Muscle-derived Cells Research Articles

Adipose-derived cells surpass muscle-derived cells in primary cell isolation efficacy.

Adipose-derived cells surpass muscle-derived cells in primary cell isolation efficacy.

Tissue-engineered sub-urethral sling with muscle-derived cells for urethral sphincter regeneration in an animal model of stress urinary incontinence.

Stress urinary incontinence (SUI) is a widespread condition affecting more than 200 million people worldwide. Common treatments for this condition include retropubic colposuspension, and pelvic sling methods, which use autologous grafts or synthetic materials to support the bladder neck and urethral sphincter. Although these treatments have a cure rate of over 80%, adverse effects and recurrence may still occur. Several studies have focused on the potential of cell therapy. Muscle-derived cells (MDCs) can be easily obtained from small biopsied striated muscular tissues and possess superior multi-lineage differentiation and self-renewal capacity. Based on the unique characteristics of MDCs and previous favorable results in muscle regeneration, we fabricated a chitosan-gelatin hydrogel sling loaded with MDCs in a rat model of SUI. Leak point pressure and histological indices regarding inflammation, muscular atrophy, and collagen density were assessed to compare the effectiveness of cell injection and cell-laden sling. The level of LPP was significantly reduced in the MODEL group versus the control animals. The LPP level was considerably higher in CELL INJECTION, SLING, and CELL/SLING groups compared to the MODEL group but did not reach the significance threshold. The inflammation rate was significantly lower in the CELL/SLING group compared to the SLING group. The CELL/SLING group showed less atrophy compared to the other experimental groups, indicating that the cells may have higher viability on SLING than through injection. This also suggests that in long-term studies, as the degradation rate of hydrogels increases, the function of cells will become more apparent.

Duchenne muscular dystrophy skeletal muscle cells derived from human induced pluripotent stem cells recapitulate various calcium dysregulation pathways

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease, caused by mutations in the dystrophin gene and resulting in premature death. As a major secondary event, an abnormal elevation of the intracellular calcium concentration in the dystrophin-deficient muscle contributes to disease progression in DMD. In this study, we investigated the specific functional features of induced pluripotent stem cell-derived muscle cells (hiPSC-skMCs) generated from DMD patients to regulate intracellular calcium concentration. As compared to healthy hiPSC-skMCs, DMD hiPSC-skMCs displayed specific spontaneous calcium signatures with high levels of intracellular calcium concentration. Furthermore, stimulations with electrical field or with acetylcholine perfusion induced higher calcium response in DMD hiPSC-skMCs as compared to healthy cells. Finally, Mn2+ quenching experiments demonstrated high levels of constitutive calcium entries in DMD hiPSC-skMCs as compared to healthy cells. Our findings converge on the fact that DMD hiPSC-skMCs display intracellular calcium dysregulation as demonstrated in several other models. Observed calcium disorders associated with RNAseq analysis on these DMD cells highlighted some mechanisms, such as spontaneous and activated sarcoplasmic reticulum (SR) releases or constitutive calcium entries, known to be disturbed in other dystrophin-deficient models. However, store operated calcium entries (SOCEs) were not found to be dysregulated in our DMD hiPSC-skMCs model. These results suggest that all the mechanisms of calcium impairment observed in other animal models may not be as pronounced in humans and could point to a preference for certain mechanisms that could correspond to major molecular targets for DMD therapies.

Culture and Immunomodulation of Equine Muscle-Derived Mesenchymal Stromal Cells: A Comparative Study of Innovative 2D versus 3D Models Using Equine Platelet Lysate.

