Generation of an Immortalized Human Adipose-Derived Mesenchymal Stromal Cell Line Suitable for Wound Healing Therapy.

Adipose-derived mesenchymal stromal cells (ADSC) are a promising source for cellular therapy of chronic wounds. However, the limited life span during in vitro expansion impedes their extensive use in clinical applications and basic research. We hypothesize that by introduction of an ectopic expression of telomerase into ADSC, the cells' lifespans could be significantly extended. To test this hypothesis, we aimed at engineering an immortalized human ADSC line using a lentiviral transduction with human telomerase (hTERT). ADSC were transduced with a third-generation lentiviral system and a hTERT codifying plasmid (pLV-hTERT-IRES-hygro). A population characterized by increased hTERT expression, extensive proliferative potential and remarkable (potent) multilineage differentiation capacity was selected. The properties for wound healing of this immortalized ADSC line were assessed after 17 passages. Their secretome induced the proliferation and migration of keratinocytes, dermal fibroblasts, and endothelial cells similarly to untransduced ADSC. Moreover, they sustained the complete re-epithelialization of a full thickness wound performed on a skin organotypic model. In summary, the engineered immortalized ADSC maintain the beneficial properties of parent cells and could represent a valuable and suitable tool for wound healing in particular, and for skin regenerative therapy in general.

Human Mesenchymal Stromal Cell-Derived Exosomes Promote In Vitro Wound Healing by Modulating the Biological Properties of Skin Keratinocytes and Fibroblasts and Stimulating Angiogenesis.

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.

Mesenchymal stromal cell-derived factors promote the colonization of collagen 3D scaffolds with human skin cells.

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.

Heterogeneity of human fibroblasts isolated from hypertrophic scar.

Pathological wound healing states, such as hypertrophic scarring and keloids, represent a huge clinical and financial burden on healthcare system. The complex biological mechanisms occurring in hypertrophic scarring are still barely understood. To date, there is no satisfactory description of hypertrophic fibroblasts. Therefore, in the present study we focused on the comparatively characterization of the fibroblasts residing in different regions of hypertrophic scars. To achieve this aim, fibroblasts were isolated from normal skin samples (n=4) and hypertrophic scars (n=4). These cell populations were further were used for the evaluation of proliferation and migration capacity, for the gene and protein expression of extracellular matrix protein type I collagen and fibronectin and for the presence of myofibroblasts. Our results demonstrated that perilesional and intralesional fibroblasts isolated from hypertrophic scars could be considered as distinct populations, having different properties. Thus, the intralesional fibroblasts had an increased proliferation capacity and increased gene and protein expression of collagen I and fibronectin. However, the perilesional fibroblasts had augmented mobility as revealed by in vitro scratch test and contained a higher percentage of myofibroblasts [alpha-smooth muscle actin (α-SMA)high cells], in comparison to the intralesional population. In conclusion, our data could provide an explanation regarding the inconsistent efficacy of topic therapies for hypertrophic scars.

Mesenchymal stromal cells derived exosomes as tools for chronic wound healing therapy.

In modern society, the healing of chronic wounds is still a major cause of discomfort for the patients and a financial burden for the care system. Current approaches use either organic tissue-engineered skin substitutes or stem cells based therapy. It has been shown that mesenchymal stem cells (MSCs) are able to improve the wound healing process by secreting factors with anti-inflammatory, anti-fibrotic and pro-angiogenic activities either as soluble molecules (growth factors, cytokines) or encapsulated within membrane vesicles (microparticles, exosomes). It has been shown that exosomes, the small membrane vesicles originating from the endocytic pathway, are the main mediators of MSCs paracrine effect. Their complex cargo (mRNA, microRNA and various anti-apoptotic and pro-angiogenic factors) has been found to induce migration and proliferation of fibroblasts as well as collagen synthesis. Thus, the combination of MSCs derived exosomes and organic biomaterials in order to enhance the healing process represents a novel approach for chronic wounds therapy, involving a cell-free use of MSCs paracrine activity.

Collagen regulates the ability of endothelial progenitor cells to protect hypoxic myocardium through a mechanism involving miR-377/VE-PTP axis.

The possibility to employ stem/progenitor cells in the cardiovascular remodelling after myocardial infarction is one of the main queries of regenerative medicine. To investigate whether endothelial progenitor cells (EPCs) participate in the restoration of hypoxia-affected myocardium, we used a co-culture model that allowed the intimate interaction between EPCs and myocardial slices, mimicking stem cell transplantation into the ischaemic heart. On this model, we showed that EPCs engrafted to some extent and only transiently survived into the host tissue, yet produced visible protective effects, in terms of angiogenesis and protection against apoptosis and identified miR-377-VE-PTP axis as being involved in the protective effects of EPCs in hypoxic myocardium. We also showed that collagen, the main component of the myocardial scar, was important for these protective effects by preserving VE-PTP levels, which were otherwise diminished by miR-377. By this, a good face of the scar is revealed, which was so far perceived as having only detrimental impact on the exogenously delivered stem/progenitor cells by affecting not only the engraftment, but also the general protective effects of stem cells.

