Skeletal Muscle Progenitor Cells Research Articles

Derivation and long-term maintenance of porcine skeletal muscle progenitor cells

Culture of muscle cells from livestock species has typically involved laborious enzyme-based approaches that yield heterogeneous populations with limited proliferative and myogenic differentiation capacity, thus limiting their use in physiologically-meaningful studies. This study reports the use of a simple explant culture technique to derive progenitor cell populations from porcine muscle that could be maintained and differentiated long-term in culture. Fragments of semitendinosus muscle from 4 to 8 week-old piglets (n = 4) were seeded on matrigel coated culture dishes to stimulate migration of muscle-derived progenitor cells (MDPCs). Cell outgrowths appeared within a few days and were serially passaged and characterised using RT-qPCR, immunostaining and flow cytometry. MDPCs had an initial mean doubling time of 1.4 days which increased to 2.5 days by passage 14. MDPC populations displayed steady levels of the lineage-specific markers, PAX7 and MYOD, up until at least passage 2 (positive immunostaining in about 40% cells for each gene), after which the expression of myogenic markers decreased gradually. Remarkably, MDPCs were able to readily generate myotubes in culture up until passage 8. Moreover, a decrease in myogenic capacity during serial passaging was concomitant with a gradual increase in the expression of the pre-adipocyte markers, CD105 and PDGFRA, and an increase in the ability of MDPCs to differentiate into adipocytes. In conclusion, explant culture provided a simple and efficient method to harvest enriched myogenic progenitors from pig skeletal muscle which could be maintained long-term and differentiated in vitro, thus providing a suitable system for studies on porcine muscle biology and applications in the expanding field of cultured meat.

Abstract 136: Determining the genomic basis of PAX3-FOXO1 mediated transformation in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Despite rigorous scientific advances and clinical trials, the survival rate for high-risk RMS has not increased above 20% in the last three decades. A deeper understanding of the basic biology driving RMS tumorigenesis is needed to discover novel therapeutic targets. RMS can be divided into two major histologic subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS). ARMS accounts for 20% of pediatric cases and has poorer survival. The majority of ARMS tumors harbor t(2;13)(q35;q14) or t(1;13)(p36;q14) chromosomal translocations resulting in the PAX3-FOXO1 (P3F) and PAX7-FOXO1 (P7F) fusion oncoproteins, respectively. ARMS tumors that lack oncofusions more closely resemble ERMS. Classifying on fusion status more accurately describes the disease and highlights the significance of P3/7F. ARMS tumor genomes only contain an average of 6.4 somatic mutations suggesting the oncofusion protein is the key oncogenic driver and a potential candidate for therapeutic intervention. RMS is commonly thought to arise from skeletal muscle progenitor cells that fail to terminally differentiate. However, RMS occurs throughout the body in sites devoid of skeletal muscle, underscoring the potential for alternative cells of origin. Our lab established that endothelial progenitors are indeed a cell of origin for ARMS by generating a genetically engineered mouse model with endothelial cell specific P3F expression coupled with Cdkn2a loss. We further generated a human model system derived from TP53-null human induced pluripotent stem cells (iPSCs), in which we direct differentiation to endothelial cell fate and then force P3F expression. P3F expression during endothelial cell directed differentiation blocked endothelial maturation and instead reprogrammed cells shifting cell fate to skeletal muscle-like cells that form ARMS tumors in mice. Generation of iPSC-derived ARMS (iARMS) occurs over 15 days and provides a unique model to determine the kinetics and specific genomic contributions facilitated by P3F during RMS transformation. To investigate the genomic changes occurring during iARMS transformation I utilized CUT&RUN for P3F and H3K27ac to assess P3F occupancy and enhancer landscape, ATAC-seq to assess chromatin accessibility, and RNA-seq to assess gene expression changes at timepoints throughout the transformation event. These results define the fundamental genomic changes needed for transformation into FP-RMS by P3F and uncovers the key determinants of ARMS cell identity. The iARMS system provides a unique platform to uncover how P3F establishes and maintains RMS cell state and to dissect the specific dependencies required to maintain the ARMS cell state. Understanding the fundamental mechanism of P3F-driven tumorigenesis could provide greater resolution into the key disease determinants to focus pre-clinical efforts on pivotal targets. Citation Format: Bradley T. Stevens, Yang Zhang, Randolph K. Larsen, Grace E. Adkins, Jack D. Hopkins, Darden W. Kimbrough, Matthew R. Garcia, Brian J. Abraham, Mark E. Hatley. Determining the genomic basis of PAX3-FOXO1 mediated transformation in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 136.