Muscle-derived mesenchymal stromal cells (mdMSCs) hold great promise in regenerative medicine due to their immunomodulatory properties, multipotent differentiation capacity and ease of collection. However, traditional in vitro expansion methods use fetal bovine serum (FBS) and have numerous limitations including ethical concerns, batch-to-batch variability, immunogenicity, xenogenic contamination and regulatory compliance issues. This study investigates the use of 10% equine platelet lysate (ePL) obtained by plasmapheresis as a substitute for FBS in the culture of mdMSCs in innovative 2D and 3D models. Using muscle microbiopsies as the primary cell source in both models showed promising results. Initial investigations indicated that small variations in heparin concentration in 2D cultures strongly influenced medium coagulation with an optimal proliferation observed at final heparin concentrations of 1.44 IU/mL. The two novel models investigated showed that expansion of mdMSCs is achievable. At the end of expansion, the 3D model revealed a higher total number of cells harvested (64.60 ± 5.32 million) compared to the 2D culture (57.20 ± 7.66 million). Trilineage differentiation assays confirmed the multipotency (osteoblasts, chondroblasts and adipocytes) of the mdMSCs generated in both models with no significant difference observed. Immunophenotyping confirmed the expression of the mesenchymal stem cell (MSC) markers CD-90 and CD-44, with low expression of CD-45 and MHCII markers for mdMSCs derived from the two models. The generated mdMSCs also had great immunomodulatory properties. Specific immunological extraction followed by enzymatic detection (SIEFED) analysis demonstrated that mdMSCs from both models inhibited myeloperoxidase (MPO) activity in a strong dose-dependent manner. Moreover, they were also able to reduce reactive oxygen species (ROS) activity, with mdMSCs from the 3D model showing significantly higher dose-dependent inhibition compared to the 2D model. These results highlighted for the first time the feasibility and efficacy of using 10% ePL for mdMSC expansion in novel 2D and 3D approaches and also that mdMSCs have strong immunomodulatory properties that can be exploited to advance the field of regenerative medicine and cell therapy instead of using FBS with all its drawbacks.

Crude polysaccharides of Helvella leucopus induces myogenic differentiation of C2C12 cells and raises levels of genes involved in mitochondrial dynamics

Age-related skeletal muscle weakness deteriorate the prognosis of underlying diseases. Previous studies revealed that crude polysaccharides of Helvella leucopus (HLP) exhibited immunomodulatory, anti-aging and antioxidant activities. We studied the effect of HLP on myogenic differentiation of mouse myoblasts (C2C12). Cellular immunofluorescence staining was used to observe the changes of C2C12 myotubes after different doses of HLP intervention. qRT-PCR was conducted to quantitatively analyze the mRNA expression levels of myogenesis, mitochondrial quality control and peroxisome proliferator-activated receptor γ coactivator 1-alpha (PGC1-α) after HLP treatment. Western blotting was conducted to quantitatively analyze the protein expression levels after HLP treatment. The increased differentiation of myoblasts and MyHC levels were found. It is worth noting that the expression of MFN2, Drp1 in mitochondrial dynamic of C2C12 cell-derived muscle fibers were observed to be increased. HLP stimulates myogenic differentiation in C2C12 cells which is associated with increased levels of genes involved in mitochondrial dynamics.

Myocardin related transcription factor and galectin-3 drive lipid accumulation in human blood vessels

ObjectiveDiabetes and hypertension are important risk factors for vascular disease, including atherosclerosis. A driving factor in this process is lipid accumulation in smooth muscle cells of the vascular wall. The glucose- and mechano-sensitive transcriptional coactivator, myocardin-related transcription factor A (MRTF-A/MKL1) can promote lipid accumulation in cultured human smooth muscle cells and contribute to the formation of smooth muscle-derived foam cells. The purpose of this study was to determine if intact human blood vessels ex vivo can be used to evaluate lipid accumulation in the vascular wall, and if this process is dependent on MRTF and/or galectin-3/LGALS3. Galectin-3 is an early marker of smooth muscle transdifferentiation and a potential mediator for foam cell formation and atherosclerosis. Approach and resultsHuman mammary arteries and saphenous veins were exposed to altered cholesterol and glucose levels in an organ culture model. Accumulation of lipids, quantified by Oil Red O, was increased by cholesterol loading and elevated glucose concentrations. Pharmacological inhibition of MRTF with CCG-203971 decreased lipid accumulation, whereas adenoviral-mediated overexpression of MRTF-A had the opposite effect. Cholesterol-induced expression of galectin-3 was decreased after inhibition of MRTF. Importantly, pharmacological inhibition of galectin-3 with GB1107 reduced lipid accumulation in the vascular wall after cholesterol loading. ConclusionEx vivo organ culture of human arteries and veins can be used to evaluate lipid accumulation in the intact vascular wall, as well as adenoviral transduction and pharmacological inhibition. Although MRTF and galectin-3 may have beneficial, anti-inflammatory effects under certain circumstances, our results, which demonstrate a significant decrease in lipid accumulation, support further evaluation of MRTF- and galectin-3-inhibitors for therapeutic intervention against atherosclerotic vascular disease.

