Francesca Diomede

Human Periodontal Stem Cells Release Specialized Proresolving Mediators and Carry Immunomodulatory and Prohealing Properties Regulated by Lipoxins

Unresolved inflammation and tissue destruction are underlying mechanisms of periodontitis, which is linked to dysregulated polymorphonuclear neutrophil (PMN) functions. Lipoxin A4 (LXA4) is a specialized proresolving lipid mediator (SPM) that dampens excessive inflammation, promotes resolution, and protects from leukocyte‐mediated tissue damage. Human periodontal ligament stem cells (hPDLSCs) represent key players during tissue regeneration and may contribute to resolution of inflammation; thus, they may represent a promising tool in regenerative dentistry. In the present study, we investigated the actions of hPDLSCs on PMN apoptosis and antimicrobial functions, and determined the impact of LXA4 on hPDLSCs. hPDLSCs significantly reduced apoptosis and stimulated microbicidal activity of human PMNs, via both cell‐cell interactions and paracrine mechanisms. Lipid mediator metabololipidomics analysis demonstrated that hPDLSCs biosynthesize SPMs, including resolvin D1, D2, D5, and D6; protectin D1; maresins; and LXB4; as well as prostaglandins D2, E2, and F2α. LXA4 significantly enhanced proliferation, migration, and wound healing capacity of hPDLSCs through the activation of its cognate receptor ALX/FPR2, expressed on hPDLSCs. Together, these results demonstrate that hPDLSCs modulate PMN functions, and provide the first evidence that stem cells generate SPM and that the LXA4‐ALX/FPR2 axis regulates regenerative functions of hPDLSCs by a novel receptor‐mediated mechanism.

The secretome of periodontal ligament stem cells from MS patients protects against EAE

Manipulation of stem cells or stem cells-derived secretome has emerged as a novel alternative therapeutic option for multiple sclerosis (MS). Here we show that human periodontal ligament stem cells (hPDLSCs)-derived conditioned medium (hPDLSCs-CM) and purified exosomes/microvesicles (hPDLSCs-EMVs) obtained from Relapsing Remitting (RR)-MS patients and healthy donors block experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by inducing anti-inflammatory and immunosuppressive effects in spinal cord and spleen, and reverse disease progression by restoring tissue integrity via remyelination in the spinal cord. We show that hPDLSCs-CM and hPDLSCs-EMVs reduce pro-inflammatory cytokines IL-17, IFN-γ, IL-1β, IL-6, TNF-α, and induce anti-inflammatory IL-10. In addition, apoptosis related STAT1, p53, Caspase 3, and Bax expressions were attenuated. Our findings unravel the immunosuppressive effects of hPDLSCs-CM and hPDLSCs-EMVs in EAE mice, and suggest simple alternative autologous source for patient-customized cell-free targeting treatment in MS patients.

Osteogenic potential of dualblocks cultured with human periodontal ligament stem cells: in vitro and synchrotron microtomography study

BACKGROUND AND OBJECTIVE In the present study, the early stages of in vitro bone formation in collagenated porcine scaffolds cultured with human periodontal ligament cells were investigated. The comparison between the osteogenic potential of this structure in basal and differentiating culture media was explored to predict the mechanism of its biological behavior as graft in human defect. Results were validated by synchrotron radiation X-Ray phase contrast computed microtomography (micro-CT). As the periodontal disease plays a key role in systemic and oral diseases, it is crucial to find advanced therapeutic clinical interventions to repair periodontal defects. This has been recently explored using cells and tissues developed in vitro that should ideally be immunologically, functionally, structurally and mechanically identical to the native tissue. MATERIAL AND METHODS In vitro cultures of human periodontal ligament cells, easily obtained by scraping of alveolar crestal and horizontal fibers of the periodontal ligament, were seeded on to collagenated porcine blocks constituted by natural cancellous and cortical bone. 3D images were obtained by synchrotron radiation micro-CT and processed with a phase-retrieval algorithm based on the transport of intensity equation. RESULTS Starting from the second week of culture, newly formed mineralized bone was detected in all the scaffolds, both in basal and differentiating media. Bone mineralization was proved to occur preferentially in the trabecular portion and in differentiating media. CONCLUSION The chosen method, supported by phase contrast micro-CT analysis, successfully and quantitatively monitored the early stages of bone formation and the rate of the bioscaffold resorption in basal and differentiating culture media.

