Characterization of neural stem cells derived from human stem cells from the apical papilla undergoing three-dimensional neurosphere induction.

OBJECTIVES: The endogenous repairing based on the activation of neural stem cells (NSCs) is impaired by neurodegenerative diseases. The present study aims to characterize human stem cells from the apical papilla (hSCAPs) with features of mesenchymal stem cells (MSCs) and to demonstrate the neuronal differentiation of hSCAPs into NSCs through the formation of three-dimensional (3D) neurospheres, verifying the structural, immunophenotyping, self-renewal, gene expression and neuronal activities of these cells to help further improve NSCs transplantation. METHODOLOGY: The hSCAPs were isolated from healthy impacted human third molar teeth and characterized as MSCs. They were then induced into 3D-neurospheres using a specific neural induction medium. Subsequently, the intra-neurospheral cells were confirmed to be NSCs by the identification of Nissl substance and the analysis of immunofluorescence staining, self-renewal ability, and gene expression of the cells. Moreover, the neuronal activity was investigated using intracellular calcium oscillation. RESULTS: The isolated cells from the human apical papilla expressed many markers of MSCs, such as self-renewal ability and multilineage differentiation. These cells were thus characterized as MSCs, specifically as hSCAPs. The neurospheres induced from hSCAPs exhibited a 3D-floating spheroidal shape and larger neurospheres, and consisted of a heterogeneous population of intra-neurospheral cells. Further investigation showed that these intra-neurospheral cells had Nissl body staining and also expressed both Nestin and SOX2. They presented a self-renewal ability as well, which was observed after their disaggregation. Their gene expression profiling also exhibited a significant amount of NSC markers (NES, SOX1, and PAX6). Lastly, a large and dynamic change of the fluorescent signal that indicated calcium ions (Ca2+) was detected in the intracellular calcium oscillation, which indicated the neuronal activity of NSCs-derived hSCAPs. CONCLUSIONS: The hSCAPs exhibited properties of MSCs and could differentiate into NSCs under 3D-neurosphere generation. The present findings suggest that NSCs-derived hSCAPs may be used as an alternative candidates for cell-based therapy, which uses stem cell transplantation to further treat neurodegenerative diseases.

Characterization of neural stem cells derived from human stem cells from the apical papilla undergoing three-dimensional neurosphere induction

Abstract Objectives The endogenous repairing based on the activation of neural stem cells (NSCs) is impaired by neurodegenerative diseases. The present study aims to characterize human stem cells from the apical papilla (hSCAPs) with features of mesenchymal stem cells (MSCs) and to demonstrate the neuronal differentiation of hSCAPs into NSCs through the formation of three-dimensional (3D) neurospheres, verifying the structural, immunophenotyping, self-renewal, gene expression and neuronal activities of these cells to help further improve NSCs transplantation. Methodology The hSCAPs were isolated from healthy impacted human third molar teeth and characterized as MSCs. They were then induced into 3D-neurospheres using a specific neural induction medium. Subsequently, the intra-neurospheral cells were confirmed to be NSCs by the identification of Nissl substance and the analysis of immunofluorescence staining, self-renewal ability, and gene expression of the cells. Moreover, the neuronal activity was investigated using intracellular calcium oscillation. Results The isolated cells from the human apical papilla expressed many markers of MSCs, such as self-renewal ability and multilineage differentiation. These cells were thus characterized as MSCs, specifically as hSCAPs. The neurospheres induced from hSCAPs exhibited a 3D-floating spheroidal shape and larger neurospheres, and consisted of a heterogeneous population of intra-neurospheral cells. Further investigation showed that these intra-neurospheral cells had Nissl body staining and also expressed both Nestin and SOX2. They presented a self-renewal ability as well, which was observed after their disaggregation. Their gene expression profiling also exhibited a significant amount of NSC markers (NES, SOX1, and PAX6). Lastly, a large and dynamic change of the fluorescent signal that indicated calcium ions (Ca2+) was detected in the intracellular calcium oscillation, which indicated the neuronal activity of NSCs-derived hSCAPs. Conclusions The hSCAPs exhibited properties of MSCs and could differentiate into NSCs under 3D-neurosphere generation. The present findings suggest that NSCs-derived hSCAPs may be used as an alternative candidates for cell-based therapy, which uses stem cell transplantation to further treat neurodegenerative diseases.

Rho kinase inhibitor induced human dental pulp stem cells to differentiate into neurons.