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.

Optimization of the Amplification of Equine Muscle-Derived Mesenchymal Stromal Cells in a Hollow-Fiber Bioreactor.

The main causes of mortality in horses are the gastrointestinal pathologies associated with septic shock. Stem cells have shown, through systemic injection, a capacity to decrease inflammation and to regenerate injured tissue faster. Nevertheless, to achieve this rapid and total regeneration, systemic injections of 1 to 2 million cells per kilogram of body weight must be considered. Here, we demonstrate for the first time the feasibility and expansion capacity of equine muscle-derived mesenchymal stromal cells (mdMSCs) in a functionally closed, automated, perfusion-based, hollow-fiber bioreactor (HFBR) called the Quantum™ Cell Expansion System (Terumo Blood and Cell Technologies). This feature greatly increases the number of generated cells with a surface area of 1.7 m2. The expansion of mdMSCs is very efficient in this bioreactor. The maximum expansion generated twenty times more cells than the initial seeding in nine days. The best returns were observed with an optimal seeding between 10 and 25 million mdMSCs, using the Bull's eye loading method and with a run duration between 7 and 10 days. Moreover, all the generated cells kept their stem properties: the ability to adhere to plastic and to differentiate into chondroblasts, osteoblasts and adipocytes. They also showed the expression of CD-44 and CD-90 markers, with a positive rate above 93%, while CD-45 and MHCII were non-expressed, with a positive rate below 0.5%. By capitalizing on the scalability, automation and 3D culture capabilities of the Quantum™, it is possible to generate large quantities of high-quality equine mdMSCs for gastrointestinal disorders and other clinical applications.

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair.

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

Establishment and Characterization of a Skeletal Muscle-Derived Myogenic Cell Line from Black Sea Bream (Acanthopagrus schlegelii)

Establishing muscle lineage cell lines from fish will provide a great opportunity to study muscle development, which can eventually contribute to the improvement of the fish quality in the aquaculture industry. However, there has been a lack of the development of proper fish muscle lineage cell lines so far. Here, we report the establishment of a skeletal muscle-derived myogenic cell line from black sea bream (Acanthopagrus schlegelii). For this, we first attempted to find the optimal conditions for the primary explant culture of A. schlegelii muscle tissues and then established muscle-derived cell lines. After that, cell lines were characterized for their muscle-specific gene expression, growth, and myogenic differentiation. We found that the primary explant culture was effective when the tissue fragments were cultured in Dulbecco’s Modified Eagle Medium supplemented with fetal bovine serum and antibiotics on gelatin-coated dishes. Additionally, we confirmed that the addition of basic fibroblast growth factor was necessary to establish the cell lines. One of three cell lines established was capable of long-term culture, expressed three major myogenic regulatory genes including Pax7, MyoD, and Myog, and differentiated to myotubes in the condition using low concentration of horse serum, demonstrating that this cell line was a skeletal muscle-derived myogenic cell line.

Conductive Microgel Annealed Scaffolds Enhance Myogenic Potential of Myoblastic Cells.

Conductive biomaterials may capture native or exogenous bioelectric signaling, but incorporation of conductive moieties is limited by cytotoxicity, poor injectability, or insufficient stimulation. Microgel annealed scaffolds are promising as hydrogel-based materials due to their inherent void space that facilitates cell migration and proliferation better than nanoporous bulk hydrogels. Conductive microgels aregenerated from poly(ethylene) glycol (PEG and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT: PSS) to explore the interplay of void volume and conductivity on myogenic differentiation. PEDOT: PSS increasesmicrogel conductivity two-fold while maintaining stiffness, annealing strength, and viability of associated myoblastic cells. C2C12 myoblasts exhibitincreases in the late-stage differentiation marker myosin heavy chain as a function of both porosity and conductivity. Myogenin, an earlier marker, isinfluenced only by porosity. Human skeletal muscle-derived cells exhibitincreased Myod1, insulin like growth factor-1, and insulin-like growth factor binding protein 2 at earlier time points on conductive microgel scaffolds compared to non-conductive scaffolds. They also secretemore vascular endothelial growth factor at early time points and expressfactors that led to macrophage polarization patterns observe during muscle repair. These data indicate that conductivity aids myogenic differentiation of myogenic cell lines and primary cells, motivating the need for future translational studies to promote muscle repair.