Evaluation of the Ability of Nanostructured PEI-Coated Iron Oxide Nanoparticles to Incorporate Cisplatin during Synthesis.

Nanoparticles (NPs) have a high potential for biological applications as they can be used as carriers for the controlled release of bioactive factors. Here we focused on poly(ethylenimine) (PEI)-coated iron oxide hybrid NPs obtained by hydrothermal synthesis in high pressure conditions and evaluated their behavior in culture medium in the presence or absence of cells, as well as their ability to incorporate antitumor drug cisplatin. Our results showed that the hydrothermal conditions used for Fe-PEI NPs synthesis allowed the incorporation of cisplatin, which even increased its anti-tumor effects. Furthermore, the commonly occurring phenomenon of NPs aggregation in culture medium was exploited for further entrapment of other active molecules, such as the fluorescent dye DiI and valinomycin. The molecules bound to NPs during synthesis or during aggregation process were delivered inside various cells after in vitro and in vivo direct contact between cells and NPs and their biological activity was preserved, thus supporting the therapeutic value of Fe-PEI NPs as drug delivery tools.

Emerging Role of Stem Cells - Derived Exosomes as Valuable Tools for Cardiovascular Therapy.

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.

Synergic effects of VEGF-A and SDF-1 on the angiogenic properties of endothelial progenitor cells.

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.

Repair of the Orbital Wall Fractures in Rabbit Animal Model Using Nanostructured Hydroxyapatite-Based Implant.

Cellular uptake and cytotoxicity of nanostructured hydroxyapatite (nanoHAp) are dependent on its physical parameters. Therefore, an understanding of both surface chemistry and morphology of nanoHAp is needed in order to be able to anticipate its in vivo behavior. The aim of this paper is to characterize an engineered nanoHAp in terms of physico-chemical properties, biocompatibility, and its capability to reconstitute the orbital wall fractures in rabbits. NanoHAp was synthesized using a high pressure hydrothermal method and characterized by physico-chemical, structural, morphological, and optical techniques. X-ray diffraction revealed HAp crystallites of 21 nm, while Scanning Electron Microscopy (SEM) images showed spherical shapes of HAp powder. Mean particle size of HAp measured by DLS technique was 146.3 nm. Biocompatibility was estimated by the effect of HAp powder on the adhesion and proliferation of mesenchymal stem cells (MSC) in culture. The results showed that cell proliferation on powder-coated slides was between 73.4% and 98.3% of control cells (cells grown in normal culture conditions). Computed tomography analysis of the preformed nanoHAp implanted in orbital wall fractures, performed at one and two months postoperative, demonstrated the integration of the implants in the bones. In conclusion, our engineered nanoHAp is stable, biocompatible, and may be safely considered for reconstruction of orbital wall fractures.

Pre-stimulation with FGF-2 increases in vitro functional coupling of mesenchymal stem cells with cardiac cells.

The functional coupling of transplanted cells with host myocardial cells is a significant challenge in mesenchymal stem cell (MSC) cardiomyoplasty being related to cell survival and therapeutic outcomes. Priming of MSCs with growth factors has been reported to improve their therapeutic efficacy through gap junction-mediated mechanisms. However, the expression pattern of Connexin43 (Cx43) in growth factor-stimulated MSC was not previously addressed. In this study we investigated how the pre-treatment with growth factors modulates MSC ability to integrate into the host tissue after transplantation, with particular focus on the expression of Cx43 and its cellular distribution. Our results showed that stimulation of MSCs with IGF-1, FGF-2, but not TGFß, increased the level of Cx43 at both mRNA and protein levels. IGF-1 stimulation resulted in a shift of the fibroblast morphology into an epithelial morphology in several well-defined areas of stimulated cells. Confocal microscopy examination revealed that the increase of Cx43 was restricted to the epithelial-like cells and did not occur in other cells. In variance, FGF-2 induced a rod-shape morphology of every single cell, which achieved an extremely low cell index. FGF-2 stimulation also induced a time-dependent increase in Cx43, with a regular distribution pattern in all cells. Dye transfer assay coupled with confocal microscopy and flow cytometry analysis demonstrated functional in vitro cell coupling between FGF-2-stimulated MSCs as well as between FGF-2-stimulated cells and H9c2 cardiomyoblasts, a scenery that mimick MSC transplantation into the myocardium. We conclude that the stimulation of MSCs with FGF-2 prior to transplantation may facilitate their access among the myocardial cells and increase the functional coupling between transplanted and host cells.