Modeling Lysosomal Storage Disorders in an Innovative Way: Establishment and Characterization of Stem Cell Lines from Human Exfoliated Deciduous Teeth of Mucopolysaccharidosis Type II Patients.

Among the many lysosomal storage disorders (LSDs) that would benefit from the establishment of novel cell models, either patient-derived or genetically engineered, is mucopolysaccharidosis type II (MPS II). Here, we present our results on the establishment and characterization of two MPS II patient-derived stem cell line(s) from deciduous baby teeth. To the best of our knowledge, this is the first time a stem cell population has been isolated from LSD patient samples obtained from the dental pulp. Taking into account our results on the molecular and biochemical characterization of those cells and the fact that they exhibit visible and measurable disease phenotypes, we consider these cells may qualify as a valuable disease model, which may be useful for both pathophysiological assessments and in vitro screenings. Ultimately, we believe that patient-derived dental pulp stem cells (DPSCs), particularly those isolated from human exfoliated deciduous teeth (SHEDs), may represent a feasible alternative to induced pluripotent stem cells (iPSCs) in many labs with standard cell culture conditions and limited (human and economic) resources.

RICK regulates the odontogenic differentiation of dental pulp stem cells through activation of TNF-α via the ERK and not through NF-κB signaling pathway.

Dental pulp stem cells (DPSCs) are capable of both self-renewal and multilineage differentiation, which play a positive role in dentinogenesis. Studies have shown that tumor necrosis factor-α (TNF-α) is involved in the differentiation of DPSCs under pro-inflammatory stimuli, but the mechanism of action of TNF-α is unknown. Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) is a biomarker of an early inflammatory response that plays a key role in modulating cell differentiation, but the role of RICK in DPSCs is still unclear. In this study, we identified that RICK regulates TNF-α-mediated odontogenic differentiation of DPSCs via the ERK signaling pathway. The expression of the biomarkers of odontogenic differentiation dental matrix protein-1 (DMP-1), dentin sialophosphoprotein (DSPP), biomarkers of odontogenic differentiation, increased in low concentration (1-10 ng/ml) of TNF-α and decreased in high concentration (50-100 ng/ml). Odontogenic differentiation increased over time in the odontogenic differentiation medium. In the presence of 10 ng/L TNF-α, the expression of RICK increased gradually over time, along with odontogenic differentiation. Genetic silencing of RICK expression reduced the expression of odontogenic markers DMP-1 and DSPP. The ERK, but not the NF-κB signaling pathway, was activated during the odontogenic differentiation of DPSCs. ERK signaling modulators decreased when RICK expression was inhibited. PD98059, an ERK inhibitor, blocked the odontogenic differentiation of DPSCs induced by TNF-α. These results provide a further theoretical and experimental basis for the potential use of RICK in targeted therapy for dentin regeneration.

Basic fibroblast growth factor promotes human dental pulp stem cells cultured in 3D porous chitosan scaffolds to neural differentiation.

AIM/PURPOSE: Dental pulp stem cells (DPSCs) were widely used as seed cells in the field of tissue engineering and regenerative medicine, including spinal cord injury (SCI) repair and other neuronal degenerative diseases, due to their easy isolation, multiple differentiation potential, low immunogenicity and low rates of rejection during transplantation. Various studies have shown that bFGF can enhance peripheral nerve regeneration after injury, and phospho-ERK (p-ERK) activation as a major mediator may be involved in this process. Previous studies also have proved that a suitable biomaterial scaffold can carry and transport the therapeutic cells effectively to the recipient area. It has showed in our earlier experiments that 3D porous chitosan scaffolds exhibited a suitable circumstance for survival and neural differentiation of DPSCs in vitro. The purpose of the study was to evaluate the influence of chitosan scaffolds and bFGF on differentiation of DPSCs. MATERIALS AND METHODS: In current study, DPSCs were cultured in chitosan scaffolds and treated with neural differentiation medium for 7 days. The neural genes and protein markers were analyzed by western blot and immunofluorescence. Meanwhile, the relevant signaling pathway involved in this process was also tested. RESULTS: Our study revealed that the viability of DPSCs was not influenced by co-culture with the chitosan scaffolds as well as bFGF. Compared with the control and DPSC/chitosan-scaffold groups, the levels of GFAP, S100ß and ß-tubulin III significantly increased in the DPSC/chitosan-scaffold+bFGF group. CONCLUSION: Chitosan scaffolds were non-cytotoxic to the survival of DPSCs, and chitosan scaffolds combined with bFGF facilitated the neural differentiation of DPSCs. The transplantation of DPSCs/chitosan-scaffold+bFGF might be a secure and effective method of treating SCI and other neuronal diseases.

