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.
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 cells1-8; 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 transplantation6,9,10. 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
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 are identified by their negative expression of hematopoietic antigens (e.g., CD45, CD34, CD14), and positive expression of CD90, CD29, CD73, CD105, CD44 and STRO-1.19,20
Easy obtained potential with minimum pain & morbidity make human DPSCs as a valuable source of MSCs compared to the ordinary sources, such as bone marrow mesenchymal stem cells21. In general, DPSCs have been isolated by either outgrowth method, i.e., migration of stem cells from pulp tissue explants (DPSC-OG)22-24, and/or by enzymatic digestion (DPSC-ED)4,6,25. Previous studies have shown that the isolation method and culture conditions can induce different populations or lineages under in vitro passages26,27. In the case of permanent teeth (pDPSCs), Huang et al revealed that enzymatic digested pDPSCs have higher proliferation potential compared to the outgrown DPSCs.26 Moreover, in the case of deciduous teeth (dDPSCs), it was demonstrated that STRO-1 & CD34 markers expressed more in dDPSC-ED in comparison with dDPSC-OG. In addition, dDPSC-ED displayed higher mineralization rate in defined osteo/odonto medium.27 Therefore, due to the outstanding potential of DPSCs in regenerative medicine, more studies will be required for better understanding of possible various populations which are derived from different isolation methods.
Here, it was attempt to introduce easy way of pulp extraction, by using one-step dental diamond disk to facilitate the process of pulp extraction. Moreover, after the isolation of human pulp-derived stem cells by applying ED or OG methods, general properties & differentiation capacity between two groups were also investigated.
1. Prepare the Enzyme Solution and Proliferation Medium (PM)
2. Prepare Human Dental Pulp Tissue for Dental Pulp Stem Cell Isolation
3. Human Dental Pulp Isolation
4. Immunophenotyping
5. