Myogenic Progenitors Research Articles

Real‐time and Multichannel Measurement of Contractility of hiPSC‐Derived 3D Skeletal Muscle using Fiber Optics‐Based Sensing

AbstractAs the field of cardiac and skeletal muscle tissue engineering expands, so does the need for accurate and reliable systems to generate in vitro 3D tissues and analyze their functional properties. In this study, the Cuore is introduced, a system that integrates sensors based on optical fibers and uses the principle of light interferometry to detect the contraction of 3D Tissue Engineered Skeletal Muscles (3D‐TESMs). The technology employed in the Cuore allows for reproducible and multichannel force measurements down to a nano‐Newtons resolution while maintaining sterility and permitting continuous non‐invasive recording within and outside standard tissue culture incubators. Thanks to the integrated electrodes for electrical pulse stimulation (EPS), 3D‐TESMs generated from three independent hiPSC‐derived myogenic progenitors (MPs) lines are stimulated and the contractility is recorded over the course of a week. Through the modulation of different EPS parameters, the optimal combination to induce the 3D‐TESMs in producing fully fused tetani without causing damage is determined. Furthermore, 3D‐TESMs from different lines exhibit characteristic signatures of spontaneous contractility and response to caffeine, verapamil, and the β‐agonist clenbuterol. The ease of use, high sensitivity, and the integrated electrodes and sensors make the Cuore an ideal technology to investigate the biology of contractile tissues and their response to drugs.

Highly contractile 3D tissue engineered skeletal muscles from human iPSCs reveal similarities with primary myoblast-derived tissues.

Skeletal muscle research is transitioning toward 3D tissue engineered invitro models reproducing muscle's native architecture and supporting measurement of functionality. Human induced pluripotent stem cells (hiPSCs) offer high yields of cells for differentiation. It has been difficult to differentiate high-quality, pure 3D muscle tissues from hiPSCs that show contractile properties comparable to primary myoblast-derived tissues. Here, we present a transgene-free method for the generation of purified, expandable myogenic progenitors (MPs) from hiPSCs grown under feeder-free conditions. We defined a protocol with optimal hydrogel and medium conditions that allowed production of highly contractile 3D tissue engineered skeletal muscles with forces similar to primary myoblast-derived tissues. Gene expression and proteomic analysis between hiPSC-derived and primary myoblast-derived 3D tissues revealed a similar expression profile of proteins involved in myogenic differentiation and sarcomere function. The protocol should be generally applicable for the study of personalized human skeletal muscle tissue in health and disease.

The mesodermal and myogenic specification of hESCs depend on ZEB1 and are inhibited by ZEB2

Human embryonic stem cells (hESCs) can differentiate into any cell lineage. Here, we report that ZEB1 and ZEB2 promote and inhibit mesodermal-to-myogenic specification of hESCs, respectively. Knockdown and/or overexpression experiments of ZEB1, ZEB2, or PAX7 in hESCs indicate that ZEB1 is required for hESC Nodal/Activin-mediated mesodermal specification and PAX7+ human myogenic progenitor (hMuP) generation, while ZEB2 inhibits these processes. ZEB1 downregulation induces neural markers, while ZEB2 downregulation induces mesodermal/myogenic markers. Mechanistically, ZEB1 binds to and transcriptionally activates the PAX7 promoter, while ZEB2 binds to and activates the promoter of the neural OTX2 marker. Transplanting ZEB1 or ZEB2 knocked down hMuPs into the muscles of a muscular dystrophy mouse model, showing that hMuP engraftment and generation of dystrophin-positive myofibers depend on ZEB1 and are inhibited by ZEB2. The mouse model results suggest that ZEB1 expression and/or downregulating ZEB2 in hESCs may also enhance hESC regenerative capacity for human muscular dystrophy therapy.

A Self‐Renewing Biomimetic Skeletal Muscle Construct Engineered using Induced Myogenic Progenitor Cells

AbstractSkeletal muscle represents a highly organized tissue that primarily regenerates by myogenic stem cells. Mimicking an in vitro skeletal muscle differentiation program that contains self‐renewing muscle stem cells and aligned myotubes is considered challenging. This study presents the engineering of a biomimetic muscle construct that can self‐regenerate and produce aligned myotubes using induced myogenic progenitor cells (iMPCs), a heterogeneous culture consisting of skeletal muscle stem, progenitor, and differentiated cells. Utilizing electrospinning, polycaprolactone (PCL) substrates are fabricated to facilitate iMPC‐differentiation into aligned myotubes by controlling PCL fiber orientation. Newly‐conceived constructs contain organized multinucleated myotubes alongside self‐renewing stem cells, whose differentiation capacity is augmented by Matrigel supplementation. Furthermore, this work utilizes single‐cell RNA‐sequencing (scRNA‐seq) to demonstrate that iMPC‐derived constructs faithfully recapitulate a step‐wise myogenic differentiation program. Notably, when subjected to a damaging myonecrotic agent, self‐renewing stem cells rapidly differentiate into aligned myotubes within the constructs, akin to skeletal muscle repair in vivo. Finally, this study demonstrates that the iMPC derivation protocol can be adapted to engineer human myoblast‐derived muscle constructs containing aligned myotubes, showcasing potential for translational applicability. Taken together, this work reports a novel in vitro system that mirrors myogenic regeneration and skeletal muscle alignment for basic research and regenerative medicine.

FLII Modulates the Myogenic Differentiation of Progenitor Cells via Actin Remodeling-Mediated YAP1 Regulation.

The dynamic rearrangement of the actin cytoskeleton plays an essential role in myogenesis, which is regulated by diverse mechanisms, such as mechanotransduction, modulation of the Hippo signaling pathway, control of cell proliferation, and the influence of morphological changes. Despite the recognized importance of actin-binding protein Flightless-1 (FLII) during actin remodeling, the role played by FLII in the differentiation of myogenic progenitor cells has not been explored. Here, we investigated the roles of FLII in the proliferation and differentiation of myoblasts. FLII was found to be enriched in C2C12 myoblasts, and its expression was stable during the early stages of differentiation but down-regulated in fully differentiated myotubes. Knockdown of FLII in C2C12 myoblasts resulted in filamentous actin (F-actin) accumulation and inhibited Yes-associated protein 1 (YAP1) phosphorylation, which triggers its nuclear translocation from the cytoplasm. Consequently, the expressions of YAP1 target genes, including PCNA, CCNB1, and CCND1, were induced, and the cell cycle and proliferation of myoblasts were promoted. Moreover, FLII knockdown significantly inhibited the expression of myogenic regulatory factors, i.e., MyoD and MyoG, thereby impairing myoblast differentiation, fusion, and myotube formation. Thus, our findings demonstrate that FLII is crucial for the differentiation of myoblasts via modulation of the F-actin/YAP1 axis and suggest that FLII is a putative novel therapeutic target for muscle wasting.

Sox11 is enriched in myogenic progenitors but dispensable for development and regeneration of the skeletal muscle

Transcription factors (TFs) play key roles in regulating differentiation and function of stem cells, including muscle satellite cells (MuSCs), a resident stem cell population responsible for postnatal regeneration of the skeletal muscle. Sox11 belongs to the Sry-related HMG-box (SOX) family of TFs that play diverse roles in stem cell behavior and tissue specification. Analysis of single-cell RNA-sequencing (scRNA-seq) datasets identify a specific enrichment of Sox11 mRNA in differentiating but not quiescent MuSCs. Consistent with the scRNA-seq data, Sox11 levels increase during differentiation of murine primary myoblasts in vitro. scRNA-seq data comparing muscle regeneration in young and old mice further demonstrate that Sox11 expression is reduced in aged MuSCs. Age-related decline of Sox11 expression is associated with reduced chromatin contacts within the topologically associating domains. Unexpectedly, Myod1Cre-driven deletion of Sox11 in embryonic myoblasts has no effects on muscle development and growth, resulting in apparently healthy muscles that regenerate normally. Pax7CreER- or Rosa26CreER- driven (MuSC-specific or global) deletion of Sox11 in adult mice similarly has no effects on MuSC differentiation or muscle regeneration. These results identify Sox11 as a novel myogenic differentiation marker with reduced expression in quiescent and aged MuSCs, but the specific function of Sox11 in myogenesis remains to be elucidated.

Transgene-free direct conversion of murine fibroblasts into functional muscle stem cells

Transcription factor-based cellular reprogramming provides an attractive approach to produce desired cell types for regenerative medicine purposes. Such cellular conversions are widely dependent on viral vectors to efficiently deliver and express defined factors in target cells. However, use of viral vectors is associated with unfavorable genomic integrations that can trigger deleterious molecular consequences, rendering this method a potential impediment to clinical applications. Here, we report on a highly efficient transgene-free approach to directly convert mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by overexpression of synthetic MyoD-mRNA in concert with an enhanced small molecule cocktail. First, we performed a candidate compound screen and identified two molecules that enhance fibroblast reprogramming into iMPCs by suppression of the JNK and JAK/STAT pathways. Simultaneously, we developed an optimal transfection protocol to transiently overexpress synthetic MyoD-mRNA in fibroblasts. Combining these two techniques enabled robust and rapid reprogramming of fibroblasts into Pax7 positive iMPCs in as little as 10 days. Nascent transgene-free iMPCs proliferated extensively in vitro, expressed a suite of myogenic stem cell markers, and could differentiate into highly multinucleated and contractile myotubes. Furthermore, using global and single-cell transcriptome assays, we delineated gene expression changes associated with JNK and JAK/STAT pathway inhibition during reprogramming, and identified in iMPCs a Pax7+ stem cell subpopulation resembling satellite cells. Last, transgene-free iMPCs robustly engrafted skeletal muscles of a Duchenne muscular dystrophy mouse model, restoring dystrophin expression in hundreds of myofibers. In summary, this study reports on an improved and clinically safer approach to convert fibroblasts into myogenic stem cells that can efficiently contribute to muscle regeneration in vivo.

Tetraspanin CD82 Associates with Trafficking Vesicle in Muscle Cells and Binds to Dysferlin and Myoferlin.

Tetraspanins organize protein complexes at the cell membrane and are responsible for assembling diverse binding partners in changing cellular states. Tetraspanin CD82 is a useful cell surface marker for prospective isolation of human myogenic progenitors and its expression is decreased in Duchenne muscular dystrophy (DMD) cell lines. The function of CD82 in skeletal muscle remains elusive, partly because the binding partners of this tetraspanin in muscle cells have not been identified. CD82-associated proteins are sought to be identified in human myotubes via mass spectrometry proteomics, which identifies dysferlin and myoferlin as CD82-binding partners. In human dysferlinopathy (Limb girdle muscular dystrophy R2, LGMDR2) myogenic cell lines, expression of CD82 protein is near absent in two of four patient samples. In the cell lines where CD82 protein levels are unaffected, increased expression of the ≈72kDa mini-dysferlin product is identified using an antibody recognizing the dysferlin C-terminus. These data demonstrate that CD82 binds dysferlin/myoferlin in differentiating muscle cells and its expression can be affected by loss of dysferlin in human myogenic cells.

Chemokine/ITGA4 Interaction Directs iPSC-Derived Myogenic Progenitor Migration to Injury Sites in Aging Muscle for Regeneration.

The failure of muscle to repair after injury during aging may be a major contributor to muscle mass loss. We recently generated muscle progenitor cells (MPCs) from human-induced pluripotent stem-cell (iPSC) cell lines using small molecules, CHIR99021 and Givinostat (Givi-MPCs) sequentially. Here, we test whether the chemokines overexpressed in injured endothelial cells direct MPC migration to the site by binding to their receptor, ITGA4. ITGA4 was heavily expressed in Givi-MPCs. To study the effects on the mobilization of Givi-MPCs, ITGA4 was knocked down by an ITGA4 shRNA lentiviral vector. With and without ITGA4 knocked down, cell migration in vitro and cell mobilization in vivo using aged NOD scid gamma (NSG) mice and mdx/scid mice were analyzed. The migration of shITGA4-Givi-MPCs was significantly impaired, as shown in a wound-healing assay. The knockdown of ITGA4 impaired the migration of Givi-MPCs towards human aortic endothelial cells (HAECs), in which CX3CL1 and VCAM-1 were up-regulated by the treatment of TNF-α compared with scramble ones using a transwell system. MPCs expressing ITGA4 sensed chemokines secreted by endothelial cells at the injury site as a chemoattracting signal to migrate to the injured muscle. The mobilization of Givi-MPCs was mediated by the ligand-receptor interaction, which facilitated their engraftment for repairing the sarcopenic muscle with injury.

