scholarly journals Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms

2021 ◽  
Vol 22 (20) ◽  
pp. 10970
Author(s):  
Ugnė Jonavičė ◽  
Diana Romenskaja ◽  
Karolina Kriaučiūnaitė ◽  
Akvilė Jarmalavičiūtė ◽  
Justina Pajarskienė ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Vesicles ◽  
Molecular Mechanisms ◽  
Atp Release ◽  
Microglial Cells ◽  
Deciduous Teeth ◽  
Human Microglia ◽  
P2x4 Receptor ◽  
Pharmacological Inhibition ◽  
Microglial Migration

Extracellular vesicles (EVs) effectively suppress neuroinflammation and induce neuroprotective effects in different disease models. However, the mechanisms by which EVs regulate the neuroinflammatory response of microglia remains largely unexplored. Here, we addressed this issue by testing the action of EVs derived from human exfoliated deciduous teeth stem cells (SHEDs) on immortalized human microglial cells. We found that EVs induced a rapid increase in intracellular Ca2+ and promoted significant ATP release in microglial cells after 20 min of treatment. Boyden chamber assays revealed that EVs promoted microglial migration by 20%. Pharmacological inhibition of different subtypes of purinergic receptors demonstrated that EVs activated microglial migration preferentially through the P2X4 receptor (P2X4R) pathway. Proximity ligation and co-immunoprecipitation assays revealed that EVs promote association between milk fat globule-epidermal growth factor-factor VIII (MFG-E8) and P2X4R proteins. Furthermore, pharmacological inhibition of αVβ3/αVβ5 integrin suppressed EV-induced cell migration and formation of lipid rafts in microglia. These results demonstrate that EVs promote microglial motility through P2X4R/MFG-E8-dependent mechanisms. Our findings provide novel insights into the molecular mechanisms through which EVs target human microglia that may be exploited for the development of new therapeutic strategies targeting disease-associated neuroinflammation.

scholarly journals Extracellular Vesicles Trigger Atp Release and Promote Migration of Human Microglia through the p2x4 Receptor / Mfg-e8 – Dependent Mechanisms

2021 ◽  
Author(s):  
Ugnė Jonavičė ◽  
Diana Romenskaja ◽  
Karolina Kriaučiūnaitė ◽  
Akvilė Jarmalavičiūtė ◽  
Justina Pajarskienė ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Milk Fat ◽  
Molecular Mechanisms ◽  
Atp Release ◽  
Deciduous Teeth ◽  
Boyden Chamber ◽  
Human Microglia ◽  
P2x4 Receptor ◽  
Pharmacological Inhibition ◽  
Microglial Migration

Extracellular vesicles (EVs) effectively suppress neuroinflammation and induce neuroprotective effects in different disease models. However, the mechanisms by which EVs regulate neuroinflammatory response of microglia remain largely unexplored. Here, we addressed this issue by testing the action of EVs derived from human exfoliated deciduous teeth stem cells (SHEDs) on immortalized human microglial cells. We found that EVs induced a rapid increase in intracellular Ca2+ and promoted a significant ATP release in microglial after 20 min of treatment. Boyden chamber assays revealed that EVs promoted microglial migration by 20 %. Pharmacological inhibition of different subtypes of purinergic receptors demonstrated that EVs activated microglial migration preferentially through the P2X4R pathway. Proximity ligation and co-immunoprecipitation assays revealed that EVs promote association between milk fat globule-epidermal growth factor-factor VIII (MFG-E8) and P2X4 receptor proteins. Furthermore, pharmacological inhibition of αVβ3/αVβ5 integrin suppressed EV -induced cell migration and formation of lipid rafts in microglia. These results demonstrate that EVs promote microglial motility through P2X4 R/ MFG-E8 – dependent mechanisms. Our findings provide novel insights into the molecular mechanisms through which EVs target human microglia that may be exploited for the development of new therapeutic strategies targeting disease associated neuroinflammation.

