Osteoblastic differentiation improved by bezafibrate-induced mitochondrial biogenesis in deciduous tooth-derived pulp stem cells from a child with Leigh syndrome

Leigh syndrome is a highly heterogeneous condition caused by pathological mutations in either nuclear or mitochondrial DNA regions encoding molecules involved in mitochondrial oxidative phosphorylation, in which many organs including the brain can be affected. Among these organs, a high incidence of poor bone health has been recognized in primary mitochondrial diseases including Leigh syndrome. However, the direct association between mitochondrial dysfunction and poor bone health has not been fully elucidated. Mitochondrial biosynthesis is a potential therapeutic target for this syndrome, as it can ameliorate the impairment of oxidative phosphorylation without altering these gene mutations. A recent study has shown the impaired osteogenesis in the dental pulp stem cells derived from the deciduous teeth of a child with Leigh syndrome, harboring the heteroplasmic mutation G13513A in the mitochondrial DNA region encoding the ND5 subunit of the respiratory chain complex I. The present study aimed to investigate whether mitochondrial biogenesis could be a therapeutic target for improving osteogenesis, using the same stem cells in a patient-specific cellular model. For this purpose, bezafibrate was used because it has been reported to induce mitochondrial biogenesis as well as to improve bone metabolism and osteoporosis. Bezafibrate clearly improved the differentiation of patient-derived stem cells into osteoblasts and the mineralization of differentiated osteoblasts. The mRNA expression of peroxisome proliferator-activated receptor-gamma coactivator-1α, ATP production, and mitochondrial Ca2+ levels were all significantly increased by bezafibrate in the patient-derived cells. In addition, the increased amount and morphological shift from the fragmentary to network shape associated with DRP1 downregulation were also observed in the bezafibrate-treated patient-derived cells. These results suggest that mitochondrial biogenesis may be a potential therapeutic target for improving osteogenesis in patients with Leigh syndrome, and bezafibrate may be one of the candidate treatment agents. Keywords: Bezafibrate, Dental pulp stem cell, Leigh syndrome, Mitochondrial biogenesis, Osteogenesi

Dental Pulp-Derived Mesenchymal Stem Cells for Modeling Genetic Disorders

A subpopulation of mesenchymal stem cells, developmentally derived from multipotent neural crest cells that form multiple facial tissues, resides within the dental pulp of human teeth. These stem cells show high proliferative capacity in vitro and are multipotent, including adipogenic, myogenic, osteogenic, chondrogenic, and neurogenic potential. Teeth containing viable cells are harvested via minimally invasive procedures, based on various clinical diagnoses, but then usually discarded as medical waste, indicating the relatively low ethical considerations to reuse these cells for medical applications. Previous studies have demonstrated that stem cells derived from healthy subjects are an excellent source for cell-based medicine, tissue regeneration, and bioengineering. Furthermore, stem cells donated by patients affected by genetic disorders can serve as in vitro models of disease-specific genetic variants, indicating additional applications of these stem cells with high plasticity. This review discusses the benefits, limitations, and perspectives of patient-derived dental pulp stem cells as alternatives that may complement other excellent, yet incomplete stem cell models, such as induced pluripotent stem cells, together with our recent data

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

Mitochondrial Calcium-Triggered Oxidative Stress and Developmental Defects in Dopaminergic Neurons Differentiated from Deciduous Teeth-Derived Dental Pulp Stem Cells with MFF Insufficiency

Mitochondrial fission factor (MFF) is an adapter that targets dynamin-related protein 1 from the cytosol to the mitochondria for fission. Loss-of-function MFF mutations cause encephalopathy due to defective mitochondrial and peroxisomal fission 2 (EMPF2). To elucidate the molecular mechanisms that were involved, we analyzed the functional effects of MFF depletion in deciduous teeth-derived dental pulp stem cells differentiating into dopaminergic neurons (DNs). When treated with MFF-targeting small interfering RNA, DNs showed impaired neurite outgrowth and reduced mitochondrial signals in neurites harboring elongated mitochondria. MFF silencing also caused mitochondrial Ca2+ accumulation through accelerated Ca2+ influx from the endoplasmic reticulum (ER) via the inositol 1,4,5-trisphosphate receptor. Mitochondrial Ca2+ overload led DNs to produce excessive reactive oxygen species (ROS), and downregulated peroxisome proliferator-activated receptor-gamma co-activator-1 alpha (PGC-1α). MFF was co-immunoprecipitated with voltage-dependent anion channel 1, an essential component of the ER-mitochondrial Ca2+ transport system. Folic acid supplementation normalized ROS levels, PGC-1α mediated mitochondrial biogenesis, and neurite outgrowth in MFF depleted DNs, without affecting their mitochondrial morphology or Ca2+ levels. We propose that MFF negatively regulates the mitochondrial Ca2+ influx from the ER. MFF-insufficiency recapitulated the EMPF2 neuropathology with increased oxidative stress and suppressed mitochondrial biogenesis. ROS and mitochondrial biogenesis might be potential therapeutic targets for EMPF2

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

Abstract 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

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. Keywords: Autism spectrum disorder, Dopaminergic neurons, Mitochondria, Stem cells from human exfoliated deciduous teet