Isolation and characterization of muscle satellite cells from various species

Mature functional myofibers are terminally differentiated cells without ability to proliferate. However, skeletal muscle has remarkable regenerative potential. Regeneration of skeletal muscle mainly comes from muscle satellite cells, stem cells that lie on the surface of the myofibres between the plasma lemma and the overlying basal lamina. Satellite cells are quiescent cells that activate to become myoblasts, proliferating progenitor cells. Myoblasts have tendency to fuse either into an existing myofibres to become myonuclei, or fuse together to form myotubes. During myogenesis, myogenic cell repopulate satellite cell poll. Each stage of the myogenic pathway is represented with specific cells with certain gene expression profile and cell surface markers. Satellite cells express Pax7, the hallmark gene for quiescent cell. In this stage cells has unique combination of cell surface markers, CD34+CXCR4+α-7 integrin+ and sca1- . When they become activated they become Myf5 and MyoD positive, and surface marker profile change to CD34-CXCR4-α-7 integrin+ and sca1+. Knowing the surface character of the cells gives us opportunity for their direct isolation and with that exploring their physiology or use those as a source for cell therapy. In this presentation we will speak about specific characteristic of myogenic spaces in various experimental animal models

Fibroblast cell line with inducible cassette exchange system- tool to study gain and loss of gene function

We generated fibroblast cell line with single copy of targeting Cre/Lox cassette exchange. The cassette is targeted with circular plasmid P2lox/cre caring the gene of interest. Successful lox/cre recombination provides reconstitution of NEO gene within the cassette that allows positive selection with neomycin. The whole cassette is under doxycyline inducible (TRE) promoter. To evaluate the effectiveness of our inducible fibroblast cell line (i3T3) we recombined the exchange locus with luciferase. The generated luciferase inducible cell line (i3T3-Luciferase) was inducible in time and dose dependent manner to doxycyline treatment. Positive luciferase signal was detected as early as 30 min post induction. Levels of luciferase can be titrated with dilutions of doxycyline from 0.5 to 500 ng/ml. For functionally evaluation of the cell line we recombined the inducible locus with MyoD and Myf5, two main myogenic transcription factors. MyoD and My5 initiated myogenic differentiation in fibroblast. Myogenesis was evident by induction of myogenic pathway and formation of terminally differentiated myotubes. Out inducible cell line allowed us to study direct targets of MyoD and Myf5 and to dissect the process of myogenic reprogramming of fibroblast. We engendered fibroblast cell line with doxycyline inducible cassette exchange system. The cassette can be efficiently targeted with any gene o

Genetically modified systems to study muscular dystrophies

We developed different approaches to utilize genetically modified cells and animal models to understand molecular mechanism of Facioscapulohumeral muscular dystrophy (FSHD).Main obstacle with primary cell lines is to obtain sufficient number of tissue specific progenitors that will be able extensively to proliferate and maintain the phenotype to be used for relevant in vitro studies. As an alternative solution for obtaining appropriate cells are induced pluripotent stem (IPS) cells. IPS cells can be made from different somatic cells by cell reprogramming. Patient specific IPS cells retain the genotype of the patient, they are good models to study molecular mechanism of diseases and they are suitable for autologous cell transplantations. We generated IPS cells from myoblasts from FSHD patients using common reprogramming factors (Oct4, Sox2, KLF4 and c-Myc), incorporated in viral vectors. Mesenchymal stem cells were obtained by differentiation of IPS cells in 3D culture conditions in presence of cocktail of growth factors. Myogenic progenitors were obtained by overexpressing Myf5 in mesenchymal cell fraction. Generated cells revealed typical characteristics of myoblast and were able functionally to recover damaged mouse muscle. Furthermore, we were able to genetically correct affected locus in FSHD IPS cells by removing 4qA161 allele using zinc finger nucleases. Gene corrected cells did not express DUX4 and displayed normal myogenic properties compared to the affected ones.To be able to study FSHD in vivo and test various therapy approaches we developed transgenic mouse model that has incorporated DUX4 gene on X chromosome. IDUX4 mouse expressed DUX4 in different tissue. Male pups have runt phenotype, with prominent skin, testis and retina pathological changes.In this talk, advantages and methods for cell manipulations for generating in vitro and in vivo diseases models will be discussed.

