Gonadotropin Cell Transduction Mechanisms

The intention of this Special Edition was to collect review and original research articles that illustrate and stimulate the growing efforts to highlight the mechanisms of action of gonadotropins, as well as deepen our understanding of their biological roles in health and disease, aiming at revealing novel therapeutic opportunities in reproductive and regenerative medicine [...]

Effect of Estradiol and Progesterone on ovine Amniotic Epithelial Cells

This study was designed to clarify Estradiol (E2) and Progesterone (P4) steroid effects on ovine Amniotic Epithelial Cells (oAECs) that has a conserved plasticity and highly self-renewable capacity (Parolini et al., Stem Cells, 26(2), 300–311, 2008; Barboni et al., Stem Cell Rev Rep, 10:725–741, 2014). Based on their conserved immunomodulatory properties, oAECs are suitable for allo and xeno-transplantation (Barboni et al., Cell Transplant, 21(11), 2377–2395, 2012; Muttini et al., Res Vet Sci, 94(1),158–169, 2013). To date, no information is present on the effects of prolonged steroid exposition on AECs. oAECs were cultured as previously reported (Barboni et al., Cell Transplant. 21(11), 2377–2395, 2012) and treated with 12.5μM and 25μM of E2 or P4 (Sigma-Aldrich, Milan, Italy), alone and in both combinations, for three passages. Untreated cells were marked control (CTR). At 70% confluency, cells were detached for doubling time (DT) evaluation. Cells at fourth passage were differentiated for 21 days in osteogenic media (DM) (Mattioli et al., Cell Biol Int 36(1):7-19, 2012) without steroid. Alizarin Red and Alcian-Blue (Sigma-Aldrich, Milano, Italy) stainings were performed. RNA and cDNA were obtained as previously reported (Barboni et al., Cell Transplant. 21(11), 2377–2395, 2012). Real Time for NANOG, SOX2 ,OCT4 stemness genes expression were performed by SensiFast SYBR (Bioline, Aurogene, Rome, Italy) using specific primers (Mattioli et al., Cell Biol Int. 36(1):7-19, 2012).The protocol was: 5 min at 95°C, 30 cycles at 95°C for 15 sec, 60°C for 30 sec, 72°C for 15 sec. Comparative Ct 2-ΔΔC(t) normalization to GAPDH was applied. IHC analyses were carried out for Cytokeratin 8 and αSMA expression as previously reported (Barboni et al. PLoS ONE 7(2): e30974, 2012). Data expressed as mean (±SD), compared by one-way ANOVA followed by Tukey’s test (GraphPad Prism 5). Significant values for P < 0.05. Steroids treated ovine AECs proliferate with significant differences between concentrations. While P4 treated cells showed cuboidal shape and Cytokeratin expression until third passage, CTR and E2 treated cells showed a rapid downregulation of Cytokeratin and increased αSMA expression. oAECs with E2+P4 showed both cell type morphology. Steroids modified stemness genes based on the concentration. 12.5 μM E2, 25μM P4 and 25μM of both E2+P4 treatments maintained higher OCT4, NANOG and SOX2 expressions in treated cells despite their progressive downregulation in the CTR. Moreover, compared to CTR, after Alizarin staining, steroid pretreated cells suffered morphological changes under DM acquiring Alcian Blue-positive chondrogenic-like morphology. AECs stemness properties and plasticity can be modified by prolonged steroidal treatment. These data improve our knowledge, opening new prospective on oAEC use in stem cell-based therapy. Acknowledgments. Research supported by H2020-MSCA ITN EJD-REP BIOTECH 675526

In Vivo Model of Osteoarthritis to Compare Allogenic Amniotic Epithelial Stem Cells and Autologous Adipose Derived Cells

