Horizontal mitochondrial transfer as a novel bioenergetic tool for mesenchymal stromal/stem cells: molecular mechanisms and therapeutic potential in a variety of diseases.

Intercellular mitochondrial transfer (MT) is a newly discovered form of cell-to-cell signalling involving the active incorporation of healthy mitochondria into stressed/injured recipient cells, contributing to the restoration of bioenergetic profile and cell viability, reduction of inflammatory processes and normalisation of calcium dynamics. Recent evidence has shown that MT can occur through multiple cellular structures and mechanisms: tunneling nanotubes (TNTs), via gap junctions (GJs), mediated by extracellular vesicles (EVs) and other mechanisms (cell fusion, mitochondrial extrusion and migrasome-mediated mitocytosis) and in different contexts, such as under physiological (tissue homeostasis and stemness maintenance) and pathological conditions (hypoxia, inflammation and cancer). As Mesenchimal Stromal/ Stem Cells (MSC)-mediated MT has emerged as a critical regulatory and restorative mechanism for cell and tissue regeneration and damage repair in recent years, its potential in stem cell therapy has received increasing attention. In particular, the potential therapeutic role of MSCs has been reported in several articles, suggesting that MSCs can enhance tissue repair after injury via MT and membrane vesicle release. For these reasons, in this review, we will discuss the different mechanisms of MSCs-mediated MT and therapeutic effects on different diseases such as neuronal, ischaemic, vascular and pulmonary diseases. Therefore, understanding the molecular and cellular mechanisms of MT and demonstrating its efficacy could be an important milestone that lays the foundation for future clinical trials.

Mesenchymal Stem Cells and Their Role in Neurodegenerative Diseases.

Mesenchymal stem cells (MSCs) have garnered significant interest in the field of regenerative medicine for their ability to potentially treat various diseases, especially neurodegenerative disorders [...].

Analogies and Differences Between Dental Stem Cells: Focus on Secretome in Combination with Scaffolds in Neurological Disorders.

Mesenchymal stem cells (MSCs) are well known for their beneficial effects, differentiation capacity and regenerative potential. Dental-derived MSCs (DSCs) are more easily accessible and have a non-invasive isolation method rather than MSCs isolated from other sources (umbilical cord, bone marrow, and adipose tissue). In addition, DSCs appear to have a relevant neuro-regenerative potential due to their neural crest origin. However, it is now known that the beneficial effects of MSCs depend, at least in part, on their secretome, referring to all the bioactive molecules (neurotrophic factors) released in the conditioned medium (CM) or in the extracellular vesicles (EVs) in particular exosomes (Exos). In this review, we described the similarities and differences between various DSCs. Our focus was on the secretome of DSCs and their applications in cell therapy for neurological disorders. For neuro-regenerative purposes, the secretome of different DSCs has been tested. Among these, the secretome of dental pulp stem cells and stem cells from human exfoliated deciduous teeth have been the most widely studied. Both CM and Exos obtained from DSCs have been shown to promote neurite outgrowth and neuroprotective effects as well as their combination with scaffold materials (to improve their functional integration in the tissue). For these reasons, the secretome obtained from DSCs in combination with scaffold materials may represent a promising tissue engineering approach for neuroprotective and neuro-regenerative treatments.

Lipid rafts mediate multilineage differentiation of human dental pulp-derived stem cells (DPSCs).

Cell outer membranes contain glycosphingolipids and protein receptors, which are integrated into glycoprotein domains, known as lipid rafts, which are involved in a variety of cellular processes, including receptor-mediated signal transduction and cellular differentiation process. In this study, we analyzed the lipidic composition of human Dental Pulp-Derived Stem Cells (DPSCs), and the role of lipid rafts during the multilineage differentiation process. The relative quantification of lipid metabolites in the organic fraction of DPSCs, performed by Nuclear Magnetic Resonance (NMR) spectroscopy, showed that mono-unsaturated fatty acids (MUFAs) were the most representative species in the total pool of acyl chains, compared to polyunsatured fatty acids (PUFAs). In addition, the stimulation of DPSCs with different culture media induces a multilineage differentiation process, determining changes in the gangliosides pattern. To understand the functional role of lipid rafts during multilineage differentiation, DPSCs were pretreated with a typical lipid raft affecting agent (MßCD). Subsequently, DPSCs were inducted to differentiate into osteoblast, chondroblast and adipoblast cells with specific media. We observed that raft-affecting agent MßCD prevented AKT activation and the expression of lineage-specific mRNA such as OSX, PPARγ2, and SOX9 during multilineage differentiation. Moreover, this compound significantly prevented the tri-lineage differentiation induced by specific stimuli, indicating that lipid raft integrity is essential for DPSCs differentiation. These results suggest that lipid rafts alteration may affect the signaling pathway activated, preventing multilineage differentiation.

