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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.

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.

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.

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.

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.

Induced Pluripotent Stem Cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell-like state. Fibroblast (FB) is the adult somatic cell most commonly used for reprogramming and is defined by spindle-shape morphology, plastic-adherence and expression of several markers. Nonetheless, such properties also define Mesenchymal Stem Cells (MSCs), that also share with dermal FB the ability to differentiate into osteoblasts, adipocytes and chondroblasts. Moving from the hypothesis that the most striking difference between FBs and MSCs is in the name, we isolated and cultured human dermal and visceral FBs from tissue waste fragments of patients undergoing surgical procedures, and evaluated their morphology and expression of markers typical of MSCs, at gene and protein level. Additionally, we analyzed synthesis and release of specific growth factors in culture medium and, finally, we tested FB ability to differentiate into osteoblasts, adipocytes and chondroblasts. Interestingly, all FBs in culture adhered to plastic culture dishes, expressed markers typical of MSCs, like CD90, CD105, CD146,SSEA-4, ECM2, ID-1, and were morphologically undistinguishable. As reported for MSCs, FBs released EGF, FGF-4, GDNF, HGF, IGF, TGF-b and VEGF, and were capable of differentiating into cells of mesodermal origin. Our findings raise the question of whether FBs and MSCs are not admittedly the same population.We might infer that previously described differences are due to different stages of differentiation, which also account for reports on high variability in both expression of mesenchymal markers by MSCs and efficiency of FB reprogramming to iPSCs.

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 40x28x6mm, 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. Hence, according to our evidence, our protocol is simple, fast, effective and is worth improving for clinical translation.

Induced Pluripotent Stem cells (iPSC) are adult somatic cells genetically reprogrammed to an embryonic stem cell-like state. Due to their autologous origin from adult somatic cells, iPSCs are considered a tremendously valuable tool for regenerative medicine, disease modeling, drug discovery and testing. iPSCs were first obtained by introducing specific transcription factors through retroviral transfection. However, cell reprogramming obtained by integrating methods prevent clinical application of iPSC because of potential risk for infection, teratomas and genomic instability. Therefore, several integration-free alternate methods have been developed and tested thus far to overcome safety issues. The present chapter provides an overview and a critical analysis of advantages and disadvantages of non-integrating methods used to generate iPSCs.