Muscle-derived Cells Research Articles

Abstract 13681: Single-cell Gene Profiling Revealed Heterogeneity of Human Skeletal Muscle Derived Stem Cells and Their Paracrine Factors

Background: Transplantation of sheet-shaped autologous skeletal muscle-derived cells (SMDCs) has been shown to produce therapeutic effects via paracrine factors in ischemic cardiomyopathy. SMDCs are heterogeneous cell population consist of myoblasts and non-myogenic cells, and their detailed characteristic and paracrine factors has not been fully analyzed. We herein applied single-cell RNA sequencing (scRNA-seq) to profile the population and paracrine factors of SMDCs. Methods and Results: Primary SMDCs of human origin were subjected to scRNA-seq and divided into 5 populations, expressing myogenic markers differently. These included four myogenic populations with higher expression of MYF5 (myoblasts), myogenin (early differentiated cells), mitosis-related gene (proliferating cells), and MYL3 (moderately differentiated cells) and non-myogenic population with lower expression of myogenic genes. On the other hand, PAX7 , quiescent satellite cell marker, and MYH4 , terminally differentiated marker, were not expressed in any populations. Furthermore, regarding the expression of key angiogenic factors, VEGF and HGF were highly expressed in the non-myogenic population and HMGB1 was expressed in both populations. Moreover, analysis of the gene expression of angiogenic factors differentially expressed in each population indicated that myoblast population highly expressed PDGFA and CCN (Cellular communication network factor) 1 , and early differentiated population highly expressed CCN2 . Although these factors were expressed in other populations, their expression tended to be lower in the moderately differentiated cell population. Conclusion: Analysis of SMDCs using scRNA-seq revealed that they are heterogeneous cells consisting of multiple myogenic populations with different degrees of differentiation and cytokine production, suggesting that single-cell gene profiling may be useful for the evaluation on characters of effector cells.

Abstract 13895: Machine Learning-based Prediction Model With Contribute the Optimal Recipient Selection for the Regenerative Therapy in ICM and Non-ICM Patients

Background: Although transplantation of patch-shaped autologous skeletal muscle-derived cells has been introduced to ischemic cardiomyopathy (ICM) or non-ICM patients, we found responders or non-responders to this treatment exist and how to predict the responsiveness to this treatment is crucial for the improvement of the effectiveness. In this study, we revealed the clinical features associated with the responders using a machine learning based-model, which was discriminated between the responder and the non-responders in this treatment. Methods and Results: We used the retrospective databases of 23 ICM patients and 23 non-ICM patients undergoing autologous myoblast patch transplantation to develop machine learning models to discriminate 3-year VAD free survival. Sixty-nine pre-transplantation clinical futures were selected to train the models. In ICM models, there were 4 VADs or deaths, and in non-ICM models, there were 10 VADs or deaths during the 3-year follow-up periods. Using these databases, we trained multiple machine learning models and evaluated the models with the leave-one-out method. In ICM, k nearest neighbor demonstrated the best performance, showing the accuracy was 95.7%, and the AUC was 0.95. The features associated with 3-year VAD free survival in ICM were NYHA classification, cardiac index, and left ventricular ejection fraction. In non-ICM, k nearest neighbor demonstrated the best performance among the trained classifiers, demonstrating the accuracy was 95.7%, and the AUC was 0.93. The features associated with 3-year VAD free survival in non-ICM were pulmonary capillary wedge pressure, pulmonary vascular resistance, and albumin. Conclusion: We found the features associated with 3-year VAD free survival in autologous myoblast patch transplantation in ICM and non-ICM. Focusing on these features may facilitate optimal candidate selection in ICM and non-ICM for regenerative therapy.

