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Cardiac tissue engineering (CTE) is a promising approach to replace and regenerate the injured and not functional cardiac tissue. Injectable self-assembling scaffolds consisting of biomaterials mimicking the native extracellular environment are an attractive option to promote specific homing and cell retention with minimal invasiveness. Among biological injectable materials, fibrin has been tested in several pathological conditions, either in combination with autologous stem cells or not, to stimulate wound healing through tissue regeneration, as it offers the unrivalled advantages of being autologous, enriched in growth factors and capable of gelling at 37 °C. In this study we combined fibrin with human cardiac decellularized extracellular matrix (d-ECM) and human Cardiac Progenitor Cells (hCPCs) to prepare an injectable hybrid three-dimensional scaffold serving as a natural cardiac platform for the delivery and engraftment of hCPCs. To this aim, samples of adult human hearts of recipients of heart transplantation were either decellularized to obtain the native cardiac d-ECM or disaggregated mechanically and by enzymatic digestion to obtain hCPCs. d-ECM was then lyophilized and solubilized to be incorporated along with the hCPCs into fibrin gels that were prepared by combining fibrinogen and thrombin. We tested three ratios of fibrin:d-ECM to determine the ideal combination. Specifically, we prepared 1:1, 1:2 and 2:1 fibrin:d-ECM gels and we allowed them to gel at 37°C. Gelling occurred in all preparations, but differences in the time needed for gelling and organization of gels occurred. The 2:1 preparation gelled after one hour, but solubilized d-ECM and fibrin did not combine well as the fibrin gel floated into the solubilized d-ECM. The 1:1 and 2:1 preparations, instead, gelled in a time ranging between eight and twenty hours, respectively, and produced homogenous gels. Gels were processed for histological analysis after three days of culture and hematoxylin-Eosin staining showed the presence and distribution of hCPCs throughout the entire thickness of the gels with 1:1 ratio, while for 1:2 and 2:1 ratios hCPCs coated the surface but were not visible inside the gels. Furthermore, the presence of collagen and ECM glycoproteins was detected by Sirius Red, Periodic Acid Schiff and Masson's Trichrome stainings exclusively in the gel with 1:1 ratio. Our results suggest that the combination of fibrin and d-ECM at 1:1 ratio yields a well-structured self-assembling scaffold that might be used as a minimally invasive method to deliver hCPCs and cardiac d-ECM in the injured myocardium to boost cardiac regeneration.

Exosomes are nano-vesicles released in body fluids and culture media by several types of cells, including stem cells. These small extracellular vesicles are loaded with a cargo of proteins, lipids, mRNA, miRNA, DNA and specific factors that they convey from one cell to another. Therefore, exosomes are involved in cellular intercommunication and signaling and can influence numerous biological mechanisms like cell death, proliferation and differentiation. We hypothesize that cardiac progenitor cells (CPCs) exert the paracrine effects that direct cardiac regeneration and remodeling by delivering exosomes to the injured myocardium. To test our hypothesis, we isolated CPCs (CPC-N) from the heart of donors, and CPCs (CPC-P) and fibroblasts (CF-P) from the heart of patients undergoing heart transplantation. First, we cultured CPC-N and collected the exosomes they released in the culture medium (Exo-CPC-N). Then, we characterized Exo-CPC-N cargo, in terms of mRNA and growth factors, and analyzed their effects on proliferation, migration and apoptosis of CPC-P as well as on extracellular matrix synthesis and deposition by CF-P, by administering Exo-CPC-N to CPC-P and CF-P in vitro. Real-Time PCR analysis revealed that Exo-CPC-N carry specific transcripts for proteins involved in cardiac (Nkx2.5, Mef2C, ACTC1, TBX, GATA6, ACTA2) and mesenchymal differentiation (TM4SF1, Sox9, CD90, CD105), while growth factor array showed the presence of specific factors as HGF, IGF, TGF that are known to enhance cell proliferation, growth and migration. When administrated to CPC-P, Exo-CPC-N not only increased CPC-P proliferation rate and resistance to apoptosis induced by oxidative stress, but also induced the differentiation towards cardiac myocytes as testified by the expression of a well-structured alpha-sarcomeric actin resulting at the immunofluorescence analysis. Additionally, Exo-CPC-N modulated in vitro the CF-P deposition of ECM proteins typical of the cardiac matrix, like collagen IV, fibronectin and laminin. Our study strengthens the hypothesis that exosomes are mediators of CPCs regenerative effects with the potential to empower the regenerative potential of resident cells through the spread of tailored molecules, and are, then, to be considered as a promising alternative to cardiac cell therapy.