scholarly journals Human adipose tissue-derived mesenchymal stem cells and their extracellular vesicles modulate lipopolysaccharide activated human microglia

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Marta Garcia-Contreras ◽  
Avnesh S. Thakor
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Extracellular Vesicles ◽  
Therapeutic Potential ◽  
Cell Communication ◽  
Human Adipose Tissue ◽  
Microglial Cells ◽  
Human Microglia ◽  
Oxide Synthase

AbstractNeurodegenerative diseases (NDs), such as Alzheimer’s disease (AD), are driven by neuroinflammation triggered by activated microglial cells; hence, the phenotypic regulation of these cells is an appealing target for intervention. Human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) may be a potential therapeutic candidate to treat NDs given their immunomodulatory properties. Evidence suggests that the mechanism of action of hAD-MSCs is through their secretome, which includes secreted factors such as cytokines, chemokines, or growth factors as well as extracellular vesicles (EVs). Recently, EVs have emerged as important mediators in cell communication given, they can transfer proteins, lipids, and RNA species (i.e., miRNA, mRNA, and tRNAs) to modulate recipient cells. However, the therapeutic potential of hAD-MSCs and their secreted EVs has not been fully elucidated with respect to human microglia. In this study, we determined the therapeutic potential of different hAD-MSCs doses (200,000, 100,000, and 50,000 cells) or their secreted EVs (50, 20, or 10 µg/ml), on human microglial cells (HMC3) that were activated by lipopolysaccharides (LPS). Upregulation of inducible nitric oxide synthase (iNOS), an activation marker of HMC3 cells, was prevented when they were cocultured with hAD-MSCs and EVs. Moreover, hAD-MSCs inhibited the secretion of proinflammatory factors, such as IL-6, IL-8, and MCP-1, while their secreted EVs promoted the expression of anti-inflammatory mediators such as IL-10 or TIMP-1 in activated microglia. The present data therefore support a role for hAD-MSCs and their secreted EVs, as potential therapeutic candidates for the treatment of NDs.

scholarly journals Extracellular vesicles from  deciduous pulp stem cells recover bone loss by regulating telomerase activity in an osteoporosis mouse model

2020 ◽  
Author(s):  
Soichiro Sonoda ◽  
Sara Murata ◽  
Kento Nishida ◽  
Hiroki Kato ◽  
Norihisa Uehara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Loss ◽  
Extracellular Vesicles ◽  
Osteoblast Differentiation ◽  
De Novo ◽  
Telomerase Activity ◽  
Trophic Factors ◽  
Deciduous Teeth ◽  
Mrna Levels ◽  
Ovariectomized Mice

Abstract Background: Systemic transplantation of stem cells from human exfoliated deciduous teeth (SHED) recover bone loss in animal models of osteoporosis; however, the mechanisms underlying this remain unclear. Here, we hypothesized that trophic factors within SHED-releasing extracellular vesicles (SHED-EVs) rescue osteoporotic phenotype. Methods: EVs were isolated from culture supernatant of SHED. SHED-EVs were treated with or without ribonuclease and systemically administrated into ovariectomized mice, followed by the function of recipient bone marrow mesenchymal stem cells (BMMSCs) including telomerase activity, osteoblast differentiation, and sepmaphorine-3A (SEMA3A) secretion. Subsequently, human BMMSCs were stimulated by SHED-EVs with or without ribonuclease treatment, and then human BMMSCs were examined the function of telomerase activity, osteoblast differentiation, and SEMA3A secretion. Furthermore, SHED-EVs treated human BMMSCs were subcutaneously transplanted into dorsal skin of immunocompromised mice with hydroxyapatite tricalcium phosphate (HA/TCP) careers and analyzed the de novo bone forming ability.Results: We revealed that systemic SHED-EV-infusion recovered bone volume in ovariectomized mice and improved the function of recipient BMMSCs by rescuing the mRNA levels of Tert and telomerase activity, osteoblast differentiation, and SEMA3A secretion. Ribonuclease treatment depleted RNAs, including microRNAs, within SHED-EVs and these RNA-depleted SHED-EVs attenuated SHED-EV-rescued function of recipient BMMSCs in the ovariectomized mice. These findings were supported by in vitro assays using human BMMSCs incubated with SHED-EVs. Conclusion: Collectively, our findings suggest that SHED-secreted RNAs, such as microRNAs, play a crucial role in treating postmenopausal osteoporosis by targeting the telomerase activity of recipient BMMSCs.

