Myogenic Cells Research Articles

In-ovo feeding with creatine monohydrate: implications for chicken energy reserves and breast muscle development during the pre-post hatching period.

The most dynamic period throughout the lifespan of broiler chickens is the pre-post-hatching period, entailing profound effects on their energy status, survival rate, body weight, and muscle growth. Given the significance of this pivotal period, we evaluated the effect of in-ovo feeding (IOF) with creatine monohydrate on late-term embryos' and hatchlings' energy reserves and post-hatch breast muscle development. The results demonstrate that IOF with creatine elevates the levels of high-energy-value molecules (creatine and glycogen) in the liver, breast muscle and yolk sac tissues 48h post IOF, on embryonic day 19 (p < 0.03). Despite this evidence, using a novel automated image analysis tool on day 14 post-hatch, we found a significantly higher number of myofibers with lower diameter and area in the IOF creatine group compared to the control and IOF NaCl groups (p < 0.004). Gene expression analysis, at hatch, revealed that IOF creatine group had significantly higher expression levels of myogenin (MYOG) and insulin-like growth factor 1 (IGF1), related to differentiation of myogenic cells (p < 0.01), and lower expression of myogenic differentiation protein 1 (MyoD), related to their proliferation (p < 0.04). These results imply a possible effect of IOF with creatine on breast muscle development through differential expression of genes involved in myogenic proliferation and differentiation. The findings provide valuable insights into the potential of pre-hatch enrichment with creatine in modulating post-hatch muscle growth and development.

MEK-inhibitors decrease Nfix in muscular dystrophy but induce unexpected calcifications, partially rescued with Cyanidin diet

Muscular dystrophies (MDs) are incurable genetic myopathies characterized by progressive degeneration of skeletal muscles. Dystrophic mice lacking the transcription factor Nfix display morphological and functional improvements of the disease. Recently, we demonstrated that MAPK signaling pathway positively regulates Nfix in muscle development and that Cyanidin, a natural antioxidant molecule, strongly ameliorates the pathology. To explore a synergistic approach aimed at treating MDs, we administered Trametinib, a clinically approved MEK inhibitor, alone or combined with Cyanidin to adult Sgca null mice. We observed that chronic treatment with Trametinib and Cyanidin reduced Nfix in myogenic cells but, unexpectedly, caused ectopic calcifications exclusively in dystrophic muscles. The combined treatment with Cyanidin resulted in histological improvements by preventing Trametinib-induced calcifications in Diaphragm and Soleus. Collectively, this first pilot study revealed that Nfix is modulated by the MAPK pathway in MDs, and that Cyanidin partly rescued the unexpected ectopic calcifications caused by MEK inhibition.

Inhibiting the MAPK pathway improves heart failure with preserved ejection fraction induced by salt-sensitive hypertension

Heart failure (HF) preserved ejection fraction (HFpEF) accounts for almost 50% of HF, and hypertension is one of the pathogenies. The MAPK signaling pathway is closely linked to heart failure and hypertension; however, its function in HEpEF resulting from salt-sensitive hypertension is not well understood. In this work, a salt-sensitive hypertension-induced HEpEF model was established based on deoxycorticosterone acetate-salt (DOCA-salt) hypertension mice. The impact of the MAPK inhibitor (Doramapimod) on HEpEF induced by salt-sensitive hypertension was assessed through various measures, such as blood pressure, transthoracic echocardiography, running distance, and histological analysis, to determine its therapeutic effectiveness on cardiac function. In addition, the effects of high salt on myogenic cells were also evaluated in vitro using qRTPCR. The LV ejection fractions (LVEF) in DOCA-salt hypertension mice were over 50%, indicating that the salt-sensitive hypertension-induced HFpEF model was successful. RNA-seq revealed that the MAPK signaling pathway was upregulated in the HFpEF model compared with the normal mice, accompanied by hypertension, impaired running distance, restricted cardiac function, increased cross-sectional and fibrosis area, and upregulation of heart failure biomarkers, including GAL-3, LDHA and BNP. The application of Doramapimod could improve blood pressure, cardiomyocyte hypertrophy, and myocardial fibrosis, as well as decrease the aforementioned heart failure biomarkers. The qRTPCR results showed similar findings to these observations. Our findings suggest that the use of a MAPK inhibitor (Doramapimod) could be a potential treatment for salt-sensitive hypertension-induced HFpEF.

