Senescence and degeneration of human nucleus pulposus cells from in vivo and in vitro culture: A single-cell RNA sequencing study

Background Senescence and degeneration contribute to intervertebral disc disorders in the nucleus pulposus (NP) but are often conflated. This study aimed to delineate the distinct characteristics of senescence and degeneration in NP, examine the effects of in vitro culture on NP cells, and clarify the involvement of NP progenitor cells. Methods NP samples from six patients were analyzed by single-cell RNA sequencing, with p16 levels and Pfirrmann grades representing senescence and degeneration, respectively. Results NP cells were heterogeneous, including progenitor NP cells (ProNPC), regulatory NP cells (RegNPC), effector NP cells (EffNPC), homeostatic NP cells (HomoNPC), and fibrosis NP cells (FibroNPC). Senescence increased proliferation and immune activity, whereas degeneration decreased them; RegNPC was the most perturbed subtype. In vitro culture elevated p16 levels, primarily in ProNPC. In primary cells, the level of p16 in ProNPC decreased with degeneration, which was mainly mediated by E2F1 , whose level increased with degeneration. After culture, the proportion of macrophages was extremely low, with only ProNPC and FibroNPC remaining. Conclusions These findings indicated that senescence and degeneration are distinct, in vitro culture alters NP cell properties, and ProNPCs are closely associated with degeneration, as reflected in p16 expression patterns.