scholarly journals Rapid Morphologic and Molecular Activation of Microglial Cells by Stimulation of the P2X7 Receptor Correlates with Neuron Loss

2021 ◽  
Author(s):  
Keith E Campagno ◽  
Wennan Lu ◽  
Assraa Hassan Jassim ◽  
Farraj Albalawi ◽  
Aurora Cenaj ◽  
...  
Keyword(s):  
Cell Body ◽  
Microglial Activation ◽  
Morphological Changes ◽  
Mechanical Strain ◽  
Atp Release ◽  
Microglial Cells ◽  
Microglial Migration ◽  
Process Retraction

Abstract Background: The endogenous signals leading to microglial activation represent central components of neuroinflammatory cascades. Given ATP release accompanies mechanical strain to neural tissue, and the P2X7R for ATP is expressed on microglial cells, we examined the morphological and molecular consequences of P2X7R stimulation in vivo and in vitro in detail to enhance understanding of the response. Methods: IL-1β release was determined with ELISA. Expression of mRNA used qPCR. ATP release was determined with the luciferin/luciferase assay while fura-2 indicated cytoplasmic calcium. Microglial migration used Boyden chambers. Morphological changes were quantified from Iba1-immunostained cells. Results: Sholl analysis of Iba1-stained cells showed retraction of microglial ramifications one day after injection of P2X7R agonist BzATP into mouse retinae. Mean branch length also decreased, while cell body size and expression of Nos2, Tnfa, Arg1, Chil3 increased. BzATP induced similar morphological changes in ex vivo tissue isolated from Cx3CR1-GFP mice, suggesting cell recruitment was unnecessary. Primary microglial cultures were developed to investigate the autonomous nature of the response. Isolated microglial cells expressed P2X7R, while increased intracellular Ca 2+ triggered by BzATP and blocked by antagonist A839977 confirmed functional expression. BzATP induced process retraction and cell body enlargement within minutes in isolated microglial cells, and increased expression of Nos2 and Arg1 . BzATP both increased expression of IL-1β, and triggered a substantial release, suggesting P2X7R both primes and activates the NLRP3 inflammasome. ATP increased microglial migration, but this required P2Y12R, not P2X7R involvement. As ATP release often accompanies mechanical strain, responses to intraocular pressure elevation were determined. Transient elevation increased ATP release and led to microglial process retraction, cell body enlargement and gene upregulation resembling the responses to BzATP injection. These pressure-dependent changes to microglia were reduced in P2X7R -/- mice. Critically, the loss of retinal ganglion cell neurons accompanying increased pressure was correlated with microglial activation in C57Bl/6J, but not P2X7R -/- mice.Conclusions: P2X7R stimulation induced morphological and molecular markers of activation in retinal microglial cells in vivo and in vitro , affecting IL-1β release and rapid process retraction but not cell migration. Parallel responses accompanied transient pressure elevation, suggesting ATP release and P2X7R stimulation contribute to the microglial response to rising pressure.

scholarly journals Extracellular Vesicles: The New Frontier of Stem Cell Regenerative Medicine?

