Fibrotic extracellular matrix impacts cardiomyocyte phenotype and function in an iPSC-derived isogenic model of cardiac fibrosis.

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-ß1) 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 (α-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.

YAP Signaling Regulates the Cellular Uptake and Therapeutic Effect of Nanoparticles.

Interactions between living cells and nanoparticles are extensively studied to enhance the delivery of therapeutics. Nanoparticles size, shape, stiffness, and surface charge are regarded as the main features able to control the fate of cell-nanoparticle interactions. However, the clinical translation of nanotherapies has so far been limited, and there is a need to better understand the biology of cell-nanoparticle interactions. This study investigates the role of cellular mechanosensitive components in cell-nanoparticle interactions. It is demonstrated that the genetic and pharmacologic inhibition of yes-associated protein (YAP), a key component of cancer cell mechanosensing apparatus and Hippo pathway effector, improves nanoparticle internalization in triple-negative breast cancer cells regardless of nanoparticle properties or substrate characteristics. This process occurs through YAP-dependent regulation of endocytic pathways, cell mechanics, and membrane organization. Hence, the study proposes targeting YAP may sensitize triple-negative breast cancer cells to chemotherapy and increase the selectivity of nanotherapy.

TGF-ß induces matrisome pathological alterations and EMT in patient-derived prostate cancer tumoroids.

Extracellular matrix (ECM) tumorigenic alterations resulting in high matrix deposition and stiffening are hallmarks of adenocarcinomas and are collectively defined as desmoplasia. Here, we thoroughly analysed primary prostate cancer tissues obtained from numerous patients undergoing radical prostatectomy to highlight reproducible structural changes in the ECM leading to the loss of the glandular architecture. Starting from patient cells, we established prostate cancer tumoroids (PCTs) and demonstrated they require TGF-ß signalling pathway activity to preserve phenotypical and structural similarities with the tissue of origin. By modulating TGF-ß signalling pathway in PCTs, we unveiled its role in ECM accumulation and remodelling in prostate cancer. We also found that TGF-ß-induced ECM remodelling is responsible for the initiation of prostate cell epithelial-to-mesenchymal transition (EMT) and the acquisition of a migratory, invasive phenotype. Our findings highlight the cooperative role of TGF-ß signalling and ECM desmoplasia in prompting prostate cell EMT and promoting tumour progression and dissemination.

Graphene-based 3D-Printed nanocomposite bioelectronics for monitoring breast cancer cell adhesion.

This work examines the suitability of graphene-based 3D-printed nanocomposite bioelectronics as innovative systems to in situ monitor and evaluate both breast cancer cell adhesion and the chemosensitivity of anti-cancer drugs. With this aim, 3D-printed nanocomposite graphene electrodes (3D-nGEs) -made of a commercially available graphene/polylactic acid filament- have been covalently biofunctionalized with an extracellular matrix protein (i.e., fibronectin) by exploiting the carbon reactivity of 3D-nGEs. The specificity and selectivity of the developed electrochemical system to monitor breast cancer cell adhesion has been tested via electrochemical impedance spectroscopy (EIS). Importantly, the resulting 3D-printed bioelectronic system displayed excellent accuracy for the rapid screening of anti-cancer drugs, which exactly corresponded with the results achieved by the standard optical method, while having the advantage of employing a label-free approach. In light of the current state-of-the-art in the field, this proof-of-concept connects electronics to biological systems within 3D printing technology, providing the bases for the sustainable and cost-effective manufacturing of graphene-based 3D-printed nanocomposite bioelectronics to simulate in vivo microenvironments using in situ and real time electronic output signals.

Generation and maturation of human iPSC-derived 3D organotypic cardiac microtissues in long-term culture.