Motor neurons and endothelial cells additively promote development and fusion of human iPSC-derived skeletal myocytes

BackgroundNeurovascular cells have wide-ranging implications on skeletal muscle biology regulating myogenesis, maturation, and regeneration. Although several in vitro studies have investigated how motor neurons and endothelial cells interact with skeletal myocytes independently, there is limited knowledge about the combined effect of neural and vascular cells on muscle maturation and development.MethodsHere, we report a triculture system comprising human-induced pluripotent stem cell (iPSC)-derived skeletal myocytes, human iPSC-derived motor neurons, and primary human endothelial cells maintained under controlled media conditions. Briefly, iPSCs were differentiated to generate skeletal muscle progenitor cells (SMPCs). These SMPCs were seeded at a density of 5 × 104 cells/well in 12-well plates and allowed to differentiate for 7 days before adding iPSC-derived motor neurons at a concentration of 0.5 × 104 cells/well. The neuromuscular coculture was maintained for another 7 days in coculture media before addition of primary human umbilical vein endothelial cells (HUVEC) also at 0.5 × 104 cells/well. The triculture was maintained for another 7 days in triculture media comprising equal portions of muscle differentiation media, coculture media, and vascular media. Extensive morphological, genetic, and molecular characterization was performed to understand the combined and individual effects of neural and vascular cells on skeletal muscle maturation.ResultsWe observed that motor neurons independently promoted myofiber fusion, upregulated neuromuscular junction genes, and maintained a molecular niche supportive of muscle maturation. Endothelial cells independently did not support myofiber fusion and downregulated expression of LRP4 but did promote expression of type II specific myosin isoforms. However, neurovascular cells in combination exhibited additive increases in myofiber fusion and length, enhanced production of Agrin, along with upregulation of several key genes like MUSK, RAPSYN, DOK-7, and SLC2A4. Interestingly, more divergent effects were observed in expression of genes like MYH8, MYH1, MYH2, MYH4, and LRP4 and secretion of key molecular factors like amphiregulin and IGFBP-4.ConclusionsNeurovascular cells when cultured in combination with skeletal myocytes promoted myocyte fusion with concomitant increase in expression of various neuromuscular genes. This triculture system may be used to gain a deeper understanding of the effects of the neurovascular niche on skeletal muscle biology and pathophysiology.

Fluorescent characterization of differentiated myotubes using flow cytometry.

Flow cytometry is routinely used in the assessment of skeletal muscle progenitor cell (myoblast) populations. However, a full gating strategy, inclusive of difficult to interpret forward and side scatter data, which documents cytometric analysis of differentiated myoblasts (myotubes) has not been reported. Beyond changes in size and shape, there are substantial metabolic and protein changes in myotubes allowing for their potential identification within heterogenous cell suspensions. To establish the utility of flow cytometry for determination of myoblasts and myotubes, C2C12 murine cell populations were assessed for cell morphology and metabolic reprogramming. Laser scatter, both forward (FSC; size) and side (SSC; granularity), measured cell morphology, while mitochondrial mass, reactive oxygen species (ROS) generation and DNA content were quantified using the fluorescent probes, MitoTracker green, CM-H2 DCFDA and Vybrant DyeCycle, respectively. Immunophenotyping for myosin heavy chain (MyHC) was utilized to confirm myotube differentiation. Cellular viability was determined using Annexin V/propidium iodide dual labelling. Fluorescent microscopy was employed to visualize fluorescence and morphology. Myotube and myoblast populations were resolvable through non-intuitive interpretation of laser scatter-based morphology assessment and mitochondrial mass and activity assessment. Myotubes appeared to have similar sizes to the myoblasts based on laser scatter but exhibited greater mitochondrial mass (159%, p < 0.0001), ROS production (303%, p < 0.0001), DNA content (18%, p < 0.001) and expression of MyHC (147%, p < 0.001) compared to myoblasts. Myotube sub-populations contained a larger viable cluster of cells which were unable to be fractionated from myoblast populations and a smaller population cluster which likely contains apoptotic bodies. Imaging of differentiated myoblasts that had transited through the flow cytometer revealed the presence of intact, 'rolled-up' myotubes, which would alter laser scatter properties and potential transit through the laser beam. Our results indicate that myotubes can be analyzed successfully using flow cytometry. Increased mitochondrial mass, ROS and DNA content are key features that correlate with MyHC expression but due to myotubes 'rolling up' during flow cytometric analysis, laser scatter determination of size is not positively correlated; a phenomenon observed with some size determination particles and related to surface properties of said particles. We also note a greater heterogeneity of myotubes compared to myoblasts as evidenced by the 2 distinct sub-populations. We suggest that acoustic focussing may prove effective in identifying myotube sub populations compared to traditional hydrodynamic focussing.