Decline of DNA damage response along with myogenic differentiation.

DNA integrity is incessantly confronted to agents inducing DNA lesions. All organisms are equipped with a network of DNA damage response mechanisms that will repair DNA lesions and restore proper cellular activities. Despite DNA repair mechanisms have been revealed in replicating cells, still little is known about how DNA lesions are repaired in postmitotic cells. Muscle fibers are highly specialized postmitotic cells organized in syncytia and they are vulnerable to age-related degeneration and atrophy after radiotherapy treatment. We have studied the DNA repair capacity of muscle fiber nuclei and compared it with the one measured in proliferative myoblasts here. We focused on the DNA repair mechanisms that correct ionizing radiation (IR)-induced lesions, namely the base excision repair, the nonhomologous end joining, and the homologous recombination (HR). We found that in the most differentiated myogenic cells, myotubes, these DNA repair mechanisms present weakened kinetics of recruitment of DNA repair proteins to IR-damaged DNA. For base excision repair and HR, this decline can be linked to reduced steady-state levels of key proteins involved in these processes.

Age-related nitration/dysfunction of myogenic stem cell activator HGF.

Mechanical perturbation triggers activation of resident myogenic stem cells to enter the cell cycle through a cascade of events including hepatocyte growth factor (HGF) release from its extracellular tethering and the subsequent presentation to signaling-receptor c-met. Here, we show that with aging, extracellular HGF undergoes tyrosine-residue (Y) nitration and loses c-met binding, thereby disturbing muscle homeostasis. Biochemical studies demonstrated that nitration/dysfunction is specific to HGF among other major growth factors and is characterized by its locations at Y198 and Y250 in c-met-binding domains. Direct-immunofluorescence microscopy of lower hind limb muscles from three age groups of rat, provided direct invivo evidence for age-related increases in nitration of ECM-bound HGF, preferentially stained for anti-nitrated Y198 and Y250-HGF mAbs (raised in-house) in fast IIa and IIx myofibers. Overall, findings highlight inhibitory impacts of HGF nitration on myogenic stem cell dynamics, pioneering a cogent discussion for better understanding age-related muscle atrophy and impaired regeneration with fibrosis (including sarcopenia and frailty).

Magnetic resonance imaging analysis of embryonal rhabdomyosarcoma of the prostate.

Rhabdomyosarcoma (RMS) is a highly malignant tumor that originates from myogenic progenitor cells. To investigate the magnetic resonance imaging (MRI) characteristics of prostate embryonal rhabdomyosarcoma (ERMS). We retrospectively analyzed the clinical and MRI imaging data of 9 cases of prostate ERMS that were confirmed pathologically. The patients were aged between 14∼49 years with a median age of 27 years, and they all underwent MRI, diffusion-weighted imaging (DWI), and dynamic contrast-enhanced MRI (DCE-MRI). The MRI scan of the lesions showed an irregular shape, mixed signals, uneven equal/long T1 signal and an equal/long T2 signal, cystic necrosis in 9 cases and hemorrhage in 6 cases; DWI and ADC images showed a mixed high/low signal, and the tumor parenchyma showed ADC low signal, with an average ADC value of 0.666 × 10-3 mm2/s. There were 5 cases of DCE-MRI TIC type II and 4 cases of DCE-MRI TIC type I. The average value of Tpeak was 120 s and the average value of MCER was 172.3%. After the enhancement, the signal of tumor enhancement was uneven, and showed patchy and reticular enhancement, however, the cyst degeneration, necrosis area, and hemorrhage focus were not enhanced. There were 3 cases with multiple pelvic lymph nodes and 1 case with multiple bone metastases. The MRI manifestations of prostate ERMS have certain characteristics, and the combination of DWI and DCE-MRI are helpful in the diagnosis.

