Skeletal Myoblasts Research Articles

Morphological changes in intraepithelial and stromal telocytes in Cyprinus carpio in response to salinity stress

Telocytes establish connections and communicate with various types of cells and structures. Few experimental studies have been performed on telocytes. In this study, we investigated the effect of salinity stress on telocytes in relation to osmoregulatory, immune, and stem cells. After exposing the common carp to 0.2 (control), 6, 10, or 14 ppt salinity, we extracted and fixed gill samples in glutaraldehyde, processed and embedded the samples in resin, and prepared semi-thin and ultrathin sections. Two types of telocytes were identified: intraepithelial and stromal telocytes. Intraepithelial telocytes were found to form part of the cellular lining of the lymphatic space and shed secretory vesicles into this space. Stromal telocytes were observed to shed their secretory vesicles into the secondary circulatory vessels. Both intraepithelial and stromal telocytes were enlarged and exhibited increased secretory activities as salinity increased. They exerted their effects via direct contact and paracrine signaling. The following changes were observed in samples from fish exposed to high salinity levels: chloride cells underwent hypertrophy, and their mitochondria became cigar-shaped; pavement cells were enlarged, and their micro-ridges became thin and elongated; stromal telocytes established contact with stem cells and skeletal myoblasts; skeletal muscle cells underwent hypertrophy; and macrophages and rodlet cells increased in number. In conclusion, our findings indicate that intraepithelial and stromal telocytes respond to salinity stress by activating cellular signaling and that they play major roles in osmoregulation, immunity, and regeneration.

Effect of Uncaria tomentosa aqueous extract on the response to palmitate-induced lipotoxicity in cultured skeletal muscle cells

BackgroundType 2 diabetes mellitus (T2DM) is frequently associated with dyslipidemia, which corresponds to the increase in the triglycerides and fatty acid concentrations in tissues, such as the skeletal muscle. Also, T2DM molecular mechanism involves increasing in reactive oxygen species (ROS) production and oxidative stress. The use of herbal medicines such as Uncaria tomentosa (Ut) has been proposed as an auxiliary treatment for patients with T2DM. In this study, it was evaluated the effect of Ut aqueous extract on cell viability and ROS production, in skeletal myoblasts from C2C12 lineage exposed to the free fatty acid palmitate (PA).MethodsCells were incubated with PA in different concentrations ranging from 10 to 1000 μM, for 24 or 48 h, for cytotoxicity assay. Cell death, DNA fragmentation and ROS production assays were performed in cell cultures incubated with PA for 24 h, in the pre (preventive condition) or post treatment (therapeutic condition) with 250 μg/ml Ut aqueous extract, for 2 or 6 h. Cell death was evaluated by MTT method or flow cytometry. ROS generation was measured by fluorescence spectroscopy using the DCFDA probe.ResultsCell viability was reduced to approximately 44% after the incubation with PA for 24 h from the concentration of 500 µM. In the incubation of cells with 500 μM PA and Ut extract for 6 h, in both conditions (preventive or therapeutic), it was observed an increase of 27 and 70% in cell viability respectively, in comparison to the cultures incubated with only PA. Also, the incubation of cultures with 500 μM PA, for 24 h, increased 20-fold the ROS formation, while the treatment with Ut extract, for 6 h, both in the preventive or therapeutic conditions, promoted decrease of 21 and 55%, respectively.ConclusionThe Ut extract was efficient in promoting cell protection against PA lipotoxicity and ROS generation, potentially preventing oxidative stress in C2C12 skeletal muscle cells. Since T2DM molecular mechanism involves oxidative stress condition and it is often associated with dyslipidemia and fatty acid accumulation in muscle tissue, these results open perspectives for the use of Ut as an auxiliary strategy for T2DM management.

Retraction: Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model.

Retraction: Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model.

