Discovery of Muscle-Tendon Progenitor Subpopulation in Human Myotendinous Junction at Single-Cell Resolution

Abstract

The myotendinous junction (MTJ) is a complex and special anatomical area that connects muscles and tendons, and it is also the key to repairing tendons. Nevertheless, the anatomical structure and connection structure of MTJ, the cluster and distribution of cells, and which cells are involved in repairing the tissue are still unclear. Here, we analyzed the cell subtype distribution and function of human MTJ at single-cell level. We identified four main subtypes, including stem cell, muscle, tendon, and muscle-tendon progenitor cells (MTP). The MTP subpopulation, which remains the characteristics of stem cells and also expresses muscle and tendon marker genes simultaneously, may have the potential for bidirectional differentiation. We also found the muscle-tendon progenitor cells were distributed in the shape of a transparent goblet; muscle cells first connect to the MTP and then to the tendon. And after being transplanted in the MTJ injury model, MTP exhibited strong regenerative capability. Finally, we also demonstrated the importance of mTOR signaling for MTP maintenance by in vitro addition of rapamycin and in vivo validation using mTOR-ko mice. Our research conducted a comprehensive analysis of the heterogeneity of myotendinous junction, discovered a special cluster called MTP, provided new insights into the biological significance of myotendinous junction, and laid the foundation for future research on myotendinous junction regeneration and restoration.

Distinct myofibre domains of the human myotendinous junction revealed by single nucleus RNA-seq

“…Furthermore, FibTen_Sub5 (and to a lesser extent FibTen_Sub3) clearly mapped to the tendon proper, which unequivocally identifies FibTen_Sub5 as a population of pure tenocyte nuclei (Figure 5B, Supplementary Figure S9). This is in line with the lack of concordance between FibTen_Sub5 with previously reported cell clusters derived from pure skeletal muscle (Supplementary Figure S5A-B), while appearing in human scRNA-seq data from muscle/tendon/MTJ (Supplementary Fig S5B, see Yan et al 2022) and human scRNA-seq data from pure tendon (Supplementary Fig S5B, see Kendall et al 2020). For the remaining cell clusters (endothelial cells, smooth muscle cells, and immune cells), the spatial analysis showed a ubiquitous distribution across muscle and tendon tissue (Supplementary Figure S9).…”

“…The spatial transcriptomics and cross-comparison with cell-clusters in human snRNA/scRNA-seq data clearly pointed towards FibTen_Sub5 as a pure tenocyte cluster. FibTen_Sub3 transcripts seemed to be more spatially concentrated at the periphery of the tendon (Supplementary Figure S9), and was the best match to a recently reported muscle-tendon progenitor subpopulation (Supplementary Figure S5B) (Yan et al 2022). The FibTen_Sub3 also demonstrated a high representation of the MTJ-enriched proteins we discovered by LCMS-proteomics, of which CILP, ITGA10, and THBS4 were confirmed by IF analysis (Karlsen et al 2022), as well as HMCN1 recently shown at the MTJ (Welcker et al 2021), and the muscle fibre specific LAMA2 (Supplementary Data S3).…”

“…This gave rise to a total of 30 subclusters including 4 MTJ-myonuclei subclusters; 26 were new subclusters (indicated by ‘Sub’ in the cluster names) and 4 original (Cytoplasm, Myo_9, Myo_10, SatCell) (Figure 4A, Supplementary Figure S4, Supplementary Data S3). To compare cell-cluster markers we include in Supplementary Figure S5 a comparison between our 30 nuclei-subclusters and cell-types or nuclei-types reported in the literature (Harvey et al 2019; Dos Santos et al 2020; Kendal et al 2020; Rubenstein et al 2020; De Micheli et al 2020a, b; Barruet et al 2020; Chemello et al 2020; He et al 2020; Kim et al 2020; Petrany et al 2020; Perez et al 2021; Wen et al 2021; Eraslan et al 2022; Scripture-Adams et al 2022; Yan et al 2022). This showed a good alignment between our clusters compared to snRNA-seq (Perez et al 2021; Eraslan et al 2022) and scRNA-seq (Rubenstein et al 2020; He et al 2020) in human skeletal muscle, and in snRNA-seq (Dos Santos et al 2020; Chemello et al 2020; Wen et al 2021) and scRNA-seq (De Micheli et al 2020b) in mice.…”

Limb connective tissue is organized in a continuum of promiscuous fibroblast identities during development

“…However, Tcf4 and Hoxa11 are also expressed in muscle cells (Murphy et al, 2011, Asfour et al, 2023) or in muscle lineage (Flynn et al, 2023), where they could directly act on muscle patterning. It has been recently shown that a subpopulation of CT fibroblasts integrates muscle fibres at the muscle/tendon interface during development and postnatal stages (Esteves de Lima et al, 2021, Yaseen et al, 2021, Yan et al, 2022, Flynn et al, 2023). This unexpected fibroblastic origin of myonuclei at muscle tips close to tendon provides us with a cellular mechanism mediating fibroblast-driven muscle patterning.…”

Effects of Myostatin on Nuclear Morphology at the Myotendinous Junction

“…However, unlike reports describing the development of the MTJ, only a few studies have focused on its regeneration. Yan et al [ 11 ] have identified muscle–tendon progenitor cells that have the capacity to regenerate the MTJ. These progenitor cells co-expressing muscle and tendon markers are located in the MTJ.…”