Urine-derived renal epithelial cells isolated after kidney transplant are sensitive to neutrophil gelatinase-associated lipocalin exposure during in vitro culture.

Urine-derived renal epithelial cells (URECs) are highly voided after kidney transplant and express typical kidney markers, including markers of kidney epithelial progenitor cells. Recently URECs have shown promising immunomodulatory properties when cultured with Peripheral Blood Mononuclear Cells (PBMCs), promoting an increase in the T regulatory cells. In vivo, kidney cells are highly exposed to damage associated molecules during both acute and chronic kidney injury. Neutrophil gelatinase-associated lipocalin (NGAL) is one of the most -known early marker of acute and chronic kidney damage. However, its role on the evolution of renal damage has not yet been fully described, nor has its impact on the characteristics of renal-derived cells during in vitro culture. The aim of this study is to investigate the effect of NGAL on the characteristics of URECs isolated after kidney transplant, by exposing these cells to the treatment with NGAL during in vitro culture and evaluating its effect on UREC viability, proliferation, and immunomodulatory potential. The exposure of URECs to NGAL reduced their viability and proliferative capacity, promoting the onset of apoptosis. The immunomodulatory properties of URECs were partially inhibited by NGAL, without affecting the increase of Treg cells observed during UREC-PBMCs coculture. These results suggest that the exposure to NGAL may compromise some features of kidney stem and specialized cell types, reducing their viability, increasing apoptosis, and partially altering their immunomodulatory properties. Thus, NGAL could represent a target for approaches acting on its inhibition or reduction to improve functional recovery.

State of the Art and New Trends from the Second International StemNet Meeting.

The Second International StemNet (Federation of Stem Cell Research Associations) meeting took place on 18-20 October 2023 in Brescia (Italy), with the support of the University of Brescia and the Zooprophylactic Institute of Lombardy and Emilia Romagna. The program of the meeting was articulated in nine sections: (1) Biomedical Communication in Italy: Critical Aspects; (2) StemNet Next Generation Session; (3) Cell-Free Therapies; (4) Tips and Tricks of Research Valorisation; (5) Stem Cells and Cancer; (6) Stem Cells in Veterinary Applications; (7) Stem Cells in Clinical Applications; (8) Organoids and 3D Systems; (9) induced pluripotent stem cells (iPCS) and Gene Therapy. National and International speakers presented their scientific works, inspiring debates and discussions among the attendees. The participation in the meeting was high, especially because of the young researchers who animated all the sessions and the rich poster session.

Revolutionizing the Use of Honeybee Products in Healthcare: A Focused Review on Using Bee Pollen as a Potential Adjunct Material for Biomaterial Functionalization.

The field of biomedical engineering highly demands technological improvements to allow the successful engraftment of biomaterials requested for healing damaged host tissues, tissue regeneration, and drug delivery. Polymeric materials, particularly natural polymers, are one of the primary suitable materials employed and functionalized to enhance their biocompatibility and thus confer advantageous features after graft implantation. Incorporating bioactive substances from nature is a good technique for expanding or increasing the functionality of biomaterial scaffolds, which may additionally encourage tissue healing. Our ecosystem provides natural resources, like honeybee products, comprising a rich blend of phytochemicals with interesting bioactive properties, which, when functionally coupled with biomedical biomaterials, result in the biomaterial exhibiting anti-inflammatory, antimicrobial, and antioxidant effects. Bee pollen is a sustainable product recently discovered as a new functionalizing agent for biomaterials. This review aims to articulate the general idea of using honeybee products for biomaterial engineering, mainly focusing on describing recent literature on experimental studies on biomaterials functionalized with bee pollen. We have also described the underlying mechanism of the bioactive attributes of bee pollen and shared our perspective on how future biomedical research will benefit from the fabrication of such functionalized biomaterials.

Mitigating Oxidative Stress in Perinatal Cells: A Critical Step toward an Optimal Therapeutic Use in Regenerative Medicine.

Oxidative stress (OS) occurs when the production of reactive oxygen species (ROS) is not balanced by the body's antioxidant defense system. OS can profoundly affect cellular health and function. ROS can have a profound negative impact on cells that undergo a predestined and time-regulated process of proliferation or differentiation, such as perinatal stem cells. Due to the large-scale employment of these immunotolerant stem cells in regenerative medicine, it is important to reduce OS to prevent them from losing function and increase their application in the regenerative medicine field. This goal can be achieved through a variety of strategies, such as the use of antioxidants and other compounds that can indirectly modulate the antioxidant defense system by enhancing cellular stress response pathways, including autophagy and mitochondrial function, thereby reducing ROS levels. This review aims to summarize information regarding OS mechanisms in perinatal stem cells and possible strategies for reducing their deleterious effects.

