Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus

Abstract

IntroductionLoss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors.MethodsNon-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ).ResultsThe AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections.ConclusionWe report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.

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

“…In support of our data, it was recently shown by RNA in situ hybridization that NP and transition zone cells, which did not have a NC morphology, express bKrt19 [26]. Moreover, we previously found that Brachyury/T mRNA and protein levels do not necessarily match in mature human NP cells [10]. A detailed age range should be performed on the histological level to determine the rate of NC depletion in the bovine tail IVD.…”

“…Previously we used six NP specific marker genes to discriminate cultured human NP from AF cells [10]. We hypothesized that these genes would be able to distinguish the NP from inner AF tissue.…”

“…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].…”

Annulus fibrosus cell phenotypes in homeostasis and injury: implications for regenerative strategies

“…The glycosaminoglycan keratan sulfate was identified in the inner and outer AF of the developing rat IVD, and has also been identified in human AF tissue as a specific matrix protein of annulocytes . A transcriptional signature of COL1A1, COL5A1, COL12A1 , and SFRP2 was proposed for a population of annulocytes isolated from young human tissue after culture . The association of type XII collagen with type I collagen is well established and is thought to modify the interactions between collagen I fibrils and surrounding matrix.…”

“…The association of type XII collagen with type I collagen is well established and is thought to modify the interactions between collagen I fibrils and surrounding matrix. SFRP2 , a modulator of Wnt signaling, is a putative AF marker specific to outer annulocytes in human and bovine tissue . Outer annulocytes share similar markers to other dense, fibrous connective tissue cells, such as tenocytes and ligamentocytes, including the genes Tnmd, Mkx , and Scx in human, bovine, and murine AF …”