Role of Nano-miRNAs in Diagnostics and Therapeutics.

MicroRNAs (miRNA) are key regulators of gene expression, controlling different biological processes such as cellular development, differentiation, proliferation, metabolism, and apoptosis. The relationships between miRNA expression and the onset and progression of different diseases, such as tumours, cardiovascular and rheumatic diseases, and neurological disorders, are well known. A nanotechnology-based approach could match miRNA delivery and detection to move beyond the proof-of-concept stage. Different kinds of nanotechnologies can have a major impact on the diagnosis and treatment of miRNA-related diseases such as cancer. Developing novel methodologies aimed at clinical practice represents a big challenge for the early diagnosis of specific diseases. Within this context, nanotechnology represents a wide emerging area at the forefront of research over the last two decades, whose potential has yet to be fully attained. Nanomedicine, derived from nanotechnology, can exploit the unique properties of nanometer-sized particles for diagnostic and therapeutic purposes. Through nanomedicine, specific treatment to counteract only cancer-cell proliferation will be improved, while leaving healthy cells intact. In this review, we dissect the properties of different nanocarriers and their roles in the early detection and treatment of cancer.

Nanomaterials in Skin Regeneration and Rejuvenation.

Skin is the external part of the human body; thus, it is exposed to outer stimuli leading to injuries and damage, due to being the tissue mostly affected by wounds and aging that compromise its protective function. The recent extension of the average lifespan raises the interest in products capable of counteracting skin related health conditions. However, the skin barrier is not easy to permeate and could be influenced by different factors. In the last decades an innovative pharmacotherapeutic approach has been possible thanks to the advent of nanomedicine. Nanodevices can represent an appropriate formulation to enhance the passive penetration, modulate drug solubility and increase the thermodynamic activity of drugs. Here, we summarize the recent nanotechnological approaches to maintain and replace skin homeostasis, with particular attention to nanomaterials applications on wound healing, regeneration and rejuvenation of skin tissue. The different nanomaterials as nanofibers, hydrogels, nanosuspensions, and nanoparticles are described and in particular we highlight their main chemical features that are useful in drug delivery and tissue regeneration.

Fibroblast Proliferation and Migration in Wound Healing by Phytochemicals: Evidence for a Novel Synergic Outcome.

Wound-healing is a dynamic skin reparative process that results in a sequence of events, including inflammation, proliferation, and migration of different cell types as fibroblasts. Fibroblasts play a crucial role in repairing processes, from the late inflammatory phase until the fully final epithelization of the injured tissue. Within this context, identifying tools able to implement cell proliferation and migration could improve tissue regeneration. Recently, plants species from all over the world are coming out as novel tools for therapeutic applications thanks to their phytochemicals, which have antioxidant properties and can promote wound healing. In this paper, we aimed at investigating antioxidant activity of waste extracts from different medicinal plants, endemic of the Mediterranean area, on fibroblast proliferation and wound healing. We determined the amount of total phenols and anti-oxidant activity by ABTS assay. We then evaluated the cytotoxicity of the compounds and the proliferative capabilities of fibroblasts by scratch assay. Our results showed that waste extracts retain antioxidant and regenerative properties, inducing tissue re-establishment after environmental stress exposure. Taken together, our findings suggest that waste material could be used in the future also in combinations to stimulate wound healing processes and antioxidant responses in damaged skin.

Tuning Adipogenic Differentiation in ADSCs by Metformin and Vitamin D: Involvement of miRNAs.

Fat tissue represents an important source of adipose-derived stem cells (ADSCs), which can differentiate towards several phenotypes under certain stimuli. Definite molecules as vitamin D are able to influence stem cell fate, acting on the expression of specific genes. In addition, miRNAs are important modulating factors in obesity and numerous diseases. We previously identified specific conditioned media able to commit stem cells towards defined cellular phenotypes. In the present paper, we aimed at evaluating the role of metformin on ADSCs differentiation. In particular, ADSCs were cultured in a specific adipogenic conditioned medium (MD), in the presence of metformin, alone or in combination with vitamin D. Our results showed that the combination of the two compounds is able to counteract the appearance of an adipogenic phenotype, indicating a feedforward regulation on vitamin D metabolism by metformin, acting on CYP27B1 and CYP3A4. We then evaluated the role of specific epigenetic modulating genes and miRNAs in controlling stem cell adipogenesis. The combination of the two molecules was able to influence stem cell fate, by modulating the adipogenic phenotype, suggesting their possible application in clinical practice in counteracting uncontrolled lipogenesis and obesity-related diseases.