Abstract 18748: Prevention of Skeletal Muscle Atrophy and Fibrosis in Congestive Heart Failure by Skeletal Myoblast-Targeted Overexpression of Angiotensin II Type 2 Receptor

Introduction: We have previously shown that angiotensin II (Ang II) type 2 receptor (AT2R) positively regulates skeletal myoblast differentiation. Systemic angiotensin II (Ang II) increase or congestive heart failure suppress AT2R expression, likely contributing to the development of skeletal muscle atrophy and fibrosis associated with these conditions. Hypothesis: We hypothesized that AT2R overexpression in skeletal myoblasts could prevent skeletal muscle atrophy and fibrosis associated with congestive heart failure (CHF). Approach: Two mouse models, systemic Ang II infusion and left anterior descending coronary artery (LAD) ligation were used in this study. Myoblast differentiation, muscle atrophy and muscle fibrosis were assessed by analyzing gene expressions (e.g. myogenin and atrogin-1), myofiber cross sectional area, and the collagen deposition (picrosirius red staining). Primary myoblasts were isolated from mouse hindlimb muscles. Myoblast-specific AT2R-overexpressing transgenic mice were generated by crossing Pax7 CreER/+ mice (tamoxifen-inducible cre recombinase is expressed under Pax7 promoter) and CAG-stop flox -AT2R mice (AT2R is expressed under CMV early enhancer element, chicken β-actin gene promoter upon cre-mediated excision of floxed stop codon). Results: AT2R expression was robustly increased during myoblast differentiation both in vitro and in vivo . siRNA-mediated AT2R knockdown significantly suppressed myoblast differentiation in vitro and skeletal muscle regeneration in vivo . In both Ang II infusion and LAD ligation models, AT2R induction during myoblast differentiation was significantly suppressed, associated with reduced muscle regeneration and increased atrophy and fibrosis. Myoblast-specific AT2R overexpression in vivo restored muscle regenerative capacity, and prevented muscle atrophy and fibrosis. Conclusions: AT2R induction in skeletal myoblast is suppressed in CHF, possibly via increased Ang II. Decreased AT2R expression and its downstream signaling in myoblast lead to lowered muscle regenerative capacity and increased muscle atrophy and fibrosis. Restoration of myoblast AT2R expression could lead to a novel therapy to prevent muscle atrophy and fibrosis in CHF condition.

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

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

Palmitate-induced insulin resistance causes actin filament stiffness and GLUT4 mis-sorting without altered Akt signaling.

Skeletal muscle insulin resistance, a major contributor to Type 2 Diabetes, is linked to the consumption of saturated fats. This insulin resistance arises from failure of insulin-induced translocation of glucose transporter type 4 (GLUT4) to the plasma membrane to facilitate glucose uptake into muscle. The mechanisms of defective GLUT4 translocation are poorly understood, limiting development of insulin-sensitizing therapies targeting muscle glucose uptake. While many studies identify early insulin signaling defects and suggest they are responsible for insulin resistance, their cause-effect has been debated. Here, we find that the saturated fat palmitate (PA) causes insulin resistance of GLUT4 translocation in skeletal muscle myoblasts and myotubes without impairing signaling to Akt or AS160. Instead, PA altered two basal-state events: a) the intracellular localization of GLUT4 and its sorting towards a perinuclear storage compartment, and b) actin filament stiffness that prevents Rac1-dependent actin remodeling. These defects were triggered by distinct mechanisms, respectively protein palmitoylation and endoplasmic reticulum (ER) stress. Our findings highlight that saturated fats elicit muscle cell-autonomous dysregulation of the basal-state machinery required for GLUT4 translocation that 'primes' cells for insulin resistance.