10th Anniversary of Biomedicines-Advances in Mesenchymal Stem Cells.

Mesenchymal stromal cells (MSCs) are non-specialized adult stem cells (ASCs), cells that reproduce to provide specific cytotypes [...].

The Importance of Stem Cells Isolated from Human Dental Pulp and Exfoliated Deciduous Teeth as Therapeutic Approach in Nervous System Pathologies.

Despite decades of research, no therapies are available to halt or slow down the course of neuro-degenerative disorders. Most of the drugs developed to fight neurodegeneration are aimed to alleviate symptoms, but none has proven adequate in altering the course of the pathologies. Cell therapy has emerged as an intriguing alternative to the classical pharmacological approach. Cell therapy consists of the transplantation of stem cells that can be obtained from various embryonal and adult tissues. Whereas the former holds notable ethical issue, adult somatic stem cells can be obtained without major concerns. However, most adult stem cells, such as those derived from the bone marrow, are committed toward the mesodermal lineage, and hence need to be reprogrammed to induce the differentiation into the neurons. The discovery of neural crest stem cells in the dental pulp, both in adults' molar and in baby teeth (dental pulp stem cells and stem cells from human exfoliated deciduous teeth, respectively) prompted researchers to investigate their utility as therapy in nervous system disorders. In this review, we recapitulate the advancements on the application of these stem cells in preclinical models of neurodegenerative diseases, highlighting differences and analogies in their maintenance, differentiation, and potential clinical application.

Dental Pulp Stem Cells (DPSCs) and Tissue Regeneration: Mechanisms Mediated by Direct, Paracrine, or Autocrine Effects.

Among mesenchymal stem cells, dental pulp stem cells (DPSCs) were discovered most recently [...].

Gangliosides and Their Role in Multilineage Differentiation of Mesenchymal Stem Cells.

Gangliosides (GGs) are a glycolipid class present on Mesenchymal Stem Cells (MSCs) surfaces with a critical appearance role in stem cell differentiation, even though their mechanistic role in signaling and differentiation remains largely unknown. This review aims to carry out a critical analysis of the predictive role of gangliosides as specific markers of the cellular state of undifferentiated and differentiated MSCs, towards the osteogenic, chondrogenic, neurogenic, and adipogenic lineage. For this reason, we analyzed the role of GGs during multilineage differentiation processes of several types of MSCs such as Umbilical Cord-derived MSCs (UC-MSCs), Bone Marrow-derived MSCs (BM-MSCs), Dental Pulp derived MSCs (DPSCs), and Adipose derived MSCs (ADSCs). Moreover, we examined the possible role of GGs as specific cell surface markers to identify or isolate specific stem cell isotypes and their potential use as additional markers for quality control of cell-based therapies.

Antioxidant Properties of Cerium Oxide Nanoparticles Prevent Retinal Neovascular Alterations In Vitro and In Vivo.

In this study, we investigated whether cerium oxide nanoparticles (CeO2-NPs), a promising antioxidant nanomaterial, may contrast retinal vascular alterations induced by oxidative damage in vitro and in vivo. For the in vivo experiments, the light damage (LD) animal model of Age-Related Macular Degeneration (AMD) was used and the CeO2-NPs were intravitreally injected. CeO2-NPs significantly decreased vascular endothelial growth factor (VEGF) protein levels, reduced neovascularization in the deep retinal plexus, and inhibited choroidal sprouting into the photoreceptor layer. The in vitro experiments were performed on human retinal pigment epithelial (ARPE-19) cells challenged with H2O2; we demonstrated that CeO2-NPs reverted H2O2-induced oxidative stress-dependent effects on this cell model. We further investigated the RPE-endothelial cells interaction under oxidative stress conditions in the presence or absence of CeO2-NPs through two experimental paradigms: (i) treatment of human umbilical vein endothelial cells (HUVECs) with conditioned media from ARPE-19 cells, and (ii) coculture of ARPE-19 and HUVECs. In both experimental conditions, CeO2-NPs were able to revert the detrimental effect of H2O2 on angiogenesis in vitro by realigning the level of tubule formation to that of the control. Altogether, our results indicate, for the first time, that CeO2-NPs can counteract retinal neovascularization and may be a new therapeutic strategy for the treatment of wet AMD.