Generation of highly pure pluripotent stem cell-derived myogenic progenitor cells and myotubes

Driving efficient and pure skeletal muscle cell differentiation from pluripotent stem cells (PSCs) has been challenging. Here, we report an optimized protocol that generates skeletal muscle progenitor cells with high efficiency and purity in a short period of time. Human induced PSCs (hiPSCs) and murine embryonic stem cells (mESCs) were specified into the mesodermal myogenic fate using distinct and species-specific protocols. We used a specific maturation medium to promote the terminal differentiation of both human and mouse myoblast populations, and generated myotubes associated with a large pool of cell-cycle arrested PAX7+ cells. We also show that myotube maturation is modulated by dish-coating properties, cell density, and percentage of myogenic progenitor cells. Given the high efficiency in the generation of myogenic progenitors and differentiated myofibers, this protocol provides an attractive strategy for tissue engineering, modeling of muscle dystrophies, and evaluation of new therapeutic approaches invitro.

Generation of three-dimensional meat-like tissue from stable pig epiblast stem cells

Cultured meat production has emerged as a breakthrough technology for the global food industry with the potential to reduce challenges associated with environmental sustainability, global public health, animal welfare, and competition for food between humans and animals. The muscle stem cell lines currently used for cultured meat cannot be passaged in vitro for extended periods of time. Here, we develop a directional differentiation system of porcine pre-gastrulation epiblast stem cells (pgEpiSCs) with stable cellular features and achieve serum-free myogenic differentiation of the pgEpiSCs. We show that the pgEpiSCs-derived skeletal muscle progenitor cells and skeletal muscle fibers have typical muscle cell characteristics and display skeletal muscle transcriptional features during myogenic differentiation. Importantly, we establish a three-dimensional differentiation system for shaping cultured tissue by screening plant-based edible scaffolds of non-animal origin, followed by the generation of pgEpiSCs-derived cultured meat. These advances provide a technical approach for the development of cultured meat.

CRISPR/Cas9 Genome Editing in LGMD2A/R1 Patient-Derived Induced Pluripotent Stem and Skeletal Muscle Progenitor Cells.

Large numbers of Calpain 3 (CAPN3) mutations cause recessive forms of limb-girdle muscular dystrophy (LGMD2A/LGMDR1) with selective atrophy of the proximal limb muscles. We have generated induced pluripotent stem cells (iPSC) from a patient with two mutations in exon 3 and exon 4 at the calpain 3 locus (W130C, 550delA). Two different strategies to rescue these mutations are devised: (i) on the level of LGMD2A-iPSC, we combined CRISPR/Cas9 genome targeting with a FACS and Tet transactivator-based biallelic selection strategy, which resulted in a new functional chimeric exon 3-4 without the two CAPN3 mutations. (ii) On the level of LGMD2A-iPSC-derived CD82+/Pax7+ myogenic progenitor cells, we demonstrate CRISPR/Cas9 mediated rescue of the highly prevalent exon 4 CAPN3 mutation. The first strategy specifically provides isogenic LGMD2A corrected iPSC for disease modelling, and the second strategy can be further elaborated for potential translational approaches.

Regenerating human skeletal muscle forms an emerging niche in vivo to support PAX7 cells

Skeletal muscle stem and progenitor cells including those derived from human pluripotent stem cells (hPSCs) offer an avenue towards personalized therapies and readily fuse to form human–mouse myofibres in vivo. However, skeletal muscle progenitor cells (SMPCs) inefficiently colonize chimeric stem cell niches and instead associate with human myofibres resembling foetal niches. We hypothesized competition with mouse satellite cells (SCs) prevented SMPC engraftment into the SC niche and thus generated an SC ablation mouse compatible with human engraftment. Single-nucleus RNA sequencing of SC-ablated mice identified the absence of a transient myofibre subtype during regeneration expressing Actc1. Similarly, ACTC1+ human myofibres supporting PAX7+ SMPCs increased in SC-ablated mice, and after re-injury we found SMPCs could now repopulate into chimeric niches. To demonstrate ACTC1+ myofibres are essential to supporting PAX7 SMPCs, we generated caspase-inducible ACTC1 depletion human pluripotent stem cells, and upon SMPC engraftment we found a 90% reduction in ACTC1+ myofibres and a 100-fold decrease in PAX7 cell numbers compared with non-induced controls. We used spatial RNA sequencing to identify key factors driving emerging human niche formation between ACTC1+ myofibres and PAX7+ SMPCs in vivo. This revealed that transient regenerating human myofibres are essential for emerging niche formation in vivo to support PAX7 SMPCs.

