preparation-adipose-progenitor-cells-from-mouse-epididymal-adipose

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Mayo Clinic

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 transcriptome analysis of muscle satellite cells reveals widespread transcriptional heterogeneity.

Muscle wasting is a decline in skeletal muscle mass and function that is associated with aging, obesity, and a spectrum of pathologies including cancer. Cancer-associated wasting not only reduces quality of life, but also directly impacts cancer mortality, chemotherapeutic efficacy, and surgical outcomes. There is an incomplete understanding of the role of tumor-derived factors in muscle wasting and sparse knowledge of how these factors impact in vivo muscle regeneration. Here, we identify several cytokines/chemokines that negatively impact in vitro myogenic differentiation. We show that one of these cytokines, CXCL1, potently antagonizes in vivo muscle regeneration and interferes with in vivo muscle satellite cell homeostasis. Strikingly, CXCL1 triggers a robust and specific neutrophil/M2 macrophage response that likely underlies or exacerbates muscle repair/regeneration defects. Taken together, these data highlight the pleiotropic nature of a novel tumor-derived cytokine and underscore the importance of cytokines in muscle progenitor cell regulation.

Tumor-derived cytokines impair myogenesis and alter the skeletal muscle immune microenvironment.

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.

A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD Decline.

More than 80% of patients with advanced cancer develop weight loss. Because preclinical data suggest poly (ADP-ribose) polymerase (PARP) inhibitors can treat this weight loss, this study was undertaken to explore the PARP inhibitor veliparib for this indication.

Does the Poly (ADP-Ribose) Polymerase Inhibitor Veliparib Merit Further Study for Cancer-Associated Weight Loss? Observations and Conclusions from Sixty Prospectively Treated Patients.

Duchenne muscular dystrophy (DMD) is a musculoskeletal disorder that causes severe morbidity and reduced lifespan. Individuals with DMD have an X-linked mutation that impairs their ability to produce functional dystrophin protein in muscle. No cure exists for this disease and the few therapies that are available do not dramatically delay disease progression. Thus, there is a need to better understand the mechanisms underlying DMD which may ultimately lead to improved treatment options. The muscular dystrophy (MDX) mouse model is frequently used to explore DMD disease traits. Though some studies of metabolism in dystrophic mice exist, few have characterized metabolic profiles of supporting cells in the diseased environment. Using nontargeted metabolomics we characterized metabolic alterations in muscle satellite cells (SCs) and serum of MDX mice. Additionally, live-cell imaging revealed MDX-derived adipose progenitor cell (APC) defects. Finally, metabolomic studies revealed a striking elevation of acylcarnitines in MDX APCs, which we show can inhibit APC proliferation. Together, these studies highlight widespread metabolic alterations in multiple progenitor cell types and serum from MDX mice and implicate dystrophy-associated metabolite imbalances in APCs as a potential contributor to adipose tissue disequilibrium in DMD.

Metabolomic Analyses Reveal Extensive Progenitor Cell Deficiencies in a Mouse Model of Duchenne Muscular Dystrophy.

Tissue-specific stem cells contribute to adult tissue maintenance, repair, and regeneration. In skeletal muscle, many different mononuclear cell types are capable of giving rise to differentiated muscle. Of these tissue stem-like cells, satellite cells (SCs) are the most studied muscle stem cell population and are widely considered the main cellular source driving muscle repair and regeneration in adult tissue. Within the satellite cell pool, many distinct subpopulations exist, each exhibiting differential abilities to exit quiescence, expand, differentiate, and self-renew. In this chapter, we discuss the different stem cell types that can give rise to skeletal muscle tissue and then focus on satellite cell heterogeneity during the process of myogenesis/muscle regeneration. Finally, we highlight emerging opportunities to better characterize muscle stem cell heterogeneity, which will ultimately deepen our appreciation of stem cells in muscle development, repair/regeneration, aging, and disease.

Skeletal Muscle Progenitor Cell Heterogeneity.

Muscle wasting, a cardinal feature of cancer-associated cachexia (CAC), is a major clinical problem with few therapeutic options. In this Forum article we discuss cellular mechanisms of CAC, focusing on impaired muscle regeneration. We highlight muscle progenitor cell dysfunction and metabolism as two variables contributing to impaired regeneration in CAC.

Impaired Muscle Regeneration in Cancer-Associated Cachexia.

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.

Refining the adipose progenitor cell landscape in healthy and obese visceral adipose tissue using single-cell gene expression profiling.

Cancer-associated muscle wasting (CAW), a symptom of cancer cachexia, is associated with approximately 20% of lung cancer deaths and remains poorly characterized on a mechanistic level. Current animal models for lung cancer-associated cachexia are limited in that they (1) primarily employ flank transplantation methods, (2) have short survival times not reflective of the patient condition, and (3) are typically performed in young mice not representative of mean patient age. This study investigates a new model for lung cancer-associated cachexia that can address these issues and also implicates muscle regeneration as a contributor to CAW.

A novel transplantable model of lung cancer-associated tissue loss and disrupted muscle regeneration.

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.

Article	Year
Single cell transcriptome analysis of muscle satellite cells reveals widespread transcriptional heterogeneity. Gene\| PubMed ID: 28893664	2017
Tumor-derived cytokines impair myogenesis and alter the skeletal muscle immune microenvironment. Cytokine\| PubMed ID: 29153940	2018
A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD Decline. Cell metabolism\| PubMed ID: 29719225	2018
Does the Poly (ADP-Ribose) Polymerase Inhibitor Veliparib Merit Further Study for Cancer-Associated Weight Loss? Observations and Conclusions from Sixty Prospectively Treated Patients. Journal of palliative medicine\| PubMed ID: 29792535	2018
Metabolomic Analyses Reveal Extensive Progenitor Cell Deficiencies in a Mouse Model of Duchenne Muscular Dystrophy. Metabolites\| PubMed ID: 30282911	2018
Skeletal Muscle Progenitor Cell Heterogeneity. Advances in experimental medicine and biology\| PubMed ID: 31487024	2019
Impaired Muscle Regeneration in Cancer-Associated Cachexia. Trends in cancer\| PubMed ID: 31706505	2019
Refining the adipose progenitor cell landscape in healthy and obese visceral adipose tissue using single-cell gene expression profiling. Life science alliance\| PubMed ID: 31767614	2019
A novel transplantable model of lung cancer-associated tissue loss and disrupted muscle regeneration. Skeletal muscle\| PubMed ID: 32151276	2020
Single-cell deconstruction of post-sepsis skeletal muscle and adipose tissue microenvironments. Journal of cachexia, sarcopenia and muscle\| PubMed ID: 32643301	2020

Jason D. Doles

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