Dead muscle tissue promotes dystrophic calcification by lowering circulating TGF-β1 level

AimsDystrophic calcification (DC) is the abnormal appearance of calcified deposits in degenerating tissue, often associated with injury. Extensive DC can lead to heterotopic ossification (HO), a pathological condition of ectopic bone formation. The highest rate of HO was found in combat-related blast injuries, a polytrauma condition with severe muscle injury. It has been noted that the incidence of HO significantly increased in the residual limbs of combat-injured patients if the final amputation was performed within the zone of injury compared to that which was proximal to the zone of injury. While aggressive limb salvage strategies may maximize the function of the residual limb, they may increase the possibility of retaining non-viable muscle tissue inside the body. In this study, we hypothesized that residual dead muscle tissue at the zone of injury could promote HO formation.MethodsWe tested the hypothesis by investigating the cellular and molecular consequences of implanting devitalized muscle tissue into mouse muscle pouch in the presence of muscle injury induced by cardiotoxin.ResultsOur findings showed that the presence of devitalized muscle tissue could cause a systemic decrease in circulating transforming growth factor-beta 1 (TGF-β1), which promoted DC formation following muscle injury. We further demonstrated that suppression of TGF-β signalling promoted DC in vivo, and potentiated osteogenic differentiation of muscle-derived stromal cells in vitro.ConclusionTaken together, these findings suggest that TGF-β1 may play a protective role in dead muscle tissue-induced DC, which is relevant to understanding the pathogenesis of post-traumatic HO.Cite this article: Bone Joint Res 2020;9(11):742–750.

Bta-miR-23a Regulates the Myogenic Differentiation of Fetal Bovine Skeletal Muscle-Derived Progenitor Cells by Targeting MDFIC Gene.

miR-23a, a member of the miR-23a/24-2/27a cluster, has been demonstrated to play pivotal roles in many cellular activities. However, the mechanisms of how bta-miR-23a controls the myogenic differentiation (MD) of PDGFRα− bovine progenitor cells (bPCs) remain poorly understood. In the present work, bta-miR-23a expression was increased during the MD of PDGFRα− bPCs. Moreover, bta-miR-23a overexpression significantly promoted the MD of PDGFRα− bPCs. Luciferase reporter assays showed that the 3’-UTR region of MDFIC (MyoD family inhibitor domain containing) could be a promising target of bta-miR-23a, which resulted in its post-transcriptional down-regulation. Additionally, the knockdown of MDFIC by siRNA facilitated the MD of PDGFRα− bPCs, while the overexpression of MDFIC inhibited the activating effect of bta-miR-23a during MD. Of note, MDFIC might function through the interaction between MyoG transcription factor and MEF2C promoter. This study reveals that bta-miR-23a can promote the MD of PDGFRα− bPCs through post-transcriptional downregulation of MDFIC.

Effects of Mini-Dystrophin on Dystrophin-Deficient, Human Skeletal Muscle-Derived Cells.

Background: We are developing a novel therapy for Duchenne muscular dystrophy (DMD), involving the transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin. Dystrophin is normally expressed in activated satellite cells and in differentiated muscle fibres. However, in past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells. Aims: Our aims are to determine if mini-dystrophin expression affects the proliferation or myogenic differentiation of DMD skeletal muscle-derived cells. Methods: Skeletal muscle-derived cells from a DMD patient were transduced with lentivirus coding for mini-dystrophins (R3–R13 spectrin-like repeats (ΔR3R13) or hinge2 to spectrin-like repeats R23 (ΔH2R23)) with EGFP (enhanced green fluorescence protein) fused to the C-terminus, driven by a constitutive promoter, spleen focus-forming virus (SFFV). Transduced cells were purified on the basis of GFP expression. Their proliferation and myogenic differentiation were quantified by ethynyl deoxyuridine (EdU) incorporation and fusion index. Furthermore, dystrophin small interfering ribonucleic acids (siRNAs) were transfected to the cells to reverse the effects of the mini-dystrophin. Finally, a phospho-mitogen-activated protein kinase (MAPK) array assay was performed to investigate signalling pathway changes caused by dystrophin expression. Results: Cell proliferation was not affected in cells transduced with ΔR3R13, but was significantly increased in cells transduced with ΔH2R23. The fusion index of myotubes derived from both ΔR3R13- and ΔH2R23 -expressing cells was significantly compromised in comparison to myotubes derived from non-transduced cells. Dystrophin siRNA transfection restored the differentiation of ΔH2R23-expressing cells. The Erk1/2- signalling pathway is altered in cells transduced with mini-dystrophin constructs. Conclusions: Ectopic expression of dystrophin in cultured human skeletal muscle-derived cells may affect their proliferation and differentiation capacity. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter.

