Single nucleus and spatial transcriptomic profiling of human healthy hamstring tendon

Abstract

The molecular and cellular basis of health in human tendons remains poorly understood. Amongst human tendons, the hamstrings are the least likely to be injured or degenerate, providing a prototypic healthy tendon reference. The aim of this study was to define the transcriptome and location of all cell types in healthy hamstring tendon. We profiled the transcriptomes of 10,533 nuclei from 4 healthy donors using single-nucleus RNA sequencing (snRNA-seq) and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and revealed the presence of cell types previously unreported for tendon sites, including different skeletal muscle cell types, satellite cells, adipocytes, and nerve cells, which are undefined nervous system cells. Location of these cell types within tendon was defined using spatial transcriptomics and imaging, and transcriptional networks and cell-cell interactions were identified. We demonstrate that fibroblasts have a high number of potential cell-cell interactions, are present throughout the whole tendon tissue, and play an important role in the production and organisation of extracellular matrix, thus confirming their role as key regulators of hamstring tendon tissue homeostasis. Overall, our findings highlight the highly complex cellular networks underpinning tendon function and underpins the importance of fibroblasts as key regulators of hamstring tendon tissue homeostasis.

Current and emerging technologies for defining and validating tendon cell fate

“…Recent studies have revealed heterogeneity in adult tendon cell populations as well as identified noninstrinsic cell types in the tissue such as pericytes, immune, and endothelial cells 28,29,43,44 . A commonality across species with these datasets is the presence of tendon cell clusters with more prototypical tendon markers (i.e., Scx , Mkx , Tnmd ) and those enriched for matrix components such as Col1a1/a2 , but devoid of these characteristic tendon markers 45,46 . Some of these non Scx / Mkx expressing cell clusters have characteristics of fibro‐adipogenic progenitors (FAPs), including increased expression of Col3a1 , Ly6e , and Pdgfra 28,45 .…”

“…There are several current platforms, including 10X Genomics Visium Spatial gene expression, Merscope (MerFISH; Vizgen), and GeoMx/CosMx (Nanostring), each with differing strengths (reviewed in Williams et al 48 ). Published and preprint studies in human and mouse have utilized the 10X Visium platform to show spatial distributions of cell types, including in mouse, cell cluster localization over healing stages 49 and in humans, specific fibroblast cell subtypes (MKX vs. PDGFRA) or myotendinous junction cells 44,46,50 …”

“…28,29,43,44 A commonality across species with these datasets is the presence of tendon cell clusters with more prototypical tendon markers (i.e., Scx, Mkx, Tnmd) and those enriched for matrix components such as Col1a1/a2, but devoid of these characteristic tendon markers. 45,46 Some of these nonScx/Mkx expressing cell clusters have characteristics of fibro-adipogenic progenitors (FAPs), including increased expression of Col3a1, Ly6e, and Pdgfra. 28,45 Pericytes/perivascular smooth muscle (CD90 + ; CD146 + ) cells were enriched for Tagln, Acta2a, Rgs5, and Itga7, which are consistent with mural (pericyte and vascular smooth muscle) cell types.…”

“…Published and preprint studies in human and mouse have utilized the 10X Visium platform to show spatial distributions of cell types, including in mouse, cell cluster localization over healing stages 49 and in humans, specific fibroblast cell subtypes (MKX vs. PDGFRA) or myotendinous junction cells. 44,46,50 Complementing RNA-seq analyses are studies examining potential upstream regulatory regions using either ATAC-seq or ChIP-seq, which can expand our understanding of potential upstream transcription factors through motif-analysis of bound regions and of downstream target pathways. ChIP-seq provides comprehensive information about the genomic regions bound by a transcription factor in specific conditions.…”