Overexpression of GPX2 gene regulates the development of porcine preadipocytes and skeletal muscle cells through MAPK signaling pathway.

Glutathione peroxidase 2 (GPX2) is a selenium-dependent enzyme and protects cells against oxidative damage. Recently, GPX2 has been identified as a candidate gene for backfat and feed efficiency in pigs. However, it is unclear whether GPX2 regulates the development of porcine preadipocytes and skeletal muscle cells. In this study, adenoviral gene transfer was used to overexpress GPX2. Our findings suggest that overexpression of GPX2 gene inhibited proliferation of porcine preadipocytes. And the process is accompanied by the reduction of the p-p38. GPX2 inhibited adipogenic differentiation and promoted lipid degradation, while ERK1/2 was reduced and p-p38 was increased. Proliferation of porcine skeletal muscle cells was induced after GPX2 overexpression, was accompanied by activation in JNK, ERK1/2, and p-p38. Overexpression methods confirmed that GPX2 has a promoting function in myoblastic differentiation. ERK1/2 pathway was activated and p38 was suppressed during the process. This study lays a foundation for the functional study of GPX2 and provides theoretical support for promoting subcutaneous fat reduction and muscle growth.

PHF2 regulates sarcomeric gene transcription in myogenesis.

Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications. However, the epigenetic regulation of myogenesis is poorly understood. Here, we focused on the epigenomic modification enzyme, PHF2, which demethylates histone 3 lysine 9 dimethyl (H3K9me2) during myogenesis. Phf2 mRNA was expressed during myogenesis, and PHF2 was localized in the nuclei of myoblasts and myotubes. We generated Phf2 knockout C2C12 myoblasts using the CRISPR/Cas9 system and analyzed global transcriptional changes via RNA-sequencing. Phf2 knockout (KO) cells 2 d post differentiation were subjected to RNA sequencing. Gene ontology (GO) analysis revealed that Phf2 KO impaired the expression of the genes related to skeletal muscle fiber formation and muscle cell development. The expression levels of sarcomeric genes such as Myhs and Mybpc2 were severely reduced in Phf2 KO cells at 7 d post differentiation, and H3K9me2 modification of Mybpc2, Mef2c and Myh7 was increased in Phf2 KO cells at 4 d post differentiation. These findings suggest that PHF2 regulates sarcomeric gene expression via epigenetic modification.

An engineered in vitro model of the human myotendinous junction.

The myotendinous junction (MTJ) is a vulnerable region at the interface of skeletal muscle and tendon that forms an integrated mechanical unit. This study presents a technique for the spatially restrictive co-culture of human embryonic stem cell (hESC)-derived skeletal myocytes and primary tenocytes for two-dimensional modeling of the MTJ. Micropatterned lanes of extracellular matrix and a 2-well culture chamber define the initial regions of occupation. On day 1, both lines occupy less than 20 % of the initially vacant interstitial zone, referred to henceforth as the junction. Myocyte-tenocyte interdigitations are observed by day 7. Immunocytochemistry reveals enhanced organization and alignment of patterned myocyte and tenocyte features, as well as differential expression of multiple MTJ markers. On day 24, electrically stimulated junction myocytes demonstrate negative contractile strains, while positive tensile strains are exhibited by mechanically passive tenocytes at the junction. Unpatterned tenocytes distal to the junction experience significantly decreased strains in comparison to cells at the interface. Unpatterned myocytes have impaired organization and uncoordinated contractile behavior. These findings suggest that this platform is capable of inducing myocyte-tenocyte junction formation and mechanical coupling similar to the native MTJ, showing transduction of force across the cell-cell interface. STATEMENT OF SIGNIFICANCE: The myotendinous junction (MTJ) is an integrated structure that transduces force across the muscle-tendon boundary, making the region vulnerable to strain injury. Despite the clinical relevance, previous in vitro models of the MTJ lack the structure and mechanical accuracy of the native tissue and have difficulty transmitting force across the cell-cell interface. This study demonstrates an in vitro model of the MTJ, using spatially restrictive cues to inform human myocyte-tenocyte interactions and architecture. The model expressed MTJ markers and developed anisotropic myocyte-tenocyte integrations that resemble the native tissue and allow for force transduction from contracting myocytes to passive tenocyte regions. As such, this study presents a system capable of investigating development, injury, and pathology in the human MTJ.

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.

