Type II collagen-positive embryonic progenitors are the major contributors to spine and intervertebral disc development and repair

Abstract

Basic mechanism of spine development is poorly understood. Type II collagen positive (Col2+) cells have been reported to encompass early mesenchymal progenitors that continue to become chondrocytes, osteoblasts, stromal cells, and adipocytes in long bone. However, the function of Col2+ cells in spine and intervertebral disc (IVD) development is largely unknown. To further elucidate the function of Col2+ progenitors in spine, we generated the mice with ablation of Col2+ cells either at embryonic or at postnatal stage. Embryonic ablation of Col2+ progenitors caused the mouse die at newborn with the absence of all spine and IVD. Moreover, postnatal deletion Col2+ cells in spine resulted in a shorter growth plate and endplate cartilage, defected inner annulus fibrosus, a less compact and markedly decreased gel-like matrix in the nucleus pulposus and disorganized cell alignment in each compartment of IVD. Genetic lineage tracing IVD cell populations by using inducible Col2-creERT;tdTomato reporter mice and non-inducible Col2-cre;tdTomato reporter mice revealed that the numbers and differentiation ability of Col2+ progenitors decreased with age. Moreover, immunofluorescence staining showed type II collagen expression changed from extracellular matrix to cytoplasm in nucleus pulposus between 6 month and 1-year-old mice. Finally, fate-mapping studies revealed that Col2+ progenitors are essential for IVD repair in IVD injured model. In summary, embryonic Col2+ cells are the major source of spine development and Col2+ progenitors are the important contributors for IVD repair and regeneration.

Keywords: intervertebral disc; lineage-tracing; nucleus pulposus; stem cell; type II collagen.

FIGURE 1

Ablation of embryonic Col2+ progenitors in mice causes absence of the spine. A, Gross appearance of Col2‐cre, and Col2‐cre;DTA^fl/− mice at P0. B, X‐ray images for P0 Col2‐cre, and Col2‐cre;DTA^fl/− mice. Yellow arrow: spine is completely absent in mutant newborn compared to Col2‐cre control. C, Alizarin red and Alcian blue staining of newborn Col2‐cre and Col2‐cre;DTA^fl/− mouse. Yellow arrow: spine complete loss in mutant newborn. Scale bar = 1 mm. D, The tail of Col2‐cre and Col2‐cre;DTA^fl/− newborns mice stained by Goldner's trichrome. Scale bar = 300 μm. E,F, Higher magnification of Golden staining in tail vertebra and skin. Yellow arrow: vertebral and intervertebral disc (IVD) complete loss in mutant newborn. Six mice evaluated in each group. Scale bar = 100 μm

FIGURE 2

Postnatal deletion of Col2+ cells disrupt the pattern of spine. A, X‐ray of spine in 4‐week‐old Col2‐creERT and Col2‐creERT;DTA^fl/fl mice. Scale bar = 1 mm. B, The quantity analysis of mice L3‐L5 length of (A) (n = 6 mice per condition from three independent experiments). Data are mean ± SD. C, Alizarin red and Alcian blue staining for the tail from the 4‐week‐old Col2‐creERT and Col2‐creERT;DTA^fl/fl mice. Scale bar = 500 μm. D, Safranin O/fast green staining of coronal sections of L4‐5 spine in 4‐week‐old Col2‐creERT and Col2‐creERT;DTA^fl/fl mice. Scale bar = 200 μm. E‐H, High magnification pictures showing growth plate (GP), AF, and NP in 4‐week‐old Col2‐creERT and Col2‐creERT;DTA^fl/fl mice. Scale bar = 20 μm. I, Immunofluorescence staining for Col2α1 in intervertebral disc (IVD) of 4‐week‐old Col2‐creERT and Col2‐creERT;DTA^fl/fl mice. Scale bar = 50 μm. J‐L, High magnification immunofluorescence pictures showing Col2α1 protein expression in NP, AF, and GP from 4‐week‐old Col2‐creERT and Col2‐creERT;DTA^fl/fl mice. M,N, Phalloidin staining show the cytoskeleton of NP and AF in 4‐week‐old Col2‐creERT and Col2‐creERT;DTA^fl/fl mice. Statistical significance was determined by one‐way ANOVA and Student's t test. *P < .05, **P < .01, ***P < .0001. NS, not statistically significant. Scale bar = 20 μm

FIGURE 3

Spatial distribution of embryonic and postnatal Col2+ cells in the mouse intervertebral disc (IVD) and vertebral bone. A, Representative images from lineage tracing of embryonic Col2+ cells in IVD at different time point (P0, P6, P14, P30, P90, P187, and P365) in Col2‐cre;tdTomato mice. Six mice evaluated in each group. B, Representative images from lineage tracing of Col2 + cells in IVD at different time point (P6, P8, P14, P30, P90, P187, and P365) in Col2‐creERT;tdTomato mice. Tamoxifen (75 mg TM/kg body weight) were i.p. injected into P3 mice. Six mice evaluated in each group. C, Representative images from lineage tracing tdTomato+ cells in IVD from Col2‐creERT;tdTomato and Aggrecan‐creERT;tdTomato mice at P30. Tamoxifen (75 mg TM/kg body weight) were i.p. injected into P3 mice. Six mice evaluated in each group. Scale bar = 200 μm

