title: Annulus cells from more degenerated human discs show modified gene expression in 3D culture compared with expression in cells from healthier discs.
abstract: BACKGROUND
  Understanding gene expression patterns of disc cells in culture is important as we develop biologic therapies for disc degeneration. The objective of the present study was to determine if cells from more degenerated discs expressed different genes, or differed in their expression patterns, compared with patterns of cells from healthier discs.
  
  
  OBJECTIVE
  To determine if annulus cells from more degenerated discs expressed different gene expression patterns compared with patterns of cells from healthier discs using genome-wide analysis.
  
  
  METHODS
  Cells from human annulus tissue were grown in three-dimensional (3D) culture and their gene expression patterns analyzed with Affymetrix microarray analysis. Gene expression patterns of cells from more degenerated discs (Thompson Grades IV and V) were compared with patterns from cells from healthier discs (Thompson Grades I, II, and III).
  
  
  METHODS
  After approval by our human subjects institutional review board, annulus cells were obtained from lumbar discs of seven subjects with Thompson Grades I, II, or III and from five subjects with discs of Thompson Grades IV and V. Cells were grown in 3D culture for 2 weeks; 3D cultures were used because this microenvironment more closely mimics the in vivo condition. mRNA was harvested, processed for Affymetrix genome-wide gene analysis, and data analyzed with p values adjusted so as to compensate for false discovery rates.
  
  
  RESULTS
  GeneSifter analyses showed that cells from more degenerated discs had 320 genes significantly upregulated, and 104 genes significantly downregulated compared with cells from healthier discs. Important genes included those related to: 1) the extracellular matrix (ECM) (keratin-associated protein 1-1, hyaluronan synthase 2, and nexin were upregulated; biglycan, collagen type VI alpha 2, thrombospondin 3, laminen alpha 1, fibronectin type III domain-containing protein 1, elastin microfibril interfacer 2, fibulin 2, and nidogen 1 and 2 were downregulated); 2) ECM proteolysis (ADAMTS6 was upregulated); 3) cell proliferation (never in mitosis gene 1-related kinase 3, cell division cycle 2-like 5 [cholinesterase-related cell division controller], RAB42 [member of RAS oncogene family], and cyclin-dependent kinase 6 were upregulated; RAS-like GTP-binding 1 was downregulated); 4) apoptosis (BCL2-like 11 and p53-inducible nuclear protein 1 were upregulated; caspase recruitment domain family, member 10, caspase-1 dominant-negative inhibitor pseudo-ICE, and caspase 9 and FADD-like apoptosis regulator were downregulated); and 5) growth factors, inflammatory mediators, and other genes (fibroblast growth factor 1, pregnancy-associated plasma protein-A, interleukin 1 alpha, and interleukin 7 were upregulated; TGF-beta-induced transcript 1, interleukin 26 and interleukin 1 receptor-like 1, tumor necrosis factor, alpha-induced protein 2, and chemokine (C-X3-C motif) ligand 1 were downregulated).
  
  
  CONCLUSIONS
  Data presented here show that annulus cells from more degenerated discs show modified gene expression in 3D culture. Important gene variations involved expression of interleukins, cytokines, ECM components, and apoptosis regulators. Results presented here have potential application in future cell-based biologic therapies for disc degeneration.
authors.affiliation: Department of Orthopaedic Surgery, Carolinas Medical Center, PO Box 32861, Charlotte, NC 28232, USA. helen.gruber@carolinashealthcare.org
authors.forename: Helen E
authors.initials: HE
authors.lastname: Gruber
authors.forename: Gretchen L
authors.initials: GL
authors.lastname: Hoelscher
authors.forename: Edward N
authors.initials: EN
authors.lastname: Hanley
mesh_terms: D000328:Adult; D002478:Cells, Cultured; D005260:Female; D015870:Gene Expression; D006801:Humans; D007403:Intervertebral Disc / Q000473:pathology*; D055959:Intervertebral Disc Degeneration / Q000235:genetics* / Q000473:pathology*; D008159:Lumbar Vertebrae / Q000473:pathology; D008297:Male; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis; D013904:Thoracic Vertebrae / Q000473:pathology
chemical_list: D012333:RNA, Messenger