Staff member

Elena Garreta Bahima

Senior Researcher
Pluripotent Stem Cells and Activation of Endogenous Tissue Programs for Organ Regeneration
+34 934 020 424
Staff member publications

Garreta, Elena, Oria, Roger, Tarantino, Carolina, Pla-Roca, Mateu, Prado, Patricia, Fernández-Avilés, Francisco, Campistol, Josep Maria, Samitier, Josep, Montserrat, Nuria, (2017). Tissue engineering by decellularization and 3D bioprinting Materials Today 20, (4), 166-178

Discarded human donor organs have been shown to provide decellularized extracellular matrix (dECM) scaffolds suitable for organ engineering. The quest for appropriate cell sources to satisfy the need of multiple cells types in order to fully repopulate human organ-derived dECM scaffolds has opened new venues for the use of human pluripotent stem cells (hPSCs) for recellularization. In addition, three-dimensional (3D) bioprinting techniques are advancing towards the fabrication of biomimetic cell-laden biomaterial constructs. Here, we review recent progress in decellularization/recellularization and 3D bioprinting technologies, aiming to fabricate autologous tissue grafts and organs with an impact in regenerative medicine.

Garreta, Elena, Marco, Andres, Izpisua Belmonte, Juan Carlos, Montserrat, Nuria, (2016). Genome editing in human pluripotent stem cells: a systematic approach unrevealing pancreas development and disease Stem Cell Investigation 4, (11), 1-4

Although mouse models have represented a major tool for understanding and predicting molecular mechanisms responsible for several human genetic diseases, still species-specific differences between mouse and humans in their biochemical and physiological characteristics represent a major hurdle when translating promising findings into the human setting (1). For instance, in several types of maturity onset diabetes of the young (MODY; autosomal dominant), mice with heterozygous mutations do not develop diabetes (2). In this regard, the derivation of human embryonic stem cells (hESCs) in 1998 represented an unprecedented opportunity for human disease modelling, and a promising source for cell replacement therapies (3). Later on, the possibility to generate patient-derived induced pluripotent stem cells (iPSCs) has opened new venues for the potential translation of stem-cell related studies into the clinic (4).

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

Montserrat, N., Garreta, E., Izpisua Belmonte, J. C., (2016). Regenerative strategies for kidney engineering FEBS Journal 283, (18), 3303-3324

The kidney is the most important organ for water homeostasis and waste excretion. It performs several important physiological functions for homeostasis: it filters the metabolic waste out of circulation, regulates body fluid balances, and acts as an immune regulator and modulator of cardiovascular physiology. The development of in vitro renal disease models with pluripotent stem cells (both human embryonic stem cells and induced pluripotent stem cells) and the generation of robust protocols for in vitro derivation of renal-specific-like cells from patient induced pluripotent stem cells have just emerged. Here we review major findings in the field of kidney regeneration with a major focus on the development of stepwise protocols for kidney cell production from human pluripotent stem cells and the latest advances in kidney bioengineering (i.e. decellularized kidney scaffolds and bioprinting). The possibility of generating renal-like three-dimensional structures to be recellularized with renal-derived induced pluripotent stem cells may offer new avenues to develop functional kidney grafts on-demand.

Keywords: Induced pluripotent stem cells, Kidney disease, Kidney engineering, Pluripotent stem cells, Renal differentiation

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

Melo, E., Garreta, E., Luque, T., Cortiella, J., Nichols, J., Navajas, D., Farré, R., (2014). Effects of the decellularization method on the local stiffness of acellular lungs Tissue Engineering Part C: Methods 20, (5), 412-422