Epigenetics, Stem Cells, and Autophagy: Exploring a Path Involving miRNA.

MiRNAs, a small family of non-coding RNA, are now emerging as regulators of stem cell pluripotency, differentiation, and autophagy, thus controlling stem cell behavior. Stem cells are undifferentiated elements capable to acquire specific phenotype under different kind of stimuli, being a main tool for regenerative medicine. Within this context, we have previously shown that stem cells isolated from Wharton jelly multipotent stem cells (WJ-MSCs) exhibit gender differences in the expression of the stemness related gene OCT4 and the epigenetic modulator gene DNA-Methyltransferase (DNMT1). Here, we further analyze this gender difference, evaluating adipogenic and osteogenic differentiation potential, autophagic process, and expression of miR-145, miR-148a, and miR-185 in WJ-MSCs derived from males and females. These miRNAs were selected since they are involved in OCT4 and DNMT1 gene expression, and in stem cell differentiation. Our results indicate a difference in the regulatory circuit involving miR-148a/DNMT1/OCT4 autophagy in male WJ-MSCs as compared to female cells. Moreover, no difference was detected in the expression of the two-differentiation regulating miRNA (miR-145 and miR-185). Taken together, our results highlight a different behavior of WJ-MSCs from males and females, disclosing the chance to better understand cellular processes as autophagy and stemness, usable for future clinical applications.

Myrtus Polyphenols, from Antioxidants to Anti-Inflammatory Molecules: Exploring a Network Involving Cytochromes P450 and Vitamin D.

Inflammatory response represents one of the main mechanisms of healing and tissue function restoration. On the other hand, chronic inflammation leads to excessive secretion of pro-inflammatory cytokines involved in the onset of several diseases. Oxidative stress condition may contribute in worsening inflammatory state fall, increasing reactive oxygen species (ROS) production and cytokines release. Polyphenols can counteract inflammation and oxidative stress, modulating the release of toxic molecules and interacting with physiological defenses, such as cytochromes p450 enzymes. In this paper, we aimed at evaluating the anti-inflammatory properties of different concentrations of Myrtus communis L. pulp and seeds extracts, derived from liquor industrial production, on human fibroblasts. We determined ROS production after oxidative stress induction by H2O2 treatment, and the gene expression of different proinflammatory cytokines. We also analyzed the expression of CYP3A4 and CYP27B1 genes, in order to evaluate the capability of Myrtus polyphenols to influence the metabolic regulation of other molecules, including drugs, ROS, and vitamin D. Our results showed that Myrtus extracts exert a synergic effect with vitamin D in reducing inflammation and ROS production, protecting cells from oxidative stress damages. Moreover, the extracts modulate CYPs expression, preventing chronic inflammation and suggesting their use in development of new therapeutic formulations.

Delay in maternal transcript degradation in ovine embryos derived from low competence oocytes.

Oocytes from prepubertal animals have a reduced ability to undergo embryo development and produce viable offspring. The present work used an ovine model consisting of oocytes derived from adult and prepubertal donors to assess the molecular status of oocytes and preimplantation embryos with different developmental competence. The lower potential of oocytes of young donors was confirmed in terms of in vitro developmental capabilities and kinetics. A panel of genes including maternal effect (DPPA3, GDF9, NMP2, ZAR1) and housekeeping genes (ACTB, RPL19, SDHA, YWHAZ, ATP1A1), genes involved in DNA methylation (DNMT1, DNMT3A, DNMT3B), genomic imprinting (IGF2R), pluripotency (NANOG, POU5F1) and cell cycle regulation (CCNB1, CDK1, MELK) was relatively quantified. Temporal analysis during oocyte maturation and preimplantation embryo development evidenced patterns associated with donor age. With a few gene-specific exceptions, the differential model showed a reduced transcript abundance in immature prepubertal oocytes that completely reversed trend after fertilization, when higher mRNA levels were consistently observed in early embryos, indicating a delay in maternal transcript degradation. We propose that the molecular shortage in the prepubertal oocyte may affect its developmental potential and impair the early pathways of maternal mRNA clearance in the embryo. While confirming the different potential of oocytes derived from adult and prepubertal donors, our work showed for the first time a consistent delay in maternal transcript degradation in embryos derived from low competence oocytes that interestingly recalls the delayed developmental kinetics. Such abnormal transcript persistence may hinder further development and represents a novel perspective on the complexity of developmental competence.

