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

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 αVß3/αVß5 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.

Exosomes as a potential novel therapeutic tools against neurodegenerative diseases.

Exosomes are extracellular vesicles that can transfer biological information over long distances affecting normal and pathological processes throughout organism. It is known that very often composition and therapeutic properties of exosomes depends on cell type and its physiological state. Thus, depending on tissue of origin and physiological context exosomes may act as promoters, or suppressors of pathological processes in CNS. From the therapeutic perspective, the most promising cellular sources of exosomes are mesenchymal stem cells, dendritic cells and inducible pluripotent stem cells. In this review, we will summarize the current state of knowledge on the molecular mechanisms underlying neuroprotective actions of exosomes derived from these cells. New therapies for the neurodegenerative disorders are often halted by the inability of drugs to cross blood-brain barrier. In this respect exosomes have a critical advantage, because they can cross blood-brain barrier. Despite the great importance, surprisingly little is known about mechanistic details of this process. Therefore we will discuss some recent findings that may explain mechanisms of exosomal entry into the brain.

Exosomes from dental pulp stem cells rescue human dopaminergic neurons from 6-hydroxy-dopamine-induced apoptosis.

BACKGROUND AIMS: Stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) have unique neurogenic properties that could be potentially exploited for therapeutic use. The importance of paracrine SHED signaling for neuro-regeneration has been recognized, but the exact mechanisms behind these effects are presently unknown. In the present study, we investigated the neuro-protective potential of exosomes and micro-vesicles derived from SHEDs on human dopaminergic neurons during oxidative stress-induced by 6-hydroxy-dopamine (6-OHDA). METHODS: ReNcell VM human neural stem cells were differentiated into dopaminergic neurons and treated with 100 µmol/L of 6-OHDA alone or in combination with exosomes or micro-vesicles purified by ultracentrifugation from SHEDs cultivated in serum-free medium under two conditions: in standard two-dimensional culture flasks or on laminin-coated micro-carriers in a bioreactor. Real-time monitoring of apoptosis was performed with the use of time-lapse confocal microscopy and the CellEvent Caspase-3/7 green detection reagent. RESULTS: Exosomes but not micro-vesicles derived from SHEDs grown on the laminin-coated three-dimensional alginate micro-carriers suppressed 6-OHDA-induced apoptosis in dopaminergic neurons by approximately 80% throughout the culture period. Strikingly, no such effects were observed for the exosomes derived from SHEDs grown under standard culture conditions. CONCLUSIONS: Our results suggest that exosomes derived from SHEDs are considered as new potential therapeutic tool in the treatment of Parkinson's disease.

Extracellular vesicles can act as a potent immunomodulators of human microglial cells.

Functional impairments of microglia have been recently associated with several neurological conditions. Therefore, modulation of anti-inflammatory and phagocytic properties of microglial cells could represent a novel therapeutic approach. In the present study, we investigated the effects of extracellular vesicles (EVs) derived from stem cells from the dental pulp of human exfoliated deciduous teeth (SHEDs) on the inflammatory response and functional properties of immortalized human microglial cells. NFκB reporter assays demonstrated that EVs suppressed LPS-induced activation of NFκB signalling pathway in human microglial cells. The effect was similar to that obtained with anti-TLR4 blocking antibody. We also show that EVs differentially affected phagocytic activity of unpolarized (M0) and polarized (M1 and M2) microglial cells. EVs induced significant upregulation of phagocytic activity in M0 cells (by 39%), slight decrease in M1 cells, and moderate increase (by 21%) in M2 cells. The Seahorse XF Glycolysis Stress Test revealed that EVs induced an immediate and sustained increase of glycolytic activity in M0, M1, and M2 cells. Interestingly, EVs acted in an inverse dose-dependent manner. These findings indicate that EVs can induce glycolytic reprogramming of unpolarized and polarized human microglial cells. In conclusion, our pilot study demonstrates that EVs derived from SHEDs can act as a potent immunomodulators of human microglial cells. These findings could be potentially exploited for the development of new therapeutic strategies targeting neuroinflammatory microglia.

