Mechanical compression creates a quiescent muscle stem cell niche

ARTICLE https://doi.org/10.1038/s42003-023-04411-2 OPEN Mechanical compression creates a quiescent muscle stem cell niche 1234567890():,; Jiaxiang Tao1, Mohammad Ikbal Choudhury2,3,7, Debonil Maity2,3,7, Taeki Kim4, Sean X. Sun Chen-Ming Fan 1,6 ✉ 2,3,5 & Tissue stem cell niches are regulated by their mechanical environment, notably the extracellular matrix (ECM). Skeletal muscles consist of bundled myofibers for force transmission. Within this macroscopic architecture, quiescent Pax7-expressing (Pax7+) muscle stem cells (MuSCs) are compressed between ECM basally and myofiber apically. Muscle injury causes MuSCs to lose apical compression from the myofiber and re-enter the cell cycle for regeneration. While ECM elasticities have been shown to affect MuSC’s renewal, the significance of apical compression remains unknown. To investigate the role of apical compression, we simulate the MuSCs’ in vivo mechanical environment by applying physical compression to MuSCs’ apical surface. We demonstrate that compression drives activated MuSCs back to a quiescent stem cell state, regardless of basal elasticities and chemistries. By mathematical modeling and cell tension manipulation, we conclude that low overall tension combined with high axial tension generated by compression leads to MuSCs’ stemness and quiescence. Unexpectedly, we discovered that apical compression results in up-regulation of Notch downstream genes, accompanied by the increased levels of nuclear Notch1&3 in a Delta ligand (Dll) and ADAM10/17 independent manner. Our results fill a knowledge gap on the role of apical compression for MuSC fate and have implications to stem cells in other tissues. 1 Embryology Department, Carnegie Institution for Science, 3520 San Martin Drive, Baltimore, MD 21218, USA. 2 Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA. 3 Institution for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA. 4 Department of Civil & Systems Engineering, Johns Hopkins University, Baltimore, MD 21218, USA. 5 Center for Cell Dynamics (CCD), Johns Hopkins School of Medicine, Baltimore, MD 21205, USA. 6 Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA. 7These authors contributed equally: Mohammad Ikbal Choudhury, Debonil Maity. ✉email: COMMUNICATIONS BIOLOGY | (2023)6:43 | https://doi.org/10.1038/s42003-023-04411-2 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04411-2 S keletal muscles consist of post-mitotic syncytial myofibrils that generate contractile forces for body movement1. They have a tremendous ability to regenerate after injury mainly owing to the resident Pax7-expressing (Pax7+) muscle stem cells (MuSCs)2–5, also known as satellite cells1,2. Within the uninjured muscle, MuSCs are mostly quiescent and sandwiched between the basement membrane (i.e., ECM) and myofiber. Upon muscle damage, MuSCs lose apical contact with the myofiber but retain contact with the ECM of the dead myofiber (i.e., the ghost fiber6). They then re-enter the cell cycle, become myogenically committed, and later differentiate and fuse to form new myofibers7. Some proliferative MuSCs self-renew and return to quiescence to maintain the stem cell pool as the regenerative cycle completes8. Molecular and genetic studies have uncovered genes and pathways regulating progressive states of MuSCs during the regenerative cycle9. On the other hand, mechanical stimuli have also been shown to regulate MuSCs’ fate. Mechanisms underlying how forces can trigger relevant endogenous signaling pathways are currently limited to cell–ECM interaction. For example, laminincoated hydrogels with a stiffness of 12 kPa, close to the physiological elasticity10, provide an optimal condition for MuSC selfrenewal in culture10–13; whereas culturing them on 2 kPa collagen I fibrils in synthetic media has been shown to prevent their activation14. Here, we address the role of understudied apical compression for the quiescence state of MuSCs. Results Quiescent MuSCs change morphology during the transition to activated state. From the intravital imaging data of yellow fluorescent protein (YFP)-marked Pax7+ MuSCs6, MuSCs display a flat and elongated shape in uninjured muscle: cell dimension perpendicular to myofiber (height) is ~4 μm, and cell dimension parallel to myofiber (length) is ~17 μm. At 1-day postinjury (dpi), they are ~5.5 μm in cell height and ~6 μm in cell length (Supplementary Fig. 1a–c). Compression release from the myofiber after injury likely contributes to such morphological changes. At 3 dpi, many YFP-marked cells are proliferating and/ or migrating, and their cell height and length are both ~10 μm (with dynamically changing lengths), which are both larger compared to their uninjured counterparts (Supplementary Fig. 1d, e). We were particularly intrigued by the cell shape change at 1 dpi before a detectable MuSC proliferation at 2 dpi6 and that MuSCs on uninjured myofibers immediately adjacent to an injury site does not appear activated6. We hypothesize that compression force exerted on MuSCs by intact myofibers keeps them quiescent and tested our hypothesis using cultured MuSCs as described below. Apical compression increases quiescent MuSCs. To investigate the link between cellular morphology/mechanical tension and MuSC state, we established a system to simulate the apical loading on quiescent MuSCs. We microfabricated a polydimethylsiloxane (PDMS) device consisting of a thin top film with vertical pillars of ~4 μm in height underneath (Supplementary Fig. 2a–c; see the “Methods” section)15,16. MuSCs cultured under this device was presumably limited to a height of 4 μm, mimicking their intact physical niche in vivo. Pax7+ MuSCs were isolated from transgenic Pax7-ZsGreen mice17 by fluorescenceactivated cell sorting (FACS, Fig. 1a). They were seeded on Matrigel/fibronectin-coated plastic dish and cultured for 2 days in growth media (see the “Methods” section). At this time, the cell height was measured at ~8.7 μm using confocal imaging. After adding the compression device, we confirmed that cells were at the expected height of ~4 μm (Supplementary Fig. 3a, b). 2 We evaluated the role of compression on MuSCs’ fate, by comparing Pax7 and MyoD expression between freshly isolated, compressed, and uncompressed MuSCs: Pax7+MyoD− for stem cells, Pax7+MyoD+ for progenitors, and Pax7−MyoD+ for differentiation committed cells18. Freshly isolated MuSCs (F) were all Pax7+, with some also expressing MyoD (Fig. 1a; Supplementary Fig. 4a). After the initial 2 days of culture without compression (2d-U), most cells expressed MyoD (~70%) with some Pax7− cells, indicative of MuSC activation and differentiation. MuSCs were then either left uncompressed for an additional 3 days (5d-U) or subjected to compression for the same period (2 + 3d-C). The Pax7+MyoD− cells were almost depleted in the 5d-U. By contrast, 2 + 3d-C MuSCs contained a sign (...truncated)