Recent data on Duchenne muscular dystrophy (DMD) show myocyte progenitor's involvement in the disease pathology often leading to the DMD patient's death. The molecular mechanism underlying stem cell impairment in DMD has not been described. We created dystrophin-deficient human pluripotent stem cell (hPSC) lines by reprogramming cells from two DMD patients, and also by introducing dystrophin mutation into human embryonic stem cells via CRISPR/Cas9. While dystrophin is expressed in healthy hPSC, its deficiency in DMD hPSC lines induces the release of reactive oxygen species (ROS) through dysregulated activity of all three isoforms of nitric oxide synthase (further abrev. as, NOS). NOS-induced ROS release leads to DNA damage and genomic instability in DMD hPSC. We were able to reduce both the ROS release as well as DNA damage to the level of wild-type hPSC by inhibiting NOS activity.

• Klíčová slova
• DMD, NO synthases, ROS, dystrophin, genome stability, pluripotent stem cells,
• MeSH
• buněčné linie MeSH
• Duchennova muskulární dystrofie genetika   MeSH
• dystrofin nedostatek   genetika   MeSH
• indukované pluripotentní kmenové buňky metabolismus   patologie   MeSH
• lidé MeSH
• nestabilita genomu * MeSH
• oxidační stres MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• synthasa oxidu dusnatého, typ I metabolismus   MeSH
• synthasa oxidu dusnatého, typ II metabolismus   MeSH
• synthasa oxidu dusnatého, typ III metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dystrofin MeSH
• NOS1 protein, human MeSH Prohlížeč
• NOS2 protein, human MeSH Prohlížeč
• NOS3 protein, human MeSH Prohlížeč
• reaktivní formy kyslíku MeSH
• synthasa oxidu dusnatého, typ I MeSH
• synthasa oxidu dusnatého, typ II MeSH
• synthasa oxidu dusnatého, typ III MeSH

Cryopreservation of cells (mouse embryonic fibroblasts) is a fundamental task for wide range of applications. In practice, cells are protected against damage during freezing by applications of specific cryoprotectants and freezing/melting protocols. In this study by using AFM and fluorescence microscopy we showed how selected cryoprotectants (dimethyl sulfoxide and polyethylene glycol) affected the cryopreserved cells mechanical properties (stiffness) and how these parameters are correlated with cytoskeleton damage and reconstruction. We showed how cryopreserved (frozen and thawed) cells' stiffness change according to type of applied cryoprotectant and its functionality in extracellular or intracellular space. We showed that AFM can be used as technique for investigation of cryopreserved cells surfaces state and development ex vivo. Our results offer a new perspective on the monitoring and characterization of frozen cells recovery by measuring changes in elastic properties by nanoindentation technique. This may lead to a new and detailed way of investigating the post-thaw development of cryopreserved cells which allows to distinguish between different cell parts.

• Klíčová slova
• AFM, DMSO, PEG, cell stiffness, cryopreservation, fluorescence microscopy,
• Publikační typ
• časopisecké články MeSH

The possibility of replacing the originally discovered and widely used DNA reprogramming transcription factors is stimulating enormous effort to identify more effective compounds that would not alter the genetic information. Here, we describe the generation of induced pluripotent stem cells (iPSc) from head-derived primary culture of mouse embryonic cells using small chemical inhibitors of the MEK and TGF-beta pathways without delivery of exogenous transcription factors. These iPSc express standard pluripotency markers and retain their potential to differentiate into cells of all germ layers. Our data indicate that head-derived embryonic neural cells might have the reprogramming potential while neither the same primary cells cultivated over five passages in vitro nor a cell population derived from adult brain possesses this capacity. Our results reveal the potential for small molecules to functionally replace routinely used transcription factors and lift the veil on molecular regulation controlling pluripotency. The conditions described here could provide a platform upon which other genome non integrative and safer reprogramming processes could be developed. This work also shows novel potential for developing embryonic neural cells.

• MeSH
• diferenciační antigeny biosyntéza   MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• MAP kinasový signální systém * MeSH
• myši MeSH
• přeprogramování buněk * MeSH
• transformující růstový faktor beta metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diferenciační antigeny MeSH
• transformující růstový faktor beta MeSH

In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes (CMs). Protocols for cardiac differentiation of hESCs and hiPSCs include formation of the three-dimensional cell aggregates called embryoid bodies (EBs). The traditional suspension method for EB formation from clumps of cells results in an EB population heterogeneous in size and shape. In this study we show that forced aggregation of a defined number of single cells on AggreWell plates gives a high number of homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors in the media. For cardiac differentiation, we used three hESC lines and one hiPSC line. Our contracting EBs and the resulting CMs express cardiac markers, namely myosin heavy chain α and β, cardiac ryanodine receptor/calcium release channel, and cardiac troponin T, shown by real-time polymerase chain reaction and immunocytochemistry. Using Ca(2+) imaging and atomic force microscopy, we demonstrate the functionality of RyR2 to release Ca(2+) from the sarcoplasmic reticulum as well as reliability in contractile and beating properties of hESC-EBs and hiPSC-EBs upon the stimulation or inhibition of the β-adrenergic pathway.

• MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• embryoidní tělíska fyziologie   MeSH
• indukované pluripotentní kmenové buňky fyziologie   MeSH
• kardiomyocyty * cytologie   fyziologie   MeSH
• lidé MeSH
• reprodukovatelnost výsledků MeSH
• ryanodinový receptor vápníkového kanálu metabolismus   MeSH
• sarkoplazmatické retikulum metabolismus   MeSH
• těžké řetězce myosinu metabolismus   MeSH
• troponin T metabolismus   MeSH
• tvar buňky MeSH
• vápník metabolismus   MeSH
• velikost buňky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ryanodinový receptor vápníkového kanálu MeSH
• těžké řetězce myosinu MeSH
• troponin T MeSH
• vápník MeSH

BACKGROUND AND PURPOSE: Paulownia tomentosa is a rich source of geranylated flavanones, some of which we have previously shown to have cytotoxic activity. To identify members of this class of compounds with cytostatic effects, we assessed the effects of the geranylated flavanone tomentodiplacone B (TOM B) on cell cycle progression and cell cycle regulatory pathways of THP-1 human monocytic leukaemia cells. EXPERIMENTAL APPROACH: Cell viability was measured by dye exclusion and proliferation by WST-1 assays; cell cycle was monitored by flow cytometry. Regulatory proteins were assessed by immunoprecipitation and kinase assays, and Western blotting. KEY RESULTS: Tomentodiplacone B had no effect during the first 24 h of cell growth at concentrations between 1 and 2.5 µM, but inhibited cell growth in a dose-dependent manner at concentrations of 5 µM or higher. Growth inhibition during the first 24 h of exposure to TOM B was not accompanied by cytotoxicity as cells were accumulated in G1 phase dose-dependently. This G1 phase accumulation was associated with down-regulation of cyclin-dependent kinase 2 activity and also protein levels of cyclins E1 and A2. However, key stress-related molecules (γ-H2AX, p53 and p21) were not induced, suggesting that TOM B acts by directly inhibiting the cyclin-dependent kinase 2 signalling pathway rather than initiating DNA damage or cellular stress. CONCLUSIONS AND IMPLICATIONS: Our study provides the first evidence that TOM B directly inhibits proliferation of human monocytic leukaemia cells, and thus is a potential anticancer agent, preventing leukaemia cells from progressing from G1 phase into DNA synthesis.

• MeSH
• akutní monocytární leukemie MeSH
• antikarcinogenní látky farmakologie   MeSH
• cyklin D metabolismus   MeSH
• cyklin-dependentní kinasa 2 antagonisté a inhibitory   biosyntéza   metabolismus   MeSH
• cykliny metabolismus   MeSH
• down regulace účinky léků   MeSH
• flavanony farmakologie   MeSH
• G1 fáze účinky léků   MeSH
• lidé MeSH
• monocyty cytologie   účinky léků   metabolismus   MeSH
• monoterpeny farmakologie   MeSH
• nádorové buněčné linie MeSH
• proliferace buněk účinky léků   MeSH
• signální transdukce účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antikarcinogenní látky MeSH
• CDK2 protein, human MeSH Prohlížeč
• cyklin D MeSH
• cyklin-dependentní kinasa 2 MeSH
• cykliny MeSH
• flavanony MeSH
• monoterpeny MeSH
• tomentodiplacone B MeSH Prohlížeč

The transcription program that is responsible for the pluripotency of human ESCs (hESCs) is believed to be comaintained by exogenous fibroblast growth factor-2 (FGF-2), which activates FGF receptors (FGFRs) and stimulates the mitogen-activated protein kinase (MAPK) pathway. However, the same pathway is stimulated by insulin receptors, insulin-like growth factor 1 receptors, and epidermal growth factor receptors. This mechanism is further complicated by intracrine FGF signals. Thus, the molecular mechanisms by which FGF-2 promotes the undifferentiated growth of hESCs are unclear. Here we show that, in undifferentiated hESCs, exogenous FGF-2 stimulated the expression of stem cell genes while suppressing cell death and apoptosis genes. Inhibition of autocrine FGF signaling caused upregulation of differentiation-related genes and downregulation of stem cell genes. Thus, exogenous FGF-2 reinforced the pluripotency maintenance program of intracrine FGF-2 signaling. Consistent with this hypothesis, expression of endogenous FGF-2 decreased during hESC differentiation and FGF-2 knockdown-induced hESC differentiation. In addition, FGF-2 signaling via FGFR2 activated MAPK kinase/extracellular signal-regulated kinase and AKT kinases, protected hESC from stress-induced cell death, and increased hESC adhesion and cloning efficiency. This stimulation of self-renewal, cell survival, and adhesion by exogenous and endogenous FGF-2 may synergize to maintain the undifferentiated growth of hESCs.

• MeSH
• aktivace enzymů MeSH
• buněčná adheze účinky léků   fyziologie   MeSH
• buněčná diferenciace účinky léků   fyziologie   MeSH
• buněčné linie MeSH
• buňky - růstové procesy účinky léků   fyziologie   MeSH
• down regulace MeSH
• embryonální kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• exprese genu MeSH
• fibroblastový růstový faktor 2 genetika   metabolismus   farmakologie   MeSH
• fosforylace MeSH
• imunoblotting MeSH
• lidé MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• onkogenní protein v-akt metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• receptor fibroblastových růstových faktorů, typ 2 metabolismus   MeSH
• signální transdukce MeSH
• viabilita buněk účinky léků   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fibroblastový růstový faktor 2 MeSH
• mitogenem aktivované proteinkinasy MeSH
• onkogenní protein v-akt MeSH
• receptor fibroblastových růstových faktorů, typ 2 MeSH