-
Methods in Molecular Biology (Clifton,... 2021The epicardium is a multipotent cell layer that is vital to myocardial development and regeneration. Epicardial cells contribute to cardiac fibroblast and smooth muscle...
The epicardium is a multipotent cell layer that is vital to myocardial development and regeneration. Epicardial cells contribute to cardiac fibroblast and smooth muscle populations of the heart and secrete paracrine factors that promote cardiomyocyte proliferation and angiogenesis. Despite a central role in cardiac biology, the mechanisms by which epicardial cells influence cardiac growth are largely unknown, and robust models of the epicardium are needed. Here, we review our protocol for differentiating induced pluripotent stem cells (iPSCs) into epicardial-like cells through temporal modulation of canonical Wnt signaling. iPSC-derived epicardial cells (iECs) resemble in vivo epicardial cells morphologically and display markers characteristic of the developing epicardium. We also review our protocol for differentiating iECs into fibroblasts and smooth muscle cells through treatment with bFGF and TGF-β1, respectively. iECs provide a platform for studying fundamental epicardial biology and can inform strategies for therapeutic heart regeneration.
Topics: Cell Differentiation; Fibroblasts; Humans; Induced Pluripotent Stem Cells; Myocytes, Cardiac; Myocytes, Smooth Muscle; Organogenesis; Pericardium; Transforming Growth Factor beta1
PubMed: 32857371
DOI: 10.1007/978-1-0716-0668-1_11 -
Molecular Biology Reports Jul 2023Labeo rohita represents the most dominant fish species in Indian aquaculture and the fish cell lines have been used as an excellent in vitro platform for performing...
BACKGROUND
Labeo rohita represents the most dominant fish species in Indian aquaculture and the fish cell lines have been used as an excellent in vitro platform for performing various biological research.
METHODS AND RESULTS
The LRM cell culture developed from the muscle tissue of L. rohita was used to study the in vitro applications. The developed muscle cells were maintained in a Leibovitz's-15 (L-15) supplemented with 10% FBS (Fetal Bovine Serum) and 10 ng/ml bFGF at 28 C temperature. The LRM cells showed fibroblastic-like morphology and was authenticated by sequencing mitochondrial gene 16S rRNA. The expression of myogenic regulatory factors (MRFs) was studied in different stages of LRM cells; however, the expression patterns varied at different passages. The MEF2A, Mrf-4, and Myogenin expressions were higher in passage 25, while the expression of MyoD was maximum in passage 15, and the expression of Myf-5 was highest in passage 1. The transfection efficiency of LRM cells revealed 14 % of the GFP expression with a pmaxGFP vector DNA. The LRM cells were susceptible to the extracellular products prepared from Aeromonas hydrophilla and Edwardsiella tarda. The acute cytotoxicity of six heavy metals (Hg, Cd, Zn, Cu, Pb, Ni) was assessed in LRM cells by a dose-dependent manner in comparison to IC values obtained from MTT and NR assays. A revival rate of 70-75% was achieved when the LRM cells were cryopreserved at - 196 °C using liquid nitrogen.
CONCLUSION
The developed muscle cells serve as an functional in vitro tool for toxicological and biotechnological studies.
