Publications

Cardiac fibrosis occurs following insults to the myocardium and is characterized by the abnormal accumulation of non-compliant extracellular matrix (ECM), which compromises cardiomyocyte contractile activity and eventually leads to heart failure. This phenomenon is driven by the activation of cardiac fibroblasts (cFbs) to myofibroblasts and results in changes in ECM biochemical, structural and mechanical properties. The lack of predictive in vitro models of heart fibrosis has so far hampered the search for innovative treatments, as most of the cellular-based in vitro reductionist models do not take into account the leading role of ECM cues in driving the progression of the pathology. Here, we devised a single-step decellularization protocol to obtain and thoroughly characterize the biochemical and micro-mechanical properties of the ECM secreted by activated cFbs differentiated from human induced pluripotent stem cells (iPSCs). We activated iPSC-derived cFbs to the myofibroblast phenotype by tuning basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-beta 1) signalling and confirmed that activated cells acquired key features of myofibroblast phenotype, like SMAD2/3 nuclear shuttling, the formation of aligned alpha-smooth muscle actin (alpha- SMA)-rich stress fibres and increased focal adhesions (FAs) assembly. Next, we used Mass Spectrometry, nanoindentation, scanning electron and confocal microscopy to unveil the characteristic composition and the visco-elastic properties of the abundant, collagen-rich ECM deposited by cardiac myofibroblasts in vitro. Finally, we demonstrated that the fibrotic ECM activates mechanosensitive pathways in iPSC-derived cardiomyocytes, impacting on their shape, sarcomere assembly, phenotype, and calcium handling properties. We thus propose human bio-inspired decellularized matrices as animal-free, isogenic cardiomyocyte culture substrates recapitulating key pathophysiological changes occurring at the cellular level during cardiac fibrosis.

JTD Keywords: Adhesio, Cardiac fibrosis modelling, Decellularized extracellular matrix, Differentiation, Expression, Fibroblast activation, Fibronectin, Heart, Induced pluripotent stem cells, Ipsc-derived-cardiac fibroblasts, Ipsc-derived-cardiomyocyte, Myocardial-infarction, Neonatal cardiomyocytes, Smooth muscle actin, Substrate stiffness

JTD Keywords: 3d printing, Biofabrication, Cardiac tissue engineering, Human heart, Human induced pluripotent stem cells

In situ tissue engineering strategies are a promising approach to activate the endogenous regenerative potential of the cardiac tissue helping the heart to heal itself after an injury. However, the current use of complex reprogramming vectors for the activation of reparative pathways challenges the easy translation of these therapies into the clinic. Here, we evaluated the response of mouse neonatal and human induced pluripotent stem cell-derived cardiomyocytes to the presence of exogenous lactate, thus mimicking the metabolic environment of the fetal heart. An increase in cardiomyocyte cell cycle activity was observed in the presence of lactate, as determined through Ki67 and Aurora-B kinase. Gene expression and RNA-sequencing data revealed that cardiomyocytes incubated with lactate showed upregulation of BMP10, LIN28 or TCIM in tandem with downregulation of GRIK1 or DGKK among others. Lactate also demonstrated a capability to modulate the production of inflammatory cytokines on cardiac fibroblasts, reducing the production of Fas, Fraktalkine or IL-12p40, while stimulating IL-13 and SDF1a. In addition, the generation of a lactate-rich environment improved ex vivo neonatal heart culture, by affecting the contractile activity and sarcomeric structures and inhibiting epicardial cell spreading. Our results also suggested a common link between the effect of lactate and the activation of hypoxia signaling pathways. These findings support a novel use of lactate in cardiac tissue engineering, modulating the metabolic environment of the heart and thus paving the way to the development of lactate-releasing platforms for in situ cardiac regeneration.Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.

JTD Keywords: cardiac regeneration, cardiac tissue engineering, cell cycle, failure, growth, heart regeneration, induced pluripotent stem cells, ischemia, lactate, metabolic environment, metabolism, mouse, proliferation, repair, Bone morphogenetic protein-10, Cardiac tissue engineering, Cardiomyocytes, Cell cycle, Induced pluripotent stem cells, Lactate, Metabolic environment

In vitro surrogate models of human cardiac tissue hold great promise in disease modeling, cardiotoxicity testing, and future applications in regenerative medicine. However, the generation of engineered human cardiac constructs with tissue-like functionality is currently thwarted by difficulties in achieving efficient maturation at the cellular and/or tissular level. Here, we report on the design and implementation of a platform for the production of engineered cardiac macrotissues from human pluripotent stem cells (PSCs), which we term “CardioSlice.” PSC-derived cardiomyocytes, together with human fibroblasts, are seeded into large 3D porous scaffolds and cultured using a parallelized perfusion bioreactor with custom-made culture chambers. Continuous electrical stimulation for 2 weeks promotes cardiomyocyte alignment and synchronization, and the emergence of cardiac tissue-like properties. These include electrocardiogram-like signals that can be readily measured on the surface of CardioSlice constructs, and a response to proarrhythmic drugs that is predictive of their effect in human patients.

