Strategies to Counteract Oxidative Stress and Inflammation in Chronic-Degenerative Diseases 2.0.

Oxidative stress and inflammation are recognized as pivotal contributors and common features of several chronic degenerative diseases, including cancer, metabolic syndrome, type 2 diabetes, cardiovascular diseases and neurodegenerative disorders, affecting a high percentage of the population [...].

Amniotic Fluid and Placental Membranes as Sources of Stem Cells: Progress and Challenges 2.0.

The aim of the second edition of this Special Issue was to collect both review and original research articles that investigate and elucidate the possible therapeutic role of perinatal stem cells in pathological conditions, such as cardiovascular and metabolic diseases, as well as inflammatory, autoimmune, musculoskeletal, and degenerative diseases [...].

Identification of Anti-Neuroinflammatory Bioactive Compounds in Essential Oils and Aqueous Distillation Residues Obtained from Commercial Varieties of Cannabis sativa L.

Neuroinflammation, which is mainly triggered by microglia, is a key contributor to multiple neurodegenerative diseases. Natural products, and in particular Cannabis sativa L., due to its richness in phytochemical components, represent ideal candidates to counteract neuroinflammation. We previously characterized different C. sativa commercial varieties which showed significantly different chemical profiles. On these bases, the aim of this study was to evaluate essential oils and aqueous distillation residues from the inflorescences of three different hemp varieties for their anti-neuroinflammatory activity in BV-2 microglial cells. Cells were pretreated with aqueous residues or essential oils and then activated with LPS. Unlike essential oils, aqueous residues showed negligible effects in terms of anti-inflammatory activity. Among the essential oils, the one obtained from 'Gorilla Glue' was the most effective in inhibiting pro-inflammatory mediators and in upregulating anti-inflammatory ones through the modulation of the p38 MAPK/NF-κB pathway. Moreover, the sesquiterpenes (E)-caryophyllene, α-humulene, and caryophyllene oxide were identified as the main contributors to the essential oils' anti-inflammatory activity. To our knowledge, the anti-neuroinflammatory activity of α-humulene has not been previously described. In conclusion, our work shows that C. sativa essential oils characterized by high levels of sesquiterpenes can be promising candidates in the prevention/counteraction of neuroinflammation.

Human Neuromuscular Junction on a Chip: Impact of Amniotic Fluid Stem Cell Extracellular Vesicles on Muscle Atrophy and NMJ Integrity.

Neuromuscular junctions (NMJs) are specialized synapses, crucial for the communication between spinal motor neurons (MNs) and skeletal muscle. NMJs become vulnerable in degenerative diseases, such as muscle atrophy, where the crosstalk between the different cell populations fails, and the regenerative ability of the entire tissue is hampered. How skeletal muscle sends retrograde signals to MNs through NMJs represents an intriguing field of research, and the role of oxidative stress and its sources remain poorly understood. Recent works demonstrate the myofiber regeneration potential of stem cells, including amniotic fluid stem cells (AFSC), and secreted extracellular vesicles (EVs) as cell-free therapy. To study NMJ perturbations during muscle atrophy, we generated an MN/myotube co-culture system through XonaTM microfluidic devices, and muscle atrophy was induced in vitro by Dexamethasone (Dexa). After atrophy induction, we treated muscle and MN compartments with AFSC-derived EVs (AFSC-EVs) to investigate their regenerative and anti-oxidative potential in counteracting NMJ alterations. We found that the presence of EVs reduced morphological and functional in vitro defects induced by Dexa. Interestingly, oxidative stress, occurring in atrophic myotubes and thus involving neurites as well, was prevented by EV treatment. Here, we provided and validated a fluidically isolated system represented by microfluidic devices for studying human MN and myotube interactions in healthy and Dexa-induced atrophic conditions-allowing the isolation of subcellular compartments for region-specific analyses-and demonstrated the efficacy of AFSC-EVs in counteracting NMJ perturbations.