Ouassima EL Kadiri, Samiha Sabri, Raja Ousalm, Salma Chabli, Ibtissam Zrara Touiti, Mouna Darfaoui, Abdelhamid El Omrani, Mouna Khouchani

Rhabdomyosarcoma (RMS) is a malignant neoplasm that originates from undifferentiated myogenic progenitor cells. It is predominantly a pediatric disease, and its occurrence in adults is exceedingly rare. Adult primary RMS of gynecologic origin is an uncommon phenomenon, and the cervix is the most frequently involved site. The incidence of adult primary uterine RMS is extremely scarce. Herein, we present the case of primary uterine RMS in a 59-year-old Moroccan postmenopausal woman who presented to clinic attention with a two-month history of metrorrhagia. The patient received a multimodal therapy comprising a combination of surgery, chemotherapy and radiotherapy with a good clinical and radiological evolution.

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System.

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with thissystem allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Loss of the Tbx5 gene in the ventricular conduction system induces spontaneous ventricular arrhythmias

The ventricular conduction system (VCS) and the working myocardium (WM) derive from the same myogenic progenitors during the embryonic development. The VCS development is regulated by numerous genes including the T-box transcription factor 5 (Tbx5). In human, Tbx5 mutations cause Holt-Oram syndrome, an autosomal dominant disease associated with heart abnormalities such as arrhythmias. In adult mice, the deletion of the Tbx5 gene provoked a slowing of ventricular conduction due to a loss of the gap junction Connexin 40 and dysfunction of cardiac channels. However, it is still unknown how Tbx5 deletion affects the development and the function of the VCS. The aim is to study the consequences of the Tbx5 deletion in embryonic progenitors of the ventricular conduction system on cardiac function in adult mice. Conditional deletion of Tbx5 alleles in mice was induced by expressing the Cre recombinase under the control of the Connexin 40 (Cx40) gene. We studied two groups of mutants: Tbx5 deletion at embryonic days E13-14 targeting both the ventricular conduction system and the working myocardium (“VCS + WM” group; n = 40) and mice deleted at E18-P2 targeting only the ventricular conduction system (“VCS” group; n = 17). Surface ECGs were achieved monthly until 3 months of age, when immunostaining was performed on the left VCS. Immunostaining showed a reduced expression of the two major connexines in the ventricle (Cx40 and Cx43) and a reduced number of conductive cells in both mutants, suggesting a defective maturation of the VCS (Fig. 1). Furthermore, the loss of Cx43 was correlated with that of Tbx5, reflecting a direct transcriptional effect. Surface ECG revealed a significant increase of the ventricular activation duration in both mutants (P < 0.01). Moreover, premature ventricular contractions (PVCs) and a left QRS axis deviation were observed in both mutants. Finally, these PVCs disappeared with age in both mutants under resting conditions, and are unmasked when the heart is challenged with a β-1 adrenergic agonist. Our results provide new insights into Tbx5 targets in the VCS. Tbx5 deletion impacts the maturation and the anatomy of the VCS as proved by the deviation of the ventricular electrical axis, which even occurs when the VCS is already formed. Tbx5 appears to have a direct impact on the Gja1 gene, encoding the gap junction Cx43, which is reduced at the junction between the VCS and the WM in both mutants and may be responsible for PVCs.

