Molecular insights into human intervertebral disc degeneration by single-cell RNA sequencing

Abstract

Low back pain shows a strong correlation with intervertebral disc degeneration. Under normal conditions, intervertebral discs are highly hydrated to facilitate backbone movement. Upon aging or pathological conditions, intervertebral discs undergo degeneration. At the center of the intervertebral disc, the nucleus pulposus (NP) absorbs compression and distributes biomechanical forces. In humans, NP cells are thought to be derived from the embryonic notochord and demonstrate morphological changes throughout the lifespan. At birth, most NP cells contain cytoplasmic vacuoles and appear notochordal-like (notochordal-like cells, NCLs). By adolescence, few NCLs remain in the NP; instead, cells resembling chondrocytes (chondrocyte-like cells, CLCs) become prominent. Later, fibroblastic-like cells (FBLs) accompany degenerative changes. Both environmental and genetic risk factors are involved in disc degeneration, albeit with unclear mechanisms. Many questions arise. Do NP cells have different lineage origin from NCLs or they are derived from further differentiation? Are these cell phenotypes a reflection of the changing environment during aging and degeneration, such as biomechanical loading and cellular stress? To gain insights into these questions, I sequenced 2,059 NP cells with or without disc degeneration. The principal component analysis showed seven NP cell populations, namely, NCLs, CLCs, degenerative CLCs expressing genes implicating inflammation pathways (i-CLCs) or myofibroblasts (f-CLCs), FBLs, myofibroblasts, and macrophages. Novel markers of each population were identified by analyzing differentially expressed genes and were validated by immunostaining. Among these genes, HOPX was found to be specifically expressed in a subpopulation of NCLs, characterized by a transitional state between NCLs and CLCs. The newly identified i-CLCs were found to be characterized by genes, implicating activation of the integrated stress response (ISR), mechanical stress, and apoptosis. In contrast, f-CLCs expressed genes characteristic of increased proliferation. To determine lineage relationships among NP cells, I utilized Cre-loxP recombinase system to label HOPX-expressing cells in NP spheroids with tdTomato. Upon attaching to plastic surface, cells at the boundary of the spheroids no longer expressed HOPX but retain expression of tdTomato and CLC markers. Cells migrated to the proximities of spheroids express tdTomato and FBL markers. Expression of tdTomato in NCLs, CLCs, and FBLs suggests that they may be originated from HOPX lineage. Loss-/Gain-of-function of HOPX in JHC7 cells revealed its role in cell fate specification through multiple pathways, including the ISR, BMP/TGF-β, and p53. Overexpression of notochord-specific genes HOPX, T, ZNF385B, and RAPGEF5 in degenerative NP cells showed increased levels of genes characteristic of NCLs, decreased levels of ISR, BMP/TGF-β, and p53 signaling. Inhibition of the ISR by a small molecule ISRIB showed decreased levels of HOPX, and increased levels of ATF3 and p53, suggesting that HOPX may be critical in the maintenance of NCLs. The findings of this study provide new insights into the characteristics of each NP cell population in humans and lineage relationships among NCLs, CLCs, and FBLs. HOPX, which was validated as a specific NCL marker, may play a critical role in cell fate specification through orchestrating multiple pathways, mainly including the ISR, BMP/TGF-β signaling, and p53 signaling.