SIMPLE SUMMARY: An early resolution of osteoarthritis (OA), through minimally invasive orthobiological solutions, would be important to enable a return to daily and sport activities, and delay prosthesis solutions. No study has yet evaluated amniotic epithelial stem cells (AECs) in OA. They could be considered a valid alternative to adipose derived cells, expanded or concentrated, because they differentiate into three lineages and express mesenchymal and embryonic markers, without a tumorigenic phenotype. The innovative aspects of this study are the comparison of three injective orthobiological treatments, the in vivo use of AECs in OA, and the evaluation of structural and inflammatory fronts of OA for up to six months. ABSTRACT: The challenge of osteoarthritis (OA) is to find a minimally invasive orthobiological therapy to contrast OA progression, on inflammatory and structural fronts. The aim of the present study is to compare the effects of an intra-articular injection of three orthobiological treatments, autologous culture expanded adipose-derived mesenchymal stromal cells (ADSCs), autologous stromal vascular fraction (SVF) and allogenic culture expanded amniotic epithelial stem cells (AECs), in an animal model of OA. OA was induced in 24 sheep by bilateral lateral meniscectomy and, at 3 and 6 months post-treatment, the results were analyzed with macroscopy, histology, histomorphometry, and biochemistry. All the three treatments showed better results than control (injection of NaCl), but SVF and AECs showed superiority over ADSCs, because they induced higher cartilage regeneration and lower inflammation. SVF showed better results than AECs at 3 and 6 months. To conclude, SVF seems to be more favorable than the other biological options, because it is easily obtained and rapidly used after harvesting, with good healing potential. AECs cause no discomfort and could be also considered for the treatment of OA joints

Effects of P4 Antagonist RU486 on VEGF and Its Receptors' Signaling during the In Vivo Transition from the Preovulatory to Periovulatory Phase of Ovarian Follicles

The development of an adequate blood vessel network is crucial for the accomplishment of ovarian follicle growth and ovulation, which is necessary to support the proliferative and endocrine functions of the follicular cells. Although the Vascular Endothelial Growth Factor (VEGF) through gonadotropins guides ovarian angiogenesis, the role exerted by the switch on of Progesterone (P4) during the periovulatory phase remains to be clarified. The present research aimed to investigate in vivo VEGF-mediated mechanisms by inducing the development of periovulatory follicles using a pharmacologically validated synchronization treatment carried out in presence or absence of P4 receptor antagonist RU486. Spatio-temporal expression profiles of VEGF, FLT1, and FLK1 receptors and the two major MAPK/ERKs and PI3K/AKT downstream pathways were analyzed on granulosa and on theca compartment. For the first time, the results demonstrated that in vivo administration of P4 antagonist RU486 inhibits follicular VEGF receptors' signaling mainly acting on the theca layer by downregulating the activation of ERKs and AKTs. Under the effect of RU486, periovulatory follicles' microarchitecture did not move towards the periovulatory stage. The present evidence provides new insights on P4 in vivo biological effects in driving vascular and tissue remodeling during the periovulatory phase

Biomimetic PLGA 3D Scaffold Potentiate Amniotic Epithelial Stem Cells Biological Capability for Tendon Tissue Engineering Applications

INTRODUCTION: Tendon tissue engineering represents a promising solution to deal with tendinopathies and aims to develop effective implantable 3D biomimetic scaffolds with ideally native tissue’s physical, mechanical, biological, and functional qualities. These constructs can be engineered with stem cells to potentiate their teno-inductive and immunomodulatory properties (El Khatib, Mauro, Di Mattia, et al., 2020; El Khatib, Mauro, Wyrwa, et al., 2020; Russo et al., 2020). In this context, amniotic epithelial stem cells (AECs) have recently received much attention in the field of regenerative medicine due to their capacity to differentiate into the tenogenic lineage and to their immunomodulatory profile (Barboni et al., 2012, 2018; Mauro et al., 2016). The focus of this research was to create bundle tendon-like PLGA 3D scaffolds, which mimic tendon macro and micro-architecture and biomechanics, and to assess their impacts on AECs’ biological potential. METHODS: PLGA fleeces, with highly aligned fibers, were fabricated via electrospinning technique through a rotatory collector. The obtained fleeces were then wrapped manually to form 3D tendon-like scaffolds, which were evaluated in terms of structure, mechanical characteristics, and biological influence on AECs by conducting in vitro experiments. Indeed, ovine AECs, seeded on the PLGA 3D scaffolds and fleeces, were compared for their morphological changes and for the cytoplasmic expression of TNMD, a mature tendon protein, respect to cells cultured on Petri dishes (CTR), after 48h and 7d of culture through a confocal microscope. Moreover, the teno-differentiative potential and immunomodulatory properties of the produced constructs were assessed by analyzing the gene expression of tendon related markers (early: SCX, late: COL1 and TNMD) and of anti- (IL10) and pro- (IL12) inflammatory cytokines respectively. Moreover, the present research evaluated YAP protein activation in the engineered AECs through immunofluorescence assay by assessing its cellular localization. RESULTS: The produced PLGA 3D scaffolds, analyzed though a scanning electron microscope, showed high fiber alignment, which closely resemble the architecture, both macroscopically and microscopically, and the biomechanical properties of native tendon tissue. AECs seeded on the produced constructs exhibited an elongated tenocyte-like morphology already after 24 hours, while AECs cultivated on petri dishes (CTR) retained their characteristic polygonal morphology. The engineered AECs' phenotypic change was also confirmed by visualizing the cytoplasmic expression of TNMD protein and supported by tendon-related genes (SCX, COL1, and TNMD) upregulation at 7-day culture respect to CTR cells (p<0.05), which showed no TNMD protein expression or significant increase in tendon-related genes. Moreover, AECs seeded on 3D PLGA scaffolds showed an anti-inflammatory profile, with a significant higher IL10/IL12 ratio respect to the CTR (p<0.05). Finally, 3D scaffolds with highly aligned fibers stimulated AECs in terms of cell cytoskeleton stress, activating their mechanosensitive YAP pathway by significantly increasing YAP nuclear localization compared to the CTR (p<0.05), in which YAP was instead localized in the cytoplasm. DISCUSSION & CONCLUSIONS: Overall, these results support the biomimicry of the fabricated scaffolds in terms of structure and biomechanics and reveal their great teno/immuno-inductive potential and mechanosensing stimulus on AECs, thus standing biomimetic PLGA 3D scaffolds as a potential candidate for tendon regeneration