Amniotic Fluid and Placental Membranes as Sources of Stem Cells: Progress and Challenges.

The intention of this special edition is to collect review and original research articles that illustrate and stimulate growing efforts to understand the implication of perinatal stem cells in pathological conditions such as cardiovascular and metabolic diseases and inflammatory, autoimmune, musculoskeletal, and degenerative diseases [...].

Strategies to Counteract Oxidative Stress and Inflammation in Chronic-Degenerative Diseases.

The great increase in life expectancy is linked to the necessity of counteracting chronic-degenerative diseases, e [...].

Exosomes Derived from Human Amniotic Fluid Mesenchymal Stem Cells Preserve Microglia and Neuron Cells from Aß.

BACKGROUND: Neuroinflammation is involved in neuronal cell death that occurs in neurodegenerative diseases such as Alzheimer's disease (AD). Microglia play important roles in regulating the brain amyloid beta (Aß) levels, so immunomodulatory properties exerted by mesenchymal stem cells may be exploited to treat this pathology. The evidence suggests that the mechanism of action of human amniotic fluid stem cells (hAFSCs) is through their secretome, which includes exosomes (exo). METHODS: We examined the effect of exosomes derived from human amniotic fluid stem cells (hAFSCs-exo) on activated BV-2 microglia cells by lipopolysaccharide (LPS) as a neuroinflammation model. To investigate the exo effect on the interplay between AD neurons and microglia, SH-SY5Y neuroblastoma cells treated with Aß were exposed to a conditioned medium (CM) obtained from activated BV-2 or co-culture systems. RESULTS: We found that the upregulation of the markers of pro-inflammatory microglia was prevented when exposed to hAFSC-exo whereas the markers of the anti-inflammatory macrophage phenotype were not affected. Interestingly, the hAFSC-exo pretreatment significantly inhibited the oxidative stress rise and apoptosis occurring in the neurons in presence of both microglia and Aß. CONCLUSION: We demonstrated that hAFSC-exo mitigated an inflammatory injury caused by microglia and significantly recovered the neurotoxicity, suggesting that hAFSC-exo may be a potential therapeutic agent for inflammation-related neurological conditions, including AD.

The Interplay between HGF/c-met Axis and Nox4 in BRAF Mutated Melanoma.

BACKGROUND: Melanoma is the leading cause of death due to cutaneous malignancy and its incidence is on the rise. Several signaling pathways, including receptor tyrosine kinases, have a role in the development and progression of melanocytic lesions and malignant melanoma. Among those, the hepatocyte growth factor (HGF)/c-met axis is emerging as a critical player because it can play a role in drug resistance. Indeed, 50% of melanoma patients present BRAF mutations, however, all responders develop resistance to the inhibitors typically within one year of treatment. Interestingly, BRAF inhibitors induce reactive oxygen species (ROS) in melanoma cells, therefore, the aim of this study was to investigate a possible interplay between HGF/c-met and ROS sources, such as NADPH oxidases (Nox). METHODS: The expression of c-met and Nox were quantified in 60 patients with primary cutaneous melanoma. In vitro experiments on melanoma primary cells and the cell line were performed to dissect the underpinned molecular mechanism. RESULTS: The outcome of interest was the correlation between the high positivity for both Nox4 and c-met and metastasis occurring at least 1 year later than melanoma diagnosis in BRAF mutated patients, in contrast to nonmutated. In vitro experiments demonstrated that the axis HGF/c-met/Nox4/ROS triggers the epithelial-mesenchymal transition. CONCLUSIONS: The observed correlation suggests an interplay between c-met and Nox4 in promoting the onset of metastasis. This study suggests that Nox4 inhibitors could be associated to the current therapy used to treat melanoma patients with BRAF mutations.

Role of Mesenchymal Stem Cells in Counteracting Oxidative Stress-Related Neurodegeneration.

