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Nowadays, the yeast Saccharomyces cerevisiae is the platform of choice for demonstrating the proof of concept of the production of metabolites with a complex structure. However, introducing heterologous genes and rewiring the endogenous metabolism is still not standardized enough, affecting negatively the readiness-to-market of such metabolites. We developed the Easy Modular Integrative fuSion-ready Expression (Easy-MISE) toolkit, which is a novel combination of synthetic biology tools based on a single Golden Gate multiplasmid assembly meant to further ameliorate the rational predictability and flexibility of the process of yeast engineering. Thanks to an improved cloning screening strategy, double and independent transcription units are easily assembled and subsequently integrated into previously characterized loci. Moreover, the devices can be tagged for localization. This design allows for a higher degree of modularity and increases the flexibility of the engineering strategy. We show with a case study how the developed toolkit accelerates the construction and the analysis of the intermediate and the final engineered yeast strains, leaving space to better characterize the heterologous biosynthetic pathway in the final host and, overall, to improve the fermentation performances. Different S. cerevisiae strains were built harboring different versions of the biochemical pathway toward glucobrassicin (GLB) production, an indolyl-methyl glucosinolate. In the end, we could demonstrate that in the tested conditions the best-producing strain leads to a final concentration of GLB of 9.80 ± 0.267 mg/L, a titer 10-fold higher than the best result previously reported in the literature.

The tight regulation of cell function in vivo requires the integration of biological and mechanical signals, as evoked by the surrounding extracellular matrix (ECM) or by neighboring cells. In this chapter, we describe the current understanding of the intracellular molecular processes through which physical cues generated at the ECM are turned into a biological response. These processes, which are the subject of intense investigation in the interdisciplinary field of mechanobiology, are needed for cellular timely adaptation to the continuous dynamic modifications of the microenvironment. By starting from the most recent findings in the field, we conceive a centripetal model of mechanotransduction whereby macromolecular complexes sitting at the interphase between ECM and the cell cytoplasm serve as the primary hub for the cell to perceive mechanical stress. Following the prompt rearrangement of the cellular membrane and focal adhesions, the inward transmission of the mechanical signal is ensured by the dynamic fine-tuning of cytoskeleton tension and the linker of nucleoskeleton and cytoskeleton (LINC) complex, the latter spanning through the nuclear envelope and thus bridging ECM-generated signals to the nucleus. LINC rearrangement deforms the nucleus, hence making cryptic DNA domains accessible to stage-specific transcription factors, whose activity is instructed by shuttling mechanosensitive cofactors.

Yes-associated protein 1 (YAP1) is a transcriptional co-activator downstream of Hippo pathway. The pathway exerts crucial roles in organogenesis and its dysregulation is associated with the spreading of different cancer types. YAP1 gene encodes for multiple protein isoforms, whose specific functions are not well defined. We demonstrate the splicing of isoform-specific mRNAs is controlled in a stage- and tissue-specific fashion. We designed expression vectors encoding for the most-represented isoforms of YAP1 with either one or two WW domains and studied their specific signaling activities in YAP1 knock-out cell lines. YAP1 isoforms display both common and unique functions and activate distinct transcriptional programs, as the result of their unique protein interactomes. By generating TEAD-based transcriptional reporter cell lines, we demonstrate individual YAP1 isoforms display unique effects on cell proliferation and differentiation. Finally, we illustrate the complexity of the regulation of Hippo-YAP1 effector in physiological and in pathological conditions of the 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 TGFβ1 (transforming growth factor β1), 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.

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 and self-organized human cardiac organoids (hCO) in vitro from hiPSCs that can be cultured for long term. This is achieved by differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated human cardiac organoids (hCOs) containing multiple cell types that physiologically compose the heart, gradually self-organize and beat without external stimuli for more than 50 days. We have shown that 3D hCOs display improved cardiac specification, survival and maturation as compared to standard monolayer cardiac differentiation. We also confirmed the functionality of hCOs by their response to cardioactive drugs in long term culture. Furthermore, we demonstrated that hCOs can be used to study chemotherapy-induced cardiotoxicity.This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.

