Reduction of Cardiac Fibrosis by Interference With YAP-Dependent Transactivation.

BACKGROUND: Conversion of cardiac stromal cells into myofibroblasts is typically associated with hypoxia conditions, metabolic insults, and/or inflammation, all of which are predisposing factors to cardiac fibrosis and heart failure. We hypothesized that this conversion could be also mediated by response of these cells to mechanical cues through activation of the Hippo transcriptional pathway. The objective of the present study was to assess the role of cellular/nuclear straining forces acting in myofibroblast differentiation of cardiac stromal cells under the control of YAP (yes-associated protein) transcription factor and to validate this finding using a pharmacological agent that interferes with the interactions of the YAP/TAZ (transcriptional coactivator with PDZ-binding motif) complex with their cognate transcription factors TEADs (TEA domain transcription factors), under high-strain and profibrotic stimulation. METHODS: We employed high content imaging, 2-dimensional/3-dimensional culture, atomic force microscopy mapping, and molecular methods to prove the role of cell/nuclear straining in YAP-dependent fibrotic programming in a mouse model of ischemia-dependent cardiac fibrosis and in human-derived primitive cardiac stromal cells. We also tested treatment of cells with Verteporfin, a drug known to prevent the association of the YAP/TAZ complex with their cognate transcription factors TEADs. RESULTS: Our experiments suggested that pharmacologically targeting the YAP-dependent pathway overrides the profibrotic activation of cardiac stromal cells by mechanical cues in vitro, and that this occurs even in the presence of profibrotic signaling mediated by TGF-ß1 (transforming growth factor beta-1). In vivo administration of Verteporfin in mice with permanent cardiac ischemia reduced significantly fibrosis and morphometric remodeling but did not improve cardiac performance. CONCLUSIONS: Our study indicates that preventing molecular translation of mechanical cues in cardiac stromal cells reduces the impact of cardiac maladaptive remodeling with a positive effect on fibrosis.

Firefly luciferase offers superior performance to AkaLuc for tracking the fate of administered cell therapies.

INTRODUCTION: A novel, red-shifted bioluminescence imaging (BLI) system called AkaBLI has been recently developed for cell tracking in preclinical models and to date, limited data is available on how it performs in relation to existing systems. PURPOSE: To systematically compare the performance of AkaBLI and the standard Firefly luciferase (FLuc) systems to monitor the biodistribution and fate of cell therapies in rodents. METHODS: Umbilical cord mesenchymal stromal cells (MSCs) were transduced to produce two genetically engineered populations, expressing either AkaLuc or the engineered FLuc luc2. The bioluminescence of AkaLuc+ and FLuc+ cells was assessed both in vitro (emission spectra, saturation kinetics and light emission per cell) and in vivo (substrate kinetics following intraperitoneal and subcutaneous administration and biodistribution of the cells up to day 7). RESULTS: Introduction of the reporter genes has no effect on MSC phenotype. For BLI, the FLuc system is superior to AkaBLI in terms of (i) light output, producing a stronger signal after subcutaneous substrate delivery and more consistent signal kinetics when delivered intraperitoneally; (ii) absence of hepatic background; and (iii) safety, where the AkaLuc substrate was associated with a reaction in the skin of the mice in vivo. CONCLUSION: We conclude that there is no advantage in using the AkaBLI system to track the biodistribution of systemically administered cell-based regenerative medicine therapies in vivo.

Optical Tissue Clearing to Study the Intra-Pulmonary Biodistribution of Intravenously Delivered Mesenchymal Stromal Cells and Their Interactions with Host Lung Cells.

Mesenchymal stromal cells (MSCs) injected intravenously are trapped in the capillaries of the lungs and die within the first 24 h. Studying the biodistribution and fate of labelled therapeutic cells in the 3D pulmonary context is important to understand their function in this organ and gain insights into their mechanisms of action. Optical tissue clearing enables volumetric cell tracking at single-cell resolution. Thus, we compared three optical tissue-clearing protocols (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC), modified stabilised 3D imaging of solvent-cleared organs (s-DISCO) and ethyl cinnamate (ECi)) to evaluate their potential to track the biodistribution of human umbilical cord MSCs expressing the tdTomato fluorescence reporter and investigate how they interact with host cells in the mouse lung. The results showed that although CUBIC clearing is the only method that enables direct imaging of fluorescently labelled MSCs, combining s-DISCO or ECi with immunofluorescence or dye labelling allows the interaction of MSCs with endothelial and immune cells to be studied. Overall, this comparative study offers guidance on selecting an optical tissue-clearing method for cell tracking applications.

