Spheroid culture induces a quiescent state in nucleus pulposus stem cells by inhibition of the PI3K-Akt-mTOR pathway and activation of autophagy

Intervertebral disc degeneration (IVDD) is a primary cause of low back pain, and stemness exhaustion of nucleus pulposus stem cells (NPSCs) represents a key underlying mechanism. Thus, maintaining the quiescent state of NPSCs is of great significance in delaying disc degeneration. This study aimed to investigate the inductive effect of in vitro three-dimensional (3D) spheroid culture on the quiescent state of NPSCs and its regulatory mechanism, thereby providing experimental evidence for targeted intervention in IVDD.

Intervertebral disc tissues were collected from juvenile and adult patients undergoing lumbar surgery in our department between December 2025 and January 2026. Tissue sections were prepared, and morphological differences were observed through HE staining. NPSCs were isolated from 24 male SD rats (8 weeks old, weighing 350±50 g). Stem cell immunophenotypes were identified by flow cytometry, and the stemness of NPSCs was verified by trilineage differentiation assay. NPSCs cultured under routine 2D conditions served as the monolayer culture (ML) group, while NPSCs cultured using the 3D spheroid culture system were established as the spheroid culture (SP) group. Cell morphology was observed under a microscope, cell cycle distribution was analyzed by flow cytometry, and the quiescent state was identified by EdU staining. Bulk transcriptome sequencing and bioinformatic analysis were performed on NPSCs from the ML and SP groups. The expression of autophagy-related molecules and key molecules in the PI3K-Akt-mTOR signaling pathway, as well as the ultrastructure of autolysosomes, were detected by RT-qPCR, Western blotting and transmission electron microscopy (TEM), respectively.

Clinical histological observation showed that nucleus pulposus cells in juvenile patients were scattered in distribution, whereas those in adult patients exhibited clustered distribution, and the proportion of clustered nucleus pulposus cells was significantly higher in adult patients than in juvenile patients (P<0. 01). The successfully isolated rat NPSCs highly expressed mesenchymal stem cell-positive markers CD90 (99. 4%), CD44 (99. 8%) and CD73 (94. 2%), and lowly expressed hematopoietic stem cell-negative markers CD34 (0. 21%), CD45 (0. 45%) and CD11 (1. 08%) as determined by flow cytometry. Trilineage differentiation assays confirmed that NPSCs possessed osteogenic, chondrogenic and adipogenic differentiation potentials, consistent with the phenotypic characteristics of mesenchymal stem cells. In vitro spheroid culture induced NPSCs to form regular spheroids with smooth edges, with an average diameter of 52. 3±11. 6 μm. Cell cycle analysis revealed that the proportion of G0-phase cells in the SP group was significantly higher than that in the ML group (P<0. 05). Moreover, EdU-positive cell proportion was markedly lower in the SP group than in the ML group (P<0. 05), indicating a typical quiescent state. RT-qPCR showed that the expression of P27, SOX2 and CD34 in the SP group were significantly higher than those in the ML group (P<0. 05). Bulk RNA-sequencing demonstrated significant differentially expressed genes between the SP and ML groups, with significant enrichment of the autophagy pathway and the PI3K-Akt-mTOR pathway. Western blotting demonstrated that, compared with the ML group, the ratio of LC3-Ⅱ/LC3-Ⅰ was significantly increased, while the expression of p62 was notably decreased in the SP group. The expression levels of key molecules in the PI3K-Akt-mTOR pathway, such as PI3K, p-Akt and p-mTOR were all significantly downregulated (P<0. 05). TEM revealed a markedly higher number of autolysosomes in the SP group than in the ML group. Rescue experiments further verified the regulatory relationship between the PI3K-Akt-mTOR pathway and autophagy (P<0. 05). Clinical sample detection showed that the expression levels of Ki67 and p-mTOR in nucleus pulposus tissues were significantly higher in juvenile patients than in adult patients, whereas the content of LC3B was significantly lower (P<0. 05).

In vitro 3D spheroid culture can effectively induce NPSCs to enter a quiescent state and maintain their stemness. The underlying mechanism may involve the activation of autophagy via inhibition of the PI3K-Akt-mTOR pathway.