PAX3-FOXO1 dictates myogenic reprogramming and rhabdomyosarcoma identity in endothelial progenitors

Fusion-positive rhabdomyosarcoma (FP-RMS) driven by the expression of the PAX3-FOXO1 (P3F) fusion oncoprotein is an aggressive subtype of pediatric rhabdomyosarcoma. FP-RMS histologically resembles developing muscle yet occurs throughout the body in areas devoid of skeletal muscle highlighting that FP-RMS is not derived from an exclusively myogenic cell of origin. Here we demonstrate that P3F reprograms mouse and human endothelial progenitors to FP-RMS. We show that P3F expression in aP2-Cre expressing cells reprograms endothelial progenitors to functional myogenic stem cells capable of regenerating injured muscle fibers. Further, we describe a FP-RMS mouse model driven by P3F expression and Cdkn2a loss in endothelial cells. Additionally, we show that P3F expression in TP53-null human iPSCs blocks endothelial-directed differentiation and guides cells to become myogenic cells that form FP-RMS tumors in immunocompromised mice. Together these findings demonstrate that FP-RMS can originate from aberrant development of non-myogenic cells driven by P3F.

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.

Differentiation-dependent chromosomal organization changes in normal myogenic cells are absent in rhabdomyosarcoma cells.

Myogenesis, the progression of proliferating skeletal myoblasts to terminally differentiated myotubes, regulates thousands of target genes. Uninterrupted linear arrays of such genes are differentially associated with specific chromosomes, suggesting chromosome specific regulatory roles in myogenesis. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle, shares common features with normal muscle cells. We hypothesized that RMS and myogenic cells possess differences in chromosomal organization related to myogenic gene arrangement. We compared the organizational characteristics of chromosomes 2 and 18, chosen for their difference in myogenic gene arrangement, in cultured RMS cell lines and normal myoblasts and myotubes. We found chromosome-specific differences in organization during normal myogenesis, with increased area occupied and a shift in peripheral localization specifically for chromosome 2. Most strikingly, we found a differentiation-dependent difference in positioning of chromosome 2 relative to the nuclear axis, with preferential positioning along the major nuclear axis present only in myotubes. RMS cells demonstrated no preference for such axial positioning, but induced differentiation through transfection of the pro-myogenic miRNA miR-206 resulted in an increase of major axial positioning of chromosome 2. Our findings identify both a differentiation-dependent, chromosome-specific change in organization in normal myogenesis, and highlight the role of chromosomal spatial organization in myogenic differentiation.

SnRNA-seq analysis in multinucleated myogenic FSHD cells identifies heterogeneous FSHD transcriptome signatures associated with embryonic-like program activation and oxidative stress-induced apoptosis.

The sporadic nature of DUX4 expression in FSHD muscle challenges comparative transcriptome analyses between FSHD and control samples. A variety of DUX4 and FSHD-associated transcriptional changes have been identified, but bulk RNA-seq strategies prohibit comprehensive analysis of their spatiotemporal relation, interdependence and role in the disease process. In this study, we used single-nucleus RNA-sequencing of nuclei isolated from patient- and control-derived multinucleated primary myotubes to investigate the cellular heterogeneity in FSHD. Taking advantage of the increased resolution in snRNA-sequencing of fully differentiated myotubes, two distinct populations of DUX4-affected nuclei could be defined by their transcriptional profiles. Our data provides insights into the differences between these two populations and suggests heterogeneity in two well-known FSHD-associated transcriptional aberrations: increased oxidative stress and inhibition of myogenic differentiation. Additionally, we provide evidence that DUX4-affected nuclei share transcriptome features with early embryonic cells beyond the well-described cleavage stage, progressing into the 8-cell and blastocyst stages. Altogether, our data suggests that the FSHD transcriptional profile is defined by a mixture of individual and sometimes mutually exclusive DUX4-induced responses and cellular state-dependent downstream effects.

Aligned skeletal muscle assembly on a biofunctionalized plant leaf scaffold.

