Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc

Abstract

Despite the high prevalence of intervertebral disc disease, little is known about changes in intervertebral disc cells and their regenerative potential with ageing and intervertebral disc degeneration. Here we identify populations of progenitor cells that are Tie2 positive (Tie2+) and disialoganglioside 2 positive (GD2+), in the nucleus pulposus from mice and humans. These cells form spheroid colonies that express type II collagen and aggrecan. They are clonally multipotent and differentiated into mesenchymal lineages and induced reorganization of nucleus pulposus tissue when transplanted into non-obese diabetic/severe combined immunodeficient mice. The frequency of Tie2+ cells in tissues from patients decreases markedly with age and degeneration of the intervertebral disc, suggesting exhaustion of their capacity for regeneration. However, progenitor cells (Tie2+GD2+) can be induced from their precursor cells (Tie2+GD2-) under simple culture conditions. Moreover, angiopoietin-1, a ligand of Tie2, is crucial for the survival of nucleus pulposus cells. Our results offer insights for regenerative therapy and a new diagnostic standard.

Figure 1. Surface markers to select putative progenitor cells.

(a) Two types of colonies derived from mouse primary NP cells in methylcellulose medium at 10 days (top panels): mNP-CFU-S (left) and mouse mNP-CFU-F (right). Immunocytochemical staining for the ECM indicates that mNP-CFU-S are robustly positive for type II collagen and aggrecan, whereas mNP-CFU-F are almost completely negative. Colonies were retrieved using 2 mM EDTA without enzymatic digestion and attached to slides using the Cytospin method. Scale bars, 100 μm. (b) One thousand mNP cells from each sorted population and unsorted cells were subjected to a CFA for 10 days. The frequency of mNP-CFU-S was higher in GD2⁺ sorted cells than in unsorted mNP cells. n=5, **P<0.01 (ANOVA with Mann–Whitney U-test). (c) One thousand human NP (hNP) cells from each sorted population were subjected to CFA for 10 days. The populations were Tie2⁺GD2^–CD24^– (Tie2 single-positive (T/sp) cells), Tie2⁺GD2⁺CD24^– (Tie2 and GD2 double-positive (TG/dp) cells), Tie2^–GD2⁺CD24^– (GD2 single-positive (G/sp) cells) and Tie2^–GD2^–CD24⁺ (CD24 single-positive (24/sp) cells). TG/dp cells had the greatest hNP-CFU-S formation among the four populations. n=5, *P<0.05, **P<0.01 (ANOVA with Mann–Whitney U-test). (d) Comparison of ECM production between hNP-CFU-S and hNP-CFU-F. hNP-CFU-S were immunopositive for type II collagen and aggrecan, in contrast to the low expression in hNP-CFU-F. Scale bars, 50 μm. (e) Quantitative comparison of ECM production between hNP-CFU-S and hNP-CFU-F by flow cytometry. The cytoplasm of enzymatically dispersed and membrane-permeabilized cells from the colonies was stained. A higher percentage of immunopositive cells was detected in the cells from hNP-CFU-S than in those from hNP-CFU-F. n=3, **P<0.01 (ANOVA with Mann–Whitney U-test). Data are represented as mean±s.d. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 2. Single-cell-derived multipotent cells in TG/dp cells.

