, aggrecan, and SOX9. In time-lapsed cell tracking analysis, notochordal cells were slower in population doubling time than chondrocyte-like cells and differentiated into 3 morphologically distinct cell types: vacuolated cells (area: 2392 ± 507.1 μm², velocity: 0.09 ± 0.01 μm/min); giant cells (area: 12678 ± 1637.0 μm², velocity: 0.08 ± 0.01 μm/min) which grew rapidly without cell division; polygonal cells (area: 3053 ± 751.2 μm², 0.14 ± 0.01 μm/min) morphologically similar to typical differentiation type of chondrocyte-like cells (area: 2671 ± 235.6 μm², 0.19 ± 0.01 μm/min). Rarely, notochordal cells formed clusters analogous to that observed in vivo. Conclusion. These studies demonstrate a chondrocyte phenotype of notochordal cells and are the first direct evidence of notochordal cell differentiation, suggesting that they may act as progenitor cells, which has the potential to lead to their use in novel approaches to regeneration of degenerative intervertebral disc.