Decellularized plant scaffolds have drawn attention as alternative tissue culture platforms due to their wide accessibility, biocompatibility, and diversity of innate microstructures. Particularly, in this work, monocot leaves with innate uniaxial micropatterned topography were utilized to promote cell alignment and elongation. The leaf scaffold was biofunctionalized with poly(PEGMEMA-r-VDM-r-GMA) copolymer that prevented non-specific protein adsorption and was modified with cell adhesive RGD peptide to enable cell adhesion and growth in serum-free media. The biofunctionalized leaf supported the adhesion, growth, and alignment of various human cells including embryonic stem cells (hESC) derived muscle cells. The hESC-derived myogenic progenitor cells cultured on the biofunctionalized leaf scaffold adopted a parallel orientation and were elongated along the leaf topography. These cells showed significant early myogenic differentiation and muscle-like bundled myotube formation. The aligned cells formed compact myotube assemblies and showed uniaxial muscle contraction under chemical stimulation, a critical requirement for developing functional skeletal muscle tissue. Polymer-functionalized plant leaf scaffolds offer a novel human cell culture platform and have potential in human tissue engineering applications that require parallel alignment of cells. STATEMENT OF SIGNIFICANCE: Plant scaffolds are plentiful sources in nature and present a prefabricated construct to present topographical cues to cells. Their feature width is ideal for human cell alignment and elongation, especially for muscle cells. However, plant scaffolds lack proteins that support mammalian cell culture. We have developed a polymer coated leaf scaffold that enables cell adhesion and growth in serum-free media. Human muscle cells cultured on the biofunctionalized leaf, aligned along the natural parallel micro-patterned leaf topography, and formed muscle-like bundled myotube assemblies. These assemblies showed uniaxial muscular contraction, a critical requirement for developing functional skeletal muscle tissue. The biodiversity of the plant materials offers a novel human cell culture platform with potential in human tissue engineering.

Foxk1 stimulates adipogenic differentiation via a peroxisome proliferator-activated receptor gamma 2-dependent mechanism.

Adipogenesis is a tightly regulated process, and its dysfunction has been linked to metabolic disorders such as obesity. Forkhead box k1 (Foxk1) is known to play a role in the differentiation of myogenic precursor cells and tumorigenesis of different types of cancers; however, it is not clear whether and how it influences adipocyte differentiation. Here, we found that Foxk1 was induced in mouse primary bone marrow stromal cells (BMSCs) and established mesenchymal progenitor/stromal cell lines C3H/10T1/2 and ST2 after adipogenic treatment. In addition, obese db/db mice have higher Foxk1 expression in inguinal white adipose tissue than nonobese db/m mice. Foxk1 overexpression promoted adipogenic differentiation of C3H/10T1/2, ST2 cells and BMSCs, along with the enhanced expression of CCAAT/enhancer binding protein-α, peroxisome proliferator-activated receptor γ (Pparγ), and fatty acid binding protein 4. Moreover, Foxk1 overexpression enhanced the expression levels of lipogenic factors during adipogenic differentiation in both C3H/10T1/2 cells and BMSCs. Conversely, Foxk1 silencing impaired these cells from fully differentiating. Furthermore, adipogenic stimulation induced the nuclear translocation of Foxk1, which depended on the mTOR and PI3-kinase signaling pathways. Subsequently, Foxk1 is directly bound to the Pparγ2 promoter, stimulating its transcriptional activity and promoting adipocyte differentiation. Collectively, our study provides the first evidence that Foxk1 promotes adipocyte differentiation from progenitor cells by promoting nuclear translocation and upregulating the transcriptional activity of the Pparγ2 promoter during adipogenic differentiation.

Circular RNA circIGF2BP3 Promotes the Proliferation and Differentiation of Chicken Primary Myoblasts.

The quality and quantity of animal meat are closely related to the development of skeletal muscle, which, in turn, is determined by myogenic cells, including myoblasts and skeletal muscle satellite cells (SMSCs). Circular RNA, an endogenous RNA derivative formed through specific reverse splicing in mRNA precursors, has the potential to influence muscle development by binding to miRNAs or regulating gene expression involved in muscular growth at the transcriptional level. Previous high-throughput sequencing of circRNA in chicken liver tissue revealed a circular transcript, circIGF2BP3, derived from the gene encoding insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3). In this study, we confirmed the presence of the natural circular molecule of circIGF2BP3 through an RNase R enzyme tolerance assay. RT-qPCR results showed high circIGF2BP3 expression in the pectoral and thigh muscles of Yuexi frizzled feather chickens at embryonic ages 14 and 18, as well as at 7 weeks post-hatch. Notably, its expression increased during embryonic development, followed by a rapid decrease after birth. As well as using RT-qPCR, Edu, CCK-8, immunofluorescence, and Western blot techniques, we demonstrated that overexpressing circIGF2BP3 could promote the proliferation and differentiation of chicken primary myoblasts through upregulating genes such as proliferating cell nuclear antigen (PCNA), cyclin D1 (CCND1), cyclin E1 (CCNE1), cyclin dependent kinase 2 (CDK2), myosin heavy chain (MyHC), myoblast-determining 1 (MyoD1), myogenin (MyoG), and Myomaker. In conclusion, circIGF2BP3 promotes the proliferation and differentiation of myoblasts in chickens. This study establishes a foundation for further investigation into the biological functions and mechanisms of circIGF2BP3 in myoblasts proliferation and differentiation.