DNA methylation across the genome in aged human skeletal muscle tissue and muscle-derived cells: the role of HOX genes and physical activity

Skeletal muscle tissue demonstrates global hypermethylation with age. However, methylome changes across the time-course of differentiation in aged human muscle derived cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 ± 4.4 years) and aged (83 ± 4 years) human skeletal muscle and that of young/aged heterogenous muscle-derived human primary cells (HDMCs) over several time points of differentiation (0, 72 h, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; pathways-in-cancer (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), rap1-signaling, axon-guidance and hippo-signalling. Aged cells also demonstrated a hypermethylated profile in pathways; axon-guidance, adherens-junction and calcium-signaling, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alterations in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in focal adhesion and PI3K-AKT signalling pathways. While the methylomes were vastly different between muscle tissue and HDMCs, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle tissue and cells on genes KIF15, DYRK2, FHL2, MRPS33, ABCA17P. Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6–8 CpGs in the HOX family of genes altered with age. With HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1, HOXA3 and HOXC-AS3. Finally, increased physical activity in young adults was associated with oppositely regulating HOXB1 and HOXA3 methylation compared with age. Overall, we demonstrate that a considerable number of HOX genes are differentially epigenetically regulated in aged human skeletal muscle and HDMCs and increased physical activity may help prevent age-related epigenetic changes in these HOX genes.

Transient and Stable Overexpression of Extracellular Superoxide Dismutase Is Positively Associated with the Myogenic Function of Human Skeletal Muscle-Derived Stem/Progenitor Cells.

In the present study, the genetic modification of human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) was investigated to identify the optimal protocol for myogenic cell preparation for use in post-infarction heart therapy. We used two types of modifications: GFP-transfection (using electroporation) and SOD3 transduction (using a lentiviral vector). SkMDS/PCs were cultured under different in vitro conditions, including standard (21% oxygen) and hypoxic (3% oxygen), the latter of which corresponded to the prevailing conditions in the post-infarction heart. Transfection/transduction efficacy, skeletal myogenic cell marker expression (CD56), cellular senescence, and apoptosis, as well as the expression of antioxidant (SOD1, SOD2, and SOD3), anti-aging (SIRT1 and FOXO), anti-apoptotic (BCL2), and myogenic (MyoD and MyoG) genes, were evaluated. The percentage of GFP-positive SkMDS/PCs was determined as an indicator of the efficacy of transfection, which reached 55%, while transduction showed better efficiency, reaching approximately 85% as estimated by fluorescence microscopy. The CD56-positive SkMDS/PCs were present in approximately 77% of the tested cells after transient transfection and approximately 96% after transduction. Under standard in vitro culture conditions, the ability of the differentiated, transfected SkMDS/PCs to form myotubes was greater than that of the wild type (WT) cell population (p < 0.001), while the cells transduced with the SOD3 gene exhibited an increase in cell fusion under both standard (p < 0.05) and hypoxic conditions (p < 0.001). In transduced SkMDS/PCs, we observed a positive influence of SOD3 overexpression on cell ageing and apoptosis. We observed an increase in the percentage of young cells under standard (p < 0.05) and hypoxic (p < 0.001) in vitro culture conditions, with a notable decrease in the percentage of senescent and advanced senescent cells in the SOD3-overexpressing cell population detected compared to that observed for the untransduced muscle-derived cells. A lower percentage of apoptotic cells was observed for transduced SkMDS/PCs than that for WT cells under hypoxic in vitro culture conditions. In transiently transfected SkMDS/PCs, we observed significantly higher gene expression levels of SOD2 (almost 40-fold) (p < 0.001) and FOXO (p < 0.05) (approximately 3-fold) under both normoxic and hypoxic culture conditions and of BCL2 under hypoxia compared to those observed in untreated cells (WT). In addition, myogenic genes showed a significant increase in MyoD (almost 18-fold) expression under standard culture conditions (p < 0.0001) and decreased MyoG expression (approximately 2-fold) after transfection (p < 0.05) compared with that detected in the WT skeletal muscle-derived cell control. Taken together, these results demonstrate that SOD3-tranduced skeletal muscle-derived cells may have potential for use in the regenerative treatment of the post-infarction heart.

Retraction Statement.