Amorphous calcium carbonate enhances osteogenic differentiation and myotube formation of human bone marrow derived mesenchymal stem cells and primary skeletal muscle cells under microgravity conditions.

Astronauts are exposed to severely stressful physiological conditions due to microgravity and increased space radiation. Space environment affects every organ and cell in the body and the significant adverse effects of long-term weightlessness include muscle atrophy and deterioration of the skeleton (spaceflight osteopenia). Amorphous Calcium Carbonate (ACC) emerges as a promising candidate for prevention of these effects, owing to its unique physicochemical properties and its potential to address the intricately linked nature of bone-muscle crosstalk. Reported here are two studies carried out on the International Space Station (ISS). The first, performed in 2018 as a part of the Ramon-Spacelab project, was a preliminary experiment, in which stromal murine cells were differentiated into osteoblasts when ACC was added to the culture medium. A parallel experiment was done on Earth as a control. The second study was part of Axiom-1's Rakia project mission launched to the ISS on 2022 utilizing organ-on-a-chip methodology with a specially designed autonomous module. In this experiment, human bone-marrow derived mesenchymal stem cells (hBM-MSCs) and human primary muscle cells were cultured in the presence or absence of ACC, in duplicates. The results showed that ACC enhanced differentiation of human primary skeletal muscle cells into myotubes. Similarly, hBM-MSCs were differentiated significantly better into osteocytes in the presence of ACC leading to increased calcium deposits. The results, combined with previous data, support the use of ACC as an advantageous supplement for preventing muscle and bone deterioration in outer space conditions, facilitating extended extraterrestrial voyages and colonization.

Generation of Chicken Contractile Skeletal Muscle Structure Using Decellularized Plant Scaffolds.

Cultured meat is a meat analogue produced by in vitro cell culture, which can replace the conventional animal production system. Tissue engineering using myogenic cells and biomaterials is a core technology for cultured meat production. In this study, we provide an efficient and economical method to produce skeletal muscle tissue-like structures by culturing chicken myoblasts in a fetal bovine serum (FBS)-free medium and plant-derived scaffolds. An FBS-free medium supplemented with 10% horse serum (HS) and 5% chick embryo extract (CEE) was suitable for the proliferation and differentiation of chicken myoblasts. Decellularized celery scaffolds (Decelery), manufactured using 1% sodium dodecyl sulfate (SDS), were nontoxic to cells and supported myoblast proliferation and differentiation. Decelery could support the 3D culture of chicken myoblasts, which could adhere and coagulate to the surface of the Decelery and form MYH1E+ and F-actin+ myotubes. After 2 weeks of culture on Decelery, fully grown myoblasts completely covered the surface of the scaffolds and formed fiber-like myotube structures. They further differentiated to form spontaneously contracting myofiber-like myotubes on the scaffold surface, indicating that the Decelery scaffold system could support the formation of a functional mature myofiber structure. In addition, as the spontaneously contracting myofibers did not detach from the surface of the Decelery, the Decelery system is a suitable biomaterial for the long-term culture and maintenance of the myofiber structures.

In Adult Skeletal Muscles, the Co-Receptors of Canonical Wnt Signaling, Lrp5 and Lrp6, Determine the Distribution and Size of Fiber Types, and Structure and Function of Neuromuscular Junctions.

Canonical Wnt signaling is involved in skeletal muscle cell biology. The exact way in which this pathway exerts its contribution to myogenesis or neuromuscular junctions (NMJ) is a matter of debate. Next to the common co-receptors of canonical Wnt signaling, Lrp5 and Lrp6, the receptor tyrosine kinase MuSK was reported to bind at NMJs WNT glycoproteins by its extracellular cysteine-rich domain. Previously, we reported canonical Wnt signaling being active in fast muscle fiber types. Here, we used conditional Lrp5 or Lrp6 knockout mice to investigate the role of these receptors in muscle cells. Conditional double knockout mice died around E13 likely due to ectopic expression of the Cre recombinase. Phenotypes of single conditional knockout mice point to a very divergent role for the two receptors. First, muscle fiber type distribution and size were changed. Second, canonical Wnt signaling reporter mice suggested less signaling activity in the absence of Lrps. Third, expression of several myogenic marker genes was changed. Fourth, NMJs were of fragmented phenotype. Fifth, recordings revealed impaired neuromuscular transmission. In sum, our data show fundamental differences in absence of each of the Lrp co-receptors and suggest a differentiated view of canonical Wnt signaling pathway involvement in adult skeletal muscle cells.

