J Korean Assoc Oral Maxillofac Surg.  2014 Aug;40(4):173-180. 10.5125/jkaoms.2014.40.4.173.

Neurogenic differentiation of human dental stem cells in vitro

• Lee JH
• Um S
• Song IS
• Kim HY
• Seo BM

• KMID: 2005442
• DOI: http://doi.org/10.5125/jkaoms.2014.40.4.173

Abstract

OBJECTIVES
The purpose of this study was to investigate the neurogenic differentiation of human dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), and stem cells from apical papilla (SCAP).
MATERIALS AND METHODS
After induction of neurogenic differentiation using commercial differentiation medium, expression levels of neural markers, microtubule-associated protein 2 (MAP2), class III beta-tubulin, and glial fibrillary acidic protein (GFAP) were identified using reverse transcriptase polymerase chain reaction (PCR), real-time PCR, and immunocytochemistry.
RESULTS
The induced cells showed neuron-like morphologies, similar to axons, dendrites, and perikaryons, which are composed of neurons in DPSCs, PDLSCs, and SCAP. The mRNA levels of neuronal markers tended to increase in differentiated cells. The expression of MAP2 and beta-tubulin III also increased at the protein level in differentiation groups, even though GFAP was not detected via immunocytochemistry.
CONCLUSION
Human dental stem cells including DPSCs, PDLSCs, and SCAP may have neurogenic differentiation capability in vitro. The presented data support the use of human dental stem cells as a possible alternative source of stem cells for therapeutic utility in the treatment of neurological diseases.

Keyword

Dental pulp; Periodontal ligament; Dental papilla; Stem cells; Neurogenic differentiation

MeSH Terms

Axons
Dendrites
Dental Papilla
Dental Pulp
Glial Fibrillary Acidic Protein
Humans
Immunohistochemistry
Microtubule-Associated Proteins
Neurons
Periodontal Ligament
Real-Time Polymerase Chain Reaction
Reverse Transcriptase Polymerase Chain Reaction
RNA, Messenger
Stem Cells*
Tubulin
Glial Fibrillary Acidic Protein
Microtubule-Associated Proteins
RNA, Messenger
Tubulin

Figure

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  Fig. 1 Human third molars provide three different cell sources. A. Apical papilla from the developing root-tip was gently separated from the surface of the root (arrow). B. Dental pulp was isolated from the cracked crown (arrow). C. The periodontal ligament was removed from the middle root surface (arrow).

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  Fig. 2 Morphological changes of the humand dental stem cells were noticeable during induction of neurogenic differentiation. Upon stimulation by neurogenic differentiation medium, spindle-shaped cells changed into neuron-like cells, as identified via microscopy. Arrows indicate neurogenic differentiation of dental stem cells that extended neurite-like projections. Scale bars=100 µm. (SCAP: stem cells from apical papilla, DPSCs: dental pulp stem cells, PDLSCs: periodontal ligament stem cells)

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  Fig. 3 Differentiated gene expression of dental stem cells upon neurogenic induction analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) (A), and real-time PCR (B) of microtubule-associated protein 2 (MAP2), β-tubulin III, and glial fibrillary acidic protein (GFAP) genes. Neuron markers (MAP2 and β-tubulin III) were upregulated with various intensities under basal conditions. The expression of GFAP strongly increased in dental pulp stem cells (DPSCs) after stimulation by induction medium. Neuronal markers increased as neurogenic differentiation progressed, though MAP2 did not significantly increase compared to the control. Expression of β-tubulin III increased in stem cells from apical papilla (SCAP) and periodontal ligament stem cells (PDLSCs), while the mRNA level of GFAP increased significantly in DPSCs and PDLSCs after induction of neurogenic differentiation. Results are expressed as fold-change value relative to the normalized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. *P<0.05, **P<0.01, ***P<0.001.

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  Fig. 4 Immunofluorescence staining for neural markers after neurogenic induction. Microtubule-associated protein 2 (MAP2) and β-tubulin III were expressed in stem cells from apical papilla (SCAP), dental pulp stem cells (DPSCs), and periodontal ligament stem cells (PDLSCs) upon culturing in neurogenic medium; however, cells in neurogenic differentiation culture conditions did not express glial fibrillary acidic protein (GFAP). Scale bars=20 µm.

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