Retraction Statement.

SPARCL1 Influences Bovine Skeletal Muscle-Derived Satellite Cell Migration and Differentiation through an ITGB1-Mediated Signaling Pathway.

As an extracellular matrix protein, secreted protein acidic and rich in cysteine (SPARC)-like 1 (SPARCL1) is involved in various cell functions. It was previously implicated in bovine skeletal muscle-derived satellite cell (MDSC) differentiation; however, the underlying mechanism remains unknown. In this study, immunoprecipitation and mass spectrometry revealed that integrin β1 (ITGB1) combines with SPARCL1. Further, co-immunoprecipitation demonstrated that SPARCL1 interacts with ITGB1. Cell scratch assays explored the influence of SPARCL1 on MDSC migration through ITGB1. In addition, desmin staining for myotube fusion rate and MyoD protein expression results showed that SPARCL1 promotes MDSC early differentiation through ITGB1. Furthermore, Western blotting results demonstrated that SPARCL1 regulates the expression of p-FAK, p-paxillin, vinculin, Cdc42, and Arp2/3 through ITGB1. These findings indicate that SPARCL1 may influence bovine MDSC migration and differentiation through an ITGB1-mediated cell signaling pathway. Herein, we elucidated the mechanism through which SPARCL1 affects MDSC differentiation. Our results provide insight into the molecular mechanism of muscle development and may in the future facilitate skeletal muscle regeneration and treatment.

An in vitro study to assess the effect of hyaluronan-based gels on muscle-derived cells: Highlighting a new perspective in regenerative medicine.

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has been widely used for biomedical applications. Here, we have analyzed the effect of HA on the rescue of primary cells under stress as well as its potential to recover muscle atrophy and validated the developed model in vitro using primary muscle cells derived from rats. The potentials of different HAs were elucidated through comparative analyses using pharmaceutical grade a) high (HHA) and b) low molecular weight (LHA) hyaluronans, c) hybrid cooperative complexes (HCC) of HA in three experimental set-ups. The cells were characterized based on the expression of myogenin, a muscle-specific biomarker, and the proliferation was analyzed using Time-Lapse Video Microscopy (TLVM). Cell viability in response to H2O2 challenge was evaluated by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the expression of the superoxide dismutase enzyme (SOD-2) was assessed by western blotting. Additionally, in order to establish an in vitro model of atrophy, muscle cells were treated with tumor necrosis factor-alpha (TNF-α), along with hyaluronans. The expression of Atrogin, MuRF-1, nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB), and Forkhead-box-(Fox)-O-3 (FoxO3a) was evaluated by western blotting to elucidate the molecular mechanism of atrophy. The results showed that HCC and HHA increased cell proliferation by 1.15 and 2.3 folds in comparison to un-treated cells (control), respectively. Moreover, both pre- and post-treatments of HAs restored the cell viability, and the SOD-2 expression was found to be reduced by 1.5 fold in HA-treated cells as compared to the stressed condition. Specifically in atrophic stressed cells, HCC revealed a noteworthy beneficial effect on the myogenic biomarkers indicating that it could be used as a promising platform for tissue regeneration with specific attention to muscle cell protection against stressful agents.