Cardiovascular diseases remain the leading cause of death worldwide; hence there is an increasing focus on developing physiologically relevant in vitro cardiovascular tissue models suitable for studying personalized medicine and pre-clinical tests. Despite recent advances, models that reproduce both tissue complexity and maturation are still limited. We have established a scaffold-free protocol to generate multicellular, beating human cardiac microtissues in vitro from hiPSCs-namely human organotypic cardiac microtissues (hOCMTs)-that show some degree of self-organization and can be cultured for long term. This is achieved by the differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated hOCMTs contain multiple cell types that physiologically compose the heart and beat without external stimuli for more than 100 days. We have shown that 3D hOCMTs display improved cardiac specification, survival and metabolic maturation as compared to standard monolayer cardiac differentiation. We also confirmed the functionality of hOCMTs by their response to cardioactive drugs in long-term culture. Furthermore, we demonstrated that they could be used to study chemotherapy-induced cardiotoxicity. Due to showing a tendency for self-organization, cellular heterogeneity, and functionality in our 3D microtissues over extended culture time, we could also confirm these constructs as human cardiac organoids (hCOs). This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.

Multiscale Analysis of Extracellular Matrix Remodeling in the Failing Heart.

RATIONALE: Cardiac ECM (extracellular matrix) comprises a dynamic molecular network providing structural support to heart tissue function. Understanding the impact of ECM remodeling on cardiac cells during heart failure (HF) is essential to prevent adverse ventricular remodeling and restore organ functionality in affected patients. OBJECTIVES: We aimed to (1) identify consistent modifications to cardiac ECM structure and mechanics that contribute to HF and (2) determine the underlying molecular mechanisms. METHODS AND RESULTS: We first performed decellularization of human and murine ECM (decellularized ECM) and then analyzed the pathological changes occurring in decellularized ECM during HF by atomic force microscopy, 2-photon microscopy, high-resolution 3-dimensional image analysis, and computational fluid dynamics simulation. We then performed molecular and functional assays in patient-derived cardiac fibroblasts based on YAP (yes-associated protein)-transcriptional enhanced associate domain (TEAD) mechanosensing activity and collagen contraction assays. The analysis of HF decellularized ECM resulting from ischemic or dilated cardiomyopathy, as well as from mouse infarcted tissue, identified a common pattern of modifications in their 3-dimensional topography. As compared with healthy heart, HF ECM exhibited aligned, flat, and compact fiber bundles, with reduced elasticity and organizational complexity. At the molecular level, RNA sequencing of HF cardiac fibroblasts highlighted the overrepresentation of dysregulated genes involved in ECM organization, or being connected to TGFß1 (transforming growth factor ß1), interleukin-1, TNF-α, and BDNF signaling pathways. Functional tests performed on HF cardiac fibroblasts pointed at mechanosensor YAP as a key player in ECM remodeling in the diseased heart via transcriptional activation of focal adhesion assembly. Finally, in vitro experiments clarified pathological cardiac ECM prevents cell homing, thus providing further hints to identify a possible window of action for cell therapy in cardiac diseases. CONCLUSIONS: Our multiparametric approach has highlighted repercussions of ECM remodeling on cell homing, cardiac fibroblast activation, and focal adhesion protein expression via hyperactivated YAP signaling during HF.

YAP-TEAD1 control of cytoskeleton dynamics and intracellular tension guides human pluripotent stem cell mesoderm specification.

The tight regulation of cytoskeleton dynamics is required for a number of cellular processes, including migration, division and differentiation. YAP-TEAD respond to cell-cell interaction and to substrate mechanics and, among their downstream effects, prompt focal adhesion (FA) gene transcription, thus contributing to FA-cytoskeleton stability. This activity is key to the definition of adult cell mechanical properties and function. Its regulation and role in pluripotent stem cells are poorly understood. Human PSCs display a sustained basal YAP-driven transcriptional activity despite they grow in very dense colonies, indicating these cells are insensitive to contact inhibition. PSC inability to perceive cell-cell interactions can be restored by tampering with Tankyrase enzyme, thus favouring AMOT inhibition of YAP function. YAP-TEAD complex is promptly inactivated when germ layers are specified, and this event is needed to adjust PSC mechanical properties in response to physiological substrate stiffness. By providing evidence that YAP-TEAD1 complex targets key genes encoding for proteins involved in cytoskeleton dynamics, we suggest that substrate mechanics can direct PSC specification by influencing cytoskeleton arrangement and intracellular tension. We propose an aberrant activation of YAP-TEAD1 axis alters PSC potency by inhibiting cytoskeleton dynamics, thus paralyzing the changes in shape requested for the acquisition of the given phenotype.