Melatonin and Vitamin D Orchestrate Adipose Derived Stem Cell Fate by Modulating Epigenetic Regulatory Genes.

Melatonin, that regulates many physiological processes including circadian rhythms, is a molecule able to promote osteoblasts maturation in vitro and to prevent bone loss in vivo, while regulating also adipocytes metabolism. In this regard, we have previously shown that melatonin in combination with vitamin D, is able to counteract the appearance of an adipogenic phenotype in adipose derived stem cells (ADSCs), cultured in an adipogenic favoring condition. In the present study, we aimed at evaluating the specific phenotype elicited by melatonin and vitamin D based medium, considering also the involvement of epigenetic regulating genes. ADSCs were cultured in a specific adipogenic conditioned media, in the presence of melatonin alone or with vitamin D. The expression of specific osteogenic related genes was evaluated at different time points, together with the histone deacetylases epigenetic regulators, HDAC1 and Sirtuins (SIRT) 1 and 2. Our results show that melatonin and vitamin D are able to modulate ADSCs commitment towards osteogenic phenotype through the upregulation of HDAC1, SIRT 1 and 2, unfolding an epigenetic regulation in stem cell differentiation and opening novel strategies for future therapeutic balancing of stem cell fate toward adipogenic or osteogenic phenotype.

Sperm telomere length in donor samples is not related to ICSI outcome.

PURPOSE: Variations in sperm telomere length (STL) have been associated with altered sperm parameters, poor embryo quality, and lower pregnancy rates, but for normozoospermic men, STL relevance in IVF/ICSI is still uncertain. Moreover, in all studies reported so far, each man's STL was linked to the corresponding female partner characteristics. Here, we study STL in sperm donor samples, each used for up to 12 women, in order to isolate and determine the relationship between STL and reproductive outcomes. METHODS: Relative STL was determined by qPCR in 60 samples used in a total of 676 ICSI cycles. Univariable and multivariable statistical analyses were used to study the STL effect on fertilization rate; embryo morphology; biochemical, clinical, and ongoing pregnancy rates; and live birth (LB) rates. RESULTS: The average STL value was 4.5 (relative units; SD 1.9; range 2.4-14.2). Locally weighted scatterplot smoothing regression and the rho-Spearman test did not reveal significant correlations between STL and the outcomes analyzed. STL was not different between cycles resulting or not in pregnancy and LB (Mann-Whitney U test, p > 0.05). No significant effect of STL on reproductive outcomes was found, with the OR for each unit increase in STL (95% CI) of 0.94 (0.86-1-04), 0.99 (0.9-1.09), 0.98 (0.89-1.09), and 0.93 (0.8-1.06) for biochemical, clinical, and ongoing pregnancy and LB, respectively. The multilevel analysis confirmed that the effect of STL on fertilization; biochemical, clinical, and ongoing pregnancy; and LB was not significant (p > 0.05). CONCLUSION: After addressing STL independently from female variables, results show that STL measurement is not useful to predict reproductive outcomes in ICSI cycles using donor semen.

Methylation dynamics during folliculogenesis and early embryo development in sheep.

Genome-wide DNA methylation reprogramming occurs during mammalian gametogenesis and early embryogenesis. Post-fertilization demethylation of paternal and maternal genomes is considered to occur by an active and passive mechanism respectively, in most mammals but sheep; in this species no loss of methylation was observed in either pronucleus. Post-fertilization reprogramming relies on methylating and demethylating enzymes and co-factors that are stored during oocyte growth, concurrently with the re-methylation of the oocyte itself. The crucial remodelling of the oocyte epigenetic baggage often overlaps with potential interfering events such as exposure to assisted reproduction technologies or environmental changes. Here, we report a temporal analysis of methylation dynamics during folliculogenesis and early embryo development in sheep. We characterized global DNA methylation and hydroxymethylation by immunofluorescence and relatively quantified the expression of the enzymes and co-factors mainly responsible for their remodelling (DNA methyltransferases (DNMTs), ten-eleven translocation (TET) proteins and methyl-CpG-binding domain (MBD) proteins). Our results illustrate for the first time the patterns of hydroxymethylation during oocyte growth. We observed different patterns of methylation and hydroxymethylation between the two parental pronuclei, suggesting that male pronucleus undergoes active demethylation also in sheep. Finally, we describe gene-specific accumulation dynamics for methylating and demethylating enzymes during oocyte growth and observe patterns of expression associated with developmental competence in a differential model of oocyte potential. Our work contributes to the understanding of the methylation dynamics during folliculogenesis and early embryo development and improves the overall picture of early rearrangements that will originate the embryo epigenome.