Cells in vivo are surrounded by a specialized and intricate microenvironment mainly consisting of extracellular matrix (ECM). The ECM affects cell behavior and can support organ function and repair, with an appropriate architecture and a molecular composition unique for each tissue and specific for the role played by the resident cells. During the last decade, the increasing interest for decellularized ECM (d-ECM) as natural scaffold for cell delivery and tissue regeneration has boosted the search for the optimal protocol to decellularize the human myocardium. In this study we propose an innovative, reproducible and effective decellularization method to produce a well-preserved cardiac d-ECM. Cardiac samples harvested from the free wall of both ventricles of adult human hearts were scaled down to fit embedding cassettes and decellularized by immersion in a solution containing SDS, Triton X-100 and antibiotics under constant agitation. To avoid the random suspension within the beaker and the irregular and uncontrolled solution transport through the samples, a plexiglass sample-holder was specifically designed and hand-made to hold up to four cassettes in a steady position while equally exposing samples to decellularizing detergents. After decellularization, samples were fixed and processed for histological analyses or snap-frozen for molecular biology analyses. The effectiveness of the decellularization procedure was confirmed by the absence of nuclei at the Hematoxylin and Eosin staining and by the quantification of DNA content that resulted well below the accepted threshold of 50 ng/mg of dry tissue. Masson’s and Mallory’s trichrome stainings revealed the preservation of the three-dimensional architecture of cardiac d-ECM, while Van Gieson, Alcian Blue, Periodic Acid Schiff and Sirius Red stainings documented the retention of elastic fibers, glycosaminoglycans (GAGs), non-collagenous proteins and collagen, respectively. Further, immunohistochemistry revealed in the cardiac d-ECM the retention of proteins typical of the native cardiac ECM, like fibronectin, tenascin and laminin, while quantitative dye binding assay and specific protein array documented the retention of elastin and growth factors, respectively. These results prove that our method, combined with a hand-made sample-holder specifically designed for decellularization procedures under constant agitation, yields a cardiac d-ECM well preserved in its composition and architecture.

Elasticity of myocardium is mostly due to elasticity of cardiomyocytes and is essential for cardiomyocyte alignment and differentiation. Cardiac decellularized ECM (d-ECM) is emerging as natural scaffold to promote and support myocardial regeneration. It is noteworthy that cardiac d-ECM is obtained through complete removal of cardiomyocytes with loss of elasticity. We hypothesize that decellularized skin might be an easily accessible, viable alternative for myocardium regeneration, as decellularization is unlikely to cause loss of skin elasticity, provided by elastic fibers rather than by resident cells. Skin fragments from patients undergoing plastic surgery were decellularized through novel simple and fast protocol. Decellularized Human Skin (d-HuSk) obtained was assayed in quantitative dye-binding method to measure content of elastin, while elastin distribution was evaluated on histological sections by Paraldehyde Fuchsin Gomori and Weigert Van Gieson stainings. d-Husk was then sectioned and used as scaffold to prepare three-dimensional culture of cardiac primitive cells (CPCs). Then, survival and ability of CPC cultured on d-HuSk to differentiate towards cardiac myocytes was evaluated at gene and protein level. Histological and quantitative analysis provided evidence of effective decellularization, preserved tissue architecture and retention of elastin. CPCs engrafted onto d-Husk, survived, and retained expression of markers specific for cardiac myocytes at gene and protein level.Our study provides compelling evidence that common signals act in cardiac and skin microenvironment to maintain CPC ability to differentiate towards cardiac muscle and that skin holds promise as an alternate biological scaffold for cardiovascular regenerative medicine.

Cancer associated fibroblasts (CAFs) are the main stromal cell type of solid tumour microenvironment and undergo an activation process associated with secretion of growth factors, cytokines, and paracrine interactions. One of the important features of solid tumours is the metabolic reprogramming that leads to changes of bioenergetics and biosynthesis in both tumour cells and CAFs. In particular, CAFs follow the evolution of tumour disease and acquire a catabolic phenotype: in tumour tissues, cancer cells and tumour microenvironment form a network where the crosstalk between cancer cells and CAFs is associated with cell metabolic reprogramming that contributes to CAFs activation, cancer growth, and progression and evasion from cancer therapies. In this regard, the study of CAFs metabolic reprogramming could contribute to better understand their activation process, the interaction between stroma, and cancer cells and could offer innovative tools for the development of new therapeutic strategies able to eradicate the protumorigenic activity of CAFs. Therefore, this review focuses on CAFs metabolic reprogramming associated with both differentiation process and cancer and stromal cells crosstalk. Finally, therapeutic responses and potential anticancer strategies targeting CAFs metabolic reprogramming are reviewed.