AIMS: Neurological diseases due to neuron loss have become major public health problems. Current treatment reduces symptoms; however, there is no cure for neurological diseases. Therefore, stem cells may be an alternative therapy. Human dental pulp stem cells (hDPSCs) are an attractive source for cell-based approaches due to their high regenerative potential. The Rho kinase (ROCK) inhibitor Y-27632 promoted the neuronal differentiation of several stem cell types. However, its neuronal-inductive effect on hDPSCs has not been reported. Thus, the aim of our study was to investigate whether Y-27632 can induce the neuronal differentiation of hDPSCs. MAIN METHODS: hDPSCs were isolated from human third molars using an enzymatic method and were subsequently characterized. Cytotoxicity was evaluated using an MTT assay. The optimal concentration to induce neural differentiation was assessed using 1-50 µM Y-27632 as evaluated by Cresyl violet and immunofluorescence staining of neurofilaments and ßIII-tubulin, respectively. Ten µM Y-27632 was used for neuronal induction for 72 h, and differentiation was confirmed based on the expression of neurogenic markers (MAP2, Brn3a, and ChAT) and intracellular calcium activity. KEY FINDINGS: Our findings indicate that Y-27632 was not cytotoxic to hDPSCs and 10 µM Y-27632 was the lowest concentration that induced the morphological changes of hDPSCs into neuronal cells with Cresyl violet-positive staining and significantly enhanced the fluorescence intensity of neurofilament and ßIII-tubulin. The neuronal genes' expression and intracellular calcium activity were upregulated after induction with Y-27632. SIGNIFICANCE: At the optimal concentration and time, Rho kinase inhibitor induces hDPSC differentiation into neuronal cells.

Optimal culture conditions for neurosphere formation and neuronal differentiation from human dental pulp stem cells.

OBJECTIVES: Human dental pulp stem cells (DPSCs) have been used to regenerate damaged nervous tissues. However, the methods of committing DPSCs into neural stem/progenitor cells (NSPCs) or neurospheres are highly diverse, resulting in many neuronal differentiation outcomes. This study aims to validate an optimal protocol for inducing DPSCs into neurospheres and neurons. METHODOLOGY: After isolation and characterization of mesenchymal stem cell identity, DPSCs were cultured in a NSPC induction medium and culture vessels. The durations of the culture, dissociation methods, and passage numbers of DPSCs were varied. RESULTS: Neurosphere formation requires a special surface that inhibits cell attachment. Five-days was the most appropriate duration for generating proliferative neurospheres and they strongly expressed Nestin, an NSPC marker. Neurosphere reformation after being dissociated by the Accutase enzyme was significantly higher than other methods. Passage number of DPSCs did not affect neurosphere formation, but did influence neuronal differentiation. We found that the cells expressing a neuronal marker, ß-tubulin III, and exhibiting neuronal morphology were significantly higher in the early passage of the DPSCs. CONCLUSION: These results suggest a guideline to obtain a high efficiency of neurospheres and neuronal differentiation from DPSCs for further study and neurodegeneration therapeutics.

Optimal culture conditions for neurosphere formation and neuronal differentiation from human dental pulp stem cells

Abstract Objectives Human dental pulp stem cells (DPSCs) have been used to regenerate damaged nervous tissues. However, the methods of committing DPSCs into neural stem/progenitor cells (NSPCs) or neurospheres are highly diverse, resulting in many neuronal differentiation outcomes. This study aims to validate an optimal protocol for inducing DPSCs into neurospheres and neurons. Methodology After isolation and characterization of mesenchymal stem cell identity, DPSCs were cultured in a NSPC induction medium and culture vessels. The durations of the culture, dissociation methods, and passage numbers of DPSCs were varied. Results Neurosphere formation requires a special surface that inhibits cell attachment. Five-days was the most appropriate duration for generating proliferative neurospheres and they strongly expressed Nestin, an NSPC marker. Neurosphere reformation after being dissociated by the Accutase enzyme was significantly higher than other methods. Passage number of DPSCs did not affect neurosphere formation, but did influence neuronal differentiation. We found that the cells expressing a neuronal marker, β-tubulin III, and exhibiting neuronal morphology were significantly higher in the early passage of the DPSCs. Conclusion These results suggest a guideline to obtain a high efficiency of neurospheres and neuronal differentiation from DPSCs for further study and neurodegeneration therapeutics.

Potential of resveratrol in enrichment of neural progenitor-like cell induction of human stem cells from apical papilla.

