CDKN2A-associated senescence-like and degeneration-related changes in human nucleus pulposus cells and culture-associated alterations: a single-cell RNA sequencing study

Background CDKN2A -associated senescence-like changes and degeneration-related changes frequently coexist in the nucleus pulposus (NP), but their cellular and transcriptional features may not fully overlap. This study investigated donor-level patterns associated with CDKN2A expression and Pfirrmann grade in human NP, examined changes accompanying short-term in vitro culture, and further characterized a putative progenitor-like cycling NP cell population. Methods NP tissues from six donors were analyzed by single-cell RNA sequencing, with the donor treated as the biological replicate. CDKN2A expression was evaluated both as a continuous donor-level variable and using post hoc CDKN2A -intensity groups, whereas degeneration was assessed using Pfirrmann grades II, III, and IV. Cell composition and cell-cycle distributions were summarized at the donor level, transcriptomic differences were evaluated using donor-aware pseudobulk analyses and Gene Ontology Biological Process GSEA, and subtype-level differences were explored using donor-blocked Augur analyses. Three donors had matched freshly isolated and first passage cells. Transcription-factor regulon activity was further assessed using pySCENIC/AUCell. Additional independent samples were used for histological and flow-cytometric assessments. Results Five major NP cell subtypes were identified: ProNPC, RegNPC, EffNPC, HomoNPC, and FibroNPC. Across the six donors, CDKN2A -associated and Pfirrmann-associated patterns in cell composition, cell-cycle distribution, and pathway activity were heterogeneous and not consistently concordant. In donor-blocked Augur analyses, FibroNPC ranked highest across post hoc CDKN2A -intensity groups, whereas RegNPC ranked highest across Pfirrmann grades. In the three matched donors, first passage was accompanied by increased overall CDKN2A expression in all three donors. Culture was also accompanied by descriptive remodeling of NP cell composition, with increased FibroNPC and ProNPC proportions and decreased EffNPC, HomoNPC, and RegNPC proportions in the paired samples. ProNPC showed relatively high CDKN2A expression together with prominent cell-cycle features, and donor-aware regulon analysis identified nine candidate regulons with consistently higher activity in ProNPC across all six donors. These analyses did not establish direct regulation of CDKN2A or causal control of the ProNPC state. Conclusions The findings suggest that CDKN2A -associated senescence-like changes and Pfirrmann-associated degeneration in human NP are not fully concordant. First passage culture was accompanied by changes in CDKN2A expression and NP cell composition that may reflect culture-associated selection and adaptation. The identified ProNPC population showed progenitor-like and cycling features together with a distinct regulatory program, but its functional identity requires further validation. Clinical trial number Not applicable.