The aim of this study was to extensively characterise natal dental pulp stem cells (nDPSC) and assess their efficiency to generate human induced pluripotent stem cells (hiPSC). A number of distinguishing features prompted us to choose nDPSC over normal adult DPSC, in that they differed in cell surface marker expression and initial doubling time. In addition, nDPSC expressed 17 out of 52 pluripotency genes we analysed, and the level of expression was comparable to human embryonic stem cells (hESC). Ours is the first group to report comprehensive characterization of nDPSC followed by directed reprogramming to a pluripotent stem cell state. nDPSC yielded hiPSC colonies upon transduction with Sendai virus expressing the pluripotency transcription factors POU5F1, SOX2, c-MYC and KLF4. nDPSC had higher reprogramming efficiency compared to human fibroblasts. nDPSC derived hiPSCs closely resembled hESC in terms of their morphology, expression of pluripotency markers and gene expression profiles. Furthermore, nDPSC derived hiPSCs differentiated into the three germ layers when cultured as embryoid bodies (EB) and by directed differentiation. Based on our findings, nDPSC present a unique marker expression profile compared with adult DPSC and possess higher reprogramming efficiency as compared with dermal fibroblasts thus proving to be more amenable for reprogramming.
- MeSH
- biologické markery MeSH
- buněčná diferenciace genetika MeSH
- embryoidní tělíska cytologie MeSH
- fibroblasty cytologie metabolismus MeSH
- indukované pluripotentní kmenové buňky cytologie metabolismus MeSH
- karyotyp MeSH
- kmenové buňky cytologie metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- předmléčné zuby cytologie MeSH
- přeprogramování buněk * MeSH
- transkriptom MeSH
- vývojová regulace genové exprese MeSH
- zubní dřeň cytologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
AIM: Cloning of artificial intronic sequence within the open reading frame (ORF) of DsRed2 gene. METHOD: Splice prediction software was used to analyze DsRed2 sequence to find an ideal site for cloning artificial intronic sequence. Intron was cloned within DsRed2 using cyclic ligation assembly. Flow cytometry was used to quantify the number of cells expressing red fluorescence. RESULT: Sequencing data confirmed precise cloning of intron at the desired position using cyclic ligation assembly. Successful expression of red fluorescence after cloning of intron confirmed successful intron recognition and splicing by host cell line. Cloning of intron increased the number of cells expressing red fluorescent protein. CONCLUSION: Cloning of intronic sequence within DsRed2 has helped to increase the number of cells expressing red fluorescence by approximately four percent.
- MeSH
- HeLa buňky MeSH
- introny MeSH
- klonování DNA metody MeSH
- lidé MeSH
- luminescentní proteiny genetika metabolismus MeSH
- otevřené čtecí rámce MeSH
- transfekce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The aim of the present study was to determine effect of two decellularized agents, sodium dodecyl sulphate (SDS) and Triton X-100, to the skeletal muscle tissue. Final scaffold was evaluated by several histological techniques to analyse preservation of essential structures including collagen and elastic fibres, basement membranes, glycosaminoglycans and also to confirm elimination of nuclear and cytoplasmic components which are redundant in effectively prepared decellularized scaffolds. Comparison of tissue scaffolds processed with different detergents proved that SDS is superior to Triton X-100 as it can effectively decellularize muscle tissue.
- MeSH
- barvicí látky MeSH
- dodecylsíran sodný farmakologie MeSH
- elastická tkáň diagnostické zobrazování účinky léků MeSH
- glykosaminoglykany MeSH
- kolagen účinky léků MeSH
- kosterní svaly cytologie účinky léků MeSH
- mikroskopie MeSH
- myši MeSH
- oktoxynol farmakologie MeSH
- povrchově aktivní látky farmakologie MeSH
- tkáňové podpůrné struktury * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Ensuring mycoplasma-free cell culture is of prime importance as they severely affect cellular characteristics leading to experimental artefacts and spurious results. Various methods persist for mycoplasma detection; out of the whole array of methods polymerase chain reaction (PCR) is the most favoured one because it is highly sensitive, specific and quick. The PCR-based detection procedure involves three steps: cell culture supernatant collection, DNA isolation, and PCR. We have modified this procedure so that cell culture supernatant can directly be used for PCR without the need for DNA extraction. This modification makes the procedure quicker and more sensitive because loss of mycoplasma DNA is prevented and this loss becomes more significant when the level of mycoplasma contamination is very low.
- MeSH
- buněčné linie MeSH
- kontaminace DNA * MeSH
- lidé MeSH
- Mycoplasma genetika izolace a purifikace MeSH
- myši MeSH
- polymerázová řetězová reakce metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
BACKGROUND: A biological scaffold from extracellular matrix can be produced by a variety of decellularization methods whose caveat consists in efficiently eliminating cells from the treated tissue. This scaffold can be used in diverse applications for tissue engineering and organ regeneration. Preservation of the extracellular matrix ultrastructure is highly desirable because of its unique architecture, contained growth factors and decreased immunological response. All of these properties provide attachment sites and adequate environment for cells colonizing this scaffold, reconstituting the decellularized organ. This review briefly describes chemical decellularization methods, evaluation of these protocols and the role of ECM in tissue engineering. CONCLUSION: Chemical decellularization is an often used method for scaffold preparation and makes possible a well-preserved three dimensional structure of extracellular matrix.
The cell culture became an invaluable tool for studying cell behaviour, development, function, gene expression, toxicity of compounds and efficacy of novel drugs. Although most results were obtained from cell cultivation in two-dimensional (2D) systems, in which cells are grown in a monolayer, three-dimensional (3D) cultures are more promising as they correspond closely to the native arrangement of cells in living tissues. In our study, we focused on three types of 3D in vitro systems used for cultivation of one cell type. Cell morphology, their spatial distribution inside of resulting multicellular structures and changes in time were analysed with histological examination of samples harvested at different time periods. In multilayered cultures of WRL 68 hepatocytes grown on semipermeable membranes and non-passaged neurospheres generated by proliferation of neural progenitor cells, the cells were tightly apposed, showed features of cell differentiation but also cell death that was observable in short-term cultures. Biogenic scaffolds composed of extracellular matrix of the murine tibial anterior muscle were colonized with C2C12 myoblasts in vitro. The recellularized scaffolds did not reach high cell densities comparable with the former systems but supported well cell anchorage and migration without any signs of cell regression.
- MeSH
- buněčné kultury MeSH
- buněčné linie MeSH
- buněčné sféroidy fyziologie MeSH
- hepatocyty fyziologie MeSH
- lidé MeSH
- myoblasty MeSH
- myši MeSH
- proliferace buněk fyziologie MeSH
- tkáňové inženýrství metody MeSH
- tkáňové podpůrné struktury * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