Material‐induced ossification is suggested as a suitable approach to heal large bone defects. Fiber‐reinforced composite–bioactive glasses (FRC‐BGs) display properties that could enhance the ossification of calvarial defects. Here, we analyzed the healing processes of a FRC‐BG implant in vivo from the perspective of material‐induced ossification. Histological analysis of the implant, which was removed 5 months after insertion, showed the formation of viable, noninflammatory mesenchymal tissue with newly‐formed mineralized woven bone, as well as nonmineralized connective tissue with capillaries and larger blood vessels. The presence of osteocytes was detected within the newly generated bone matrix. To expand our understanding on the osteogenic properties of FRC‐BG, we cultured human adipose tissue‐derived mesenchymal stromal cells (AD‐MSCs) in the presence of two different BGs (45S5 and S53P4) and Al2O3 control. AD‐MSCs grew and proliferated on all the scaffolds tested, as well as secreted abundant extracellular matrix, when osteogenic differentiation was appropriately stimulated. 45S5 and S53P4 induced enhanced expression of COL2A1, COL10A1, COL5A1 collagen subunits, and pro‐osteogenic genes BMP2 and BMP4. The concomitant downregulation of BMP3 was also detected. Our findings show that FRC‐BG can support the vascularization of the implant and the formation of abundant connective tissue in vivo. Specifically, BG 45S5 and BG S53P4 are suited to evoke the osteogenic potential of host mesenchymal stromal cells. In conclusion, FRC‐BG implant demonstrated material‐induced ossification both in vitro and in vivo.

Correction to: Vrbský, J., Vinarský, V., Perestrelo, A. R., De La Cruz, J. O., Martino, F., Pompeiano, A., Izzi, V., Hlinomaz, O., Rotrekl, V., Sudol, M., Pagliari, S., & Forte, G. (2021). Evidence for discrete modes of YAP1 signaling via mRNA splice isoforms in development and diseases. Genomics, 113(3), 1349–1365. https://doi.org/10.1016/j.ygeno.2021.03.009 Rinnakkaistallennettu versio / Self-archived version

Cocoa beans are one of the largest cultivated crops all over the world, producing large amount of by-products. For this reason, it is necessary to valorise cocoa by-products to obtain valuable source of bioactive compounds. In this paper, a pressurized hot water extraction process for recovery of theobromine and caffeine from cocoa by-product was developed and optimized. The extraction was carried out on ASE and all parameters affected by extraction efficiency and antioxidant capacity were optimized by a chemometric approach. Theobromine and caffeine were quantified by UPLC-UV (283 nm), whereas antioxidant capacity was evaluated by in vitro assays (ABTS and DPPH). By applying the PHWE under optimized conditions (ethanol 15%, temperature 90 °C, 5 cycles and static time 6 min), the extraction efficiency increased by 156% for theobromine and 160% for caffeine in comparison with the results obtained using ultrasound assisted liquid extraction. The effect of PHWE extract on cell viability of colorectal and breast cancer cell lines was also tested. The application of PHWE to selective recovery of theobromine and caffeine from cocoa shell by-products is a green, automatic, and rapid method, representing a valid alternative to conventional extraction methods to obtain ingredients for food industries.

Cardiac pathologies are characterized by intense remodeling of the extracellular matrix (ECM) that eventually leads to heart failure. Cardiomyocytes respond to the ensuing biomechanical stress by re-expressing fetal contractile proteins via transcriptional and post-transcriptional processes, like alternative splicing (AS). Here, we demonstrate that the heterogeneous nuclear ribonucleoprotein C (hnRNPC) is upregulated and relocates to the sarcomeric Z-disk upon ECM pathological remodeling. We show that this is an active site of localized translation, where the ribonucleoprotein associates to the translation machinery. Alterations in hnRNPC expression and localization can be mechanically determined and affect the AS of numerous mRNAs involved in mechanotransduction and cardiovascular diseases, like Hippo pathway effector YAP1. We propose that cardiac ECM remodeling serves as a switch in RNA metabolism by impacting an associated regulatory protein of the spliceosome apparatus. These findings offer new insights on the mechanism of mRNAs homeostasis mechanoregulation in pathological conditions.