2020 ◽  
Author(s):  
Maria Magdalena Barreca ◽  
Patrizia Cancemi ◽  
Fabiana Geraci
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Regenerative Medicine ◽  
Extracellular Vesicles ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Cell Communication ◽  
Self Healing ◽  
Combined Use ◽  
Organ Repair

Regenerative medicine aims to repair damaged or missing cells, tissues or organs for the treatment of various diseases, poorly managed with conventional drugs and medical procedures. To date there are different approaches to obtain these results. Multimodal regenerative methods include transplant of healthy organs, tissues, or cells, body stimulation to activate a self healing response in damaged tissues, as well as the combined use of cells and bio-degradable scaffold to obtain functional tissues. Certainly, stem cells and derived products are promising tools in regenerative medicine due to their ability to induce de novo tissue formation and/or promote tissue and organ repair and regeneration. Currently, several studies have shown that the beneficial stem cell effects in damaged tissue restore are not depending on their engraftment and differentiation on the injury site, but rather to their paracrine activity. It is now well known that paracrine action of stem cells is due to their ability to release Extracellular Vesicles (EVs). EVs play a fundamental role in cell-to cell communication and are directly involved in tissue regeneration. In the present review, we tried to summarize the molecular mechanisms trough which EVs carry out their therapeutic action and their possible application for the treatment of several diseases.

scholarly journals Extracellular vesicles derived from hypoxia-preconditioned olfactory mucosa mesenchymal stem cells enhance angiogenesis via miR-612

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Lite Ge ◽  
Chengfeng Xun ◽  
Wenshui Li ◽  
Shengyu Jin ◽  
Zuo Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Extracellular Vesicles ◽  
Tissue Repair ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Olfactory Mucosa ◽  
Luciferase Reporter ◽  
Treatment Benefit ◽  
Tissue Repair And Regeneration

AbstractMesenchymal stem cells (MSCs) play important roles in tissue repair and regeneration, such as the induction of angiogenesis, particularly under hypoxic conditions. However, the molecular mechanisms underlying hypoxic MSC activation remain largely unknown. MSC-derived extracellular vesicles (EVs) are vital mediators of cell-to-cell communication and can be directly utilized as therapeutic agents for tissue repair and regeneration. Here, we explored the effects of EVs from human hypoxic olfactory mucosa MSCs (OM-MSCs) on angiogenesis and its underlying mechanism. EVs were isolated from normoxic (N) OM-MSCs (N-EVs) and hypoxic (H) OM-MSCs (H-EVs) using differential centrifugation and identified by transmission electron microscopy and flow cytometry. In vitro and in vivo, both types of OM-MSC-EVs promoted the proliferation, migration, and angiogenic activities of human brain microvascular endothelial cells (HBMECs). In addition, angiogenesis-stimulatory activity in the H-EV group was significantly enhanced compared to the N-EV group. MicroRNA profiling revealed a higher abundance of miR-612 in H-EVs than in N-EVs, while miR-612 inactivation abolished the N-EV treatment benefit. To explore the roles of miR-612, overexpression and knock-down experiments were performed using a mimic and inhibitor or agomir and antagomir of miR-612. The miR-612 target genes were confirmed using the luciferase reporter assay. Gain- and loss-of-function studies allowed the validation of miR-612 (enriched in hypoxic OM-MSC-EVs) as a functional messenger that stimulates angiogenesis and represses the expression of TP53 by targeting its 3′-untranslated region. Further functional assays showed that hypoxic OM-MSC-EVs promote paracrine Hypoxia-inducible factor 1-alpha (HIF-1α)-Vascular endothelial growth factor (VEGF) signaling in HBMECs via the exosomal miR-612-TP53-HIF-1α-VEGF axis. These findings suggest that hypoxic OM-MSC-EVs may represent a promising strategy for ischemic disease by promoting angiogenesis via miR-612 transfer. Graphical Abstract

scholarly journals Extracellular Vesicles from Adipose Tissue Stem Cells in Diabetes and Associated Cardiovascular Disease; Pathobiological Impact and Therapeutic Potential