(a) Single TG/dp cells were purified from 7-day cultured hNP cells and inoculated into methylcellulose medium in 96-well culture plates. Single-cell-derived hNP-CFU-S were observed on day 10. (b) Population-doubling assays of TG/dp and 24/sp clones. TG/dp clones proliferated beyond 27 doublings, whereas 24/sp clones did not. (c) Clonal differentiation assays for mesenchymal lineages. Expanded clonal cultures from single TG/dp cells were cultured with induction medium for 21 days, and osteogenesis (von Kossa/ALP: Alkaline phosphatase), adipogenesis (Oil Red O) and chondrogenesis (toluidine blue, Safranin O, aggrecan and type II collagen) were detected. Scale bars, 100 μm. (d) Survival of the transplanted cells in vivo detected by bioluminescence imaging (BLI). An EGFP-labelled TG/dp clone or EGFP-labelled 24/sp clone was injected into injured tail IVDs of NOD/SCID mice (1 × 10⁵/IVD, respectively). BLI shows the survival of the TG/dp clone at day 31, whereas the 24/sp clone disappeared (upper left). Negative control mice without cell transplantation were used for calibration of autofluorescence. Superior survival of TG/dp cells is shown in the graph, n=3, *P<0.05, (ANOVA with Tukey’s post hoc test). EGFP-labelled TG/dp clone cells were detected inside the harvested disc sections (bottom left, mid-coronal; middle two panels, transverse sections). Coexpression of type II collagen with EGFP was detected by immunostaining (low magnification and high magnification, in three colours). Scale bars, 50 μm. (e) For osteogenesis in vivo, an EGFP-labelled TG/dp clone (1 × 10⁵) was injected with a hydroxyapatite scaffold into a femoral bone defect model in NOD/SCID mice. Eight weeks later, Masson’s trichrome staining (top left) of the paraffin section identified blue collagenous fibres surrounding the EGFP⁺ cells (bottom left). Immunohistochemical staining showed positive staining for osteocalcin (top right) and osteocalcin expression in the EGFP⁺ cells (bottom right) in a cavity within the hydroxyapatite (*). Straight bars, 50 μm; arrowed bars, 20 μm, data are represented as mean±s.d. DAPI, 4′,6-diamidino-2-phenylindole; Neg. cont., negative control.

Figure 3. Self-renewal capacity and NP tissue reorganization.

(a) Strategy for testing the self-renewal capacity of TG/dp cells by serial subcutaneous transplantation into NOD/SCID mice. Sorted cell populations (TG/dp, G/sp and 24/sp cells) transduced with the EGFP gene were transplanted subcutaneously with lethally irradiated (15 Gy) allogeneic hNP tissues (0.10 g) as a scaffold. FCM, Flow cytometry. (b) Transplantation of scaffold tissue alone (upper panels). Transplantation of EGFP-labelled TG/dp cells (1 × 10⁵) with tissue (lower panels). A number of EGFP⁺ cells were detected in the TG/dp transplanted specimen, and histological analyses detected robust type II collagen and proteoglycan staining (Safranin O and toluidine blue). Scale bars, 200 μm. (c) Harvested tissue weight was maintained when transplanted with TG/dp. However, tissues transplanted without cells or with other cell populations (G/sp, 24/sp cells) lost considerable weight. n=5, **P<0.01 (ANOVA with Mann–Whitney U-test). (d) Percentage of EGFP⁺ cells detected by flow cytometry in cells retrieved from the transplants: 29.0±9.3% from TG/dp transplants, 4.4±3.8% from G/sp transplants and 0.5±0.4% from 24/sp transplants (top left). The corresponding cell numbers are shown (top right). After retrieval, the growth rates of cells cultured for 2 weeks were compared. The growth rate was higher in TG/dp transplants than in G/sp or 24/sp transplants (bottom left). n=5 *P<0.05, **P<0.01 (ANOVA with Mann–Whitney U-test). (e) EGFP⁺ cells from the TG/dp transplants maintained their multipotent differentiation ability towards osteogenesis (von Kossa/ALP), adipogenesis (Oil Red O) and chondrogenesis (type II collagen, aggrecan and Safranin O). Scale bars, 100 μm. (f) The expanded EGFP⁺ cells from the primary TG/dp transplants were subjected to secondary transplantation with lethally irradiated (15 Gy) allogeneic hNP tissue (0.10 g) as a scaffold. EGFP⁺ cells and type II collagen (left) and proteoglycan staining (Safranin O and toluidine blue, middle and right) were still detected in frozen sections of the harvested tissues. Scale bars, 200 μm. (g) The ratio and cell number of EGFP⁺ cells retrieved from five individual recipients after secondary transplantation. EGFP-labelled TG/dp cells underwent two rounds of transplantation and still maintained high growth capacity for 2 weeks after retrieval. Data are represented as mean±s.d.

Figure 4. Function of Tie2–Ang-1 signalling in the hNP.

