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Novel immortal human cell lines reveal subpopulations in the nucleus pulposus

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 repl...

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Autores principales: van den Akker, Guus GH, Surtel, Don AM, Cremers, Andy, Rodrigues-Pinto, Ricardo, Richardson, Stephen M, Hoyland, Judith A, van Rhijn, Lodewijk W, Welting, Tim JM, Voncken, Jan Willem
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227062/
https://www.ncbi.nlm.nih.gov/pubmed/24972717
http://dx.doi.org/10.1186/ar4597
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author van den Akker, Guus GH
Surtel, Don AM
Cremers, Andy
Rodrigues-Pinto, Ricardo
Richardson, Stephen M
Hoyland, Judith A
van Rhijn, Lodewijk W
Welting, Tim JM
Voncken, Jan Willem
author_facet van den Akker, Guus GH
Surtel, Don AM
Cremers, Andy
Rodrigues-Pinto, Ricardo
Richardson, Stephen M
Hoyland, Judith A
van Rhijn, Lodewijk W
Welting, Tim JM
Voncken, Jan Willem
author_sort van den Akker, Guus GH
collection PubMed
description 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.
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spelling pubmed-42270622014-11-12 Novel immortal human cell lines reveal subpopulations in the nucleus pulposus van den Akker, Guus GH Surtel, Don AM Cremers, Andy Rodrigues-Pinto, Ricardo Richardson, Stephen M Hoyland, Judith A van Rhijn, Lodewijk W Welting, Tim JM Voncken, Jan Willem Arthritis Res Ther Research Article 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. BioMed Central 2014 2014-06-27 /pmc/articles/PMC4227062/ /pubmed/24972717 http://dx.doi.org/10.1186/ar4597 Text en Copyright © 2014 van den Akker et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/4.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
van den Akker, Guus GH
Surtel, Don AM
Cremers, Andy
Rodrigues-Pinto, Ricardo
Richardson, Stephen M
Hoyland, Judith A
van Rhijn, Lodewijk W
Welting, Tim JM
Voncken, Jan Willem
Novel immortal human cell lines reveal subpopulations in the nucleus pulposus
title Novel immortal human cell lines reveal subpopulations in the nucleus pulposus
title_full Novel immortal human cell lines reveal subpopulations in the nucleus pulposus
title_fullStr Novel immortal human cell lines reveal subpopulations in the nucleus pulposus
title_full_unstemmed Novel immortal human cell lines reveal subpopulations in the nucleus pulposus
title_short Novel immortal human cell lines reveal subpopulations in the nucleus pulposus
title_sort novel immortal human cell lines reveal subpopulations in the nucleus pulposus
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227062/
https://www.ncbi.nlm.nih.gov/pubmed/24972717
http://dx.doi.org/10.1186/ar4597
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