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. ispartof: CELL DEATH AND DIFFERENTIATION vol:28 issue:4 pages:1193-1207 ispartof: location:England status: published

Nanodrugs represent novel solutions to reshuffle repurposed drugs for cancer therapy. They might offer different therapeutic options by combining targeted drug delivery and imaging in unique platforms. Such nanomaterials are deemed to overcome the limitations of currently available treatments, ultimately improving patients’ life quality. However, despite these promises being made for over three decades, the poor clinical translation of nanoparticle- based therapies calls for deeper in vitro and in vivo investigations. Translational issues arise very early during the development of nanodrugs, where complex and more reliable cell models are often replaced by easily accessible and convenient 2D monocultures. This is particularly true in the field of cancer therapy. In fact, 2D monocultures provide poor information about the real impact of the nanodrugs in a complex living organism, especially given the poor mimicry of the solid Tumors Microenvironment (TME). The dense and complex extracellular matrix (ECM) of solid tumors dramatically restricts nanoparticles efficacy, impairing the successful implementation of nanodrugs in medical applications. Herein, we propose a comprehensive guideline of the 3D cell culture models currently available, including their potential and limitations for the evaluation of nanodrugs activity. Advanced culture techniques, more closely resembling the physiological conditions of the TME, might give a better prediction of the reciprocal interactions between cells and nanoparticles and eventually help reconsider the use of old drugs for new applications.

In the context of the global need to move towards circular economies, microbial cell factories can be employed thanks to their ability to use side-stream biomasses from the agro-industrial sector to obtain additional products. The valorization of residues allows for better and complete use of natural resources and, at the same time, for the avoidance of waste management to address our needs. In this work, we focused our attention on the microbial valorization of cinnamon waste material after polyphenol extraction (C-PEW) (Cinnamomum verum J.Presl), generally discarded without any additional processing. The sugars embedded in C-PEW were released by enzymatic hydrolysis, more compatible than acid hydrolysis with the subsequent microbial cultivation. We demonstrated that the yeast Rhodosporidium toruloides was able to grow and produce up to 2.00 (±0.23) mg/L of carotenoids in the resulting hydrolysate as a sole carbon and nitrogen source despite the presence of antimicrobial compounds typical of cinnamon. To further extend the potential of our finding, we tested other fungal cell factories for growth on the same media. Overall, these results are opening the possibility to develop separate hydrolysis and fermentation (SHF) bioprocesses based on C-PEW and microbial biotransformation to obtain high-value molecules.

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., The transcriptional co-activator YAP is known to operate downstream of mechanical signals arising from the cell niche. Here the authors demonstrate that YAP controls cell mechanics, force development and adhesion strength by promoting the transcription of genes related to focal adhesions.

The contribution of adult stem cells to cardiac repair is mostly ascribed to an indirect paracrine effect, rather than to their actual engraftment and differentiation into new contractile and vascular cells. This effect consists in a direct reduction of host cell death, promotion of neovascularization, and in a “bystander effect” on local inflammation. A number of cytokines secreted by adult stem/progenitor cells has been proposed to be responsible for the consistent beneficial effect reported in the early attempts to deliver different stem cell subsets to the injured myocardium. Aiming to maximize their beneficial activity on the diseased myocardium, the genetic modification of adult stem cells to enhance and/or control the secretion of specific cytokines would turn them into active drug delivery vectors. On the other hand, engineering biocompatible scaffolds as to release paracrine factors could result in multiple advantages: (1) achieve a local controlled release of the drug of interest, thus minimizing off-target effects, (2) enhance stem cell retention in the injured area and (3) boost the beneficial paracrine effects exerted by adult stem cells on the host tissue. In the present review, a critical overview of the state-of-the-art in the modification of stem cells and the functionalization of biocompatible scaffolds to deliver beneficial soluble factors to the injured myocardium is offered. Besides the number of concerns to be addressed before a clinical application can be foreseen for such concepts, this path could translate into the generation of active scaffolds as smart cell and drug delivery systems for cardiac repair.