SIX1+PAX3+ identify a progenitor for myogenic lineage commitment from hPSCs.

The earliest skeletal muscle progenitor cells (SMPCs) derived from human pluripotent stem cells (hPSCs) are often identified by factors expressed by a diverse number of progenitors. An early transcriptional checkpoint that defines myogenic commitment could improve hPSC differentiation to skeletal muscle. Analysis of several myogenic factors in human embryos and early hPSC differentiations found SIX1+PAX3+ co-expression was most indictive of myogenesis. Using dCas9-KRAB hPSCs, we demonstrate that early inhibition of SIX1 alone significantly decreased PAX3 expression, reduced PAX7+ SMPCs, and myotubes later in differentiation. Emergence of SIX1+PAX3+ precursors can be improved by manipulating seeding density, monitoring metabolic secretion and altering the concentration of CHIR99021. These modifications resulted in the co-emergence of hPSC-derived sclerotome, cardiac and neural crest that we hypothesized enhanced hPSC myogenic differentiation. Inhibition of non-myogenic lineages modulated PAX3 independent of SIX1. To better understand SIX1 expression, we compared directed differentiations to fetal progenitors and adult satellite cells by RNA-seq. Although SIX1 continued to be expressed across human development, SIX1 co-factor expression was dependent on developmental timing. We provide a resource to enable efficient derivation of skeletal muscle from hPSCs.

Mitochondrial Dysfunction: At the Nexus between Alcohol-Associated Immunometabolic Dysregulation and Tissue Injury.

Alcohol misuse, directly or indirectly as a result of its metabolism, negatively impacts most tissues, including four with critical roles in energy metabolism regulation: the liver, pancreas, adipose, and skeletal muscle. Mitochondria have long been studied for their biosynthetic roles, such as ATP synthesis and initiation of apoptosis. However, current research has provided evidence that mitochondria participate in myriad cellular processes, including immune activation, nutrient sensing in pancreatic β-cells, and skeletal muscle stem and progenitor cell differentiation. The literature indicates that alcohol impairs mitochondrial respiratory capacity, promoting reactive oxygen species (ROS) generation and disrupting mitochondrial dynamics, leading to dysfunctional mitochondria accumulation. As discussed in this review, mitochondrial dyshomeostasis emerges at a nexus between alcohol-disrupted cellular energy metabolism and tissue injury. Here, we highlight this link and focus on alcohol-mediated disruption of immunometabolism, which refers to two distinct, yet interrelated processes. Extrinsic immunometabolism involves processes whereby immune cells and their products influence cellular and/or tissue metabolism. Intrinsic immunometabolism describes immune cell fuel utilization and bioenergetics that affect intracellular processes. Alcohol-induced mitochondrial dysregulation negatively impacts immunometabolism in immune cells, contributing to tissue injury. This review will present the current state of literature, describing alcohol-mediated metabolic and immunometabolic dysregulation from a mitochondrial perspective.

Myoscaffolds reveal laminin scarring is detrimental for stem cell function while sarcospan induces compensatory fibrosis

We developed an on-slide decellularization approach to generate acellular extracellular matrix (ECM) myoscaffolds that can be repopulated with various cell types to interrogate cell-ECM interactions. Using this platform, we investigated whether fibrotic ECM scarring affected human skeletal muscle progenitor cell (SMPC) functions that are essential for myoregeneration. SMPCs exhibited robust adhesion, motility, and differentiation on healthy muscle-derived myoscaffolds. All SPMC interactions with fibrotic myoscaffolds from dystrophic muscle were severely blunted including reduced motility rate and migration. Furthermore, SMPCs were unable to remodel laminin dense fibrotic scars within diseased myoscaffolds. Proteomics and structural analysis revealed that excessive collagen deposition alone is not pathological, and can be compensatory, as revealed by overexpression of sarcospan and its associated ECM receptors in dystrophic muscle. Our in vivo data also supported that ECM remodeling is important for SMPC engraftment and that fibrotic scars may represent one barrier to efficient cell therapy.

