Table 1.
IVD donor characteristics and clones.
Table 2.
Quantitative PCR primer list.
Fig 1.
Confirmation of AF cell phenotype in vitro.
A) Representative image of a human IVD tissue specimen from a scoliosis donor. The relevant tissue zones are indicated; the dotted box indicates, by approximation, the tissues section used for AF cell isolation. B) Safranin O/Fast Green staining of AF tissue from a scoliosis donor at 25x (left image) and 100x magnification (right image). Bars indicate 200 μm. C) Phase-contrast images of primary NP and AF cell pools from donor 1 (D1) and from donor 2 (D2) at passage 5 (P5). Primary AF cells were slightly more rounded, while NP cells showed a typical wave-like patterning. Bars indicate 20 μm. D) Gene expression analysis of the putative AF markers COL1A1, COL5A1, SFRP2, COL12A1 and ADAMTS17 in primary AF (white bars) and NP (black bars) cell isolates from donor 1 D1(P5) and donor 2 D2(P5), respectively; gene expression was normalized to Cyclophillin B mRNA levels and data is presented relative to expression in NP cells. Statistical significance was assessed by Student’s t-test * p<0.05.
Fig 2.
Clonal phenotypes of AF cell lines.
A) Phase-contrast images of two representative AF cell clones 102 and 133 (D2) under normal, (i.e. non-differentiation) maintenance conditions. Cell clones showed preferential cell-cell contacts and alignment at 1–2 days post-plating. Time points in days are indicated after seeding 6,400 cells/cm2 at day zero; the t = 0 image represents 12 hours post-plating. Bars indicate 20 μm. B) Spectrometric quantification of Crystal violet staining assays, reflecting growth of eleven immortalized cell clones. Every dot represents the average value of a biological triplicate measurement for a single clone. Mean values and standard deviations are depicted for eleven independent clones. At approximately 6 days post-plating (P10), a plateau was reached due to confluence of the cell cultures. C) Gene expression analysis of COL1A1, COL2A1, COMP, ACAN, SOX9 and the novel AF markers COL5A1, COL12A1, SFRP2. Every dot represents a single clone and shows the average of a biological triplicate measurement. Mean expression values and standard deviations are depicted per marker gene for eleven clones. Gene expression values were normalized to Cyclophillin B mRNA levels.
Fig 3.
TGFβ3-induced sheet formation in a subgroup of AF clones.
A) Phase contrast images of AF-S clones 102, 115, 126 and AF-nS clones 119, 123, 133 (from D2) at t = 0 and cultured in control medium (Control) or TGFβ3 containing medium (+ TGFβ3) for 7 days. Bars represent 20 μm. Cells did not exhibit sheet formation in control medium. B) Gene expression analyses of COL1A1, COL5A1, COL12A1 and SFRP2 in immortal AF cell clones. Every dot represents a single clone and is the average of a biological triplicate measurement. Gene expression was normalized to cyclophillin B mRNA levels. Fold induction (t = 7 TGFβ3 / t = 0) was calculated for each clone separately. Mean and standard deviations are depicted for the three clones together per gene. Statistical significance was assessed by Student’s t-test; * p<0.05.
Fig 4.
Collagen processing determines the sheet forming capacity in AF clones.
A) Immunoblot analyses of Collagen type I protein in AF-S and AF-nS clones cultured in control or TGFβ3 containing medium for 7 days. Procollagen-alpha 1 (180 kDa; pro-α1) and Procollagen alpha 2 (145 kDa; pro-α2) variants of Collagen type I are indicated. The appearance of mature alpha 1 (135 kDa; α1) and alpha 2 (120 kDa; α2) variants of Collagen type I correlated well with sheet formation in AF-S clones. β-Actin (βACT) was used as loading control. B) Immunoblot analyses of Collagen type I protein in AF-S clones cultured in TGFβ3 medium with or without ascorbic acid for 7 days. Alpha-tubulin (αTUB) was used as loading control. C) Quantification of Collagen maturation as a function of time in AF-S and AF-nS clones. The ratio of mature COL1A1 over ProCOL1A1 and mature COL1A2 over ProCOL1A2 are depicted in the left and right graphs, respectively. At t = 0 no mature forms of Collagen type I were detectable. Statistical significance was assessed by Student’s t-test; * p<0.05.
Fig 5.
Collagen modifying enzymes are transcriptionally regulated by TGFβ3 in sheet forming AF clones.
Upper panels: expression analysis of P4HA1-3 genes in AF-S and AF-nS clones at t = 0 and t = 7 days of culturing in TGFβ3. Middle panels: expression analysis of PLOD1-3 genes in AF-S and AF-nS clones at t = 0 and t = 7 days of culturing in TGFβ3. Lower panels: expression analysis of genes involved in cleavage of Collagen type I pro-peptides (BMP1, TLL1) and crosslinking of Collagen fibers (LOX) in AF-S and AF-nS clones at t = 0 and t = 7 days of culturing in TGFβ3. Gene expression was normalized to cyclophillin B mRNA levels. Fold induction (t = 7 TGFβ3 / t = 0) was calculated for each clone separately. Mean and standard deviations are depicted for the three clones together per gene. Statistical significance was assessed by Student’s t-test; * p<0.05.
Fig 6.
P4HA3 positivity in AF tissue is heterogeneous.
Expression of P4HA3 was determined immunohistochemically in human AF tissue. Representative images of outer AF (left panel) and inner AF (right panel) from a 45 year old male donor with no signs of disc degeneration. Asterisks: representative examples of immune-positive cellsl circles: immune-negative cells. Black bars represent 50 μm.