Stem cell-based therapy for myocardial regeneration has reported several functional improvements that are attributed mostly to the paracrine effects stimulating angiogenesis and cell survival. This study was conducted to comparatively evaluate the potential of factors secreted by mesenchymal stem cells (MSCs) in normoxic and hypoxic conditions to promote tissue repair by sustaining endothelial cell (EC) adhesion and proliferation and conferring protection against apoptosis. To this aim, a conditioned medium (CM) was generated from MSCs after 24-h incubation in a serum-free normal or hypoxic environment. MSCs exhibited resistance to hypoxia, which induced increased secretion of vascular endothelial growth factor (VEGF) and decreased levels of other cytokines, including stromal-derived factor-1 (SDF). The CM derived from normal (nMSC-CM) and hypoxic cells (hypMSC-CM) induced similar protective effects on H9c2 cells in hypoxia. Minor differences were noticed in the potential of normal versus hypoxic CM to promote angiogenesis, which were likely connected to SDFa and VEGF levels: the nMSC-CM was more effective in stimulating EC migration, whereas the hypMSC-CM had an enhanced effect on EC adhesion. However, the factors secreted by MSCs in normoxic or hypoxic conditions supported adhesion, but not proliferation, of ECs in vitro, as revealed by impedance-based dynamic assessments. Surprisingly, factors secreted by other stem/progenitor cells, such as endothelial progenitor cells (EPCs), had complementary effects to the MSC-CM. Thus, the EPC-CM, in either a normal or hypoxic environment, supported EC proliferation, but did not sustain EC adhesion. Combined use of the MSC-CM and EPC-CM promoted both EC adhesion and proliferation, suggesting that the local angiogenesis at the site of ischemic injury might be better stimulated by simultaneous releasing of factors secreted by multiple stem/progenitor cell populations.

The treatment of cardiac diseases by cell therapy continues to be challenged by a limited supply of appropriate cells. Although stem cells can generate myocytes after local delivery into the heart, this is often accompanied by the generation of several other cell types as a consequence of environment-driven differentiation. One strategy for overcoming dysregulated differentiation is the pretreatment of stem cells with the demethylation agent 5-azacytidine. The effects of 5-azacytidine on various stem cell types vary from cardiomyogenic differentiation to failure of differentiation or from adipogenic and chondrogenic differentiation to uncontrollable expression of a variety of genes. The underlying mechanisms remain poorly understood, and the effect of 5-azacytidine on the multipotent capacity of stem cells has never been addressed. This study was designed to investigate the changes induced by 5-azacytidine in mesenchymal stem cells (MSC), with particular focus on multipotency maintenance and the capacity of 5-azacytidine to boost myogenic differentiation. Our results show that MSCs retained their multipotent capacity after one pulse with 5-azacytidine, whereas additional pulses resulted in a restricted differentiation potential with concomitant increased ability to accomplish chondrogenic commitment. The induction of cardiac differentiation of MSCs was not observed unless the transcriptional activation of several genes was induced by random hypomethylation. Nevertheless, 5-azacytidine treatment promoted cell response to subsequent stimuli and generation of myogenic differentiation under permissive environmental conditions. Therefore, we assume that one pulse with 5-azacytidine might similarly promote the subsequent cardiac differentiation of MSCs, but it is dependent on the finding of adequate conditions for myocardial differentiation.

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Bone marrow-derived mesenchymal stromal cells (MSCs) are major players in regenerative therapies for wound healing via their paracrine activity, mediated partially by exosomes. Our purpose was to test if MSC-derived exosomes could accelerate wound healing by enhancing the biological properties of the main cell types involved in the key phases of this process. Thus, the effects of exosomes on (i) macrophage activation, (ii) angiogenesis, (iii) keratinocytes and dermal fibroblasts proliferation and migration, and (iv) the capacity of myofibroblasts to regulate the turnover of the extracellular matrix were evaluated. The results showed that, although exosomes did not exhibit anti-inflammatory properties, they stimulated angiogenesis. Exposure of keratinocytes and dermal (myo)fibroblasts to exosomes enhanced their proliferation and migratory capacity. Additionally, exosomes prevented the upregulation of gene expression for type I and III collagen, α-smooth muscle actin, and MMP2 and 14, and they increased MMP13 expression during the fibroblast–myofibroblast transition. The regenerative properties of exosomes were validated using a wound healing skin organotypic model, which exhibited full re-epithelialization upon exosomes exposure. In summary, these data indicate that exosomes enhance the biological properties of keratinocytes, fibroblasts, and endothelial cells, thus providing a reliable therapeutic tool for skin regeneration.

