Abstract
IntroductionRelatively 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.MethodsNondegenerate 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.ResultsA 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.ConclusionsHere 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.
Highlights
Little is known about cellular subpopulations in the mature nucleus pulposus (NP)
To determine whether any of the NP markers described above could discriminate between the clonal subtypes (NP-R vs nR), we evaluated differences in mRNA levels of Keratin 19 (KRT19), Carbonic anhydrase XII (CA12), cluster of differentiation 24 (CD24), PTN, Forkhead box F1 (FOXF1) and Paired box 1 (PAX1)
For the first time to the best of our knowledge, that the significant differences in marker expression between immortal NP subtypes isolated from immortalized primary cultures (CA12, CD24, FOXF1, PTN, PAX1 and Cartilage oligomeric matrix protein (COMP)) discriminate human NP subtypes from two independent donors in primary monolayer cultures
Summary
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. We aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human NP could be generated using immortalization strategies. It is becoming increasingly clear that the NP comprises multiple cell subpopulations [8,9,10,11] This cellular heterogeneity may reflect different stages of proliferation, differentiation and maturation; relatively little is known about these NP cell subpopulations. Current research on primary cells is hampered by restricted availability of human cells, from nondegenerate discs, where there is a relatively inherent low cellularity within the tissue. A few NP cell lines have been independently generated by Sakai et al [12] and, more recently, by Liu and co-workers [13]
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