Perspectives on skeletal muscle stem cells

REVIEW ARTICLE https://doi.org/10.1038/s41467-020-20760-6 OPEN Perspectives on skeletal muscle stem cells 1234567890():,; F. Relaix 1,2,3,4 ✉, M. Bencze 1, M. J. Borok1, A. Der Vartanian1, F. Gattazzo1,3, D. Mademtzoglou 1, S. Perez-Diaz1, A. Prola1,2, P. C. Reyes-Fernandez1, A. Rotini1 & Taglietti V.1 Skeletal muscle has remarkable regeneration capabilities, mainly due to its resident muscle stem cells (MuSCs). In this review, we introduce recently developed technologies and the mechanistic insights they provide to the understanding of MuSC biology, including the redefinition of quiescence and Galert states. Additionally, we present recent studies that link MuSC function with cellular heterogeneity, highlighting the complex regulation of selfrenewal in regeneration, muscle disorders and aging. Finally, we discuss MuSC metabolism and its role, as well as the multifaceted regulation of MuSCs by their niche. The presented conceptual advances in the MuSC field impact on our general understanding of stem cells and their therapeutic use in regenerative medicine. R epresenting 30–40% of our body mass, skeletal muscle is a highly organized tissue made up of a large number of syncytial cells, known as myofibers, which are formed by the fusion of myogenic progenitor cells. Despite the post-mitotic nature of its myofibers, skeletal muscle has a robust regenerative capacity in response to injury. This relies on resident muscle stem cells (MuSCs), also called “satellite cells” because of their unique anatomical position at the periphery of the myofibers. MuSCs typically exist in a quiescent state but may enter the cell cycle following injury in order to regenerate the skeletal muscle tissue and replenish the stem cell pool for future needs. Several transcription factors have been identified as markers and key regulators of the quiescent state as well as of activation and progression to the myogenic lineage. Among them, the paired homeobox factors PAX3 and PAX7 as well as the so-called Myogenic Regulatory Factors – MRFs (MYF5, MYOD, MYOGENIN, MRF4) stand out for their unique and important roles in muscle formation, specification, homeostasis, and repair (for more details the reader may refer to ref. 1,2). PAX7 is commonly used as a marker of MuSCs, and a subset of them co-expresses PAX3 in adult muscle3,4. The MRFs regulate the progression of MuSCs towards myogenic determination, differentiation, and fusion to form multinucleated myofibers2. The renewal of the MuSC cellular compartment requires a tightly regulated balance between quiescence and activation that is associated with many transcriptional changes in MuSCs. Activation is accompanied by metabolic reprogramming, reinforcing the evidence of a strict interplay between MuSC function and metabolic status. Moreover, recent studies show that MuSCs are a heterogeneous stem cell population, with different abilities to support tissue regeneration. The dynamic changes in MuSC behavior are regulated by the microenvironment and by distinct tissue resident cells of the niche that provide molecular cues to regulate MuSC fate. Here, we review novel findings that have challenged our knowledge of MuSC biology, discussing the molecular mechanisms regulating MuSC quiescence and activation states and 1 Univ Paris Est Creteil, INSERM, IMRB, 94010 Creteil, France. 2 EnvA, IMRB, 94700 Maisons-Alfort, France. 3 EFS, IMRB, 94010 Creteil, France. 4 AP-HP, Hopital Mondor, Service d’histologie, 94010 Creteil, France. ✉email: NATURE COMMUNICATIONS | (2021)12:692 | https://doi.org/10.1038/s41467-020-20760-6 | www.nature.com/naturecommunications 1 REVIEW ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-20760-6 heterogeneity. Moreover, we describe the latest advances that enhance our understanding of how MuSC metabolism adapts to quiescence and differentiation, and the role of the microenvironmental niche in regulating MuSC behavior and function. Finally, we present new insights into the pathological conditions associated with MuSC dysfunction, such as muscular dystrophies and aging, showing how the deregulation of MuSCs can lead to an exacerbation of pathology. State of the art MuSC quiescence and activation Capturing the quiescence state. The ability of MuSCs to drive robust tissue regeneration is based on their ability to exit from their steady-state quiescent state and pass to an activated state following stimuli encountered notably in traumatic or pathological conditions2. Initially, the quiescent and activated states were defined at the molecular level by PAX7 expression and by rapid MRF (e.g. MYF5, MYOD) upregulation, respectively. Following activation, MuSCs start to proliferate; the majority differentiates for muscle repair, while a subpopulation of activated/proliferating MuSCs will replenish the quiescent pool. However, recent technical advances have revealed a higher level of complexity in the molecular signatures of quiescence and activation. Four groups have independently developed protocols to capture a dormant gradient state of MuSCs by in situ fixation5,6, by single-cell sequencing7 or by isolation of quiescent cells via TU-tagging8. These studies showed that during mechanical and enzymatic tissue dissociation MuSCs undergo rapid changes in transcription and histone modifications5–8 and they provide novel experimental approaches to isolate MuSCs in their native state, and to analyze both quiescence and early activation mechanisms. The early response of MuSCs to the disruption of their niche includes increased expression of AP-1 members such as Fos and Jun, rapid downregulation of Hox genes, of genes encoding zinc finger proteins or metabolism enzymes, and of Notch signaling5–8. Regulation of quiescent MuSCs. How quiescence is maintained is not fully understood, but Notch signaling plays a key role. Notch is active in quiescent MuSCs and interference with canonical Notch signaling results in depletion of MuSCs through spontaneous differentiation9,10. More recently, Notch signaling was found to induce the transcription of miR-708 (Fig. 1), which impedes MuSC proliferation and motility11. KLF7 is an additional factor that was placed downstream of Notch and was found to be necessary for maintaining MuSC quiescence. Knockdown and overexpression experiments showed that KLF7 limited MuSC cell cycle entry through upregulation of the Cyclin-Dependent Kinase Inhibitor (CDKI) p21 (Fig. 1) but did not affect differentiation12. In the same CDKI family as p21, p57 was found to migrate from the cytoplasm to the nucleus to promote cell cycle exit of activated MuSC-derived myoblasts13 (Fig. 1). Both studies demonstrated CDKI effects on proliferation but not differentiation, which are frequently concomitantly de-regulated. Regulation of MuSC proliferation. In parallel with studies focusing on the establishment and maintenance of quiescence, recent work explored factors that promote MuSC proliferation, following their activation. MuSC-sp (...truncated)