Molecular Insights and Clinical Outcomes of Drugs of Abuse Adulteration: New Trends and New Psychoactive Substances

Adulteration is a well-known practice of drug manufacturers at different stages of drug production. The intentional addition of active ingredients to adulterate the primary drug may enhance or mask pharmacological effects or may produce more potent drugs to increase the number of available doses and the dealer\u27s profit. Adulterants found in different drugs change over time in response to different factors. A systematic literature search in PubMed and Scopus databases and official international organizations\u27 websites according to PRISMA guidelines was performed. A total of 724 studies were initially screened, with 145 articles from PubMed and 462 from Scopus excluded according to the criteria described in the Method Section. The remaining 117 records were further assessed for eligibility to exclude articles without sufficient data. Finally, 79 studies were classified as non-biological (n = 35) or biological (n = 35 case reports; n = 9 case series) according to the samples investigated. Although the seized samples analyses revealed the presence of well-established adulterants such as levamisole for cocaine or paracetamol/acetaminophen for heroin, the reported data disclosed new adulteration practices, such as the use of NPS as cutting agents for classic drugs of abuse and other NPS. For example, heroin adulterated with synthetic cannabinoids or cocaine adulterated with fentanyl/fentalogues raised particular concern. Notably, adulterants play a role in some adverse effects commonly associated with the primary drug, such as levamisole-adulterated cocaine that may induce vasculitis via an autoimmune process. It is essential to constantly monitor adulterants due to their changing availability that may threaten drug consumers\u27 health

Insight into Hypoxia Stemness Control

Recently, the research on stemness and multilineage differentiation mechanisms has greatly increased its value due to the potential therapeutic impact of stem cell-based approaches. Stem cells modulate their self-renewing and differentiation capacities in response to endogenous and/or extrin- sic factors that can control stem cell fate. One key factor controlling stem cell phenotype is oxygen (O2). Several pieces of evidence demonstrated that the complexity of reproducing O2 physiological tensions and gradients in culture is responsible for defective stem cell behavior in vitro and after transplantation. This evidence is still worsened by considering that stem cells are conventionally incubated under non-physiological air O2 tension (21%). Therefore, the study of mechanisms and signaling activated at lower O2 tension, such as those existing under native microenvironments (referred to as hypoxia), represent an effective strategy to define if O2 is essential in preserving naïve stemness potential as well as in modulating their differentiation. Starting from this premise, the goal of the present review is to report the status of the art about the link existing between hypoxia and stemness providing insight into the factors/molecules involved, to design targeted strategies that, recapitulating naïve O2 signals, enable towards the therapeutic use of stem cell for tissue engineering and regenerative medicine