INTRODUCTION: Stem cell transplantation of exogenous neural progenitor cells (NPCs) derived from mesenchymal stem cells (MSCs) has emerged as a promising approach for neurodegenerative disease. Human stem cells from apical papilla (hSCAPs) are derived from migratory neural crest stem cells and exhibit a potential of neuronal differentiation. However, their neuronal differentiation is low and unpredictable. Resveratrol has been described as a sirtuin 1 (SIRT1) activator which plays an important role in enhancing neuronal differentiation. In this study, we investigate the potential of resveratrol as an enhancer on neuronal differentiation through NPCs induction of hSCAPs. METHODS: Stem cells were isolated from human apical papilla and characterized as MSCs. The cellular toxicity of resveratrol treatment to the characterized hSCAPs was investigated by MTT assay. The non-cellular toxicity concentrations of resveratrol were assessed with various pre-treatment times to select the optimal condition that highly expressed the neural progenitor gene, NES. Consequently, the optimal condition of resveratrol pre-treatment was synergistically performed with a neuronal induction medium to trigger neuronal differentiation. The differentiated cells were visualized, the genes profiling was quantified, and the percentage of neuronal differentiation was calculated. Moreover, the intracellular calcium oscillation was demonstrated. RESULTS: The cellular toxicity of resveratrol was not observed for up to 50 µM for 12 h. Interestingly, hSCAPs pre-treated with 10 µM resveratrol for 12 h (RSV-hSCAPs) significantly expressed NES, which is determined as the optimal condition. Under neuronal induction, both of hSCAPs and RSV-hSCAPs were differentiated (d-hSCAPs and RSV-d-hSCAPs) as they exhibited neuronal-like appearances with Nissl substance staining. The highest expression of NES and SOX1 was observed in RSV-d-hSCAPs. Additionally, the percentage of neuronal differentiation of RSV-d-hSCAPs was significantly higher than d-hSCAPs for 4 times. Importantly, the neuronal-like cells exhibited slightly increasing pattern of calcium intensity. CONCLUSION: This study demonstrated that pre-treatment of resveratrol strongly induces neural progenitor marker gene expression which synergistically enhances neural progenitor-like cells' induction with neuronal induction medium.

Neuronal differentiation of dental pulp stem cells from human permanent and deciduous teeth following coculture with rat auditory brainstem slices.

Sensorineural hearing loss is a common disability found worldwide which is associated with a degeneration of spiral ganglion neurons (SGN). It is a challenge to restore SGN due to the permanent degeneration and viability of SGN is requisite for patients to receive an advantage from hearing aid devices. Human dental pulp stem cells (DPSC) and stem cells from human exfoliated deciduous teeth (SHED) are self-renewing stem cells that originate from the neural crest during development. These stem cells have a high potential for neuronal differentiation. This is primarily due to their multilineage differentiation potential and their relative ease of access. Previously, we have shown the ability of these stem cell types to differentiate into spiral ganglion neuron-like cells. In this study, we induced the cells into neural precursor cells (NPC) and cocultured with auditory brainstem slice (ABS) encompassing cochlear nucleus by the Stoppini method. We also investigated their ability to differentiate after 2 weeks and 4 weeks in coculture. Neuronal differentiation of DPSC-NPC and SHED-NPC was higher expression of specific markers to SGN, TrkB, and Gata3, compared to monoculture. The cells also highly expressed synaptic vesicle protein (SV2A) and exhibited intracellular calcium oscillations. Our findings demonstrated the possibility of using DPSCs and SHEDs as an autologous stem cell-based therapy for sensorineural hearing loss patients.

Deleterious mtDNA mutations are common in mature oocytes.

Heritable mitochondrial DNA (mtDNA) mutations are common, yet only a few recurring pathogenic mtDNA variants account for the majority of known familial cases in humans. Purifying selection in the female germline is thought to be responsible for the elimination of most harmful mtDNA mutations during oogenesis. Here we show that deleterious mtDNA mutations are abundant in ovulated mature mouse oocytes and preimplantation embryos recovered from PolG mutator females but not in their live offspring. This implies that purifying selection acts not in the maternal germline per se, but during post-implantation development. We further show that oocyte mtDNA mutations can be captured and stably maintained in embryonic stem cells and then reintroduced into chimeras, thereby allowing examination of the effects of specific mutations on fetal and postnatal development.

Germline and somatic mtDNA mutations in mouse aging.