Primary cilia in satellite cells are the mechanical sensors for muscle hypertrophy.

Skeletal muscle atrophy is commonly associated with aging, immobilization, muscle unloading, and congenital myopathies. Generation of mature muscle cells from skeletal muscle satellite cells (SCs) is pivotal in repairing muscle tissue. Exercise therapy promotes muscle hypertrophy and strength. Primary cilium is implicated as the mechanical sensor in some mammalian cells, but its role in skeletal muscle cells remains vague. To determine mechanical sensors for exercise-induced muscle hypertrophy, we established three SC-specific cilium dysfunctional mouse models-Myogenic factor 5 (Myf5)-Arf-like Protein 3 (Arl3)-/-, Paired box protein Pax-7 (Pax7)-Intraflagellar transport protein 88 homolog (Ift88)-/-, and Pax7-Arl3-/--by specifically deleting a ciliary protein ARL3 in MYF5-expressing SCs, or IFT88 in PAX7-expressing SCs, or ARL3 in PAX7-expressing SCs, respectively. We show that the Myf5-Arl3-/- mice develop grossly the same as WT mice. Intriguingly, mechanical stimulation-induced muscle hypertrophy or myoblast differentiation is abrogated in Myf5-Arl3-/- and Pax7-Arl3-/- mice or primary isolated Myf5-Arl3-/- and Pax7-Ift88-/- myoblasts, likely due to defective cilia-mediated Hedgehog (Hh) signaling. Collectively, we demonstrate SC cilia serve as mechanical sensors and promote exercise-induced muscle hypertrophy via Hh signaling pathway.

RNA-Seq analysis of a Pax3-expressing myoblast clone in-vitro and effect of culture surface stiffness on differentiation.

Skeletal muscle satellite cells cultured on soft surfaces (12 kPa) show improved differentiation than cells cultured on stiff surfaces (approximately 100 kPa). To better understand the reasons for this, we performed an RNA-Seq analysis for a single satellite cell clone (C1F) derived from the H2kb-tsA58 immortomouse, which differentiates into myotubes under tightly regulated conditions (withdrawal of É£-interferon, 37 °C). The largest change in overall gene expression occurred at day 1, as cells switched from proliferation to differentiation. Surprisingly, further analysis showed that proliferating C1F cells express Pax3 and not Pax7, confirmed by immunostaining, yet their subsequent differentiation into myotubes is normal, and enhanced on softer surfaces, as evidenced by significantly higher expression levels of myogenic regulatory factors, sarcomeric genes, enhanced fusion and improved myofibrillogenesis. Levels of mRNA encoding extracellular matrix structural constituents and related genes were consistently upregulated on hard surfaces, suggesting that a consequence of differentiating satellite cells on hard surfaces is that they attempt to manipulate their niche prior to differentiating. This comprehensive RNA-Seq dataset will be a useful resource for understanding Pax3 expressing cells.

Feeder-supported in vitro exercise model using human satellite cells from patients with sporadic inclusion body myositis.

Contractile activity is a fundamental property of skeletal muscles. We describe the establishment of a "feeder-supported in vitro exercise model" using human-origin primary satellite cells, allowing highly-developed contractile myotubes to readily be generated by applying electrical pulse stimulation (EPS). The use of murine fibroblasts as the feeder cells allows biological responses to EPS in contractile human myotubes to be selectively evaluated with species-specific analyses such as RT-PCR. We successfully applied this feeder-supported co-culture system to myotubes derived from primary satellite cells obtained from sporadic inclusion body myositis (sIBM) patients who are incapable of strenuous exercise testing. Our results demonstrated that sIBM myotubes possess essentially normal muscle functions, including contractility development, de novo sarcomere formation, and contraction-dependent myokine upregulation, upon EPS treatment. However, we found that some of sIBM myotubes, but not healthy control myotubes, often exhibit abnormal cytoplasmic TDP-43 accumulation upon EPS-evoked contraction, suggesting potential pathogenic involvement of the contraction-inducible TDP-43 distribution peculiar to sIBM. Thus, our "feeder-supported in vitro exercise model" enables us to obtain contractile human-origin myotubes, potentially utilizable for evaluating exercise-dependent intrinsic and pathogenic properties of patient muscle cells. Our approach, using feeder layers, further expands the usefulness of the "in vitro exercise model".

Ginsenoside Rg3 protects glucocorticoid­induced muscle atrophy in vitro through improving mitochondrial biogenesis and myotube growth.