DEAE-Dextran Enhances the Lentiviral Transduction of Primary Human Mesenchymal Stromal Cells from All Major Tissue Sources Without Affecting Their Proliferation and Phenotype.

Genetic engineering of mesenchymal stromal cells (MSCs) is a tool widely used to explore MSC properties in vitro and in vivo. Lentiviral infection with the use of polycations as an adjuvant is a method that is commonly used to generate stably transduced cells. However, it is known that some polycations can negatively affect primary MSCs and to date, no study has explored the effect of different polycations on the transduction efficiency and properties of all main types of MSCs, namely those derived from umbilical cord, bone marrow and adipose tissue. Here we explore a range of polycations, using transduction protocols with and without spinoculation, to produce stably transduced MSCs from these three tissue sources. We identified that an overnight incubation with diethylaminoethyl-dextran (DEAE-Dextran) is the protocol associated with the best transduction efficiency without compromising the viability of the cells, and which worked consistently with lentiviral particles encoding for different transgenes. Transduced and sorted MSC populations revealed no significant changes in proliferation, morphology and expression of MSC markers compared to naïve MSCs. Following this study, we conclude that DEAE-Dextran is a polycation that can be successfully used to enhance the transduction of MSCs from all major tissue sources.

Fate of intravenously administered umbilical cord mesenchymal stromal cells and interactions with the host's immune system.

Mesenchymal stromal cells (MSCs) are multipotent cells showing promise in pre-clinical studies and currently used in many clinical trials. The regenerative potential of MSCs is mediated, at least in part, by direct and indirect immunomodulatory processes. However, the mechanism of action is not fully understood yet, and there are still concerns about possible undesired negative effects associated with the administration of living cells. In this study, we (i) compare the long-term fate and safety of umbilical cord (UC-)MSCs administered to immunocompetent and immunocompromised (severe combined immunodeficient (SCID) and non-obese diabetic (NOD)/SCID) animals, and (ii) investigate the immunological response of the host to the administered cells. Intravenous administration of firefly luciferase expressing UC-MSCs revealed that the cells get trapped in the lungs of both immunocompetent and immunocompromised animals, with > 95% of the cells disappearing within 72 h after administration. In 27% of the SCID and 45% of the NOD/SCID, a small fraction of the cells lived up to day 14 but in most cases they all disappeared earlier. One NOD/SCID mouse showed a weak signal up to day 31. Immunocompetent mice displayed elevated percentages of neutrophils in the lungs, the blood, and the spleen 2 h after the administration of the cells. The concentration of neutrophil chemoattractants (MCP1, CCL7, Gro-α and IP-10) were also increased in the plasma of the animals 2 h after the administration of the MSCs. Our results suggest that although the UC-MSCs are short-lived in mice, they still result in an immunological response that might contribute to a therapeutic effect.

Harmonised culture procedures minimise but do not eliminate mesenchymal stromal cell donor and tissue variability in a decentralised multicentre manufacturing approach.

BACKGROUND: Mesenchymal stromal cells (MSCs), commonly sourced from adipose tissue, bone marrow and umbilical cord, have been widely used in many medical conditions due to their therapeutic potential. Yet, the still limited understanding of the underlying mechanisms of action hampers clinical translation. Clinical potency can vary considerably depending on tissue source, donor attributes, but importantly, also culture conditions. Lack of standard procedures hinders inter-study comparability and delays the progression of the field. The aim of this study was A- to assess the impact on MSC characteristics when different laboratories, performed analysis on the same MSC material using harmonised culture conditions and B- to understand source-specific differences. METHODS: Three independent institutions performed a head-to-head comparison of human-derived adipose (A-), bone marrow (BM-), and umbilical cord (UC-) MSCs using harmonised culture conditions. In each centre, cells from one specific tissue source were isolated and later distributed across the network to assess their biological properties, including cell expansion, immune phenotype, and tri-lineage differentiation (part A). To assess tissue-specific function, angiogenic and immunomodulatory properties and the in vivo biodistribution were compared in one expert lab (part B). RESULTS: By implementing a harmonised manufacturing workflow, we obtained largely reproducible results across three independent laboratories in part A of our study. Unique growth patterns and differentiation potential were observed for each tissue source, with similar trends observed between centres. Immune phenotyping verified expression of typical MSC surface markers and absence of contaminating surface markers. Depending on the established protocols in the different laboratories, quantitative data varied slightly. Functional experiments in part B concluded that conditioned media from BM-MSCs significantly enhanced tubulogenesis and endothelial migration in vitro. In contrast, immunomodulatory studies reported superior immunosuppressive abilities for A-MSCs. Biodistribution studies in healthy mice showed lung entrapment after administration of all three types of MSCs, with a significantly faster clearance of BM-MSCs. CONCLUSION: These results show the heterogeneous behaviour and regenerative properties of MSCs as a reflection of intrinsic tissue-origin properties while providing evidence that the use of harmonised culture procedures can reduce but do not eliminate inter-lab and operator differences.