Here we investigated the impact of hypoxic environment on the angiogenic properties of early-outgrowth endothelial progenitor cells (EPCs), with particular focus on the role of secreted vascular endothelial growth factor-A (VEGF-A) and stromal derived factor-1 (SDF-1) in mediating these effects. We found that cultured EPCs secreted factors with paracrine effects on chemotaxis, migration, proliferation and tube formation of mature endothelial cells (ECs), and these properties were not affected by hypoxia. Depletion of VEGF-A did not change the ability of EPC-conditioned medium (CM) to promote EC migration and tube formation in vitro, suggesting that the pro-angiogenic paracrine effects of EPCs did not totally rely on the presence of VEGF-A. These findings were confirmed by in vivo experiments, on a mouse model of hind limb ischaemia, which showed that VEGF-depleted EPC-CM sustained tissue perfusion at the same level as complete EPC-CM. However, concomitant deletion of VEGF-A and SDF-1 in EPC-CM impaired the pro-angiogenic properties of EPC-CM, by inhibition of EC spreading in culture, tube-like structure formation on Matrigel support, in vivo neovessels formation and ischaemic hind limb regeneration. Taken together, our data demonstrate that: (i) hypoxia does not affect the capacity of EPCs to support the angiogenic process; (ii) the absence of either VEGF-A or SDF-1 from EPC-CM can be rescued by the presence of the other one, so that the overall angiogenic effects remain unchanged; and (iii) and the concomitant deletion of VEGF-A and SDF-1 from EPC-CM impairs its pro-angiogenic effect, both in vitro and in vivo. Copyright © 2016 John Wiley & Sons, Ltd.

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In modern society, myocardial infarction is a major cause of mortality, morbidity and deterioration of quality of life. Although various therapeutic approaches are available, none of them lead to the regeneration of infarcted tissue. The use of mesenchymal stem cells in cell therapy for myocardial infarction showed a beneficial effect consisting in reduced infarcted area and improved cardiac function, which can be explained by paracrine mechanism. It has been shown that stem cells are able to release a very complex range of factors including growth factors, cytokines and chemokines, along with an abundant mixture of membrane vesicles. These secreted elements contribute to the beneficial effect of stem cells therapy observed both in vitro and in vivo. Recent studies have shown that exosomes, which are small membrane vesicles originating in the endocytic pathway of the cells and carry a complex cargo consisting in mRNA, microRNA and various other anti-apoptotic and pro-angiogenic factors, are the main mediators of stem cells paracrine effect. In this review, we discuss the capacity of mesenchymal stem cells to protect the ischemic myocardium, the role of exosomes as protective factors secreted by stem cells and the possibility to use these vesicles in developing a novel approach in cardiovascular therapy, involving a non-cellular use of mesenchymal stem cells paracrine activity.

The development of stem cell technology in combination with advances in biomaterials has opened new ways of producing engineered tissue substitutes. In this study, we investigated whether the therapeutic potential of an acellular porous scaffold made of type I collagen can be improved by the addition of a powerful trophic agent in the form of mesenchymal stromal cells conditioned medium (MSC‐CM) in order to be used as an acellular scaffold for skin wound healing treatment. Our experiments showed that MSC‐CM sustained the adherence of keratinocytes and fibroblasts as well as the proliferation of keratinocytes. Moreover, MSC‐CM had chemoattractant properties for keratinocytes and endothelial cells, attributable to the content of trophic and pro‐angiogenic factors. Also, for the dermal fibroblasts cultured on collagen scaffold in the presence of MSC‐CM versus serum control, the ratio between collagen III and I mRNAs increased by 2‐fold. Furthermore, the gene expression for α‐smooth muscle actin, tissue inhibitor of metalloproteinase‐1 and 2 and matrix metalloproteinase‐14 was significantly increased by approximately 2‐fold. In conclusion, factors existing in MSC‐CM improve the colonization of collagen 3D scaffolds, by sustaining the adherence and proliferation of keratinocytes and by inducing a pro‐healing phenotype in fibroblasts.