Topics: Animals; RNA, Ribosomal, 16S; Cell Line; Cyprinidae; Muscles; Muscle Cells
PubMed: 37179501
DOI: 10.1007/s11033-023-08427-z -
Neurochemistry International Dec 2014Amyotrophic Lateral Sclerosis (ALS) is the most common form of adult-onset motor neuron disease. It is now considered a multi-factorial and multi-systemic disorder in... (Review)
Review
Amyotrophic Lateral Sclerosis (ALS) is the most common form of adult-onset motor neuron disease. It is now considered a multi-factorial and multi-systemic disorder in which alterations of the crosstalk between neuronal and non-neuronal cell types might influence the course of the disease. In this review, we will provide evidence that dysfunctions of affected muscle cells are not only a marginal consequence of denervation associated to motor neurons loss, but a direct consequence of cell muscle toxicity of mutant SOD1. In muscle, the misfolded state of mutant SOD1 protein, unlike in motor neurons, does not appear to have direct effects on protein aggregation and mitochondrial functionality. Muscle cells are, in fact, more capable than motor neurons to handle misfolded proteins, suggesting that mutant SOD1 toxicity in muscle is not mediated by classical mechanisms of intracellular misfolded proteins accumulation. Several recent works indicate that a higher activation of molecular chaperones and degradative systems is present in muscle cells, which for this reason are possibly able to better manage misfolded mutant SOD1. However, several alterations in gene expression and regenerative potential of skeletal muscles have also been reported as a consequence of the expression of mutant SOD1 in muscle. Whether these changes in muscle cells are causative of ALS or a consequence of motor neuron alterations is not yet clear, but their elucidation is very important, since the understanding of the mechanisms involved in mutant SOD1 toxicity in muscle may facilitate the design of treatments directed toward this specific tissue to treat ALS or at least to delay disease progression.
Topics: Amyotrophic Lateral Sclerosis; Animals; Autophagy; Humans; Motor Neurons; Muscle Cells; Proteostasis Deficiencies
PubMed: 25451799
DOI: 10.1016/j.neuint.2014.10.007 -
International Journal of Molecular... Oct 2020Many studies evaluated the short-term in vitro toxicity of nanoparticles (NPs); however, long-term effects are still not adequately understood. Here, we investigated the...
Many studies evaluated the short-term in vitro toxicity of nanoparticles (NPs); however, long-term effects are still not adequately understood. Here, we investigated the potential toxic effects of biomedical (polyacrylic acid and polyethylenimine coated magnetic NPs) and two industrial (SiO and TiO) NPs following different short-term and long-term exposure protocols on two physiologically different in vitro models that are able to differentiate: L6 rat skeletal muscle cell line and biomimetic normal porcine urothelial (NPU) cells. We show that L6 cells are more sensitive to NP exposure then NPU cells. Transmission electron microscopy revealed an uptake of NPs into L6 cells but not NPU cells. In L6 cells, we obtained a dose-dependent reduction in cell viability and increased reactive oxygen species (ROS) formation after 24 h. Following continuous exposure, more stable TiO and polyacrylic acid (PAA) NPs increased levels of nuclear factor Nrf2 mRNA, suggesting an oxidative damage-associated response. Furthermore, internalized magnetic PAA and TiO NPs hindered the differentiation of L6 cells. We propose the use of L6 skeletal muscle cells and NPU cells as a novel approach for assessment of the potential long-term toxicity of relevant NPs that are found in the blood and/or can be secreted into the urine.
Topics: Animals; Cell Line; Cell Survival; Cells, Cultured; Epithelial Cells; Muscle Cells; NF-E2-Related Factor 2; Nanoparticles; Polyesters; Rats; Reactive Oxygen Species; Swine; Titanium; Toxicity Tests; Urothelium
PubMed: 33066271
DOI: 10.3390/ijms21207545 -
Communications Biology Nov 2023Optical stimulation and control of muscle cell contraction opens up a number of interesting applications in hybrid robotic and medicine. Here we show that recently...
Optical stimulation and control of muscle cell contraction opens up a number of interesting applications in hybrid robotic and medicine. Here we show that recently designed molecular phototransducer can be used to stimulate C2C12 skeletal muscle cells, properly grown to exhibit collective behaviour. C2C12 is a skeletal muscle cell line that does not require animal sacrifice Furthermore, it is an ideal cell model for evaluating the phototransducer pacing ability due to its negligible spontaneous activity. We study the stimulation process and analyse the distribution of responses in multinuclear cells, in particular looking at the consistency between stimulus and contraction. Contractions are detected by using an imaging software for object recognition. We find a deterministic response to light stimuli, yet with a certain distribution of erratic behaviour that is quantified and correlated to light intensity or stimulation frequency. Finally, we compare our optical stimulation with electrical stimulation showing advantages of the optical approach, like the reduced cell stress.
