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

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.

Mesenchymal stem cells; Progenitor cells; Neuronal differentiation

Page	Section	Where it reads	The sentence should read
6	Characterization of hSCAPs	… and CD146+ (Figure 1G).	… and CD146+ (Figure 2G).
6	Characterization of hSCAPs	… were formed (Figure 1H).	… were formed (Figure 2H).
6	Identification of Nissl substance	… neuronal differentiation (Figure 4F).	… neuronal differentiation (Figure 2F).
6/7	Self-renewal ability	… of NSCs (Figure 7A).	… of NSCs (Figure 6A).
7	Self-renewal ability	… individual cells (Figure 7B).	… individual cells (Figure 6B).
7	Gene expression profiling	These three markers are known to indicate the presence of NSCs.	These three markers are known to indicate the presence of NSCs (Figure 7).

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[1] Corresponding address: Charoensri Thonabulsombat. Mahidol University - Faculty of Science - Department of Anatomy - 272 RAMA VI Road - Ratchathewi - Bangkok - 10400 -Thailand. Phone: +668 6610 3599. e-mail: charoensri.tho@mahidol.ac.th