Substrate mechanics controls adipogenesis through YAP phosphorylation by dictating cell spreading.

The mechanoregulated proteins YAP/TAZ are involved in the adipogenic/osteogenic switch of mesenchymal stem cells (MSCs). MSC fate decision can be unbalanced by controlling substrate mechanics, in turn altering the transmission of tension through cell cytoskeleton. MSCs have been proposed for orthopedic and reconstructive surgery applications. Thus, a tight control of their adipogenic potential is required in order to avoid their drifting towards fat tissue. Substrate mechanics has been shown to drive MSC commitment and to regulate YAP/TAZ protein shuttling and turnover. The mechanism by which YAP/TAZ co-transcriptional activity is mechanically regulated during MSC fate acquisition is still debated. Here, we design few bioengineering tools suited to disentangle the contribution of mechanical from biological stimuli to MSC adipogenesis. We demonstrate that the mechanical repression of YAP happens through its phosphorylation, is purely mediated by cell spreading downstream of substrate mechanics as dictated by dimensionality. YAP repression is sufficient to prompt MSC adipogenesis, regardless of a permissive biological environment, TEAD nuclear presence or focal adhesion stabilization. Finally, by harnessing the potential of YAP mechanical regulation, we propose a practical example of the exploitation of adipogenic transdifferentiation in tumors.

Advanced and Rationalized Atomic Force Microscopy Analysis Unveils Specific Properties of Controlled Cell Mechanics.

The cell biomechanical properties play a key role in the determination of the changes during the essential cellular functions, such as contraction, growth, and migration. Recent advances in nano-technologies have enabled the development of new experimental and modeling approaches to study cell biomechanics, with a level of insights and reliability that were not possible in the past. The use of atomic force microscopy (AFM) for force spectroscopy allows nanoscale mapping of the cell topography and mechanical properties under, nearly physiological conditions. A proper evaluation process of such data is an essential factor to obtain accurate values of the cell elastic properties (primarily Young's modulus). Several numerical models were published in the literature, describing the depth sensing indentation as interaction process between the elastic surface and indenting probe. However, many studies are still relying on the nowadays outdated Hertzian model from the nineteenth century, or its modification by Sneddon. The lack of comparison between the Hertz/Sneddon model with their modern modifications blocks the development of advanced analysis software and further progress of AFM promising technology into biological sciences. In this work, we applied a rationalized use of mechanical models for advanced postprocessing and interpretation of AFM data. We investigated the effect of the mechanical model choice on the final evaluation of cellular elasticity. We then selected samples subjected to different physicochemical modulators, to show how a critical use of AFM data handling can provide more information than simple elastic modulus estimation. Our contribution is intended as a methodological discussion of the limitations and benefits of AFM-based advanced mechanical analysis, to refine the quantification of cellular elastic properties and its correlation to undergoing cellular processes in vitro.

YAP regulates cell mechanics by controlling focal adhesion assembly.

Hippo effectors YAP/TAZ act as on-off mechanosensing switches by sensing modifications in extracellular matrix (ECM) composition and mechanics. The regulation of their activity has been described by a hierarchical model in which elements of Hippo pathway are under the control of focal adhesions (FAs). Here we unveil the molecular mechanism by which cell spreading and RhoA GTPase activity control FA formation through YAP to stabilize the anchorage of the actin cytoskeleton to the cell membrane. This mechanism requires YAP co-transcriptional function and involves the activation of genes encoding for integrins and FA docking proteins. Tuning YAP transcriptional activity leads to the modification of cell mechanics, force development and adhesion strength, and determines cell shape, migration and differentiation. These results provide new insights into the mechanism of YAP mechanosensing activity and qualify this Hippo effector as the key determinant of cell mechanics in response to ECM cues.

Distinct prostate cancer-related mRNA cargo in extracellular vesicle subsets from prostate cell lines.

