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. 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.