J Korean Med Sci.  2016 Feb;31(2):171-177. 10.3346/jkms.2016.31.2.171.

Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro

• Chun SY
• Soker S
• Jang YJ
• Kwon TG
• Yoo ES

• KMID: 2360038
• DOI: http://doi.org/10.3346/jkms.2016.31.2.171

Abstract

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

Keyword

Human Dental Pulp Stem Cells; Dopaminergic Differentiation; Tyrosine Hydroxylase; Parkinson Disease; Autologous Stem Cell Therapy; Dopaminergic Neural Cells

MeSH Terms

Animals
Brain/pathology
*Cell Differentiation/drug effects
Cells, Cultured
Culture Media/chemistry/pharmacology
Dental Pulp/*cytology
Dopaminergic Neurons/*cytology/*metabolism/pathology
Enzyme-Linked Immunosorbent Assay
Glial Fibrillary Acidic Protein/genetics/metabolism
Humans
Mice
Mice, Inbred ICR
Myelin Basic Protein/genetics/metabolism
Real-Time Polymerase Chain Reaction
Stage-Specific Embryonic Antigens/genetics/metabolism
Stem Cells/*cytology/*metabolism/pathology
Tubulin/genetics/metabolism
Tyrosine 3-Monooxygenase/analysis/genetics/metabolism
Culture Media
Glial Fibrillary Acidic Protein
Myelin Basic Protein
Stage-Specific Embryonic Antigens
Tubulin
Tyrosine 3-Monooxygenase

Figure

• 

  Fig. 1 Morphological and immunocytochemical analyses of human dental pulp stem cells (hDPSCs) throughout the various differentiation stages. (A) The neuronal characteristics of hDPSCs were verified at the protein level by immunocytochemical analysis. (B) Substantia nigra of ICR mouse brain tissue was used as the positive control for immunohistochemistry. SSEA4, stage-specific embryonic antigen 4; TuJ1, β-tubulin III; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; TH, tyrosine hydroxylase; H&E, hematoxylin & eosin.

• 

  Fig. 2 Genotypic analyses of human dental pulp stem cells (hDPSCs) according to the various differentiation stages by real-time PCR. The different letters on top of the bars show significant differences at P < 0.05. P values are for the analysis of variance Tukey’s test, n = 4.

• 

  Fig. 3 Tyrosine hydroxylase (TH) synthesis by human dental pulp stem cells (hDPSCs) at stage 1 and 5 through ELISA analysis. n = 4, t-test. *P < 0.001.

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