Types of scaffolds and their application in bone tissue engineering

Bone tissue engineering is founded on the conception of bone structure, bone mechanics and tissue creature as it aims to restore, maintain, or improve tissue functions that are defective or have been lost by different pathological conditions. Functional bone tissueengineering exact the newly reconstruct bone to be completely integrated with the host bone. Compared with small bone defects, for successful regeneration of large bone defects is necessary implantation of bone replacement in a critical defect. In recent years, the research has been focused on the producting of biomaterials, scaffolds, which are defined as three-dimension porous solid biomaterials designed for differnet applications. Materials have been developed for tissue engineering approaches and currently proposed types of scaffolds made of inorganic materials, organic or synthetic polymers, or of mixed materials (composite scaffolds). The concept of tissue engineering is being applied for treatment of salivary gland disorders, regeneration of craniofacial tissues, oral mucose, periodontium, dentin and dental pulp. Further, we review current patents on scaffold for bone regeneration and their classification, as well as biocompatibility of scaffolds and cells. Preoperative and perioperative contamination and biomaterial-associated infections pose a serious problem in tissue engineering. In order to prevent and treat bone infections, antibiotics could be incorporated into scaffold and those scaffolds could be utilized for tissue engineering

Perspectives on regeneration of alveolar bone defects

Bone atrophy of the alveolar process is an important parameter in patients undergoing dental implants. There are several methods for preserving the alveolar process, with the autologous bone graft as the gold standard. Other approaches include the use of allografts, xenografts and synthetic bone grafts. In recent years, the use of stem cells has increased in importance. Th e most common type of stem cells used are mesenchymal stem cells from various sources, including bone marrow, adipose tissue and dental pulp. The discovery of induced pluripotent stem cells and the continued research on embryonic stem cells open new possibilities in this field. However, further research is needed to optimise protocols for isolation, diff erentiation and transplantation of cells with or without appropriate scaff olds, and to determine the correct clinical and therapeutic implications

Antibacterial Activity of Selected Silver Nanoparticles Coatings - Our Initial Experience