Hypocalcemia in combination with hyperphosphatemia impairs muscle cell differentiation in vitro

PurposeHypoparathyroidism is a rare endocrine disorder characterized by low or absent secretion of parathyroid hormone (PTH), which leads to decreased calcium and increased phosphorus levels in the serum. The diagnosis of hypoparathyroidism is based on the identification of the aforementioned biochemical abnormalities, which may be accompanied by clinical manifestations. Symptoms of hypoparathyroidism, primarily attributed to hypocalcemia, include muscle cramps or spasms, facial, leg, and foot pain, seizures, and tingling in the lips or fingers. The treatment of hypoparathyroidism depends on the severity of symptoms and the underlying pathology. Over the long term, calcium supplements, active vitamin D analogs, and thiazide diuretics may be needed. In fact, in patient cohorts in which optimal disease control still remains elusive, replacement therapy with recombinant parathyroid hormone analogs may be contemplated. Despite the predominantly neuromuscular symptoms of hypoparathyroidism, further effects of parathyroid hormone deficiency at the muscle cell level remain poorly understood. Thus, the aim of our study was to evaluate the effects of hypocalcemia in combination with hyperphosphatemia on muscle cells differentiation in vitro.MethodsC2C12 cells, an in vitro model of muscle cells, were differentiated for 2 or 6 days in the presence of hypocalcemia (CaCl2 0.9 mmol/l) and moderate (PO4 1.4 mmol/l) or severe (PO4 2.9 mmol/l) hyperphosphatemia, or combinations of both conditions. Cell differentiation and expression of genes linked to muscle differentiation were evaluated.ResultsThe combination of hypocalcemia with hyperphosphatemia induced a significant reduction (50%) in differentiation marker levels, such as MyoD (protein 1 for myoblast determination) and myogenin on the 1st day of differentiation, and MHC (myosin heavy chains) after 6 days of differentiation compared to control. Furthermore, this condition induced a statistically significant reduction of insulin-like growth factor-1 (IGF-1) mRNA expression and inhibition of IGF signaling and decrease in ERK phosphorylation compared to control cells.ConclusionsOur results showed that a condition of hypocalcemia with hyperphosphatemia induced an alteration of muscle cell differentiation in vitro. In particular, we observed the reduction of myogenic differentiation markers, IGF-1 signaling pathway, and ERK phosphorylation in differentiated skeletal myoblasts. These data suggest that this altered extracellular condition might contribute to the mechanisms causing persistence of symptoms in patients affected by hypoparathyroidism.

Establishment and characterization of a skeletal myoblast cell line of grass carp (Ctenopharyngodon idellus).

Skeletal muscle myoblastic cell lines can provide a valuable new in vitro model for the exploration of the mechanisms that control skeletal muscle development and its associated molecular regulation. In this study, the skeletal muscle tissues of grass carp were digested with trypsin and collagenase I to obtain the primary myoblast cell culture. Myoblast cells were obtained by differential adherence purification and further analyzed by cryopreservation and resuscitation, chromosome analysis, immunohistochemistry, and immunofluorescence. A continuous grass carp myoblast cell line (named CIM) was established from grass carp (Ctenopharyngodon idellus) muscle and has been subcultured > 100 passages in a year and more. The CIM cells revived at 79.78-95.06% viability after 1-6 months of cryopreservation, and shared a population doubling time of 27.24 h. The number of modal chromosomes of CIM cells was 48, and the mitochondrial 12S rRNA sequence of the CIM cell line shared 99% identity with those of grass carp registered in GenBank. No microorganisms (bacteria, fungi, or mycoplasma) were detected during the whole study. The cell type of CIM cells was proven to be myoblast by immunohistochemistry of specific myogenic protein markers, including CD34, desmin, MyoD, and MyHC, as well as relative expression of key genes. And the myogenic rate and fusion index of this cell line after 10 days of induced differentiation were 8.96 ~ 9.42% and 3-24%, respectively. The telomerase activity and transfection efficiency of CIM cell line were 0.027 IU/mgprot and 23 ~ 24%, respectively. These results suggest that a myoblast cell line named CIM with normal biological function has been successfully established, which may provide a valuable tool for related in vitro studies.