Topics: Animals; Muscle Fibers, Skeletal; Muscle Contraction; Electric Stimulation; Light; Robotics
PubMed: 37952040
DOI: 10.1038/s42003-023-05538-y -
Journal of Receptor and Signal... Jun 2020This review describes the very specific role of Sigma1 receptor in different types of muscle cells. Sigma1 receptor is a transmembrane protein residing in such... (Review)
Review
This review describes the very specific role of Sigma1 receptor in different types of muscle cells. Sigma1 receptor is a transmembrane protein residing in such structures like MAM. It has chaperoning activity supporting function of many proteins, particularly ion channels, including Ca channels. This latter function is of particular meaning for muscle cells, due to their calcium-based/regulated metabolism. Here we discuss new reports pointing to participation of Sigma1 receptor in muscle specific processes like contraction, EC-coupling, calcium currents and in diseases like left ventricular hypertrophy, transverse aortic stenosis and hypertension-induced heart dysfunction.
Topics: Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cardiotonic Agents; Humans; Muscle Cells; Muscle Proteins; Receptors, sigma; Sigma-1 Receptor
PubMed: 32054378
DOI: 10.1080/10799893.2020.1727924 -
Journal of Endovascular Therapy : An... Aug 2021Abdominal aortic aneurysms (AAAs) are associated with overall high mortality in case of rupture. Since the pathophysiology is unclear, no adequate pharmacological...
INTRODUCTION
Abdominal aortic aneurysms (AAAs) are associated with overall high mortality in case of rupture. Since the pathophysiology is unclear, no adequate pharmacological therapy exists. Smooth muscle cells (SMCs) dysfunction and extracellular matrix (ECM) degradation have been proposed as underlying causes. We investigated SMC spatial organization and SMC-ECM interactions in our novel 3-dimensional (3D) vascular model. We validated our model for future use by comparing it to existing 2-dimensional (2D) cell culture. Our model can be used for translational studies of SMC and their role in AAA pathophysiology.
MATERIALS AND METHODS
SMC isolated from the medial layer of were the aortic wall of controls and AAA patients seeded on electrospun poly-lactide--glycolide scaffolds and cultured for 5 weeks, after which endothelial cells (EC) are added. Cell morphology, orientation, mechanical properties and ECM production were quantified for validation and comparison between controls and patients.
RESULTS
We show that cultured SMC proliferate into multiple layers after 5 weeks in culture and produce ECM proteins, mimicking their behavior in the medial aortic layer. EC attach to multilayered SMC, mimicking layer interactions. The novel SMC model exhibits viscoelastic properties comparable to biological vessels; cytoskeletal organization increases during the 5 weeks in culture; increased cytoskeletal alignment and decreased ECM production indicate different organization of AAA patients' cells compared with control.
CONCLUSION
We present a valuable preclinical model of AAA constructed with patient specific cells with applications in both translational research and therapeutic developments. We observed SMC spatial reorganization in a time course of 5 weeks in our robust, patient-specific model of SMC-EC organization and ECM production.
Topics: Aortic Aneurysm, Abdominal; Endothelial Cells; Extracellular Matrix; Humans; Myocytes, Smooth Muscle; Treatment Outcome
PubMed: 33902345
DOI: 10.1177/15266028211009272 -
Cells Mar 2022(1) Abdominal aortic aneurysm (AAA) is a silent, progressive disease with significant mortality from rupture. Whilst screening programmes are now able to detect this...
