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myoblast electroporation

Andrea Cerquone Perpetuini, Andrea David Re Cecconi, Michela Chiappa, Giulia Benedetta Martinelli, Claudia Fuoco, Giovanni Desiderio, Luisa Castagnoli, Cesare Gargioli, Rosanna Piccirillo, Gianni Cesareni
BACKGROUND: Skeletal muscle is characterized by an efficient regeneration potential that is often impaired during myopathies. Understanding the molecular players involved in muscle homeostasis and regeneration could help to find new therapies against muscle degenerative disorders. Previous studies revealed that the Ser/Thr kinase p21 protein-activated kinase 1 (Pak1) was specifically down-regulated in the atrophying gastrocnemius of Yoshida hepatoma-bearing rats. In this study, we evaluated the role of group I Paks during cancer-related atrophy and muscle regeneration...
May 21, 2018: Journal of Cachexia, Sarcopenia and Muscle
Takayuki Hoshino, Moto Yoshioka, Akira Wagatsuma, Hiroki Miyazako, Kunihiko Mabuchi
Electroporation, a physical transfection method to introduce genomic molecules in selective living cells, could be implemented by microelectrode devices. A local electric field generated by a finer electrode can induces cytomembrane poration in the electrode vicinity. To employ fine, high-speed scanning electrodes, we developed a fine virtual cathode pattern, which was generated on a cell adhesive surface of 100-nm-thick SiN membrane by inverted-electron beam lithography. The SiN membrane works as both a vacuum barrier and the display screen of the virtual cathode...
March 2018: IEEE Transactions on Nanobioscience
Kamil R Wierzbinski, Tomasz Szymanski, Natalia Rozwadowska, Jakub D Rybka, Agnieszka Zimna, Tomasz Zalewski, Karolina Nowicka-Bauer, Agnieszka Malcher, Magdalena Nowaczyk, Michal Krupinski, Michal Fiedorowicz, Piotr Bogorodzki, Pawel Grieb, Michal Giersig, Maciej K Kurpisz
Myocardial infarction (MI) is one of the most frequent causes of death in industrialized countries. Stem cells therapy seems to be very promising for regenerative medicine. Skeletal myoblasts transplantation into postinfarction scar has been shown to be effective in the failing heart but shows limitations such, e.g. cell retention and survival. We synthesized and investigated superparamagnetic iron oxide nanoparticles (SPIONs) as an agent for direct cell labeling, which can be used for stem cells imaging. High quality, monodisperse and biocompatible DMSA-coated SPIONs were obtained with thermal decomposition and subsequent ligand exchange reaction...
February 27, 2018: Scientific Reports
Agnieszka Zimna, Bartosz Wiernicki, Tomasz Kolanowski, Natalia Rozwadowska, Agnieszka Malcher, Wojciech Labedz, Tomasz Trzeciak, Katarzyna Chojnacka, Katarzyna Bednarek-Rajewska, Przemyslaw Majewski, Maciej Kurpisz
Cardiovascular diseases are a growing problem in developing countries; therefore, there is an ongoing intensive search for new approaches to treat these disorders. Currently, cellular therapies are focused on healing the damaged heart by implanting stem cells modified with pro-angiogenic factors. This approach ensures that the introduced cells are capable of fulfilling the complex requirements of the environment, including the replacement of the post-infarction scar with cells that are able to contract and promote the formation of new blood vessels that can supply the ischaemic region with nutrients and oxygen...
April 2018: Archivum Immunologiae et Therapiae Experimentalis
Wouter Masselink, Megumi Masaki, Daniel Sieiro, Christophe Marcelle, Peter D Currie
The migration of limb myogenic precursors from limb level somites to their ultimate site of differentiation in the limb is a paradigmatic example of a set of dynamic and orchestrated migratory cell behaviours. The homeobox containing transcription factor ladybird homeobox 1 (Lbx1) is a central regulator of limb myoblast migration, null mutations of Lbx1 result in severe disruptions to limb muscle formation, particularly in the distal region of the limb in mice (Gross et al., 2000). As such Lbx1 has been hypothesized to control lateral migration of myoblasts into the distal limb anlage...
October 15, 2017: Developmental Biology
Sonja Prpar Mihevc, Mojca Pavlin, Simona Darovic, Marko Živin, Matej Podbregar, Boris Rogelj, Tomaz Mars
Degeneration of distal axons and neuromuscular junctions is an early feature in the pathology of amyotrophic lateral sclerosis (ALS), which culminates in motor neuron loss due to axon retraction and muscle atrophy. The complex interactions in the pathogenesis of ALS between motor neurons, muscle cells and accompanying glia require an appropriate experimental model. Here, we have defined a co-culture model based on human myotubes innervated by neurons from embryonic rat spinal cord explants to investigate the pathology and treatment of ALS...
August 2017: Journal of Molecular Neuroscience: MN
Daniel Sieiro, Nadège Véron, Christophe Marcelle
The fusion of myoblasts into multinucleated myotubes is a crucial step of muscle growth during development and of muscle repair in the adult. While multiple genes were shown to play a role in this process, a vertebrate model where novel candidates can be tested and analyzed at high throughput and relative ease has been lacking. Here, we show that the early chicken embryo is a fast and robust model in which functional testing of muscle fusion candidate genes can be performed. We have used known modulators of muscle fusion, Rac1 and Cdc42, along with the in vivo electroporation of integrated, inducible vectors, to show that the chicken embryo is a suitable model in which their function can be tested and quantified...
2017: PloS One
Arnaud Perrin, Joël Rousseau, Jacques P Tremblay
Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adult human muscle, but laminin subunit alpha 1 (LAMA1) gene is expressed only during embryogenesis...
March 17, 2017: Molecular Therapy. Nucleic Acids
Ly Q Hong-Brown, C Randell Brown, Maithili Navaratnarajah, Charles H Lang
BACKGROUND: Excessive alcohol (EtOH) consumption causes an imbalance in protein metabolism. EtOH impairs protein synthesis in C2C12 myoblasts via a FoxO1-AMPK-TSC2-mTORC1 pathway and also induces protein degradation. As the underlying regulatory signaling cascades for these processes are currently poorly defined, we tested the hypothesis that alcohol-induced autophagy is mediated via activation of the PIK3C3 complex that is regulated by FoxO1-AMPK. METHODS: C2C12 myoblasts were incubated with EtOH for various periods of time, and autophagy pathway-related proteins were assessed by Western blotting and immunoprecipitation...
May 2017: Alcoholism, Clinical and Experimental Research
Cheng Ye, Duo Zhang, Lei Zhao, Yan Li, Xiaohan Yao, Hui Wang, Shengjie Zhang, Wei Liu, Hongchao Cao, Shuxian Yu, Yucheng Wang, Jingjing Jiang, Hui Wang, Xihua Li, Hao Ying
Skeletal muscle has a major role in locomotion and muscle disorders are associated with poor regenerative efficiency. Therefore, a deeper understanding of muscle regeneration is needed to provide a new insight for new therapies. CaMKK2 plays a role in the calcium/calmodulin-dependent kinase cascade; however, its role in skeletal muscle remains unknown. Here, we found that CaMKK2 expression levels were altered under physiological and pathological conditions including postnatal myogensis, freeze or cardiotoxin-induced muscle regeneration, and Duchenne muscular dystrophy...
October 24, 2016: International Journal of Molecular Sciences
Po-Ju Chen, Isabel Martinez-Pena Y Valenzuela, Mohamed Aittaleb, Mohammed Akaaboune
UNLABELLED: Rapsyn, a 43 kDa scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at synaptic sites between mammalian motor neurons and muscle cells. However, the mechanism by which rapsyn is inserted and retained at postsynaptic sites at the neuromuscular junction (NMJ) in vivo remains largely unknown. We found that neither the N-terminal myristoylation nor the cysteine-rich RING H2 domain of rapsyn is required for its stable association with the postsynaptic membrane of NMJs...
May 25, 2016: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
Jean-Paul Iyombe-Engembe, Dominique L Ouellet, Xavier Barbeau, Joël Rousseau, Pierre Chapdelaine, Patrick Lagüe, Jacques P Tremblay
The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel), a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene...
2016: Molecular Therapy. Nucleic Acids
Jasna Lojk, Katarina Mis, Sergej Pirkmajer, Mojca Pavlin
Introduction of genetic material into muscle tissue has been extensively researched, including isolation and in vitro expansion of primary myoblasts as a potential source of cells for skeletal and heart muscle tissue engineering applications. In this study, we optimized the electroporation protocol for introduction of short interfering ribonucleic acid (siRNA) against messenger RNA for Hypoxia Inducible Factor 1α (HIF-1α) into cultured primary human myoblasts. We established optimal pulsing protocol for siRNA electro transfection, and theoretically analyzed the effect of electric field and pulse duration on silencing efficiency and electrophoretic displacement of siRNA...
