Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Summary

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Abstract

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells^1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation^6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers & also their differentiation capacity.

Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,^11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 & CD44), hematopoietic/endothelial Markers (CD34, CD45 & CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) & Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE & dentin sialophosphoprotein; DSPP).¹⁴

Introduction

Stem cells are clonogenic cells which possess two remarkable features, known as multi-potency and self-renewal¹⁵. Among all stem cells with different replicative potencies, dental stem cells as the postnatal stem cells have drawn attention in recent years because of their accessibility, plasticity, and high proliferative ability in comparison with other adult stem cells¹⁶. Characteristically, similar to the mesenchymal stem cells, dental pulp stem cells are adherent clonogenic cells which have multiple differentiation capacity into mesenchyme and/or non-mesenchyme lineages, both in vitro and in vivo.^1-8,17,18 DPSCs ar....

Protocol

1. Prepare the Enzyme Solution and Proliferation Medium (PM)

1. Make Collagenase Type I Solution: Weigh out collagenase type I (12 mg/ml) and dissolve in 1 ml PBS and filter using a 0.2 μm syringe filter. Then place it 15 ml tube and keep it at -20 °C until needed.
2. Make dispase Solution: Weigh out dispase (16 mg/ml) and dissolve in 1 ml PBS and filter using a 0.2 μm syringe filter. Then place it 15 ml tube and keep it at 4 °C until needed.
3. Make enzyme solution: Add 1 ml collagenase type I solutions (12 mg/ml) and 1 ml dispase solutions (16 mg/ml) into the 2 ml sterile PBS....

Results

1. DPSCs that were obtained by enzymatic dissociation (DPSC-ED) are shown here at day 10, 15,18 (Figure 1). The colonies initiated to form on almost 3 to 5 days after the isolation.
2. Outgrown DPSCs (DPSC-OG) are shown in Figure 2 on day 5, 10, 13 & 18. Fibroblast-like cells began to migrate from pulp tissue into the flask by parallel ordering by almost 5 days after seeding.
3. DPSCs at passage 3 using both methods are shown in Figure 3. Both types of DPSCs .......

Discussion

This protocol describes the process of isolation and characterization of hDPSCs from dental pulp using two methods, enzymatic dissociation and direct outgrowth of stem cells from pulp tissue explants. In addition, in vitro differentiation of these cells into odontoblasts, was assessed by quantitative Alizarin Red S assay & QPCR.

Existing protocols for isolating pulp tissue from human tooth had been used various instruments such as pliers (bone forceps)⁹, extirpation .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
α-MEM	GIBCO	11900-073
Collagenase type I	Sigma-Aldrich	C0130-100MG
Dispase	GIBCO	17105-041
Penicillin/streptomycin	GIBCO	15140-122
Amphotericin B	GIBCO	15290-018
Fetal Bovine serum defined (FBS)	HyClone	SH30070.03
L-ascorbic acid 2-phosphate	Sigma	A8960-5G
L-glutamine	GIBCO	25030-024
Dexamethasone	Sigma	D4902
β-Glycerol phosphate disodium salt hydrate, BioUltra	Sigma	G9422-100G
Potassium phosphate monobasic	Sigma-Aldrich	P5655
Osteogenesis Assay Kit	Millipore	PS01802031
Mouse IgG2b-PE isotype control	BD pharmingen	50808088029
FITC mouse IgG2b isotype control	BD pharmingen	559532
FITC mouse IgG1 κ isotype	BD pharmingen	11471471
FITC/PE mouse anti-human CD34/CD45	BD pharmingen	341071
PE anti-human CD146	BD pharmingen	550315
Monoclonal mouse anti-human CD90/FITC	Daka	00034418
PE mouse anti-human CD73	BD pharmingen	550257
Anti-h CD105/Endoglin PE	BD pharmingen	FAB10971P
PE mouse anti-human CD11b/Mac1	BD pharmingen	5553888
CD44 PE mouse anti human	BD pharmingen	555479
Phosphate buffer Solution (PBS)	GIBCO	003000
70-μm cell strainer	Falcon	352360
0.2 μm syringe filter	Millex-GV	SLGV033RB
25 cm2 culture flask	Sigma-Aldrich	Z707481
EQUIPMENT
BD FACSCalibur	BD	342975
multiskan microplate spectrophotometer	Thermo scientific	51119200
Fleurcense Microscope	Olympus
Flowing Software version 2.3.1