Neurodegenerative diseases include a variety of pathologies such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and so forth, which share many common characteristics such as oxidative stress, glycation, abnormal protein deposition, inflammation, and progressive neuronal loss. The last century has witnessed significant research to identify mechanisms and risk factors contributing to the complex etiopathogenesis of neurodegenerative diseases, such as genetic, vascular/metabolic, and lifestyle-related factors, which often co-occur and interact with each other. Apart from several environmental or genetic factors, in recent years, much evidence hints that impairment in redox homeostasis is a common mechanism in different neurological diseases. However, from a pharmacological perspective, oxidative stress is a difficult target, and antioxidants, the only strategy used so far, have been ineffective or even provoked side effects. In this review, we report an analysis of the recent literature on the role of oxidative stress in Alzheimer's and Parkinson's diseases as well as in amyotrophic lateral sclerosis, retinal ganglion cells, and ataxia. Moreover, the contribution of stem cells has been widely explored, looking at their potential in neuronal differentiation and reporting findings on their application in fighting oxidative stress in different neurodegenerative diseases. In particular, the exposure to mesenchymal stem cells or their secretome can be considered as a promising therapeutic strategy to enhance antioxidant capacity and neurotrophin expression while inhibiting pro-inflammatory cytokine secretion, which are common aspects of neurodegenerative pathologies. Further studies are needed to identify a tailored approach for each neurodegenerative disease in order to design more effective stem cell therapeutic strategies to prevent a broad range of neurodegenerative disorders.

Amniotic Fluid Stem Cell-Derived Extracellular Vesicles Counteract Steroid-Induced Osteoporosis In Vitro.

Background-Osteoporosis is characterized by defects in both quality and quantity of bone tissue, which imply high susceptibility to fractures with limitations of autonomy. Current therapies for osteoporosis are mostly concentrated on how to inhibit bone resorption but give serious adverse effects. Therefore, more effective and safer therapies are needed that even encourage bone formation. Here we examined the effect of extracellular vesicles secreted by human amniotic fluid stem cells (AFSC) (AFSC-EV) on a model of osteoporosis in vitro. Methods-human AFSC-EV were added to the culture medium of a human pre-osteoblast cell line (HOB) induced to differentiate, and then treated with dexamethasone as osteoporosis inducer. Aspects of differentiation and viability were assessed by immunofluorescence, Western blot, mass spectrometry, and histological assays. Since steroids induce oxidative stress, the levels of reactive oxygen species and of redox related proteins were evaluated. Results-AFSC-EV were able to ameliorate the differentiation ability of HOB both in the case of pre-osteoblasts and when the differentiation process was affected by dexamethasone. Moreover, the viability was increased and parallelly apoptotic markers were reduced. The presence of EV positively modulated the redox unbalance due to dexamethasone. Conclusion-these findings demonstrated that EV from hAFSC have the ability to recover precursor cell potential and delay local bone loss in steroid-related osteoporosis.

Development of a novel method for amniotic fluid stem cell storage.

BACKGROUND AIMS: Current procedures for collection of human amniotic fluid stem cells (hAFSCs) indicate that cells cultured in a flask for 2 weeks can then be used for research. However, hAFSCs can be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of this study was to determine whether direct freezing of amniotic fluid cells is able to maintain or improve the potential of a sub-population of stem cells. METHODS: We compared the potential of the hAFSCs regarding timing of freezing, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in a flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis and differentiation potential of C and D samples were compared. RESULTS: hAFSCs isolated from D samples expressed mesenchymal stem cells markers until later passages, had a good proliferation rate and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, direct freezing induced a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. CONCLUSIONS: This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration, which affect stem cell properties. This technique might be a cost-effective and reasonable approach to the process of Good Manufacturing Process accreditation for stem-cell banks.

Human biliary tree stem/progenitor cells immunomodulation: Role of hepatocyte growth factor.