Establishment of fibroblast and myofibroblast phenotypes for use in in vitro co-culture models

Fibroblasts function to secrete and modify components of the extracellular matrix. During wound healing, fibroblasts migrate to the site of injury and differentiate into contractile myofibroblasts; this differentiation is characterised by an increased contractile capacity. Fully differentiated myofibroblasts can be distinguished from fibroblasts via the higher expression of α-smooth muscle actin as well as a denser cytoskeleton. Impaired wound healing has been characterised by a lack of myofibroblasts; as a result, tissue does not fully regain its strength and function. Under pathological conditions, this may be associated with the effect that a pro-inflammatory microenvironment has on fibroblast and skeletal muscle progenitor cell migration and differentiation.Given their distinct roles in tissue maintenance and repair, the communication between fibroblasts versus myofibroblasts with other cellular mediators of repair is likely to influence cell behaviour and the outcome of wound repair. An in vitro test model is required to investigate this intercellular influence, but the establishment of such a model is hampered by the difficulty in retaining the dedifferentiated fibroblastic phenotype under regular serum-containing cell culture conditions. We present a model that supports the establishment and retention in culture of fibroblast and myofibroblast phenotypes for use in a simple, inexpensive, yet relevant in vitro 2D assay. This model is then applied in a co-culture setting to determine whether the presence of myoblasts affects the ability of fibroblasts versus myofibroblasts to close an in vitro wound. Our results emphasize the importance of considering the impact of paracrine communication between all cells during wound healing.

I.09 Induced pluripotent stem cells for modeling neuromuscular disorders: development of disease-specific assays, live cells functional testing and drug design

Induced pluripotent stem cells (iPSC) represent an innovative, easily obtained and renewable stem cell source. This somatic cell-derived model that can be derived without considerable ethical issues has generated significant enthusiasm for its potential application in basic and translational research or development of new therapeutics. In particular, patient-specific iPS-derived tissues and latterly, organoids offer attractive platform to model a wide range of diseases but also personalized or regenerative medicine. Compared to other tissues, only a limited number of large-scale protocols describe the production of mature skeletal muscle fibers from human iPSCs (hiPSCs). We developed a procedure for simultaneous differentiation of hiPSC into muscle cells and motor neurons, that generates innervated and contractile multinucleated skeletal muscle fibers with sarcomeric organization and formation of neuromuscular junction. Our protocol permits the production of expandable skeletal muscle progenitor cells and mature skeletal muscle fibers that can be maintained and regenerates on the long-term. These in vitro muscle constructs can be used for the exploration of skeletal muscle differentiation for basic research, disease modeling and drug discovery. Our “muscle in a dish” model has been used to successfully model a number of neuromuscular disorders. By combining transcriptomics with artificial intelligence, we were able to design specific pipelines for a comprehensive understanding of the molecular, cell biological and functional properties of hiPSC-derived muscle tissue in diseases. Overall, this strategy opens a wide rage of opportunities for disease modeling, personalized medicine for patients in diagnosis deadlocks, drug design or tissue bioengineering and bioprinting. Induced pluripotent stem cells (iPSC) represent an innovative, easily obtained and renewable stem cell source. This somatic cell-derived model that can be derived without considerable ethical issues has generated significant enthusiasm for its potential application in basic and translational research or development of new therapeutics. In particular, patient-specific iPS-derived tissues and latterly, organoids offer attractive platform to model a wide range of diseases but also personalized or regenerative medicine. Compared to other tissues, only a limited number of large-scale protocols describe the production of mature skeletal muscle fibers from human iPSCs (hiPSCs). We developed a procedure for simultaneous differentiation of hiPSC into muscle cells and motor neurons, that generates innervated and contractile multinucleated skeletal muscle fibers with sarcomeric organization and formation of neuromuscular junction. Our protocol permits the production of expandable skeletal muscle progenitor cells and mature skeletal muscle fibers that can be maintained and regenerates on the long-term. These in vitro muscle constructs can be used for the exploration of skeletal muscle differentiation for basic research, disease modeling and drug discovery. Our “muscle in a dish” model has been used to successfully model a number of neuromuscular disorders. By combining transcriptomics with artificial intelligence, we were able to design specific pipelines for a comprehensive understanding of the molecular, cell biological and functional properties of hiPSC-derived muscle tissue in diseases. Overall, this strategy opens a wide rage of opportunities for disease modeling, personalized medicine for patients in diagnosis deadlocks, drug design or tissue bioengineering and bioprinting.