Inhibition of p53-MDM2 binding reduces senescent cell abundance and improves the adaptive responses of skeletal muscle from aged mice.

Skeletal muscle adaptation to external stimuli, such as regeneration following injury and hypertrophy in response to resistance exercise, are blunted with advanced age. The accumulation of senescent cells, along with defects in myogenic progenitor cell (MPC) proliferation, have been strongly linked as contributing factors to age-associated impairment in muscle adaptation. p53 plays an integral role in all these processes, as upregulation of p53 causes apoptosis in senescent cells and prevents mitotic catastrophe in MPCs from old mice. The goal of this study was to determine if a novel pharmaceutical agent (BI01), which functions by upregulating p53 through inhibition of binding to MDM2, the primary p53 regulatory protein, improves muscle regeneration and hypertrophy in old mice. BI01 effectively reduced the number of senescent cells in vitro but had no effect on MPC survival or proliferation at a comparable dose. Following repeated oral gavage with 2mg/kg of BI01 (OS) or vehicle (OV), old mice (24months) underwent unilateral BaCl2 injury in the tibialis anterior (TA) muscle, with PBS injections serving as controls. After 7days, satellite cell number was higher in the TA of OS compared to OV mice, as was the expression of genes involved in ATP production. By 35days, old mice treated with BI01 displayed reduced senescent cell burden, enhanced regeneration (higher muscle mass and fiber cross-sectional area) and restoration of muscle function relative to OV mice. To examine the impact of 2mg/kg BI01 on muscle hypertrophy, the plantaris muscle was subjected to 28days of mechanical overload (MOV) in OS and OV mice. In response to MOV, OS mice had larger plantaris muscles and muscle fibers than OV mice, particularly type 2b + x fibers, associated with reduced senescent cells. Together our data show that BI01 is an effective senolytic agent that may also augment muscle metabolism to enhance muscle regeneration and hypertrophy in old mice.

Klotho regulates the myogenic response of muscle to mechanical loading and exercise.

What is the central question of this study? Does the hormone Klotho affect the myogenic response of muscle cells to mechanical loading or exercise? What is the main finding and its importance? Klotho prevents direct, mechanical activation of genes that regulate muscle differentiation, including genes that encode the myogenic regulatory factor myogenin and proteins in the canonical Wnt signalling pathway. Similarly, elevated levels of klotho expression in vivo prevent the exercise-induced increase in myogenin-expressing cells and reduce exercise-induced activation of the Wnt pathway. These findings demonstrate a new mechanism through which the responses of muscle to the mechanical environment are regulated. Muscle growth is influenced by changes in the mechanical environment that affect the expression of genes that regulate myogenesis. We tested whether the hormone Klotho could influence the response of muscle to mechanical loading. Applying mechanical loads to myoblasts in vitro increased RNA encoding transcription factors that are expressed in activated myoblasts (Myod) and in myogenic cells that have initiated terminal differentiation (Myog). However, application of Klotho to myoblasts prevented the loading-induced activation of Myog without affecting loading-induced activation of Myod. This indicates that elevated Klotho inhibits mechanically-induced differentiation of myogenic cells. Elevated Klotho also reduced the transcription of genes encoding proteins involved in the canonical Wnt pathway or their target genes (Wnt9a, Wnt10a, Ccnd1). Because the canonical Wnt pathway promotes differentiation of myogenic cells, these findings indicate that Klotho inhibits the differentiation of myogenic cells experiencing mechanical loading. We then tested whether these effects of Klotho occurred in muscles of mice experiencing high-intensity interval training (HIIT) by comparing wild-type mice and klotho transgenic mice. The expression of a klotho transgene combined with HIIT synergized to tremendously elevate numbers of Pax7+ satellite cells and activated MyoD+ cells. However, transgene expression prevented the increase in myogenin+ cells caused by HIIT in wild-type mice. Furthermore, transgene expression diminished the HIIT-induced activation of the canonical Wnt pathway in Pax7+ satellite cells. Collectively, these findings show that Klotho inhibits loading- or exercise-induced activation of muscle differentiation and indicate a new mechanism through which the responses of muscle to the mechanical environment are regulated.