Among biomaterials tested so far in cardiac tissue engineering, the decellularized native ECM (d-ECM) appears by far the most promising and appealing[1]. In order to obtain intact scaffold of d-ECM while preserving its composition, we compared the human cardiac d-ECM produced through four different protocols (named Pr1, Pr2, Pr3 and Pr4) in terms of efficiency of decellularization, composition of scaffolds and their suitability for repopulation with cardiac stem cells to develop myocardial patches. As regards efficiency of decellularization, DNA measurement revealed ineffectiveness of Pr1 and Pr3 in producing thoroughly acellular d-ECM, as they yielded scaffolds containing 46,33+5,3 and 96,7+12,2 ng of DNA/mg of tissue, respectively, while DNA content was as low as 9+2,1 in d-ECMs produced with Pr2, which includes DNase treatment, and 17,33+2.22 in the ones obtained with Pr4. Hematoxylin and Eosin staining confirmed the effectiveness of the decellularization procedure for Pr2 and Pr4, while Masson's staining revealed that Pr4 produced a d-ECM with more preserved architecture. Sirius Red, PAS and Gomori stainings documented the retention of collagen, non-collagenous proteins and elastic fibers in d-ECM from all protocols. Immunodetection of fibronectin, tenascin and laminin confirmed the retention of such ECM proteins in d-ECM, while assay for insoluble collagen and sulphated glycosaminoglycan confirmed the presence of collagen and GAGs in all protocols. Mostly due to ineffective decellularization and, as such, to mild treatment, ECM obtained through Pr3 retained more collagen and GAGs. Nevertheless, collagen content was only 1,5-fold higher than in d-ECM obtained with Pr4, which retained the highest amount of GAGs as well. Notably, GAGs protect growth factors against proteolysis, and protein array to investigate the retention of growth factors by d-ECM proved the highest efficiency of Pr4 in retaining growth factors as HGF, VEGF, TGF and IGF-1 in d-ECM. Finally, to assess suitability of d-ECM scaffolds for cell repopulation, we cultured cardiac stem cells (CSCs) on d-ECM to evaluate their viability and differentiation trend. Among all, Pr2 and Pr4 better supported cellular viability, but Pr4 provided the best environment to promote terminal differentiation towards myocardium, endothelium and smooth muscle cell lineages. Interestingly, we previously reported the inability of human cardiac stem cells to acquire fully differentiated state in vitro[2], but d-ECM supports stem cell terminal differentiation prompting progenitor cells to further proceed in differentiation towards precursor and mature phenotype. In conclusion, the composition and architecture of scaffolds of human cardiac d-ECM depend on decellularization protocol. We report here an adaptation of previously described protocol which yields highly preserved d-ECM, in terms of composition and architectures, that might be successfully be employed in regenerative medicine as biological scaffold.