Lung bioengineering, a novel approach to obtain organs potentially available for transplantation, is based on decellularizing donor lungs and seeding natural scaffolds with stem cells. Various physicochemical protocols have been used to decellularize lungs, and their performance has been evaluated in terms of efficient decellularization and matrix preservation. No data are available, however, on the effect of different decellularization procedures on the local stiffness of the acellular lung. This information is important since stem cells directly sense the rigidity of the local site they are engrafting to during recellularization, and it has been shown that substrate stiffness modulates cell fate into different phenotypes. The aim of this study was to assess the effects of the decellularization procedure on the inhomogeneous local stiffness of the acellular lung on five different sites: alveolar septa, alveolar junctions, pleura, and vessels' tunica intima and tunica adventitia. Local matrix stiffness was measured by computing Young's modulus with atomic force microscopy after decellularizing the lungs of 36 healthy rats (Sprague-Dawley, male, 250–300 g) with four different protocols with/without perfusion through the lung circulatory system and using two different detergents (sodium dodecyl sulfate [SDS] and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate [CHAPS]). The local stiffness of the acellular lung matrix significantly depended on the site within the matrix (p<0.001), ranging from ∼15 kPa at the alveolar septum to ∼60 kPa at the tunica intima. Acellular lung stiffness (p=0.003) depended significantly, albeit modestly, on the decellularization process. Whereas perfusion did not induce any significant differences in stiffness, the use of CHAPS resulted in a ∼35% reduction compared with SDS, the influence of the detergent being more important in the tunica intima. In conclusion, lung matrix stiffness is considerably inhomogeneous, and conventional decellularization procedures do not result in substantially different local stiffness in the acellular lung.

Melo, E., Cárdenes, N., Garreta, E., Luque, T., Rojas, M., Navajas, D., Farré, R., (2014). Inhomogeneity of local stiffness in the extracellular matrix scaffold of fibrotic mouse lungs Journal of the Mechanical Behavior of Biomedical Materials 37, 186-195

Lung disease models are useful to study how cell engraftment, proliferation and differentiation are modulated in lung bioengineering. The aim of this work was to characterize the local stiffness of decellularized lungs in aged and fibrotic mice. Mice (2- and 24-month old; 14 of each) with lung fibrosis (N=20) and healthy controls (N=8) were euthanized after 11 days of intratracheal bleomycin (fibrosis) or saline (controls) infusion. The lungs were excised, decellularized by a conventional detergent-based (sodium-dodecyl sulfate) procedure and slices of the acellular lungs were prepared to measure the local stiffness by means of atomic force microscopy. The local stiffness of the different sites in acellular fibrotic lungs was very inhomogeneous within the lung and increased according to the degree of the structural fibrotic lesion. Local stiffness of the acellular lungs did not show statistically significant differences caused by age. The group of mice most affected by fibrosis exhibited local stiffness that were ~2-fold higher than in the control mice: from 27.2±1.64 to 64.8±7.1. kPa in the alveolar septa, from 56.6±4.6 to 99.9±11.7. kPa in the visceral pleura, from 41.1±8.0 to 105.2±13.6. kPa in the tunica adventitia, and from 79.3±7.2 to 146.6±28.8. kPa in the tunica intima. Since acellular lungs from mice with bleomycin-induced fibrosis present considerable micromechanical inhomogeneity, this model can be a useful tool to better investigate how different degrees of extracellular matrix lesion modulate cell fate in the process of organ bioengineering from decellularized lungs.

Keywords: Ageing, Atomic force microscopy, Decellularization, Lung fibrosis, Tissue engineering, Atomic force microscopy, Biological organs, Peptides, Sodium dodecyl sulfate, Sodium sulfate, Tissue engineering, Ageing, Decellularization, Extracellular matrices, Healthy controls, Inhomogeneities, Lung fibrosis, Micro-mechanical, Statistically significant difference, Mammals, bleomycin, adventitia, animal experiment, animal model, article, atomic force microscopy, bleomycin-induced pulmonary fibrosis, cell fate, controlled study, extracellular matrix, female, intima, lung alveolus, lung fibrosis, lung mechanics, mechanical probe, microenvironment, mouse, nonhuman, pleura, priority journal, rigidity, tissue engineering

Nonaka, P. N., Campillo, N., Uriarte, J. J., Garreta, E., Melo, E., de Oliveira, L. V. F., Navajas, D., Farré, R., (2014). Effects of freezing/thawing on the mechanical properties of decellularized lungs Journal of Biomedical Materials Research - Part A 102, (2), 413-419

