ABSTRACT
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.
ABSTRACT
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.