Organometallic N‐acylhydrazones of 5‐nitrofuran and 5‐nitrothiophene based: Synthesis, electrochemical, antiparasitic evaluation, and computational study

In searching for new therapeutic agents for treating American trypanosomiasis and Human African trypanosomiasis, four nitroheterocyclic acylhydrazones of general formulae [R1‐CH=N‐NH‐C(O)‐(5‐C4H2X)] (where R1 = ferrocenyl or cyrhetrenyl, and X = O or S) have been synthesized and characterized by spectroscopic techniques. Comparative studies of their stability by 1H‐NMR and UV–Vis experiments were reported. Single‐crystal X‐ray diffraction confirmed the molecular structures of NF‐1 and NT‐2. Their X‐ray crystal structures reveal that both adopt an E‐configuration on the C=N moiety. Regarding the ‐NH‐C(O)‐ bond, the structure of NF‐1 confirmed a trans conformation, while NT‐2 exhibited a cis‐amide conformation. The cyclic voltammetry and electron spin resonance (ESR) experiments were conducted to study the electrochemical behavior of N‐acylhydrazones. The antiparasitic activities of compounds against Trypanosoma cruzi (epimastigotes) and Trypanosoma brucei (trypomastigotes) revealed that cyrhetrenyl complexes were more effective than their ferrocenyl analogs. The cyrhetrenyl derivative NT‐2 (EC50 = 2.25 μM) showed activity against T. brucei comparable to the standard drug nifurtimox (Nfx, EC50 = 3.56 μM). The ferrocenyl compound NT‐1 (>200 μM) was at least two times less cytotoxic than the Nfx (88.7 μM) against the L6 rat skeletal myoblast cell line and exhibited a selectivity like Nfx toward T. brucei. Density functional theory (DFT) calculations were utilized as an approximation to explain the impact of organometallic and heterocyclic rings on antiparasitic activities. This study supported the experimental results, confirming that the cyrhetrenyl fragment in N‐acylhydrazone derivatives plays a significant role in the antitrypanosomal activity, which can be attributed to an increase in positive charge on the metal.

B-175 Phytochemicals in K. pinnata and the Antioxidant Activity of Combined K. pinnata and Metformin Preparations on Normal and Diabetic Skeletal Muscle Cells

Abstract Background Diabetes Mellitus is associated with oxidative stress, which may play a central role in developing diabetic complications. With the rise in healthcare costs, more people globally are choosing to complement their pharmacological regimen with dietary supplements from natural products. In this study, the compound composition of K. pinnata and the effects of combined preparations of K. pinnata and metformin on antioxidant activity in human skeletal muscle myoblasts (HSMM) and human diabetic skeletal muscle myoblasts (DHSMM) were investigated. Methods K. pinnata leaves were crushed and extracted with hot water, then gravity filtered and freeze-dried. A 1 mL aliquot of the Kalanchoe pinnata extract solution was utilized to determine the chemical composition of the herbal extract. Liquid chromatography-mass spectrometry (LC-MS) was run in negative ion mode, with the K. pinnata extract analyzed in triplicate. A computer program further analyzed the LC-MS data to determine the chemical components in K. pinnata aqueous extract. A database algorithm within Compound Discoverer was used to identify the compounds in K. pinnata aqueous extract. For subsequent cell culture experiments, the K. pinnata leaf extract was weighed and combined with aqueous metformin solutions to prepare various combinatorial treatments. Human skeletal muscle myoblast cells and diabetic human skeletal muscle myoblast cells (Lonza Inc, Walkersville, MD) were cultured and incubated at 37 °C with 5% CO2. A CCK-8 cytotoxicity assay was performed to determine cellular viability, while trypan blue was used to assess the cellular proliferation of in-vitro cells. The effects of combinatorial preparations of K. pinnata and metformin on antioxidant indices were determined in HSMM, DHSMM, and HSMM H2O2 stress-induced cells. Results Liquid chromatography-mass spectrometry (LC-MS) showed that K. pinnata contains biologically active flavanols. The main compounds identified in locally grown K. pinnata were quercetin, kaempferol, apigenin, epigallocatechin gallate (EGCG), and avicularin. Antioxidant results indicated that a combinatorial preparation of K. pinnata with metformin might modulate antioxidant responses by increasing the enzymatic activity of superoxide dismutase and increasing levels of reduced glutathione. A combination of 50 μM and 150 μg/mL of metformin and K. pinnata resulted in a significant increase in reduced glutathione levels in non-diabetic and diabetic human skeletal muscle myoblasts and H2O2 stress-induced human skeletal muscle myoblasts. Additionally, a K. pinnata treatment (400 µg/mL) alone significantly increased catalase (CAT) activity for non-diabetic and diabetic human skeletal muscle myoblasts and an H2O2 stress-induced human skeletal muscle myoblast cell line while significantly lowering malondialdehyde (MDA) levels. The antioxidant activity of the treatment options was more effective at promoting cell viability after 24 h of treatment vs 72 h. Conclusion These treatment options show promise, especially after 24 h for treating oxidative stress-mediated pathophysiological complications associated with type II diabetes.