Abstract A02: YAP1 opposes differentiation in mesenchymal tumors

Abstract Soft tissue sarcomas are aggressive mesenchymal malignancies diagnosed in ~15,000 Americans annually. Unlike in epithelial cancers, where novel targeted therapies have improved patient survival, the treatment approach for sarcomas has not changed significantly in 25 years. Our work revealed that deregulation of the Hippo pathway enhances sarcomagenesis in an aggressive muscle tumor called undifferentiated pleomorphic sarcoma (UPS). UPS is a commonly diagnosed and metastatic sarcoma subtype found most frequently in adult muscle tissues. We have observed that expression of Angiomotin (AMOT), a crucial mediator of Hippo-associated growth restriction, is commonly silenced in UPS. Among human tissues AMOT is most highly expressed in differentiated human muscle tissue. Ectopic re-expression of the p130 isoform of AMOT in muscle-derived UPS cells significantly inhibits proliferation in vitro. This finding is consistent with the central function of AMOT in cancer cells, which is to sequester the Hippo pathway effector YAP1 and facilitate its degradation, thereby inhibiting growth. YAP1 is a proproliferation transcriptional regulator whose deletion in an autochthonous genetically engineered mouse model (GEMM) of UPS significantly decreased tumorigenesis. Together these data suggest that AMOT loss promotes YAP1-mediated sarcomagenesis in muscle-derived UPS. We investigated the downstream effects of YAP1 signaling in UPS by gene expression analysis of control and Yap1-deficient murine tumors. Mechanistically, we found that Yap1 controls NF-κB transcriptional activity in UPS by inhibiting expression of Usp31, a negative regulator of NF-κB activity. Furthermore, using ChIP-seq of patient samples, we found that both YAP1 and NF-κB activity are substantially upregulated in human UPS. Consistent with these findings, UPS cell proliferation is highly sensitive to YAP1 and NF-κB inhibition. Importantly, loss of NF-κB completely prevents the formation in our murine UPS GEMM. In our effort to identify key transcriptional outputs of YAP1/NF-κB and determine how this signaling pathway controls sarcomagenesis, we found that it represses circadian clock gene expression (PER1, CRY2, ARNTL) and activity. Intriguingly, clock function is activated during muscle differentiation, whereas YAP1 and NF-κB inhibit differentiation is known to enhance proliferation of muscle precursor cells. These observations highlight a role for the YAP1/NF-κB axis in circadian regulation of muscle differentiation. Further studies revealed that YAP1/NF-κB-mediated suppression of the clock leads to downregulation of the unfolded protein response (UPR), a critical component of muscle cell differentiation. UPR transcriptional targets including ATF6 and CHOP are necessary for pruning of myoblasts and apoptosis of differentiation-incompetent muscle precursor cells. Together, our findings suggest a mechanism underlying YAP1-mediated sarcomagenesis via suppression of normal muscle differentiation pathways. Citation Format: Ying Liu, Gabrielle Ciotti, T.S. Karin Eisinger-Mathason. YAP1 opposes differentiation in mesenchymal tumors [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A02.

Stimulating Factors and Origins of Precursor Cells in Traumatic Heterotopic Ossification Around the Temporomandibular Joint in Mice.

The contributing factors and the origins of precursor cells in traumatic heterotopic ossification around the temporomandibular joint (THO-TMJ), which causes obvious restriction of mouth opening and maxillofacial malformation, remain unclear. In this study, our findings demonstrated that injured chondrocytes in the condylar cartilage, but not osteoblasts in the injured subchondral bone, played definite roles in the development of THO-TMJ in mice. Injured condylar chondrocytes without articular disc reserves might secrete growth factors, such as IGF1 and TGFβ2, that stimulate precursor cells, such as endothelial cells and muscle-derived cells, to differentiate into chondrocytes or osteoblasts and induce THO-TMJ. Preserved articular discs can alleviate the pressure on the injured cartilage and inhibit the development of THO-TMJ by inhibiting the secretion of these growth factors from injured chondrocytes. However, the exact molecular relationships among trauma, the injured condylar cartilage, growth factors such as TGFβ2, and pressure need to be explored in detail in the future.

Generation of myogenic progenitor cell-derived smooth muscle cells for sphincter regeneration

