Dental Pulp-Derived Stem Cells Preserve Astrocyte Health During Induced Gliosis by Modulating Mitochondrial Activity and Functions.

Astrocytes have been implicated in the onset and complication of various central nervous system (CNS) injuries and disorders. Uncontrolled astrogliosis (gliosis), while a necessary process for recovery after CNS trauma, also causes impairments in CNS performance and functions. The ability to preserve astrocyte health and better regulate the gliosis process could play a major role in controlling damage in the aftermath of acute insults and during chronic dysfunction. Here in, we demonstrate the ability of dental pulp-derived stem cells (DPSCs) in protecting the health of astrocytes during induced gliosis. First of all, we have characterized the expression of genes in primary astrocytes that are relevant to the pathological conditions of CNS by inducing gliosis. Subsequently, we found that astrocytes co-cultured with DPSCs reduced ROS production, NRF2 and GCLM expressions, mitochondrial membrane potential, and mitochondrial functions compared to the astrocytes that were not co-cultured with DPSCs in gliosis condition. In addition, hyperactive autophagy was also decreased in astrocytes that were co-cultured with DPSCs compared to the astrocytes that were not co-cultured with DPSCs during gliosis. This reversal and mitigation of gliosis in astrocytes were partly due to induction of neurogenesis in DPSCs through enhanced expressions of the neuronal genes like GFAP, NeuN, and Synapsin in DPSCs and by secretion of higher amounts of neurotropic factors, such as BDNF, GDNF, and TIMP-2. Protein-Protein docking analysis suggested that BDNF and GDNF can bind with CSPG4 and block the downstream signaling. Together these findings demonstrate novel functions of DPSCs to preserve astrocyte health during gliosis.

Dental pulp-derived stem cells inhibit osteoclast differentiation by secreting osteoprotegerin and deactivating AKT signalling in myeloid cells.

Osteoclasts (OCs) differentiate from the monocyte/macrophage lineage, critically regulate bone resorption and remodelling in both homeostasis and pathology. Various immune and non-immune cells help initiating activation of myeloid cells for differentiation, whereas hyper-activation leads to pathogenesis, and mechanisms are yet to be completely understood. Herein, we show the efficacy of dental pulp-derived stem cells (DPSCs) in limiting RAW 264.7 cell differentiation and underlying molecular mechanism, which has the potential for future therapeutic application in bone-related disorders. We found that DPSCs inhibit induced OC differentiation of RAW 264.7 cells when co-cultured in a contact-free system. DPSCs reduced expression of key OC markers, such as NFATc1, cathepsin K, TRAP, RANK and MMP-9 assessed by quantitative RT-PCR, Western blotting and immunofluorescence detection methods. Furthermore, quantitative RT-PCR analysis revealed that DPSCs mediated M2 polarization of RAW 264.7 cells. To define molecular mechanisms, we found that osteoprotegerin (OPG), an OC inhibitory factor, was up-regulated in RAW 264.7 cells in the presence of DPSCs. Moreover, DPSCs also constitutively secrete OPG that contributed in limiting OC differentiation. Finally, the addition of recombinant OPG inhibited OC differentiation in a dose-dependent manner by reducing the expression of OC differentiation markers, NFATc1, cathepsin K, TRAP, RANK and MMP9 in RAW 264.7 cells. RNAKL and M-CSF phosphorylate AKT and activate PI3K-AKT signalling pathway during osteoclast differentiation. We further confirmed that OPG-mediated inhibition of the downstream activation of PI3K-AKT signalling pathway was similar to the DPSC co-culture-mediated inhibition of OC differentiation. This study provides novel evidence of DPSC-mediated inhibition of osteoclastogenesis mechanisms.

Cellular and Molecular Mechanisms Mediated by recPrPC Involved in the Neuronal Differentiation Process of Mesenchymal Stem Cells.

