Antoine de Morree

Cells are the basic unit of life. In multicellular organisms, cells are organized into tissues. This enables a division of labor where tissues perform complex tasks via coordinated actions of specialized cell types. Accordingly, the cellular collective determines tissue function. A comprehensive overview of which cell types exist in solid tissues is lacking. Using skeletal muscle as a model, we discuss basic principles for cell type classification, summarize 62 unified definitions of cell types in terms of lineage, molecular signatures, and function from the literature, and discuss how these cell types contribute to muscle function. For cell types for which quantitative data was available, we compare abundances in immunohistology and single cell and single-nucleus RNA sequencing data, revealing cell types that are commonly over- or underrepresented in each method. The result is a cell type resource that will serve as a benchmark for single-cell studies of skeletal muscle.

Skeletal muscle fibrosis, as occurs with age, in response to injury, or in the setting of degenerative diseases, results in impairments of muscle regeneration and function. Fibro-adipogenic progenitors (FAPs), a distinct population of muscle-resident mesenchymal progenitor cells that reside in the muscle interstitium, play a crucial role in normal muscle regeneration by supporting muscle stem cell proliferation. However, in pathological conditions such as severe or recurrent muscle injury, FAPs can aberrantly differentiate into fibrogenic cells, resulting in excessive deposition of extracellular matrix and fibrosis. In this study, we explore the molecular regulation of FAP differentiation along the fibrogenic lineage to gain insights into the mechanisms of fibrosis in aged muscle in response to injury. Our findings reveal that aging is associated with an increased expression of the complement component 1q (C1q) in muscle-resident macrophages and elevated expression of the complement proteins C1r and C1s in FAPs. Exposure of proliferating FAPs to C1q results in the activation of the Wnt signaling pathway, elevated expression of collagen genes, and FAP fibrogenic differentiation, leading to increased tissue fibrosis. We demonstrate that either pharmacological inhibition of the complement pathway or genetic ablation of C1s in FAPs in aged mice reduces fibrogenic differentiation of FAPs by suppressing Wnt signaling. This reduction in FAP differentiation attenuates the fibrotic response to injury in aged animals as well as in a mouse model of muscular dystrophy. Our study supports the inhibition of complement signaling as a potential therapeutic strategy for mitigating fibrosis in skeletal muscle injury or degeneration.

Mouse lemurs are the smallest and fastest reproducing primates, as well as one of the most abundant, and they are emerging as a model organism for primate biology, behaviour, health and conservation. Although much has been learnt about their ecology and phylogeny in Madagascar and their physiology, little is known about their cellular and molecular biology. Here we used droplet-based and plate-based single-cell RNA sequencing to create Tabula Microcebus, a transcriptomic atlas of 226,000 cells from 27 mouse lemur organs opportunistically obtained from four donors clinically and histologically characterized. Using computational cell clustering, integration and expert cell annotation, we define and biologically organize more than 750 lemur molecular cell types and their full gene expression profiles. This includes cognates of most classical human cell types, including stem and progenitor cells, and differentiating cells along the developmental trajectories of spermatogenesis, haematopoiesis and other adult tissues. We also describe dozens of previously unidentified or sparsely characterized cell types. We globally compare expression profiles to define the molecular relationships of cell types across the body, and explore primate cell and gene expression evolution by comparing lemur transcriptomes to those of human, mouse and macaque. This reveals cell-type-specific patterns of primate specialization and many cell types and genes for which the mouse lemur provides a better human model than mouse¹. The atlas provides a cellular and molecular foundation for studying this model primate and establishes a general approach for characterizing other emerging model organisms.

The development of non-human primate models is essential for the fields of developmental and regenerative biology because those models will more closely approximate human biology than do murine models. Based on single cell RNAseq and fluorescence-activated cell sorting, we report the identification and functional characterization of two quiescent stem cell populations (skeletal muscle stem cells (MuSCs) and mesenchymal stem cells termed fibro-adipogenic progenitors (FAPs)) in the non-human primate Microcebus murinus (the gray mouse lemur). We demonstrate in vivo proliferation, differentiation, and self-renewal of both MuSCs and FAPs. By combining cell phenotyping with cross-species molecular profiling and pharmacological interventions, we show that mouse lemur MuSCs and FAPs are more similar to human than to mouse counterparts. We identify unexpected gene targets involved in regulating primate MuSC proliferation and primate FAP adipogenic differentiation. Moreover, we find that the cellular composition of mouse lemur muscle better models human muscle than does macaque (Macaca fascicularis) muscle. Finally, we note that our approach presents as a generalizable pipeline for the identification, isolation, and characterization of stem cell populations in new animal models.

The development of non-human primate models is essential for the fields of developmental and regenerative biology because those models will more closely approximate human biology than do murine models. Based on single cell RNAseq and fluorescence-activated cell sorting, we report the identification and functional characterization of two quiescent stem cell populations (skeletal muscle stem cells (MuSCs) and mesenchymal stem cells termed fibro-adipogenic progenitors (FAPs)) in the non-human primate Microcebus murinus (the gray mouse lemur). We demonstrate in vivo proliferation, differentiation, and self-renewal of both MuSCs and FAPs. By combining cell phenotyping with cross-species molecular profiling and pharmacological interventions, we show that mouse lemur MuSCs and FAPs are more similar to human than to mouse counterparts. We identify unexpected gene targets involved in regulating primate MuSC proliferation and primate FAP adipogenic differentiation. Moreover, we find that the cellular composition of mouse lemur muscle better models human muscle than does macaque ( Macaca fascicularis ) muscle. Finally, we note that our approach presents as a generalizable pipeline for the identification, isolation, and characterization of stem cell populations in new animal models.

