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.

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.

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.

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.

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.

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.