Supraclavicular brown adipocytes arise from myogenic Tbx1-lineage progenitors

Brown adipose tissue (BAT) is a thermogenic organ that promotes metabolic fitness. In humans, there are two spatiotemporally distinct phases of BAT ontogeny: the prenatally developed dorsal-anterior-located interscapular BAT (iBAT); and the postnatally emerged supraclavicular BAT (sBAT). It has been established in rodents that the dorsal-anterior-located iBAT depots predominately arise from a Pax3 +/ Myf5 + lineage in the epaxial dermomyotome that also forms the trunk muscle. However, the developmental origins of the supraclavicular BAT that adult humans have remain unknown. In this study, using lineage tracing mouse models, we found that these supraclavicular BAT depots are not derived from the Pax3 + or Myf5 + precursor cells. Knock out of PPARγ, the master regulator of adipogenesis, in Myf5 + cells caused severe atrophy of iBAT, but did not affect the supraclavicular depots. Instead, we identified that most of the supraclavicular brown adipocytes are progeny cells of the developmental Tbx1 + lineage that also gives rise to neck muscle cells. PPARγ deficiency in this lineage did not perturb iBAT development, but caused paucity in the supraclavicular regions, thus sensitizing mice to cold-induced decrease in body temperature.In summary, our results suggest that there are region-specific myogenic precursors that contribute to BAT development at different anatomical locations. Future studies to understand the pathophysiological regulation of these developmentally distinct BAT depots could inspire new approaches to measure BAT mass and to activate BAT activity in humans for the treatment of obesity, diabetes and related metabolic disorders. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Expression of Myomaker and Myomerger in myofibers causes muscle pathology

BackgroundSkeletal muscle development and regeneration depend on cellular fusion of myogenic progenitors to generate multinucleated myofibers. These progenitors utilize two muscle-specific fusogens, Myomaker and Myomerger, which function by remodeling cell membranes to fuse to each other or to existing myofibers. Myomaker and Myomerger expression is restricted to differentiating progenitor cells as they are not detected in adult myofibers. However, Myomaker remains expressed in myofibers from mice with muscular dystrophy. Ablation of Myomaker from dystrophic myofibers results in reduced membrane damage, leading to a model where persistent fusogen expression in myofibers, in contrast to myoblasts, is harmful.MethodsDox-inducible transgenic mice were developed to ectopically express Myomaker or Myomerger in the myofiber compartment of skeletal muscle. We quantified indices of myofiber membrane damage, such as serum creatine kinase and IgM+ myofibers, and assessed general muscle histology, including central nucleation, myofiber size, and fibrosis.ResultsMyomaker or Myomerger expression in myofibers independently caused membrane damage at acute time points. This damage led to muscle pathology, manifesting with centrally nucleated myofibers and muscle atrophy. Dual expression of both Myomaker and Myomerger in myofibers exacerbated several aspects of muscle pathology compared to expression of either fusogen by itself.ConclusionsThese data reveal that while myofibers can tolerate some level of Myomaker and Myomerger, expression of a single fusogen above a threshold or co-expression of both fusogens is damaging to myofibers. These results explain the paradigm that their expression in myofibers can have deleterious consequences in muscle pathologies and highlight the need for their highly restricted expression during myogenesis and fusion.

Establishment of Skeletal Myogenic Progenitors from Non-Human Primate Induced Pluripotent Stem Cells.

Pluripotent stem (PS) cells enable the scalable production of tissue-specific derivatives with therapeutic potential for various clinical applications, including muscular dystrophies. Given the similarity to human counterparts, the non-human primate (NHP) is an ideal preclinical model to evaluate several questions, including delivery, biodistribution, and immune response. While the generation of human-induced PS (iPS)-cell-derived myogenic progenitors is well established, there have been no data for NHP counterparts, probably due to the lack of an efficient system to differentiate NHP iPS cells towards the skeletal muscle lineage. Here, we report the generation of three independent Macaca fascicularis iPS cell lines and their myogenic differentiation using PAX7 conditional expression. The whole-transcriptome analysis confirmed the successful sequential induction of mesoderm, paraxial mesoderm, and myogenic lineages. NHP myogenic progenitors efficiently gave rise to myotubes under appropriate in vitro differentiation conditions and engrafted in vivo into the TA muscles of NSG and FKRP-NSG mice. Lastly, we explored the preclinical potential of these NHP myogenic progenitors in a single wild-type NHP recipient, demonstrating engraftment and characterizing the interaction with the host immune response. These studies establish an NHP model system through which iPS-cell-derived myogenic progenitors can be studied.

Induction of miR-665-3p Impairs the Differentiation of Myogenic Progenitor Cells by Regulating the TWF1-YAP1 Axis.