The critical impact of traumatic muscle loss on fracture healing: Basic science and clinical aspects.

Musculoskeletal trauma, specifically fractures, is a leading cause of patient morbidity and disability worldwide. In approximately 20% of cases with fracture and related traumatic muscle loss, bone healing is impaired leading to fracture nonunion. Over the past few years, several studies have demonstrated that bone and the surrounding muscle tissue interact not only anatomically and mechanically but also through biochemical pathways and mediators. Severe damage to the surrounding musculature at the fracture site causes an insufficiency in muscle-derived osteoprogenitor cells that are crucial for fracture healing. As an endocrine tissue, skeletal muscle produces many myokines that act on different bone cells, such as osteoblasts, osteoclasts, osteocytes, and mesenchymal stem cells. Investigating how muscle influences fracture healing at cellular, molecular, and hormonal levels provides translational therapeutic solutions to this clinical challenge. This review provides an overview about the contributions of surrounding muscle tissue in directing fracture healing. The focus of the review is on describing the interactions between bone and muscle in both healthy and fractured environments. We discuss current progress in identifying the bone-muscle molecular pathways and strategies to harness these pathways as cues for accelerating fracture healing. In addition, we review the existing challenges and research opportunities in the field.

Smooth muscle-derived adventitial progenitor cells direct atherosclerotic plaque composition complexity in a Klf4-dependent manner.

We previously established that vascular smooth muscle-derived adventitial progenitor cells (AdvSca1-SM) preferentially differentiate into myofibroblasts and contribute to fibrosis in response to acute vascular injury. However, the role of these progenitor cells in chronic atherosclerosis has not been defined. Using an AdvSca1-SM cell lineage tracing model, scRNA-Seq, flow cytometry, and histological approaches, we confirmed that AdvSca1-SM-derived cells localized throughout the vessel wall and atherosclerotic plaques, where they primarily differentiated into fibroblasts, smooth muscle cells (SMC), or remained in a stem-like state. Krüppel-like factor 4 (Klf4) knockout specifically in AdvSca1-SM cells induced transition to a more collagen-enriched fibroblast phenotype compared with WT mice. Additionally, Klf4 deletion drastically modified the phenotypes of non-AdvSca1-SM-derived cells, resulting in more contractile SMC and atheroprotective macrophages. Functionally, overall plaque burden was not altered with Klf4 deletion, but multiple indices of plaque composition complexity, including necrotic core area, macrophage accumulation, and fibrous cap thickness, were reduced. Collectively, these data support that modulation of AdvSca1-SM cells through KLF4 depletion confers increased protection from the development of potentially unstable atherosclerotic plaques.

Nutrient Rescue of Early Maturing and Deteriorating Satellite Cell-Derived Engineered Muscle Tissue.

Engineered human muscle tissue is a promising tool for tissue models to better understand muscle physiology and diseases, since they can replicate many biomimetic structures and functions of skeletal muscle in vitro. We have developed a method to produce contractile muscle sheet tissues from human myoblasts, based on our cell sheet fabrication technique. This study reports that our tissue engineering technique allowed us to discover unique characteristics of human muscle satellite cells as a cell source for our muscle sheet tissue. The tissues engineered from satellite cells functionally matured within several days, which is earlier than those created from myoblasts. On the other hand, satellite cell-derived muscle sheet tissues were unable to maintain the contractile ability, whereas the myoblast-derived tissues showed muscle contractions for several weeks. The sarcomere structures and membrane-like structures of laminin and dystrophin were lost along with early functional deterioration. Based on a hypothesis that an insufficiency of nutrients caused a shortened lifetime, we supplemented the culture medium for the satellite cell-derived muscle sheet tissues with 10% serum, although a lower serum medium is commonly used to produce muscle tissues. Further combined with the transforming growth factor (TGF-β1) receptor inhibitor, SB431542, the contractile ability of the muscle tissues was increased remarkably and the tissue microstructures were maintained for a longer term, while retaining the early functionalization and the enriched culture conditions prevented early deterioration. These results strengthened our understanding of the biology of myoblasts and satellite cells in muscle tissue formation and provided new insights into the applications of muscle tissue engineering.

Single-cell analysis of bovine muscle-derived cell types for cultured meat production.