Mesenchymal stromal cells: what have we learned so far about their therapeutic potential and mechanisms of action?

Mesenchymal stromal cells (MSCs) have been found to be safe and effective in a wide range of animal models of human disease. MSCs have been tested in thousands of clinical trials, but results show that while these cells appear to be safe, they tend to lack efficacy. This has raised questions about whether animal models are useful for predicting efficacy in patients. However, a problem with animal studies is that there is a lack of standardisation in the models and MSC therapy regimes used; there appears to be publication bias towards studies reporting positive outcomes; and the reproducibility of results from animal experiments tends not to be confirmed prior to clinical translation. A further problem is that while some progress has been made towards investigating the mechanisms of action (MoA) of MSCs, we still fail to understand how they work. To make progress, it is important to ensure that prior to clinical translation, the beneficial effects of MSCs in animal studies are real and can be repeated by independent research groups. We also need to understand the MoA of MSCs to assess whether their effects are likely to be beneficial across different species. In this review, we give an overview of the current clinical picture of MSC therapies and discuss what we have learned from animal studies. We also give a comprehensive update of what we know about the MoA of MSCs, particularly in relation to their role in immunomodulation.

Culture Into Perfusion-Assisted Bioreactor Promotes Valve-Like Tissue Maturation of Recellularized Pericardial Membrane.

Derivation of tissue-engineered valve replacements is a strategy to overcome the limitations of the current valve prostheses, mechanical, or biological. In an effort to set living pericardial material for aortic valve reconstruction, we have previously assessed the efficiency of a recellularization strategy based on a perfusion system enabling mass transport and homogenous distribution of aortic valve-derived "interstitial" cells inside decellularized pericardial material. In the present report, we show that alternate perfusion promoted a rapid growth of valve cells inside the pericardial material and the activity of a proliferation-supporting pathway, likely controlled by the YAP transcription factor, a crucial component of the Hippo-dependent signaling cascade, especially between 3 and 14 days of culture. Quantitative mass spectrometry analysis of protein content in the tissue constructs showed deposition of valve proteins in the decellularized pericardium with a high variability at day 14 and a reproducible tissue maturation at 21 days. These results represent a step forward in the definition of strategies to produce a fully engineered tissue for replacing the calcified leaflets of failing aortic valves.

Automated Segmentation of Fluorescence Microscopy Images for 3D Cell Detection in human-derived Cardiospheres.

The 'cardiosphere' is a 3D cluster of cardiac progenitor cells recapitulating a stem cell niche-like microenvironment with a potential for disease and regeneration modelling of the failing human myocardium. In this multicellular 3D context, it is extremely important to decrypt the spatial distribution of cell markers for dissecting the evolution of cellular phenotypes by direct quantification of fluorescent signals in confocal microscopy. In this study, we present a fully automated method, named CARE ('CARdiosphere Evaluation'), for the segmentation of membranes and cell nuclei in human-derived cardiospheres. The proposed method is tested on twenty 3D-stacks of cardiospheres, for a total of 1160 images. Automatic results are compared with manual annotations and two open-source software designed for fluorescence microscopy. CARE performance was excellent in cardiospheres membrane segmentation and, in cell nuclei detection, the algorithm achieved the same performance as two expert operators. To the best of our knowledge, CARE is the first fully automated algorithm for segmentation inside in vitro 3D cell spheroids, including cardiospheres. The proposed approach will provide, in the future, automated quantitative analysis of markers distribution within the cardiac niche-like environment, enabling predictive associations between cell mechanical stresses and dynamic phenotypic changes.