Inherited myogenic abilities in muscle precursor cells defined by the mitochondrial complex I-encoding protein

Skeletal muscle comprises different muscle fibers, including slow- and fast-type muscles, and satellite cells (SCs), which exist in individual muscle fibers and possess different myogenic properties. Previously, we reported that myoblasts (MBs) from slow-type enriched soleus (SOL) had a high potential to self-renew compared with cells derived from fast-type enriched tibialis anterior (TA). However, whether the functionality of myogenic cells in adult muscles is attributed to the muscle fiber in which they reside and whether the characteristics of myogenic cells derived from slow- and fast-type fibers can be distinguished at the genetic level remain unknown. Global gene expression analysis revealed that the myogenic potential of MBs was independent of the muscle fiber type they reside in but dependent on the region of muscles they are derived from. Thus, in this study, proteomic analysis was conducted to clarify the molecular differences between MBs derived from TA and SOL. NADH dehydrogenase (ubiquinone) iron-sulfur protein 8 (Ndufs8), a subunit of NADH dehydrogenase in mitochondrial complex I, significantly increased in SOL-derived MBs compared with that in TA-derived cells. Moreover, the expression level of Ndufs8 in MBs significantly decreased with age. Gain- and loss-of-function experiments revealed that Ndufs8 expression in MBs promoted differentiation, self-renewal, and apoptosis resistance. In particular, Ndufs8 suppression in MBs increased p53 acetylation, followed by a decline in NAD/NADH ratio. Nicotinamide mononucleotide treatment, which restores the intracellular NAD+ level, could decrease p53 acetylation and increase myogenic cell self-renewal ability in vivo. These results suggested that the functional differences in MBs derived from SOL and TA governed by the mitochondrial complex I-encoding gene reflect the magnitude of the decline in SC number observed with aging, indicating that the replenishment of NAD+ is a possible approach for improving impaired cellular functions caused by aging or diseases.

Stem cell-based strategies and challenges for production of cultivated meat.

Cultivated meat scale-up and industrial production will require multiple stable cell lines from different species to recreate the organoleptic and nutritional properties of meat from livestock. In this Review, we explore the potential of stem cells to create the major cellular components of cultivated meat. By using developments in the fields of tissue engineering and biomedicine, we explore the advantages and disadvantages of strategies involving primary adult and pluripotent stem cells for generating cell sources that can be grown at scale. These myogenic, adipogenic or extracellular matrix-producing adult stem cells as well as embryonic or inducible pluripotent stem cells are discussed for their proliferative and differentiation capacity, necessary for cultivated meat. We examine the challenges for industrial scale-up, including differentiation and culture protocols, as well as genetic modification options for stem cell immortalization and controlled differentiation. Finally, we discuss stem cell-related safety and regulatory challenges for bringing cultivated meat to the marketplace.

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.

MRNA in situ hybridization exhibits unbalanced nuclear/cytoplasmic dystrophin transcript repartition in Duchenne myogenic cells and skeletal muscle biopsies

To gain insight on dystrophin (DMD) gene transcription dynamics and spatial localization, we assayed the DMD mRNA amount and defined its compartmentalization in myoblasts, myotubes, and skeletal muscle biopsies of Duchenne muscular dystrophy (DMD) patients. Using droplet digital PCR, Real-time PCR, and RNAscope in situ hybridization, we showed that the DMD transcript amount is extremely reduced in both DMD patients’ cells and muscle biopsies and that mutation-related differences occur. We also found that, compared to controls, DMD transcript is dramatically reduced in the cytoplasm, as up to 90% of it is localized in nuclei, preferentially at the perinuclear region. Using RNA/protein colocalization experiments, we showed that about 40% of nuclear DMD mRNA is localized in the nucleoli in both control and DMD myogenic cells. Our results clearly show that mutant DMD mRNA quantity is strongly reduced in the patients’ myogenic cells and muscle biopsies. Furthermore, mutant DMD mRNA compartmentalization is spatially unbalanced due to a shift in its localization towards the nuclei. This abnormal transcript repartition contributes to the poor abundance and availability of the dystrophin messenger in cytoplasm. This novel finding also has important repercussions for RNA-targeted therapies.

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.