The stemness state is characterized by self-renewal and differentiation properties. However, stem cells are not able to preserve these characteristics in long-term culture because of the intrinsic fragility of their phenotype easily undergoing senescence or neoplastic transformation. Furthermore, although isolated from the same original tissue using similar protocols, adult stem cells can display dissimilar phenotypes and important cell clone/species contamination. Finally, the lack of a clear standardization contributes to complicate the comprehension about the stemness condition. In this context, cell lines displaying a particularly stable phenotype must be identified to define one or multiple benchmarks against which other stem cell lines could be reliably assessed. The present paper demonstrates that it is possible to isolate from the rat dental pulp a stem cell line (MUR-1) that does not display neoplastic transformation in long-term culture. MUR-1 cells stably express a broad range of stemness markers and are able to differentiate into adipogenic, osteogenic, chondrogenic, neurogenic, and cardiomyogenic lineages independently of the culture passages. Moreover, serial in vitro passages have not changed their immunophenotype, proliferation capacity, or differentiation potential. The uniqueness of these characteristics candidates MUR-1 as a model to reliably improve the understanding of the mechanisms governing the stem cell fate in the same as well as in other stem cell populations.

Cardiac progenitor cells (CPCs) are a promising autologous source of cells for cardiac regenerative medicine. However, CPC culture in vitro requires the presence of microenvironmental conditions (a complex array of bioactive substance concentration, mechanostructural factors, and physicochemical factors) closely mimicking the natural cell surrounding in vivo, including the capability to uphold reactive oxygen species (ROS) within physiological levels in vitro. Cerium oxide nanoparticles (nanoceria) are redox-active and could represent a potent tool to control the oxidative stress in isolated CPCs. Here, we report that 24 h exposure to 5, 10, and 50 μg/mL of nanoceria did not affect cell growth and function in cardiac progenitor cells, while being able to protect CPCs from H(2)O(2)-induced cytotoxicity for at least 7 days, indicating that nanoceria in an effective antioxidant. Therefore, these findings confirm the great potential of nanoceria for controlling ROS-induced cell damage.

International audience

Cardiovascular diseases represent the main cause of morbidity and mortality worldwide. Millions of people are affected by such diseases in the industrialized countries, with hundreds of thousands new cases diagnosed every year. Among cardiac diseases, heart failure is the most common end-stage pathology, leading to impaired cardiac output and cardiac performance as a result of the irreversible loss of contractile cardiomyocytes. Tissue engineering holds the promise to provide personalized solutions to the problem of cardiac muscle repair. Indeed, the identification of little reservoirs of stem and progenitor cells within every body district opened new perspectives to the setup of patient-specific protocols for cardiac diseases. Nonetheless, the results of the first pre-clinical and clinical trials in which adult stem/progenitor cells were adopted pointed at the route of delivery to the injured organ as well as at the cell source as the main issues for cardiac tissue engineers. In fact, when adult stem cells were directly injected into the myocardium or delivered through bloodstream to the heart, no or few cells could be found engrafted within host tissue few days after the administration. Renewed enthusiasm was generated by the techniques set up to enrich cardiomyocytes obtained by embryonic stem cells and by the recent disclosure of the protocols to obtain reprogrammed pluripotent cells or reprogrammed cardiomyocytes out of patients' own somatic cells. In this context, additional efforts to setup efficient systems to deliver stem cells to the injured site are required. The application of forefront technologies to fabricate synthetic and hybrid scaffolds to be employed as cell delivery systems and the acknowledgement that surface physical, mechanical, chemical properties can exert specific effects on stem cells per se prompted new enthusiasm in the field. In this respect, a cardiac-specific scaffold should be able to comply with cardiac muscle architecture, be deformable as to indulge and possibly sustain cardiac contraction. As expected, such a scaffold should favor stem cell electromechanical coupling with host tissue, while promoting the vascularization of the newly-formed tissue.