Plasticizer Di-(2-ethylhexyl) Phthalate and Its Metabolite Mono(2-ethylhexyl) Phthalate Inhibit Myogenesis in Differentiating Mouse and Human Skeletal Muscle Cell Models

The relationship between plasticizer di(2-ethylhexyl) phthalate (DEHP) and low birth weight in neonates has been reported. Immature muscle differentiation may be involved in low birth weight. The myotoxic characteristics of chemicals have been observed in differentiating immortalized and primary muscle cells. Here, we explored the myotoxic effects of DEHP and its metabolite mono(2-ethylhexyl) phthalate (MEHP) in vitro using the immortalized mouse skeletal myoblasts C2C12 and primary human skeletal muscle progenitor cell (HSMPC) models. We found that both DEHP and MEHP at the concentrations of 10–100 μM, which were non- and low-cytotoxicity concentrations, significantly and dose-dependently inhibited the creatine kinase activity, myotube formation with multiple nuclei, and myogenin and myosin heavy chain (muscle differentiation markers) protein expression in C2C12 and HSMPCs under differentiation medium. Both DEHP and MEHP significantly decreased Akt phosphorylation in C2C12 and HSMPCs during differentiation. Taken together, DEHP and its metabolite MEHP are capable of inhibiting Akt-regulated myogenesis in myoblasts/myogenic progenitors during differentiation. These findings suggest the possibility of DEHP as an environmental risk factor affecting skeletal myogenic differentiation. Moreover, these in vitro muscle cell models may be a possible alternative method to animal myotoxicity testing.

Skeletal Muscle Cells Derived From Old Donors Show Altered Mitochondrial Morphology And Reduced Respiratory Capacity

With age, skeletal muscle mitochondria lose their oxidative capacity and their ability to respond to energy demand. These phenomena can lead to a reduction in skeletal muscle oxygen consumption, atrophy, and increased risk of developing age-related diseases such as sarcopenia. Whether age-derived changes in mitochondrial function correspond to structural changes in the mitochondrial reticulum remains unknown. PURPOSE: Investigate age-related changes in mitochondrial morphology and function using primary skeletal muscle cells derived from healthy young and old men. METHODS: Primary skeletal muscle progenitor cells (SkM) derived from the Rectus abdominis muscle of healthy active 18-19-year-old men (SkM Young), and 66-69-year-old men (SkM Old) were obtained from Cook MyoSite Inc. (Pittsburgh, PA). The mitochondrial network was analyzed in live cells using confocal microscopy. Oxygen Consumption Rate (OCR) was measured in intact cells using extracellular flux assays and a Seahorse analyzer (Agilent Technologies; Santa Clara, CA). RESULTS: Primary cells derived from SkM Young revealed a higher Basal and Maximal OCR compared to SkM Old (Basal: 23.20 ± 1.49 vs. 11.34 ± 2.31; Maximal: 41.50 ± 4.72 vs. 18.36 ± 1.65 pmol/min/protein). SkM Young also had higher ATP production and Spare Respiratory Capacity (ATP: 19.08 ± 1.21 vs. 9.19 ± 2.10; SRC: 18.30 ± 3.47 vs. 7.03 ± 1.29 pmol/min/protein). No differences were revealed in morphology (Individuals: 37.80 ± 23.18 vs. 46.67 ± 22.43 counts; Networks: 10.20 ± 2.48 vs. 15.33 ± 5.75 counts; Mean Branches per Network: 15.52 ± 6.41 vs. 11.27 ± 3.67 counts). CONCLUSIONS: These preliminary results show primary skeletal muscle progenitor cells derived from healthy young donors have higher respiration, produce more ATP, and have a greater capacity to adapt to energy demands when compared to SkM derived from Old donors. Further analyses can give us an insight into human skeletal muscle-derived cellular physiological capacity. Technology to observe human muscle mitochondrial dysfunction in vitro helps us understand the effects of aging on skeletal muscle mitochondria and whole-body declines of resting and exercise metabolic rates. Additional studies will be required on aging individuals to determine if predicted decrements in energy coupling efficiency occur with aging in vivo.

New Insights into the Diversity of Branchiomeric Muscle Development: Genetic Programs and Differentiation.

