A dual-color PAX7 and MYF5 in vivo reporter to investigate muscle stem cell heterogeneity in regeneration and aging.

Increasing evidence suggests that the muscle stem cell (MuSC) pool is heterogeneous. In particular, a rare subset of PAX7-positive MuSCs that has never expressed the myogenic regulatory factor MYF5 displays unique self-renewal and engraftment characteristics. However, the scarcity and limited availability of protein markers make the characterization of these cells challenging. Here, we describe the generation of StemRep reporter mice enabling the monitoring of PAX7 and MYF5 proteins based on equimolar levels of dual nuclear fluorescence. High levels of PAX7 protein and low levels of MYF5 delineate a deeply quiescent MuSC subpopulation with an increased capacity for asymmetric division and distinct dynamics of activation, proliferation, and commitment. Aging primarily reduces the MYF5Low MuSCs and skews the stem cell pool toward MYF5High cells with lower quiescence and self-renewal potential. Altogether, we establish the StemRep model as a versatile tool to study MuSC heterogeneity and broaden our understanding of mechanisms regulating MuSC quiescence and self-renewal in homeostatic, regenerating, and aged muscles.

Denervation alters the secretome of myofibers and thereby affects muscle stem cell lineage progression and functionality.

Skeletal muscle function crucially depends on innervation while repair of skeletal muscle relies on resident muscle stem cells (MuSCs). However, it is poorly understood how innervation affects MuSC properties and thereby regeneration of skeletal muscle. Here, we report that loss of innervation causes precocious activation of MuSCs concomitant with the expression of markers of myogenic differentiation. This aberrant activation of MuSCs after loss of innervation is accompanied by profound alterations on the mRNA and protein level. Combination of muscle injury with loss of innervation results in impaired regeneration of skeletal muscle including shifts in myogenic populations concomitant with delayed maturation of regenerating myofibers. We further demonstrate that loss of innervation leads to alterations in myofibers and their secretome, which then affect MuSC behavior. In particular, we identify an increased secretion of Osteopontin and transforming growth factor beta 1 (Tgfb1) by myofibers isolated from mice which had undergone sciatic nerve transection. The altered secretome results in the upregulation of early activating transcription factors, such as Junb, and their target genes in MuSCs. However, the combination of different secreted factors from myofibers after loss of innervation is required to cause the alterations observed in MuSCs after loss of innervation. These data demonstrate that loss of innervation first affects myofibers causing alterations in their secretome which then affect MuSCs underscoring the importance of proper innervation for MuSC functionality and regeneration of skeletal muscle.

Remodeling of the Cardiac Extracellular Matrix Proteome During Chronological and Pathological Aging.

Impaired extracellular matrix (ECM) remodeling is a hallmark of many chronic inflammatory disorders that can lead to cellular dysfunction, aging, and disease progression. The ECM of the aged heart and its effects on cardiac cells during chronological and pathological aging are poorly understood across species. For this purpose, we first used mass spectrometry-based proteomics to quantitatively characterize age-related remodeling of the left ventricle (LV) of mice and humans during chronological and pathological (Hutchinson-Gilford progeria syndrome (HGPS)) aging. Of the approximately 300 ECM and ECM-associated proteins quantified (named as Matrisome), we identified 13 proteins that were increased during aging, including lactadherin (MFGE8), collagen VI α6 (COL6A6), vitronectin (VTN) and immunoglobulin heavy constant mu (IGHM), whereas fibulin-5 (FBLN5) was decreased in most of the data sets analyzed. We show that lactadherin accumulates with age in large cardiac blood vessels and when immobilized, triggers phosphorylation of several phosphosites of GSK3B, MAPK isoforms 1, 3, and 14, and MTOR kinases in aortic endothelial cells (ECs). In addition, immobilized lactadherin increased the expression of pro-inflammatory markers associated with an aging phenotype. These results extend our knowledge of the LV proteome remodeling induced by chronological and pathological aging in different species (mouse and human). The lactadherin-triggered changes in the proteome and phosphoproteome of ECs suggest a straight link between ECM component remodeling and the aging process of ECs, which may provide an additional layer to prevent cardiac aging.

Extracellular matrix: Brick and mortar in the skeletal muscle stem cell niche.

The extracellular matrix (ECM) is an interconnected macromolecular scaffold occupying the space between cells. Amongst other functions, the ECM provides structural support to tissues and serves as a microenvironmental niche that conveys regulatory signals to cells. Cell-matrix adhesions, which link the ECM to the cytoskeleton, are dynamic multi-protein complexes containing surface receptors and intracellular effectors that control various downstream pathways. In skeletal muscle, the most abundant tissue of the body, each individual muscle fiber and its associated muscle stem cells (MuSCs) are surrounded by a layer of ECM referred to as the basal lamina. The core scaffold of the basal lamina consists of self-assembling polymeric laminins and a network of collagens that tether proteoglycans, which provide lateral crosslinking, establish collateral associations with cell surface receptors, and serve as a sink and reservoir for growth factors. Skeletal muscle also contains the fibrillar collagenous interstitial ECM that plays an important role in determining tissue elasticity, connects the basal laminae to each other, and contains matrix secreting mesenchymal fibroblast-like cell types and blood vessels. During skeletal muscle regeneration fibroblast-like cell populations expand and contribute to the transitional fibronectin-rich regenerative matrix that instructs angiogenesis and MuSC function. Here, we provide a comprehensive overview of the role of the skeletal muscle ECM in health and disease and outline its role in orchestrating tissue regeneration and MuSC function.