Hybrid 2D polymeric-ceramic biosupports are fabricated by mixing a nanostructured CeO2 powder with 85:15 poly(D,L-lactic-co-glycolic acid) (PLGA)/dichloromethane solutions at specific concentrations, followed by solvent casting onto pre-patterned molds. The mold patterning allows the orientation of ceramic nanoparticles into parallel lines within the composite scaffold. The ability of the produced films to host and address cell growth is evaluated after 1,3, and 6 days of culturing with murine derived cardiac and mesenchymal stem cells (CSCs and MSCs), and compared with PLGA films without ceramics and loaded with nanostructured TiO2. Aligned cell growth is observed only for scaffolds that incorporate oriented ceramic nanoparticles, attributed to the nanoceramic ability to modulate the roughness pitch, thus improving cell sensitivity towards the host surface features. Better CSC and MSC proliferative activity is observed for CeO2 composites with respect to either TiO2-added or unfilled PLCA films. This evidence may be related to the nanostructured CeO2 antioxidative properties. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel (scalable) electrospinning process was developed to fabricate bio-inspired multiscale three-dimensional scaffolds endowed with a controlled multimodal distribution of fiber diameters and geared towards soft tissue engineering. The resulting materials finely mingle nano- and microscale fibers together, rather than simply juxtaposing them, as is commonly found in the literature. A detailed proof of concept study was conducted on a simpler bimodal poly(epsilon-caprolactone) (PCL) scaffold with modes of fiber distribution at 600 nm and 3.3 microm. Three conventional unimodal scaffolds with mean diameters of 300 nm and 2.6 and 5.2 microm, respectively, were used as controls to evaluate the new materials. Characterization of the microstructure (i.e. porosity, fiber distribution and pore structure) and mechanical properties (i.e. stiffness, strength and failure mode) indicated that the multimodal scaffold had superior mechanical properties (Young's modulus approximately 40MPa and strength approximately 1MPa) in comparison with the controls, despite the large porosity ( approximately 90% on average). A biological assessment was conducted with bone marrow stromal cell type (mesenchymal stem cells, mTERT-MSCs). While the new material compared favorably with the controls with respect to cell viability (on the outer surface), it outperformed them in terms of cell colonization within the scaffold. The latter result, which could neither be practically achieved in the controls nor expected based on current models of pore size distribution, demonstrated the greater openness of the pore structure of the bimodal material, which remarkably did not come at the expense of its mechanical properties. Furthermore, nanofibers were seen to form a nanoweb bridging across neighboring microfibers, which boosted cell motility and survival. Lastly, standard adipogenic and osteogenic differentiation tests served to demonstrate that the new scaffold did not hinder the multilineage potential of stem cells.

An immortalized murine mesenchymal stem cell line (mTERT-MSC) enriched forLinneg/Sca-1posfraction has been obtained through the transfection of MSC with murine TERT and single-cell isolation. Such cell line maintained the typical MSC self-renewal capacity and continuously expressed MSC phenotype. Moreover, mTERT-MSC retained the functional features of freshly isolated MSC in culture without evidence of senescence or spontaneous differentiation events. Thus, mTERT-MSC have been cultured onto PLA films, 30 and 100 μm PLA microbeads, and onto unpressed and pressed HYAFF-11 scaffolds. While the cells adhered preserving their morphology on PLA films, clusters of mTERT-MSC were detected on PLA beads and unpressed fibrous scaffolds. Finally, mTERT-MSC were not able to colonize the inner layers of pressed HYAFF-11. Nevertheless, such cell line displayed the ability to preserve Sca-1 expression and to retain multilineage potential when appropriately stimulated on all the scaffolds tested.

Stem cell responsiveness to extracellular matrix (ECM) composition and mechanical cues has been the subject of a number of investigations so far, yet the molecular mechanisms underlying stem cell mechano-biology still need full clarification. Here we demonstrate that the paralog proteins YAP and TAZ exert a crucial role in adult cardiac progenitor cell mechano-sensing and fate decision. Cardiac progenitors respond to dynamic modifications in substrate rigidity and nanopattern by promptly changing YAP/TAZ intracellular localization. We identify a novel activity of YAP and TAZ in the regulation of tubulogenesis in 3D environments and highlight a role for YAP/TAZ in cardiac progenitor proliferation and differentiation. Furthermore, we show that YAP/TAZ expression is triggered in the heart cells located at the infarct border zone. Our results suggest a fundamental role for the YAP/TAZ axis in the response of resident progenitor cells to the modifications in microenvironment nanostructure and mechanics, thereby contributing to the maintenance of myocardial homeostasis in the adult heart. These proteins are indicated as potential targets to control cardiac progenitor cell fate by materials design.