Correction: Bellu et al. Smart Nanofibers with Natural Extracts Prevent Senescence Patterning in a Dynamic Cell Culture Model of Human Skin. Cells 2020, 9, 2530.

In the original publication [...].

Myrtle-Functionalized Nanofibers Modulate Vaginal Cell Population Behavior While Counteracting Microbial Proliferation.

Vaginal infections affect millions of women annually worldwide. Therapeutic options are limited, moreover drug-resistance increases the need to find novel antimicrobials for health promotion. Recently phytochemicals were re-discovered for medical treatment. Myrtle (Myrtus communis L.) plant extracts showed in vitro antioxidant, antiseptic and anti-inflammatory properties thanks to their bioactive compounds. The aim of the present study was to create novel nanodevices to deliver three natural extracts from leaves, seeds and fruit of myrtle, in vaginal milieu. We explored their effect on human cells (HeLa, Human Foreskin Fibroblast-1 line, and stem cells isolated from skin), resident microflora (Lactobacillus acidophilus) and on several vaginal pathogens (Trichomonas vaginalis, Escherichia coli, Staphylococcus aureus, Candida albicans, Candida kefyr, Candida glabrata, Candida parapsilosis, Candida krusei). Polycaprolactone-Gelatin nanofibers encapsulated with leaves extract and soaked with seed extracts exhibited a different capability in regard to counteracting microbial proliferation. Moreover, these nanodevices do not affect human cells and resident microflora viability. Results reveal that some of the tested nanofibers are interesting candidates for future vaginal infection treatments.

Natural Compounds and PCL Nanofibers: A Novel Tool to Counteract Stem Cell Senescence.

Tissue homeostasis mainly depends on the activity of stem cells to replace damaged elements and restore tissue functions. Within this context, mesenchymal stem cells and fibroblasts are essential for maintaining tissue homeostasis in skin, in particular in the dermis. Modifications in collagen fibers are able to affect stem cell features. Skin properties can be significantly reduced after injuries or with aging, and stem cell niches, mainly comprising extracellular matrix (ECM), may be compromised. To this end, specific molecules can be administrated to prevent the aging process induced by UV exposure in the attempt to maintain a youngness phenotype. NanoPCL-M is a novel nanodevice able to control delivery of Mediterranean plant myrtle (Myrtus communis L.) extracts. In particular, we previously described that myrtle extracts, rich in bioactive molecules and nutraceuticals, were able to counteract senescence in adipose derived stem cells. In this study, we analyzed the effect of NanoPCL-M on skin stem cells (SSCs) and dermal fibroblasts in a dynamic cell culture model in order to prevent the effects of UV-induced senescence on proliferation and collagen depot. The BrdU assay results highlight the significantly positive effect of NanoPCL-M on the proliferation of both fibroblasts and SSCs. Our results demonstrate that-M is able to preserve SSCs features and collagen depot after UV-induced senescence, suggesting their capability to retain a young phenotype.

Adipose-Derived Stem Cell Features and MCF-7.

Human adipose tissue-derived stem cells (hADSCs) are highly suitable for regeneration therapies being easily collected and propagated in vitro. The effects of different external factors and culturing conditions are able to affect hADSC proliferation, senescence, differentiation, and migration, even at the molecular level. In the present paper, we exposed hADSCs to an exhausted medium from the breast cancer cell line (MCF-7) to evaluate whether the soluble factors released by these cells may be able to induce changes in stem cell behavior. In particular, we investigated the expression of stemness-related genes (OCT4; Sox 2; Nanog), the cell-cycle regulators p21 (WAF1/CIP1) p53, epigenetic markers (DNMT1 and Sirt1), and autophagy-related proteins. From our results, we can infer that the exhausted medium from MCF-7 is able to influence the hADSCs behavior increasing the expression of stemness-related genes, cell proliferation, and autophagy. Polyamines detectable in MCF-7 exhausted medium could be related to the higher proliferation capability observed in hADSCs, suggesting direct crosstalk between these molecules and the observed changes in stem cell potency.