December 2015: Bioelectromagnetics
Rosie J Naylor, Richard J Piercy
OBJECTIVE: To produce a clonal equine myoblast cell line that retains the ability to divide for multiple passages and differentiate into multinucleated myotubes during specific conditions. SAMPLE: Cultured primary equine skeletal muscle-derived cells from a healthy Thoroughbred. PROCEDURES: Cell cultures were transfected by electroporation with a plasmid (pNIT) that expresses the temperature-sensitive simian vacuolating virus 40 large T antigen (TAg), which can be controlled by a doxycycline-responsive promoter...
July 2015: American Journal of Veterinary Research
Jieun Hong, Eunjee A Lee, Eun-Seo Lee, Giyoung Jung, Hansaem Jeong, Hwajin Lee, Hyukjin Lee, Nathaniel S Hwang
Lineage conversion from one somatic cell type to another is an attractive approach for deriving specific therapeutic cell generation. In order to bypass inducing pluripotent stage, transdifferentiation/direct conversion technologies have been recently developed. We report the development of a direct conversion methodology in which cells are transdifferentiated through a plastic intermediate state induced by exposure to non-integrative minicircle DNA (MCDNA)-based reprogramming factors, followed by differentiation into myoblasts...
February 28, 2015: Journal of Controlled Release: Official Journal of the Controlled Release Society
Tomaz Mars, Marusa Strazisar, Katarina Mis, Nejc Kotnik, Katarina Pegan, Jasna Lojk, Zoran Grubic, Mojca Pavlin
Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared...
April 2015: Journal of Membrane Biology
Amit Kumar Majhi, Greeshma Thrivikraman, Bikramjit Basu, V Venkataraman
In order to study cell electroporation in situ, polymer devices have been fabricated from poly-dimethyl siloxane with transparent indium tin oxide parallel plate electrodes in horizontal geometry. This geometry with cells located on a single focal plane at the interface of the bottom electrode allows a longer observation time in both transmitted bright-field and reflected fluorescence microscopy modes. Using propidium iodide (PI) as a marker dye, the number of electroporated cells in a typical culture volume of 10-100 μl was quantified in situ as a function of applied voltage from 10 to 90 V in a series of ~2-ms pulses across 0...
February 2015: European Biophysics Journal: EBJ
Sudheer Shenoy P, Bipasha Bose, Mridula Sharma, Craig McFarlane, Ravi Kambadur
Conversion of skin fibroblasts into myoblasts by transducing the cells with myogenic master regulator MyoD has been in practice for more than two decades. The purpose of such conversion is due to scarcity of muscle biopsies during muscle wasting, hence conversion of fibroblasts to myogenic lineage from various genetic backgrounds offers a great alternative for cell therapies. Here, we have investigated if eliminating Myostatin, a potent negative regulator of myogenesis, could improve the myogenic conversion of fibroblasts...
November 2014: Journal of Cellular Biochemistry
A Zimna, A Janeczek, N Rozwadowska, M Fraczek, P Kucharzewska, M Rucinski, T Mietkiewski, M Kurpisz
Myocardial infarction results in cardiomyocyte loss and may eventually lead to cardiac failure. Skeletal myoblast transplantation into the scar area may compensate for this observed cell loss by strengthening the weakened myocardium and inducing myogenesis. Moreover, skeletal myoblasts may serve as potential transgene carriers for the myocardium (i.e., delivering pro-angiogenic factors, which may potentially improve blood perfusion in infarcted heart). We examined the influence of the simultaneous overexpression of two potent pro-angiogenic factors, fibroblast growth factor-4 (FGF-4) and vascular endothelial growth factor (VEGF), on human primary myoblast proliferation, cell cycle, resistance to hypoxic stress conditions and myogenic gene expression, as well as the induction of pro-angiogenic activities...
April 2014: Journal of Physiology and Pharmacology: An Official Journal of the Polish Physiological Society
Zhaoyong Hu, Janet D Klein, William E Mitch, Liping Zhang, Ivan Martinez, Xiaonan H Wang
The mechanisms underlying the development of aging-induced muscle atrophy are unclear. By microRNA array and individual qPCR analyses, we found significant up-regulation of miR-29 in muscles of aged rodents vs. results in young. With aging, p85α, IGF-1 and B-myb muscle levels were lower while the expression of certain cell arrest proteins (p53, p16 and pRB) increased. When miR-29 was expressed in muscle progenitor cells (MPC), their proliferation was impaired while SA-βgal expression increased signifying the development of senescence...
March 2014: Aging
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