BackgroundDegeneration of smooth muscles in sphincters can cause debilitating diseases such as fecal incontinence. Skeletal muscle-derived cells have been effectively used in clinics for the regeneration of the skeletal muscle sphincters, such as the external anal or urinary sphincter. However, little is known about the in vitro smooth muscle differentiation potential and in vivo regenerative potential of skeletal muscle-derived cells.MethodsMyogenic progenitor cells (MPC) were isolated from the skeletal muscle and analyzed by flow cytometry and in vitro differentiation assays. The differentiation of MPC to smooth muscle cells (MPC-SMC) was evaluated by immunofluorescence, flow cytometry, patch-clamp, collagen contraction, and microarray gene expression analysis. In vivo engraftment of MPC-SMC was monitored by transplanting reporter protein-expressing cells into the pyloric sphincter of immunodeficient mice.ResultsMPC derived from human skeletal muscle expressed mesenchymal surface markers and exhibit skeletal myogenic differentiation potential in vitro. In contrast, they lack hematopoietic surface marker, as well as adipogenic, osteogenic, and chondrogenic differentiation potential in vitro. Cultivation of MPC in smooth muscle differentiation medium significantly increases the fraction of alpha smooth muscle actin (aSMA) and smoothelin-positive cells, while leaving the number of desmin-positive cells unchanged. Smooth muscle-differentiated MPC (MPC-SMC) exhibit increased expression of smooth muscle-related genes, significantly enhanced numbers of CD146- and CD49a-positive cells, and in vitro contractility and express functional Cav and Kv channels. MPC to MPC-SMC differentiation was also accompanied by a reduction in their skeletal muscle differentiation potential. Upon removal of the smooth muscle differentiation medium, a major fraction of MPC-SMC remained positive for aSMA, suggesting the definitive acquisition of their phenotype. Transplantation of murine MPC-SMC into the mouse pyloric sphincter revealed engraftment of MPC-SMC based on aSMA protein expression within the host smooth muscle tissue.ConclusionsOur work confirms the ability of MPC to give rise to smooth muscle cells (MPC-SMC) with a well-defined and stable phenotype. Moreover, the engraftment of in vitro-differentiated murine MPC-SMC into the pyloric sphincter in vivo underscores the potential of this cell population as a novel cell therapeutic treatment for smooth muscle regeneration of sphincters.

WISP1 promotes bovine MDSC differentiation via recruitment of ANXA1 for the regulation of the TGF-β signalling pathway.

Skeletal muscle is one of the most important tissues of the human body necessary for sporting activities. The differentiation of muscle-derived satellite cells (MDSCs) plays an important role in the development and regeneration of skeletal muscles. Similarly, the Wnt/β-catenin signalling pathway plays an important role in the process of muscle differentiation. Wnt1-inducible signalling pathway protein-1 (WISP1), a downstream protein of the Wnt/β-catenin signalling pathway and a member of the CCN family that also plays an important role in the differentiation process, and its expression increase during the differentiation of bovine MDSCs. However, its role in MDSC differentiation is poorly understood. Therefore, we investigated the mechanisms regulating this process via Western blot and immunofluorescence staining. Immunoprecipitation and mass spectrometry detected annexin A1 (ANXA1), a protein that interacts with WISP1. To determine whether WISP1 influences TGF-β signalling and differentiation independently of ANXA1, the latter was knocked down, while WISP1 was activated. WISP1 expression increased significantly during bovine MDSC differentiation. However, WISP1 did not affect the TGF-β signalling pathway protein marker when ANXA1 was inhibited. Taken together, WISP1 regulates the TGF-β signalling pathway through ANXA1 recruitment, thereby promoting bovine MDSC differentiation, suggesting the Wnt/β-catenin signalling pathway as another target to promote cell differentiation.

Association Analysis of Single-Cell RNA Sequencing and Proteomics Reveals a Vital Role of Ca2+ Signaling in the Determination of Skeletal Muscle Development Potential.

This study is aimed at exploring the mechanism underlying the homeostasis between myogenesis and adipogenesis in skeletal muscle using a special porcine model with a distinct phenotype on muscle growth rate and intramuscular fat deposition. Differentiation potential of muscle-derived Myo-lineage cells of lean-type pigs was significantly enhanced relative to obese-type pigs, while that of their Adi-lineage cells was similar. Single-cell RNA sequencing revealed that lean-type pigs reserved a higher proportion of Myo-lineage cells in skeletal muscle relative to obese-type pigs. Besides, Myo-lineage cells of the lean-type pig settled closer to the original stage of muscle-derived progenitor cells. Proteomics analysis found that differentially expressed proteins between two sources of Myo-lineage cells are mainly involved in muscle development, cell proliferation and differentiation, ion homeostasis, apoptosis, and the MAPK signaling pathway. The regulation of intracellular ion homeostasis, Ca2+ in particular, significantly differed between two sources of Myo-lineage cells. Ca2+ concentration in both cytoplasm and endoplasmic reticulum was lower in Myo-lineage cells of lean-type pigs relative to obese-type pigs. In conclusion, a higher proportion and stronger differentiation capacity of Myo-lineage cells are the main causes for the higher capability of myogenic differentiation and lower intramuscular fat deposition. Relative low concentration of cellular Ca2+ is advantageous for Myo-lineage cells to keep a potent differentiation potential.