Plumbagin, a Natural Compound with Several Biological Effects and Anti-Inflammatory Properties.

Phytochemicals from various medicinal plants are well known for their antioxidant properties and anti-cancer effects. Many of these bioactive compounds or natural products have demonstrated effects against inflammation, while some showed a role that is only approximately described as anti-inflammatory. In particular, naphthoquinones are naturally-occurring compounds with different pharmacological activities and allow easy scaffold modification for drug design approaches. Among this class of compounds, Plumbagin, a plant-derived product, has shown interesting counteracting effects in many inflammation models. However, scientific knowledge about the beneficial effect of Plumbagin should be comprehensively reported before candidating this natural molecule into a future drug against specific human diseases. In this review, the most relevant mechanisms in which Plumbagin plays a role in the process of inflammation were summarized. Other relevant bioactive effects were reviewed to provide a complete and compact scenario of Plumbagin's potential therapeutic significance.

Non-Permissive Parvovirus B19 Infection: A Reservoir and Questionable Safety Concern in Mesenchymal Stem Cells.

Mesenchymal stromal/stem cells (MSCs) are multipotent cells with differentiation, immunoregulatory and regenerative properties. Because of these features, they represent an attractive tool for regenerative medicine and cell-based therapy. However, MSCs may act as a reservoir of persistent viruses increasing the risk of failure of MSCs-based therapies and of viral transmission, especially in immunocompromised patients. Parvovirus B19V (B19V) is a common human pathogen that infects bone marrow erythroid progenitor cells, leading to transient or persistent anemia. Characteristics of B19V include the ability to cross the placenta, infecting the fetus, and to persist in several tissues. We thus isolated MSCs from bone marrow (BM-MSCs) and fetal membrane (FM-MSCs) to investigate their permissiveness to B19V infection. The results suggest that both BM- and FM- MSCs can be infected by B19V and, while not able to support viral replication, allow persistence over time in the infected cultures. Future studies are needed to understand the potential role of MSCs in B19V transmission and the conditions that can favor a potential reactivation of the virus.

By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model.

Castanea sativa is very common in Italy, and the large amount of waste material generated during chestnut processing has a high environmental impact. Several studies demonstrated that chestnut by-products are a good source of bioactive compounds, mainly endowed with antioxidant properties. This study further investigates the anti-neuroinflammatory effect of chestnut leaf and spiny bur extracts, together with the deepest phytochemical characterisation (by NMR and MS) of active biomolecules contained in leaf extracts, which resulted in being more effective than spiny bur ones. BV-2 microglial cells stimulated with lipopolysaccharide (LPS) were used as a model of neuroinflammation. In BV-2 cells pre-treated with chestnut extracts, LPS signalling is partially blocked via the reduced expression of TLR4 and CD14 as well as the expression of LPS-induced inflammatory markers. Leaf extract fractions revealed the presence of specific flavonoids, such as isorhamnetin glucoside, astragalin, myricitrin, kaempferol 3-rhamnosyl (1-6)(2″-trans-p-coumaroyl)hexoside, tiliroside and unsaturated fatty acids, all of which could be responsible for the observed anti-neuroinflammatory effects. Interestingly, the kaempferol derivative has been identified in chestnut for the first time. In conclusion, the exploitation of chestnut by-products is suitable for the achievement of two goals: satisfaction of consumers' demand for new, natural bio-active compounds and valorisation of by-products.

Characterization of Perinatal Stem Cell Spheroids for the Development of Cell Therapy Strategy.

Type 1 diabetes mellitus (T1DM) is a complex metabolic disease characterized by a massive loss of insulin-producing cells due to an autoimmune reaction. Currently, daily subcutaneous administration of exogenous insulin is the only effective treatment. Therefore, in recent years considerable interest has been given to stem cell therapy and in particular to the use of three-dimensional (3D) cell cultures to better reproduce in vivo conditions. The goal of this study is to provide a reliable cellular model that could be investigated for regenerative medicine applications for the replacement of insulin-producing cells in T1DM. To pursue this aim we create a co-culture spheroid of amniotic epithelial cells (AECs) and Wharton's jelly mesenchymal stromal cells (WJ-MSCs) in a one-to-one ratio. The resulting co-culture spheroids were analyzed for viability, extracellular matrix production, and hypoxic state in both early- and long-term cultures. Our results suggest that co-culture spheroids are stable in long-term culture and are still viable with a consistent extracellular matrix production evaluated with immunofluorescence staining. These findings suggest that this co-culture may potentially be differentiated into endo-pancreatic cells for regenerative medicine applications in T1DM.

