Digitally Enhanced Methods for the Diagnosis and Monitoring of Treatment Responses in Actinic Keratoses: A New Avenue in Personalized Skin Care.

Non-melanocytic skin cancers represent an important public health problem due to the increasing incidence and the important local destructive potential. Thus, the early diagnosis and treatment of precancerous lesions (actinic keratoses) is a priority for the dermatologist. In recent years, non-invasive skin imaging methods have seen an important development, moving from simple observational methods used in clinical research, to true diagnostic and treatment methods that make the dermatologist's life easier. Given the frequency of these precancerous lesions, their location on photo-exposed areas, as well as the long treatment periods, with variable, imprecise end-points, the need to use non-invasive imaging devices is increasingly evident to complete the clinical observations in the diagnosis and treatment of these lesions, with the aim of increasing accuracy and decreasing the adverse effects due to long treatment duration. This is the first review that brings together all skin imaging methods (dermoscopy, reflectance confocal microscopy, ultrasonography, dermoscopy-guided high frequency ultrasonography, and optical coherence tomography) used in the evaluation of actinic keratoses and their response to different treatment regimens.

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.

Patient-specific induced pluripotent stem cells as "disease-in-a-dish" models for inherited cardiomyopathies and channelopathies - 15 years of research.

Among inherited cardiac conditions, a special place is kept by cardiomyopathies (CMPs) and channelopathies (CNPs), which pose a substantial healthcare burden due to the complexity of the therapeutic management and cause early mortality. Like other inherited cardiac conditions, genetic CMPs and CNPs exhibit incomplete penetrance and variable expressivity even within carriers of the same pathogenic deoxyribonucleic acid variant, challenging our understanding of the underlying pathogenic mechanisms. Until recently, the lack of accurate physiological preclinical models hindered the investigation of fundamental cellular and molecular mechanisms. The advent of induced pluripotent stem cell (iPSC) technology, along with advances in gene editing, offered unprecedented opportunities to explore hereditary CMPs and CNPs. Hallmark features of iPSCs include the ability to differentiate into unlimited numbers of cells from any of the three germ layers, genetic identity with the subject from whom they were derived, and ease of gene editing, all of which were used to generate "disease-in-a-dish" models of monogenic cardiac conditions. Functionally, iPSC-derived cardiomyocytes that faithfully recapitulate the patient-specific phenotype, allowed the study of disease mechanisms in an individual-/allele-specific manner, as well as the customization of therapeutic regimen. This review provides a synopsis of the most important iPSC-based models of CMPs and CNPs and the potential use for modeling disease mechanisms, personalized therapy and deoxyribonucleic acid variant functional annotation.

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.

Stem/progenitor cells and obstructive sleep apnea syndrome - new insights for clinical applications.

Obstructive sleep apnea syndrome (OSAS) is a widespread disorder, characterized by recurrent upper airway obstruction during sleep, mostly as a result of complete or partial pharyngeal obstruction. Due to the occurrence of frequent and regular hypoxic events, patients with OSAS are at increased risk of cardiovascular disease, stroke, metabolic disorders, occupational errors, motor vehicle accidents and even death. Thus, OSAS has severe consequences and represents a significant economic burden. However, some of the consequences, as well as their costs can be reduced with appropriate detection and treatment. In this context, the recent advances that were made in stem cell biology knowledge and stem cell - based technologies hold a great promise for various medical conditions, including respiratory diseases. However, the investigation of the role of stem cells in OSAS is still recent and rather limited, requiring further studies, both in animal models and humans. The goal of this review is to summarize the current state of knowledge regarding both lung resident as well as circulating stem/progenitor cells and discuss existing controversies in the field in order to identify future research directions for clinical applications in OSAS. Also, the paper highlights the requisite for inter-institutional, multi-disciplinary research collaborations in order to achieve breakthrough results in the field.

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.

Factors secreted by mesenchymal stem cells and endothelial progenitor cells have complementary effects on angiogenesis in vitro.

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 SDFα 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.

Cardiomyocyte apoptosis in ischaemia-reperfusion due to the exogenous oxidants at the time of reperfusion.

