Tissue specific stem cells are indispensable contributors to adult tissue maintenance, repair, and regeneration. In skeletal muscle, satellite cells (SCs) are the resident muscle stem cell population ...and are required to maintain skeletal muscle homeostasis throughout life. Increasing evidence suggests that SCs are a heterogeneous cell population with substantial biochemical and functional diversity. A major limitation in the field is an incomplete understanding of the nature and extent of this cellular heterogeneity. Single cell analyses are well suited to addressing this issue, especially when coupled to unbiased profiling paradigms such as high throughout RNA sequencing. We performed single cell RNA sequencing (scRNA-seq) on freshly isolated muscle satellite cells and found a surprising degree of heterogeneity at multiple levels, from muscle-specific transcripts to the broader SC transcriptome. We leveraged several comparative bioinformatics techniques and found that individual SCs enrich for unique transcript clusters. We propose that these gene expression “fingerprints” may contribute to observed functional SC diversity. Overall, these studies underscore the importance of several established SC signaling pathways/processes on a single cell level, implicate novel regulators of SC heterogeneity, and lay the groundwork for further investigation into SC heterogeneity in health and disease.
•Single cell analyses of skeletal muscle satellite cells (SCs) reveal substantial variability in muscle transcript expression.•Non-negative matrix factorization (NMF) analyses fail to reveal robust SC subpopulations.•Unsupervised hierarchical clustering analyses uncover unique transcript clusters in individual SCs.•RNA regulatory pathways may play an important role in SC homeostasis.
Aging is characterized by the development of metabolic dysfunction and frailty. Recent studies show that a reduction in nicotinamide adenine dinucleotide (NAD+) is a key factor for the development of ...age-associated metabolic decline. We recently demonstrated that the NADase CD38 has a central role in age-related NAD+ decline. Here we show that a highly potent and specific thiazoloquin(az)olin(on)e CD38 inhibitor, 78c, reverses age-related NAD+ decline and improves several physiological and metabolic parameters of aging, including glucose tolerance, muscle function, exercise capacity, and cardiac function in mouse models of natural and accelerated aging. The physiological effects of 78c depend on tissue NAD+ levels and were reversed by inhibition of NAD+ synthesis. 78c increased NAD+ levels, resulting in activation of pro-longevity and health span-related factors, including sirtuins, AMPK, and PARPs. Furthermore, in animals treated with 78c we observed inhibition of pathways that negatively affect health span, such as mTOR-S6K and ERK, and attenuation of telomere-associated DNA damage, a marker of cellular aging. Together, our results detail a novel pharmacological strategy for prevention and/or reversal of age-related NAD+ decline and subsequent metabolic dysfunction.
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•Highly potent and specific CD38 inhibitor, 78c, prevents age-related NAD+ decline•Treatment of old mice with 78c improved physiological and metabolic parameters•Inhibition of CD38 promotes an increase in NAD+ and its precursors in tissue•78c is a novel NAD+-boosting therapy to prevent age-related NAD+ decline
A reduction in nicotinamide adenine dinucleotide (NAD+) is associated with aging. Tarragó et al. show physiological and metabolic improvements of aging in old mice given the small molecule 78c, which inhibits the NADase enzyme CD38. Mechanistically, mTORS6K/ERK and telomere-associated DNA damage pathways mitigate the NAD+ decline.
Many chronic disease patients experience a concurrent loss of lean muscle mass. Skeletal muscle is a dynamic tissue maintained by continuous protein turnover and progenitor cell activity. Muscle stem ...cells, or satellite cells, differentiate (by a process called myogenesis) and fuse to repair and regenerate muscle. During myogenesis, satellite cells undergo extensive metabolic alterations; therefore, pathologies characterized by metabolic derangements have the potential to impair myogenesis, and consequently exacerbate skeletal muscle wasting. How disease-associated metabolic disruptions in satellite cells might be contributing to wasting is an important question that is largely neglected. With this review we highlight the impact of various metabolic disruptions in disease on myogenesis and skeletal muscle regeneration. We also discuss metabolic therapies with the potential to improve myogenesis, skeletal muscle regeneration, and ultimately muscle mass.