Intranasal Administration of Extracellular Vesicles Derived from Human Teeth Stem Cells Improves Motor Symptoms and Normalizes Tyrosine Hydroxylase Expression in the Substantia Nigra and Striatum of the 6-Hydroxydopamine-Treated Rats.

Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting millions of people worldwide. At present, there is no effective cure for PD; treatments are symptomatic and do not halt progression of neurodegeneration. Extracellular vesicles (EVs) can cross the blood-brain barrier and represent promising alternative to the classical treatment strategies. In the present study, we examined therapeutic effects of intranasal administration of EVs derived from human exfoliated deciduous teeth stem cells (SHEDs) on unilateral 6-hydroxydopamine (6-OHDA) medial forebrain bundle (MFB) rat model of PD. CatWalk gait tests revealed that EVs effectively suppressed 6-OHDA-induced gait impairments. All tested gait parameters (stand, stride length, step cycle, and duty cycle) were significantly improved in EV-treated animals when compared with 6-OHDA-lesion group rats. Furthermore, EVs slowed down numbers of 6-OHDA-induced contralateral rotations in apomorphine test. Improvements in motor function correlated with normalization of tyrosine hydroxylase expression in the striatum and substantia nigra. In conclusion, we demonstrated, for the first time, the therapeutic efficacy of intranasal administration of EVs derived from SHEDs in a rat model of PD induced by 6-OHDA intra-MFB lesion. Our findings could be potentially exploited for the development of new treatment strategies against PD.

Microcarrier culture enhances osteogenic potential of human periodontal ligament stromal cells.

Regeneration of periodontal tissue represents a major challenge to modern tissue engineering, since cell-based therapies require large amounts of periodontal ligament stromal cells (PLSC), which can be obtained only by in vitro expansion. Ideally, the period of the in vitro expansion should be optimized for the generation of large enough numbers of pre-specified progenitor cells ready to contribute to the restoration of periodontal tissues. In the present study, we used a commercially available, three-dimensional culturing platform and alginate microcarrier cell culture system for the propagation of human PLSCs, which were derived using the explant outgrowth method. Induction of osteogenic differentiation resulted in rapid and robust mineralization of the extracellular matrix in PLSCs grown on microcarriers, but not in PLSCs grown under standard culture conditions. Gene expression studies revealed upregulation of osteogenesis-related genes, BMP2, ALP, RUNX2, MSX2, cementum protein 23, bone sialoprotein, osteopontin and periostin, in undifferentiated and differentiating microcarrier cultures of PLSCs. In addition, the microcarrier culture enhanced the expression of ß-catenin, intermediate filament protein vimentin and focal adhesion proteins vinculin and paxillin. Our study shows that microcarrier culture allows rapid generation of large numbers of PLSCs pre-specified towards an osteogenic-like phenotype. This method may be useful for the development of new tissue engineering protocols for the reconstruction of periodontal tissues.

Exosomes from Human Dental Pulp Stem Cells Suppress Carrageenan-Induced Acute Inflammation in Mice.