(1) Abdominal aortic aneurysm (AAA) is a silent, progressive disease with significant mortality from rupture. Whilst screening programmes are now able to detect this pathology early in its development, no therapeutic intervention has yet been identified to halt or retard aortic expansion. The inability to obtain aortic tissue from humans at early stages has created a necessity for laboratory models, yet it is essential to create a timeline of events from EARLY to END stage AAA progression. (2) We used a previously validated ex vivo porcine bioreactor model pre-treated with protease enzyme to create "aneurysm" tissue. Mechanical properties, histological changes in the intact vessel wall, and phenotype/function of vascular smooth muscle cells (SMC) cultured from the same vessels were investigated. (3) The principal finding was significant hyperproliferation of SMC from EARLY stage vessels, but without obvious histological or SMC aberrancies. END stage tissue exhibited histological loss of α-smooth muscle actin and elastin; mechanical impairment; and, in SMC, multiple indications of senescence. (4) Aortic SMC may offer a therapeutic target for intervention, although detailed studies incorporating intervening time points between EARLY and END stage are required. Such investigations may reveal mechanisms of SMC dysfunction in AAA development and hence a therapeutic window during which SMC differentiation could be preserved or reinstated.
Topics: Animals; Aortic Aneurysm, Abdominal; Cell Differentiation; Myocytes, Smooth Muscle; Phenotype; Swine
PubMed: 35326494
DOI: 10.3390/cells11061043 -
International Journal of Molecular... Sep 2021Muscular dystrophies are a heterogeneous group of inherited diseases characterized by the progressive degeneration and weakness of skeletal muscles, leading to... (Review)
Review
Muscular dystrophies are a heterogeneous group of inherited diseases characterized by the progressive degeneration and weakness of skeletal muscles, leading to disability and, often, premature death. To date, no effective therapies are available to halt or reverse the pathogenic process, and meaningful treatments are urgently needed. From this perspective, it is particularly important to establish reliable in vitro models of human muscle that allow the recapitulation of disease features as well as the screening of genetic and pharmacological therapies. We herein review and discuss advances in the development of in vitro muscle models obtained from human induced pluripotent stem cells, which appear to be capable of reproducing the lack of myofiber proteins as well as other specific pathological hallmarks, such as inflammation, fibrosis, and reduced muscle regenerative potential. In addition, these platforms have been used to assess genetic correction strategies such as gene silencing, gene transfer and genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as well as to evaluate novel small molecules aimed at ameliorating muscle degeneration. Furthermore, we discuss the challenges related to in vitro drug testing and provide a critical view of potential therapeutic developments to foster the future clinical translation of preclinical muscular dystrophy studies.
Topics: Animals; Cell Differentiation; Drug Discovery; Dystrophin; Genetic Therapy; Humans; Induced Pluripotent Stem Cells; Muscle Cells; Muscular Dystrophies; Muscular Dystrophy, Animal
PubMed: 34502539
DOI: 10.3390/ijms22179630 -
Toxins May 2018Chronic kidney disease (CKD) is associated with profound vascular remodeling, which accelerates the progression of cardiovascular disease. This remodeling is... (Review)
Review
Chronic kidney disease (CKD) is associated with profound vascular remodeling, which accelerates the progression of cardiovascular disease. This remodeling is characterized by intimal hyperplasia, accelerated atherosclerosis, excessive vascular calcification, and vascular stiffness. Vascular smooth muscle cell (VSMC) dysfunction has a key role in the remodeling process. Under uremic conditions, VSMCs can switch from a contractile phenotype to a synthetic phenotype, and undergo abnormal proliferation, migration, senescence, apoptosis, and calcification. A growing body of data from experiments in vitro and animal models suggests that uremic toxins (such as inorganic phosphate, indoxyl sulfate and advanced-glycation end products) may directly impact the VSMCs' physiological functions. Chronic, low-grade inflammation and oxidative stress-hallmarks of CKD-are also strong inducers of VSMC dysfunction. Here, we review current knowledge about the impact of uremic toxins on VSMC function in CKD, and the consequences for pathological vascular remodeling.
Topics: Animals; Humans; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Toxins, Biological; Uremia
PubMed: 29844272
DOI: 10.3390/toxins10060218