Early time course of change in angiogenic proteins in human skeletal muscle and vascular cells with endurance training.

Angiogenic, mitochondrial, and related transcriptional proteins were assessed in human skeletal muscle and isolated vascular cells during the early phase of endurance training. Thigh muscle biopsies were obtained in healthy young subjects, after one acute bout (n=9) and after 3, 5, 7, and 14days (n=9) of cycle ergometer training. Whole muscle homogenates were analyzed for angiogenic, mitochondrial, and regulatory mRNA and protein levels. Angiogenic proteins were determined in muscle-derived endothelial cells and pericytes sorted by fluorescence-activated cell sorting. Acute exercise induced an increase in whole muscle mRNA of peroxisome proliferator-activated receptor gamma coactivator 1α (4.5-fold; P=.002) and vascular endothelial growth factor (VEGF) (2.4-fold; P=.001) at 2hours post. After 14days of training, there was an increase in CD31 protein (63%; P=.010) in whole muscle indicating capillary growth. There was also an increase in muscle VEGF receptor 2 (VEGFR2) (1.5-fold; P=.013), in OXPHOS proteins (complex I, II, IV, V; 1.4- to 1.9-fold; P<.05) after 14days of training and an increase in estrogen-related receptorα protein (1.5-fold; P=.039) at 14days compared to 5days of training. Both endothelial cells and pericytes expressed VEGF and other angiogenic factors at the protein level but with a distinctively lower expression of VEGFR2 and thrombospondin-1 (TSP-1) in pericytes. The findings illustrate that initiation of capillary and mitochondrial adaptations occurs within 14days of training and suggest that sustained changes in angiogenic proteins including VEGF and TSP-1 are moderate in whole muscle and vascular cells.

Isolation and Characterization of Extraocular Muscle-Derived Muscle Progenitor Cells from Normal and Graves' Orbitopathy Patients.

Mesenchymal stem cells (MSCs) are useful for various purposes, including tissue engineering, regeneration, and gene therapy. MSCs isolated from extraocular muscles (EOMs) can be easily expanded in vitro, and can undergo multilineage differentiations involving adipogenesis, chondrogenesis, osteogenesis, and even neuronal or myogenic differentiation. This study aimed to isolate, characterize, and compare extraocular muscle-derived muscle progenitor cells (EOM-MPCs) from normal subjects and patients with Graves' orbitopathy (GO). EOM was obtained during strabismus surgery. Flow cytometry was conducted to identify CD surface antigens such as CD34, CD45, CD44, CD59, CD73, and CD90. We quantitated various cytokines secreted from MSCs, including interleukin (IL)-1α, IL-2, IL-6, IL-8, IL-10, IL-12, IL17A, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, using a multi-analysis enzyme-linked immunosorbent assay array kit. We performed Oil Red O staining for adipogenesis, Alzarin Red staining for osteogenesis, Alcian blue staining for chondrogenesis, and polymerase chain reaction to measure messenger RNA expression during myogenesis. Our results show that EOM-MPCs from normal subjects and GO patients had similar levels of surface antigen expression and cytokine secretion. There was also no significant difference in the multilineage differentiation of adipocytes, chondrocytes, osteocytes, and myoblasts from EOM-MPCs taken from normal subjects and GO patients. However, hyaluronic acid synthetase 2 expression was higher after induction with tafluprost in EOM-MPCs from GO patients when compared with normal subjects. Together, these results show that EOM-MPCs derived from normal subjects are a good source for stem cell-based therapy for various disorders.

