title: Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Orthopaedic Surgery, Maastricht University Medical Centre, Maastricht, the Netherlands.|Department of Molecular Genetics, Maastricht University Medical Centre, Maastricht, the Netherlands.
authors.forename: Guus G H
authors.initials: GG
authors.lastname: van den Akker
authors.affiliation: Department of Orthopaedic Surgery, Maastricht University Medical Centre, Maastricht, the Netherlands.
authors.forename: Don A M
authors.initials: DA
authors.lastname: Surtel
authors.affiliation: Department of Orthopaedic Surgery, Maastricht University Medical Centre, Maastricht, the Netherlands.
authors.forename: Andy
authors.initials: A
authors.lastname: Cremers
authors.affiliation: Centre for Tissue Injury and Repair, Institute of Inflammation and Repair, The University of Manchester, Manchester, United Kingdom.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Richardson
authors.affiliation: Centre for Tissue Injury and Repair, Institute of Inflammation and Repair, The University of Manchester, Manchester, United Kingdom.
authors.forename: Judith A
authors.initials: JA
authors.lastname: Hoyland
authors.affiliation: Department of Orthopaedic Surgery, Maastricht University Medical Centre, Maastricht, the Netherlands.
authors.forename: Lodewijk W
authors.initials: LW
authors.lastname: van Rhijn
authors.affiliation: Department of Molecular Genetics, Maastricht University Medical Centre, Maastricht, the Netherlands.
authors.forename: Jan Willem
authors.initials: JW
authors.lastname: Voncken
authors.affiliation: Department of Orthopaedic Surgery, Maastricht University Medical Centre, Maastricht, the Netherlands.
authors.forename: Tim J M
authors.initials: TJ
authors.lastname: Welting
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism; D000071096:ADAMTS Proteins; D000293:Adolescent; D015415:Biomarkers / Q000378:metabolism; D064236:Cartilage Oligomeric Matrix Protein / Q000378:metabolism; D002461:Cell Line, Transformed / Q000187:drug effects / Q000378:metabolism; D024042:Collagen Type I / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D024062:Collagen Type V / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology* / Q000502:physiology*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053782:Transforming Growth Factor beta3 / Q000494:pharmacology; D000094742:Secreted Frizzled-Related Proteins
chemical_list: D051722:ADAM Proteins; D000071096:ADAMTS Proteins; D015415:Biomarkers; D064236:Cartilage Oligomeric Matrix Protein; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D024043:Collagen Type II; D024062:Collagen Type V; D008565:Membrane Proteins; D011393:Procollagen-Proline Dioxygenase; D016212:Transforming Growth Factor beta; D053782:Transforming Growth Factor beta3; C499177:COL2A1 protein, human; C530725:COL5A1 protein, human; C109618:SFRP2 protein, human; D000094742:Secreted Frizzled-Related Proteins; C477143:P4HA3 protein, human; C579149:ADAMTS17 protein, human; C577553:TSP5 protein, human