BACKGROUND: Multiple types of extracellular vesicles (EVs), including microvesicles (MVs) and exosomes (EXOs), are released by all cells constituting part of the cellular EV secretome. The bioactive cargo of EVs can be shuffled between cells and consists of lipids, metabolites, proteins, and nucleic acids, including multiple RNA species from non-coding RNAs to messenger RNAs (mRNAs). In this study, we hypothesized that the mRNA cargo of EVs could differ based on the EV cellular origin and subpopulation analyzed. METHODS: We isolated MVs and EXOs from PC-3 and LNCaP prostate cancer cells by differential centrifugation and compared them to EVs derived from the benign PNT2 prostate cells. The relative mRNA levels of 84 prostate cancer-related genes were investigated and validated using quantitative reverse transcription PCR arrays. RESULTS: Based on the mRNA abundance, MVs rather than EXOs were enriched in the analyzed transcripts, providing a snapshot of the tumor transcriptome. LNCaP MVs specifically contained significantly increased mRNA levels of NK3 Homeobox 1 (NKX3-1), transmembrane protease serine 2 (TMPRSS2), and tumor protein 53 (TP53) genes, whereas PC-3 MVs carried increased mRNA levels of several genes including, caveolin-2 (CAV2), glutathione S-transferase pi 1 (GSTP1), pescadillo ribosomal biogenesis factor 1 (PES1), calmodulin regulated spectrin associated protein 1 (CAMSAP1), zinc-finger protein 185 (ZNF185), and others compared to PNT2 MVs. Additionally, ETS variant 1 (ETV1) and fatty acid synthase (FASN) mRNAs identified in LNCaP- and PC-3- derived MVs highly correlated with prostate cancer progression. CONCLUSIONS: Our study provides new understandings of the variability of the mRNA cargo of MVs and EXOs from different cell lines despite same cancer origin, which is essential to better understand the the proportion of the cell transcriptome that can be detected within EVs and to evaluate their role in disease diagnosis.

Cancer stem cells from human glioblastoma resemble but do not mimic original tumors after in vitro passaging in serum-free media.

Human gliomas harbour cancer stem cells (CSCs) that evolve along the course of the disease, forming highly heterogeneous subpopulations within the tumour mass. These cells possess self-renewal properties and appear to contribute to tumour initiation, metastasis and resistance to therapy. CSC cultures isolated from surgical samples are considered the best preclinical in vitro model for primary human gliomas. However, it is not yet well characterized to which extent their biological and functional properties change during in vitro passaging in the serum-free culture conditions. Here, we demonstrate that our CSC-enriched cultures harboured from one to several CSC clones from the human glioma sample. When xenotransplanted into mouse brain, these cells generated tumours that reproduced at least three different dissemination patterns found in original tumours. Along the passages in culture, CSCs displayed increased expression of stem cell markers, different ratios of chromosomal instability events, and a varied response to drug treatment. Our findings highlight the need for better characterization of CSC-enriched cultures in the context of their evolution in vitro, in order to uncover their full potential as preclinical models in the studies aimed at identifying molecular biomarkers and developing new therapeutic approaches of human gliomas.

Heart valve tissue engineering: how far is the bedside from the bench?

Heart disease, including valve pathologies, is the leading cause of death worldwide. Despite the progress made thanks to improving transplantation techniques, a perfect valve substitute has not yet been developed: once a diseased valve is replaced with current technologies, the newly implanted valve still needs to be changed some time in the future. This situation is particularly dramatic in the case of children and young adults, because of the necessity of valve growth during the patient's life. Our review focuses on the current status of heart valve (HV) therapy and the challenges that must be solved in the development of new approaches based on tissue engineering. Scientists and physicians have proposed tissue-engineered heart valves (TEHVs) as the most promising solution for HV replacement, especially given that they can help to avoid thrombosis, structural deterioration and xenoinfections. Lastly, TEHVs might also serve as a model for studying human valve development and pathologies.

Role of mitochondria ROS generation in ethanol-induced NLRP3 inflammasome activation and cell death in astroglial cells.