Role and therapeutic effects of skeletal muscle-derived non-myogenic cells in a rat myocardial infarction model

BackgroundTransplantation of skeletal myoblast sheets is a promising strategy for the treatment of heart failure, and its therapeutic effects have already been proven in both animal disease models and clinical trials. Myoblast sheets reportedly demonstrate their therapeutic effects by producing many paracrine factors. Although the quality of processed cells for transplantation can be evaluated by the positive ratio of CD56, a myoblast marker, it is unclear which cell populations from isolated cells produce paracrine factors that have an impact on therapeutic effects, and whether these therapeutic effects are closely correlated with CD56-positive cells isolated from the skeletal muscle is also unclear. Therefore, we hypothesized that CD56-negative cells as well as CD56-positive cells isolated from the skeletal muscle produce paracrine factors and have therapeutic effects in skeletal muscle-derived cell sheet therapy for heart failure.MethodsCell surface and intracellular markers of CD56-negative non-myogenic cells (NMCs) and CD56-positive myoblasts were evaluated. We also analyzed cytokine expression, tube formation ability, and stem cell mobilization in both cell populations. Finally, we assessed the therapeutic effects of the cell populations in a rat myocardial infarction model.ResultsAnalysis of cell surface and intracellular markers revealed that CD56-negative NMCs expressed fibroblast markers and a higher level of mesenchymal cell markers, such as CD49b and CD140a, than myoblasts. Both NMCs and myoblasts expressed various cytokines in vitro with different expression patterns. In addition, NMCs induced tube formation (control vs. myoblasts vs. NMCs: 100 ± 11.2 vs. 142 ± 8.3 vs. 198 ± 7.4%) and stem cell mobilization (control vs. myoblasts vs. NMCs: 100 ± 6.8 vs. 210 ± 22.9 vs. 351 ± 36.0%) to a higher degree in vitro than did myoblasts. The effect of NMCs and myoblasts on the improvement of cardiac function and suppression of myocardial fibrosis in rat myocardial infarction model was comparable.ConclusionThese results indicate that NMCs exhibit therapeutic effects in skeletal muscle-derived cell sheet therapy for heart failure. Thus, accurate parameters correlating with therapeutic effects need to be further explored.

TCP11L2 promotes bovine skeletal muscle-derived satellite cell migration and differentiation via FMNL2.

T-complex 11 like 2 (TCP11L2) is a protein containing a serine-rich region in its N-terminal region. However, the function of TCP11L2 is unclear. Here, we showed that TCP11L2 expression gradually increased during muscle-derived satellite cell(MDSC) differentiation in vitro, reaching a peak on Day 3, which is the migration and fusion stage of MDSCs. Using CRISPR/dCas9 gene-editing technology to elevate or repress the expression of TCP11L2, we also showed that TCP11L2 promoted MDSC differentiation. Moreover, wound-healing assays showed that TCP11L2 promoted the migration of MDSCs during differentiation. Additionally, immunofluorescence analyses showed that TCP11L2 was mainly distributed around the microfilament and microtubules. Furthermore, the expression of TCP11L2 affected the expression of actin-related protein 2/3 (ARP2/3) complex. Co-immunoprecipitation assays and immunofluorescence analysis showed that TCP11L2 interacted with formin-like 2 (FMNL2). This protein promoted migration of bovine MDSCs by affecting the expression of ARP2/3. Finally, the activities of TCP11L2 during MDSC differentiation and migration were blocked when FMNL2 was inhibited. Taken together, our data established that TCP11L2 interacted with FMNL2 to promote MDSC migration and differentiation.

Characterising hyperinsulinemia-induced insulin resistance in human skeletal muscle cells.

Hyperinsulinaemia potentially contributes to insulin resistance in metabolic tissues, such as skeletal muscle. The purpose of these experiments was to characterise glucose uptake, insulin signalling and relevant gene expression in primary human skeletal muscle-derived cells (HMDCs), in response to prolonged insulin exposure (PIE) as a model of hyperinsulinaemia-induced insulin resistance. Differentiated HMDCs from healthy human donors were cultured with or without insulin (100 nM) for 3 days followed by an acute insulin stimulation. HMDCs exposed to PIE were characterised by impaired insulin-stimulated glucose uptake, blunted IRS-1 phosphorylation (Tyr612) and Akt (Ser473) phosphorylation in response to an acute insulin stimulation. Glucose transporter 1 (GLUT1), but not GLUT4, mRNA and protein increased following PIE. The mRNA expression of metabolic (PDK4) and inflammatory markers (TNF-α) was reduced by PIE but did not change lipid (SREBP1 and CD36) or mitochondrial (UCP3) markers. These experiments provide further characterisation of the effects of PIE as a model of hyperinsulinaemia-induced insulin resistance in HMDCs.