J Korean Assoc Oral Maxillofac Surg.  2010 Jun;36(3):186-196. 10.5125/jkaoms.2010.36.3.186.

Isolation and characterization of human dental tissue-derived stem cells in the impacted wisdom teeth: comparison of dental follicle, dental pulp, and root apical papilla-derived cells

• Song JH
• Park BW
• Byun JH
• Kang EJ
• Rho GJ
• Shin SH
• Kim UK
• Kim JR

• KMID: 2189946
• DOI: http://doi.org/10.5125/jkaoms.2010.36.3.186

Abstract

INTRODUCTION
The first aim of this study was to isolate the dental tissue-derived stem cells from the dental follicle (DF), dental pulp (DP), and root apical papilla (RAP) of the extracted wisdom teeth. Second was to evaluate their characterization with the expressions of transcription factors and cell surface markers. Finally, their ability of the in vitro multi-lineage differentiations into osteogenic and adipogenic cells were compared, respectively.
MATERIALS AND METHODS
Dental tissues, including dental follicle, dental pulp, and root apical papilla, were separated in the extracted wisdom teeth. These three dental tissues were cultured in Dulbecco's modified Eagle's medium (DMEM) with supplements, respectively. After passage 3, the homogeneous shaped dental tissue-derived cells were analyzed the expression of transcription factors (Oct-4, Nanog and Sox-2) and cell surface markers (CD44, CD90 and CD105) with reverse transcription polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS) analysis. In order to evaluate in vitro multi-lineage differentiations, the culture media were changed to the osteogenic and adipogenic induction mediums when the dental tissue-derived cells reached to passage 3. The characteristics of these three dental tissue-derived cells were compared with immunohistochemistry.
RESULTS
During primary culture, heterogenous and colony formatted dental tissue-derived cells were observed in the culture plates. After passage 2 or 3, homogenous spindle-like cells were observed in all culture plates. Transcription factors and mesenchymal stem cell markers were positively observed in all three types of dental tissue-derived cells. However, the quantity of expressed transcription factors was most large in RAP-derived cells. In all three types of dental tissue-derived cells, osteogenic and adipogenic differentiations were observed after treatment of specific induction media. In vitro adipogenic differentiation was similar among these three types of cells. In vitro osteogenic differentiation was most strongly and frequently observed in the RAP-derived cells, whereas rarely osteogenic differentiation was observed in the DP-derived cells.
CONCLUSION
These findings suggest that three types of human dental tissue-derived cells from extracted wisdom teeth were multipotent mesenchymal stem cells, have the properties of multi-lineage differentiations. Especially, stem cells from root apical papilla (SCAP) have much advantage in osteogenic differentiation, whereas dental follicle cells (DFCs) have a characteristic of easy adipogenic differentiation.

Keyword

Human dental tissue-derived stem cells; Dental pulp stem cells (DPSCs); Dental follicle cells (DFCs); Stem cells from root apical papilla (SCAP); Mesenchymal stem cells (MSCs)

MeSH Terms

Culture Media
Dental Pulp
Dental Sac
Durapatite
Flow Cytometry
Humans
Imidazoles
Immunohistochemistry
Mesenchymal Stromal Cells
Molar, Third
Nitro Compounds
Polymerase Chain Reaction
Reverse Transcription
Stem Cells
Transcription Factors
Culture Media
Durapatite
Imidazoles
Nitro Compounds
Transcription Factors

Figure

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  Fig. 1. A. Schematic illustrations of dental pulp (DP), root apical papilla (RAP), and dental follicle (DF) in the extracted impacted wisdom tooth. B. Photograph of the obtained dental tissues after treatment of mechanical dissociation and enzyme (collagenase I) digestion.(Pa: root apical papilla tissue, F: dental follicle tissue, Pu: dental pulp tissue)

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  Fig. 2. Isolation and primary culture of dental tissue-derived cells. Microphotographs of dental follicle-derived cells (DF), root apical papilla-derived cells (RAP), and dental pulp-derived cells (DP) at 1st and 10th days later of primary culture (passage 0). At 1st day of primary culture, round shaped unattached floating primary cells were rarely observed in the culture media. When the culturing cells reached nearly 70–80% confluence of primary culture (10th day of primary culture), remarkably increased cell number and irregular heterogeneous shaped dental tissue-derived cells were observed. In this primary culture period, there are no definitely morphological differences between these three dental tissue-derived cells.

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  Fig. 3. Sequential cell proliferations of the third passaged dental tissue-derived cells.(original magnification x100) RAP-derived cells showed the most prominent proliferation rate, especially between 3rd and 6th days of subculture, there is remarkable increase of cell number. However, DF and DP-derived cells showed the remarkable cell number increasing between 6th and 9th days of subculture. (DP: dental pulp, RAP: root apical papilla, DF: dental follicle)

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  Fig. 4. Graphs showed the increasing numbers of in vitro cultured cells as time passed. The most prominent cell proliferation rate was observed in the RAP-derived cells. (DP: dental pulp, RAP: root apical papilla, DF: dental follicle)

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  Fig. 5. Expression of transcription factors, Oct-4, Nanog, and Sox-2 in the cultured dental tissue-derived cells of passage 3 by RT-PCR. A. Although Sox-2 gene was weakly observed, these transcription factors were all detected in the dental tissue-derived cells. B. Quantification of transcription factors expressions. Transcription factors were most strongly detected in the root apical papilla-derived cells.(RAP) (GAPDH: glyceraldehyde-3-phosphate dehydrogenase, DP: dental pulp, RAP: DF: dental follicle, RT-PCR: reverse transcription polymerase chain reaction)

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  Fig. 6. Expressions of cell surface markers with FACS analysis. Mesenchymal stem cell marker proteins were detected in the all three types of dental tissue-derived cells. A. Dental follicle cells. B. Dental pulp cells. C. Root apical papilla cells. (FACS: fluorescence-activated cell sorting)

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  Fig. 7. Osteogenic differentiations of dental tissue-derived stem cells for 4 weeks. The most abundant bone matrix formation was observed in the RAP cells, whereas it was rarely detected in the DP cells by von Kossa and alizarin red stainings. (DP: dental pulp, RAP: root apical papilla)

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  Fig. 8. Adipogenic differentiation of dental tissue-derived stem cells for 4 weeks.(original magnification x100) Small red color stained lipoid nodules were detected in all three types of dental stem cells by oil-red O staining.(arrows) Most actively adipogenic differentiation was showed in the dental follicle-derived cells.

Reference

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