Conditioned medium of periodontal ligament mesenchymal stem cells exert anti-inflammatory effects in lipopolysaccharide-activated mouse motoneurons

Conditioned medium derived from mesenchymal stem cells (MSCs) shows immunomodulatory and neuroprotective effects in preclinical models. Given the difficulty to harvest MSCs from bone marrow and adipose tissues, research has been focused to find alternative resources for MSCs, such as oral-derived tissues. Recently, we have demonstrated the protective effects of MSCs obtained from healthy human periodontal ligament tissue (hPDLSCs) in murine experimental autoimmune encephalomyelitis model. In the present in vitro study, we have investigated the immunomodulatory and neuroprotective effects of conditioned medium obtained from hPDLSCs of Relapsing Remitting- Multiple sclerosis (RR-MS) patients on NSC34 mouse motoneurons stimulated with lipopolysaccharide (LPS). Immunocytochemistry and western blotting were performed. Increased level of TLR4 and NFκB, and reduced level of IκB-α were observed in LPS-stimulated motoneurons, which were modulated by pre-conditioning with hPDLSC-conditioned medium. Inflammatory cytokines (TNF-α, IL-10), neuroprotective markers (Nestin, NFL 70, NGF, GAP43), and apoptotic markers (Bax, Bcl-2, p21) were modulated. Moreover, extracellular vesicles of hPDLSC-conditioned medium showed the presence of anti-inflammatory cytokines IL-10 and TGF-β. Our results demonstrate the immunosuppressive properties of hPDLSC-conditioned medium of RR-MS patients in motoneurons subjected to inflammation. Our findings warrant further preclinical and clinical studies to elucidate the autologous therapeutic efficacy of hPDLSC-conditioned medium in neurodegenerative diseases.

Gingival Stromal Cells as an In Vitro Model: Cannabidiol Modulates Genes Linked With Amyotrophic Lateral Sclerosis.

Research in recent years has extensively investigated the therapeutic efficacy of mesenchymal stromal cells in regenerative medicine for many neurodegenerative diseases at preclinical and clinical stages. However, the success rate of stem cell therapy remains less at translational phase. Lack of relevant animal models that potentially simulate the molecular etiology of human pathological symptoms might be a reason behind such poor clinical outcomes associated with stem cell therapy. Apparently, self‐renewal and differentiation ability of mesenchymal stem cells may help to study the early developmental signaling pathways connected with the diseases, such as Alzheimers disease, Amyotrophic lateral sclerosis (ALS), etc., at in vitro level. Cannabidiol, a non‐psychotrophic cannabinoid, has been demonstrated as a potent anti‐inflammatory and neuroprotective agent in neurological preclinical models. In the present study, we investigated the modulatory role of cannabidiol on genes associated with ALS using human gingiva‐derived mesenchymal stromal cells (hGMSCs) as an in vitro model system. Next generation transcriptomic sequencing analysis demonstrated considerable modifications in the expression of genes connected with ALS pathology, oxidative stress, mitochondrial dysfunction, and excitotoxicity in hGMSCs treated with cannabidiol. Our results suggest the efficacy of cannabidiol to delineate the unknown molecular pathways, which may underlie ALS pathology at an early stage using hGMSCs as a compelling in vitro system. J. Cell. Biochem. 118: 819–828, 2017.

Nuclear translocation of PKCα isoenzyme is involved in neurogenic commitment of human neural crest-derived periodontal ligament stem cells.

Stem cells isolated from human adult tissue niche represent a promising source for neural differentiation. Human Periodontal Ligament Stem Cells (hPDLSCs) originating from the neural crest are particularly suitable for induction of neural commitment. In this study, under xeno-free culture conditions, in undifferentiated hPDLSCs and in hPDLSCs induced to neuronal differentiation by basic Fibroblast Growth Factor, the level of some neural markers have been analyzed. The hPDLSCs spontaneously express Nestin, a neural progenitor marker. In these cells, the neurogenic process induced to rearrange the cytoskeleton, form neurospheres and express higher levels of Nestin and Tyrosine Hydroxylase, indicating neural induction. Protein Kinase C (PKC) is highly expressed in neural tissue and has a key role in neuronal functions. In particular the Ca(2+) and diacylglycerol-dependent activation of PKCα isozyme is involved in the regulation of neuronal differentiation. Another main component of the pathways controlling neuronal differentiation is the Growth Associated Protein-43 (GAP-43), whose activity is strictly regulated by PKC. The aim of this study is to investigate the role of PKCα/GAP-43 nuclear signal transduction pathway during neuronal commitment of hPDLSCs. During hPDLSCs neurogenic commitment the levels of p-PKC and p-GAP-43 increased both in cytoplasmic and nuclear compartment. PKCα nuclear translocation induced GAP-43 movement to the cytoplasm, where it is known to regulate growth cone dynamics and neuronal differentiation. Moreover, the degree of cytosolic Ca(2+) mobilization appeared to be more pronounced in differentiated hPDLSCs than in undifferentiated cells. This study provides evidences of a new PKCα/GAP-43 nuclear signalling pathway that controls neuronal differentiation in hPDLSCs, leading the way to a potential use of these cells in cell-based therapy in neurodegenerative diseases.