Ginsenoside Rg3 (Rg3), amplified by iterative heating processing with fresh ginseng, has a broad range of pharmacological activities and improves mitochondrial biogenesis in skeletal muscle. However, thus far no study has examined how Rg3 affects myotube growth or muscle atrophy, to the best of the authors' knowledge. The present study was conducted to examine the myogenic effect of Rg3 on dexamethasone (DEX)­induced myotube atrophy and the underlying molecular mechanisms. Rg3 activated Akt/mammalian target of rapamycin signaling to prevent DEX­induced myotube atrophy thereby stimulating the expression of muscle­specific genes, including myosin heavy chain and myogenin, and suppressing muscle­specific ubiquitin ligases as demonstrated by immunoblotting and immunostaining assays. Furthermore, Rg3 efficiently prevented DEX­triggered mitochondrial dysfunction of myotubes through peroxisome proliferator­activated receptor­Î³ coactivator1α activities and its mitochondrial biogenetic transcription factors, nuclear respiratory factor­1 and mitochondrial transcription factor A. These were confirmed by immunoblotting, luciferase assays, RT­qPCR and mitochondrial analysis measuring the levels of ROS, ATP and membrane potential. By providing a mechanistic insight into the effect of Rg3 on myotube atrophy, the present study suggested that Rg3 has potential as a therapeutic or nutraceutical remedy to intervene in muscle aging or diseases including cancer cachexia.

Satellite Cells Exhibit Decreased Numbers and Impaired Functions on Single Myofibers Isolated from Vitamin B6-Deficient Mice.

Emerging research in human studies suggests an association among vitamin B6, sarcopenia, and muscle strength. However, very little is known regarding its potential role at the cellular level, especially in muscle satellite cells. Therefore, to determine whether vitamin B6 affects the satellite cells, we isolated single myofibers from muscles of vitamin B6-deficient and vitamin B6-supplemented mice. Subsequently, we subjected them to single myofiber culture and observed the number and function of the satellite cells, which remained in their niche on the myofibers. Prior to culture, the vitamin B6-deficient myofibers exhibited a significantly lower number of quiescent satellite cells, as compared to that in the vitamin B6-supplemented myofibers, thereby suggesting that vitamin B6 deficiency induces a decline in the quiescent satellite cell pool in mouse muscles. After 48 and 72 h of culture, the number of proliferating satellite cells per cluster was similar between the vitamin B6-deficient and -supplemented myofibers, but their numbers decreased significantly after culturing the myofibers in vitamin B6-free medium. After 72 h of culture, the number of self-renewing satellite cells per cluster was significantly lower in the vitamin B6-deficient myofibers, and the vitamin B6-free medium further decreased this number. In conclusion, vitamin B6 deficiency appears to reduce the number of quiescent satellite cells and suppress the proliferation and self-renewal of satellite cells during myogenesis.

ERK1/2 inhibition promotes robust myotube growth via CaMKII activation resulting in myoblast-to-myotube fusion.

Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes. CaMKII activation results in myotube growth via fusion with mononucleated myoblasts at a fusogenic synapse. Mechanistically, CaMKII interacts with and regulates MYMK and Rac1, and CaMKIIδ/γ knockout mice exhibit smaller regenerated myofibers following injury. In addition, the expression of a dominant negative CaMKII inhibits the formation of large multinucleated myotubes. Finally, we demonstrate the evolutionary conservation of the pathway in chicken myoblasts. We conclude that ERK1/2 represses a signaling cascade leading to CaMKII-mediated fusion of myoblasts to myotubes, providing an attractive target for the cultivated meat industry and regenerative medicine.

Intermittent bolus feeding does not enhance protein synthesis, myonuclear accretion, or lean growth more than continuous feeding in a premature piglet model.