2020 ◽  
Vol 21 (24) ◽  
pp. 9598
Author(s):  
Alina Constantin ◽  
Alexandru Filippi ◽  
Nicoleta Alexandru ◽  
Miruna Nemecz ◽  
Adriana Georgescu
Keyword(s):  
Cardiovascular Disease ◽  
Stem Cells ◽  
Adipose Tissue ◽  
Extracellular Vesicles ◽  
Molecular Mechanisms ◽  
Bioactive Molecules ◽  
Target Cells ◽  
Long Distance ◽  
Differentiation Potential ◽  
Cardiovascular Tissue

Adipose tissue-derived stem cells (ADSCs) are pluripotent mesenchymal stem cells found in relatively high percentages in the adipose tissue and able to self-renew and differentiate into many different types of cells. “Extracellular vesicles (EVs), small membrane vesicular structures released during cell activation, senescence, or apoptosis, act as mediators for long distance communication between cells, transferring their specific bioactive molecules into host target cells”. There is a general consensus on how to define and isolate ADSCs, however, multiple separation and characterization protocols are being used in the present which complicate the results’ integration in a single theory on ADSCs’ and their derived factors’ way of action. Metabolic syndrome and type 2 diabetes mellitus (T2DM) are mainly caused by abnormal adipose tissue size, distribution and metabolism and so ADSCs and their secretory factors such as EVs are currently investigated as therapeutics in these diseases. Moreover, due to their relatively easy isolation and propagation in culture and their differentiation ability, ADSCs are being employed in preclinical studies of implantable devices or prosthetics. This review aims to provide a comprehensive summary of the current knowledge on EVs secreted from ADSCs both as diagnostic biomarkers and therapeutics in diabetes and associated cardiovascular disease, the molecular mechanisms involved, as well as on the use of ADSC differentiation potential in cardiovascular tissue repair and prostheses.

scholarly journals Mesenchymal and Induced Pluripotent Stem Cells-Derived Extracellular Vesicles: The New Frontier for Regenerative Medicine?

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1163 ◽  
Author(s):  
Maria Magdalena Barreca ◽  
Patrizia Cancemi ◽  
Fabiana Geraci
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Induced Pluripotent Stem Cells ◽  
Extracellular Vesicles ◽  
Pluripotent Stem Cells ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Cell Communication ◽  
Self Healing ◽  
Induced Pluripotent

Regenerative medicine aims to repair damaged, tissues or organs for the treatment of various diseases, which have been poorly managed with conventional drugs and medical procedures. To date, multimodal regenerative methods include transplant of healthy organs, tissues, or cells, body stimulation to activate a self-healing response in damaged tissues, as well as the combined use of cells and bio-degradable scaffold to obtain functional tissues. Certainly, stem cells are promising tools in regenerative medicine due to their ability to induce de novo tissue formation and/or promote organ repair and regeneration. Currently, several studies have shown that the beneficial stem cell effects, especially for mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) in damaged tissue restore are not dependent on their engraftment and differentiation on the injury site, but rather to their paracrine activity. It is now well known that paracrine action of stem cells is due to their ability to release extracellular vesicles (EVs). EVs play a fundamental role in cell-to-cell communication and are directly involved in tissue regeneration. In the present review, we tried to summarize the molecular mechanisms through which MSCs and iPSCs-derived EVs carry out their therapeutic action and their possible application for the treatment of several diseases.

scholarly journals Biliary atresia-specific deciduous pulp stem cells feature biliary deficiency

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Soichiro Sonoda ◽  
Koichiro Yoshimaru ◽  
Haruyoshi Yamaza ◽  
Ratih Yuniartha ◽  
Toshiharu Matsuura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Carbon Tetrachloride ◽  
Liver Fibrosis ◽  
Biliary Atresia ◽  
Molecular Mechanisms ◽  
Deciduous Teeth ◽  
Intrahepatic Bile Ducts ◽  
Bile Drainage