Behavioral Changes in Stem-Cell Potency by HepG2-Exhausted Medium.

Wharton jelly mesenchymal stem cells (WJ-MSCs) are able to differentiate into different cell lineages upon stimulation. This ability is closely related to the perfect balance between the pluripotency-related genes, which control stem-cell proliferation, and genes able to orchestrate the appearance of a specific phenotype. Here we studied the expression of stemness-related genes, epigenetic regulators (DNMT1, SIRT1), miRNAs (miR-145, miR-148, and miR-185) related to stemness, exosomes, the cell-cycle regulators p21 (WAF1/CIP1) and p53, and the senescence-associated genes (p16, p19, and hTERT). Cells were cultured in the presence or absence of the human hepatocarcinoma cell line HepG2-exhausted medium, to evaluate changes in stemness, differentiation capability, and senescence sensibility. Our results showed the overexpression of SIRT1 and reduced levels of p21 mRNA. Moreover, we observed a downregulation of DNMT1, and a simultaneous overexpression of Oct-4 and c-Myc. These findings suggest that WJ-MSCs are more likely to retain a stem phenotype and sometimes to switch to a highly undifferentiable proliferative-like behavior if treated with medium exhausted by human HepG2 cell lines.

Smart Nanofibers with Natural Extracts Prevent Senescence Patterning in a Dynamic Cell Culture Model of Human Skin.

Natural cosmetic products have recently re-emerged as a novel tool able to counteract skin aging and skin related damages. In addition, recently achieved progress in nanomedicine opens a novel approach yielding from combination of modern nanotechnology with traditional treatment for innovative pharmacotherapeutics. In the present study, we investigated the antiaging effect of a pretreatment with Myrtus communis natural extract combined with a polycaprolactone nanofibrous scaffold (NanoPCL-M) on skin cell populations exposed to UV. We set up a novel model of skin on a bioreactor mimicking a crosstalk between keratinocytes, stem cells and fibroblasts, as in skin. Beta-galactosidase assay, indicating the amount of senescent cells, and viability assay, revealed that fibroblasts and stem cells pretreated with NanoPCL-M and then exposed to UV are superimposable to control cells, untreated and unexposed to UV damage. On the other hand, cells only exposed to UV stress, without NanoPCL-M pretreatment, exhibited a significantly higher yield of senescent elements. Keratinocyte-based 3D structures appeared disjointed after UV-stress, as compared to NanoPCL-M pretreated samples. Gene expression analysis performed on different senescence associated genes, revealed the activation of a molecular program of rejuvenation in stem cells pretreated with NanoPCL-M and then exposed to UV. Altogether, our results highlight a future translational application of NanoPCL-M to prevent skin aging.

Lessons from human umbilical cord: gender differences in stem cells from Wharton's jelly.

OBJECTIVE: To study the molecular features of mesenchymal stem cells from Wharton Jelly (WJ-MSCs) of umbilical cord to predict their differentiation capacity. DESIGN: Comparison of gene expression from mesenchymal stem cells of male and female umbilical cord SETTING: University hospital PATIENT (S): umbilical cords (n = 12, 6 males and 6 females) retrieved from spontaneous full-term vaginal delivery of healthy women INTERVENTION: we analyzed the expression of the stemness related genes C-MYC, OCT4, SOX2 and NANOG and of the epigenetic modulating gene DNA-methyltransferase 1 (DNMT1). MEAN OUTCOME MEASURE: WJ-MSCs were isolated by standard procedures and immunophenotypically characterized. Gene expression analysis of stemness related genes and the epigenetic modulating gene DNMT1 were performed by real-time PCR RESULTS: expression of the OCT4 and DNMT1 genes was significantly higher in WJ- MSCs isolated from male subjects, as compared to MSCs isolated from female-derived WJ. The resulting higher expression of OCT4 and DNMT1 in WJ-MSCs from males as compared with female WJ-MSCs for the first time identifies a specific relationship between stemness genes, an epigenetic modulator, and gender differences. CONCLUSION: our findings disclose novel biomedical implications in WJ-MSCs related to the sex of the donor, thus providing additional cues to exploit their regenerative potential in allogenic transplantation.