Visualization of the Skeletal Muscle Stem Cell Niche in Fiber Bundles.

Skeletal muscle stem cells (MuSCs) reside in a complex niche composed of the muscle fiber plasma membrane and the laminin-rich basal lamina surrounded by the microvasculature, as well as different supportive cell types such as fibro-adipogenic progenitors residing in the interstitial extracellular matrix. Within the first few hours after tissue damage, MuSCs undergo cytoskeletal rearrangements and transcriptional changes that prime the cells for activation. Due to their time-consuming nature, enzymatic methods for liberation of single muscle fibers with fully quiescent MuSCs are challenging. Moreover, during enzymatic digestion, important niche components including the microvasculature and the collagenous interstitial matrix are destroyed. Here, we provide a method for the visualization of MuSCs on muscle fibers in their intact niche. Our method relies on mechanical teasing of fiber bundles from fixed skeletal muscles. We demonstrate that teased muscle fiber bundles allow the investigator to capture a representative snapshot of the MuSC niche in skeletal muscle, and outline how stem cell morphology and different microenvironmental components can be visualized. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of fiber bundles Basic Protocol 2: Immunofluorescence staining of MuSCs on fiber bundles Support Protocol: Preparation of Sylgard dishes.

Extensive remodeling of the extracellular matrix during aging contributes to age-dependent impairments of muscle stem cell functionality.

During aging, the regenerative capacity of skeletal muscle decreases due to intrinsic changes in muscle stem cells (MuSCs) and alterations in their niche. Here, we use quantitative mass spectrometry to characterize intrinsic changes in the MuSC proteome and remodeling of the MuSC niche during aging. We generate a network connecting age-affected ligands located in the niche and cell surface receptors on MuSCs. Thereby, we reveal signaling by integrins, Lrp1, Egfr, and Cd44 as the major cell communication axes perturbed through aging. We investigate the effect of Smoc2, a secreted protein that accumulates with aging, primarily originating from fibro-adipogenic progenitors. Increased levels of Smoc2 contribute to the aberrant Integrin beta-1 (Itgb1)/mitogen-activated protein kinase (MAPK) signaling observed during aging, thereby causing impaired MuSC functionality and muscle regeneration. By connecting changes in the proteome of MuSCs to alterations of their niche, our work will enable a better understanding of how MuSCs are affected during aging.

Stem cell aging: The upcoming era of proteins and metabolites.

So far, the investigation of stem cell aging has been primarily carried out at the genomic level to follow transcriptome changes, clonal dominance, epigenetic changes, and, more recently, population heterogeneity by single cell sequencing. Here, we review recent findings in the field of stem cell aging that include failure of proteostasis, the impact of age-related metabolic changes on stem cell differentiation and heterogeneity of stem cell niches. These emerging concepts highlight the need of a paradigm shift to move forward our understanding of stem cell aging to the next level, in particular, the need of investigating cell intrinsic changes in stem cells and their niche by looking in a quantitative manner to proteins and metabolites.

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo.

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

Adult stem cells at work: regenerating skeletal muscle.

Skeletal muscle regeneration is a finely tuned process involving the activation of various cellular and molecular processes. Satellite cells, the stem cells of skeletal muscle, are indispensable for skeletal muscle regeneration. Their functionality is critically modulated by intrinsic signaling pathways as well as by interactions with the stem cell niche. Here, we discuss the properties of satellite cells, including heterogeneity regarding gene expression and/or their phenotypic traits and the contribution of satellite cells to skeletal muscle regeneration. We also summarize the process of regeneration with a specific emphasis on signaling pathways, cytoskeletal rearrangements, the importance of miRNAs, and the contribution of non-satellite cells such as immune cells, fibro-adipogenic progenitor cells, and PW1-positive/Pax7-negative interstitial cells.

Isolation and Culture of Individual Myofibers and Their Adjacent Muscle Stem Cells from Aged and Adult Skeletal Muscle.

The isolation and culture of single floating myofibers with their adjacent muscle stem cells allow the analysis and comparison of muscle stem cells from aged and young mice. This method has the advantage that muscle stem cells are cultured on the myofiber, thereby culturing them in conditions as close to their endogenous niche as possible. Here we describe the isolation, culture, transfection with siRNA, and subsequent immunostaining for muscle stem cells on their adjacent myofibers from aged and young mice.