Nanoparticles exhibits chemical and physical properties significantly different to their macro-scale counterparts composed of the same substance (due higher surface/volume ratio, colour, solubility, diffusivity,material strength, toxicity, thermodynamic, magnetic, optical and other properties) and may have unique impacts on health and environment. Extremelly small size (1-100 nm) enables them to enter the human body through usual or unusual routes, pass through cell membranes, or cross the blood-brain barrier. Gravity loses impact and importance, surface tension and Van der Waals constants has more importance in system of nano particles. Nanotechnology is already used in various applications, with potential tobe applied at any stage in food industry: production, preservation, processing, packaging,transport, nanobarcodes for food authenticity and tracing, labelling, keeping the quality of food products, extend the products shelf-life, removal of undesirable tastes, flavours or allergens from food products, nano (bio)sensors for food safety, water filtration. Risks of nanotechnology are still unknown and unpredictable. Initial scientific studies showed negative effects on living organisms and a potential for serious threat to human health. Authorities of the most developed countries, have set up, guidance documents and procedures for nanoenabled products based on existing regulations, appropriate in vitro and in vivo ADME studies (absorption, distribution, metabolism and excretion) and requirements for standardised and harmonised analytical test methods for proper risk assesments, clear identification and characterization of nano-hazards. Nanotechnology in medicine (Nanomedicine) apply for rapid and sensitive detection of pathogenic bacteria and low levels of viruses, in small sample volumes, at lower costs than current in-use technologies. This advance in early detection enables accurate and prompt treatment. Nano-robots to make repairs at the cellular level are under development. Rapid and sensitive detection methods, based on nano (bio) senzors, are developed for food-borne pathogens E. coli, especially E. coli O157:H7, S. aureus, S. typhimurium, C. jejuni, E. cloacae, B. subtilis, L. monocytogenes. Detection sensor to detect bacterial biofilm formation on surfaces are under development. New strategies to combat multydrug resistant microorganisms (MDR) are urgently needed and nanomaterials are very promising approach. Small size provides large surface of nanoparticles and at least 50% of molecules will react to the microorganisms. Metal nanomaterials (silver, gold, copper, titanium, zinc, magnesium,cadmium, and alumina) possess advantage of unique antimicrobial activities. Scientists offers also new complex antibacterial and antiviral nano systems on the basis of metal oxides or intermetallic oxide compounds (such as TiO2, ZrO2, SnO and SiO2). Inside the human body ionic silver quickly combines with chloride to form an insoluble compound called silver chloride which is far less reactive than metallic silver nanoparticles. Some studies established that silver ions has strongest bactericidal effect, cooper and gold weaker one. In our preliminary study on antibacterial activity of several different compositions of nanoparticle coatings (titanium, inox and silver), we found antimicrobial activity of silver, double composition of titanium and silver against Staphylococcus aureus and Staphylococcus epidermidis, but not for E. coli, Pseudomonas aeruginosa, Listeria monocytogenes and Candida albicans. More extensive studies will follow. Key words: Nanoscience, nanoparticles, silver, ions, biofilm, antimicrobial, antibiotic, resistance

Biphasic Myopathic Phenotype of Mouse DUX, an ORF within Conserved FSHD-Related Repeats

Facioscapulohumeral muscular dystrophy (FSHD) is caused by contractions of D4Z4 repeats at 4q35.2 thought to induce misregulation of nearby genes, one of which, DUX4, is actually localized within each repeat. A conserved ORF (mDUX), embedded within D4Z4-like repeats, encoding a double-homeodomain protein, was recently identified on mouse chromosome 10. We show here that high level mDUX expression induces myoblast death, while low non-toxic levels block myogenic differentiation by down-regulating MyoD and Myf5. Toxicity and MyoD/Myf5 expression changes were competitively reversed by overexpression of Pax3 or Pax7, implying mechanistic similarities with the anti-myogenic activity of human DUX4. We tested the effect of mDUX expression on Xenopus development, and found that global overexpression led to abnormalities in gastrulation. When targeted unilaterally into blastomeres fated to become tail muscle in 16-cell embryos, mDUX caused markedly reduced tail myogenesis on the injected side. These novel cell and animal models highlight the myopathic nature of sequences within the FSHD-related repeat array

Decreased Proliferation Kinetics of Mouse Myoblasts Overexpressing FRG1

Although recent publications have linked the molecular events driving facioscapulohumeral muscular dystrophy (FSHD) to expression of the double homeobox transcription factor DUX4, overexpression of FRG1 has been proposed as one alternative causal agent as mice overexpressing FRG1 present with muscular dystrophy. Here, we characterize proliferative defects in two independent myoblast lines overexpressing FRG1. Myoblasts isolated from thigh muscle of FRG1 transgenic mice, an affected dystrophic muscle, exhibit delayed proliferation as measured by decreased clone size, whereas myoblasts isolated from the unaffected diaphragm muscle proliferated normally. To confirm the observation that overexpression of FRG1 could impair myoblast proliferation, we examined C2C12 myoblasts with inducible overexpression of FRG1, finding increased doubling time and G1-phase cells in mass culture after induction of FRG1 and decreased levels of pRb phosphorylation. We propose that depressed myoblast proliferation may contribute to the pathology of mice overexpressing FRG1 and may play a part in FSHD