Actin dynamics are known to orchestrate various myogenic processes in progenitor cells. Twinfilin-1 (TWF1) is an actin-depolymerizing factor that plays a crucial role in the differentiation of myogenic progenitor cells. However, little is known about the mechanisms underlying the epigenetic regulation of TWF1 expression and impaired myogenic differentiation in the background of muscle wasting. This study investigated how miR-665-3p affects TWF1 expression, actin filaments' modulation, proliferation, and myogenic differentiation in progenitor cells. Palmitic acid, the most prevalent saturated fatty acid (SFA) in food, suppressed TWF1 expression and inhibited the myogenic differentiation of C2C12 cells while increasing the level of miR-665-3p expression. Interestingly, miR-665-3p inhibited TWF1 expression by targeting TWF1 3'UTR directly. In addition, miR-665-3p accumulated filamentous actin (F-actin) and enhanced the nuclear translocation of Yes-associated protein 1 (YAP1), consequently promoting cell cycle progression and proliferation. Furthermore, miR-665-3p suppressed the expressions of myogenic factors, i.e., MyoD, MyoG, and MyHC, and consequently impaired myoblast differentiation. In conclusion, this study suggests that SFA-inducible miR-665-3p suppresses TWF1 expression epigenetically and inhibits myogenic differentiation by facilitating myoblast proliferation via the F-actin/YAP1 axis.

Abstract 3666: A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle

Abstract Skeletal muscle dysfunction due to the effects of cancer secretome is observed in multiple cancer types and extreme dysfunction is manifested as cachexia. Major preclinical studies on cancer-associated muscle defects utilized mouse models or mouse C2C12 mouse myoblast cell line for in vitro studies. Because of species specificity of certain cytokines/chemokines in the secretome, a human model system is required to fully comprehend the effects of cancer secretome on skeletal muscle. Here, we report a simple method to establish skeletal muscle stem cell line (hMuSC), which can be differentiated into myotubes. Using single nuclei ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq), we document chromatin accessibility and transcriptomic changes associated with hMuSCs to myotube transition. Cancer cell line derived factors accelerated stem to myotube differentiation with accompanying changes including an increase in PAX7+/MyoD+ myogenic progenitor cells. Among the pathways activated by cancer-derived factors include inflammatory pathway involving CXCL8 (also called IL-8), glucocorticoid receptor (GR) pathway, and wound healing pathway. Furthermore, cancer-derived factors significantly altered splicing machinery in hMuSCs. Additionally, AKT and p53 pathways that function in metabolic/survival pathways of the skeletal muscle were adversely affected when hMuSCs were exposed to cancer cell-derived factors. Cancer-derived factors increased the expression levels of previously known cachexia-associated genes such as MT-2, ZIP14, and PDK4. Thus, the model system not only recapitulates results of previous mouse studies but also provides much needed human model system that can easily be adapted for large scale studies to explore the epigenomic changes during hMuSC differentiation and to screen for drugs that restore skeletal muscle function in various diseases. Citation Format: Ruizhong Wang, Brijesh Kumar, Poornima Bhat-Nakshatri, Aditi Sanjay Khatpe, Michael P. Murphy, Kristen E. Wanczyk, Emma H. Doud, Amber L. Mosley, Yunlong Liu, Duojiao Chen, Ed Simpson, Hongyu Gao, Harikrishna Nakshatri. A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3666.

MicroRNA control of the myogenic cell transcriptome and proteome: the role of miR-16.

MicroRNAs (miRs) control stem cell biology and fate. Ubiquitously expressed and conserved miR-16 was the first miR implicated in tumorigenesis. miR-16 is low in muscle during developmental hypertrophy and regeneration. It is enriched in proliferating myogenic progenitor cells but is repressed during differentiation. The induction of miR-16 blocks myoblast differentiation and myotube formation, whereas knockdown enhances these processes. Despite a central role for miR-16 in myogenic cell biology, how it mediates its potent effects is incompletely defined. In this investigation, global transcriptomic and proteomic analyses after miR-16 knockdown in proliferating C2C12 myoblasts revealed how miR-16 influences myogenic cell fate. Eighteen hours after miR-16 inhibition, ribosomal protein gene expression levels were higher relative to control myoblasts and p53 pathway-related gene abundance was lower. At the protein level at this same time point, miR-16 knockdown globally upregulated tricarboxylic acid (TCA) cycle proteins while downregulating RNA metabolism-related proteins. miR-16 inhibition induced specific proteins associated with myogenic differentiation such as ACTA2, EEF1A2, and OPA1. We extend prior work in hypertrophic muscle tissue and show that miR-16 is lower in mechanically overloaded muscle in vivo. Our data collectively point to how miR-16 is implicated in aspects of myogenic cell differentiation. A deeper understanding of the role of miR-16 in myogenic cells has consequences for muscle developmental growth, exercise-induced hypertrophy, and regenerative repair after injury, all of which involve myogenic progenitors.