Lung bioengineering based on decellularized organ scaffolds is a potential alternative for transplantation. Freezing/thawing, a usual procedure in organ decellularization and storage could modify the mechanical properties of the lung scaffold and reduce the performance of the bioengineered lung when subjected to the physiological inflation-deflation breathing cycles. The aim of this study was to determine the effects of repeated freezing/thawing on the mechanical properties of decellularized lungs in the physiological pressure-volume regime associated with normal ventilation. Fifteen mice lungs (C57BL/6) were decellularized using a conventional protocol not involving organ freezing and based on sodium dodecyl sulfate detergent. Subsequently, the mechanical properties of the acellular lungs were measured before and after subjecting them to three consecutive cycles of freezing/thawing. The resistance (RL) and elastance (EL) of the decellularized lungs were computed by linear regression fitting of the recorded signals (tracheal pressure, flow, and volume) during mechanical ventilation. RL was not significantly modified by freezing-thawing: from 0.88 ± 0.37 to 0.90 ± 0.38 cmH2O·s·mL-1 (mean ± SE). EL slightly increased from 64.4 ± 11.1 to 73.0 ± 16.3 cmH2O·mL-1 after the three freeze-thaw cycles (p = 0.0013). In conclusion, the freezing/thawing process that is commonly used for both organ decellularization and storage induces only minor changes in the ventilation mechanical properties of the organ scaffold.

Keywords: Elastance, Freezing/thawing, Lung bioengineering, Lung decellularization, Mechanical ventilation, Organ scaffold

Luque, T., Melo, E., Garreta, E., Cortiella, J., Nichols, J., Farré, R., Navajas, D., (2013). Local micromechanical properties of decellularized lung scaffolds measured with atomic force microscopy Acta Biomaterialia 9, (6), 6852-6859

Bioartificial lungs re-engineered from decellularized organ scaffolds are a promising alternative to lung transplantation. Critical features for improving scaffold repopulation depend on the mechanical properties of the cell microenvironment. However, the mechanics of the lung extracellular matrix (ECM) is poorly defined. The local mechanical properties of the ECM were measured in different regions of decellularized rat lung scaffolds with atomic force microscopy. Lungs excised from rats (n = 11) were decellularized with sodium dodecyl sulfate (SDS) and cut into ∼7 μm thick slices. The complex elastic modulus (G*) of lung ECM was measured over a frequency band ranging from 0.1 to 11.45 Hz. Measurements were taken in alveolar wall segments, alveolar wall junctions and pleural regions. The storage modulus (G′, real part of G*) of alveolar ECM was ∼6 kPa, showing small changes between wall segments and junctions. Pleural regions were threefold stiffer than alveolar walls. G′ of alveolar walls and pleura increased with frequency as a weak power law with exponent 0.05. The loss modulus (G″, imaginary part of G*) was 10-fold lower and showed a frequency dependence similar to that of G′ at low frequencies (0.1-1 Hz), but increased more markedly at higher frequencies. Local differences in mechanical properties and topology of the parenchymal site could be relevant mechanical cues for regulating the spatial distribution, differentiation and function of lung cells.

Tsapikouni, T., Garreta, E., Melo, E., Navajas, D., Farré, R., (2012). A bioreactor for subjecting cultured cells to fast-rate intermittent hypoxia Respiratory Physiology & Neurobiology 182, (1), 47-52

High frequency intermittent hypoxia is one of the most relevant injurious stimuli experienced by patients with obstructive sleep apnea (OSA). Given that the conventional setting for culturing cells under intermittent hypoxia conditions is limited by long equilibration times, we designed a simple bioreactor capable of effectively subjecting cultured cells to controlled high-frequency hypoxic/normoxic stimuli. The bioreactor's operation is based on exposing cells to a medium that is bubbled with the appropriate mixture of gases into two separate containers, and from there it is directed to the cell culture dish with the aid of two bidirectional peristaltic pumps. The device was tested on human alveolar epithelial cells (A549) and mouse melanoma cells (B16-F10), subjecting them to patterns of intermittent hypoxia (20s at 5% O 2 and 50s at 20% O 2), which realistically mimic OSA of up to severe intensity as defined by the apnea hypopnea index. The proposed bioreactor can be easily and inexpensively assembled and is of practical use for investigating the effects of high-rate changes in oxygen concentration in the cell culture medium.

Keywords: Hypoxia-reoxygenation, Obstructive sleep apnea, Oxygen partial pressure

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