The primary goal of this study was to examine the effects of human dental pulp stem cell-derived exosomes on the carrageenan-induced acute inflammation in mice. Exosomes were purified by differential ultracentrifugation from the supernatants of stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) cultivated in serum-free medium. At 1 h post-carrageenan injection, exosomes derived from supernatants of 2 × 10(6) SHEDs were administered by intraplantar injection to BALB/c mice; 30 mg/kg of prednisolone and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. Edema was measured at 6, 24, and 48 h after carrageenan injection. For the in vivo imaging experiments, AngioSPARK750, Cat B 750 FAST, and MMPSense 750 FAST were administered into the mouse tail vein 2 h post-carrageenan injection. Fluorescence images were acquired at 6, 24, and 48 h after edema induction by IVIS Spectrum in vivo imaging system. Exosomes significantly reduced the carrageenan-induced edema at all the time points studied (by 39.5, 41.6, and 25.6% at 6, 24, and 48 h after injection, respectively), to similar levels seen with the positive control (prednisolone). In vivo imaging experiments revealed that, both exosomes and prednisolone suppress activities of cathepsin B and matrix metalloproteinases (MMPs) at the site of carrageenan-induced acute inflammation, showing more prominent effects of prednisolone at the early stages, while exosomes exerted their suppressive effects gradually and at later time points. Our study demonstrates for the first time that exosomes derived from human dental pulp stem cells suppress carrageenan-induced acute inflammation in mice.

A New Experimental Model for Neuronal and Glial Differentiation Using Stem Cells Derived from Human Exfoliated Deciduous Teeth.

Stem cells isolated from human adult tissues represent a promising source for neural differentiation studies in vitro. We have isolated and characterized stem cells from human exfoliated deciduous teeth (SHEDs). These originate from the neural crest and therefore particularly suitable for induction of neural differentiation. We here established a novel three-stage protocol for neural differentiation of SHEDs cells. After adaptation to a serum-free and neurogenic environment, SHEDs were induced to differentiate. This resulted in the formation of stellate or bipolar round-shaped neuron-like cells with subpopulations expressing markers of sensory neurons (Brn3a, peripherin) and glia (myelin basic protein). Commercial PCR array analyses addressed the expression profiles of genes related to neurogenesis and cAMP/calcium signalling. We found distinct evidence for the upregulation of genes regulating the specification of sensory (MAF), sympathetic (midkine, pleitrophin) and dopaminergic (tyrosine hydroxylase, Nurr1) neurons and the differentiation and support of myelinating and non-myelinating Schwann cells (Krox24, Krox20, apolipoprotein E). Moreover, for genes controlling major developmental signalling pathways, there was upregulation of BMP (TGF ß-3, BMP2) and Notch (Notch 2, DLL1, HES1, HEY1, HEY2) in the differentiating SHEDs. SHEDs treated according to our new differentiation protocol gave rise to mixed neuronal/glial cell cultures, which opens new possibilities for in vitro studies of neuronal and glial specification and broadens the potential for the employment of such cells in experimental models and future treatment strategies.

Effects of major human antiprotease alpha-1-antitrypsin on the motility and proliferation of stromal cells from human exfoliated deciduous teeth.

AIM: Intrinsic tissue regeneration mechanisms are still not fully understood. The destruction/reconstruction processes are usually in fine balance; however, this can be easily destroyed, for example in the environment of chronic inflammation. One of the major proteins present at the inflammatory sites is the multifunctional protein alpha-1-antitrypsin (AAT). In this study, potential therapeutic effects of this major human antiprotease on progenitor cells are assessed. MATERIALS & METHODS: Stromal cells from human exfoliated deciduous teeth (SHEDs) were used, which are similar to the mesenchymal stromal cells isolated from other tissues. SHEDs were cultivated in the presence of subphysiological, physiological and inflammatory concentrations of AAT, and their proliferation and motility traits were assayed. Some intracellular signaling pathways, AAT internalization by SHEDs and their matrix metalloprotease profile were studied in parallel. RESULTS: Physiologic and inflammatory concentrations of AAT significantly increased the cell proliferation rate, induced phosphorylation of several key protein kinases and increased the amount of secreted active gelatinases. Moreover, cells exposed to physiologic and inflammatory levels of AAT were able to invade and migrate more efficiently. Subphysiologic AAT levels did not change cell behavior significantly. CONCLUSION: AAT at physiologic and inflammatory concentrations positively modulates the proliferation and motility of SHEDs in vitro. These results suggest the importance of AAT in the maintenance and regulation of tissue progenitor cells in vivo.