Various studies performed on different models have demonstrated that apoptosis occurs in ischaemic-reperfused myocardium in vivo; however, the individual contribution of ischaemia and reperfusion to CMC (cardiomyocyte) apoptosis remains uncertain. We have determined the main inducer of CMC apoptosis in ischaemia-reperfusion by exposing CMCs to either 30 min ischaemia followed by reperfusion or to 25-OH-cholesterol (25-hydroxycholesterol) for 1-3 days. Both ischaemia-reperfusion and exogenous oxidants increased the Bax/Bcl-2 ratio, a favourable effect for the apoptotic process. However, apoptosis was not observed in ischaemic CMCs in the absence of reperfusion. Moreover, reperfusion after 30 min ischaemia did not make an important contribution to CMC apoptosis in culture in terms of caspase 3 activation. In contrast, 25-OH-cholesterol promoted CMC apoptosis by a caspase 3-dependent mechanism that involved the transcriptional activation of the pro-apoptotic protein, Bax and post-translational degradation of the anti-apoptotic protein, Bcl-2. From these results, we conclude that CMC apoptosis is not induced by ischaemia per se, but by the oxidants from the surrounding environment at the time of reperfusion. These exogenous oxidants exacerbate the alterations induced by ischaemia and complete the apoptotic process at the time when ATP and glucose levels are restored.

Effect of 5-azacytidine: evidence for alteration of the multipotent ability of mesenchymal stem cells.

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.

Isolation of a mouse bone marrow population enriched in stem and progenitor cells by centrifugation on a Percoll gradient.

Given the complex composition of bone marrow, a cell separation technique that results in populations enriched in progenitor cells is required for cellular differentiation and transplantation studies. In the present study, we designed a method that allows for the isolation of a progenitor-enriched population of bone marrow by exploiting the physical properties of these cells. Bone marrow aspirate was separated on a discontinuous Percoll gradient (ranging from 1.050 to 1.083 g/cm3) that resulted in the recovery of six cell fractions. The fractions were characterized by FACS and RT-PCR (reverse transcription-PCR) analyses and evaluated for their capacity to differentiate into haematopoietic and mesenchymal cells. Fraction IV, including cells with a density of 1.070-1.076 g/ml, contained 11.68% of total bone marrow cells and was enriched in c-kit+ and Sca-1+ (stem cell antigen-1) progenitor cells as compared with total bone marrow. This fraction demonstrated an increase in clonogenic capacity under specific conditions as well as a potential to generate a mesenchymal stem cell culture in a shorter period than that using bone marrow aspirate. Furthermore, this fraction lacked differentiated cell types and contained cells positive for endothelial markers, which further increases its value in cellular transplant. In conclusion, a bone marrow subpopulation that is enriched in progenitor cells and may be valuable in cellular transplant therapy can be isolated by exploiting the physical properties of these cells.

Promoting effect of 5-azacytidine on the myogenic differentiation of bone marrow stromal cells.

Bone marrow stromal cells (BMSC) can differentiate into various cell types including myocytes, which may be valuable in cellular therapy of myocardial infarction. In an attempt to increase the myogenic commitment of BMSC, we investigated the extent of conversion induced by the demethylation agent 5-azacytidine. BMSC isolated from the adult rat tibia were exposed in culture to 5microM 5-azacytidine for 24h, 1 day after seeding. The treatment was repeated at weekly intervals and the expression of muscle-specific proteins and genes was assessed. The results revealed that cultured cells lost the native expression of osteocalcin and alkaline phosphatase as a function of time and began to express connexin 43. Exposure to 5-azacytidine of BMSC induced, at 14 days, a myocyte-resembling phenotype that included the expression of muscle-specific proteins (sarcomeric alpha-actin, troponin T, desmin, alpha-actinin, and GATA-4) and genes (GATA-4, myoD, desmin, and alpha-actinin), numerous mitochondria and myofilaments; however, the latter did not form sarcomeres. Although some of these myogenic markers also appeared in untreated cells, exposure to 5-azacytidine induced an enhanced response of calcium channels, as well as a threefold increase in desmin and myoD gene expression and a twofold increase in alpha-actinin gene and protein expression above the control values. In conclusion, the results demonstrate a promoting effect of 5-azacytidine on the expression of muscle-specific proteins and genes in BMSC in culture. Notably, the myogenic differentiation takes place over a short period of time. Priming of mesenchymal cells to cardiomyogenic differentiation may have significant applications in cellular approaches to ameliorate muscle loss after myocardial ischemia.