Staff member

Elena Martínez Fraiz

Junior Group Leader
Biomimetic Systems for Cell Engineering
+34 934 037 177
Staff member publications

Garreta, E., de Oñate, L., Fernández-Santos, M. E., Oria, R., Tarantino, C., Climent, A. M., Marco, A., Samitier, M., Martínez, E., Valls-Margarit, M., Matesanz, R., Taylor, D. A., Fernández-Avilés, F., Izpisua Belmonte, J. C., Montserrat, N., (2016). Myocardial commitment from human pluripotent stem cells: Rapid production of human heart grafts Biomaterials 98, 64-78

Genome editing on human pluripotent stem cells (hPSCs) together with the development of protocols for organ decellularization opens the door to the generation of autologous bioartificial hearts. Here we sought to generate for the first time a fluorescent reporter human embryonic stem cell (hESC) line by means of Transcription activator-like effector nucleases (TALENs) to efficiently produce cardiomyocyte-like cells (CLCs) from hPSCs and repopulate decellularized human heart ventricles for heart engineering. In our hands, targeting myosin heavy chain locus (MYH6) with mCherry fluorescent reporter by TALEN technology in hESCs did not alter major pluripotent-related features, and allowed for the definition of a robust protocol for CLCs production also from human induced pluripotent stem cells (hiPSCs) in 14 days. hPSCs-derived CLCs (hPSCs-CLCs) were next used to recellularize acellular cardiac scaffolds. Electrophysiological responses encountered when hPSCs-CLCs were cultured on ventricular decellularized extracellular matrix (vdECM) correlated with significant increases in the levels of expression of different ion channels determinant for calcium homeostasis and heart contractile function. Overall, the approach described here allows for the rapid generation of human cardiac grafts from hPSCs, in a total of 24 days, providing a suitable platform for cardiac engineering and disease modeling in the human setting.

Keywords: Cardiac function, Extracellular matrix, Gene targeting, Pluripotent stem cells

Estévez, M., Martínez, E., Yarwood, S. J., Dalby, M. J., Samitier, J., (2015). Adhesion and migration of cells responding to microtopography Journal of Biomedical Materials Research - Part A 103, (5), 1659-1668

It is known that cells respond strongly to microtopography. However, cellular mechanisms of response are unclear. Here, we study wild-type fibroblasts responding to 25 μm2 posts and compare their response to that of FAK-/- fibroblasts and fibroblasts with PMA treatment to stimulate protein kinase C (PKC) and the small g-protein Rac. FAK knockout cells modulated adhesion number and size in a similar way to cells on topography; that is, they used more, smaller adhesions, but migration was almost completely stalled demonstrating the importance of FAK signaling in contact guidance and adhesion turnover. Little similarity, however, was observed to PKC stimulated cells and cells on the topography. Interestingly, with PKC stimulation the cell nuclei became highly deformable bringing focus on these surfaces to the study of metastasis. Surfaces that aid the study of cellular migration are important in developing understanding of mechanisms of wound healing and repair in aligned tissues such as ligament and tendon.

Keywords: Adhesion, Cell migration, Cell morphology, Focal adhesion kinase, Microstructures

Comelles, J., Hortigüela, V., Martínez, E., Riveline, D., (2015). Methods for rectifying cell motions in vitro: Breaking symmetry using microfabrication and microfluidics Methods in Cell Biology - Biophysical Methods in Cell Biology (ed. Wilson, L., Tran, P.), Academic Press (Santa Barbara, USA) 125, 437-452

Cell motility is an important phenomenon in cell biology, developmental biology, and cancer. Here we report methods that we designed to identify and characterize external factors which direct cell motions by breaking locally the symmetry. We used microfabrication and microfluidics techniques to impose and combine mechanical and chemical cues to moving fibroblasts. Gradients can thereby be engineered at the cellular scale and this approach has allowed to disentangle roles of the nucleus and protrusion activity in setting cell directions.

Keywords: Adhesion, Biological physics, Cell motility, Gradient, Ratchet

de Oñate, L., Garreta, E., Tarantino, C., Martínez, E., Capilla, E., Navarro, I., Gutiérrez, J., Samitier, J., Campistol, J.M., Muñoz-Cánovas, P., Montserrat, N., (2015). Research on skeletal muscle diseases using pluripotent stem cells Muscle Cell and Tissue (ed. Sakuma, K.), InTech (Rijeka, Croatia) , 333-357

The generation of induced pluripotent stem cells (iPSCs), especially the generation of patient-derived pluripotent stem cells (PSCs) suitable for disease modelling in vitro, opens the door for the potential translation of stem-cell related studies into the clinic. Successful replacement, or augmentation, of the function of damaged cells by patient-derived differentiated stem cells would provide a novel cell-based therapy for skeletal muscle-related diseases. Since iPSCs resemble human embryonic stem cells (hESCs) in their ability to generate cells of the three germ layers, patient-specific iPSCs offer definitive solutions for the ethical and histo-incompatibility issues related to hESCs. Indeed human iPSC (hiPSC)-based autologous transplantation is heralded as the future of regenerative medicine. Interestingly, during the last years intense research has been published on disease-specific hiPSCs derivation and differentiation into relevant tissues/organs providing a unique scenario for modelling disease progression, to screen patient-specific drugs and enabling immunosupression-free cell replacement therapies. Here, we revise the most relevant findings in skeletal muscle differentiation using mouse and human PSCs. Finally and in an effort to bring iPSC technology to the daily routine of the laboratory, we provide two different protocols for the generation of patient-derived iPSCs.

Keywords: Pluripotent stem cells, Myogenic differentiation, Disease modelling, Patient-specific induced pluripotent stem cells, Muscular dystrophy

Comelles, J., Caballero, D., Voituriez, ., Hortigüela, V., Wollrab, V., Godeau, A. L., Samitier, J., Martínez, E., Riveline, D., (2014). Cells as active particles in asymmetric potentials: Motility under external gradients Biophysical Journal 107, (7), 1513-1522

Cell migration is a crucial event during development and in disease. Mechanical constraints and chemical gradients can contribute to the establishment of cell direction, but their respective roles remain poorly understood. Using a microfabricated topographical ratchet, we show that the nucleus dictates the direction of cell movement through mechanical guidance by its environment. We demonstrate that this direction can be tuned by combining the topographical ratchet with a biochemical gradient of fibronectin adhesion. We report competition and cooperation between the two external cues. We also quantitatively compare the measurements associated with the trajectory of a model that treats cells as fluctuating particles trapped in a periodic asymmetric potential. We show that the cell nucleus contributes to the strength of the trap, whereas cell protrusions guided by the adhesive gradients add a constant tunable bias to the direction of cell motion.

Lagunas, A., Comelles, J., Oberhansl, S., Hortigüela, V., Martínez, E., Samitier, J., (2013). Continuous bone morphogenetic protein-2 gradients for concentration effect studies on C2C12 osteogenic fate Nanomedicine: Nanotechnology, Biology, and Medicine 9, (5), 694-701

Cells can respond to small changes in a varying concentration of exogenous signaling molecules. Here we propose the use of continuous surface chemical gradients for the in-depth study of dose-dependent effects on cells. A continuous surface gradient of bone morphogenetic protein-2 (BMP-2) is presented. The gradient covers a narrow range of surface densities (from 1.4 to 2.3 pmol/cm2) with a shallow slope (0.9 pmol/cm3). These characteristics represent a quasi-homogeneous surface concentration at the cell scale, which is crucial for cell screening studies. Cell fate evaluation at early stages of osteogenesis in C2C12 cells, indicates the potential of continuous gradients for in vitro screening applications.

Prats-Alfonso, E., Oberhansl, S., Lagunas, A., Martínez, E., Samitier, J., Albericio, F., (2013). Effective and versatile strategy for the total solid-phase synthesis of alkanethiols for biological applications European Journal of Organic Chemistry 2013, (7), 1233-1239

Biological applications increasingly demand tailored surfaces with a range of functional groups. Herein we describe a straightforward and inexpensive method based exclusively on solid-phase synthesis for the preparation of a variety of customized alkanethiols (ATs). The technique overcomes all the difficulties encountered during the preparation of these molecules in solution. The procedure allows the use of ATs without further purification for the preparation of self-assembled monolayers on gold, typically used to achieve functional group diversity on this surface. This paper describes a straightforward and inexpensive method based exclusively on solid-phase synthesis for the preparation of a variety of customized alkanethiols (ATs). The technique allows a variety of ATs to be obtained in only three steps, overcoming the difficulties encountered during their preparation in solution.

Diéguez, Lorena, Caballero, David, Calderer, Josep, Moreno, Mauricio, Martínez, Elena, Samitier, Josep, (2012). Optical gratings coated with thin Si3N4 layer for efficient immunosensing by optical waveguide lightmode spectroscopy Biosensors 2, (2), 114-126

New silicon nitride coated optical gratings were tested by means of Optical Waveguide Lightmode Spectroscopy (OWLS). A thin layer of 10 nm of transparent silicon nitride was deposited on commercial optical gratings by means of sputtering. The quality of the layer was tested by x-ray photoelectron spectroscopy and atomic force microscopy. As a proof of concept, the sensors were successfully tested with OWLS by monitoring the concentration dependence on the detection of an antibody-protein pair. The potential of the Si3N4 as functional layer in a real-time biosensor opens new ways for the integration of optical waveguides with microelectronics.