Bleomycin-treated myoblasts undergo p21-associated cellular senescence and have severely impaired differentiation.

As we age, the ability to regenerate and repair skeletal muscle damage declines, partially due to increasing dysfunction of muscle resident stem cells-satellite cells (SC). Recent evidence implicates cellular senescence, which is the irreversible arrest of proliferation, as a potentiator of SC impairment during aging. However, little is known about the role of senescence in SC, and there is a large discrepancy in senescence classification within skeletal muscle. The purpose of this study was to develop a model of senescence in skeletal muscle myoblasts and identify how common senescence-associated biomarkers respond. Low-passage C2C12 myoblasts were treated with bleomycin or vehicle and then evaluated for cytological and molecular senescence markers, proliferation status, cell cycle kinetics, and differentiation potential. Bleomycin treatment caused double-stranded DNA breaks, which upregulated p21 mRNA and protein, potentially through NF-κB and senescence-associated super enhancer (SASE) signaling (p < 0.01). Consequently, cell proliferation was abruptly halted due to G2/M-phase arrest (p < 0.01). Bleomycin-treated myoblasts displayed greater senescence-associated β-galactosidase staining (p < 0.01), which increased over several days. These myoblasts remained senescent following 6days of differentiation and had significant impairments in myotube formation (p < 0.01). Furthermore, our results show that senescence can be maintained despite the lack of p16 gene expression in C2C12 myoblasts. In conclusion, bleomycin treatment provides a valid model of damage-induced senescence that was associated with elevated p21, reduced myoblast proliferation, and aberrant cell cycle kinetics, while confirming that a multi-marker approach is needed for the accurate classification of senescence within skeletal muscle.

Highly selective, sensitive and biocompatible rhodamine-based isomers for Al3+ detection: A comparative study

Selective detection of a metal ion with high selectivity is of great importance to understand its existence and its role in many chemical and biological processes. We report here the synthesis, characterization and Al3+ sensing properties of two rhodamine-based isomers, (E)-2-((2-(allyloxy)benzylidene)amino)ethyl)-3′,6′-bis(ethylamine)-2′,7′-dimethylspiro[isoindoline-1,9′-xanthen]-3-one (L-2-oxy) and (E)-2-((4-(allyloxy)benzylidene)amino)ethyl)-3′,6′-bis(ethylamine)-2′,7′-dimethylspiro[isoindoline-1,9′-xanthen]-3-one (L-4-oxy). L-2-oxyand L-4-oxy show pink coloration, significant enhancement in absorbance at 530 nm and fluorescence intensity at 553 nm in the presence of Al3+ among several cations. Quantum yield and lifetime of the probes increase in the presence of Al3+. LOD values have been determined as low as ∼1.0 nM for both the isomers. DFT study suggests that the cation induces opening of spirolactam ring resulting in the changes of the rhodamine dyes. Additional reason could be Chelation Enhanced Fluorescence (CHEF) effect due to the subsequent chelation of the metal ion. Between two isomers, L-2-oxy displays better sensing ability towards Al3+ in terms of fluorescence enhancement, limit of detection, lifetime enhancement. Both the probes have been utilized in cell imaging studies using rat skeletal myoblast cell line (L6 cell line).

