Download Cell models for the human intervertebral disc: nucleus pulposus and

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