Toll-like receptors (TLRs) and NOD-like receptors (NLRs) are innate immunity sensors that provide an early/effective response to pathogenic or injury conditions. We have reported that ethanol-induced TLR4 activation triggers signaling inflammatory responses in glial cells, causing neuroinflammation and brain damage. However, it is uncertain if ethanol is able to activate NLRs/inflammasome in astroglial cells, which is the mechanism of activation, and whether there is crosstalk between both immune sensors in glial cells. Here we show that chronic ethanol treatment increases the co-localization of caspase-1 with GFAP(+) cells, and up-regulates IL-1ß and IL-18 in the frontal medial cortex in WT, but not in TLR4 knockout mice. We further show that cultured cortical astrocytes expressed several inflammasomes (NLRP3, AIM2, NLRP1, and IPAF), although NLRP3 mRNA is the predominant form. Ethanol, as ATP and LPS treatments, up-regulates NLRP3 expression, and causes caspase-1 cleavage and the release of IL-1ß and IL-18 in astrocytes supernatant. Ethanol-induced NLRP3/caspase-1 activation is mediated by mitochondrial (m) reactive oxygen species (ROS) generation because when using a specific mitochondria ROS scavenger, the mito-TEMPO (500 µM) or NLRP3 blocking peptide (4 µg/ml) or a specific caspase-1 inhibitor, Z-YVAD-FMK (10 µM), abrogates mROS release and reduces the up-regulation of IL-1ß and IL-18 induced by ethanol or LPS or ATP. Confocal microscopy studies further confirm that ethanol, ATP or LPS promotes NLRP3/caspase-1 complex recruitment within the mitochondria to promote cell death by caspase-1-mediated pyroptosis, which accounts for ≈73% of total cell death (≈22%) and the remaining (≈25%) die by caspase-3-dependent apoptosis. Suppression of the TLR4 function abrogates most ethanol effects on NLRP3 activation and reduces cell death. These findings suggest that NLRP3 participates, in ethanol-induced neuroinflammation and highlight the NLRP3/TLR4 crosstalk in ethanol-induced brain injury.

SOX2+ cell population from normal human brain white matter is able to generate mature oligodendrocytes.

OBJECTIVES: A number of neurodegenerative diseases progress with a loss of myelin, which makes them candidate diseases for the development of cell-replacement therapies based on mobilisation or isolation of the endogenous neural/glial progenitor cells, in vitro expansion, and further implantation. Cells expressing A2B5 or PDGFRA/CNP have been isolated within the pool of glial progenitor cells in the subcortical white matter of the normal adult human brain, all of which demonstrate glial progenitor features. However, the heterogeneity and differentiation potential of this pool of cells is not yet well established. METHODS: We used diffusion tensor images, histopathology, and immunostaining analysis to demonstrate normal cytoarchitecture and the absence of abnormalities in human temporal lobe samples from patients with mesial temporal sclerosis. These samples were used to isolate and enrich glial progenitor cells in vitro, and later to detect such cells in vivo. RESULTS: We have identified a subpopulation of SOX2+ cells, most of them co-localising with OLIG2, in the white matter of the normal adult human brain in vivo. These cells can be isolated and enriched in vitro, where they proliferate and generate immature (O4+) and mature (MBP+) oligodendrocytes and, to a lesser extent, astrocytes (GFAP+). CONCLUSION: Our results demonstrate the existence of a new glial progenitor cell subpopulation that expresses SOX2 in the white matter of the normal adult human brain. These cells might be of use for tissue regeneration procedures.

Cancer-initiating enriched cell lines from human glioblastoma: preparing for drug discovery assays.

Glioblastoma multiforme (GBM) is the most lethal type of brain tumour in the adult humans. The cancer-initiating cell (CIC) hypothesis supports the notion that failures in current approaches to GBM treatment might be attributed to the survival of the CIC subpopulation. Recent evidence shows the idea that using CIC-enriched cell lines derived from human GBM as new targets for drug discovery programs, may improve the chance of successfully translating the basic research findings into clinical trials. Although this approach appears promising, many important biological and technical issues (characterization of functional CIC markers, inter- and intra-tumoral CIC heterogeneity, and isolation and maintenance inconsistency) need to be resolved.