Hypoxia Induces DPSC Differentiation versus a Neurogenic Phenotype by the Paracrine Mechanism.

As previously described by several authors, dental pulp stem cells (DPSCs), when adequately stimulated, may acquire a neuronal-like phenotype acting as a favorable source of stem cells in the generation of nerves. Besides, it is known that hypoxia conditioning is capable of stimulating cell differentiation as well as survival and self-renewal, and that multiple growth factors, including Epidermal Growth factor (EGF) and basic fibroblast growth factor (bFGF), are often involved in the induction of the neuronal differentiation of progenitor cells. In this work, we investigated the role of hypoxia in the commitment of DPSCs into a neuronal phenotype. These cells were conditioned with hypoxia (O2 1%) for 5 and 16 days; subsequently, we analyzed the proliferation rate and morphology, and tested the cells for neural and stem markers. Moreover, we verified the possible autocrine/paracrine role of DPSCs in the induction of neural differentiation by comparing the secretome profile of the hypoxic and normoxic conditioned media (CM). Our results showed that the hypoxia-mediated DPSC differentiation was time dependent. Moreover, conditioned media (CM derived from DPSCs stimulated by hypoxia were able, in turn, to induce the neural differentiation of SH-SY5Y neuroblastoma cells and undifferentiated DPSCs. In conclusion, under the herein-mentioned conditions, hypoxia seems to favor the differentiation of DPSCs into neuron-like cells. In this way, we confirm the potential clinical utility of differentiated neuronal DPSCs, and we also suggest the even greater potential of CM-derived-hypoxic DPSCs that could more readily be used in regenerative therapies.

Hypoxia-Mimetic CoCl2 Agent Enhances Pro-Angiogenic Activities in Ovine Amniotic Epithelial Cells-Derived Conditioned Medium.

Amniotic epithelial stem cells (AECs) are largely studied for their pro-regenerative properties. However, it remains undetermined if low oxygen (O2) levels that AECs experience in vivo can be of value in maintaining their biological properties after isolation. To this aim, the present study has been designed to evaluate the effects of a hypoxia-mimetic agent, cobalt chloride (CoCl2), on AECs' stemness and angiogenic activities. First, a CoCl2 dose-effect was performed to select the concentration able to induce hypoxia, through HIF-1α stabilization, without promoting any cytotoxicity effect assessed through the analysis of cell vitality, proliferation, and apoptotic-related events. Then, the identified CoCl2 dose was evaluated on the expression and angiogenic properties of AECs' stemness markers (OCT-4, NANOG, SOX-2) by analysing VEGF expression, angiogenic chemokines' profiles, and AEC-derived conditioned media activity through an in vitro angiogenic xeno-assay. Results demonstrated that AECs are sensitive to the cytotoxicity effects of CoCl2. The unique concentration leading to HIF-1α stabilization and nuclear translocation was 10 µM, preserving cell viability and proliferation up to 48 h. CoCl2 exposure did not modulate stemness markers in AECs while progressively decreasing VEGF expression. On the contrary, CoCl2 treatment promoted a significant short-term release of angiogenic chemokines in culture media (CM). The enrichment in bio-active factors was confirmed by the ability of CoCl2-derived CM to induce HUVEC growth and the cells' organization in tubule-like structures. These findings demonstrate that an appropriate dose of CoCl2 can be adopted as a hypoxia-mimetic agent in AECs. The short-term, chemical-induced hypoxic condition can be targeted to enhance AECs' pro-angiogenic properties by providing a novel approach for stem cell-free therapy protocols.

Multiple Antitumor Molecular Mechanisms Are Activated by a Fully Synthetic and Stabilized Pharmaceutical Product Delivering the Active Compound Sulforaphane (SFX-01) in Preclinical Model of Human Glioblastoma.