Cultured meat technologies leverage the proliferation and differentiation of animal-derived stem cells ex vivo to produce edible tissues for human consumption in a sustainable fashion. However, skeletal muscle is a dynamic and highly complex tissue, involving the interplay of numerous mono- and multinucleated cells, including muscle fibers, satellite cells (SCs) and fibro-adipogenic progenitors (FAPs), and recreation of the tissue in vitro thus requires the characterization and manipulation of a broad range of cell types. Here, we use a single-cell RNA sequencing approach to characterize cellular heterogeneity within bovine muscle and muscle-derived cell cultures over time. Using this data, we identify numerous distinct cell types, and develop robust protocols for the easy purification and proliferation of several of these populations. We note overgrowth of undesirable cell types within heterogeneous proliferative cultures as a barrier to efficient cultured meat production, and use transcriptomics to identify conditions that favor the growth of SCs in the context of serum-free medium. Combining RNA velocities computed in silico with time-resolved flow cytometric analysis, we characterize dynamic subpopulations and transitions between active, quiescent, and committed states of SCs, and demonstrate methods for modulation of these states during long-term proliferative cultures. This work provides an important reference for advancing our knowledge of bovine skeletal muscle biology, and its application in the development of cultured meat technologies.

Vitamin A injection at birth improves muscle growth in lambs.

Vitamin A and its metabolite, retinoic acid (RA) play important roles in regulating skeletal muscle development. This study was conducted to investigate the effects of early intramuscular vitamin A injection on the muscle growth of lambs. A total of 16 newborn lambs were given weekly intramuscular injections of corn oil (control group, n=8) or 7,500 IU vitamin A palmitate (vitamin A group, n=8) from birth to 3wk of age (4 shots in total). At 3wk of age and weaning, biceps femoris muscle samples were taken to analyze the effects of vitamin A on the myogenic capacity of skeletal muscle cells. All lambs were slaughtered at 8 months of age. The results suggest that vitamin A treatment accelerated the growth rate of lambs and increased the loin eye area (P<0.05). Consistently, vitamin A increased the diameter of myofibers in longissimus thoracis muscle (P<0.01) and increased the final body weight of lambs (P<0.05). Vitamin A injection did not change the protein kinase B/mammalian target of rapamycin and myostatin signaling (P>0.05). Moreover, vitamin A upregulated the expression of PAX7 (P<0.05) and the myogenic marker genes including MYOD and MYOG (P<0.01). The skeletal muscle-derived mononuclear cells from vitamin A-treated lambs showed higher expression of myogenic genes (P<0.05) and formed more myotubes (P<0.01) when myogenic differentiation was induced invitro. In addition, invitro analysis showed that RA promoted myogenic differentiation of the skeletal muscle-derived mononuclear cells in the first 3d(P<0.05) but not at the later stage (P>0.05) as evidenced by myogenic gene expression and fusion index. Taken together, neonatal intramuscular vitamin A injection promotes lamb muscle growth by promoting the myogenic potential of satellite cells.

Thermal Effect on Heat Shock Protein 70 Family to Prevent Atherosclerotic Cardiovascular Disease.

Heat shock protein 70 (HSP70) is a chaperone protein induced by various stresses on cells and is involved in various disease mechanisms. In recent years, the expression of HSP70 in skeletal muscle has attracted attention for its use as a prevention of atherosclerotic cardiovascular disease (ASCVD) and as a disease marker. We have previously reported the effect of thermal stimulation targeted to skeletal muscles and skeletal muscle-derived cells. In this article, we reported review articles including our research results. HSP70 contributes to the improvement of insulin resistance as well as chronic inflammation which are underlying pathologies of type 2 diabetes, obesity, and atherosclerosis. Thus, induction of HSP70 expression by external stimulation such as heat and exercise may be useful for ASCVD prevention. It may be possible to induce HSP70 by thermal stimulus in those who have difficulty in exercise because of obesity or locomotive syndrome. It requires further investigation to determine whether monitoring serum HSP70 concentration is useful for ASCVD prevention.

Developing In Vitro Models to Define the Role of Direct Mitochondrial Toxicity in Frequently Reported Drug-Induced Rhabdomyolysis.

