Stromal cell-derived factor-1α promotes recruitment and differentiation of nucleus pulposus-derived stem cells

Abstract

BACKGROUND Intervertebral disc (IVD) degeneration is a condition characterized by a reduction in the water and extracellular matrix content of the nucleus pulposus (NP) and is considered as one of the dominating contributing factors to low back pain. Recent evidence suggests that stromal cell-derived factor 1α (SDF-1α) and its receptor C-X-C chemokine receptor type 4 (CXCR4) direct the migration of stem cells associated with injury repair in different musculoskeletal tissues. AIM To investigate the effects of SDF-1α on recruitment and chondrogenic differentiation of nucleus pulposus-derived stem cells (NPSCs). METHODS We performed real-time RT-PCR and enzyme-linked immunosorbent assay to examine the expression of SDF-1α in nucleus pulposus cells after treatment with pro-inflammatory cytokines in vitro . An animal model of IVD degeneration was established using annular fibrosus puncture in rat coccygeal discs. Tissue samples were collected from normal control and degeneration groups. Differences in the expression of SDF-1α between the normal and degenerative IVDs were analyzed by immunohistochemistry. The migration capacity of NPSCs induced by SDF-1α was evaluated using wound healing and transwell migration assays. To determine the effect of SDF-1α on chondrogenic differentiation of NPSCs, we conducted cell micromass culture and examined the expression levels of Sox-9, aggrecan, and collagen II. Moreover, the roles of SDF-1/CXCR4 axis in the migration and chondrogenesis differentiation of NPSCs were analyzed by immunofluorescence, immunoblotting, and real-time RT-PCR. RESULTS SDF-1α was significantly upregulated in the native IVD cells cultured in vitro with pro-inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, mimicking the degenerative settings. Immunohistochemical staining showed that the level of SDF-1α was also significantly higher in the degenerative group than in the normal group. SDF-1α enhanced the migration capacity of NPSCs in a dose-dependent manner. In addition, SDF-1α induced chondrogenic differentiation of NPSCs, as evidenced by the increased expression of chondrogenic markers using histological and immunoblotting analyses. Real-time RT-PCR, immunoblotting, and immunofluorescence showed that SDF-1α not only increased CXCR4 expression but also stimulated translocation of CXCR4 from the cytoplasm to membrane, accompanied by cytoskeletal rearrangement. Furthermore, blocking CXCR4 with AMD3100 effectively suppressed the SDF-1α-induced migration and differentiation capacities of NPSCs. CONCLUSION These findings demonstrate that SDF-1α has the potential to enhance recruitment and chondrogenic differentiation of NPSCs via SDF-1/CXCR4 chemotaxis signals that contribute to IVD regeneration.

Granulocyte‐colony stimulating factor improves intervertebral disc degeneration in experimental adult male rats: A microscopic and radiological study

“…In this study, IVDD resulted in an increased SDF‐1α expression detected by immunohistochemistry in both AF and NP tissues that was decreased after GCSF treatment. These results were in accordance with those of Ying et al (2019), who induced IVDD by needle puncture in rat tails and evaluated SDF‐1α immune expression 4 weeks after surgery. They reported that SDF‐1α immune expression was significantly higher in the IVDD group than the control one.…”

“…For SDF‐1α immune staining, the sections were incubated overnight with SDF‐1α primary antibodies (1:100 dilution) at 4°C. The next day, IVD sections were washed with phosphate‐buffered saline and then incubated for 20 min with horseradish peroxidase‐conjugated goat anti‐rabbit IgG (1:200 dilution) as a secondary antibody at 37°C (Ying et al, 2019).…”

“…Visualizations of SDF‐1α and caspase‐3 expressions were developed by 0.06% 3,3′‐diaminobenzidine staining, and counterstaining was then performed using hematoxylin (de Campos et al, 2016; Ying et al, 2019). A positive expression was indicated by a brown color under light microscope examination.…”

“…Thus, once the IVDD process is triggered, it is difficult to slow it down, leading to an irreversible condition (Tzaan & Chen, 2011). In such degenerative changes, it was proven that stromal cell‐derived factor‐1α (SDF‐1 α) and its receptor C‐X‐C chemokine receptor‐4 (CXCR‐4) are upregulated in the IVD tissue (Ying, Wen, Pei, Su, & Ruan, 2019). SDF‐1α is the unique strong chemoattractant for HSCs in human and rat (Vagima et al, 2011).…”

Therapeutic Strategies for IVD Regeneration through Hyaluronan/SDF-1-Based Hydrogel and Intravenous Administration of MSCs

“…In recent years, promoting cell recruitment of endogenous cell populations for the IVD has been explored as a potential therapeutic strategy [4,5]. Rather than cell transplantation, this strategy proposes the use of chemokines to enhance cell recruitment, avoiding in vitro manipulation.…”