Frequent relapses and therapeutic resistance make the management of glioblastoma (GBM, grade IV glioma), extremely difficult. Therefore, it is necessary to develop new pharmacological compounds to be used as a single treatment or in combination with current therapies in order to improve their effectiveness and reduce cytotoxicity for non-tumor cells. SFX-01 is a fully synthetic and stabilized pharmaceutical product containing the α-cyclodextrin that delivers the active compound 1-isothiocyanato-4-methyl-sulfinylbutane (SFN) and maintains biological activities of SFN. In this study, we verified whether SFX-01 was active in GBM preclinical models. Our data demonstrate that SFX-01 reduced cell proliferation and increased cell death in GBM cell lines and patient-derived glioma initiating cells (GICs) with a stem cell phenotype. The antiproliferative effects of SFX-01 were associated with a reduction in the stemness of GICs and reversion of neural-to-mesenchymal trans-differentiation (PMT) closely related to epithelial-to-mesenchymal trans-differentiation (EMT) of epithelial tumors. Commonly, PMT reversion decreases the invasive capacity of tumor cells and increases the sensitivity to pharmacological and instrumental therapies. SFX-01 induced caspase-dependent apoptosis, through both mitochondrion-mediated intrinsic and death-receptor-associated extrinsic pathways. Here, we demonstrate the involvement of reactive oxygen species (ROS) through mediating the reduction in the activity of essential molecular pathways, such as PI3K/Akt/mTOR, ERK, and STAT-3. SFX-01 also reduced the in vivo tumor growth of subcutaneous xenografts and increased the disease-free survival (DFS) and overall survival (OS), when tested in orthotopic intracranial GBM models. These effects were associated with reduced expression of HIF1α which, in turn, down-regulates neo-angiogenesis. So, SFX-01 may have potent anti-glioma effects, regulating important aspects of the biology of this neoplasia, such as hypoxia, stemness, and EMT reversion, which are commonly activated in this neoplasia and are responsible for therapeutic resistance and glioma recurrence. SFX-01 deserves to be considered as an emerging anticancer agent for the treatment of GBM. The possible radio- and chemo sensitization potential of SFX-01 should also be evaluated in further preclinical and clinical studies.

Anti-osteoblastogenic, pro-inflammatory and pro-angiogenic effect of extracellular vesicles isolated from the human osteosarcoma cell line MNNG/HOS.

Extracellular Vesicles (EVs) are becoming increasingly recognized as integral signaling vehicles in several types of cancers, including bone malignancies. However, the specific mechanisms by which EVs influence osteosarcoma progression have not been fully determined. We evaluated the effects of EVs derived from the human osteosarcoma cell line MNNG/HOS (MNNG/HOS-EVs) on bone resident cells. We found that MNNG/HOS-EVs are internalized by osteoblasts and osteoclasts in vitro, with potent inhibitory effects on osteoblast metabolic activity, cell density and alkaline phosphatase activity. Consistently, MNNG/HOS-EVs reduced the expression of cell cycle and pro-osteoblastogenic genes, whilst increasing transcriptional expression and protein release of pro-osteoclastogenic/inflammatory cytokines (RankL, Il1b, Il6 and Lcn2), pro-tumoral cytokines (CCL2,5,6,12 and CXCL1,2,5) and the metalloproteinase MMP3. MNNG/HOS-EVs did not induce osteoclast differentiation, while promoting in vitro and in vivo angiogenesis. Intriguingly, EVs derived from another osteosarcoma cell line (U2OS) reduced ALP activity but had no other effect on osteoblast phenotype. MNNG/HOS-EVs were also found to dramatically increase Serpin b2 expression in osteoblasts. To evaluate the significance of this finding, osteoblasts were forced to overexpress Serpin b2, which however did not affect osteoblast differentiation, while Il6 and Lcn2 mRNAs were up regulated. Overall, we shed light on the interactions of osteosarcoma EVs with the cells of the bone microenvironment, identifying key anti-osteoblastogenic, pro-inflammatory and pro-angiogenic factors that could contribute to osteosarcoma expansion.

