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Cell models for the human intervertebral disc: nucleus pulposus and annulus fibrosis +1,2Van den Akker, GGH; 1Welting, TJM; 1Surtel, D; 1Cremers, A; 2Voncken, JW; 1Van Rhijn, LW; +1Maastricht University Medical Centre, department of Orthopaedic Surgery, the Netherlands, 2Maastricht University Medical Centre, department of Molecular Genetics, the Netherlands. [email protected] Introduction Degenerative disc disease (DDD) is an increasing socio-economic burden for developed countries (Maniadakis et al, 2000). Importantly, loss of nucleus pulposus cellularity is one of the first hallmarks of DDD which may progress to disc herniation and low back pain. Currently, InterVertebral Disc (IVD) research is hampered by a lack of good in vitro cell models. In sharp contrast, the availability of experimental models for chondrocytes (ATDC5 (Atsumi et al 1990), SW1353 (ATCC #HB-94), MCT (Lefebvre et al, 1995)) has greatly expanded our fundamental understanding of chondrocyte function, homeostasis and arthritis. The unique properties of the human IVD compared to animal model organisms (Lotz et al 2004) further emphasizes the need for human IVD models. In this study we aimed to generate clonal cell lines representing the human Nucleus Pulposus (NP) and Annulus Fibrosis (AF). Materials and methods Non-degenerated healthy disc material from young adolescent scoliosis patients was obtained as surplus material from correction surgery. We immortalized early passage monolayer cell cultures by retroviral expression of the SV40Lt and hTERT genes and by selection for coexpressed antibiotic resistance markers. Immortalized cell pools were subjected to clonal expansion and characterized on the basis of Collagen type I, II and Sox9 protein expression and a subset of published in vivo RNA markers (Minogue et al, 2010l Rutges et al, 2009; Sakai et al 2009). We generated 60 NP clones, and 40 AF clones. Chondrogenic differentiation was carried out using ITS, Ascorbic acid and TGFβ3 (Barbero et al, 2007). Results Clones were characterized for expression of immortalization markers (SV40Lt, hTERT), in addition to telomerase activity and cell biological assessment of proliferation rate and genetic stability (i.e. karyotyping). We identified two distinct morphological subclones in the NP cell pools (Fig 1A, B) that differ in Collagen type II expression (Fig 2). The first subtype, characterized by a cobble stone phenotype (Fig 1A), continuously synthesizes Collagen type II (fig2) and expresses several established NP markers such as CD24 (Fig 3D and data not shown). The second subtype displays a wave-like cell organization (Fig 1B), and produces high levels of Sox9 and small quantities of Collagen type II (Fig 2) upon differentiation. In addition these clones tend to express many NP markers (Fig3 clone 115, KRT19, FoxF1, CA12). AF cell pools predominantly yielded a third clonal phenotype, morphologically distinct from NP clones. These cells initially show a wave-like arrangement at subconfluency, but have a strong tendency to migrate and become organized in patterned alignments. Upon reaching confluency these clones revert to a cobble stone phenotype, comparable to the first NP clone subtype. Marker gene analysis for the AF subclones is ongoing. Figure 1: Subclone morphology of immortal human NP and AF clones. Representative phase contrast images of NP (A, B) and AF subclones (C) after 7 days of differentiation. Percentages of clonal morphology per tissue type (NP or AF) are indicated. Figure 2: Differential marker expression in NP subtypes. Protein analysis of Collagen Types I, II, Sox9 and β-Actin (loading control). Clones were differentiated for 7 days or kept undifferentiated. Cobble stone NP clones (105, 119) continuously express Col2AI and -II. Cleaved collagen species appear upon differentiation. Wave-like NP clones (115, 108) strongly upregulate Sox9 levels in response to differentiation. A moderate increase in Collagen type II is observed. Discussion We report for the first time the generation of clonal cell lines from nondegenerated human IVD tissue and present extensively characterized IVD cell lines. The in vitro propagated NP clones show expression of several established in vivo NP markers. Morphologically distinct NP subclones were obtained, representing chondrocyte-like NP cells and a possible progenitor (notochordal) cell type, the latter supported by KRT19 expression (Rutges et al.). We are currently evaluating marker gene expression of AF subclones and exploring the possibilities for 3D culturing. Additionally we intend to develop high throughput screening methods aimed at finding novel therapeutics for DDD. Significance These cell lines provide a promising tool to explore differential characteristics between IVD cells and articular chondrocytes. In addition the cell lines will contribute to the development of new therapeutic interventions and the elucidation of the underlying molecular mechanisms causing DDD. Figure 3: Differential NP marker expression. Representative overview of NP clones. KRT19 (A) FoxF1 (B) and CA12 (D) are mostly expressed in wave-like cultures; CD24 is expressed in both clone types. Not all clones are positive for NP markers (e.g. 116 and 118) despite similar Collagen type 2 and Sox 9 expression. Poster No. 1198 • ORS 2012 Annual Meeting