Novel immortal human cell lines reveal subpopulations in the nucleus pulposus

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.

Transcriptional profiling distinguishes inner and outer annulus fibrosus from nucleus pulposus in the bovine intervertebral disc

“…In addition, we find that bFoxf1 and bPax1 are not differentially expressed in the bovine tail IVD. ADAMTS17, SFRP2, COL5A1 and COL12A1 were previously identified as (outer) AF markers compared to NP cells [9]. To test if these marker genes distinguish inner from outer AF tissue, they were evaluated in the bovine tail IVD.…”

“…The AF marker genes ADAMTS17, SFRP2, COL5A1 and COL12A1, previously used for cultured NP and AF cells, could be used to distinguish bovine AF from NP tissue [9]. Few studies have addressed inner and outer AF cell phenotypes [27][28][29].…”

“…Previously, we used a subset of genes specifically expressed in the AF or NP to confirm correct isolation of cell populations and identified functional cellular subpopulations in human NP and AF cell lines [9,10]. In addition, NP specific marker genes are increasingly used as readout to develop stem cell differentiation protocols for NP regeneration [11,12].…”

Improvement of Bovine Nucleus Pulposus Cells Isolation Leads to Identification of Three Phenotypically Distinct Cell Subpopulations

“…7 Cells isolated from degenerated and non-degenerated human discs can differentiate into adipogenic, osteogenic, and chondrogenic lineages, indicating that the IVD may include progenitor cells within its population. [8][9][10][11][12][13][14] In addition, expression of markers associated with progenitors (e.g., Tie2, GD2, Stro-1, Oct3/4, CD105, CD90, and Notch-1) suggests that precursor cell populations might be present within degenerated human discs. 8,9 These findings indicate that degenerated human discs may count on an endogenous regenerative capacity, although the exact nature and origin of the participating cells remains unknown.…”

PHD/HIF-1 upregulates CA12 to protect against degenerative disc disease: a human sample, in vitro and ex vivo study

“…13,14 Several research groups have begun to use CA12 as an NP marker. [15][16][17] Thus, we hypothesize that CA12 has a role in NP physiology. Although Power et al 14 found that CA inhibition disrupts pH and leads to lactate accumulation in NP cells, the exact function of CA12 in NP remains unknown.…”