Nucleus Pulposus Cell Conditioned Medium Promotes Mesenchymal Stem Cell Differentiation into Nucleus Pulposus-Like Cells under Hypoxic Conditions

Abstract

Low back pain (LBP) is a major physical and socioeconomic challenge worldwide. Nucleus pulposus (NP) is directly associated with LBP due to intervertebral disc (IVD) degeneration. IVD degeneration is mainly caused by structural and matrix-related changes within the IVD occurring during aging and degeneration. Mesenchymal stem cells (MSCs) can differentiate into multiple mesenchymal lineages under specific stimulatory conditions. This study is aimed at evaluating the effectiveness of the nucleus pulposus cell (NPC) conditioned medium for promoting the expression of MSCs and at confirming the expression of healthy NP phenotypic markers recently recommended by the Spine Research Interest Group. Expression was investigated using quantitative polymerase chain reaction (qPCR) and western blotting under normoxic and hypoxic conditions. qPCR and western blotting demonstrated significant upregulation of NP marker expression in MSCs cultured under hypoxic conditions and treated with the 50% or 100% NPC conditioned medium, compared with those cultured under normoxic conditions. Upregulation was highest in the presence of the 100% NPC conditioned medium compared with the control group (aggrecan, p < 0.01; brachyury, p < 0.05; collagen II, p < 0.001; KRT8, p < 0.01; KRT19, p < 0.001; and Shh, p < 0.01). The expression levels of genes in MSCs treated with the 50% NPC conditioned medium also showed upregulation compared with the control group (collagen II, p < 0.05; KRT8, p < 0.05; and KRT19, p < 0.01). These findings suggested that the NPC conditioned medium stimulated MSC differentiation into an NP-like phenotype with distinct characteristics. The results could inform strategies for IVD regeneration.

Figure 1

Markers recommended for defining the young healthy NP phenotype. Relative gene expression by qPCR in NPCs cultured under normoxic and hypoxic conditions. Gene expression levels for each sample were normalized against the housekeeping gene β-actin. (a) Aggrecan, (b) brachyury, (c) carbonic anhydrase 3, (d) carbonic anhydrase 12, (e) CD24, (f) collagen II, (g) Glut-1, (h) HIF-1α, (i) HIF-2α, (j) KRT8, (k) KRT18, (l) KRT19, and (m) Shh. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Figure 2

(a) Relative protein expression by western blotting in NPCs cultured under normoxic and hypoxic conditions. Protein expression levels for each sample were normalized against the housekeeping gene β-actin. (b) Collagen II, (c) Glut-1, (d) CD24, (e) carbonic anhydrase 12, (f) brachyury, (g) aggrecan, (h) HIF-1α, (i) HIF-2α, (j) carbonic anhydrase 3, (k) KRT8, (l) KRT18, (m) KRT19, and (n) Shh. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Figure 3

Relative protein expression in NPCs cultured under normoxic and hypoxic conditions, measured by immunofluorescence. (a) Aggrecan, (b) brachyury, (c) carbonic anhydrase 3, (d) CD24, (e) collagen II, (f) Glut-1, (g) HIF-1α, (h) HIF-2α, (i) KRT8, (j) KRT18, (k) KRT19, and (l) Shh.

Figure 4

qPCR assessment of relative gene expression in MSCs under normoxic and hypoxic conditions with the 50% or 100% NPC conditioned medium. Gene expression levels for each sample were normalized against the housekeeping gene β-actin. (a) Aggrecan: (i) normoxia, (ii) hypoxia; (b) brachyury: (i) normoxia, (ii) hypoxia; (c) collagen II: (i) normoxia, (ii) hypoxia; (d) KRT8: (i) normoxia, (ii) hypoxia; (e) KRT18: (i) normoxia, (ii) hypoxia; (f) KRT19: (i) normoxia, (ii) hypoxia; and (g) Shh: (i) normoxia, (ii) hypoxia. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Figure 5

(a–c) Western blotting assessment of relative protein expression in MSCs under normoxic and hypoxic conditions with the 50% or 100% NPC conditioned medium. Protein expression levels for each sample were normalized against the housekeeping gene β-actin. (d) Aggrecan: (i) normoxia, (ii) hypoxia; (e) brachyury: (i) normoxia, (ii) hypoxia; (f) collagen II: (i) normoxia, (ii) hypoxia; (g) KRT8: (i) normoxia, (ii) hypoxia; (h) KRT18: (i) normoxia, (ii) hypoxia; (i) KRT19: (i) normoxia, (ii) hypoxia; and (j) Shh: (i) normoxia, (ii) hypoxia. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.