AIM: Human biliary tree stem/progenitor cells (hBTSC) are multipotent epithelial stem cells with the potential for allogenic transplant in liver, biliary tree, and pancreatic diseases. Human mesenchymal stem cells, but also epithelial stem cells, are able to modulate immune responses with different types of secretion molecules. METHODS: The initial aim of the present study was to develop for the first time a culture protocol in order to expand hBTSC in vitro through passages, allowing to maintain a similar stem cell and secretome profile. Furthermore, we investigated the secretome profile of the hBTSC to assess the production of molecules capable of affecting immune feedback. RESULTS: We found that hepatocyte growth factor produced by hBTSC exerts its cytoprotective role inducing apoptosis in human immune cells, such as lymphocytes. CONCLUSIONS: The present study, therefore, supports the hypothesis that hBTSC can be useful for the purpose of regenerative medicine, as they can be banked and expanded, and they can secrete immunoregulatory factors.

Apoptosis and inflammatory response in human astrocytes are induced by a transmissible cytotoxic agent of neurological origin.

We demonstrated the presence of an in vitro transmissible cytotoxic agent (TCA) in the cerebrospinal fluid (CSF) of patients with different acute neurological diseases. The nature of this agent is still a matter of study since repeated attempts have failed to identify it as a conventional infectious agent. Here, we describe the mechanisms through which TCA affects human astrocytes, demonstrating: a late apoptotic process, mediated by caspases 9 and 3 activation, involving the Bcl2-Bak-axis; an early and late p38 MAPK activation; an interference with the IL-8 and MCP-1 secretory response. These in vitro data provide initial evidence of TCA involvement as a pro-apoptotic and pro-inflammatory signal, directly affecting astrocytic behavior. The implications of these findings in certain neurological diseases will be discussed.

Human amniotic fluid stem cells: neural differentiation in vitro and in vivo.

The successful integration of stem cells after their implantation into the brain has become a central issue in modern neuroscience. In this study, we test the neural differentiation potential of c-Kit(+)/Oct-4(+) human amniotic fluid stem cells (hAFSCs) in vitro and their survival and integration in vivo. hAFSCs were induced towards neural differentiation and specific markers (GFAP, ß-III tubulin, CNPase, MAP2, NeuN, synapsines, S100, PMP22) were detected by immunofluorescence and Western blot analysis. Glial proteins were expressed as early as 2 weeks after the initial differentiation stimulus, whereas neuronal markers started to appear from the third week of differentiation under culturing conditions of high cell density. This timeline suggested that glial cells possessed a promoting role in the differentiation of hAFSCs towards a neuronal fate. hAFSCs were then implanted into the lateral ventricle of the brain of 1-day-old rats, since neuronal development occurs up to 1 month after birth in this animal model. Our data showed that hAFSCs survived for up to 6 weeks post-implantation, were integrated into various areas of the central nervous system and migrated away from the graft giving rise to mature neurons and oligodendrocytes. We conclude that hAFSCs are able to differentiate and integrate into nervous tissue during development in vivo.

Neurotoxic effects of coronavirus: Potential implications in Alzheimer's onset and progression.