Perinatal Stem Cell Therapy to Treat Type 1 Diabetes Mellitus: A Never-Say-Die Story of Differentiation and Immunomodulation.

Human term placenta and other postpartum-derived biological tissues are promising sources of perinatal cells with unique stem cell properties. Among the massive current research on stem cells, one medical focus on easily available stem cells is to exploit them in the design of immunotherapy protocols, in particular for the treatment of chronic non-curable human diseases. Type 1 diabetes is characterized by autoimmune destruction of pancreatic beta cells and perinatal cells can be harnessed both to generate insulin-producing cells for beta cell replenishment and to regulate autoimmune mechanisms via immunomodulation capacity. In this study, the strong points of cells derived from amniotic epithelial cells and from umbilical cord matrix are outlined and their potential for supporting cell therapy development. From a basic research and expert stem cell point of view, the aim of this review is to summarize information regarding the regenerative medicine field, as well as describe the state of the art on possible cell therapy approaches for diabetes.

A Tailored Lipid Supplement Restored Membrane Fatty Acid Composition and Ameliorates In Vitro Biological Features of Human Amniotic Epithelial Cells.

Cell culture conditions influence several biological and biochemical features of stem cells (SCs), including the membrane lipid profile, thus limiting the use of SCs for cell therapy approaches. The present study aims to investigate whether the in vitro culture may alter the membrane fatty acid signature of human Amniotic Epithelial Cells (hAECs). The analysis of the membrane fatty acid composition of hAECs cultured in basal medium showed a loss in polyunsaturated fatty acids (PUFA), in particular in omega-6 (ω-6) content, compared to freshly isolated hAECs. The addition to the basal culture medium of a chemically defined and animal-free tailored lipid supplement, namely Refeed®, partially restored the membrane fatty acid signature of hAECs. Although the amelioration of the membrane composition did not prolong hAECs culture lifespan, Refeed® influenced cell morphology, counteracted the onset of senescence, and increased the migratory capacity as well as the ability of hAECs to inhibit Peripheral Blood Mononuclear Cell (PBMC) proliferation. This study provides new information on hAEC features during culture passages and demonstrates that the maintenance of the membrane fatty acid signature preserved higher cell quality during in vitro expansion, suggesting the use of lipid supplementation for SC expansion in cell-based therapies.

Host-Pathogen Interactions: Organotypic Cultures to Unravel the Mysteries of the Primordial Hostility among Organisms.

The interaction of humans with microorganisms represents a subtle balance between harm and good [...].

Effective Label-Free Sorting of Multipotent Mesenchymal Stem Cells from Clinical Bone Marrow Samples.

Mesenchymal stem cells (MSC) make up less than 1% of the bone marrow (BM). Several methods are used for their isolation such as gradient separation or centrifugation, but these methodologies are not direct and, thus, plastic adherence outgrowth or magnetic/fluorescent-activated sorting is required. To overcome this limitation, we investigated the use of a new separative technology to isolate MSCs from BM; it label-free separates cells based solely on their physical characteristics, preserving their native physical properties, and allows real-time visualization of cells. BM obtained from patients operated for osteochondral defects was directly concentrated in the operatory room and then analyzed using the new technology. Based on cell live-imaging and the sample profile, it was possible to highlight three fractions (F1, F2, F3), and the collected cells were evaluated in terms of their morphology, phenotype, CFU-F, and differentiation potential. Multipotent MSCs were found in F1: higher CFU-F activity and differentiation potential towards mesenchymal lineages compared to the other fractions. In addition, the technology depletes dead cells, removing unwanted red blood cells and non-progenitor stromal cells from the biological sample. This new technology provides an effective method to separate MSCs from fresh BM, maintaining their native characteristics and avoiding cell manipulation. This allows selective cell identification with a potential impact on regenerative medicine approaches in the orthopedic field and clinical applications.

Acid Sphingomyelinase Controls Early Phases of Skeletal Muscle Regeneration by Shaping the Macrophage Phenotype.

