Inducirane pluripotentne matične celice in možnosti njihove uporabe pri zdravljenju dednih bolezni kože

Leta 1962 je Gurdon odkril, da je specializacija celic reverzibilna; jedro v jajčni celici žabe je nadomestil z jedrom iz črevesne celice. Iz spremenjene jajčne celice se je razvil paglavec. Leta 2006 je Yamanaka odkril, kako je mogoče z uporabo le nekaj genov reprogramirati odrasle mišje celice v stanje, ki je podobno embrionalnim celicam. Tako pridobljene celice so poimenovali inducirane pluripotentne matične celice, ki se lahko razvijejo v vse vrste celic v telesu, zato so obetavno orodje za različne pristope zdravljenja, ki temeljijo na celični terapiji. Gurdon in Yamanaka sta za odkritje, da je mogoče dozorele celice reprogramirati v pluripotentno stanje, leta 2012 prejela Nobelovo nagrado za fiziologijo. V tem prispevku bomo prikazali kratek pregled dognanj na tem področju, pri čemer bomo poudarili področje skupine redkih dednih bolezni krhkosti kože

Adhesion and stiffness of detached breast cancer cells in vitro

Metastatic cancer cells can overcome detachment-induced cell death and can proliferate in anchorage-independent conditions. A recent study revealed that a co-treatment with two drugs that interfere with cell metabolism, metformin and 2-deoxy-D-glucose, promotes detachment of viable MDA-MB-231 breast cancer cells. In the present study, we analyzed if these detached viable MDA-MB-231 cells also exhibit other features related to cancer metastatic potential, i.e., if they are softer and more prone to adhere to epithelial cells. The cell mechanics of attached cells and floating cells were analyzed by optical tweezers and cell deformability cytometry, respectively. The adhesion was assessed on a confluent monolayer of HUVEC cells, with MDA-MB-231 cells either in static conditions or in a microfluidic flow. Additionally, to test if adhesion was affected by the state of the epithelial glycocalyx, HUVEC cells were treated with neuraminidase and tunicamycin. It was found that the treated MDA-MB-231 cells were more prone to adhere to HUVEC cells and that they were softer than the control, both in the floating state and after re-seeding to a substrate. The changes in the HUVEC glycocalyx, however, did not increase the adhesion potential of MDA-MB-231

The pore-forming action of polyenes

The incidence of resistant fungal pathogens has been increasing, especially in immuno-compromised people. As such, considerable research has been focused on discovering anti-fungal agents with new mechanisms of action and on optimizing the use of existing agents. In this context, interest in the polyene group of anti-fungals has recently been renewed, since they are known to be effective against a broad spectrum of fungal pathogens that only rarely develop a resistance to them. In the past 10 years considerable efforts have been made to improve their efficacy and, simultaneously, to reduce their toxicity. Knowledge about the basic mechanisms of their action will be of crucial importance to further optimizing their use. The mechanisms of polyene action at the membrane level are reviewed here, focusing primarily on their pore-forming activity and on the resulting osmotic responses of artificial lipid vesicles and different eukaryotic cells

Keratin dynamics and spatial distribution in wild-type and K14 R125P mutant cells—a computational model

Keratins are one of the most abundant proteins in epithelial cells. They form a cytoskeletal filament network whose structural organization seriously conditions its function. Dynamic keratin particles and aggregates are often observed at the periphery of mutant keratinocytes related to the hereditary skin disorder epidermolysis bullosa simplex, which is due to mutations in keratins 5 and 14. To account for their emergence in mutant cells, we extended an existing mathematical model of keratin turnover in wild-type cells and developed a novel 2D phase-field model to predict the keratin distribution inside the cell. This model includes the turnover between soluble, particulate and filamentous keratin forms. We assumed that the mutation causes a slowdown in the assembly of an intermediate keratin phase into filaments, and demonstrated that this change is enough to account for the loss of keratin filaments in the cell\u27s interior and the emergence of keratin particles at its periphery. The developed mathematical model is also particularly tailored to model the spatial distribution of keratins as the cell changes its shape