Establishment and Characterization of Continuous Satellite Muscle Cells from Olive Flounder (Paralichthys olivaceus): Isolation, Culture Conditions, and Myogenic Protein Expression.

Olive flounder (Paralichthys olivaceus) muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached 80% confluence within 8 days, compared to 15 days for the explant method. Optimal OFMC growth was observed in 20% fetal bovine serum at 28 °C with 0.8 mM CaCl2 and the basic fibroblast growth factor (BFGF) to enhance cell growth. OFMCs have become permanent cell lines through the spontaneous immortalization crisis at the 20th passage. Olive flounder skeletal muscle myoblasts were induced into a mitogen-poor medium containing 2% horse serum for differentiation; they fused to form multinucleate myotubes. The results indicated complete differentiation of myoblasts into myotubes; we also detected the expression of the myogenic regulatory factors myoD, myogenin, and desmin. Upregulation (Myogenin, desmin) and downregulation (MyoD) of muscle regulation factors confirmed the differentiation in OFMCs.

Myogenetic Oligodeoxynucleotide Induces Myocardial Differentiation of Murine Pluripotent Stem Cells.

An 18-base myogenetic oligodeoxynucleotide (myoDN), iSN04, acts as an anti-nucleolin aptamer and induces myogenic differentiation of skeletal muscle myoblasts. This study investigated the effect of iSN04 on murine embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In the undifferentiated state, iSN04 inhibited the proliferation of ESCs and iPSCs but did not affect the expression of pluripotent markers. In the differentiating condition, iSN04 treatment of ESCs/iPSCs from day 5 onward dramatically induced differentiation into Nkx2-5+ beating cardiomyocytes with upregulation of Gata4, Isl1, and Nkx2-5, whereas iSN04 treatment from earlier stages completely inhibited cardiomyogenesis. RNA sequencing revealed that iSN04 treatment from day 5 onward contributes to the generation of cardiac progenitors by modulating the Wnt signaling pathway. Immunostaining showed that iSN04 suppressed the cytoplasmic translocation of nucleolin and restricted it to the nucleoli. These results demonstrate that nucleolin inhibition by iSN04 facilitates the terminal differentiation of cardiac mesoderm into cardiomyocytes but interferes with the differentiation of early mesoderm into the cardiac lineage. This is the first report on the generation of cardiomyocytes from pluripotent stem cells using a DNA aptamer. Since iSN04 did not induce hypertrophic responses in primary-cultured cardiomyocytes, iSN04 would be useful and safe for the regenerative therapy of heart failure using stem cell-derived cardiomyocytes.

Regulation of HDAC11 gene expression in early myogenic differentiation.

Histone acetylation and deacetylation affect the patterns of gene expression in cellular differentiation, playing pivotal roles in tissue development and maintenance. For example, the intrinsic histone acetyltransferase activity of transcriptional coactivator p300 is especially required for the expression of myogenic regulatory factors including Myf5 and MyoD, and consequently for skeletal myogenesis. On the other hand, histone deacetylases (HDACs) remove the acetyl group from histones, which is critical for gene repression in stem cell fate transition. Through integrative omic analyses, we found that while some HDACs were differentially expressed at the early stage of skeletal myoblast differentiation, Hdac11 gene expression was significantly enhanced by nuclear receptor signaling. In addition, p300 and MyoD control Hdac11 expression in milieu of normal and signal-enhanced myoblast differentiation. Thus, HDAC11 may be essential to differential gene expression at the onset of myoblast differentiation.