The United States Food and Drug Administration Adverse Event Reporting System (FAERS) logged 27,140 rhabdomyolysis cases from 2004 to 31 March 2020. We used FAERS to identify 14 drugs frequently reported in 6583 rhabdomyolysis cases and to investigate whether mitochondrial toxicity is a common pathway of drug-induced rhabdomyolysis by these drugs. Preliminary screening for mitochondrial toxicity was performed using the acute metabolic switch assay, which is adapted here for use in murine L6 cells. Fenofibrate, risperidone, pregabalin, propofol, and simvastatin lactone drugs were identified as mitotoxic and underwent further investigation, using real-time respirometry (Seahorse Technology) to provide more detail on the mechanism of mitochondrial-induced toxicity. To confirm the human relevance of the findings, fenofibrate and risperidone were evaluated in primary human skeletal muscle-derived cells (HSKMDC), using the acute metabolic switch assay and real-time respirometry, which confirmed this designation, although the toxic effects on the mitochondria were more pronounced in HSKMDC. Overall, these studies demonstrate that the L6 model of acute modification may find utility as an initial, cost-effective screen for identifying potential myotoxicants with relevance to humans and, importantly, that drug-induced mitochondrial dysfunction may be a common mechanism shared by some drugs that induce myotoxicity.

Sheep-derived cell immortalization through the expression of cell cycle regulators and biological characterization using transcriptomes.

Sheep are important domestic animals for the production of wool and meat. Although numerous cultured cell lines from humans and mice have been established, the number of cell lines derived from sheep is limited. To overcome this issue, the efficient establishment of a sheep-derived cell line and its biological characterization is reported. Mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase were introduced into sheep muscle-derived cells in an attempt to immortalize primary cells using the K4DT method. Furthermore, the SV40 large T oncogene was introduced into the cells. The successful immortalization of sheep muscle-derived fibroblasts was shown using the K4DT method or SV40 large T antigen. Furthermore, the expression profile of established cells showed close biological characteristics of ear-derived fibroblasts. This study provides a useful cellular resource for veterinary medicine and cell biology.

Contribution of smooth muscle-derived vascular progenitor cells to atherosclerosis

Purpose: Atherosclerosis is a major cause of morbidity and mortality worldwide, but current therapies fail to adequately meet clinical needs. Emerging evidence implicates the outer layer of the blood vessel, the adventitia, in the pathogenesis of atherosclerosis. Specifically, it has been suggested that expansion of adventitial microvessels, the vasa vasorum (VV), drives atherosclerosis by facilitating inflammatory cell infiltration. Our group previously identified a unique population of multipotent resident vascular stem cells (AdvSca1-SM cells) that derive from mature vascular smooth muscle cells (SMCs) and reside in the vessel adventitia, where they are poised to respond to vascular injury. The goal of this project was to determine the contribution of AdvSca1-SM cells to the chronic vascular injury response. We hypothesized that in the setting of atherosclerosis, AdvSca1-SM cells contribute to plaque growth or VV expansion to drive disease progression. Methods: We generated a highly specific lineage tracing mouse model to track AdvSca1-SM cells in vivo over time. Lineage tracing mice were placed on either normal chow or modified Western diet (combined with PCSK9 AAV) for 8 to 30 weeks, then vascular tissue was analyzed using IF microscopy, scRNA-Seq, and flow cytometry. Results: scRNA-Seq and flow cytometry revealed a large reservoir of AdvSca1-SM cells in a stem-like state in the setting of atherosclerosis, but these cells showed significant phenotypic shifts compared to control conditions. Additionally, genetic modulation of the AdvSca1-SM cells influenced the transcriptional profiles of non-AdvSca1-SM-derived cells, indicating a signaling role for AdvSca1-SM cells in the vascular microenvironment. When AdvSca1-SM cells differentiate into other cell types, they primarily become mature SMCs, modulated SMCs, and myofibroblasts. These AdvSca1-SM-derived SMCs and myofibroblasts can be found in the core of the plaque, the fibrous cap, and the media wall. Additionally, these cells can be associated with adventitial microvessels in plaque regions, suggesting a role in plaque neovascularization. Contrary to our preliminary data, AdvSca1-SM cells very rarely differentiate into endothelial cells or macrophages. Conclusions: These studies emphasize the dynamic nature of AdvSca1-SM cells in the vascular injury response, demonstrating the potential of these cells to both differentiate into a variety of cell types as well as signal to other cells in the vascular wall. Ongoing studies will further characterize the distinct contributions of AdvSca1-SM cells to atherosclerosis. NHLBI F31 (Dubner, HL160149-01); NHLBI R01 (Weiser-Evans, HL121877) 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.