FIGURE 4

Lineage tracing Col2+ cells in spine and intervertebral disc (IVD) at different time points. A, Lineage tracing Col2+ cells in spine and IVD at P0, P6, P14, P30, and P90 in Col2‐cre;tdTomato mice. (Since all the Col2+ cells activated from embryonic stage Col2‐cre;tdTomato mice, we only include the date for harvesting mice sample in each group. For example, P6 means: in Col2‐cre;tdTomato mice, the date for harvest is P6.) B, Quantitative measurements of the percentage of tdTomato+ cells to the total DAPI+ cells in (A) (n = 6 mice per condition from three independent experiments). Data are mean ± SD. C, Lineage tracing Col2+ cells in spine and IVD at P6, P14, P30, and P90 in Col2‐creERT;tdTomato mice. Tamoxifen (75 mg TM/kg body weight) were i.p. injected into P3 mice. Six mice evaluated in each group. (The data presented: harvest time (tamoxifen injected date + the tracing time.) For example, P6 (3 + 3) means: in Col2‐creERT;tdTomato mice, the date for harvest is P6 (TM injected date is P3 and tracing for 3 days). D, Quantitative measurements of the percentage of tdTomato+ cells to the total DAPI+ cells in (C) (n = 6 mice per condition from three independent experiments). Data are mean ± SD. Statistical significance determined by one‐way ANOVA and Student's t test. *P < .05, **P < .01, ***P < .0001. NS, not statistically significant. Scale bar = 500 μm

FIGURE 5

The decreased numbers and differentiation ability of Col2+ progenitors during aging. A, Lineage tracing of postnatal Col2+ cells for 3 days in each component of intervertebral disc (IVD) which activated at different time points (P3, P21, P27, P87, P184, and P362). 75 mg/kg tamoxifen were i.p. injected into mice at above indicated different time points. The mice were harvested at day 3 following TM injection. B, Lineage tracing of Col2+ cells in IVD at P90, which was activated at different time points (since embryo, P3, P30, P60, and P87). C, Lineage tracing Col2+ cells in IVD for 1 month while activated at different time point (since embryo, P3, P60). 75 mg/kg tamoxifen were i.p. injected into mice at above indicated different points. D, Quantitative measurements of the percentage of tdTomato+ cells to the total DAPI+ cells in (A) (n = 6 mice per condition from three independent experiments). Data are mean ± SD. E, Quantitative measurements of the percentage of tdTomato+ cells to the total DAPI+ cells in (B) (n = 6 mice per condition from three independent experiments). Data are mean ± SD. F, Quantitative measurements of the percentage of the cells with tdTomato+ to the total DAPI+ cells per view of (C) (n = 6 mice per condition from three independent experiments). Data are mean ± SD. The percentage calculation in each tissue were measured at least 1000 cells each sample. Statistical significance was determined by one‐way ANOVA and Student's t test. *P < .05, **P < .01, ***P < .0001. NS, not statistically significant. Scale bar = 200 μm

FIGURE 6

The inconsistence of Col2+ cells and type II collagen expression in mice intervertebral disc (IVD). A‐D, The expression Col2+ cells by lineage tracing of Col2‐creERT;tdTomato, Col2‐cre;tdTomato mice, and Col2α1 expression examined by IF staining in IVD at P6 (A), P30 (B), P187 (C), and P365 (D). Six mice were evaluated in each group. Scale bar = 200 μm

FIGURE 7

Col2+ progenitors are important for intervertebral disc (IVD) repair. A, The illustration of the experiment design. B, Representative of X‐ray images in Col2‐creERT;tdTomato and Col2‐creERT;DTA^fl/fl mice. Yellow arrow: the injured IVD in each group. Scale bar = 5 mm. C, Measurement protocol for the determination of the DHI calculated as [(d + e + f)/(a + b + c + g + h + i) ×100%]. D, DHI at different time‐points following injury. Values are expressed as the mean ± SD (n = 10). DHI, disc height index (n = 6 mice per condition from three independent experiments). Data are mean ± SD. E, Representative fluorescent images of coronal sections of IVD in intact Col2‐creERT;tdTomato, injured Col2‐creERT;tdTomato, and Col2‐creERT;DTA^fl/fl;tdTomato mice. Scale bar = 20 μm. F, Representative H&E images of coronal sections of IVD in intact Col2‐creERT, injured Col2‐creERT and Col2‐creERT;DTA^fl/fl mice. Scale bar = 20 μm. G, CFU‐F assay in Col2+ IAF progenitor. Scale bar = 20 μm. H, Trilineage differentiation of Col2+ progenitors. Osteogenic (left), chondrogenic (center), and adipogenic (right) differentiation conditions. Statistical significance was determined by one‐way ANOVA and Student's t test. *P < .05, **P < .01, ***P < .0001. NS, not statistically significant. Scale bar = 10 μm