The accumulation of acquired mitochondrial genome (mtDNA) mutations with aging in somatic cells has been implicated in mitochondrial dysfunction and linked to age-onset diseases in humans. Here, we asked if somatic mtDNA mutations are also associated with aging in the mouse. MtDNA integrity in multiple organs and tissues in young and old (2-34 months) wild type (wt) mice was investigated by whole genome sequencing. Remarkably, no acquired somatic mutations were detected in tested tissues. However, we identified several non-synonymous germline mtDNA variants whose heteroplasmy levels (ratio of normal to mutant mtDNA) increased significantly with aging suggesting clonal expansion of inherited mtDNA mutations. Polg mutator mice, a model for premature aging, exhibited both germline and somatic mtDNA mutations whose numbers and heteroplasmy levels increased significantly with age implicating involvement in premature aging. Our results suggest that, in contrast to humans, acquired somatic mtDNA mutations do not accompany the aging process in wt mice.

Differentiation of stem cells from human deciduous and permanent teeth into spiral ganglion neuron-like cells.

OBJECTIVE: Stem cells from pulp tissue are a promising cell-based therapy for neurodegenerative patients based on their origin in the neural crest. The aim of this study was to differentiate and evaluate the ability of human dental pulp stem cells from permanent teeth (DPSC) and stem cells from human exfoliated deciduous teeth (SHED) to differentiate into spiral ganglion neurons. DESIGN: After isolation and characterization of mesenchymal stem cell properties, DPSC and SHED were treated with the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial cell-derived neurotrophic factor (GDNF). The differentiation was identified by immunostaining and qRT-PCR analysis of neuronal markers and measuring intracellular calcium activity. RESULTS: After 2 weeks of induction, morphological changes were observed in both DPSC and SHED. The differentiated cells expressed neuron-specific class III beta-tubulin, GATA binding protein 3 (GATA3) and tropomyosin receptor kinase B, protein markers of spiral ganglion neurons. These cells also showed upregulation of the genes encoding these proteins, namely GATA3 and neurotrophic receptor tyrosine kinase 2. Intracellular calcium dynamics that reflect neurotransmitter release were observed in differentiated DPSC and SHED. CONCLUSION: These results demonstrate that dental pulp stem cells from permanent and deciduous teeth can differentiate into spiral ganglion neuron-like cells.

In vitro study of morphological changes of the cultured otocyst isolated from the chick embryo / Estudio in vitro de los cambios morfológicos del otocisto cultivado aislado del embrión de pollo

The aim of this study was to observe morphological changes of the cultured otocysts isolated from various stages of the chick embryo. Isolated otocysts were dissected from embryonic day, E2.5-4.5 of incubation (HH stage 16-26) according to stages of developing inner ear. Morphology of the chick otocyst exhibited an ovoid shape. The width and height of the otocyst were 0.2 mm and 0.3 mm, respectively. Elongation of a tube-like structure, the endolymphatic duct, was found at the dorsal aspect of the otocyst. The cultured otocyst is lined by the otic epithelium and surrounding periotic mesenchymal cells started to migrate outwards the lateral aspect of such epithelium. Notably, the acoustic-vestibular ganglion (AVG) was observed at the ventrolateral aspect of the otocyst. Appearance of AVG in vitro can be applied for studying chemical-induced ototoxicity and sensorineural hearing loss. It was concluded that the organ-cultured otocyst of the chick embryo could be used as a model to study sensory organ development of avian inner ear.

El objetivo de este estudio fue observar los cambios morfológicos de otocistos cultivados aislados en las diversas etapas del desarrollo del embrión de pollo. Otocistos aislados fueron obtenidos de embriones día, E2.5-4.5 de incubación (HH etapa 16-26) de acuerdo a las etapas de desarrollo del oído interno. El otocisto de pollo presentó una morfología ovoide. El ancho y la altura del otocisto fue de 0,2 mm y 0,3 mm, respectivamente. En la cara dorsal del otocisto se visualizó el alargamiento de una estructura similar a un tubo, el conducto endolinfático. El otocisto cultivado está revestido por epitelio ótico y células mesenquimatosas perióticas que comienzan a migrar hacia el exterior de la cara lateral en búsqueda del epitelio. En particular, el ganglio acústico-vestibular (GAV) fue observado en la parte ventrolateral del otocisto. La aparición de GAV in vitro puede ser aplicado para el estudio de la ototoxicidad inducida por productos químicos y la pérdida de audición neurosensorial. Se concluyó que el otocisto cultivado de embrión de pollo podría ser utilizado como un modelo para estudiar el desarrollo de órganos sensoriales del oído interno aviar.