The vascularization of tissue engineered products represents a key issue in regenerative medicine which needs to be addressed before the translation of these protocols to the bedside can be foreseen. Here we propose a multistep procedure to prepare pre-vascularized three-dimensional (3D) cardiac bio-substitutes using dynamic cell cultures and highly porous biocompatible gelatin scaffolds. The strategy adopted exploits the peculiar differentiation potential of two distinct subsets of adult stem cells to obtain human vascularized 3D cardiac tissues. In the first step of the procedure, human mesenchymal stem cells (hMSCs) are seeded onto gelatin scaffolds to provide interconnected vessel-like structures, while human cardiomyocyte progenitor cells (hCMPCs) are stimulated in vitro to obtain their commitment towards the cardiac phenotype. The use of a modular bioreactor allows the perfusion of the whole scaffold, providing superior performance in terms of cardiac tissue maturation and cell survival. Both the cell culture on natural-derived polymers and the continuous medium perfusion of the scaffold led to the formation of a densely packaged proto-tissue composed of vascular-like and cardiac-like cells, which might complete maturation process and interconnect with native tissue upon in vivo implantation. In conclusion, the data obtained through the approach here proposed highlight the importance to provide stem cells with complementary signals in vitro able to resemble the complexity of cardiac microenvironment.

The identification of different pools of cardiac progenitor cells resident in the adult mammalian heart opened a new era in heart regeneration as a means to restore the loss of functional cardiac tissue and overcome the limited availability of donor organs. Indeed, resident stem cells are believed to participate to tissue homeostasis and renewal in healthy and damaged myocardium although their actual contribution to these processes remain unclear. The poor outcome in terms of cardiac regeneration following tissue damage point out at the need for a deeper understanding of the molecular mechanisms controlling CPC behavior and fate determination before new therapeutic strategies can be developed. The regulation of cardiac resident stem cell fate and function is likely to result from the interplay between pleiotropic signaling pathways as well as tissue- and cell-specific regulators. Such a modular interaction – which has already been described in the nucleus of a number of different cells where transcriptional complexes form to activate specific gene programs - would account for the unique responses of cardiac progenitors to general and tissue-specific stimuli.The study of the molecular determinants involved in cardiac stem/progenitor cell regulatory mechanisms may shed light on the processes of cardiac homeostasis in health and disease and thus provide clues on the actual feasibility of cardiac cell therapy through tissue-specific progenitors.

Putative cardiac progenitor cells (CPCs) have been identified in the myocardium and are regarded as promising candidates for cardiac cell-based therapies. Although two distinct populations of CPCs reached the clinical setting, more detailed studies are required to portray the optimal cell type and therapeutic setting to drive robust cell engraftment and cardiomyogenesis after injury. Owing to the scarcity of the CPCs and the need for reproducibility, the generation of faithful cellular models would facilitate this scrutiny. Here, we evaluate whether immortalized Lin(-)Sca-1(+) CPCs (iCPC(Sca-1)) represent their native-cell counterpart, thereby constituting a robust in vitro model system for standardized investigation in the cardiac field. iCPC(Sca-1) were established in vitro as plastic adherent cells endowed with robust self-renewal capacity while preserving a stable phenotype in long-term culture. iCPC(Sca-1) differentiated into cardiomyocytic-, endothelial-, and smooth muscle-like cells when subjected to appropriate stimuli. The cell line consistently displayed features of Lin(-)Sca-1(+) CPCs in vitro, as well as in vivo after intramyocardial delivery in the onset of myocardial infarction (MI). Transplanted iCPC(Sca-1) significantly attenuated the functional and anatomical alterations caused by MI while promoting neovascularization. iCPC(Sca-1) are further shown to engraft, establish functional connections, and differentiate in loco into cardiomyocyte- and vasculature-like cells. These data validate iCPC(Sca-1) as an in vitro model system for Lin(-)Sca-1(+) progenitors and for systematic dissection of mechanisms underlying CPC subsets engraftment/differentiation in vivo. Moreover, iCPC(Sca-1) can be regarded as a ready-to-use CPCs source for pre-clinical bioengineering studies toward the development of novel strategies for restoration of the damaged myocardium.

To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ε-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.

Human heart harbors a population of resident progenitor cells that can be isolated by stem cell antigen-1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, less than 1%-5% survive and differentiate. Among the major causes of this failure are the distressing protocols used to culture in vitro and implant progenitor cells into damaged hearts. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature-responsive surfaces. In the engineered tissue, progenitor cells established proper three-dimensional intercellular relationships and were embedded in self-produced extracellular matrix preserving their phenotype and multipotency in the absence of significant apoptosis. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the murine myocardium and in the vascular walls, where they integrated in the respective textures. The study demonstrates the suitability of such an approach to deliver stem cells to the myocardium. Interestingly, the successful delivery of cells in murine healthy hearts suggests that myocardium displays a continued cell cupidity that is strictly regulated by the limited release of progenitor cells by the adopted source. When an unregulated cell source is added to the system, cells are delivered to the myocardium. The exploitation of this novel concept may pave the way to the setup of new protocols in cardiac cell therapy.

