Phenotypic changes of stem cells after culture in the intervertebral disc niche

Abstract

Introduction: The intervertebral disc (IVD) is a highly specialised niche that is rich in proteoglycans and water. The loss of these factors are signs of IVD degeneration, in which the resulting biochemical imbalance and inability to maintain the integrity of the tissues can lead to structural and biomechanical changes to the disc, ultimately affecting their function. We hypothesise that the localised disc environment (the IVD niche) itself can contribute to the development and upkeep of the IVD by aiding in the differentiation and/or activities of progenitors/stem cells into IVD cells. The aim of this study was to introduce pluripotent stem cells into a decellularised IVD scaffold, and study how the IVD environment can alter the behaviour and phenotype of the stem cells. Materials and Methods: Human Embryonic Stem cell line 2 (HES2) cells were labelled with a fluorescent dye and then either injected into whole decellularised bovine IVD via the annulus or seeded onto the surface of decellularised nucleus pulposus (NP) tissue, and cultured in DMEM for 7 or 14 days. The samples were then cryosectioned to examine their morphology, viability and cell surface expression of particular NP and stem cell markers by immunostaining. Results: The HES2 cells that were introduced into the IVD niche were observed to be viable after 7 and 14 days of culture. The cells that were injected into the bovine IVD did not migrate or distribute evenly throughout the tissue and were observed to cluster around the needle track that was created during cell injection. The ESCs that were seeded onto the decellularised NP tissues exhibited mixed morphology with the presence of spherical-shaped and star-shaped cells. Additionally, the cells that were seeded onto the decellularised tissues had limited penetration into the tissues. There was a loss of pluripotency factors Oct3/4 and Nanog observed by day 7 and expression of NP marker cadherin 2 after 14 days. Conclusion: HES2 cells were able to survive on the decellularised IVD scaffold however there was limited penetration of the cells into the tissues, which was possibly due to a limited migratory ability of the cells. It also appeared that the injection of cells into the 3D whole IVD scaffold versus the seeding on top of the surface of IVD tissues gave rise to cells of differing morphologies, with the cells in the 3D scaffold exhibiting a more uniform morphology. This indicated that despite the use of the same type of scaffold, the way in which the cells are cultured (a 3D environment versus a 2D environment) can potentially alter cellular morphology. The stem cells were positive for NP marker cadherin 2 at 14 days, an indication that the scaffold could aid and/or maintain its expression, but as to whether or not these stem cells had truly become NP-like requires further characterisation of the gene and protein expression of these cells.