Aortic valve cell seeding into decellularized animal pericardium by perfusion-assisted bioreactor.

Animal-derived pericardium is the elective tissue employed in manufacturing heart valve prostheses. The preparation of this tissue for biological valve production consists of fixation with aldehydes, which reduces, but not eliminates, the xenoantigens and the donor cellular material. As a consequence, especially in patients below 65-70 years of age, the employment of valve substitutes contaning pericardium is not indicated due to progressive calcification that causes tissue degeneration and recurrence of valve insufficiency. Decellularization with ionic or nonionic detergents has been proposed as an alternative procedure to prepare aldehyde- or xenoantigen-free pericardium for biological valve manufacturing. In the present contribution, we optimized a decellularization procedure that is permissive for seeding and culturing valve competent cells able to colonize and reconstitute a valve-like tissue. A high-efficiency cellularization was achieved by forcing cell penetration inside the pericardium matrix using a perfusion bioreactor. Because the decellularization procedure was found not to alter the collagen composition of the pericardial matrix and cells seeded in the tissue constructs consistently grew and acquired the phenotype of "quiescent" valve interstitial cells, our investigation sets a novel standard in pericardium application for tissue engineering of "living" valve implants.

Activation of human aortic valve interstitial cells by local stiffness involves YAP-dependent transcriptional signaling.

Differentiation of valve interstitial cells (VICs) into pro-calcific cells is one of the central events in calcific aortic valve (AoV) disease (CAVD). While the paracrine pathways and the responsivity of VICs to mechanical compliance of the surrounding environment are well characterized, the molecular programming related to variations in local stiffness, and its link to cytoskeleton dynamics, is less consolidated. By using a simple method to produce 2D poly-acrylamide gels with stiffness controlled with atomic force microscopy (AFM), we manufactured adhesion substrates onto which human VICs from stenotic valves were plated, and subsequently investigated for cytoskeleton dynamics and activation of the mechanosensing-related transcription factor YAP. As a comparison, we employed VICs from patients undergoing valve substitution for valve insufficiency, a non-calcific AoV disease, which does not involve extensive inflammation. While the two VICs types did not differ for basic responses onto substrates with different stiffness values (e.g. adhesion and proliferation), they were subject to a different dynamics of stiffness-dependent YAP nuclear shuttling, revealing for the first time an intracellular force transduction mechanism distinctive for calcific aortic valve disease. In VICs from stenotic valves, YAP nuclear translocation occurred in concert with an increase in cytoskeleton tensioning and loading of the myofibroblast-specific protein αSMA onto the F-actin cytoskeleton. AFM force mapping performed along radial sections of human calcific valve leaflets identified, finally, areas with high and low levels of rigidity within a similar range to those controlling YAP nuclear translocation in vitro. Since VICs juxtaposed to these areas exhibited nuclear localized YAP, we conclude that subtle variations in matrix stiffness are involved in mechanosensing-dependent VICs activation and pathological differentiation in CAVD.

Acrylate-based materials for heart valve scaffold engineering.

Calcific aortic valve disease (CAVD) is the most frequent cardiac valve pathology. Its standard treatment consists of surgical replacement either with mechanical (metal made) or biological (animal tissue made) valve prostheses, both of which have glaring deficiencies. In the search for novel materials to manufacture artificial valve tissue, we have conducted a high-throughput screening with subsequent up-scaling to identify non-degradable polymer substrates that promote valve interstitial cells (VICs) adherence/growth and, at the same time, prevent their evolution toward a pro-calcific phenotype. Here, we provide evidence that one of the two identified 'hit' polymers, poly(methoxyethylmethacrylate-co-diethylaminoethylmethacrylate), provided robust VICs adhesion and maintained the healthy VICs phenotype without inducing pro-osteogenic differentiation. This ability was also maintained when the polymer was used to coat a non-woven poly-caprolactone (PCL) scaffold using a novel solvent coating procedure, followed by bioreactor-assisted VICs seeding. Since we observed that VICs had an increased secretion of the elastin-maturing component MFAP4 in addition to other valve-specific extracellular matrix components, we conclude that valve implants constructed with this polyacrylate will drive the biological response of human valve-specific cells.