Skeletal muscle regeneration is a complex process involving crosstalk between immune cells and myogenic precursor cells, i.e., satellite cells. In this scenario, macrophage recruitment in damaged muscles is a mandatory step for tissue repair since pro-inflammatory M1 macrophages promote the activation of satellite cells, stimulating their proliferation and then, after switching into anti-inflammatory M2 macrophages, they prompt satellite cells' differentiation into myotubes and resolve inflammation. Here, we show that acid sphingomyelinase (ASMase), a key enzyme in sphingolipid metabolism, is activated after skeletal muscle injury induced in vivo by the injection of cardiotoxin. ASMase ablation shortens the early phases of skeletal muscle regeneration without affecting satellite cell behavior. Of interest, ASMase regulates the balance between M1 and M2 macrophages in the injured muscles so that the absence of the enzyme reduces inflammation. The analysis of macrophage populations indicates that these events depend on the altered polarization of M1 macrophages towards an M2 phenotype. Our results unravel a novel role of ASMase in regulating immune response during muscle regeneration/repair and suggest ASMase as a supplemental therapeutic target in conditions of redundant inflammation that impairs muscle recovery.

Are the Organoid Models an Invaluable Contribution to ZIKA Virus Research?

In order to prevent new pathogen outbreaks and avoid possible new global health threats, it is important to study the mechanisms of microbial pathogenesis, screen new antiviral agents and test new vaccines using the best methods. In the last decade, organoids have provided a groundbreaking opportunity for modeling pathogen infections in human brains, including Zika virus (ZIKV) infection. ZIKV is a member of the Flavivirus genus, and it is recognized as an emerging infectious agent and a serious threat to global health. Organoids are 3D complex cellular models that offer an in-scale organ that is physiologically alike to the original one, useful for exploring the mechanisms behind pathogens infection; additionally, organoids integrate data generated in vitro with traditional tools and often support those obtained in vivo with animal model. In this mini-review the value of organoids for ZIKV research is examined and sustained by the most recent literature. Within a 3D viewpoint, tissue engineered models are proposed as future biological systems to help in deciphering pathogenic processes and evaluate preventive and therapeutic strategies against ZIKV. The next steps in this field constitute a challenge that may protect people and future generations from severe brain defects.

Microfluidic Tools for Enhanced Characterization of Therapeutic Stem Cells and Prediction of Their Potential Antimicrobial Secretome.

Antibiotic resistance is creating enormous attention on the development of new antibiotic-free therapy strategies for bacterial diseases. Mesenchymal stromal stem cells (MSCs) are the most promising candidates in current clinical trials and included in several cell-therapy protocols. Together with the well-known immunomodulatory and regenerative potential of the MSC secretome, these cells have shown direct and indirect anti-bacterial effects. However, the low reproducibility and standardization of MSCs from different sources are the current limitations prior to the purification of cell-free secreted antimicrobial peptides and exosomes. In order to improve MSC characterization, novel label-free functional tests, evaluating the biophysical properties of the cells, will be advantageous for their cell profiling, population sorting, and quality control. We discuss the potential of emerging microfluidic technologies providing new insights into density, shape, and size of live cells, starting from heterogeneous or 3D cultured samples. The prospective application of these technologies to studying MSC populations may contribute to developing new biopharmaceutical strategies with a view to naturally overcoming bacterial defense mechanisms.

Antioxidant and Anti-Inflammatory Profiles of Spent Coffee Ground Extracts for the Treatment of Neurodegeneration.

Spent coffee grounds (SCGs), waste products of coffee beverage production, are rich in organic compounds such as phenols. Different studies have demonstrated phenol beneficial effects in counteracting neurodegenerative diseases. These diseases are associated with oxidative stress and neuroinflammation, which initiates the degeneration of neurons by overactivating microglia. Unfortunately, to date, there are no pharmacological therapies to treat these pathologies. The aim of this study was to evaluate the phenolic content of 4 different SCG extracts and their ability to counteract oxidative stress and neuroinflammation. Caffeine and 5-O-caffeoylquinic acid were the most abundant compounds in all extracts, followed by 3-O-caffeoylquinic acid and 3,5-O-dicaffeoylquinic acid. The four extracts demonstrated a different ability to counteract oxidative stress and neuroinflammation in vitro. In particular, the methanol extract was the most effective in protecting neuron-like SH-SY5Y cells against H2O2-induced oxidative stress by upregulating endogenous antioxidant enzymes such as thioredoxin reductase, heme oxygenase 1, NADPH quinone oxidoreductase, and glutathione reductase. The water extract was the most effective in counteracting lipopolysaccharide-induced neuroinflammation in microglial BV-2 cells by strongly reducing the expression of proinflammatory mediators through the modulation of the TLR4/NF-κB pathway. On these bases, SCG extracts could represent valuable nutraceutical sources for the treatment of neurodegeneration.

Refining Host-Pathogen Interactions: Organ-on-Chip Side of the Coin.

