Cellular and molecular landscapes of human tendons across the lifespan revealed by spatial and single-cell transcriptomics

Abstract Tendon injuries are common and often heal poorly. While developing tendons heal without scarring, this capacity declines with age, yet the underlying cellular transitions remain poorly defined. Here, we integrate histological, single-nucleus, single-cell, and spatial transcriptomic profiling of human Achilles and quadriceps tendons across embryonic, foetal, and adult stages, including ruptured adult tendons. We identify seven embryonic progenitor states that give rise to three distinct tendon-associated fibrillar, connective tissue, and chondrogenic lineages. These populations diversify during development and occupy distinct spatial niches, adopting specialised roles in matrix synthesis, tissue remodelling, and mechanical adaptation. While non-fibroblast populations remain transcriptionally stable with age, fibroblasts undergo marked reprogramming, shifting to homeostatic or injury-responsive states. In ruptured adult tendons, a subset of fibroblasts partially reactivates developmental programs but remains transcriptionally distinct from their regenerative counterparts. These findings define the cellular architecture of human tendon development and ageing and reveal lineage-specific targets for therapeutic repair. Data description This dataset comprises the 10X Visium spatial transcriptomics data from human fetal Achilles (Dev16126_Ach_EnthMB) and Quadriceps (Dev16126_Quad_MB and Dev16126_Quad_MB2) tendons. For each sample this dataset contains: cellranger filtered_feature_bc_matrix spatial files, including Loupe files Methods: Foetal Achilles (N=1) and quadriceps tendons (N=2) (Table S1) were dissected from both legs of a single 20 pcw foetus and flash frozen in liquid nitrogen. In preparation for spatial transcriptomics (ST), the samples were cut to ≤0.65 cm² to fit the 10x Genomics Visium ST slide regions. We were able to retain enthesis-to-MTJ as well as adjacent muscle tissue regions for both types of tendons. The samples were embedded in cold OCT mounting medium (VWR) on dry ice and cut longitudinally into 10 μm sections, which were then fixed and stained with H&E to verify tissue morphology and suitability. The sections were prepared for sequencing according to the 10X Genomics recommended protocols using the Visium Gene Expression Slide and Reagent Kit alongside a Dual Index Kit TT Set A. Libraries were sequenced using Illumina NextSeq500 (paired-end) at a depth of 54,000 (Quad2 tendon), 74,000 (Ach) and 119,000 (Quad1) mean reads per spot. The data and images were processed with SpaceRanger (v1.3.1; 10X Genomics) using default settings and mapped to the GRCh38 reference genome.### 摘要 肌腱损伤十分常见，且往往愈合效果不佳。发育中的肌腱可实现无疤痕愈合，但该能力会随年龄增长逐渐衰退，其背后的细胞转变机制仍未明确。本研究整合了人类跟腱（Achilles tendon）与股四头肌肌腱（quadriceps tendon）在胚胎期、胎儿期及成年期（含断裂的成年肌腱）的组织学、单细胞核转录组分析（single-nucleus transcriptomic profiling）、单细胞转录组分析（single-cell transcriptomic profiling）及空间转录组分析（spatial transcriptomic profiling）数据。我们鉴定出7种胚胎祖细胞状态，这些祖细胞可分化为3种不同的肌腱相关纤维状、结缔组织及软骨生成细胞谱系。这些细胞群在发育过程中发生分化，并占据独特的空间微环境，在基质合成、组织重塑及机械适应中发挥特化功能。非成纤维细胞群的转录组特征随年龄保持稳定，而成纤维细胞则经历显著的重编程，转向稳态或损伤应答状态。在断裂的成年肌腱中，部分成纤维细胞会部分重激活发育程序，但在转录组层面仍与再生型对应细胞存在显著差异。本研究明确了人类肌腱发育与衰老的细胞架构，并鉴定出可用于治疗性修复的谱系特异性靶点。 ### 数据说明 本数据集包含人类胎儿期跟腱（Dev16126_Ach_EnthMB）与股四头肌肌腱（Dev16126_Quad_MB、Dev16126_Quad_MB2）的10X Visium空间转录组（10X Visium spatial transcriptomics）数据。每个样本包含以下内容： 1. cellranger过滤后的特征条形码矩阵（cellranger filtered_feature_bc_matrix） 2. 空间组学文件，包括Loupe文件（Loupe files） ### 实验方法 本研究从1名孕20周（20 pcw，即受孕后周数）胎儿的双侧下肢分离得到1例跟腱样本与2例股四头肌肌腱样本（详见表S1），随后置于液氮中快速冷冻。为开展空间转录组测序，将样本切割至面积≤0.65 cm²以适配10x Genomics Visium空间转录组芯片区域。我们成功保留了两类肌腱的附着点至肌腱连接点区域以及相邻肌肉组织区域。将样本置于干冰上，用低温OCT包埋剂（OCT mounting medium，VWR品牌）进行包埋，随后纵向切割为10 μm厚的切片，经固定后进行苏木精-伊红染色（H&E staining）以验证组织形态与样本适用性。按照10X Genomics官方推荐流程，使用Visium基因表达芯片与试剂试剂盒及双索引试剂盒TT Set A完成切片的测序文库制备。采用Illumina NextSeq500测序仪进行双端测序，每个位点的平均测序深度分别为：Quad2肌腱54000条读段、跟腱74000条读段、Quad1肌腱119000条读段。使用SpaceRanger（v1.3.1; 10X Genomics）默认参数对数据与图像进行处理，并将测序数据比对至GRCh38参考基因组（GRCh38 reference genome）。