Optimizing enteral nutrition for premature infants may help mitigate extrauterine growth restriction and adverse chronic health outcomes. Previously, we showed in neonatal pigs born at term that lean growth is enhanced by intermittent bolus compared with continuous feeding. The objective was to determine if prematurity impacts how body composition, muscle protein synthesis, and myonuclear accretion respond to feeding modality. Following preterm delivery, pigs were fed equivalent amounts of formula delivered either as intermittent boluses (INT; n = 30) or continuously (CONT; n = 14) for 21 days. Body composition was measured by dual-energy X-ray absorptiometry (DXA) and muscle growth was assessed by morphometry, myonuclear accretion, and satellite cell abundance. Tissue anabolic signaling and fractional protein synthesis rates were determined in INT pigs in postabsorptive (INT-PA) and postprandial (INT-PP) states and in CONT pigs. Body weight gain and composition did not differ between INT and CONT pigs. Longissimus dorsi (LD) protein synthesis was 34% greater in INT-PP than INT-PA pigs (P < 0.05) but was not different between INT-PP and CONT pigs. Phosphorylation of 4EBP1 and S6K1 and eIF4E·eIF4G abundance in LD paralleled changes in LD protein synthesis. Satellite cell abundance, myonuclear accretion, and fiber cross-sectional area in LD did not differ between groups. These results suggest that, unlike pigs born at term, intermittent bolus feeding does not enhance lean growth more than continuous feeding in pigs born preterm. Premature birth attenuates the capacity of skeletal muscle to respond to cyclical surges in insulin and amino acids with intermittent feeding in early postnatal life.NEW & NOTEWORTHY Extrauterine growth restriction often occurs in premature infants but may be mitigated by optimizing enteral feeding strategies. We show that intermittent bolus feeding does not increase skeletal muscle protein synthesis, myonuclear accretion, or lean growth more than continuous feeding in preterm pigs. This attenuated anabolic response of muscle to intermittent bolus feeding, compared with previous observations in pigs born at term, may contribute to deficits in lean mass that many premature infants exhibit into adulthood.

Insulin stimulates ß-alanine uptake in skeletal muscle cells in vitro.

We evaluated whether insulin could stimulate ß-alanine uptake by skeletal muscle cells in vitro. Mouse myoblasts (C2C12) (n = 3 wells per condition) were cultured with ß-alanine (350 or 700 µmol·L-1), with insulin (100 µU·mL-1) either added to the media or not. Insulin stimulated the ß-alanine uptake at the lower (350 µmol·L-1) but not higher (700 µmol·L-1) ß-alanine concentration in culture medium, indicating that transporter saturation might blunt the stimulatory effects of insulin.

Defective autophagy and increased apoptosis contribute toward the pathogenesis of FKRP-associated muscular dystrophies.

Fukutin-related protein (FKRP) is a glycosyltransferase involved in glycosylation of alpha-dystroglycan (α-DG). Mutations in FKRP are associated with muscular dystrophies (MD) ranging from limb-girdle LGMDR9 to Walker-Warburg Syndrome (WWS), a severe type of congenital MD. Although hypoglycosylation of α-DG is the main hallmark of this group of diseases, a full understanding of the underlying pathophysiology is still missing. Here, we investigated molecular mechanisms impaired by FKRP mutations in pluripotent stem (PS) cell-derived myotubes. FKRP-deficient myotubes show transcriptome alterations in genes involved in extracellular matrix receptor interactions, calcium signaling, PI3K-Akt pathway, and lysosomal function. Accordingly, using a panel of patient-specific LGMDR9 and WWS induced PS cell-derived myotubes, we found a significant reduction in the autophagy-lysosome pathway for both disease phenotypes. In addition, we show that WWS myotubes display decreased ERK1/2 activity and increased apoptosis, which were restored in gene edited myotubes. Our results suggest the autophagy-lysosome pathway and apoptosis may contribute to the FKRP-associated MD pathogenesis.

A Pillar-Based High-Throughput Myogenic Differentiation Assay to Assess Drug Safety.

High-throughput, pillar-strip-based assays have been proposed as a drug-safety screening tool for developmental toxicity. In the assay described here, muscle cell culture and differentiation were allowed to occur at the end of a pillar strip (eight pillars) compatible with commercially available 96-well plates. Previous approaches to characterize cellular differentiation with immunostaining required a burdensome number of washing steps; these multiple washes also resulted in a high proportion of cellular loss resulting in poor yield. To overcome these limitations, the approach described here utilizes cell growth by easily moving the pillars for washing and immunostaining without significant loss of cells. Thus, the present pillar-strip approach is deemed suitable for monitoring high-throughput myogenic differentiation. Using this experimental high-throughput approach, eight drugs (including two well-known myogenic inhibitory drugs) were tested at six doses in triplicate, which allows for the generation of dose-response curves of nuclei and myotubes in a 96-well platform. As a result of comparing these F-actin (an actin-cytoskeleton protein), nucleus, and myotube data, two proposed differentiation indices-curve-area-based differentiation index (CA-DI) and maximum-point-based differentiation index (MP-DI) were generated. Both indices successfully allowed for screening of high-myogenic inhibitory drugs, and the maximum-point-based differentiation index (MP-DI) experimentally demonstrated sensitivity for quantifying drugs that inhibited myogenic differentiation.

