Single-cell analysis reveals fibroblast heterogeneity and myofibroblast conversion in ligamentum flavum hypertrophy. Single-cell analysis reveals fibroblast heterogeneity and myofibroblast conversion in ligamentum flavum hypertrophy

The ligamentum flavum (LF) is a crucial structure in maintaining spinal stability; however, hypertrophy of the LF is a significant contributor to lumbar spinal canal stenosis (LSCS). However, the mechanisms linking LF hypertrophy to the exacerbation of LSCS remain incompletely understood. In this study, we investigated the cellular proportions and signaling pathways observed in the hypertrophied LF using single-cell RNA sequencing (scRNA-seq). The LF tissues were obtained from three patients undergoing decompressive surgery at the lumbar level. These patients had been diagnosed with LSCS prior to surgery and had an LF thickness exceeding 4 mm. After single-cell dissociation of the LF tissues, scRNA-seq was performed. Fibroblasts accounted for 75% of the total cells, followed by endothelial cells, T cells, macrophages, and B cells. Among heterogeneous types of fibroblasts, we identified that a subset of fibroblasts trans-differentiated into myofibroblasts. Two types of macrophages that exhibited phenotypic plasticity akin to M1 and M2 states were observed. We also identified novel signaling pathways involved in fibroblast and immune cell interaction in the hypertrophied LF, such as GAS and GRN, as well as known signaling pathways, such as TGF-β, PDGF, CXCL, and ANGPTL. Our study highlights the transdifferentiation process from fibroblasts to myofibroblasts in the hypertrophied LF through intrinsic changes in and extrinsic influences of fibroblasts. Overall design: Human ligamentum flavum tissues diagnosed with LSCS accompanied with Ligamentum Flavum were collected during surgery involving decompressive lumbar laminectomy. LF tissue was dissociated and cells were isolated. The suspended cells were processed through 10x Genomics and were analyzed as scRNAseq.

黄韧带（ligamentum flavum, LF）是维持脊柱稳定性的关键结构，然而黄韧带肥厚是腰椎管狭窄症（lumbar spinal canal stenosis, LSCS）的重要致病因素之一，但黄韧带肥厚与腰椎管狭窄症加重之间的关联机制尚未完全阐明。本研究采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）技术，分析了肥厚黄韧带中的细胞组成与信号通路特征。研究样本取自3名术前确诊为腰椎管狭窄症、且黄韧带厚度超过4mm的腰椎减压手术患者的黄韧带组织。对获取的黄韧带组织进行单细胞解离后，开展了scRNA-seq检测。细胞组分分析结果显示，成纤维细胞占总细胞数的75%，其余依次为内皮细胞、T细胞、巨噬细胞与B细胞。在异质性成纤维细胞亚群中，我们鉴定到一类可转分化为肌成纤维细胞的成纤维细胞亚群；同时观察到两种分别具有类似M1、M2表型可塑性的巨噬细胞亚群。此外，本研究还鉴定出肥厚黄韧带中参与成纤维细胞与免疫细胞互作的新型信号通路（如GAS、GRN），以及已知信号通路（如TGF-β、PDGF、CXCL、ANGPTL）。本研究通过解析成纤维细胞的内在变化与外在调控因素，揭示了肥厚黄韧带中成纤维细胞向肌成纤维细胞的转分化过程。实验整体设计：收集腰椎减压椎板切除术中获取的、确诊为腰椎管狭窄症且伴黄韧带肥厚的人类黄韧带组织，解离组织并分离细胞，通过10x Genomics平台对悬浮细胞进行处理，后续开展scRNA-seq分析。