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Oswestry, United Kingdom

Turner S.,Spinal Studies | Turner S.,Keele University | Balain B.,Spinal Studies | Caterson B.,University of Cardiff | And 3 more authors.
European Spine Journal | Year: 2014

Purpose: There is much interest in the development of a cellular therapy for the repair or regeneration of degenerate intervertebral discs (IVDs) utilising autologous cells, with some trials already underway. Clusters of cells are commonly found in degenerate IVDs and are formed via cell proliferation, possibly as a repair response. We investigated whether these clusters may be more suitable as a source of cells for biological repair than the single cells in the IVD. Methods: Discs were obtained at surgery from 95 patients and used to assess the cell viability, growth kinetics and stem or progenitor cell markers in both the single and clustered cell populations. Results: Sixty-nine percent (±15) of cells in disc tissue were viable. The clustered cell population consistently proliferated more slowly in monolayer than single cells, although this difference was only significant at P0–1 and P3–4. Both populations exhibited progenitor or notochordal cell markers [chondroitin sulphate epitopes (3B3(−), 7D4, 4C3 and 6C3), Notch-1, cytokeratin 8 and 19] via immunohistochemical examination; stem cell markers assessed with flow cytometry (CD73, 90 and 105 positivity) were similar to those seen on bone marrow-derived mesenchymal stem cells. Conclusions: These results confirm those of previous studies indicating that progenitor or stem cells reside in adult human intervertebral discs. However, although the cell clusters have arisen via proliferation, there appear to be no greater incidence of these progenitor cells within clusters compared to single cells. Rather, since they proliferate more slowly in vitro than the single cell population, it may be beneficial to avoid the use of clustered cells when sourcing autologous cells for regenerative therapies. © 2014, Springer-Verlag Berlin Heidelberg.

Turner S.A.,Spinal Studies | Turner S.A.,Keele University | Wright K.T.,Spinal Studies | Wright K.T.,Keele University | And 5 more authors.
Stem Cells International | Year: 2016

Much emphasis has been placed recently on the repair of degenerate discs using implanted cells, such as disc cells or bone marrow derived mesenchymal stem cells (MSCs). This study examines the temporal response of bovine and human nucleus pulposus (NP) cells and MSCs cultured in monolayer following exposure to altered levels of glucose (0, 3.15, and 4.5 g/L) and foetal bovine serum (0, 10, and 20%) using an automated time-lapse imaging system. NP cells were also exposed to the cell death inducers, hydrogen peroxide and staurosporine, in comparison to serum starvation. We have demonstrated that human NP cells show an initial "shock" response to reduced nutrition (glucose). However, as time progresses, NP cells supplemented with serum recover with minimal evidence of cell death. Human NP cells show no evidence of proliferation in response to nutrient supplementation, whereas MSCs showed greater response to increased nutrition. When specifically inducing NP cell death with hydrogen peroxide and staurosporine, as expected, the cell number declined. These results support the concept that implanted NP cells or MSCs may be capable of survival in the nutrient-poor environment of the degenerate human disc, which has important clinical implications for the development of IVD cell therapies. Copyright © 2016 Sarah A. Turner et al.

Stephan S.,Spinal Studies | Stephan S.,Keele University | Stephan S.,University of Manchester | Eustace Johnson W.,Spinal Studies | And 4 more authors.
European Cells and Materials | Year: 2011

The adult human intervertebral disc (IVD) is normally avascular. Changes to the extracellular matrix in degenerative disc disease may promote vascularisation and subsequently alter cell nutrition and disc homeostasis. This study examines the influence of cell density and the presence of glucose and serum on the proliferation and survival of IVD cells in 3D culture. Bovine nucleus pulposus (NP) cells were seeded at a range of cell densities (1.25 ×105-106 cells/mL) and cultured in alginate beads under standard culture conditions (with 3.15 g/L glucose and 10% serum), or without glucose and/or 20% serum. Cell proliferation, apoptosis and cell senescence were examined after 8 days in culture. Under standard culture conditions, NP cell proliferation and cluster formation was inversely related to cell seeding density, whilst the number of apoptotic cells and enucleated "ghost" cells was positively correlated to cell seeding density. Increasing serum levels from 10% to 20% was associated with increased cluster size and also an increased prevalence of apoptotic cells within clusters. Omitting glucose produced even larger clusters and also more apoptotic and senescent cells. These studies demonstrate that NP cell growth and survival are influenced both by cell density and the availability of serum or nutrients, such as glucose. The observation of clustered, senescent, apoptotic or "ghost" cells in vitro suggests that environmental factors may influence the formation of these phenotypes that have been previously reported in vivo. Hence this study has implications for both our understanding of degenerative disc disease and also cell-based therapy using cells cultured in vitro.

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