Magnetic Nanofibrous Hydrogels for Dynamic Control of Stem Cell Differentiation.

The extracellular matrix in tissue consists of complex heterogeneous soft materials with hierarchical structure and dynamic mechanical properties dictating cell and tissue level function. In many natural matrices, there are nanofibrous structures that serve to guide cell activity and dictate the form and function of tissue. Synthetic hydrogels with integrated nanofibers can mimic the structural properties of native tissue; however, model systems with dynamic mechanical properties remain elusive. Here we demonstrate modular nanofibrous hydrogels that can be reversibly stiffened in response to applied magnetic fields. Iron oxide nanoparticles were incorporated into gelatin nanofibers through electrospinning, followed by chemical stabilization and fragmentation. These magnetoactive nanofibers can be mixed with virtually any hydrogel material and reversibly stiffen the matrix at a low fiber content (≤3%). In contrast to previous work, where a large quantity of magnetic material disallowed cell encapsulation, the low nanofiber content allows matrix stiffening with cells in 3D. Using adipose derived stem cells, we show how nanofibrous matrices are beneficial for both osteogenesis and adipogenesis, where stiffening the hydrogel with applied magnetic fields enhances osteogenesis while discouraging adipogenesis. Skeletal myoblast progenitors were used as a model of tissue morphogenesis with matrix stiffening augmenting myogenesis and multinucleated myotube formation. The ability to reversibly stiffen fibrous hydrogels through magnetic stimulation provides a useful tool for studying nanotopography and dynamic mechanics in cell culture, with a scope for stimuli responsive materials for tissue engineering.

Rab44 negatively regulates myoblast differentiation by controlling fusogenic protein transport and mTORC1 signaling.

Skeletal muscle is composed of multinucleated myotubes formed by the fusion of mononucleated myoblasts. Skeletal muscle differentiation, termed as myogenesis, have been investigated using the mouse skeletal myoblast cell line C2C12. It has been reported that several "small" Rab proteins, major membrane-trafficking regulators, possibly regulate membrane protein transport in C2C12 cells; however, the role of Rab proteins in myogenesis remains unexplored. Rab44, a member of "large" Rab GTPases, has recently been identified as a negative regulator of osteoclast differentiation. In this study, using C2C12 cells, we found that Rab44 expression was upregulated during myoblast differentiation into myotubes. Knockdown of Rab44 enhanced myoblast differentiation and myotube formation. Consistent with these results, Rab44 knockdown in myoblasts increased expression levels of several myogenic marker genes. Rab44 knockdown increased the surface accumulation of myomaker and myomixer, two fusogenic proteins required for multinucleation, implying enhanced cell fusion. Conversely, Rab44 overexpression inhibited myoblast differentiation and tube formation, accompanied by decreased expression of some myogenic markers. Furthermore, Rab44 was found to be predominantly localized in lysosomes, and Rab44 overexpression altered the number and size of lysosomes. Considering the underlying molecular mechanism, Rab44 overexpression impaired the signaling pathway of the mechanistic target of rapamycin complex1 (mTORC1) in C2C12 cells. Namely, phosphorylation levels of mTORC1 and downstream mTORC1 substrates, such as S6 and P70-S6K, were notably lower in Rab44 overexpressing cells than those in control cells. These results indicate that Rab44 negatively regulates myoblast differentiation into myotubes by controlling fusogenic protein transport and mTORC1 signaling.

3D Co-Printing and Substrate Geometry Influence the Differentiation of C2C12 Skeletal Myoblasts.