Stemness Maintenance Properties in Human Oral Stem Cells after Long-Term Passage

Background. Neural crest-derived mesenchymal stem cells (MSCs) from human oral tissues possess immunomodulatory and regenerative properties and are emerging as a potential therapeutic tool to treat diverse diseases, such as multiple sclerosis, myocardial infarction, and connective tissue damages. In addition to cell-surface antigens, dental MSCs express embryonic stem cell markers as neural crest cells originate from the ectoderm layer. In vitro passages may eventually modify these embryonic marker expressions and other stemness properties, including proliferation. In the present study, we have investigated the expression of proteins involved in cell proliferation/senescence and embryonic stem cell markers during early (passage 2) and late passages (passage 15) in MSCs obtained from human gingiva, periodontal, and dental pulp tissues. Methods. Cell proliferation assay, beta galactosidase staining, immunocytochemistry, and real-time PCR techniques were applied. Results. Cell proliferation assay showed no difference between early and late passages while senescence markers p16 and p21 were considerably increased in late passage. Embryonic stem cell markers including SKIL, MEIS1, and JARID2 were differentially modulated between P2 and P15 cells. Discussion. Our results suggest that the presence of embryonic and proliferation markers even in late passage may potentially endorse the application of dental-derived MSCs in stem cell therapy-based clinical trials.

Cannabidiol Modulates the Immunophenotype and Inhibits the Activation of the Inflammasome in Human Gingival Mesenchymal Stem Cells

Human Gingival Mesenchymal Stem Cells (hGMSCs) are multipotential cells that can expand and differentiate in culture under specific and standardized conditions. In the present study, we have investigated whether in vitro pre-treatment of hGMSCs with Cannabidiol (CBD) can influence their expression profile, improving the therapeutic potential of this cell culture. Following CBD treatment (5 μM) for 24 h, gene expression analysis through Next Generation Sequencing (NGS) has revealed several genes differentially expressed between CBD-treated hGMSCs (CBD-hGMSCs) and control cells (CTR-hGMSCs) that were linked to inflammation and apoptosis. In particular, we have demonstrated that CBD treatment in hGMSCs prevented the activation of the NALP3-inflammasome pathway by suppressing the levels of NALP3, CASP1, and IL18, and in parallel, inhibited apoptosis, as demonstrated by the suppression of Bax. CBD treatment was also able to modulate the expression of the well-known mesenchymal stem cell markers (CD13, CD29, CD73, CD44, CD90, and CD166), and other surface antigens. Specifically, CBD led to the downregulation of genes codifying for antigens involved in the activation of the immune system (CD109, CD151, CD40, CD46, CD59, CD68, CD81, CD82, CD99), while it led to the upregulation of those implicated in the inhibition of the immune responses (CD47, CD55, CD276). In conclusion, the present study will provide a new simple and reproducible method for preconditioning hGMSCs with CBD, before transplantation, as an interesting strategy for improving the hGMSCs molecular phenotype, reducing the risk of immune or inflammatory reactions in the host, and in parallel, for increasing their survival and thus, their long-term therapeutic efficacy.

