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Open Access Research article

Novel immortal human cell lines reveal subpopulations in the nucleus pulposus

Guus GH van den Akker12, Don AM Surtel1, Andy Cremers1, Ricardo Rodrigues-Pinto34, Stephen M Richardson3, Judith A Hoyland3, Lodewijk W van Rhijn1, Tim JM Welting1 and Jan Willem Voncken2*

Author Affiliations

1 Department of Orthopaedic Surgery, Maastricht University Medical Centre, Maastricht Postbox 616, 6200 MD, the Netherlands

2 Department of Molecular Genetics, Maastricht University Medical Centre, Maastricht, the Netherlands

3 Centre for Regenerative Medicine, Institute of Inflammation and Repair, University of Manchester, Oxford Road, Manchester M13 9PL, UK

4 Department of Orthopaedics, Central Hospital of Porto–St Anthony Hospital, Porto, Portugal

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Arthritis Research & Therapy 2014, 16:R135  doi:10.1186/ar4597

Published: 27 June 2014

Abstract

Introduction

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.