Methylglyoxal-Dependent Glycative Stress Is Prevented by the Natural Antioxidant Oleuropein in Human Dental Pulp Stem Cells through Nrf2/Glo1 Pathway.

Methylglyoxal (MG) is a potent precursor of glycative stress (abnormal accumulation of advanced glycation end products, AGEs), a relevant condition underpinning the etiology of several diseases, including those of the oral cave. At present, synthetic agents able to trap MG are known; however, they have never been approved for clinical use because of their severe side effects. Hence, the search of bioactive natural scavengers remains a sector of strong research interest. Here, we investigated whether and how oleuropein (OP), the major bioactive component of olive leaf, was able to prevent MG-dependent glycative stress in human dental pulp stem cells (DPSCs). The cells were exposed to OP at 50 µM for 24 h prior to the administration of MG at 300 µM for additional 24 h. We found that OP prevented MG-induced glycative stress and DPSCs impairment by restoring the activity of Glyoxalase 1 (Glo1), the major detoxifying enzyme of MG, in a mechanism involving the redox-sensitive transcription factor Nrf2. Our results suggest that OP holds great promise for the development of preventive strategies for MG-derived AGEs-associated oral diseases and open new paths in research concerning additional studies on the protective potential of this secoiridoid.

The Importance of Tumor Stem Cells in Glioblastoma Resistance to Therapy.

Glioblastoma (GBM) is known to be the most common and lethal primary malignant brain tumor. Therapies against this neoplasia have a high percentage of failure, associated with the survival of self-renewing glioblastoma stem cells (GSCs), which repopulate treated tumors. In addition, despite new radical surgery protocols and the introduction of new anticancer drugs, protocols for treatment, and technical advances in radiotherapy, no significant improvement in the survival rate for GBMs has been realized. Thus, novel antitarget therapies could be used in conjunction with standard radiochemotherapy approaches. Targeted therapy, indeed, may address specific targets that play an essential role in the proliferation, survival, and invasiveness of GBM cells, including numerous molecules involved in signal transduction pathways. Significant cellular heterogeneity and the hierarchy with GSCs showing a therapy-resistant phenotype could explain tumor recurrence and local invasiveness and, therefore, may be a target for new therapies. Therefore, the forced differentiation of GSCs may be a promising new approach in GBM treatment. This article provides an updated review of the current standard and experimental therapies for GBM, as well as an overview of the molecular characteristics of GSCs, the mechanisms that activate resistance to current treatments, and a new antitumor strategy for treating GSCs for use as therapy.

Regenerative Potential of DPSCs and Revascularization: Direct, Paracrine or Autocrine Effect?

A new source of mesenchymal stem cells has recently been discovered, the so-called dental pulp derived stem cells (DPSCs) which therefore could represent potentially tools for regenerative medicine. DPSC originate from the neural crest and are physiologically involved in dentin homeostasis; moreover, they contribute to bone remodeling and differentiation into several tissues including cartilage, bone, adipose and nervous tissues. DPSCs have also been shown to influence the angiogenesis process, for example through the release of secretory factors or by differentiating into vascular and/or perivascular cells. Angiogenesis, that has a pivotal role in tissue regeneration and repair, is defined as the formation of new vessels from preexisting vessels and is mediated by mutual and reciprocal interactions between endothelial cells and perivascular cells. It is also known that co-cultures of perivascular and endothelial cells (ECs) can form a vascular network in vitro and also in vivo. Since DPSCs seem to have characteristics similar to pericytes, understanding the possible mechanism of interaction between DPSCs and ECs during neo-angiogenesis is dramatically important for the development of advanced clinical application in the field of regeneration.

What Is Known about Theragnostic Strategies in Colorectal Cancer.

Despite the paradigmatic shift occurred in recent years for defined molecular subtypes in the metastatic setting treatment, colorectal cancer (CRC) still remains an incurable disease in most of the cases. Therefore, there is an urgent need for new tools and biomarkers for both early tumor diagnosis and to improve personalized treatment. Thus, liquid biopsy has emerged as a minimally invasive tool that is capable of detecting genomic alterations from primary or metastatic tumors, allowing the prognostic stratification of patients, the detection of the minimal residual disease after surgical or systemic treatments, the monitoring of therapeutic response, and the development of resistance, establishing an opportunity for early intervention before imaging detection or worsening of clinical symptoms. On the other hand, preclinical and clinical evidence demonstrated the role of gut microbiota dysbiosis in promoting inflammatory responses and cancer initiation. Altered gut microbiota is associated with resistance to chemo drugs and immune checkpoint inhibitors, whereas the use of microbe-targeted therapies including antibiotics, pre-probiotics, and fecal microbiota transplantation can restore response to anticancer drugs, promote immune response, and therefore support current treatment strategies in CRC. In this review, we aim to summarize preclinical and clinical evidence for the utilization of liquid biopsy and gut microbiota in CRC.