Keywords: Silicon nitride, Optical gratings, Waveguide, Biosensor

Lagunas, Anna , Comelles, Jordi, Martínez, Elena, Prats-Alfonso, Elisabet , Acosta, Gerardo A., Albericio, Fernando , Samitier, Josep , (2012). Cell adhesion and focal contact formation on linear RGD molecular gradients: study of non-linear concentration dependence effects Nanomedicine: Nanotechnology, Biology and Medicine 8, (4), 432-439

Cell adhesion onto bioengineered surfaces is affected by a number of variables, including the former substrate derivatization process. In this investigation, we studied the correlation between cell adhesion and cell–adhesive ligand surface concentration and organization due to substrate modification. For this purpose, Arg-Gly-Asp (RGD) gradient surfaces were created on poly(methyl methacrylate) substrates by continuous hydrolysis and were then grafted with biotin-PEG-RGD molecules. Cell culture showed that adhesion behavior changes in a nonlinear way in the narrow range of RGD surface densities assayed (2.8 to 4.4 pmol/cm2), with a threshold value of 4.0 pmol/cm2 for successful cell attachment and spreading. This nonlinear dependence may be explained by nonhomogeneous RGD surface distribution at the nanometre scale, conditioned by the stochastic nature of the hydrolysis process. Atomic force microscopy analysis of the gradient surface showed an evolution of surface morphology compatible with this hypothesis.

Keywords: RGD gradient, Cell adhesion, Poly(methyl methacrylate), Hydrolysis, Biotin-streptavidin

Azevedo, S., Diéguez, L., Carvalho, P., Carneiro, J. O., Teixeira, V., Martínez, E., Samitier, J., (2012). Deposition of ITO thin films onto PMMA substrates for waveguide based biosensing devices Journal of Nano Research 17, 75-83

Biosensors' research filed has clearly been changing towards the production of multifunctional and innovative design concepts to address the needs related with sensitivity and selectivity of the devices. More recently, waveguide biosensors, that do not require any label procedure to detect biomolecules adsorbed on its surface, have been pointed out as one of the most promising technologies for the production of biosensing devices with enhanced performance. Moreover the combination of optical and electrochemical measurements through the integration of transparent and conducting oxides in the multilayer structures can greatly enhance the biosensors' sensitivity. Furthermore, the integration of polymeric substrates may bring powerful advantages in comparison with silicon based ones. The biosensors will have a lower production costs being possible to disposable them after use ("one use sensor chip"). This research work represents a preliminary study about the influence of substrate temperature on the overall properties of ITO thin films deposited by DC magnetron sputtering onto 0,5 mm thick PMMA sheets.

Keywords: ITO thin films, PMMA sheets, Waveguide biosensing devices, Biosensing devices, Conducting oxides, Dc magnetron sputtering, Electrochemical measurements, Enhanced performance, Innovative design, ITO thin films, Multilayer structures, Overall properties, PMMA sheets, Polymeric substrate, Production cost, Sensor chips, Silicon-based, Substrate temperature, Biosensors, Deposition, Design, Film preparation, Optical multilayers, Thin films, Vapor deposition, Waveguides, Substrates

Martínez, E., Pla, M., Samitier, J., (2012). Micro/nanopatterning of proteins using a nanoimprint-based contact printing technique Nanotechnology in Regenerative Medicine - Methods and Protocols (Methods in Molecular Biology) (ed. Navarro, M., Planell, J. A.), Springer (New York, USA) 811, 79-87

Micro and nanoscale protein patterning based on microcontact printing technique on large substrates have often resolution problems due to roof collapse of the poly(dimethylsiloxane) (PDMS) stamps used. Here, we describe a technique that overcomes these issues by using instead a stamp made of poly(methyl methacrylate) (PMMA), a much more rigid polymer that do not collapse even using stamps with very high aspect ratios (up to 300:1). Conformal contact between the stamp and the substrate is achieved because of the homogeneous pressure applied via the nanoimprint lithography instrument, and it has allowed us to print lines of protein 150 nm wide, at a 400 nm period. This technique, therefore, provides an excellent method for the direct printing of high-density submicrometer scale patterns, or, alternatively, micro/nanopatterns spaced at large distances.

Keywords: Microcontact printing, Nanoimprint lithography, Poly(methyl methacrylate), Protein

Issle, J., Pla, M., Martínez, E., Hartmann, U., (2008). Patterning of magnetic nanobeads on surfaces by poly(dimethylsiloxane) stamps Langmuir 24, (3), 888-893

Poly(dimethylsiloxane) (PDMS) stamps are widely used in soft lithographic methods. They are powerful tools for obtaining structures of soft material in the micrometer to nanometer range by printing techniques. In this contribution, a new application of h-PDMS stamps for nanobead deposition is introduced. Magnetite-polysaccharide particles of an average diameter of 150 nm are used. They can be biologically functionalized by attaching various molecular groups. Deposition of these particles on a carrier substrate results in well-reproducible structures. This is achieved by means of PDMS stamps with different patterns using a microfluidic approach on one hand and a printing approach on the other hand. Furthermore, magnetic substrates with particular domain structures have been used. The beads can then be arranged in rather complicated but well-defined geometrical structures along the domain walls. The magnetic interaction considerably increases the adhesion of the beads to the carrier substrate. All involved materials are biocompatible. Thus the setup can be used in cell culture experiments in order to investigate influences of different particle-bound proteins and particle patterns on cell growth and vitality.

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