Human Dental Pulp Stem Cells (hDPSCs) represent a type of adult mesenchymal stem cells that have the ability to differentiate in vitro in several lineages such as odontoblasts, osteoblasts, chondrocytes, adipocytes and neurons. In the current work, we used hDPSCs as the experimental model to study the role of recombinant prion protein 23⁻231 (recPrPC) in the neuronal differentiation process, and in the signal pathway activation of ERK 1/2 and Akt. We demonstrated that recPrPC was able to activate an intracellular signal pathway mediated by extracellular-signal-regulated kinase 1 and 2 (ERK 1/2) and protein kinase B (Akt). Moreover, in order to understand whether endogenous prion protein (PrPC) was necessary to mediate the signaling induced by recPrPC, we silenced PrPC, demonstrating that the presence of endogenous PrPC was essential for ERK 1/2 and Akt phosphorylation. Since endogenous PrPC is a well-known lipid rafts component, we evaluated the role of these structures in the signal pathway induced by recPrPC. Our results suggest that lipid rafts integrity play a key role in recPrPC activity. In fact, lipid rafts inhibitors, such as fumonisin B1 and MßCD, significantly prevented ERK 1/2 and Akt phosphorylation induced by recPrPC. In addition, we investigated the capacity of recPrPC to induce hDPSCs neuronal differentiation process after long-term stimulation through the evaluation of typical neuronal markers expression such as B3-Tubulin, neurofilament-H (NFH) and growth associated protein 43 (GAP43). Accordingly, when we silenced endogenous PrPC, we observed the inhibition of neuronal differentiation induced by recPrPC. The combined data suggest that recPrPC plays a key role in the neuronal differentiation process and in the activation of specific intracellular signal pathways in hDPSCs.

Role of lipid rafts in neuronal differentiation of dental pulp-derived stem cells.

Human dental pulp-derived stem cells (hDPSCs) are characterized by a typical fibroblast-like morphology. They express specific markers for mesenchymal stem cells and are capable of differentiation into osteoblasts, adipoblasts and neurons in vitro. Previous studies showed that gangliosides are involved in the induction of early neuronal differentiation of hDPSCs. This study was undertaken to investigate the role of lipid rafts in this process. Lipid rafts are signaling microdomains enriched in glycosphingolipids, cholesterol, tyrosine kinase receptors, mono- or heterotrimeric G proteins and GPI-anchored proteins. We preliminary showed that established cells expressed multipotent mesenchymal stromal-specific surface antigens. Then, we analyzed the distribution of lipid rafts, revealing plasma membrane microdomains with GM2 and EGF-R enrichment. Following stimulation with EGF/bFGF, neuronal differentiation was observed. To analyze the functional role of lipid rafts in EGF/bFGF-induced hDPSCs differentiation, cells were preincubated with lipid raft affecting agents, i.e. [D]-PDMP or methyl-ß-cyclodextrin. These compounds significantly prevented neuronal-specific antigen expression, as well as Akt and ERK 1/2 phosphorylation, induced by EGF/bFGF, indicating that lipid raft integrity is essential for EGF/bFGF-induced hDPSCs differentiation. These results suggest that lipid rafts may represent specific chambers, where multimolecular signaling complexes, including lipids (gangliosides, cholesterol) and proteins (EGF-R), play a role in hDPSCs differentiation.

Secretome of Dental Pulp-Derived Stem Cells Reduces Inflammation and Proliferation of Glioblastoma Cells by Deactivating Mapk-Akt Pathway.