Kinetin stimulates differentiation of C2C12 myoblasts.

Kinetin or N6-furfuryladenine (K) belongs to a class of plant hormones called cytokinins, which are biologically active molecules modulating many aspects of plant growth and development. However, biological activities of cytokinins are not only limited to plants; their effects on animals have been widely reported in the literature. Here, we found that Kinetin is a potent small molecule that efficiently stimulates differentiation of C2C12 myoblasts into myotubes in vitro. The highest efficacy was achieved at 1µM and 10µM Kinetin concentrations, in both mitogen-poor and rich media. More importantly, Kinetin was able to strongly stimulate the MyoD-dependent conversion of fibroblasts into myotubes. Kinetin alone did not give rise to fibroblast conversion and required MyoD; this demonstrates that Kinetin augments the molecular repertoire of necessary key regulatory factors to facilitate MyoD-mediated myogenic differentiation. This novel Kinetin pro-myogenic function may be explained by its ability to alter intracellular calcium levels and by its potential to impact on Reactive Oxygen Species (ROS) signalling. Taken together, our findings unravel the effects of a new class of small molecules with potent pro-myogenic activities. This opens up new therapeutic avenues with potential for treating skeletal muscle diseases related to muscle aging and wasting.

Automated image segmentation method to analyse skeletal muscle cross section in exercise-induced regenerating myofibers.

Skeletal muscle is an adaptive tissue with the ability to regenerate in response to exercise training. Cross-sectional area (CSA) quantification, as a main parameter to assess muscle regeneration capability, is highly tedious and time-consuming, necessitating an accurate and automated approach to analysis. Although several excellent programs are available to automate analysis of muscle histology, they fail to efficiently and accurately measure CSA in regenerating myofibers in response to exercise training. Here, we have developed a novel fully-automated image segmentation method based on neutrosophic set algorithms to analyse whole skeletal muscle cross sections in exercise-induced regenerating myofibers, referred as MyoView, designed to obtain accurate fiber size and distribution measurements. MyoView provides relatively efficient, accurate, and reliable measurements for CSA quantification and detecting different myofibers, myonuclei and satellite cells in response to the post-exercise regenerating process. We showed that MyoView is comparable with manual quantification. We also showed that MyoView is more accurate and efficient to measure CSA in post-exercise regenerating myofibers as compared with Open-CSAM, MuscleJ, SMASH and MyoVision. Furthermore, we demonstrated that to obtain an accurate CSA quantification of exercise-induced regenerating myofibers, whole muscle cross-section analysis is an essential part, especially for the measurement of different fiber-types. We present MyoView as a new tool to quantify CSA, myonuclei and satellite cells in skeletal muscle from any experimental condition including exercise-induced regenerating myofibers.

Directed Differentiation of Human Pluripotent Stem Cells toward Skeletal Myogenic Progenitors and Their Purification Using Surface Markers.

Advancements in reprogramming somatic cells into induced pluripotent stem cells (iPSCs) have provided a strong framework for in vitro disease modeling, gene correction and stem cell-based regenerative medicine. In cases of skeletal muscle disorders, iPSCs can be used for the generation of skeletal muscle progenitors to study disease mechanisms, or implementation for the treatment of muscle disorders. We have recently developed an improved directed differentiation method for the derivation of skeletal myogenic progenitors from hiPSCs. This method allows for a short-term (2 weeks) and efficient skeletal myogenic induction (45-65% of the cells) in human pluripotent stem cells (ESCs/iPSCs) using small molecules to induce mesoderm and subsequently myotomal progenitors, without the need for any gene integration or modification. After initial differentiation, skeletal myogenic progenitors can be purified from unwanted cells using surface markers (CD10+CD24-). These myogenic progenitors have been extensively characterized using in vitro gene expression/differentiation profiling as well as in vivo engraftment studies in dystrophic (mdx) and muscle injury (VML) rodent models and have been proven to be able to engraft and form mature myofibers as well as seeding muscle stem cells. The current protocol describes a detailed, step-by-step guide for this method and outlines important experimental details and troubleshooting points for its application in any human pluripotent stem cells.