Conditioned medium from relapsing-remitting multiple sclerosis patients reduces the expression and release of inflammatory cytokines induced by LPS-gingivalis in THP-1 and MO3.13 cell lines

HighlightshPDLSCs‐conditioned medium (CM) provides anti‐inflammatory effects in THP‐1 and MO3.13 cells.hPDLSCs‐CM is also effective when obtained by Relapsing‐Remitting MS patients.hPDLSCs‐CM may provide a novel potential autologous stem cell‐free therapeutic strategy. Abstract The present research was aimed at evaluating the effect of the conditioned medium (CM) from human periodontal ligament stem cells (hPDLSCs) obtained from healthy donors (hPDLSCs‐CM) and from Relapsing‐Remitting Multiple Sclerosis patients (RR‐MS‐CM) on inflammatory response induced by Porphyromonas gingivalis lipopolysaccharide (LPS‐G) in a monocytoid human cell line (THP‐1) and human oligodendrocyte cell line (MO3.13). Human periodontal ligament biopsies were carried out from control donor patients and selected RR‐MS donors. Sample tissues were obtained from premolar teeth during root scaling and subsequently cultured. The effect of hPDLSCs‐CM and RR‐MS‐CM on cell viability in PMA differentiated THP‐1 (as a model of microglia) was measured using a 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyltetrazolium bromide (MTT) assay. The same experiments were performed in undifferentiated and differentiated MO3.13 cells used as models of progenitor cells and oligodendrocytes, respectively. The expression of tumor necrosis factor alpha (TNF)‐&agr;, interleukin (IL)‐1&bgr; and IL‐6 was evaluated by Real‐Time Polymerase Chain Reaction (RT‐PCR), and enzyme‐linked immunosorbent assay (ELISA). The expression level of the Toll‐like receptor 4 (TLR‐4), for which LPS‐G is a ligand, was evaluated by Western blot analysis. The results were analyzed by ANOVA using Graph Pad Prism software. LPS‐G significantly increased TNF&agr;, IL‐1&bgr; and IL‐6 mRNA expression and protein levels in the differentiated THP‐1 cells and oligodendrocyte MO3.13 progenitor cells. Treatment with hPDLSCs‐CM or with RR‐MS‐CM significantly attenuated the LPS‐induced expression and production of these pro‐inflammatory cytokines. The CM from both healthy donors and RR‐MS patients also reduced the LPS‐G stimulated protein levels of TLR‐4 in differentiated THP‐1 cells. On the whole our data add new evidence on the anti‐inflammatory effects of these peculiar stem cells even when derived from RR‐MS patients and open novel perspectives in the therapeutic use of autologous periodontal stem cells in neuroinflammatory/neurodegenerative diseases including MS.

Anti-inflammatory effects of hypoxia-preconditioned human periodontal ligament cell secretome in an experimental model of multiple sclerosis: a key role of IL-37

Recent research has widely investigated the anti‐inflammatory effects of mesenchymal stem cells and their secretory products, termed the secretome, in the treatment of multiple sclerosis (MS). The present study examined the capacity of the conditioned medium (CM) from human periodontal ligament stem cells (hPLSCs) under hypoxia (H‐hPDLSCs‐CM) to suppress experimental autoimmune encephalomyelitis (EAE), a murine model of MS. To induce EAE, female C57BL/6 mice were immunized with myelin oligodendroglial glycoprotein peptide35–55. At the onset of symptoms, H‐hPDLSCs‐CM was infused via the tail vein of mice. Our results demonstrate the efficacy of H‐hPDLSCs‐CM treatment in diminishing clinical and histologic disease score. A key finding from this study is the marked expression of anti‐inflammatory cytokine IL‐37, paralleled by the suppression of proinflammatory cytokines in mice with EAE that were treated with H‐hPDLSCs‐CM. In addition, a consequent modulation of oxidative stress, autophagic, and apoptotic markers was observed in mice with EAE after hPDLSCs‐CM administration. In addition, to provide additional evidence of the molecular mechanisms that underlie H‐hPDLSCs‐CM, we investigated its therapeutic action in scratch injury–exposed NSC‐34 neurons, an in vitro model of injury. This model reproduces severe inflammation and oxidative stress conditions as observed after EAE damage. In vitro results corroborate the ability of hPDLSCs‐CM to modulate inflammatory, oxidative stress, and apoptotic pathways. Taken together, our findings suggest H‐hPDLSCs‐CM as a new pharmacologic opportunity for the management of MS.—Giacoppo, S., Thangavelu, S. R., Diomede, F., Bramanti, P., Conti, P., Trubiani, O., Mazzon, E. Anti‐inflammatory effects of hypoxia‐preconditioned human periodontal ligament cell secretome in an experimental model of multiple sclerosis: a key role of IL‐37. FASEB J. 31, 5592–5608 (2017). www.fasebj.org