Oral Presentation 51st International Society for the Study of the Lumbar Spine Annual Meeting 2025

INTRODUCTION

Our research has focused on acquiring nucleus pulposus (NP) cells optimized for therapeutic application in regenerative medicinal products aimed at alleviating intervertebral disc degeneration.¹ Previously, we reported² that the use of laminin 511-coated dishes for NP cell cultures, which produces highly functional cells with enhanced Tie2 positivity and type II collagen (Col2) expression2. In this study, we aimed to investigate the regulatory mechanisms underlying the enhanced functional properties, utilizing advanced methodologies.

METHODS

Human NP tissues (n=6, age: 18.8 ± 4.0 years) were collected from patients undergoing microdiscectomy surgery after obtaining approval from the authors’ Institutional Ethics Review Board (17R173) and informed consent from the patients. Tissue fragments were collected following Sako et al.³ and applied as whole tissue fragment cultures (WTC) for two weeks in 20% fetal bovine serum at 5% O₂, 5% CO2. After the initial WTC culture, samples were collected and either (1) immediately analyzed or (2) cultured in uncoated dishes for a further week, followed by 3 x 10⁴ cells/5 mL culture in uncoated or laminin-coated plates for another week. Additionally, cultures were supplemented with 20µM c-MYC Inhibitor  (SANTA CRUZ, c-MYC (9E10): sc-40). RNA and proteins were extracted from the cultured products and analyzed using RNA-Seq (SMART-Seq v4 Ultra Low Input Kit＋Nextera XT DNA Library Prep Kit, Takara Bio Inc.) and proteomic analysis (Standard DIA proteome analysis, Kazusa DNA Research Institute). Significant differential expression in RNA-Seq and proteomic data was defined by a fold log-change ≥ 2 and p < 0.05. Findings were confirmed through Western blotting. Furthermore, the relationship between MYC/p38 and Tie2 (R&D SYSTEMS, FAB3131A) was assessed using flow cytometry and cytokine concentrations in the supernatant were assessed using ELISA (abcam). For statistical analysis, p<0.05 was considered statistically significant and the results are presented as mean ± standard deviation.

RESULTS

RNA-Seq and proteomic analyses revealed distinct expression profiles between the laminin- and non-coated cultures. (Fig 1a) Further analysis identified MYC as a key differentially expressed gene. (Fig 1b) Focusing on MYC/p38 signaling, Western blotting conformed that p38 expression was significantly suppressed following laminin-coated cultures. (Fig 1c,d) Conversely, Tie2 expression was significantly enhanced following laminin-coating regardless of c-MYC Inhibitor supplementation. The c-MYC Inhibitor in non-coated cultures significantly enhanced Tie2 expression compared to untreated controls.(Fig 1e) IL1β in the supernatant was significantly suppressed by laminin-coating, suggesting a more anabolic phenotype.

DISCUSSION

Our study revealed that the beneficial effects of laminin-coated dishes were in part mediated by suppression of MYC/p38 signaling expression and which resulted in the acquisition of juvenile and more functional NP cells. Conversely, increased MYC expression appears to be associated with the maturation of the NP cell populations. These findings support the development of cell-based therapeutics⁴ by controlling or potentially modulating MYC/p38 signaling pathways.