Background: Dental pulp-derived stem cells (DPSC) is a promising therapy as they modulate the immune response, so we evaluated the inhibitory effect of DPSC secretome (DPSC℗) on the proliferation and inflammation in human glioblastoma (GBM) cells (U-87 MG) and elucidated the concomitant mechanisms involved. Methods: The U87-MG cells were cultured with DPSC℗ for 24 h and assessed the expression of inflammatory molecules using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), generation of reactive oxygen species (ROS), and mitochondrial functionality using a seahorse flux analyzer. MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay and cell cycle analysis were performed to evaluate the proliferation and cell cycle. Finally, the protein levels were determined by western blot. Results: DPSC℗ reduced the inflammation and proliferation of U-87 MG cells by down-regulating the pro-inflammatory markers and up-regulating anti-inflammatory markers expressions through ROS-mediated signaling. Moreover, DPSC℗ significantly reduced the mitochondrial membrane potential (MMP) in the cells. The cellular bioenergetics revealed that all the parameters of oxygen consumption rate (OCAR) and the extracellular acidification rate (ECAR) were significantly decreased in the GBM cells after the addition of DPSC℗. Additionally, DPSC℗ decreased the GBM cell proliferation by arresting the cell cycle at the G1 phase through activation (phosphorylation) of checkpoint molecule CHK1. Furthermore, mechanistically, we found that the DPSC℗ impedes the phosphorylation of the mitogen-activated protein kinases (P38 MAPK) and protein kinase B (AKT) pathway. Conclusion: Our findings lend the first evidence of the inhibitory effects of DPSC℗ on proliferation and inflammation in GBM cells by altering the P38 MAPK-AKT pathway.

Dental Pulp-Derived Stem Cells Reduce Inflammation, Accelerate Wound Healing and Mediate M2 Polarization of Myeloid Cells.

This work aimed to validate the potential use of dental pulp-derived stem cells (DPSCs) for the treatment of inflammation by defining their mechanisms of action. We planned to investigate whether priming of DPSC with proinflammatory molecules had any impact on their behavior and function. In the first step of our validation in vitro, we showed that priming of DPSCs with the bioactive agents LPS, TNF-α, or IFN-γ altered DPSCs' immunologic properties by increasing their expression levels of IL-10, HGF, IDO, and IL-4 and by decreasing their mitochondrial functions. Moreover, DPSCs induced accelerated wound healing irrespective of priming, as determined by using a gut epithelial cell line in a scratch wound assay. Wound healing of gut epithelial cells was mediated by regulating the expressions of AKT, NF-κB, and ERK1/2 proteins compared to the control epithelial cells. In addition, primed DPSCs altered monocyte polarization toward an immuno-suppressive phenotype (M2), where monocytes expressed higher levels of IL-4R, IL-6, Arg1, and YM-1 compared to monocytes cultured with control DPSCs. In silico analysis revealed that this was accomplished in part by the interaction between kynurenine and PPARγ, which regulated the expression of M2 differentiation-related genes. Collectively, these data provided evidence that the DPSCs reduced inflammation, induced M2 polarization of myeloid cells, and healed damaged gut epithelial cells through inactivation of inflammation and modulating constitutively active signaling pathways.

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.

Effectiveness of Dental Pulp-derived Stem Cells and Bone Marrowderived Mesenchymal Stromal Cells Implanted into a Murine Critical Bone Defect.

BACKGROUND: While bone marrow-derived mesenchymal stromal cells (BM-MSCs) have been used for many years in bone tissue engineering applications, the procedure still has drawbacks such as painful collection methods and damage to the donor site. Dental pulp-derived stem cells (DPSCs) are readily accessible, occur in high amounts, and show a high proliferation and differentiation capability. Therefore, DPSCs may be a promising alternative for BM-MSCs to repair bone defects. OBJECTIVE: The aim of this study was to investigate the bone regenerative potential of DPSCs in comparison to BM-MSCs in vitro and in vivo. METHODS: In vitro investigations included analysis of cell doubling time as well as proliferation and osteogenic differentiation. For the in vivo study, 36 male NMRI nude mice were randomized into 3 groups: 1) control (cell-free mineralized collagen matrix (MCM) scaffold), 2) MCM + DPSCs, and 3) MCM + BMMSCs. Critical size 2 mm bone defects were created at the right femur of each mouse and stabilized by an external fixator. After 6 weeks, animals were euthanized, and microcomputed tomography scans (µCT) and histological analyses were performed. RESULTS: In vitro DPSCs showed a 2-fold lower population doubling time and a 9-fold higher increase in proliferation when seeded onto MCM scaffolds as compared to BM-MSCs, but DPSCs showed a significantly lower osteogenic capability than BM-MSCs. In vivo, the healing of the critical bone defect in NMRI nude mice was comparable among all groups. CONCLUSION: Pre-seeding of MCM scaffolds with DPSCs and BM-MSCs did not enhance bone defect healing.

Vertical ridge augmentation using guided bone regeneration procedure and dental pulp derived mesenchymal stem cells with simultaneous dental implant placement: A histologic study in a sheep model.

OBJECTIVES: To determine the effects of bone graft and dental pulp derived mesenchymal stem cells (DPMSCs) implantation with simultaneous dental implant placement on osteointegration, newly formed bone and vertical bone height histologically and histomorphometrically in a sheep model. MATERIAL AND METHODS: A total of 48 implants were divided into three groups. In Group I (n = 16), no material was placed around the implants. In Group II (n = 16), particulate deproteinized bovine bone graft (DBBG) was placed around the implant and in Group III (n = 16), 2 × 106 DPMSCs were placed around the implant with DBBG. All implants were covered with a 20 × 30 mm collagen membrane and the edges of the membrane were fixed with mini screws. The animals were sacrificed 3 and 6 weeks after surgery. Histologic and histomorphometric assessments were performed. RESULTS: The area of newly formed bone in Groups I, II, and III were calculated as percentage 2.15 ± 0.22, 11.88 ± 0.77, and 14.50 ± 0.67 respectively after 3 weeks and 3.33 ± 0.37, 18.45 ± 0.33, and 29 ± 1.07 after 6 weeks, respectively (P < 0.05). Three weeks after dental implant placement, the vertical bone length was 0.17 ± 0.02 mm in Group I, 0.89 ± 0.068 mm in Group II and 0.96 ± 0.05 mm in Group III. After 6 weeks, these values were 0.28 ± 0.03 mm, 1.34 ± 0.08 mm, and 1.49 ± 0.08 mm, respectively. There was no significant difference between Groups II and III at 3 and 6 weeks in terms of vertical bone length. CONCLUSION: Bone graft and DPMSCs application with dental implant have beneficial effects on newly formed bone and vertical bone height in this experimental sheep model.

Role of Prion protein-EGFR multimolecular complex during neuronal differentiation of human dental pulp-derived stem cells.

Cellular prion protein (PrPC) is expressed in a wide variety of stem cells in which regulates their self-renewal as well as differentiation potential. In this study we investigated the presence of PrPC in human dental pulp-derived stem cells (hDPSCs) and its role in neuronal differentiation process. We show that hDPSCs expresses early PrPC at low concentration and its expression increases after two weeks of treatment with EGF/bFGF. Then, we analyzed the association of PrPC with gangliosides and EGF receptor (EGF-R) during neuronal differentiation process. PrPC associates constitutively with GM2 in control hDPSCs and with GD3 only after neuronal differentiation. Otherwise, EGF-R associates weakly in control hDPSCs and more markedly after neuronal differentiation. To analyze the functional role of PrPC in the signal pathway mediated by EGF/EGF-R, a siRNA PrP was applied to ablate PrPC and its function. The treatment with siRNA PrP significantly prevented Akt and ERK1/2 phosphorylation induced by EGF. Moreover, siRNA PrP treatment significantly prevented neuronal-specific antigens expression induced by EGF/bFGF, indicating that cellular prion protein is essential for EGF/bFGF-induced hDPSCs differentiation. These results suggest that PrPC interact with EGF-R within lipid rafts, playing a role in the multimolecular signaling complexes involved in hDPSCs neuronal differentiation.

Immunophenotypic and Molecular Analysis of Human Dental Pulp Stem Cells Potential for Neurogenic Differentiation.