A mathematical model for the dependence of keratin aggregate formation on the quantity of mutant keratin expressed in EGFP-K14 R125P keratinocytes

We examined keratin aggregate formation and the possible mechanisms involved. With this aim, we observed the effect that different ratios between mutant and wild-type keratins expressed in cultured keratinocytes may have on aggregate formation in vitro, as well as how keratin aggregate formation affects the mechanical properties of cells at the cell cortex. To this end we prepared clones with expression rates as close as possible to 25%, 50% and 100% of the EGFP-K14 proteins (either WT or R125P and V270M mutants). Our results showed that only in the case of the 25% EGFP-K14 R125P mutant significant differences could be seen. Namely, we observed in this case the largest accumulation of keratin aggregates and a significant reduction in cell stiffness. To gain insight into the possible mechanisms behind this observation, we extended our previous mathematical model of keratin dynamics by implementing a more complex reaction network that considers the coexistence of wild-type and mutant keratins in the cell. The new model, consisting of a set of coupled, non-linear, ordinary differential equations, allowed us to draw conclusions regarding the relative amounts of intermediate filaments and aggregates in cells, and suggested that aggregate formation by asymmetric binding between wild-type and mutant keratins could explain the data obtained on cells grown in culture

Organization of the actin in blebs.

<p>In “living” cells the actin is organized in small (a) and bigger blebs (b), as depicted by fluorescent signal. Some representative blebs are indicated by arrows.</p

Osmotic Effects Induced by Pore-Forming Agent Nystatin: From Lipid Vesicles to the Cell

<div><p>The responses of Chinese hamster ovary epithelial cells, caused by the pore-forming agent nystatin, were investigated using brightfield and fluorescence microscopy. Different phenomena, i.e., the detachment of cells, the formation of blebs, the occurrence of “cell-vesicles” and cell ruptures, were observed. These phenomena were compared to those discovered in giant lipid vesicles. A theoretical model, based on the osmotic effects that occur due to the size-discriminating nystatin transmembrane pores in lipid vesicles, was extended with a term that considers the conservation of the electric charge density in order to describe the cell’s behavior. The increase of the cellular volume was predicted and correlated with the observed phenomena.</p></div

The characteristic types of tension-pore behavior.

<p>The three types of tension-pore behavior as a function of the radius and the number of nystatin pores (<i>r</i>_NP and <i>N</i>_NP). The dashed line shows the border between the type-II and type-III tension-pore behavior for a ten-times-smaller lysis tension.</p

Predicted changes in the cell volume and in the tension-pore radius.

<p>The changes in the cell volume (a) and in the tension-pore radius (b) are shown for different numbers of nystatin pores 2.5 × 10⁷ (dash-dot line), 9 × 10⁷ (dashed line) and 3 × 10⁸ (full line), characteristic for type-I, type-II and type-III tension-pore behavior. The dotted line indicates the critical volume of the cell. The cell volume is normalized to its initial volume (<i>V</i>/<i>V</i>₀) and the tension pore radius to the radius of the cell (<i>R</i>_TP/<i>R</i>_c). The radius of the nystatin pores is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165098#pone.0165098.g007" target="_blank">Fig 7</a>. The numerical procedure is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165098#pone.0165098.s001" target="_blank">S1 File</a>.</p

Organization of fluorescent actin structures in “living” cells as seen using confocal microscopy after the addition of 300 μmol/L of nystatin solution.

<p>Top view (in the middle) and side views (bottom and right) of the nystatin treated cells along the thin lines are presented at different time points as indicated (first row). As a control, the actin structures after the “methanol only” treatment at 3% volume fraction are shown (second row). The step size was equal to 0.5 μm, while the number of images in the stack was 86.</p