Clinically relevant radioresistant rhabdomyosarcoma cell lines: functional, molecular and immune-related characterization.

BACKGROUND: The probability of local tumor control after radiotherapy (RT) remains still miserably poor in pediatric rhabdomyosarcoma (RMS). Thus, understanding the molecular mechanisms responsible of tumor relapse is essential to identify personalized RT-based strategies. Contrary to what has been done so far, a correct characterization of cellular radioresistance should be performed comparing radioresistant and radiosensitive cells with the same isogenic background. METHODS: Clinically relevant radioresistant (RR) embryonal (RD) and alveolar (RH30) RMS cell lines have been developed by irradiating them with clinical-like hypo-fractionated schedule. RMS-RR cells were compared to parental isogenic counterpart (RMS-PR) and studied following the radiobiological concept of the "6Rs", which stand for repair, redistribution, repopulation, reoxygenation, intrinsic radioresistance and radio-immuno-biology. RESULTS: RMS-RR cell lines, characterized by a more aggressive and in vitro pro-metastatic phenotype, showed a higher ability to i) detoxify from reactive oxygen species; ii) repair DNA damage by differently activating non-homologous end joining and homologous recombination pathways; iii) counteract RT-induced G2/M cell cycle arrest by re-starting growth and repopulating after irradiation; iv) express cancer stem-like profile. Bioinformatic analyses, performed to assess the role of 41 cytokines after RT exposure and their network interactions, suggested TGF-ß, MIF, CCL2, CXCL5, CXCL8 and CXCL12 as master regulators of cancer immune escape in RMS tumors. CONCLUSIONS: These results suggest that RMS could sustain intrinsic and acquire radioresistance by different mechanisms and indicate potential targets for future combined radiosensitizing strategies.

Nanoceria Particles Are an Eligible Candidate to Prevent Age-Related Macular Degeneration by Inhibiting Retinal Pigment Epithelium Cell Death and Autophagy Alterations.

Retinal pigment epithelium (RPE) dysfunction and degeneration underlie the development of age-related macular degeneration (AMD), which is the leading cause of blindness worldwide. In this study, we investigated whether cerium oxide nanoparticles (CeO2-NPs or nanoceria), which are anti-oxidant agents with auto-regenerative properties, are able to preserve the RPE. On ARPE-19 cells, we found that CeO2-NPs promoted cell viability against H2O2-induced cellular damage. For the in vivo studies, we used a rat model of acute light damage (LD), which mimics many features of AMD. CeO2-NPs intravitreally injected three days before LD prevented RPE cell death and degeneration and nanoceria labelled with fluorescein were found localized in the cytoplasm of RPE cells. CeO2-NPs inhibited epithelial-mesenchymal transition of RPE cells and modulated autophagy by the down-regulation of LC3B-II and p62. Moreover, the treatment inhibited nuclear localization of LC3B. Taken together, our study demonstrates that CeO2-NPs represent an eligible candidate to counteract RPE degeneration and, therefore, a powerful therapy for AMD.

Generation of CCM Phenotype by a Human Microvascular Endothelial Model.

Cerebral cavernous malformations (CCMs) is a disorder of endothelial cells predominantly localized in the brain. Although a complete inactivation of each CCM protein has been found in the affected endothelium of diseased patients and a necessary and additional role of microenvironment has been demonstrated to determine in vivo the occurrence of vascular lesions, a microvascular endothelial model based on knockdown of a CCM gene represents today a convenient method to study some of critical signaling events regulating pathogenesis of CCM. For these reasons, in our laboratory we developed a microvascular cerebral endothelial model of Krit1 deficiency performing silencing experiments of CCM1 gene (Krit1) with siRNA procedure.