Cells are influenced by several biomechanical aspects of their microenvironment, such as substrate geometry. According to the literature, substrate geometry influences the behavior of muscle cells; in particular, the curvature feature improves cell proliferation. However, the effect of substrate geometry on the myogenic differentiation process is not clear and needs to be further investigated. Here, we show that the 3D co-printing technique allows the realization of substrates. To test the influence of the co-printing technique on cellular behavior, we realized linear polycaprolactone substrates with channels in which a fibrinogen-based hydrogel loaded with C2C12 cells was deposited. Cell viability and differentiation were investigated up to 21 days in culture. The results suggest that this technology significantly improves the differentiation at 14 days. Therefore, we investigate the substrate geometry influence by comparing three different co-printed geometries-linear, circular, and hybrid structures (linear and circular features combined). Based on our results, all structures exhibit optimal cell viability (>94%), but the linear pattern allows to increase the in vitro cell differentiation, in particular after 14 days of culture. This study proposes an endorsed approach for creating artificial muscles for future skeletal muscle tissue engineering applications.

Efficacy of Autologous Skeletal Myoblast Cell Sheet Transplantation for Liver Regeneration in Liver Failure.

No effective therapies have yet been established for liver regeneration in liver failure. Autologous skeletal myoblast cell sheet transplantation has been proven to improve cardiac function in patients with heart failure, and one of the mechanisms has been reported to be a paracrine effect by various growth factors associated with liver regeneration. Therefore, the present study focused on the effect of myoblast cells on liver regeneration in vitro and in vivo. We assessed the effect of myoblast cells on the cells comprising the liver in vitro in association with liver regeneration. In addition, we examined in vivo effect of skeletal myoblast cell sheet transplantation in C57/BL/6 mouse models of liver failure, such as liver fibrosis induced by thioacetamide and hepatectomy. In vitro, the myoblast cells exhibited a capacity to promote the proliferation of hepatic epithelial cells and the angiogenesis of liver sinusoidal endothelial cells, and suppress the activation of hepatic stellate cells. In vivo, sheet transplantation significantly suppressed liver fibrosis in the induced liver fibrosis model and accelerated liver regeneration in the hepatectomy model. Autologous skeletal myoblast cell sheet transplantation significantly improved the liver failure in the in vitro and in vivo models. Sheet transplantation is expected to have the potential to be a clinically therapeutic option for liver regeneration in liver failure.

Tea polyphenols coated sodium alginate-gelatin 3D edible scaffold for cultured meat

This study aimed to use edible scaffolds as a platform for animal stem cell expansion, thus constructing block-shaped cell culture meat. The tea polyphenols (TP)-coated 3D scaffolds were constructed of sodium alginate (SA) and gelatin (Gel) with good biocompatibility and mechanical support. Initially, the physicochemical properties and mechanical properties of SA-Gel-TP scaffolds were measured, and the biocompatibility of the scaffolds was evaluated by C2C12 cells. SEM results showed that the scaffold had a porous laminar structure with TP particles attached to the surface, while FT-IR results also demonstrated the encapsulation of TP coating on the scaffold. In addition, the porosity of all scaffolds was higher than 40% and the degradation rate during the incubation cycle was less than 40% and the S2-G1-TP0.1–3 h scaffold has excellent cell adhesion and extension. Subsequently, we inoculated rabbit skeletal muscle myoblasts (RbSkMC) on the scaffold and induced differentiation. The results showed good adhesion and extension behavior of RbSkMC on S2-G1-TP0.1–3 h scaffolds with high expression of myogenic differentiation proteins and genes, and SEM results confirmed the formation of myotubes. Additionally, the adhesion rate of cells on scaffolds with TP coating was 1.5 times higher than that on scaffolds without coating, which significantly improved the cell proliferation rate and the morphology of cells with extension on the scaffolds. Furthermore, rabbit-derived cultured meat had similar appearance and textural characteristics to fresh meat. These conclusions indicate the high potential of the scaffolds with TP coating as a platform for the production of cultured meat products.