Simple SummaryWe review the transcription factors and signaling molecules driving differentiation of a subset of head muscles known as the branchiomeric muscles due to their origin in the pharyngeal arches. We provide novel data on the distinct myogenic programs within these muscles and explore how the cranial neural crest cell regulates branchiomeric muscle patterning and differentiation.Branchiomeric skeletal muscles are a subset of head muscles originating from skeletal muscle progenitor cells in the mesodermal core of pharyngeal arches. These muscles are involved in facial expression, mastication, and function of the larynx and pharynx. Branchiomeric muscles have been the focus of many studies over the years due to their distinct developmental programs and common origin with the heart muscle. A prerequisite for investigating these muscles’ properties and therapeutic potential is understanding their genetic program and differentiation. In contrast to our understanding of how branchiomeric muscles are formed, less is known about their differentiation. This review focuses on the differentiation of branchiomeric muscles in mouse embryos. Furthermore, the relationship between branchiomeric muscle progenitor and neural crest cells in the pharyngeal arches of chicken embryos is also discussed. Additionally, we summarize recent studies into the genetic networks that distinguish between first arch-derived muscles and other pharyngeal arch muscles.

Characterization of the Three Dimensional Genome in Human Skeletal Muscle Progenitor Cells

Duchenne Muscular Dystrophy (DMD) is a devastating disease with no cure affecting approximately 1 in 3,500‐5,000 boys. Stem cell treatments using skeletal muscle stem cells (or satellite cells, SCs) provide great potential for regenerating new muscle and we have developed directed differentiation strategies to generate skeletal muscle cells from human induced pluripotent stem cells (hiPSCs). Our work has shown that hiPSCs generate PAX7+ skeletal muscle progenitor cells (SMPCs) resembling early myogenic cells that align closer to week 7‐12 in human development and are not equivalent to adult SCs. We are interested in understanding the key molecular and functional differences that control SMPC versus SC cell states. Recently, there has been an intense interest in the three dimensional (3D) organization of the genome and its involvement in cell specific gene regulation. This has led to the discovery of chromatin loops between gene enhancers and promoters as well as self‐interacting domains termed topologically associating domains (TADs). Recently published data support a role for the 3D genome in cellular differentiation, however, the differences between the 3D genome in human SMPCs compared to adult SCs is unknown. High throughput chromosome conformation capture (Hi‐C) was used to characterize the 3D genome of SMPCs. We found genome‐wide 3D configurations were different between hPSCs and SMPCs as was the number of TADs. Interestingly, TAD size was also different between cell types, suggesting dynamic control of TADs during differentiation. When focusing on the PAX7 locus, TAD boundaries differ between cell types further highlighting the dynamic nature of TADs with respect to cell specific gene expression. Not all muscle specific loci were different between cell types, however, suggesting that some TADs may be pre‐established early in the differentiation process while others are established de‐novo. With respect to chromatin looping at the PAX7 locus, we found SMPC specific looping between the PAX7 promoter and downstream sequences that were unique in SMPCs. Considering that PAX7 enhancer sequences have yet to be determined, these sequences may serve as candidate enhancers for PAX7. These data, for the first time, characterize the 3D genome of human SMPCs using Hi‐C. Moreover, these data provide candidate enhancer sequences for that could provide unique candidates for support of PAX7 SMPCs.

Mitochondria from Skeletal Muscle Cells Derived from Old Donors Show Reduced Energy Production and Respiratory Capacity

