Dataset Information

A Membranome-Centered Approach Defines Novel Biomarkers for Cellular Subtypes in the Intervertebral Disc.

ABSTRACT:

Objective

Lack of specific marker-sets prohibits definition and functional distinction of cellular subtypes in the intervertebral disc (IVD), such as those from the annulus fibrosus (AF) and the nucleus pulposus (NP).

Design

We recently generated immortalized cell lines from human NP and AF tissues; these comprise a set of functionally distinct clonal subtypes. Whole transcriptome analyses were performed of 12 phenotypically distinct clonal cell lines (4× NP-Responder, 4× NP-nonResponder, 2× AF-Sheet forming, and 2× AF-nonSheet forming). Data sets were filtered for membrane-associated marker genes and compared to literature.

Results

Comparison of our immortal cell lines to published primary NP, AF, and articular chondrocytes (AC) transcriptome datasets revealed preservation of AF and NP phenotypes. NP-specific membrane-associated genes were defined by comparison to AF cells in both the primary dataset (46 genes) and immortal cell-lines (161 genes). Definition of AF-specific membrane-associated genes yielded 125 primary AF cell and 92 immortal cell-line markers. Overlap between primary and immortal NP cells yielded high-confidence NP-specific marker genes for NP-R (CLDN11, TMEFF2, CA12, ANXA2, CD44) and NP-nR (EFNA1, NETO2, SLC2A1). Overlap between AF and immortal AF subtypes yielded specific markers for AF-S (COLEC12, LPAR1) and AF-nS (CHIC1).

Conclusions

The current study provides a reference platform for preclinical evaluation of novel membrane-associated cell type-specific markers in the IVD. Future research will focus on their biological relevance for IVD function in development, homeostasis, and degenerate conditions.

SUBMITTER: van den Akker GGH

PROVIDER: S-EPMC7097986 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Publications

A Membranome-Centered Approach Defines Novel Biomarkers for Cellular Subtypes in the Intervertebral Disc.

van den Akker Guus G H GGH Eijssen Lars M T LMT Richardson Stephen M SM Rhijn Lodewijk W van LWV Hoyland Judith A JA Welting Tim J M TJM Voncken Jan Willem JW

Cartilage 20180409 2

<h4>Objective</h4>Lack of specific marker-sets prohibits definition and functional distinction of cellular subtypes in the intervertebral disc (IVD), such as those from the annulus fibrosus (AF) and the nucleus pulposus (NP).<h4>Design</h4>We recently generated immortalized cell lines from human NP and AF tissues; these comprise a set of functionally distinct clonal subtypes. Whole transcriptome analyses were performed of 12 phenotypically distinct clonal cell lines (4× NP-Responder, 4× NP-nonRe ...[more]

PMID: 29629573

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Project description:Tissue-engineered intervertebral discs (IVDs) have been proposed as a useful therapeutic strategy for the treatment of intervertebral disc degeneration (IDD). However, most studies have focused on fabrication and assessment of tissue-engineered IVDs in small animal models and the mechanical properties of the scaffolds are far below those of native human IVDs. The aim of this study was to produce a novel tissue-engineered IVD for IDD regeneration in the porcine lumbar spine. Firstly, a novel whole tissue-engineered IVD scaffold was fabricated using chitosan hydrogel to simulate the central nucleus pulposus (NP) structure, surrounded with a poly(butylene succinate-co-terephthalate) (PBST) fiber film for inner annulus fibrosus (IAF). And, a poly(ether ether ketone) (PEEK) ring was used to stimulate the outer annulus fibrosus (OAF). Then, the scaffolds were seeded with IVD cells and the cell-scaffold hybrids were transplanted into the porcine damaged spine and harvested at 4 and 8 weeks. In vitro cell experiments showed that IVD cells distributed and grew well in the scaffolds including porous hydrogel and PBST fibers. After implantation into pigs, radiographic and MRI images indicated that the tissue-engineered IVD construct could preserve the disc height in the case of discectomy as the normal disc height and maintain a large extracellular matrix and water content in the NP. Combined with the histological and gene expression results, it was concluded that the tissue-engineered IVD had similar morphological and histological structure to the natural IVD. Moreover, after implantation for 8 weeks, the tissue-engineered IVD showed a good compressive stress and elastic moduli, approaching those of natural porcine IVD. Therefore, the prepared tissue-engineered IVD construct had similar morphological and biofunctional properties to the native tissue. Also, the tissue-engineered IVD construct with excellent biocompatibility and mechanical properties provides a promising candidate for human IDD regeneration.

Project description:BackgroundDuring the intervertebral disc (IVD) degeneration process, initial degenerative events occur at the extracellular matrix level, with the appearance of neoepitope peptides formed by the cleavage of aggrecan and collagen. This study aims to elucidate the spatial and temporal alterations of aggrecan and collagen neoepitope level during IVD degeneration.MethodsBovine caudal IVDs were cultured under four different conditions to mimic different degenerative situations. Samples cultured after 1- or 8-days were collected for analysis. Human IVD samples were obtained from patients diagnosed with lumbar disc herniation (LDH) or adolescent idiopathic scoliosis (AIS). After immunohistochemical (IHC) staining of Aggrecanase Cleaved C-terminus Aggrecan Neoepitope (NB100), MMP Cleaved C-terminus Aggrecan Neoepitope (MMPCC), Collagen Type 1α1 1/4 fragment (C1α1) and Collagenase Cleaved Type I and II Collagen Neoepitope (C1,2C), staining optical density (OD)/area in extracellular matrix (OECM) and pericellular zone (OPCZ) were analyzed. Conditioned media of the bovine IVD was collected to measure protein level of inflammatory cytokines and C1,2C.ResultsFor the bovine IVD sections, the aggrecan MMPCC neoepitope was accumulated in nucleus pulposus (NP) and cartilage endplate (EP) regions following mechanical overload in the one strike model after long-term culture; as for the TNF-α induced degeneration, the OECM and OPCZ of collagen C1,2C neoepitope was significantly increased in the outer AF region after long-term culture; moreover, the C1,2C was only detected in conditioned medium from TNF-α injection + Degenerative loading group after 8 days of culture. LDH patients showed higher MMPCC OECM in NP and higher C1,2C OECM in AF region compared with AIS patients.ConclusionsIn summary, aggrecan and collagen neoepitope profiles showed degeneration induction trigger- and region-specific differences in the IVD organ culture models. Different IVD degeneration types are correlated with specific neoepitope expression profiles. These neoepitopes may be helpful as biomarkers of ECM degradation in early IVD degeneration and indicators of different degeneration phenotypes.

Dataset Information

A Membranome-Centered Approach Defines Novel Biomarkers for Cellular Subtypes in the Intervertebral Disc.

Objective

Design

Results

Conclusions

Publications

A Membranome-Centered Approach Defines Novel Biomarkers for Cellular Subtypes in the Intervertebral Disc.

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