Skeletal muscle wasting drives negative clinical outcomes and is associated with a spectrum of pathologies including cancer. Cancer cachexia is a multi-factorial syndrome that encompasses skeletal ...muscle wasting and remains understudied, despite being a frequent and serious co-morbidity. Deviation from the homeostatic balance between breakdown and regeneration leads to muscle wasting disorders, such as cancer cachexia. Muscle stem cells (MuSCs) are the cellular compartment responsible for muscle regeneration, which makes MuSCs an intriguing target in the context of wasting muscle. Molecular studies investigating MuSCs and skeletal muscle wasting largely focus on transcriptional changes, but our group and others propose that metabolic changes are another layer of cellular regulation underlying MuSC dysfunction in cancer cachexia. In the present study, we combined gene expression and non-targeted metabolomic profiling of myoblasts exposed to wasting conditions (cancer cell conditioned media, CC-CM) to derive a more complete picture of the myoblast response to wasting factors. After mapping these features to annotated pathways, we found that more than half of the mapped pathways were amino acid-related, linking global amino acid metabolic disruption to conditioned media-induced myoblast defects. Notably, arginine metabolism was a highly enriched pathway in combined metabolomic and transcriptomic data. Arginine catabolism generates nitric oxide (NO), an important signaling molecule known to have negative effects on mature muscle. We hypothesize that tumor-derived disruptions in Nitric Oxide Synthase (NOS)2-regulated arginine catabolism impair differentiation of MuSCs. The work presented here further investigates the effect of NOS2 overactivity on myoblast proliferation and differentiation. We show that NOS2 inhibition is sufficient to rescue wasting phenotypes associated with inflammatory cytokines. Ultimately, this work provides new insights into MuSC biology and opens up potential therapeutic avenues for addressing disrupted MuSC dynamics in cancer cachexia.
•Amino acid metabolism is impacted in myoblasts treated with cancer cell conditioned media.•Inflammatory cytokines induce nitric oxide synthase 2 in myoblasts.•Elevated nitric oxide synthase 2 activity impairs myoblast proliferation and differentiation.
Culturing primary muscle stem cells ex vivo is a useful method for studying this cell population in controlled environments. Primary muscle stem cells respond to external stimuli differently than ...immortalized myoblasts (C2C12 cells), making ex vivo culture of muscle stem cells an important tool in understanding cell responses to stimuli. Primary muscle stem cells cultured ex vivo retain a majority of the characteristics they possess in vivo such as the abilities to differentiate into multinucleated structures, and self-renew a stem cell-like population. In this chapter, we describe methods for isolating primary muscle stem cells, controlled differentiation into myotubes, and quantification of differentiation using IncuCyte live cell imaging and analysis software.
Obesity is a serious health concern and is associated with a reduced quality of life and a number of chronic diseases, including diabetes, heart disease, stroke, and cancer. With obesity rates on the ...rise worldwide, adipose tissue biology has become a top biomedical research priority. Despite steady growth in obesity-related research, more investigation into the basic biology of adipose tissue is needed to drive innovative solutions aiming to curtail the obesity epidemic. Adipose progenitor cells (APCs) play a central role in adipose tissue homeostasis and coordinate adipose tissue expansion and remodeling. Although APCs are well studied, defining and characterizing APC subsets remains ambiguous because of ill-defined cellular heterogeneity within this cellular compartment. In this study, we used single-cell RNA sequencing to create a cellular atlas of APC heterogeneity in mouse visceral adipose tissue. Our analysis identified two distinct populations of adipose tissue-derived stem cells (ASCs) and three distinct populations of preadipocytes (PAs). We identified novel cell surface markers that, when used in combination with traditional ASC and preadipocyte markers, could discriminate between these APC subpopulations by flow cytometry. Prospective isolation and molecular characterization of these APC subpopulations confirmed single-cell RNA sequencing gene expression signatures, and ex vivo culture revealed differential expansion/differentiation capabilities. Obese visceral adipose tissue featured relative expansion of less mature ASC and PA subpopulations, and expression analyses revealed major obesity-associated signaling alterations within each APC subpopulation. Taken together, our study highlights cellular and transcriptional heterogeneity within the APC pool, provides new tools to prospectively isolate and study these novel subpopulations, and underscores the importance of considering APC diversity when studying the etiology of obesity.
Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key ...role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies - including single-cell RNA-sequencing and single-cell proteomics - is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1
/CD31
/CD45
/Ter119
APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.
Skeletal muscle satellite cells in their niche are quiescent and upon muscle injury, exit quiescence, proliferate to repair muscle tissue, and self-renew to replenish the satellite cell population. ...To understand the mechanisms involved in maintaining satellite cell quiescence, we identified gene transcripts that were differentially expressed during satellite cell activation following muscle injury. Transcripts encoding RNA binding proteins were among the most significantly changed and included the mRNA decay factor Tristetraprolin. Tristetraprolin promotes the decay of MyoD mRNA, which encodes a transcriptional regulator of myogenic commitment, via binding to the MyoD mRNA 3' untranslated region. Upon satellite cell activation, p38α/β MAPK phosphorylates MAPKAP2 and inactivates Tristetraprolin, stabilizing MyoD mRNA. Satellite cell specific knockdown of Tristetraprolin precociously activates satellite cells in vivo, enabling MyoD accumulation, differentiation and cell fusion into myofibers. Regulation of mRNAs by Tristetraprolin appears to function as one of several critical post-transcriptional regulatory mechanisms controlling satellite cell homeostasis.
Altered stem cell homeostasis is linked to organismal aging. However, the mechanisms involved remain poorly understood. Here we report novel alterations in hair follicle stem cells during skin aging, ...including increased numbers, decreased function, and an inability to tolerate stress. Performing high-throughput RNA sequencing on aging stem cells, cytokine arrays, and functional assays, we identify an age-associated imbalance in epidermal Jak-Stat signaling that inhibits stem cell function. Collectively, this study reveals a role for the aging epidermis in the disruption of cytokine and stem cell homeostasis, suggesting that stem cell decline during aging may be part of broader tumor-suppressive mechanisms.
Background
Persistent loss of skeletal muscle mass and function as well as altered fat metabolism are frequently observed in severe sepsis survivors. Studies examining sepsis‐associated tissue ...dysfunction from the perspective of the tissue microenvironment are scarce. In this study, we comprehensively assessed transcriptional changes in muscle and fat at single‐cell resolution following experimental sepsis induction.
Methods
Skeletal muscle and visceral white adipose tissue from control mice or mice 1 day or 1 month following faecal slurry‐induced sepsis were used. Single cells were mechanically and enzymatically prepared from whole tissue, and viable cells were further isolated by fluorescence activated cell sorting. Droplet‐based single‐cell RNA‐sequencing (scRNA‐seq; 10× Genomics) was used to generate single‐cell gene expression profiles of thousands of muscle and fat‐resident cells. Bioinformatics analyses were performed to identify and compare individual cell populations in both tissues.
Results
In skeletal muscle, scRNA‐seq analysis classified 1438 single cells into myocytes, endothelial cells, fibroblasts, mesenchymal stem cells, macrophages, neutrophils, T‐cells, B‐cells, and dendritic cells. In adipose tissue, scRNA‐seq analysis classified 2281 single cells into adipose stem cells, preadipocytes, endothelial cells, fibroblasts, macrophages, dendritic cells, B‐cells, T‐cells, NK cells, and gamma delta T‐cells. One day post‐sepsis, the proportion of most non‐immune cell populations was decreased, while immune cell populations, particularly neutrophils and macrophages, were highly enriched. Proportional changes of endothelial cells, neutrophils, and macrophages were validated using faecal slurry and cecal ligation and puncture models. At 1 month post‐sepsis, we observed persistent enrichment/depletion of cell populations and further uncovered a cell‐type and tissue‐specific ability to return to a baseline transcriptomic state. Differential gene expression analyses revealed key genes and pathways altered in post‐sepsis muscle and fat and highlighted the engagement of infection/inflammation and tissue damage signalling. Finally, regulator analysis identified gonadotropin‐releasing hormone and Bay 11‐7082 as targets/compounds that we show can reduce sepsis‐associated loss of lean or fat mass.
Conclusions
These data demonstrate persistent post‐sepsis muscle and adipose tissue disruption at the single‐cell level and highlight opportunities to combat long‐term post‐sepsis tissue wasting using bioinformatics‐guided therapeutic interventions.
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