Functional Dental Pulp Regeneration: Basic Research and Clinical Translation.

Pulpal and periapical diseases account for a large proportion of dental visits, the current treatments for which are root canal therapy (RCT) and pulp revascularisation. Despite the clinical signs of full recovery and histological reconstruction, true regeneration of pulp tissues is still far from being achieved. The goal of regenerative endodontics is to promote normal pulp function recovery in inflamed or necrotic teeth that would result in true regeneration of the pulpodentinal complex. Recently, rapid progress has been made related to tissue engineering-mediated pulp regeneration, which combines stem cells, biomaterials, and growth factors. Since the successful isolation and characterisation of dental pulp stem cells (DPSCs) and other applicable dental mesenchymal stem cells, basic research and preclinical exploration of stem cell-mediated functional pulp regeneration via cell transplantation and cell homing have received considerably more attention. Some of this effort has translated into clinical therapeutic applications, bringing a ground-breaking revolution and a new perspective to the endodontic field. In this article, we retrospectively examined the current treatment status and clinical goals of pulpal and periapical diseases and scrutinized biological studies of functional pulp regeneration with a focus on DPSCs, biomaterials, and growth factors. Then, we reviewed preclinical experiments based on various animal models and research strategies. Finally, we summarised the current challenges encountered in preclinical or clinical regenerative applications and suggested promising solutions to address these challenges to guide tissue engineering-mediated clinical translation in the future.

Depletion of EREG enhances the osteo/dentinogenic differentiation ability of dental pulp stem cells via the p38 MAPK and Erk pathways in an inflammatory microenvironment.

BACKGROUND: Epiregulin (EREG) is an important component of EGF and was demonstrated to promote the osteo/dentinogenic differentiation of stem cells from dental apical papilla (SCAPs). Whether EREG can stimulate the osteo/dentinogenic differentiation of dental pulp stem cells (DPSCs) in inflammatory environment is not clear. The purpose of the present study is to investigate the role of EREG on the osteo/dentinogenic differentiation ability of DPSCs in inflammatory environment. METHODS: DPSCs were isolated from human third molars. Short hairpin RNAs (shRNAs) were used to knock down EREG expression in DPSCs. Recombinant human EREG (rhEREG) protein was used in the rescue experiment. TNF-α was employed to mimic the inflammatory environment in vitro. Alkaline phosphatase (ALP) staining, Alizarin red staining, quantitative calcium analysis, and real-time RT-PCR were performed to detect osteo/dentinogenic differentiation markers and related signalling pathways under normal and inflammatory conditions. RESULTS: EREG depletion promoted the ALP activity and mineralization ability of DPSCs. The expression of BSP, DMP-1, and DSPP was also enhanced. Moreover, 50 ng/mL rhEREG treatment decreased the osteo/dentinogenic differentiation potential of DPSCs, while treatment with 10 ng/mL TNF-α for 4 h increased the expression of EREG in DPSCs. Conversely, EREG knockdown rescued the impaired osteo/dentinogenic differentiation ability caused by TNF-α treatment. Further mechanistic studies showed that EREG depletion activated the p38 MAPK and Erk signalling pathways in DPSCs under normal and inflammatory conditions. CONCLUSIONS: Our results demonstrated that EREG could inhibit the osteo/dentinogenic differentiation potential of DPSCs via the p38 MAPK and Erk signalling pathways. Under inflammatory environment, EREG depletion enhanced osteo/dentinogenic differentiation potential of DPSCs by improving the expression of p-p38 MAPK and p-Erk.

FAM96B inhibits the senescence of dental pulp stem cells.

Dental pulp stem cells (DPSCs) are considered a remarkable source for the regeneration of dental pulp tissues, but their therapeutic effectiveness remains limited, especially in elderly people. Previous studies found that senescence has a negative effect on the proliferation and differentiation potential of DPSCs. Moreover, numerous long non-coding RNA (lncRNA) and messenger RNA were significantly differentially regulated in DPSCs from young and elderly donors. However, the changes in DPSCs protein during senescence have not been addressed. In this study, differences in DPSC protein expression profiles and coexpression of protein and lncRNA were analyzed using proteomics and bioinformatics. The results showed 75 upregulated proteins and 69 downregulated proteins in DPSCs from elderly donors. Vasopressin-regulated water reabsorption, Parkinson's disease, Alzheimer's disease, and protein export were the top four functional pathways associated with DPSCs. High mobility group N1 (HMGN1), HMGN2, UCHL1, and the family with sequence similarity 96 member B homeobox gene (FAM96B) were associated with DPSCs senescence. Then, we investigated FAM96B function in DPSCs. After FAM96B depletion, telomerase reverse transcriptase (TERT) activity decreased, but the number of senescence-associated ß-galactosidase (SA-ß-gal) positive cells and the protein levels of p16, p53 were significantly increased. Gain-of-function assays suggested that FAM96B overexpression was positively correlated with TERT activity, but negatively correlated with the number of SA-ß-gal positive cells and the protein levels of P16 and P53. Moreover, after FAM96B overexpression, the results showed a significant increase in alkaline phosphatase activity and an enhanced mineralization ability of DPSCs. The reverse-transcription polymerase chain reaction results also showed that dentin sialophosphoprotein and osteocalcin were expressed at greater levels. The carboxyfluorescein succinimidyl ester (CFSE) results displayed that FAM96B increased the proliferation potential of DPSCs. Our study revealed candidate proteins that might be related to DPSCs senescence and provided information to elucidate the mechanism of the biological changes in DPSCs' aging. Moreover, FAM96B was demonstrated to play an important role in suppressing DPSCs senescence and promoting osteogenic differentiation and proliferation.

Downregulated microRNA-488 enhances odontoblast differentiation of human dental pulp stem cells via activation of the p38 MAPK signaling pathway.

Human dental pulp stem cells (hDPSCs) are primarily derived from the pulp tissues of permanent third molar teeth. They were widely used in human bone tissue engineering. It was previously indicated that microRNA (miR) expressions are closely associated with hDPSCs development. However, the specific effect of miR-488 on hDPSCs still remains unclear. In this study, we aimed to investigate effects of miR-488 on the differentiation of hDPSCs into odontoblast cells through the p38 mitogen-activated protein kinases (MAPK) signaling pathway by binding to MAPK1. The hDPSCs were isolated and cultured in vitro. Dual-luciferase reporter gene assay was performed to test the relationship between MAPK1 (p38) and miR-488. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to detect the mRNA and protein expressions of p38 MAPK signaling pathway-related genes (MAPK1, Ras, and Mitogen-activated protein kinase kinase 3/6 [MKK3/6]), along with expressions of dentin Sialophosphoprotein (DSPP), alkaline phosphatase (ALP), and osteonectin (OCN). ALP staining and alizarin red staining were conducted to detect ALP activity and degree of mineralization. Initially, we found that MAPK1 was the target gene of miR-488. Besides, downregulation of miR-488 was observed to stimulate the p38 MAPK signaling pathway and to increase the messenger RNA and protein expressions of DSPP, ALP, and OCN. Furthermore, ALP activity and formation of a mineralized nodule in hDPSCs were enhanced upon downregulation of miR-488. The aforementioned findings provided evidence supporting that downregulation of miR-488 promotes odontoblastic differentiation of hDPSCs through the p38 MAPK signaling pathway by targeting MAPK1, paving the basis for further study about hDPSCs.

CKIP-1 suppresses odontoblastic differentiation of dental pulp stem cells via BMP2 pathway and can interact with NRP1.

AIM: Casein kinase 2 interacting protein-1 (CKIP-1) is a recently discovered intracellular regulator of bone formation, muscle cell differentiation, and tumor cell proliferation. Our study aims to identify the inhibition of BMP2-Smad1/5 signaling by CKIP-1 in odontoblastic differentiation of human dental pulp stem cells (DPSCs). MATERIALS AND METHODS: DPSCs infected CKIP-1 siRNA or transfected CKIP-1 full-length plasmid were cultured in odontoblastic differentiation medium or added noggin (200 ng/mL) for 21 days. We examined the effects of CKIP-1 on odontoblastic differentiation, mineralized nodules formation, and interaction by western blot, real-time polymerase chain reaction (RT-PCR), alkaline phosphatase (ALP) staining, alizarin red S staining, and immunoprecipitation. RESULTS: Firstly, we have demonstrated that CKIP-1 expression markedly decreased time-dependently along with cell odontoblastic differentiation. Indeed, the silence of CKIP-1 upregulated odontoblastic differentiation via BMP2-Smad1/5 signaling, while CKIP-1 over-expression had a negative effect on odontoblastic differentiation of DPSCs. Furthermore, CKIP-1 could interact with Neuropilin-1 (NRP1). CONCLUSIONS: This work provides data that advocates a novel perception on odontoblastic differentiation of DPSCs. Therefore, inhibiting the expression of CKIP-1 may be of great significance to the development of dental caries.

Enterococcus faecium L-15 Cell-Free Extract Improves the Chondrogenic Differentiation of Human Dental Pulp Stem Cells.

Hyaline cartilage is a tissue of very low regenerative capacity because of its histology and limited nutrient supply. Cell-based therapies have been spotlighted in the regeneration of damaged cartilage. Dental pulp stem cells (DPSCs) are multipotent and are easily accessible for therapeutic purposes. In human gastrointestinal tracts, Enterococcus faecium is a naturally occurring commensal species of lactic acid bacteria. In this work, the human DPSCs were differentiated into chondrocytes using a chondrogenic differentiation medium with or without L-15 extract. We observed that chondrogenic differentiation improved in an E. faecium L-15 extract (L-15)-treated DPSC group via evaluation of chondrogenic-marker mRNA expression levels. In particular, we found that L-15 treatment promoted early-stage DPSC differentiation. Cells treated with L-15 were inhibited at later stages and were less likely to transform into hypertrophic chondrocytes. In L-15-treated groups, the total amount of cartilage extracellular matrix increased during the differentiation process. These results suggest that L-15 promotes chondrogenic differentiation, and that L-15 may be used for cartilage repair or cartilage health supplements. To our knowledge, this is the first report demonstrating the beneficial effect of L-15 treatment on chondrogenic differentiation.

Clinical Potential and Current Progress of Dental Pulp Stem Cells for Various Systemic Diseases in Regenerative Medicine: A Concise Review.

Dental pulp stem cells (DPSCs) are mesenchymal stem cells (MSCs) that have multipotent differentiation and a self-renewal ability. They have been useful not only for dental diseases, but also for systemic diseases. Extensive studies have suggested that DPSCs are effective for various diseases, such as spinal cord injuries, Parkinson's disease, Alzheimer's disease, cerebral ischemia, myocardial infarction, muscular dystrophy, diabetes, liver diseases, eye diseases, immune diseases, and oral diseases. DPSCs have the potential for use in a cell-therapeutic paradigm shift to treat these diseases. It has also been reported that DPSCs have higher regenerative potential than the bone marrow-derived mesenchymal stem cells known as representative MSCs. Therefore, DPSCs have recently gathered much attention. In this review, the therapeutic potential of DPSCs, the latest progress in the pre-clinical study for treatment of these various systemic diseases, and the clinical applications of DPSCs in regenerative medicine, are all summarized. Although challenges, including mechanisms of the effects and establishment of cell processing and transplantation methods for clinical use, still remain, DPSCs could be promising stem cells sources for various clinical applications, because of their easy isolation by a noninvasive procedure without ethical concerns.

Diagnostic Cytokines and Comparative Analysis Secreted from Exfoliated Deciduous Teeth, Dental Pulp, and Bone Marrow Derived Mesenchymal Stem Cells for Functional Cell-Based Therapy.

The aim of the study was to clarify the distinctive features of stem cells for effective cell-based therapy strategies in regenerative medicine. The expression levels of cytokines secreted from stem cells from exfoliated deciduous teeth (SHED), dental pulp stem cells (DPSCs), and bone marrow derived mesenchymal stem cells (BMMSCs) were examined to identify the details of their characteristics. A total of 174 cytokines were analyzed using cytokine antibody array, and their expression levels were confirmed by an enzyme-linked immunosorbent assay. These results indicated that 11 cytokines that were related to tissue regeneration, including growth factors, chemokines, and inflammatory cytokines, were identical in SHED, DPSCs, and BMMSCs. The comparative analyses between SHED and BMMSCs revealed that hepatocyte growth factor (HGF), matrix metalloproteinase-3, and stromal cell derived factor 1 (SDF-1) were expressed 6.7-, 2.5-, and 2.1-fold higher, respectively, in SHEDs. HGF was also expressed 3.4-fold higher in DPSCs than BMMSCs. Monocyte chemoattractant protein-1, and-3 were expressed more strongly in BMMSCs. SHED contained significantly higher SDF-1 levels than DPSCs. The distinct cytokine secretion indicated that they had different character besides basic MSC features. This knowledge of diagnostic cytokines analysis secreted from SHED, DPSCs, and BMMSCs extends our understanding, and can provide a novel therapeutic paradigm shift for functional cell-based therapy.

Effects of Nerve Growth Factor and Basic Fibroblast Growth Factor Promote Human Dental Pulp Stem Cells to Neural Differentiation.

Dental pulp stem cells (DPSCs) were the most widely used seed cells in the field of neural regeneration and bone tissue engineering, due to their easily isolation, lack of ethical controversy, low immunogenicity and low rates of transplantation rejection. The purpose of this study was to investigate the role of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on neural differentiation of DPSCs in vitro. DPSCs were cultured in neural differentiation medium containing NGF and bFGF alone or combination for 7 days. Then neural genes and protein markers were analyzed using western blot and RT-PCR. Our study revealed that bFGF and NGF increased neural differentiation of DPSCs synergistically, compared with bFGF and NGF alone. The levels of Nestin, MAP-2, ßIII-tubulin and GFAP were the most highest in the DPSCs + bFGF + NGF group. Our results suggested that bFGF and NGF signifiantly up-regulated the levels of Sirt1. After treatment with Sirt1 inhibitor, western blot, RT-PCR and immunofluorescence staining showed that neural genes and protein markers had markedly decreased. Additionally, the ERK and AKT signaling pathway played a key role in the neural differentiation of DPSCs stimulated with bFGF + NGF. These results suggested that manipulation of the ERK and AKT signaling pathway may be associated with the differentiation of bFGF and NGF treated DPSCs. Our date provided theoretical basis for DPSCs to treat neurological diseases and repair neuronal damage.

Runx2 modified dental pulp stem cells (DPSCs) enhance new bone formation during rapid distraction osteogenesis (DO).

Distraction osteogenesis (DO) remains a major challenge in orthopedic and craniofacial surgery. The transplantion of mesenchymal stem cells (MSCs) could reduce the treatment period and the associated complications by increasing new bone formation during long-bone DO. Runt-related transcription factor 2 (Runx2) encodes a nuclear protein that is a pivotal regulator of osteoblast differentiation. It significantly stimulates calcium accumulation and alkaline phosphatase (ALP) activity in dental pulp stem cells (DPSCs). In this study, we investigated the effects of gene therapy using Runx2 on new bone formation during tibia DO of rabbits. The distraction gap of the rabbits was injected with adenovirus (Adv)-Runx2-green fluorescent protein (GFP)-transfected DPSCs (overexpression group, Group OE) or Adv-GFP-transfected DPSCs (negative control group, Group NC). Rabbits in the control group (Groups CON) were injected with physiologic saline. The generation of new bone tissue in the distraction gap was studied by radiographic examination, micro-computed tomography (CT) evaluation, histological analyze, and Mechanical testing at weeks 8 in the consolidation period. Excellent bone formation in the distracted callus was observed in Group OE and Group NC. Moreover, the OE group showed better bone formation and the highest bone mineral density (BMD) and bone mineral content (BMC). Group CON animals showed inadequate bone formation in the distracted callus compared to the other groups. The results suggest that gene therapy using Runx2-modified DPSCs was more effective during bone deposition and new bone formation in tibia DO.

Odontogenic differentiation and biomineralization potential of dental pulp stem cells inside Mg-based bioceramic scaffolds under low-level laser treatment.

This study aimed to investigate the potential of low-level laser irradiation (LLLI) to promote odontogenic differentiation and biomineralization by dental pulp stem cells (DPSCs) seeded inside bioceramic scaffolds. Mg-based, Zn-doped bioceramic scaffolds, synthesized by the sol-gel technique, were spotted with DPSCs and exposed to LLLI at 660 nm with maximum output power of 140 mw at fluencies (a) 2 and 4 J/cm2 to evaluate cell viability/proliferation by the MTT assay and (b) 4 J/cm2 to evaluate cell differentiation, using real-time PCR (expression of odontogenic markers) and a p-nitrophenylphosphate (pNPP)-based assay for alkaline phosphatase (ALP) activity measurement. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis were used for structural/chemical characterization of the regenerated tissues. Exposure of the DPSCs/scaffold complexes to the proposed LLLI scheme was associated with statistically significant increase of odontogenesis-related markers (bone morphogenetic protein 2 (BMP-2): 22.4-fold, dentin sialophosphoprotein (DSPP): 28.4-fold, Osterix: 18.5-fold, and Runt-related transcription factor 2 (Runx2): 3.4-fold). ALP activity was significantly increased at 3 and 7 days inside the irradiated compared to that in the non-irradiated SC/DPSC complexes, but gradually decreased until 14 days. Newly formed Ca-P tissue was formed on the SC/DPSC complexes after 28 days of culture that attained the characteristics of bioapatite. Overall, LLLI treatment proved to be beneficial for odontogenic differentiation and biomineralization of DPSCs inside the bioceramic scaffolds, making this therapeutic modality promising for targeted dentin engineering.

Chitosan scaffolds induce human dental pulp stem cells to neural differentiation: potential roles for spinal cord injury therapy.

Cell-based transplantation strategies hold great potential for spinal cord injury (SCI) repair. Chitosan scaffolds have therapeutic benefits for spinal cord regeneration. Human dental pulp stem cells (DPSCs) are abundant available stem cells with low immunological incompatibility and can be considered for cell replacement therapy. The purpose of this study is to investigate the role of chitosan scaffolds in the neural differentiation of DPSCs in vitro and to assess the supportive effects of chitosan scaffolds in an animal model of SCI. DPSCs were incubated with chitosan scaffolds. Cell viability and the secretion of neurotrophic factors were analyzed. DPSCs incubated with chitosan scaffolds were treated with neural differentiation medium for 14 days and then neural genes and protein markers were analyzed by Western blot and reverse transcription plus the polymerase chain reaction. Our study revealed a higher cell viability and neural differentiation in the DPSC/chitosan-scaffold group. Compared with the control group, the levels of BDNF, GDNF, b-NGF, and NT-3 were significantly increased in the DPSC/chitosan-scaffold group. The Wnt/ß-catenin signaling pathway played a key role in the neural differentiation of DPSCs combined with chitosan scaffolds. Transplantation of DPSCs together with chitosan scaffolds into an SCI rat model resulted in the marked recovery of hind limb locomotor functions. Thus, chitosan scaffolds were non-cytotoxic and provided a conducive and favorable microenvironment for the survival and neural differentiation of DPSCs. Transplantation of DPSCs might therefore be a suitable candidate for treating SCI and other neuronal degenerative diseases.

Connexin43-containing gap junctions potentiate extracellular Ca²âº-induced odontoblastic differentiation of human dental pulp stem cells via Erk1/2.

Extracellular Ca(2+) can promote dentin sialophosphoprotein (DSPP) expression and odontoblastic differentiation of dental pulp stem cells (DPSCs). Gap junctions mediated by connexin43 (Cx43) allow diffusion of small molecules (such as Ca(2+)) among cells to regulate cell-to-cell communications. However, it is unclear whether Cx43 is required for the Ca(2+)-induced cell differentiation. Here, we found that the influx of extracellular Ca(2+) through L-type Ca(2+) channels increases intracellular free Ca(2+) levels to promote DSPP expression. Cx43 overexpression potentiated the extracellular Ca(2+)-induced DSPP expression via Erk1/2. Flow cytometry analyses showed that Cx43 increased the percentage of p-Erk1/2 positive cells in response to Ca(2+), indicating that Cx43 in DPSCs possibly acts as a traditional gap junction channel, which permits the sharing of signals among coupled cells to make more DPSCs respond to Ca(2+). Furthermore, inhibition of Cx43 function and gap junction communication decreased Ca(2+)-induced the expression of DSPP, suggesting that cell-to-cell contacts are required for Cx43 to promote the Ca(2+)-induced cell differentiation. Similarly, the study performed on DPSCs cultured at low-density and high-density revealed that intercellular contacts are required to potentiate Erk1/2 activity and DSPP expression. In total, this study indicates that Cx43 increases Ca(2+)-induced DSPP expression and odontoblastic differentiation of DPSCs via Erk1/2 through gap junction-mediated cell-to-cell contacts.

AGS3 is involved in TNF-α medicated osteogenic differentiation of human dental pulp stem cells.

Dental pulp stem cells (DPSCs) are multipotent adult stem cells capable of differentiating along the osteoblast, adipocyte, and chondrocyte lineages. Regulating differentiation of DPSCs may be a useful tool for regenerative medicine and cell-based therapy in oral diseases. Multisignaling pathways are involved in osteogenic differentiation of DPSCs. Recent studies show that cAMP/PKA/CREB signaling could stimulate the expression of genes such as bone morphogenic proteins 2 (BMP2), inhibitor of DNA binding 2 (ID2), bone sialoprotein, osteocalcin, and type XXIV collagen, which have been implicated in osteogenesis and bone formation. Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, plays an important role in regulating the phosphorylation of cyclic AMP response element-binding protein (p-CREB). However, the involvement of AGS3 in osteogenic differentiation of DPSCs has not been explored. Our data indicated that increased expression of AGS3 would inhibit osteogenic differentiation of DPSCs exposed to inflammatory cytokine tumor necrosis factor α (TNF-α) via cAMP/PKA/CREB signaling. The negative role of AGS3 in osteogenic differentiation was further confirmed by knocking down and over expression of AGS3. Our findings may provide clinical implications for osteoporosis.

Transplantation of islet-like cell clusters derived from human dental pulp stem cells restores normoglycemia in diabetic mice.

BACKGROUND AIMS: The success of islet transplantation for diabetes depends on the availability of an adequate number of allogeneic or autologous islets. Postnatal stem cells are now considered for the generation of physiologically competent, insulin-producing cells. Our group showed earlier that it is possible to generate functional islets from human dental pulp stem cells by using a serum-free cocktail in a three-step protocol. METHODS: We compared the yield of generated islet-like cell clusters (ICCs) from stem cells from pulps of human exfoliated deciduous teeth (SHED) and dental pulp stem cells from permanent teeth (DPSCs). ICCs derived from SHED were packed in immuno-isolatory biocompatible macro-capsules and transplanted into streptozotocin (STZ)-induced diabetic mice. Non-diabetic and diabetic controls were transplanted with macro-capsules with or without islets. RESULTS: SHED were superior to DPSCs. STZ diabetic mice alone and mice transplanted with empty macro-capsules exhibited hyperglycemia throughout the experiment, whereas mice transplanted with macro-capsules containing ICCs were restored to normoglycemia within 3-4 weeks, which persisted for >60 days. CONCLUSIONS: Our results demonstrate for the first time that ICCs derived from SHED reverse STZ diabetes in mice without immunosuppression and offer an autologous and non-controversial source of human tissue that could be used for stem cell therapy in diabetes.

TNF-α triggers osteogenic differentiation of human dental pulp stem cells via the NF-κB signalling pathway.

Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cells (MSCs) characterised by self-renewal and multi-lineage differentiation, including chondrocytes, adipocytes, neural cells and osteoblasts, which make it an attractive choice for tissue engineering purposes. Tumour necrosis factor α (TNF-α) had the positive effect on the mineralisation of bone marrow MSCs and stromal cells derived from human adipose tissue. However, the effect of TNF-α on DPSCs is unclear. We found that TNF-α activated the NF-κB pathway during the osteogenic differentiation of DPSCs. TNF-α also increased mineralisation and the expression of bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and collagen type I (COL I) during this process. PDTC, an NF-κB inhibitor, blocked the osteogenic differentiation induced by TNF-α. No effect of TNF-α on proliferation of DPSCs or cell cycle was detected. In summary, TNF-α promotes mineralisation and mineralisation-related gene expression through the NF-κB signalling pathway in DPSCs, which may provide a foundation for autologous transplantation of DPSCs.

Overexpression of adrenomedullin (ADM) alleviates the senescence of human dental pulp stem cells by regulating the miR-152/CCNA2 pathway.

The limitation of human dental pulp stem cells (DPSCs), which have potential application value in regenerative medicine, is that they are prone to age in vitro. Studies have shown adrenomedullin (ADM) is believed to promote the proliferation of human DPSCs, but whether it can also affect aging remains to be investigated. A lentivirus vector was used to construct human DPSCs overexpressing ADM. Senescence tests were carried out on cells of the 7th and 15th passage. Transcriptome analysis was conducted to analyze microRNA expression regulation changes after human DPSCs overexpressed ADM. H2O2 induced the aging model of human DPSCs, and we examined the mechanism of recovery of aging through transfection experiments with miR-152 mimic, pCDH-CCNA2, and CCNA2 siRNA. Overexpression of ADM significantly upregulated the G2/M phase ratio of human DPSCs in natural passage culture (P = 0.001) and inhibited the expression of p53 (P = 0.014), P21 WAF1 (P = 0.015), and P16 INK4A (P = 0.001). Decreased ROS accumulation was observed in human DPSCs during long-term natural passage (P = 0.022). Transcriptome analysis showed that miR-152 was significantly upregulated during human DPSC senescence (P = 0.001) and could induce cell senescence by directly targeting CCNA2. Transfection with miR-152 mimic significantly reversed the inhibitory effect of ADM overexpression on p53 (P = 0.006), P21 WAF1 (P = 0.012), and P16 INK4A (P = 0.01) proteins in human DPSCs (H2O2-induced). In contrast, pCDH-CCNA2 weakened the effect of the miR-152 mimic, thus promoting cell proliferation and antiaging. ADM-overexpressing human DPSCs promote cell cycle progression and resist cellular senescence through CCNA2 expression promotion by inhibiting miR-152.