Bioinspired organ-level in vitro platforms that recapitulate human organ physiology and organ-specific responses have emerged as effective technologies for infectious disease research, drug discovery, and personalized medicine. A major challenge in tissue engineering for infectious diseases has been the reconstruction of the dynamic 3D microenvironment reflecting the architectural and functional complexity of the human body in order to more accurately model the initiation and progression of host-microbe interactions. By bridging the gap between in vitro experimental models and human pathophysiology and providing alternatives for animal models, organ-on-chip microfluidic devices have so far been implemented in multiple research areas, contributing to major advances in the field. Given the emergence of the recent pandemic, plug-and-play organ chips may hold the key for tackling an unmet clinical need in the development of effective therapeutic strategies. In this review, latest studies harnessing organ-on-chip platforms to unravel host-pathogen interactions are presented to highlight the prospects for the microfluidic technology in infectious diseases research.

The Potential of Honeybee Products for Biomaterial Applications.

The development of biomaterials required continuous improvements in their properties for new tissue engineering applications. Implants based on biocompatible materials and biomaterial-based dressings are susceptible to infection threat; moreover, target tissues can suffer injuring inflammation. The inclusion of nature-derived bioactive compounds usually offers a suitable strategy to expand or increase the functional properties of biomaterial scaffolds and can even promote tissue healing. Honey is traditionally known for its healing property and is a mixture of phytochemicals that have a proven reputation as antimicrobial, anti-inflammatory, and antioxidant agents. This review discusses on the potential of honey and other honeybee products for biomaterial improvements. Our study illustrates the available and most recent literature reporting the use of these natural products combined with different polymeric scaffolds, to provide original insights in wound healing and other tissue regenerative approaches.

Activity of Antioxidants from Crocus sativus L. Petals: Potential Preventive Effects towards Cardiovascular System.

The petals of the saffron crocus (Crocus sativus L.) are considered a waste material in saffron production, but may be a sustainable source of natural biologically active substances of nutraceutical interest. The aim of this work was to study the cardiovascular effects of kaempferol and crocin extracted from saffron petals. The antiarrhythmic, inotropic, and chronotropic effects of saffron petal extract (SPE), kaempferol, and crocin were evaluated through in vitro biological assays. The antioxidant activity of kaempferol and crocin was investigated through the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay using rat cardiomyoblast cell line H9c2. The MTT assay was applied to assess the effects of kaempferol and crocin on cell viability. SPE showed weak negative inotropic and chronotropic intrinsic activities but a significant intrinsic activity on smooth muscle with a potency on the ileum greater than on the aorta: EC50 = 0.66 mg/mL versus EC50 = 1.45 mg/mL. Kaempferol and crocin showed a selective negative inotropic activity. In addition, kaempferol decreased the contraction induced by KCl (80 mM) in guinea pig aortic and ileal strips, while crocin had no effect. Furthermore, following oxidative stress, both crocin and kaempferol decreased intracellular ROS formation and increased cell viability in a concentration-dependent manner. The results indicate that SPE, a by-product of saffron cultivation, may represent a good source of phytochemicals with a potential application in the prevention of cardiovascular diseases.

Leaves and Spiny Burs of Castanea Sativa from an Experimental Chestnut Grove: Metabolomic Analysis and Anti-Neuroinflammatory Activity.

Castanea sativa cultivation has been present in Mediterranean regions since ancient times. In order to promote a circular economy, it is of great importance to valorize chestnut groves' by-products. In this study, leaves and spiny burs from twenty-four Castanea trees were analyzed by 1H NMR metabolomics to provide an overview of their phytochemical profile. The Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) performed on these data allowed us to distinguish 'Marrone' from 'Castagna', since the latter were generally more enriched with secondary metabolites, in particular, flavonoids (astragalin, isorhamnetin glucoside, and myricitrin) were dominant. Knowing that microglia are involved in mediating the oxidative and inflammatory response of the central nervous system, the potential anti-inflammatory effects of extracts derived from leaves and spiny burs were evaluated in a neuroinflammatory cell model: BV-2 microglia cells. The tested extracts showed cytoprotective activity (at 0.1 and 0.5 mg/mL) after inflammation induction by 5 µg/mL lipopolysaccharide (LPS). In addition, the transcriptional levels of IL-1ß, TNF-α, and NF-kB expression induced by LPS were significantly decreased by cell incubation with spiny burs and leaves extracts. Taken together, the obtained results are promising and represent an important step to encourage recycling and valorization of chestnut byproducts, usually considered "waste".