In healthy human hearts, only 10-20% of the total cells are contractile cardiomyocytes and, at the age of 25 years, no more than 1% of them are annually substituted by progenitor cells, this percentage reducing to less than 0.5% at the age of 75. In total, less than 50% of cardiomyocytes are renewed during a normal human life span [1]. For this reason, the topic of cardiac repair is among the major challenges for the tissue engineers worldwide. In fact, cardiac diseases are a predominant cause of mortality and morbidity in industrialized countries, despite the recent advancements achieved in pharmacological treatment and interventional cardiology procedures. Nonetheless, end-stage heart failure management still relies on organ transplantation as unique approach, and, notwithstanding the use of massive immunosuppressive drugs, still a percentage falling within 20%-40% of patients encounters immune rejection during the first year post-transplant [2]. Among the patients not facing severe immune rejection, almost 70% is forced to retire or reduce their working activity, their survival rate falling below 70% during the first five years post organ transplantation [3]. Last, but not least, the economic impact of cardiovascular diseases and stroke has been estimated in 2010 at $503.2 billion [4]. Currently, post-infarction myocardial revascularization protocols include the administration of raw bone marrow stem cells, while a number of clinical trials have been performed or are currently in progress in which different cell subsets are implanted in the damaged tissue by means of surgical techniques. The results of such trials are still controversial. In fact, when autologous skeletal myoblasts were injected into the heart of patients suffering from ischemic cardiomyopathy, the modest functional improvement obtained was impaired by the arising of arrhythmia events, thus requiring the adoption of a pacemaker [5]. On the other side, intracoronary administration of bone marrow mesenchymal stem cells resulted in minimal improvements in cardiac contractile function in patients with dilated cardiomyopathy [6]. These mild results were mostly ascribed to a paracrine effect exerted on host tissue, rather than to a direct contribution of stem cells to the contractile activity.

Dr. S. Pagliari , Dr. G. Forte , Dr. F. Pagliari , Dr. M. Minieri , Prof. P. Di Nardo Laboratory of Molecular and Cellular Cardiology Department of Internal Medicine University of Rome “Tor Vergata”Rome 00133, Italy E-mail: dinardo@uniroma2.it Dr. S. Pagliari, Dr. G. Forte, Dr. F. Pagliari, Dr. M. Minieri, Prof. P. Di NardoJapanese-Italian Tissue Engineering Laboratory (JITEL) Tokyo Women’s Medical University-Waseda University Joint Institution for Advanced Biomedical Sciences (TWIns) Tokyo, Japan Dr. S. Pagliari, Dr. G. Forte, Dr. F. Pagliari, Dr. M. Minieri, Prof. P. Di NardoItalian Institute for Cardiovascular Research (INRC) 40126 Bologna, Italy Prof. A. C. Vilela-Silva Instituto de Ciencias Biomedicas and Laboratorio de Tecido Conjuntivo Hospital Universitario Clementino Fraga Filho Rio de Janeiro, Brazil Dr. C. Mandoli , Prof. E. Traversa International Research Center for Materials Nanoarchitectonics (MANA) National Institute for Materials Science (NIMS) 1–1 Namiki, Tsukuba, Ibaraki 305–0044, Japan E-mail: TRAVERSA.Enrico@nims.go.jp Dr. S. Pietronave , Prof. M. Prat Department of Medical Sciences University “A. Avogadro” of Piemonte Orientale 28100 Novara, Italy Dr. G. Vozzi , Prof. A. Ahluwalia Interdepartmental Research Center “E. Piaggio” University of Pisa56126 Pisa, Italy Prof. S. Licoccia , Prof. E. Traversa NAST Centre & Department of Chemical Science and Technology University of Rome “Tor Vergata” Roma 00133, Italy [†] S.P. and A.C.V.S. contributed equally to this work.