title: Novel immortal human cell lines reveal subpopulations in the nucleus pulposus.
abstract: BACKGROUND
  Relatively little is known about cellular subpopulations in the mature nucleus pulposus (NP). Detailed understanding of the ontogenetic, cellular and molecular characteristics of functional intervertebral disc (IVD) cell populations is pivotal to the successful development of cell replacement therapies and IVD regeneration. In this study, we aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human NP could be generated using immortalization strategies.
  
  
  METHODS
  Nondegenerate healthy disc material (age range, 8 to 15 years) was obtained as surplus surgical material. Early passage NP monolayer cell cultures were initially characterized using a recently established NP marker set. NP cells were immortalized by simian virus 40 large T antigen (SV40LTag) and human telomerase reverse transcriptase expression. Immortalized cells were clonally expanded and characterized based on collagen type I, collagen type II, α1 (COL2A1), and SRY-box 9 (SOX9) protein expression profiles, as well as on expression of a subset of established in vivo NP cell lineage markers.
  
  
  RESULTS
  A total of 54 immortal clones were generated. Profiling of a set of novel NP markers (CD24, CA12, PAX1, PTN, FOXF1 and KRT19 mRNA) in a representative set of subclones substantiated successful immortalization of multiple cellular subpopulations from primary isolates and confirmed their NP origin and/or phenotype. We were able to identify two predominant clonal NP subtypes based on their morphological characteristics and their ability to induce SOX9 and COL2A1 under conventional differentiation conditions. In addition, cluster of differentiation 24 (CD24)-negative NP responder clones formed spheroid structures in various culture systems, suggesting the preservation of a more immature phenotype compared to CD24-positive nonresponder clones.
  
  
  CONCLUSIONS
  Here we report the generation of clonal NP cell lines from nondegenerate human IVD tissue and present a detailed characterization of NP cellular subpopulations. Differential cell surface marker expression and divergent responses to differentiation conditions suggest that the NP subtypes may correspond to distinct maturation stages and represent distinct NP cell subpopulations. Hence, we provide evidence that the immortalization strategy that we applied is capable of detecting cell heterogeneity in the NP. Our cell lines yield novel insights into NP biology and provide promising new tools for studies of IVD development, cell function and disease.
authors: Guus G H
authors: GG
authors: van den Akker
authors: Don A M
authors: DA
authors: Surtel
authors: Andy
authors: A
authors: Cremers
authors: Ricardo
authors: R
authors: Rodrigues-Pinto
authors: Stephen M
authors: SM
authors: Richardson
authors: Judith A
authors: JA
authors: Hoyland
authors: Lodewijk W
authors: LW
authors: van Rhijn
authors: Tim J M
authors: TJ
authors: Welting
authors: Jan Willem
authors: JW
authors: Voncken
mesh_terms: D000293:Adolescent; D053674:Aggrecans / Q000378:metabolism; D051927:CD24 Antigen / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D002478:Cells, Cultured; D002648:Child; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D024042:Collagen Type I / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D015151:Immunoblotting; D016130:Immunophenotyping; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism*; D008297:Male; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism
chemical_list: C508319:ACAN protein, human; D053674:Aggrecans; D051927:CD24 Antigen; C499177:COL2A1 protein, human; D002352:Carrier Proteins; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D024043:Collagen Type II; D016207:Cytokines; C098521:FOXF1 protein, human; D051858:Forkhead Transcription Factors; D051761:Paired Box Transcription Factors; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; C066845:pleiotrophin; C070835:PAX1 transcription factor