JTD Keywords: Cardiac tissue engineering, CardioSlice, ECG-like signals, Electrical stimulation, Heart physiology, Human induced pluripotent stem cells, Perfusion bioreactor, Tissue-like properties

Gerstmann-Sträussler-Scheinker (GSS) syndrome is a fatal autosomal dominant neurodegenerative prionopathy clinically characterized by ataxia, spastic paraparesis, extrapyramidal signs and dementia. In some GSS familiar cases carrying point mutations in the PRNP gene, patients also showed comorbid tauopathy leading to mixed pathologies. In this study we developed an induced pluripotent stem (iPS) cell model derived from fibroblasts of a GSS patient harboring the Y218N PRNP mutation, as well as an age-matched healthy control. This particular PRNP mutation is unique with very few described cases. One of the cases presented neurofibrillary degeneration with relevant Tau hyperphosphorylation. Y218N iPS-derived cultures showed relevant astrogliosis, increased phospho-Tau, altered microtubule-associated transport and cell death. However, they failed to generate proteinase K-resistant prion. In this study we set out to test, for the first time, whether iPS cell-derived neurons could be used to investigate the appearance of disease-related phenotypes (i.e, tauopathy) identified in the GSS patient.

JTD Keywords: Cellular prion protein, Gerstmann-Sträussler-Scheinker, Induced pluripotent stem cells, Tau

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.

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

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.

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

Fanconi anemia (FA) is a complex genetic disease associated with a defective DNA repair pathway known as the FA pathway. In contrast to many other FA proteins, BRCA2 participates downstream in this pathway and has a critical role in homology-directed recombination (HDR). In our current studies, we have observed an extremely low reprogramming efficiency in cells with a hypomorphic mutation in Brca2 (Brca2Δ27/Δ27), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2Δ27/Δ27 induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2Δ27/Δ27 iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2 Δ27/Δ27 mouse embryonic fibroblasts. Gene-corrected Brca2Δ27/Δ27 iPSCs could be differentiated in vitro toward the hematopoietic lineage, although with a more limited efficacy than WT iPSCs or mouse embryonic stem cells, and did not engraft in irradiated Brca2Δ27/Δ27 recipients. Our results are consistent with previous studies proposing that HDR is critical for cell reprogramming and demonstrate that reprogramming defects characteristic of Brca2 mutant cells can be efficiently overcome by gene complementation. Finally, based on analysis of the phenotype, genetic stability, and hematopoietic differentiation potential of gene-corrected Brca2Δ27/Δ27 iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed.

JTD Keywords: Bone marrow aplasia, Cellular therapy, Fanconi anemia, Gene therapy, Hematopoietic stem cells, Induced pluripotent stem cells

A critical step in the development of effective therapeutics to treat Parkinson's disease (PD) is the identification of molecular pathogenic mechanisms underlying this chronically progressive neurodegenerative disease. However, while animal models have provided valuable information about the molecular basis of PD, the lack of faithful cellular and animal models that recapitulate human pathophysiology is delaying the development of new therapeutics. The reprogramming of somatic cells to induced pluripotent stem cells (iPSC) using delivery of defined combinations of transcription factors is a groundbreaking discovery that opens great opportunities for modeling human diseases, including PD, since iPSC can be generated from patients and differentiated into disease-relevant cell types, which would capture the patients' genetic complexity. Furthermore, human iPSC-derived neuronal models offer unprecedented access to early stages of the disease, allowing the investigation of the events that initiate the pathologic process in PD. Recently, human iPSC-derived neurons from patients with familial and sporadic PD have been generated and importantly they recapitulate some PD-related cell phenotypes, including abnormal α-synuclein accumulation in vitro, and alterations in the autophagy machinery. This review highlights the current PD iPSC-based models and discusses the potential future research directions of this field.

JTD Keywords: Human cellular model, Induced pluripotent stem cells, Neurodegenerative disease, Parkinson's disease