MicroRNA-501 controls myogenin+/CD74+ myogenic progenitor cells during muscle regeneration

ObjectiveSkeletal muscle regeneration is markedly impaired during aging. How adult muscle stem cells contribute to this decrease in regenerative capacity is incompletely understood. We investigated mechanisms of age-related changes in myogenic progenitor cells using the tissue-specific microRNA 501. MethodsYoung and old C57Bl/6 mice were used (3 months or 24 months of age, respectively) with or without global or tissue-specific genetic deletion of miR-501. Muscle regeneration was induced using intramuscular cardiotoxin injection or treadmill exercise and analysed using single cell and bulk RNA sequencing, qRT-PCR and immunofluorescence. Muscle fiber damage was assessed with Evan`s blue dye (EBD). In vitro analysis was performed in primary muscle cells obtained from mice and humans. ResultsSingle cell sequencing revealed myogenic progenitor cells in miR-501 knockout mice at day 6 after muscle injury that are characterized by high levels of myogenin and CD74. In control mice these cells were less in number and already downregulated after day 3 of muscle injury. Muscle from knockout mice had reduced myofiber size and reduced myofiber resilience to injury and exercise. miR-501 elicits this effect by regulating sarcomeric gene expression through its target gene estrogen-related receptor gamma (Esrrg). Importantly, in aged skeletal muscle where miR-501 was significantly downregulated and its target Esrrg significantly upregulated, the number of myog+/CD74+ cells during regeneration was upregulated to similar levels as observed in 501 knockout mice. Moreover, myog+/CD74+-aged skeletal muscle exhibited a similar decrease in the size of newly formed myofibers and increased number of necrotic myofibers after injury as observed in mice lacking miR-501. ConclusionsmiR-501 and Esrrg are regulated in muscle with decreased regenerative capacity and loss of miR-501 is permissive to the appearance of CD74+ myogenic progenitors. Our data uncover a novel link between the metabolic transcription factor Esrrg and sarcomere formation and demonstrate that stem cell heterogeneity in skeletal muscle during aging is under miRNA control. Targeting Esrrg or myog+/CD74+ progenitor cells might improve fiber size and myofiber resilience to exercise in aged skeletal muscle.

3D human induced pluripotent stem cell-derived bioengineered skeletal muscles for tissue, disease and therapy modeling.

Skeletal muscle is a complex tissue composed of multinucleated myofibers responsible for force generation that are supported by multiple cell types. Many severe and lethal disorders affect skeletal muscle; therefore, engineering models to reproduce such cellular complexity and function are instrumental for investigating muscle pathophysiology and developing therapies. Here, we detail the modular 3D bioengineering of multilineage skeletal muscles from human induced pluripotent stem cells, which are first differentiated into myogenic, neural and vascular progenitor cells and then combined within 3D hydrogels under tension to generate an aligned myofiber scaffold containing vascular networks and motor neurons. 3D bioengineered muscles recapitulate morphological and functional features of human skeletal muscle, including establishment of a pool of cells expressing muscle stem cell markers. Importantly, bioengineered muscles provide a high-fidelity platform to study muscle pathology, such as emergence of dysmorphic nuclei in muscular dystrophies caused by mutant lamins. The protocol is easy to follow for operators with cell culture experience and takes between 9 and 30 d, depending on the number of cell lineages in the construct. We also provide examples of applications of this advanced platform for testing gene and cell therapies in vitro, as well as for in vivo studies, providing proof of principle of its potential as a tool to develop next-generation neuromuscular or musculoskeletal therapies.