Myogenesis defects in a patient-derived iPSC model of hereditary GNE myopathy.

Hereditary muscle diseases are disabling disorders lacking effective treatments. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy (GNEM) is an autosomal recessive distal myopathy with rimmed vacuoles typically manifesting in late adolescence/early adulthood. GNE encodes the rate-limiting enzyme in sialic acid biosynthesis, which is necessary for the proper function of numerous biological processes. Outside of the causative gene, very little is known about the mechanisms contributing to the development of GNE myopathy. In the present study, we aimed to address this knowledge gap by querying the underlying mechanisms of GNE myopathy using a patient-derived induced pluripotent stem-cell (iPSC) model. Control and patient-specific iPSCs were differentiated down a skeletal muscle lineage, whereby patient-derived GNEM iPSC clones were able to recapitulate key characteristics of the human pathology and further demonstrated defects in myogenic progression. Single-cell RNA sequencing time course studies revealed clear differences between control and GNEM iPSC-derived muscle precursor cells (iMPCs), while pathway studies implicated altered stress and autophagy signaling in GNEM iMPCs. Treatment of GNEM patient-derived iMPCs with an autophagy activator improved myogenic differentiation. In summary, we report an in vitro, iPSC-based model of GNE myopathy and implicate defective myogenesis as a contributing mechanism to the etiology of GNE myopathy.

ACUTE AND SUSTAINED ALTERATIONS TO THE BONE MARROW IMMUNE MICROENVIRONMENT FOLLOWING POLYMICROBIAL INFECTION.

ABSTRACT: Sepsis is a highly prevalent cause of death in intensive care units. Characterized by severe immune cell derangements, sepsis is often associated with multiorgan dysfunction. For many sepsis survivors, these deficits can persist long after clinical resolution of the underlying infection. Although many studies report on the impact of sepsis on individual immune cell subtypes, a comprehensive analysis of sepsis-induced alterations within and across the immune cell landscape is lacking. In this study, we used single-cell RNA sequencing to assess sepsis-associated transcriptional changes in immune cells isolated from bone marrow at single-cell resolution. We used a high-survival fecal-induced peritonitis sepsis model using Friend leukemia virus B mice. Single-cell RNA sequencing classified 3402 single cells from control subjects into 14 clusters representing long-term hematopoietic stem cell (HSC), short-term HSC, basophil, dendritic cell, eosinophil, erythroblast, erythrocyte, macrophage, neutrophil, natural killer cell, plasma cell, plasmacytoid dendritic cell, pre-B cell, and T memory cell lineages. One day following experimentally induced sepsis, cell type compositions shifted significantly and included notable decreases in HSC and myeloid cell abundance. In addition to proportional cell composition changes, acute sepsis induced significant transcriptional alterations in most immune cell types analyzed-changes that failed to completely resolve 1 month after sepsis. Taken together, we report widespread and persistent transcriptional changes in diverse immune cells in response to polymicrobial infection. This study will serve as a valuable resource for future work investigating acute and/or long-term sepsis-associated immune cell derangements.

Anticachectic regulator analysis reveals Perp-dependent antitumorigenic properties of 3-methyladenine in pancreatic cancer.

Approximately 80% of pancreatic cancer patients suffer from cachexia, and one-third die due to cachexia-related complications such as respiratory failure and cardiac arrest. Although there has been considerable research into cachexia mechanisms and interventions, there are, to date, no FDA-approved therapies. A major contributing factor for the lack of therapy options could be the failure of animal models to accurately recapitulate the human condition. In this study, we generated an aged model of pancreatic cancer cachexia to compare cachexia progression in young versus aged tumor-bearing mice. Comparative skeletal muscle transcriptome analyses identified 3-methyladenine (3-MA) as a candidate antiwasting compound. In vitro analyses confirmed antiwasting capacity, while in vivo analysis revealed potent antitumor effects. Transcriptome analyses of 3-MA-treated tumor cells implicated Perp as a 3-MA target gene. We subsequently (a) observed significantly higher expression of Perp in cancer cell lines compared with control cells, (b) noted a survival disadvantage associated with elevated Perp, and (c) found that 3-MA-associated Perp reduction inhibited tumor cell growth. Finally, we have provided in vivo evidence that survival benefits conferred by 3-MA administration are independent of its effect on tumor progression. Taken together, we report a mechanism linking 3-MA to Perp inhibition, and we further implicate Perp as a tumor-promoting factor in pancreatic cancer.

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 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.

Single-cell deconstruction of post-sepsis skeletal muscle and adipose tissue microenvironments.

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.

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

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.

Skeletal Muscle Progenitor Cell Heterogeneity.

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.

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

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.

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

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.

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

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.

Single cell transcriptome analysis of muscle satellite cells reveals widespread transcriptional heterogeneity.

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.

Generation of induced pluripotent stem cells from Chinese hamster embryonic fibroblasts.

We derived a stable cell line from Chinese hamster embryonic fibroblasts by transduction of four mouse transcription factors (M3O, Sox2, Klf4, and n-Myc) using a lentiviral vector. The cell line possess all the characteristics of an induced pluripotent stem cell (iPSC) line. Given that Chinese hamster ovary (CHO) cells are the predominant host cells used for therapeutic protein production and no pluripotent stem cell line or other normal cell line has been isolated from Chinese hamster, this iPSC line may serve as a useful tool for research using CHO cells or even be used for deriving new cell lines.

PDGFRα+ Cells in Embryonic Stem Cell Cultures Represent the In Vitro Equivalent of the Pre-implantation Primitive Endoderm Precursors.

In early mouse pre-implantation development, primitive endoderm (PrE) precursors are platelet-derived growth factor receptor alpha (PDGFRα) positive. Here, we demonstrated that cultured mouse embryonic stem cells (mESCs) express PDGFRα heterogeneously, fluctuating between a PDGFRα+ (PrE-primed) and a platelet endothelial cell adhesion molecule 1 (PECAM1)-positive state (epiblast-primed). The two surface markers can be co-detected on a third subpopulation, expressing epiblast and PrE determinants (double-positive). In vitro, these subpopulations differ in their self-renewal and differentiation capability, transcriptional and epigenetic states. In vivo, double-positive cells contributed to epiblast and PrE, while PrE-primed cells exclusively contributed to PrE derivatives. The transcriptome of PDGFRα+ subpopulations differs from previously described subpopulations and shows similarities with early/mid blastocyst cells. The heterogeneity did not depend on PDGFRα but on leukemia inhibitory factor and fibroblast growth factor signaling and DNA methylation. Thus, PDGFRα+ cells represent the in vitro counterpart of in vivo PrE precursors, and their selection from cultured mESCs yields pure PrE precursors.

Cell Expansion During Directed Differentiation of Stem Cells Toward the Hepatic Lineage.

The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 109-1010 cells, because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage, to allow for at least an eightfold increase in cell number, with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array, and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition, our transcriptome, protein and functional studies, including albumin secretion, drug-induced CYP450 expression and urea production, all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications.