Stem cells from human exfoliated deciduous teeth attenuate mechanical allodynia in mice through distinct from the siglec-9/MCP-1-mediated tissue-repairing mechanism.

The effects of stem cells from human exfoliated deciduous teeth (SHED) on mechanical allodynia were examined in mice. A single intravenous injection of SHED and conditioned medium from SHED (SHED-CM) through the left external jugular vein significantly reversed the established mechanical allodynia induced by spinal nerve transection at 6 days after injection. SHED or SHED-CM significantly decreased the mean numbers of activating transcription factor 3-positive neurons and macrophages in the ipsilateral side of the dorsal root ganglion (DRG) at 20 days after spinal nerve transection. SHED or SHED-CM also suppressed activation of microglia and astrocytes in the ipsilateral side of the dorsal spinal cord. A single intravenous injection of secreted ectodomain of sialic acid-binding Ig-like lectin-9 and monocyte chemoattractant protein-1 had no effect on the established mechanical allodynia, whereas a single intravenous injection of protein component(s) contained in SHED-CM with molecular weight of between 30 and 50 kDa reversed the pain. Therefore, it may be concluded that protein component(s) with molecular mass of 30-50 kDa secreted by SHED could protect and/or repair DRG neurons damaged by nerve transection, thereby ameliorating mechanical allodynia.

Positive effect of exogenous brain-derived neurotrophic factor on impaired neurite development and mitochondrial function in dopaminergic neurons derived from dental pulp stem cells from children with attention deficit hyperactivity disorder.

Attention deficit hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders and is characterized by impaired attention, hyperactivity, and impulsivity. While multiple etiologies are implicated in ADHD, its underlying mechanism(s) remain unclear. Although previous studies have suggested dysregulation of dopaminergic signals, mitochondria, and brain-derived neurotrophic factor (BDNF) in ADHD, few studies have reported these associations directly. Stem cells from human exfoliated deciduous teeth (SHED) can efficiently differentiate into dopaminergic neurons (DNs) and are thus a useful disease-specific cellular model for the study of neurodevelopmental disorders associated with DN dysfunction. This study aimed to elucidate the relationships between DNs, mitochondria, and BDNF in ADHD by analyzing DNs differentiated from SHED obtained from three boys with ADHD and comparing them to those from three typically developing boys. In the absence of exogenous BDNF in the cell culture media, DNs derived from boys with ADHD (ADHD-DNs) exhibited impaired neurite outgrowth and branching, decreased mitochondrial mass in neurites, and abnormal intracellular ATP levels. In addition, BDNF mRNA was significantly decreased in ADHD-DNs. Supplementation with BDNF, however, significantly improved neurite development and mitochondrial function in ADHD-DNs. These results suggest that ADHD-DNs may have impaired neurite development and mitochondrial function associated with insufficient production of BDNF, which may be improved by exogenous BDNF supplementation. Findings such as these, from patient-derived SHED, may contribute to the future development of treatment strategies for aberrant dopaminergic signaling, mitochondrial functioning, and BDNF levels implicated in ADHD pathogenesis.

Acetylsalicylic Acid Treatment and Suppressive Regulation of AKT Accelerate Odontogenic Differentiation of Stem Cells from the Apical Papilla.

INTRODUCTION: Stem cells isolated from the root apical papilla of human teeth (stem cells from the apical papilla [SCAPs]) are capable of forming tooth root dentin and are a feasible source for bioengineered tooth root regeneration. In this study, we examined the effect of acetylsalicylic acid (ASA) on odontogenic differentiation of SCAPs in vitro and in vivo. METHODS: SCAPs were cultured under odontogenic conditions supplemented with or without ASA. ASA-treated SCAPs were also subcutaneously transplanted into immunocompromised mice. RESULTS: ASA accelerates in vitro and in vivo odontogenic differentiation of SCAPs associated with down-regulation of runt-related nuclear factor 2 and up-regulation of specificity protein 7, nuclear factor I C, and dentin phosphoprotein. ASA up-regulated the phosphorylation of AKT in the odontogenic SCAPs. Of interest, pretreatments with phosphoinositide 3-kinase inhibitor LY294402 and small interfering RNA for AKT promoted ASA-induced in vitro and in vivo odontogenic differentiation of SCAPs. LY294402 and small interfering RNA for AKT also suppressed the ASA-induced expression of runt-related nuclear factor 2 and enhanced ASA-induced expression of specificity protein 7, nuclear factor I C, and dentin phosphoprotein in SCAPs. CONCLUSIONS: These findings suggest that a combination of ASA treatment and suppressive regulation of the phosphoinositide 3-kinase-AKT signaling pathway is a novel approach for SCAP-based tooth root regeneration.

Regenerative medicine using stem cells from human exfoliated deciduous teeth (SHED): a promising new treatment in pediatric surgery.

Stem cells from human exfoliated deciduous teeth (SHEDs), being a type of mesenchymal stem cell, are an ideal cell source for regenerative medicine. They have minimal risk of oncogenesis, high proliferative capacity, high multipotency, and immunosuppressive ability. Stem cell transplantation using SHED has been found to have an anti-fibrotic effect on liver fibrosis in mice. SHED transplantation and the bio 3D printer, which can create scaffold-free 3-D images of the liver and diaphragm, provide a new innovative treatment modality for intractable pediatric surgical diseases such as biliary atresia and diaphragmatic hernia.

Therapeutic potential of hepatocyte-like-cells converted from stem cells from human exfoliated deciduous teeth in fulminant Wilson's disease.

Wilson's disease (WD) is an inherited metabolic disease arising from ATPase copper transporting beta gene (ATP7B) mutation. Orthotoropic liver transplantation is the only radical treatment of fulminant WD, although appropriate donors are lacking at the onset of emergency. Given the hepatogenic capacity and tissue-integration/reconstruction ability in the liver of stem cells from human exfoliated deciduous teeth (SHED), SHED have been proposed as a source for curing liver diseases. We hypothesized the therapeutic potential of SHED and SHED-converted hepatocyte-like- cells (SHED-Heps) for fulminant WD. SHED and SHED-Heps were transplanted into WD model Atp7b-mutated Long-Evans Cinnamon (LEC) rats received copper overloading to induce a lethal fulminant liver failure. Due to the superior copper tolerance via ATP7B, SHED-Hep transplantation gave more prolonged life-span of fulminant LEC rats than SHED transplantation. The integrated ATP7B-expressing SHED-Heps showed more therapeutic effects on to restoring the hepatic dysfunction and tissue damages in the recipient liver than the integrated naïve SHED without ATP7B expression. Moreover, SHED-Heps could reduce copper-induced oxidative stress via ATP7B- independent stanniocalcin 1 secretion in the fulminant LEC rats, suggesting a possible role for paracrine effect of the integrated SHED-Heps. Taken together, SHED-Heps offer a potential of functional restoring, bridging, and preventive approaches for treating fulminant WD.

Folic acid-mediated mitochondrial activation for protection against oxidative stress in human dental pulp stem cells derived from deciduous teeth.

Enzymatic antioxidant systems, mainly involving mitochondria, are critical for minimizing the harmful effects of reactive oxygen species, and these systems are enhanced by interactions with nonenzymatic antioxidant nutrients. Because fetal growth requires extensive mitochondrial respiration, pregnant women and fetuses are at high risk of exposure to excessive reactive oxygen species. The enhancement of the antioxidant system, e.g., by nutritional management, is therefore critical for both the mother and fetus. Folic acid supplementation prevents homocysteine accumulation and epigenetic dysregulation associated with one-carbon metabolism. However, few studies have examined the antioxidant effects of folic acid for healthy pregnancy outcomes. The purpose of this study was to elucidate the association between the antioxidant effect of folic acid and mitochondria in undifferentiated cells during fetal growth. Neural crest-derived dental pulp stem cells of human exfoliated deciduous teeth were used as a model of undifferentiated cells in the fetus. Pyocyanin induced excessive reactive oxygen species, resulting in a decrease in cell growth and migration accompanied by mitochondrial fragmentation and inactivation in dental pulp stem cells. This damage was significantly improved by folic acid, along with decreased mitochondrial reactive oxygen species, PGC-1α upregulation, DRP1 downregulation, mitochondrial elongation, and increased ATP production. Folic acid may protect undifferentiated cells from oxidative damage by targeting mitochondrial activation. These results provide evidence for a new benefit of folic acid in pregnant women and fetuses.

Suppression of AKT-mTOR signal pathway enhances osteogenic/dentinogenic capacity of stem cells from apical papilla.

BACKGROUND: Stem cells from apical papilla (SCAP) are a subpopulation of mesenchymal stem cells (MSCs) isolated from the apical papilla of the developing tooth root apex of human teeth. Because of their osteogenic/dentinogenic capacity, SCAP are considered as a source for bone and dentin regeneration. However, little is understood about the molecular mechanism of osteogenic/dentinogenic differentiation of SCAP. Phosphoinositide 3 kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) signal pathway participates in regulating the differentiation of various cell types, such as MSCs. In this study, we examined the role of the PI3K-AKT-mTOR signal pathway in the osteogenic/dentinogenic differentiation of SCAP. Moreover, we challenge to fabricate scaffold-free SCAP-based spheroidal calcified constructs. METHODS: SCAP were pretreated with or without small interfering RNA for AKT (AKT siRNA), PI3K inhibitor LY294402, and mTOR inhibitor rapamycin and were cultured under osteogenic/dentinogenic differentiation to examine in vitro and in vivo calcified tissue formation. Moreover, SCAP-based cell aggregates were pretreated with or without LY294402 and rapamycin. The cell aggregates were cultured under osteogenic/dentinogenic condition and were analyzed the calcification of the aggregates. RESULTS: Pretreatment with AKT siRNA, LY294402, and rapamycin enhances the in vitro and in vivo calcified tissue-forming capacity of SCAP. SCAP were fabricated as scaffold-free spheroids and were induced into forming calcified 3D constructs. The calcified density of the spheroidal constructs was enhanced when the spheroids were pretreated with LY294402 and rapamycin. CONCLUSIONS: Our findings indicate that the suppression of PI3K-AKT-mTOR signal pathway plays a role in not only enhancing the in vivo and in vitro osteogenic/dentinogenic differentiation of SCAP, but also promoting the calcification of scaffold-free SCAP-based calcified constructs. These findings suggest that a suppressive regulation of PI3K-AKT-mTOR signal pathway is a novel approach for SCAP-based bone and dentin regeneration.

Neurodevelopmental Outcomes in Infants With Birth Weight ≤500 g at 3 Years of Age.

OBJECTIVES: To determine neurodevelopmental outcomes at 3 years of age in children born with a birth weight (BW) of ≤500 g. METHODS: Infants who were born with a BW of ≤500 g from 2003 to 2012 in the Neonatal Research Network of Japan and survived to discharge from the NICU were eligible in this study. The study population consisted of 460 children (56.7% of 811 surviving infants) who were evaluated at 36 to 42 months of age. Neurodevelopmental impairment (NDI) was defined as having cerebral palsy, visual impairment, hearing impairment, or a developmental quotient score of <70. RESULTS: The overall proportion of NDI was 59.1% (95% confidence interval [CI]: 54.6%-63.5%). The trend revealed no significant change during the study period. In a multivariate modified Poisson regression analysis, NDI was associated with severe intraventricular hemorrhage (adjusted risk ratio [RR]: 1.42; 95% CI: 1.19-1.68; P < .01), cystic periventricular leukomalacia (adjusted RR: 1.40; 95% CI: 1.13-1.73; P < .01), severe necrotizing enterocolitis (adjusted RR: 1.31; 95% CI: 1.07-1.60; P < .01), surgical ligation for patent ductus arteriosus (adjusted RR: 1.29; 95% CI: 1.09-1.54; P < .01), and male sex (adjusted RR: 1.19; 95% CI: 1.01-2.40; P = .04). CONCLUSIONS: This cohort showed that neurodevelopmental outcomes of infants with a BW of ≤500 g have not improved from 2003 to 2012. Multivariate analysis revealed that severe intracranial hemorrhage and cystic periventricular leukomalacia were the strongest risk factors for NDIs. Our data suggested that measures aimed at reducing neurologic morbidities will be important for improving outcomes of infants with a BW of ≤500 g.

Pamidronate decreases bilirubin-impaired cell death and improves dentinogenic dysfunction of stem cells from human deciduous teeth.

BACKGROUND: Hyperbilirubinemia that occurs in pediatric liver diseases such as biliary atresia can result in the development of not only jaundice in the brain, eyes, and skin, but also tooth abnormalities including green pigmentation and dentin hypoplasia in the developing teeth. However, hyperbilirubinemia-induced tooth impairments remain after liver transplantation. No effective dental management to prevent hyperbilirubinemia-induced tooth impairments has been established. METHODS: In this study, we focused on pamidronate, which is used to treat pediatric osteopenia, and investigated its effects on hyperbilirubinemia-induced tooth impairments. We cultured stem cells from human exfoliated deciduous teeth (SHED) under high and low concentrations of unconjugated bilirubin in the presence or absence of pamidronate. We then analyzed the effects of pamidronate on the cell death, associated signal pathways, and dentinogenic function in SHED. RESULTS: We demonstrated that a high concentration of unconjugated bilirubin induced cell death in SHED via the mitochondrial pathway, and this was associated with the suppression of AKT and extracellular signal-related kinase 1 and 2 (ERK1/2) signal pathways and activation of the nuclear factor kappa B (NF-κB) signal pathway. The high concentration of unconjugated bilirubin impaired the in vitro and in vivo dentinogenic capacity of SHED, but not the low concentration. We then demonstrated that pamidronate decreased the bilirubin-induced cell death in SHED via the altered AKT, ERK1/2, and NF-κB signal pathways and recovered the bilirubin-impaired dentinogenic function of SHED. CONCLUSIONS: Our findings suggest that pamidronate may prevent tooth abnormalities in pediatric patients with hyperbilirubinemia.

Impaired neurite development associated with mitochondrial dysfunction in dopaminergic neurons differentiated from exfoliated deciduous tooth-derived pulp stem cells of children with autism spectrum disorder.

Autism spectrum disorder (ASD) is a highly heterogeneous neurodevelopmental disorder characterized by impaired social interactions, restrictive interests, and repetitive stereotypic behaviors. Among the various mechanisms underlying the pathogenesis of ASD, dysfunctions of dopaminergic signaling and mitochondria have been hypothesized to explain the core symptoms of children with ASD. However, only a few studies focusing on the pathological association between dopaminergic neurons (DN) and mitochondria in ASD have been performed using patient-derived stem cells and in vitro differentiated neurons. Stem cells from human exfoliated deciduous teeth (SHED) are neural crest-derived mesenchymal stem cells present in the dental pulp of exfoliated deciduous teeth; these cells can differentiate into dopaminergic neurons (DN) in vitro. This study aimed to investigate the pathological association between development of DN and mitochondria in ASD by using SHED as a disease- or patient-specific cellular model. The SHED obtained from three children with ASD and three typically developing children were differentiated into DN, and the neurobiology of these cells was examined. The DN derived from children with ASD showed impaired neurite outgrowth and branching, associated with decreased mitochondrial membrane potential, ATP production, number of mitochondria within the neurites, amount of mitochondria per cell area and intracellular calcium level. In addition, impaired neurite outgrowth and branching of ASD-derived DN were not improved by brain-derived neurotrophic factor (BDNF), suggesting impairment of the BDNF signaling pathway in ASD. These results imply that intracerebral dopamine production may have decreased in these children. The earliest age at which deciduous teeth spontaneously exfoliate in humans, and SHED can be noninvasively collected, is approximately 6 years. Our results suggest that in vitro analysis of SHED-derived DN obtained from children with ASD provides neurobiological information that may be useful in determining treatment strategies in the early stages of ASD.

Altered development of dopaminergic neurons differentiated from stem cells from human exfoliated deciduous teeth of a patient with Down syndrome.

BACKGROUND: Down syndrome (DS) is a common developmental disorder resulting from the presence of an additional copy of chromosome 21. Abnormalities in dopamine signaling are suggested to be involved in cognitive dysfunction, one of the symptoms of DS, but the pathophysiological mechanism has not been fully elucidated at the cellular level. Stem cells from human exfoliated deciduous teeth (SHED) can be prepared from the dental pulp of primary teeth. Importantly, SHED can be collected noninvasively, have multipotency, and differentiate into dopaminergic neurons (DN). Therefore, we examined dopamine signaling in DS at the cellular level by isolating SHED from a patient with DS, differentiating the cells into DN, and examining development and function of DN. METHODS: Here, SHED were prepared from a normal participant (Ctrl-SHED) and a patient with DS (DS-SHED). Initial experiments were performed to confirm the morphological, chromosomal, and stem cell characteristics of both SHED populations. Next, Ctrl-SHED and DS-SHED were differentiated into DN and morphological analysis of DN was examined by immunostaining. Functional analysis of DN was performed by measuring extracellular dopamine levels under basal and glutamate-stimulated conditions. In addition, expression of molecules involved in dopamine homeostasis was examined by quantitative real-time polymerase chain reaction and immunostaining. Statistical analysis was performed using two-tailed Student's t-tests. RESULTS: Compared with Ctrl-SHED, DS-SHED showed decreased expression of nestin, a neural stem-cell marker. Further, DS-SHED differentiated into DN (DS-DN) exhibiting decreased neurite outgrowth and branching compared with Ctrl-DN. In addition, DS-DN dopamine secretion was lower than Ctrl-DN dopamine secretion. Moreover, aberrant expression of molecules involved in dopaminergic homeostasis was observed in DS-DN. CONCLUSIONS: Our results suggest that there was developmental abnormality and DN malfunction in the DS-SHED donor in this study. In the future, to clarify the detailed mechanism of dopamine-signal abnormality due to DN developmental and functional abnormalities in DS, it is necessary to increase the number of patients for analysis. Non-invasively harvested SHED may be very useful in the analysis of DS pathology.

Mitochondrial dysfunction in dopaminergic neurons differentiated from exfoliated deciduous tooth-derived pulp stem cells of a child with Rett syndrome.

Rett syndrome is an X-linked neurodevelopmental disorder associated with psychomotor impairments, autonomic dysfunctions and autism. Patients with Rett syndrome have loss-of-function mutations in MECP2, the gene encoding methyl-CpG-binding protein 2 (MeCP2). Abnormal biogenic amine signaling and mitochondrial function have been found in patients with Rett syndrome; however, few studies have analyzed the association between these factors. This study investigated the functional relationships between mitochondria and the neuronal differentiation of the MeCP2-deficient stem cells from the exfoliated deciduous teeth of a child with Rett syndrome. An enrolled subject in this study was a 5-year-old girl carrying a large deletion that included the methyl-CpG-binding domain, transcriptional repression domain, and nuclear localization signal of MECP2. Using the single-cell isolation technique, we found that the two populations of MeCP2-expressing and MeCP2-deficient stem cells kept their MECP2 expression profiles throughout the stages of cell proliferation and neuronal differentiation in vitro. Neurite outgrowth and branching were attenuated in MeCP2-deficient dopaminergic neurons. MeCP2-deficient cells showed reduced mitochondrial membrane potential, ATP production, restricted mitochondrial distribution in neurites, and lower expression of a central mitochondrial fission factor, dynamin-related protein 1 than MeCP2-expressing cells. These data indicated that MeCP2-deficiency dysregulates the expression of mitochondrial factors required for the maturation of dopaminergic neurons. This study also provides insight into the pathogenic mechanism underlying dysfunction of the intracerebral dopaminergic signaling pathway in Rett syndrome.

Accelerated dentinogenesis by inhibiting the mitochondrial fission factor, dynamin related protein 1.

Undifferentiated odontogenic epithelium and dental papilla cells differentiate into ameloblasts and odontoblasts, respectively, both of which are essential for tooth development. These differentiation processes involve dramatic functional and morphological changes of the cells. For these changes to occur, activation of mitochondrial functions, including ATP production, is extremely important. In addition, these changes are closely related to mitochondrial fission and fusion, known as mitochondrial dynamics. However, few studies have focused on the role of mitochondrial dynamics in tooth development. The purpose of this study was to clarify this role. We used mouse tooth germ organ cultures and a mouse dental papilla cell line with the ability to differentiate into odontoblasts, in combination with knockdown of the mitochondrial fission factor, dynamin related protein (DRP)1. In organ cultures of the mouse first molar, tooth germ developed to the early bell stage. The amount of dentin formed under DRP1 inhibition was significantly larger than that of the control. In experiments using a mouse dental papilla cell line, differentiation into odontoblasts was enhanced by inhibiting DRP1. This was associated with increased mitochondrial elongation and ATP production compared to the control. These results suggest that DRP1 inhibition accelerates dentin formation through mitochondrial elongation and activation. This raises the possibility that DRP1 might be a therapeutic target for developmental disorders of teeth.

Direct effects of mitochondrial dysfunction on poor bone health in Leigh syndrome.

Mitochondrial diseases are the result of aberrant mitochondrial function caused by mutations in either nuclear or mitochondrial DNA. Poor bone health has recently been suggested as a symptom of mitochondrial diseases; however, a direct link between decreased mitochondrial function and poor bone health in mitochondrial disease has not been demonstrated. In this study, stem cells from human exfoliated deciduous teeth (SHED) were isolated from a child with Leigh syndrome (LS), a mitochondrial disease, and the effects of decreased mitochondrial function on poor bone health were analyzed. Compared with control SHED, LS SHED displayed decreased osteoblastic differentiation and calcium mineralization. The intracellular and mitochondrial calcium levels were lower in LS SHED than in control SHED. Furthermore, the mitochondrial activity of LS SHED was decreased compared with control SHED both with and without osteoblastic differentiation. Our results indicate that decreased osteoblast differentiation potential and osteoblast function contribute to poor bone health in mitochondrial diseases.

Mitochondria Regulate the Differentiation of Stem Cells from Human Exfoliated Deciduous Teeth.

Stem cells from human exfoliated deciduous teeth (SHED) are isolated from the dental pulp tissue of primary teeth and can differentiate into neuronal cells. Although SHED are a desirable type of stem cells for transplantation therapy and for the study of neurological diseases, a large part of the neuronal differentiation machinery of SHED remains unclear. Recent studies have suggested that mitochondrial activity is involved in the differentiation of stem cells. In the present work, we investigated the neuronal differentiation machinery of SHED by focusing on mitochondrial activity. During neuronal differentiation of SHED, we observed increased mitochondrial membrane potential, increased mitochondrial DNA, and elongated mitochondria. Furthermore, to examine the demand for mitochondrial activity in neuronal differentiation, we then differentiated SHED into neuronal cells in the presence of rotenone, an inhibitor of mitochondrial respiratory chain complex I, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupler, and found that neuronal differentiation was inhibited by treatment with rotenone and CCCP. These results indicated that increased mitochondrial activity was crucial for the neuronal differentiation of SHED.Key words: mitochondria, differentiation, stem cells, dental pulp, exfoliated deciduous teeth.

Early Mortality and Morbidity in Infants with Birth Weight of 500 Grams or Less in Japan.

OBJECTIVE: To assess the short-term prognosis of Japanese infants with a birth weight (BW) of ≤500 g. STUDY DESIGN: Demographic and clinical data were reviewed for 1473 live born infants with a BW ≤500 g at gestational age ≥22 weeks who were treated in the 204 affiliated hospitals of the Neonatal Research Network of Japan between 2003 and 2012. RESULTS: Survival to hospital discharge occurred in 811 of 1473 infants (55%; 95% CI 53%-58%). The survival rates of BW ≤300 g, 301-400 g, and 401-500 g were 18% (95% CI 10%-31%), 41% (95% CI 36%-47%), and 60% (95% CI 57%-63%), respectively. In a multivariable Cox proportional hazards analysis, antenatal corticosteroid use (adjusted hazard ratio: 0.68; 95% CI 0.58-0.81; P < .01), cesarean delivery (0.69; 95% CI 0.56-0.85; P < .01), advanced gestational age per week (0.94; 95% CI 0.89-0.99; P = .02), BW per 100-g increase (0.55; 95% CI 0.49-0.64; P < .01), Apgar score ≥4 at 5 minutes (0.51; 95% CI 0.43-0.61; P < .01), and no major congenital abnormalities (0.38; 95% CI 0.29-0.51; P < .01) were associated with survival to discharge. Despite the improved survival rate over the 10-year study period (from 40% in 2003 [95% CI 30%-51%] to 68% in 2012 [95% CI 61%-75%]), at least 1 severe morbidity was present in 81%-89% of the survivors. CONCLUSIONS: Improvements in perinatal-neonatal medicine have improved the survival, but not the rate of major morbidities, of infants with a BW ≤500 g in Japan.

Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells.

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

Dihydroorotate dehydrogenase depletion hampers mitochondrial function and osteogenic differentiation in osteoblasts.

Mutation of the dihydroorotate dehydrogenase (DHODH) gene is responsible for Miller syndrome, which is characterized by craniofacial malformations with limb abnormalities. We previously demonstrated that DHODH was involved in forming a mitochondrial supercomplex and that mutated DHODH led to protein instability, loss of enzyme activity, and increased levels of reactive oxygen species in HeLa cells. To explore the etiology of Miller syndrome in more detail, we investigated the effects of DHODH inhibition in the cells involved in skeletal structure. Dihydroorotate dehydrogenase in MC3T3-E1 cells derived from mouse calvaria osteoblast precursor cells was knocked down by specific small interfering RNAs (siRNAs), and cell proliferation, ATP production, and expression of bone-related genes were investigated in these cells. After depletion of DHODH using specific siRNAs, inhibition of cell proliferation and cell cycle arrest occurred in MC3T3-E1 cells. In addition, ATP production was reduced in whole cells, especially in mitochondria. Furthermore, the levels of runt-related transcription factor 2 (Runx2) and osteocalcin (Ocn) mRNAs were lower in DHODH siRNA-treated cells compared with controls. These data suggest that depletion of DHODH affects the differentiation and maturation of osteoblasts. This study shows that mitochondrial dysfunction by DHODH depletion in osteoblasts can be directly linked to the abnormal bone formation in Miller syndrome.

Interferon-gamma improves impaired dentinogenic and immunosuppressive functions of irreversible pulpitis-derived human dental pulp stem cells.

Clinically, irreversible pulpitis is treated by the complete removal of pulp tissue followed by replacement with artificial materials. There is considered to be a high potential for autologous transplantation of human dental pulp stem cells (DPSCs) in endodontic treatment. The usefulness of DPSCs isolated from healthy teeth is limited. However, DPSCs isolated from diseased teeth with irreversible pulpitis (IP-DPSCs) are considered to be suitable for dentin/pulp regeneration. In this study, we examined the stem cell potency of IP-DPSCs. In comparison with healthy DPSCs, IP-DPSCs expressed lower colony-forming capacity, population-doubling rate, cell proliferation, multipotency, in vivo dentin regeneration, and immunosuppressive activity, suggesting that intact IP-DPSCs may be inadequate for dentin/pulp regeneration. Therefore, we attempted to improve the impaired in vivo dentin regeneration and in vitro immunosuppressive functions of IP-DPSCs to enable dentin/pulp regeneration. Interferon gamma (IFN-γ) treatment enhanced in vivo dentin regeneration and in vitro T cell suppression of IP-DPSCs, whereas treatment with tumor necrosis factor alpha did not. Therefore, these findings suggest that IFN-γ may be a feasible modulator to improve the functions of impaired IP-DPSCs, suggesting that autologous transplantation of IFN-γ-accelerated IP-DPSCs might be a promising new therapeutic strategy for dentin/pulp tissue engineering in future endodontic treatment.

Complete resolution of a calcifying cystic odontogenic tumor with physiological eruption of a dislocated permanent tooth after marsupialization in a child with a mixed dentition: a case report.

Here, we report the complete resolution of a calcifying cystic odontogenic tumor (CCOT) in the right mandible after marsupialization in an 8-year-old girl with a mixed dentition. Clinical, radiographic, and histopathological findings showed a simple cystic variant of CCOT in the region of the deciduous second molar, with dislocation of the permanent second premolar tooth germ. Initial treatment involved marsupialization, including extraction of the involved deciduous tooth, incision of pathological tissue, and creation of a window in the extraction socket. The crown of the dislocated second premolar was exposed at the base of the cystic cavity after marsupialization. One year and nine months later, complete bone healing and spontaneous eruption of the second premolar were observed, providing evidence of the bone regeneration capacity and tooth germ eruption potential in children. No recurrence was observed after 7 years. The findings from this case suggest that marsupialization can be successfully applied for the treatment of CCOT in children with a mixed dentition.