Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus.

Autor: van den Akker GG; Department of Orthopaedic Surgery, Maastricht University Medical Centre, Maastricht, the Netherlands.; Department of Molecular Genetics, Maastricht University Medical Centre, Maastricht, the Netherlands., Surtel DA; Department of Orthopaedic Surgery, Maastricht University Medical Centre, Maastricht, the Netherlands., Cremers A; Department of Orthopaedic Surgery, Maastricht University Medical Centre, Maastricht, the Netherlands., Richardson SM; Centre for Tissue Injury and Repair, Institute of Inflammation and Repair, The University of Manchester, Manchester, United Kingdom., Hoyland JA; Centre for Tissue Injury and Repair, Institute of Inflammation and Repair, The University of Manchester, Manchester, United Kingdom., van Rhijn LW; Department of Orthopaedic Surgery, Maastricht University Medical Centre, Maastricht, the Netherlands., Voncken JW; Department of Molecular Genetics, Maastricht University Medical Centre, Maastricht, the Netherlands., Welting TJ; Department of Orthopaedic Surgery, Maastricht University Medical Centre, Maastricht, the Netherlands.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2016 Jan 21; Vol. 11 (1), pp. e0144497. Date of Electronic Publication: 2016 Jan 21 (Print Publication: 2016).
DOI: 10.1371/journal.pone.0144497
Abstrakt: Introduction: Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors.
Methods: Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ).
Results: The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections.
Conclusion: We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.
Databáze: MEDLINE
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