The COVID-19, caused by SARS-CoV-2, first affects the respiratory tract but evidence is emerging that the virus, reaching the central nervous system (CNS), can lead to severe neurological disorders. In particular, CoV infection could cause an acceleration of the neurodegenerative process. On the other hand, patients diagnosed with Alzheimer's disease (AD) develop more serious forms of COVID-19 with worse relapses. Therefore, understanding the connection between the two pathologies, AD and infection by coronavirus, could help in the development of new therapeutic approaches to counter them. We used the SH-SY5Y cell line differentiated into neurons, as widely used in studies of AD if supplemented with exogenous fibrillary ß-amyloid (Aß). As a glial counterpart, human microglia (HMC3) and astrocytic (D54MG) cell lines were used to create co-cultures with neurons via transwell systems. In these experimental models, we generated infection with the Human Coronavirus OC43 (HCoV-OC43), a low-risk model of SARS-CoV-2. Our results suggest that the infection by HCoV-OC43 leads to a neurotoxic effect not depending on an already present event of Aß deposition. Indeed, unlike microglia, neurons and even more astrocytes are susceptible to CoV infection and, although the infection does not show a cytotoxic effect in the neurons in the first few days, significant alterations at a biochemical and morphological level have been observed, suggesting that the neurons are reacting to a stressful condition, including the prodromal and neurodegenerative features of AD. Interestingly, the interaction of infected astrocytes with the neurons resulted in the manifestation of signs of neurodegeneration, such as amyloid-beta deposition. By using exogenous fibrillary Aß, as an AD in vitro model, our data suggest that there is an aggravating effect both on the infection itself and on the neurological disease progression. In conclusion, the results of this study suggest a causal interplay between HCoV-OC43 and neurological diseases and demonstrate that the co-presence of different CNS cell populations is the necessary condition to study the pathogenic effects in vitro as a whole.

Native characterization and QC profiling of human amniotic mesenchymal stromal cell vesicular fractions for secretome-based therapy.

Human amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties making them attractive candidates for regenerative applications in inflammatory diseases. Most of their beneficial properties are mediated through their secretome. The bioactive factors concurring to its therapeutic activity are still unknown. Evidence suggests synergy between the two main components of the secretome, soluble factors and vesicular fractions, pivotal in shifting inflammation and promoting self-healing. Biological variability and the absence of quality control (QC) protocols hinder secretome-based therapy translation to clinical applications. Moreover, vesicular secretome contains a multitude of particles with varying size, cargos and functions whose complexity hinders full characterization and comprehension. This study achieved a significant advancement in secretome characterization by utilizing native, FFF-based separation and characterizing extracellular vesicles derived from hAMSCs. This was accomplished by obtaining dimensionally homogeneous fractions then characterized based on their protein content, potentially enabling the identification of subpopulations with diverse functionalities. This method proved to be successful as an independent technique for secretome profiling, with the potential to contribute to the standardization of a qualitative method. Additionally, it served as a preparative separation tool, streamlining populations before ELISA and LC-MS characterization. This approach facilitated the categorization of distinctive and recurring proteins, along with the identification of clusters associated with vesicle activity and functions. However, the presence of proteins unique to each fraction obtained through the FFF separation tool presents a challenge for further analysis of the protein content within these cargoes.

Reactive Oxygen Species Regulation of Chemoresistance and Metastatic Capacity of Melanoma: Role of the Cancer Stem Cell Marker CD271.

BRAF mutations are present in 30-50% of cases of cutaneous melanoma, and treatment with selective BRAF and MEK inhibitors has been introduced. However, the development of resistance to these drugs often occurs. Chemo-resistant melanoma cells show increased expression of CD271, a stem cell marker that features increased migration. Concordantly, resistance to the selective inhibitor of oncogenic BRAFV600E/K, vemurafenib, is mediated by the increased expression of CD271. It has recently been shown that the BRAF pathway leads to an overexpression of the NADPH oxidase Nox4, which produces reactive oxygen species (ROS). Here, we examined in vitro how Nox-derived ROS in BRAF-mutated melanoma cells regulates their drug sensitivity and metastatic potential. We demonstrated that DPI, a Nox inhibitor, reduced the resistance of a melanoma cell line (SK-MEL-28) and a primary culture derived from a BRAFV600E-mutated biopsy to vemurafenib. DPI treatment affected the expression of CD271 and the ERK and Akt signaling pathways, leading to a drop in epithelial-mesenchymal transition (EMT), which undoubtedly promotes an invasive phenotype in melanoma. More importantly, the scratch test demonstrated the efficacy of the Nox inhibitor (DPI) in blocking migration, supporting its use to counteract drug resistance and thus cell invasion and metastasis in BRAF-mutated melanoma.