(a) In hNP tissues, immunohistochemistry detected Tie2⁺ cells and Ang-1 expression in the matrix and cells (top left, cavities in hNP tissue; top right, the cavity magnified). Coexpression of Tie2 and Ang-1 was also detected (middle and bottom panels). Straight bars, 50 μm; arrowed bars, 10 μm. (b) Apoptosis assay in a serum-free culture of hNP cells. Addition of Tie2-blocking polyclonal antibody (BpAb) increased the number of apoptotic hNP cells after 48 h compared with medium alone or addition of goat control Ig (CIg). Apoptotic cells were identified by flow cytometry as Annexin V-positive and PI-negative cells. n=3, *P<0.05 (ANOVA with Mann–Whitney U-test). (c) Quantitative comparison by real-time RT–PCR of Ang-1 mRNA expression in the sorted hNP cell fractions. Gene expression levels were normalized to 18S rRNA and are presented relative to the levels in Tie2^–GD2^–CD24^– cells. n=3, *P<0.05 between the sorted cell fractions, #P<0.05 compared with the Tie2^–GD2^–CD24^– cell control (ANOVA with Mann–Whitney U-test). (d) sAng1 significantly upregulated hNP-CFU-S generation. Note the significant reduction in hNP-CFU-S generation with addition of Tie2 BpAb compared with the CIg. n=5, **P<0.01, †P<0.05 compared with the no-sAng1 control (ANOVA with Mann–Whitney U-test). (e) Cocultures in contact with AHESS-5 feeder cells through a culture insert (murine stromal cells overexpressing human Ang-1). The frequency of hNP-CFU-S increased significantly in the NP cells cocultured with AHESS-5, whereas the frequency of hNP-CFU-F did not change. Addition of Tie2 BpAb to the coculture medium blocked this effect. n=3, *P<0.05, †P<0.05 compared with the HESS-5 (without overexpression of Ang-1) control (ANOVA with Mann–Whitney U-test). (f) A comparison of the three hNP cell populations (T/sp, TG/dp and G/sp) shows marked Ang-1-dependent growth in the two Tie2⁺ populations (T/sp and TG/dp). n=3, *P<0.05 between the groups, †P<0.05 compared with the HESS-5 control. Data are represented as mean±s.d.

Figure 5. Expression of Tie2 and GD2 in IVD tissues.

(a) Histological analysis of paraffin sections of 9-week-old mouse tail IVDs sectioned in the mid-coronal plane and stained with haematoxylin and eosin (HE, top left). Line A indicates the direction of the sectioning, and the location was identified in the frozen sections used for immunofluorescence. The HE image on the far left shows the AF and NP border, and the right HE image shows the central NP. Fluorescent staining results show visualization of F-actin fibres of the cytoskeleton of mNP cells by phalloidin reagent (green). Expression of Tie2 (top middle, red in mNP cells; top right, the stained cells magnified) and GD2 (bottom middle, red in mNP cells; bottom right, stained cells magnified) on the cell surface was confirmed. Yellow scale bars, 20 μm; black and white bars, 50 μm. (b) Histological paraffin section of human IVD tissue (Thompson grade II) stained with HE identifies the corresponding locations in the following frozen sections indicated in the middle and lower panels (top panel; scale bar, 1 mm). Middle (HE) and bottom (immunofluorescence) panels of sections obtained from the AF (far left) to central NP (far right). No apparent immunostaining was detected for Tie2 or GD2 in the AF area (bottom left), but GD2⁺ and Tie2⁺ cells were detected (bottom centre to far right) in other areas. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 6. Decrease of Tie2⁺ NP cells with ageing and degeneration.

hNP cells donated by patients were studied together with their clinical profiles. Cells were freshly dispersed and only cells detected by flow cytometry within the live and the PI-negative gate were analysed. (a) Representative flow cytometry data of Tie2 and GD2 cell positivity in different age groups. (b) The frequency of Tie2⁺ cells (T/sp and TG/dp hNP cells) began to decrease before 20 years of age and correlated negatively with age (n=23, R²=0.9224). (c) The frequency of hNP-CFU-S generation also decreased with age (n=23, R²=0.8665). (d) The frequency of Tie2⁺ cells (T/sp and TG/dp hNP cells) decreased in relation to the extent of disc degeneration graded by morphology and (e) with disc degeneration graded by diagnostic magnetic resonance imaging (n=11). (f) The frequency of hNP-CFU-S generation decreased in relation to the extent of disc degeneration graded by morphology (n=23). *P<0.05, **P<0.01 (ANOVA with Mann–Whitney U-test). Data are represented as mean±s.d.

Figure 7. Schematic model of NP cell differentiation.

NP cell differentiation pathway was identified by cell surface markers based on experimental results. Individual surface marker expression is shown based on the experimental results (Supplementary Fig. S6). The arrowed circle indicates self-renewal; the blue arrows indicate irreversible change; the red arrows indicate supply of Ang-1 protein.