BACKGROUND: Growing evidence shows that dental pulp (DP) tissues could be a potential source of adult stem cells for the treatment of devastating neurological diseases and several other conditions. AIMS: Exploration of the expression profile of several key molecular markers to evaluate the molecular dynamics in undifferentiated and differentiated DP-derived stem cells (DPSCs) in vitro. SETTINGS AND DESIGN: The characteristics and multilineage differentiation ability of DPSCs were determined by cellular and molecular kinetics. DPSCs were further induced to form adherent (ADH) and non-ADH (NADH) neurospheres under serum-free condition which was further induced into neurogenic lineage cells and characterized for their molecular and cellular diversity at each stage. STATISTICAL ANALYSIS USED: Statistical analysis used one-way analysis of variance, Student's t-test, Livak method for relative quantification, and R programming. RESULTS: Immunophenotypic analysis of DPSCs revealed >80% cells positive for mesenchymal markers CD90 and CD105, >70% positive for transferring receptor (CD71), and >30% for chemotactic factor (CXCR3). These cells showed mesodermal differentiation also and confirmed by specific staining and molecular analysis. Activation of neuronal lineage markers and neurogenic growth factors was observed during lineage differentiation of cells derived from NADH and ADH spheroids. Greater than 80% of cells were found to express ß-tubulin III in both differentiation conditions. CONCLUSIONS: The present study reported a cascade of immunophenotypic and molecular markers to characterize neurogenic differentiation of DPSCs under serum-free condition. These findings trigger the future analyses for clinical applicability of DP-derived cells in regenerative applications.

Control of proliferation and osteogenic differentiation of human dental-pulp-derived stem cells by distinct surface structures.

The ability to control the behavior of stem cells provides crucial benefits, for example, in tissue engineering and toxicity/drug screening, which utilize the stem cell's capacity to engineer new tissues for regenerative purposes and the testing of new drugs in vitro. Recently, surface topography has been shown to influence stem cell differentiation; however, general trends are often difficult to establish due to differences in length scales, surface chemistries and detailed surface topographies. Here we apply a highly versatile screening approach to analyze the interplay of surface topographical parameters on cell attachment, morphology, proliferation and osteogenic differentiation of human mesenchymal dental-pulp-derived stem cells (DPSCs) cultured with and without osteogenic differentiation factors in the medium (ODM). Increasing the inter-pillar gap size from 1 to 6 µm for surfaces with small pillar sizes of 1 and 2 µm resulted in decreased proliferation and in more elongated cells with long pseudopodial protrusions. The same alterations of pillar topography, up to an inter-pillar gap size of 4 µm, also resulted in enhanced mineralization of DPSCs cultured without ODM, while no significant trend was observed for DPSCs cultured with ODM. Generally, cells cultured without ODM had a larger deposition of osteogenic markers on structured surfaces relative to the unstructured surfaces than what was found when culturing with ODM. We conclude that the topographical design of biomaterials can be optimized for the regulation of DPSC differentiation and speculate that the inclusion of ODM alters the ability of the cells to sense surface topographical cues. These results are essential in order to transfer the use of this highly proliferative, easily accessible stem cell into the clinic for use in cell therapy and regenerative medicine.

Clinical grade adult stem cell banking.

There has been a great deal of scientific interest recently generated by the potential therapeutic applications of adult stem cells in human care but there are several challenges regarding quality and safety in clinical applications and a number of these challenges relate to the processing and banking of these cells ex-vivo. As the number of clinical trials and the variety of adult cells used in regenerative therapy increases, safety remains a primary concern. This has inspired many nations to formulate guidelines and standards for the quality of stem cell collection, processing, testing, banking, packaging and distribution. Clinically applicable cryopreservation and banking of adult stem cells offers unique opportunities to advance the potential uses and widespread implementation of these cells in clinical applications. Most current cryopreservation protocols include animal serum proteins and potentially toxic cryoprotectant additives (CPAs) that prevent direct use of these cells in human therapeutic applications. Long term cryopreservation of adult stem cells under good manufacturing conditions using animal product free solutions is critical to the widespread clinical implementation of ex-vivo adult stem cell therapies. Furthermore, to avoid any potential cryoprotectant related complications, reduced CPA concentrations and efficient post-thaw washing to remove CPA are also desirable. The present review focuses on the current strategies and important aspects of adult stem cell banking for clinical applications. These include current good manufacturing practices (cGMPs), animal protein free freezing solutions, cryoprotectants, freezing & thawing protocols, viability assays, packaging and distribution. The importance and benefits of banking clinical grade adult stem cells are also discussed.