Abstract Background Biliary atresia (BA) is a severe hepatobiliary disease in infants that ultimately results in hepatic failure; however, its pathological mechanism is poorly elucidated. Current surgical options, including Kasai hepatoportoenterostomy and orthotopic liver organ transplantations, are palliative; thus, innovation in BA therapy is urgent. Methods To examine whether BA-specific post-natal stem cells are feasible for autologous cell source for BA treatment, we isolated from human exfoliated deciduous teeth, namely BA-SHED, using a standard colony-forming unit fibroblast (CFU-F) method and compared characteristics as mesenchymal stem cells (MSCs) to healthy donor-derived control SHED, Cont-SHED. BA-SHED and Cont-SHED were intrasplenically transplanted into chronic carbon tetrachloride (CCl4)-induced liver fibrosis model mice, followed by the analysis of bile drainage function and donor integration in vivo. Immunohistochemical assay was examined for the regeneration of intrahepatic bile ducts in the recipient’s liver using anti-human specific keratin 19 (KRT19) antibody. Results BA-SHED formed CFU-F, expressed MSC surface markers, and exhibited in vitro mesenchymal multipotency similar to Cont-SHED. BA-SHED showed less in vitro hepatogenic potency than Cont-SHED. Cont-SHED represented in vivo bile drainage function and KRT19-positive biliary regeneration in chronic carbon tetrachloride-induced liver fibrosis model mice. BA-SHED failed to show in vivo biliary potency and bile drainage function compared to Cont-SHED. Conclusion These findings indicate that BA-SHED are not feasible source for BA treatment, because BA-SHED may epigenetically modify the underlying prenatal and perinatal BA environments. In conclusion, these findings suggest that BA-SHED-based studies may provide a platform for understanding the underlying molecular mechanisms of BA development and innovative novel modalities in BA research and treatment.

scholarly journals Extracellular vesicles from  deciduous pulp stem cells recover bone loss by regulating telomerase activity in an osteoporosis mouse model

2020 ◽  
Author(s):  
Soichiro Sonoda ◽  
Sara Murata ◽  
Kento Nishida ◽  
Hiroki Kato ◽  
Norihisa Uehara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Regeneration ◽  
Extracellular Vesicles ◽  
De Novo ◽  
Telomerase Activity ◽  
Trophic Factors ◽  
Deciduous Teeth ◽  
Mrna Levels ◽  
Ovariectomized Mice

Abstract Background: Systemic transplantation of stem cells from human exfoliated deciduous teeth (SHED) induces bone regeneration in animal models of osteoporosis; however, the mechanisms underlying this remain unclear. Here, we hypothesized that trophic factors within SHED-releasing extracellular vesicles (SHED-EVs) rescue osteoporotic phenotype.Methods: EVs were isolated from culture supernatant of SHED. SHED-EVs were treated with or without ribonuclease and systemically administrated into ovariectomized mice, followed by the measurement of bone regenerative function of recipient bone marrow mesenchymal stem cells (BMMSCs) including Tert expression. Subsequently, human BMMSCs were stimulated by SHED-EVs with or without ribonuclease treatment, and then human BMMSCs were examined osteogenic function in vitro and telomerase activity. Furthermore, SHED-EVs treated human BMMSCs were subcutaneously transplanted into dorsal skin of immunocompromised mice with hydroxyapatite tricalcium phosphate (HA/TCP) careers and analyzed the de novo bone forming ability.Results: We revealed that systemic SHED-EV-infusion recovered bone volume in ovariectomized mice and improved osteogenic function of recipient BMMSCs by rescuing the mRNA levels of Tert and telomerase activity. Ribonuclease treatment depleted RNAs, including microRNAs, within SHED-EVs and these RNA-depleted SHED-EVs attenuated SHED-EV-triggered bone regeneration and telomerase-mediated osteogenesis in the ovariectomized mice. These findings were supported by in vitro osteogenic assays using human BMMSCs incubated with SHED-EVs.Conclusion: Collectively, our findings suggest that SHED-secreted trophic factors, specifically microRNAs, play a crucial role in treating postmenopausal osteoporosis by targeting the telomerase activity of recipient BMMSCs.

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