Effect of the Enrichment in c-Kit Stem Cell Potential of Foetal Human Amniotic Fluid Cells: Characterization from Single Cell Analysis to the Secretome Content.

Human amniotic fluid cells (hAFSCs) are a fascinating foetal cell-type that have important stem cell characteristics; however, they are a heterogeneous population that ranges from totally differentiated or progenitor cells to highly multipotent stem cells. There is no single approach to isolating the stem cell component, but the selection of a subpopulation of hAFSCs expressing c-Kit is widely employed, while a deep characterization of the two populations is still lacking. Here we performed single-cell and bulk RNAseq analysis to compare the gene expression profiles of adherent amniotic fluid cells and their subpopulation c-Kit+. Information deriving from this high throughput technology on the transcriptome was then confirmed for specific targets with protein expression experiments and functional analysis. In particular, transcriptome profiling identified changes in cellular distribution among the different clusters that correlated with significant differential expression in pathways related to stemness, proliferation, and cell cycle checkpoints. These differences were validated by RT-PCR, immunofluorescence, WB, and cell cycle assays. Interestingly, the two populations produced secretomes with different immune-modulating and pro-regenerative potentials. Indeed, the presence of TGFß, HGF, IDO was higher in EVs deriving from c-Kit+ cells, unlike IL-6. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting c-Kit positive fractions with higher potential in regenerative medicine applications.

Shared and Divergent Epigenetic Mechanisms in Cachexia and Sarcopenia.

Significant loss of muscle mass may occur in cachexia and sarcopenia, which are major causes of mortality and disability. Cachexia represents a complex multi-organ syndrome associated with cancer and chronic diseases. It is often characterized by body weight loss, inflammation, and muscle and adipose wasting. Progressive muscle loss is also a hallmark of healthy aging, which is emerging worldwide as a main demographic trend. A great challenge for the health care systems is the age-related decline in functionality which threatens the independence and quality of life of elderly people. This biological decline can also be associated with functional muscle loss, known as sarcopenia. Previous studies have shown that microRNAs (miRNAs) play pivotal roles in the development and progression of muscle wasting in both cachexia and sarcopenia. These small non-coding RNAs, often carried in extracellular vesicles, inhibit translation by targeting messenger RNAs, therefore representing potent epigenetic modulators. The molecular mechanisms behind cachexia and sarcopenia, including the expression of specific miRNAs, share common and distinctive trends. The aim of the present review is to compile recent evidence about shared and divergent epigenetic mechanisms, particularly focusing on miRNAs, between cachexia and sarcopenia to understand a facet in the underlying muscle wasting associated with these morbidities and disclose potential therapeutic interventions.

NADPH Oxidases: Redox Regulators of Stem Cell Fate and Function.

One of the major sources of reactive oxygen species (ROS) generated within stem cells is the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOXs), which are critical determinants of the redox state beside antioxidant defense mechanisms. This balance is involved in another one that regulates stem cell fate: indeed, self-renewal, proliferation, and differentiation are decisive steps for stem cells during embryo development, adult tissue renovation, and cell therapy application. Ex vivo culture-expanded stem cells are being investigated for tissue repair and immune modulation, but events such as aging, senescence, and oxidative stress reduce their ex vivo proliferation, which is crucial for their clinical applications. Here, we review the role of NOX-derived ROS in stem cell biology and functions, focusing on positive and negative effects triggered by the activity of different NOX isoforms. We report recent findings on downstream molecular targets of NOX-ROS signaling that can modulate stem cell homeostasis and lineage commitment and discuss the implications in ex vivo expansion and in vivo engraftment, function, and longevity. This review highlights the role of NOX as a pivotal regulator of several stem cell populations, and we conclude that these aspects have important implications in the clinical utility of stem cells, but further studies on the effects of pharmacological modulation of NOX in human stem cells are imperative.