Induced Pluripotent Stem cells (iPSC) are pluripotent stem cells reprogrammed from adult somatic cells. Although iPSC hold great potential for applications in regenerative medicine, technical problems, mostly related to the low efficiency of reprogramming, are yet to be solved. Since the most used cells for iPSC reprogramming are skin fibroblasts (FB), and since FB preserve positional memory, we hypothesize that the anatomic origin of FB might influence iPSC reprogramming.We isolated FB from skin of five different sites (neck, arm, thigh, breast, abdomen) of 13 patients undergoing plastic surgery or from heart wall or ascending aorta wall of the explanted heart of 3 patients receiving heart transplantation. FB from different anatomic sites and control FB from neonatal foreskin, were cultured for one week to evaluate morphology, proliferation rate and proneness to apoptosis. Additionally, expression of vimentin, cadherin, smooth muscle actin and Factor VIII was investigated to exclude the presence of other cell types. Transcriptome analysis including genes involved in stemness maintenance, embryogenesis, cell growth, activation and development, was performed by real-time PCR. Despite the similar morphology of FB from different sites, and immunopositivity for vimentin, along with the absence of other cell type markers, FB isolated from abdomen and heart had 1.5-fold higher doubling time, while FB from heart, abdomen and breast were less susceptible to apoptosis. Intriguingly, Real-Time PCR revealed that in abdomen, breast, neck, arm and heart FB genes involved in cell growth, development, proliferation, and migration, as TM4SF1, GPC4, CSPG2, DDIT4, ID1 were up-regulated, while genes regulating embryogenesis and tissue morphogenesis, like VCAN, FN1, HOXA5, CD49a were up-regulated in FB isolated from abdomen, arm and heart. However, all FBs had transcripts of markers of Mesenchymal Stem Cells (MSC), as CD105 and CD90. Our results provide evidence that human adult FB from different sites have different genetic program. Therefore, FB may respond to reprogram technology in different manner, thus affecting reprogramming efficiency. While offering novel perspective of the reprogramming technology, our study also demonstrates that abdomen and breast FB share cardiac genetic signature of cardiac FB while expressing markers of MSC and they might represent the ideal cell for cardiac reprogramming., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Extracellular matrix (ECM) is an intricate mesh of collagenous and non-collagenous proteins, whose presence and amount vary according to type of tissue. ECM drew the attention of regenerative medicine scientists as natural scaffold suitable for stem cell delivery into damaged tissues. Although a multitude of protocols and combinations of chemical agents and physical methods have been tested and proved effective in the decellularization of human heart, none of the ones tried in our setting fulfilled the goal of obtaining a structurally preserved cardiac decellularized ECM (d-ECM). While testing already described procedures, we made several adjustments that led to the development of a novel, simpler and robust protocol to decellularize adult human heart. Specifically, we decellularized cardiac samples of the free wall of both ventricles of adult human hearts scaled down to fit into embedding cassettes used to avoid stirring stress and preserve structure. To shorten the procedure, a combination of SDS, Triton X-100 and antibiotics was used in simple and fast two-step protocol. After decellularization, d-ECM was fixed and processed for histological study or snap-frozen for molecular biology analysis or cytocompatibility test in vitro. Histochemistry and immunoistochemistry confirmed the absence of nuclei and the preservation of architecture and composition of d-ECM. Further, while DNA content in d-ECM was well below accepted standards, sGAG, elastin and growth factors were retained and d-ECM scaffolds supported cardiac primitive cell engraftment and survival in vitro. Hence, according to our evidence, our protocol is simple, fast, effective and is worth improving for clinical translation., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Although, when injected, human Cardiac Primitive Cells (CPC) are not retained by host myocardium, they still improve cardiac function. Emerging evidence supports the hypothesis that exosomes may be responsible for beneficial effects induced by stem cells delivered in the infarcted myocardium. Exosomes are nano-sized vesicles naturally secreted by almost all cells and ubiquitously found in cell culture supernatants and biological fluids. Transporting and transferring peptides, lipids, and nucleic acids, exosomes have the potential to modulate signaling pathways, cell growth, migration, and proliferation of recipient cells. Accordingly, CPC may deliver chemoattractive, pro-survival and differentiating signals to resident cells through exosomes. To test our hypothesis, we isolated exosomes released in culture by CPC isolated from adult human myocardium (Exo-CPC) and analyzed the composition of their cargo and the effects elicited in vitro by their administration to resident population of CPC or fibroblasts. Specifically, we searched for the presence of specific factors known to regulate CPC migration, survival and differentiation. Additionally, we tested in vitro the potential of Exo-CPC of either regulating CPC proliferation and programmed cell death, and modulating interstitial fibrosis, extracellular-matrix (ECM) synthesis and deposition. Interestingly, on one hand, signals delivered by Exo-CPC affected proliferation and survival of CPC and, on the other hand, regulated ECM protein production. Therefore, we might speculate that Exo-CPC have potential effect on both resident CPC and fibroblasts when injected in cardiac wall.

The ascending aorta and pulmonary trunk develop from the common truncus arteriosus that later becomes symmetrically divided by the aorticopulmonary septum. Normally, the systolic pressure value and the diastolic pressure gradient in the aorta is much higher than that in the pulmonary artery. In the Ross procedure, patient’s diseased aortic valve is replaced with their own pulmonary valve and as a consequence the pulmonary trunk is placed in the aortic root position. Typically, a reinforcement of transposed pulmonary trunk is necessary to avoid vessel dilation. In order to investigate how the blood flow characteristics determine the vessel wall structure we examined by immunochemistry the wall of normal aorta (NA), normal pulmonary trunk (NPT), transposed pulmonary trunk (trPT) and transposed pulmonary trunk with reinforcement (trPT-R). Throughout the tunica media of NA, elastic fibers form numerous, conspicuous and orderly arranged wavy lamellae that parallel thin layers of smooth muscle cells between the internal and external elastic membrane. In the NPT, smooth muscle cells run amid collagen fibers and form layers that are thicker and irregular, with elastic fibers arranged accordingly. In the trPT, intima denudation and media disruptions were observed. In the tunica media, smooth muscle cells were abundant, but muscle fibers, with irregular profiles and no discernible alignment, were widely spaced with intervening collagen fibers. In the trPA-R, the endothelial lining was preserved and elastic fibers formed a thick and highly organized layer of concentric lamellae in the middle third of tunica media. The structure of normal aorta and pulmonary trunk, both elastic arteries, with common embryological origin, differs significantly in terms of smooth muscle and elastic sheets number and organization. The animal model of Ross procedure with pulmonary trunk in aorta position further underscores the role of blood pressure and mechanical stress in vessel wall modification., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014