Abstract
Introduction
The intervertebral disc (IVD) has a limited regenerative potential and low back pain represents a leading cause of disability.1 IVD repair strategies require an appropriate cell source that is able to regenerate the damaged tissue such as progenitor stem cells. Recently, progenitor cells that are positive for the angiopoietin receptor (Tie2) in the nucleus pulposus were identified.2 These cells, which express type II collagen and aggrecan, were shown to have progenitor-like multipotency. Here, we isolated primary cells from bovine IVD aged 1 year and sorted bovine nucleus pulposus progenitor cells (NPPC) for the marker Tie2. Furthermore, we demonstrate that in contrast to Tie2− cells, Tie2+ expressing cells can differentiate into osteogenic and adipogenic lineages in vitro.
Materials and Methods
NP cells were obtained from 1-year-old bovine tails by sequential digestion with Pronase for 1 hour and collagenase overnight. Sorted Tie2− and Tie2+ cells were cultured in osteogenic and adipogenic medium containing 5% fetal bovine serum for 3 weeks. The formed cell layers from both subpopulations were stained for calcium deposition and fat droplets using alizarin red and red oil staining, respectively. Colony forming units (CFU) were prepared for both cell suspensions (Tie2− and Tie2 + ) in methylcellulose-based medium. The formed colonies (> 10 cells) were analyzed macroscopically and counted after 8 days.
Results
After 3 weeks of culture, sorted Tie2+ cells were able to differentiate into osteocytes and adipocytes as characterized by calcium deposition and fat droplet formation. By contrast, Tie2− cells generated a weak staining for calcium and no fat droplets were obtained (Fig. 1). Sorted Tie2− and Tie2+ subpopulations of cells, both the formed colonies, however with different morphologies. The colonies formed from Tie2+ cells were spheroid in shape, whereas those fromTie2− cells were spread and fibroblastic. In addition, the highest frequency of the formed colonies was obtained for the Tie2+ cells after 8 days of culture in the methylcellulose-based medium.
Differentiation assays of Tie2+ and Tie2− bovine nucleus pulposus cells: Osteogenesis (top row) and adipogenesis (lower row).
Conclusion
Our data showed that Tie2+ cells of the nucleus pulposus cells are progenitor-like cells that are able to differentiate into osteogenic and adipogenic lineages. Sorting of NPPC for Tie2 may represent a promising strategy with the potential to be used in the clinics for treatment of intervertebral disc damage.
Acknowledgments
This project was funded by two projects of the Swiss National Science Foundation, grant numbers IZK0Z3_154384 and 310030_153411.
Freemont AJ. The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain. Rheumatology (Oxford) 2009;48(1):5–10
Sakai D, Nakamura Y, Nakai T, et al. Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat Commun 2012;3:1264