Adult stem cells play key roles in tissue homeostasis and regeneration. Recent evidence suggests that dietary interventions can significantly impact adult stem cell function. Some of these effects depend on ketone bodies. Adult stem cells could therefore potentially be manipulated through dietary regimens or exogenous ketone body supplementation, a possibility with significant implications for regenerative medicine. In this review we discuss recent findings of the mechanisms by which ketone bodies could influence adult stem cells, including ketogenesis in adult stem cells, uptake and transport of circulating ketone bodies, receptor-mediated signaling, and changes to cellular metabolism. We also discuss the potential effects of ketone bodies on intracellular processes such as protein acetylation and post-transcriptional control of gene expression. The exploration of mechanisms underlying the effects of ketone bodies on stem cell function reveals potential therapeutic targets for tissue regeneration and age-related diseases and suggests future research directions in the field of ketone bodies and stem cells.

Adult stem cells are important for mammalian tissues, where they act as a cell reserve that supports normal tissue turnover and can mount a regenerative response following acute injuries. Quiescent stem cells are well established in certain tissues, such as skeletal muscle, brain, and bone marrow. The quiescent state is actively controlled and is essential for long-term maintenance of stem cell pools. In this Review, we discuss the importance of maintaining a functional pool of quiescent adult stem cells, including haematopoietic stem cells, skeletal muscle stem cells, neural stem cells, hair follicle stem cells, and mesenchymal stem cells such as fibro-adipogenic progenitors, to ensure tissue maintenance and repair. We discuss the molecular mechanisms that regulate the entry into, maintenance of, and exit from the quiescent state in mice. Recent studies revealed that quiescent stem cells have a discordance between RNA and protein levels, indicating the importance of post-transcriptional mechanisms, such as alternative polyadenylation, alternative splicing, and translation repression, in the control of stem cell quiescence. Understanding how these mechanisms guide stem cell function during homeostasis and regeneration has important implications for regenerative medicine.

Adult stem cells play key roles in homeostasis and tissue repair. These cells are regulated by a tight control of transcriptional programs. For example, muscle stem cells (MuSCs), located beneath the basal lamina, exist in the quiescent state but can transition to an activated, proliferative state upon injury. The control of MuSC state depends on the expression levels of myogenic transcription factors. Recent studies revealed the presence of different mRNA isoforms, with distinct biological regulation. Quantifying the exact expression levels of the mRNA isoforms encoding these myogenic transcription factors is therefore key to understanding how MuSCs switch between cell states. Previously, quantitative real-time polymerase chain reaction (qRT-PCR) has been used to quantify RNA expression levels. However, qRT-PCR depends on large amounts of RNA input and only measures relative abundance. Here, we present a protocol for the absolute quantification of mRNA isoforms using microfluidic digital PCR (mdPCR). Primary MuSCs isolated from individual skeletal muscles (gastrocnemius and masseter) are lysed, and their RNA is reverse-transcribed into cDNA and copied into double-stranded DNA. Following exonuclease I digestion to remove remaining single-stranded DNA, the samples are loaded onto a mdPCR chip with TaqMan probes targeting the mRNA isoforms of interest, whereupon target molecules are amplified in nanoliter chambers. We demonstrate that mdPCR can give exact molecule counts per cell for mRNA isoforms encoding the myogenic transcription factor Pax3. This protocol enables the absolute quantification of low abundant mRNA isoforms in a fast, precise, and reliable way.

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells (MuSCs) have the ability to make new muscle, whereas fibro-adipogenic progenitors (FAPs) contribute to stromal supporting tissues and have the ability to make fibroblasts and adipocytes. Both MuSCs and FAPs reside in a state of prolonged reversible cell cycle exit, called quiescence. The quiescent state is key to their function. Quiescent stem cells are commonly purified from multiple muscle tissues pooled together in a single sample. However, recent studies have revealed distinct differences in the molecular profiles and quiescence depth of MuSCs isolated from different muscles. The present protocol describes the isolation and study of MuSCs and FAPs from individual skeletal muscles and presents strategies to perform molecular analysis of stem cell activation. It details how to isolate and digest muscles of different developmental origin, thicknesses, and functions, such as the diaphragm, triceps, gracilis, tibialis anterior (TA), gastrocnemius (GA), soleus, extensor digitorum longus (EDL), and the masseter muscles. MuSCs and FAPs are purified by fluorescence-activated cell sorting (FACS) and analyzed by immunofluorescence staining and 5-ethynyl-2´-deoxyuridine (EdU) incorporation assay.

Significance: The replication stress response protein, ATR, is active in quiescent muscle stem cells in response to DNA:RNA hybrids, and this activity maintains quiescence by ensuring degradation of key cell-cycle transition factors by the E3 ubiquitin ligase cyclin F–SCF complex. This is critical for understanding how stem cells regulate a state of prolonged and reversible cell-cycle arrest and how genome integrity is maintained over time. Together, these studies offer a unique picture of a molecular mechanism controlling stem cell quiescence.