Confirmation of the nucleus pulposus cell phenotype in vitro. (A) Representative phase-contrast images of primary nucleus pulposus (NP) and annular fibrosus (AF) cells from donor 1 (D1) and donor 2 (D2). Passage numbers (Px) for each donor’s tissue are indicated in parentheses (for example, D1 (P0)). Black bars = 20 μm. (B) Top: Graphed results of gene expression analysis of the chondrocyte markers collagen type I, α1 (COL1A1), and collagen type II, α1 (COL2A1), in primary AF (white bars) and NP (black bars) cell isolates from D1 (P0) and D2 (P1) tissues, respectively. Gene expression was normalized to β-actin (bACT) mRNA levels. Data presented are relative to the AF values. Lower panels: Immunoblot analysis of primary AF and NP cell lysates from two independent donors, D1 (P0) and D3 (P1), respectively, for COL1A1 and COL2A1. bACT was used as a loading control. (C) Representative phase-contrast images of AF and NP cultures from D4 and D5, both at P5. Black bars = 20 μm. (D) Immunoblot analysis of COL1A1, COL2A1 and SRY-box 9 (SOX9) on cell lysates from P5 AF and NP cells. (E) P5 AF and NP cells were stimulated for 7 days with differentiation medium, and cell lysates were analysed for COL1A1, COL2A1 and SOX9. bACT was used as a loading control. (F) Gene expression (mRNA) analysis of six NP markers: keratin 19 (KRT19), Carbonic anhydrase XII (CA12), cluster of differentiation 24 (CD24), Forkhead box F1 (FoxF1), paired box 1 (Pax1) and pleiotrophin (PTN) in cultured AF (white bars) and NP cell isolates (black bars) in tissue from three independent donors: D2 (P1), D4 (P5) and D5 (P5). Marker gene expression was normalized to bACT levels. NP data presented are relative to AF values (per patient). aIndicated P-values were obtained by comparing AF and NP values combined for all three donors. Statistical significance was assessed by Student’s t-test. *P < 0.05; **P < 0.01. P-values for AF vs NP comparisons per donor are listed in Additional file 1: Table S1.