With age, skeletal muscle mitochondria lose their oxidative capacity and their ability to respond to energy demand. These phenomena can lead to a reduction in skeletal muscle oxygen consumption, atrophy, and increased risk of developing age‐related diseases such as sarcopenia. Whether age‐derived changes in mitochondrial function correspond to morphological changes in the mitochondrial reticulum remains unknown. Our objective is to investigate age‐related changes in mitochondrial morphology and function using primary skeletal muscle cells derived from healthy young and old men. Primary skeletal muscle progenitor cells (SkM) derived from the Rectus abdominis muscle of healthy active 18‐19‐year‐old men (Young), and 66‐69‐year‐old men (Old) were obtained from Cook MyoSite Inc. (Pittsburgh, PA). Mitochondrial morphology was analyzed in live cells using confocal microscopy. Oxygen Consumption Rate (OCR) was measured in intact cells using the extracellular flux assay and a Seahorse analyzer (Agilent Technologies; Santa Clara, CA). Seahorse Cell Mito Stress Test of primary cells derived from Young revealed a higher Basal and Maximal OCR compared to Old (Basal: 23.20 ± 1.49 vs. 11.34 ± 2.31; Maximal: 41.50 ± 4.72 vs. 18.36 ± 1.65 pmol/min/protein). Young also had higher ATP production and Spare Respiratory Capacity (ATP: 19.08 ± 1.21 vs. 9.19 ± 2.10; SRC: 18.30 ± 3.47 vs. 7.03 ± 1.29 pmol/min/protein). No differences were revealed in morphology (Individuals: 37.80 ± 23.18 vs. 46.67 ± 22.43 counts; Networks: 10.20 ± 2.48 vs. 15.33 ± 5.75 counts; Mean Branches per Network: 15.52 ± 6.41 vs. 11.27 ± 3.67 counts). These preliminary results show primary skeletal muscle progenitor cells derived from old donors have lower respiration, produce less ATP, and have a reduced capacity to adapt to energy demands when compared to SkM derived from young donors. Further analyses can give us an insight into human skeletal muscle‐derived cellular physiological capacity. Technology to observe human muscle mitochondrial dysfunction in vitro helps us understand the effects of aging on skeletal muscle mitochondria and whole‐body declines of resting and exercise metabolic rates. Studies on aging individuals will be required to determine if predicted decrements in energy coupling efficiency occur with aging in vivo.

CRISPR/Cas9 editing of directly reprogrammed myogenic progenitors restores dystrophin expression in a mouse model of muscular dystrophy.

SummaryGenetic mutations in dystrophin manifest in Duchenne muscular dystrophy (DMD), the most commonly inherited muscle disease. Here, we report on reprogramming of fibroblasts from two DMD mouse models into induced myogenic progenitor cells (iMPCs) by MyoD overexpression in concert with small molecule treatment. DMD iMPCs proliferate extensively, while expressing myogenic stem cell markers including Pax7 and Myf5. Additionally, DMD iMPCs readily give rise to multinucleated myofibers that express mature skeletal muscle markers; however, they lack DYSTROPHIN expression. Utilizing an exon skipping-based approach with CRISPR/Cas9, we report on genetic correction of the dystrophin mutation in DMD iMPCs and restoration of protein expression in vitro. Furthermore, engraftment of corrected DMD iMPCs into the muscles of dystrophic mice restored DYSTROPHIN expression and contributed to the muscle stem cell reservoir. Collectively, our findings report on a novel in vitro cellular model for DMD and utilize it in conjunction with gene editing to restore DYSTROPHIN expression in vivo.

Single-cell RNA Sequencing Reveals Heterogeneity of Cultured Bovine Satellite Cells

Skeletal muscle from meat-producing livestock such as cattle is a major source of food for humans. To improve skeletal muscle growth efficiency or quality in cattle, it is necessary to understand the genetic and physiological mechanisms that govern skeletal muscle composition, development, and growth. Satellite cells are the myogenic progenitor cells in postnatal skeletal muscle. In this study we analyzed the composition of bovine satellite cells with single-cell RNA sequencing (scRNA-seq). We isolated satellite cells from a 2-week-old male calf, cultured them in growth medium for a week, and performed scRNA-seq using the 10x Genomics platform. Deep sequencing of two scRNA-seq libraries constructed from cultured bovine satellite cells yielded 860 million reads. Cell calling analyses revealed that these reads were sequenced from 19,096 individual cells. Clustering analyses indicated that these reads represented 15 cell clusters that differed in gene expression profile. Based on the enriched expression of markers of satellite cells (PAX7 and PAX3), markers of myoblasts (MYOD1, MYF5), and markers of differentiated myoblasts or myocytes (MYOG), three clusters were determined to be satellite cells, two clusters myoblasts, and two clusters myocytes. Gene ontology and trajectory inference analyses indicated that cells in these myogenic clusters differed in proliferation rate and differentiation stage. Two of the remaining clusters were enriched with PDGFRA, a marker of fibro-adipogenic (FAP) cells, the progenitor cells for intramuscular fat, and are therefore considered to be FAP cells. Gene ontology analyses indicated active lipogenesis in one of these two clusters. The identity of the remaining six clusters could not be defined. Overall, the results of this study support the hypothesis that bovine satellite cells are composed of subpopulations that differ in transcriptional and myogenic state. The results of this study also support the hypothesis that intramuscular fat in cattle originates from fibro-adipogenic cells.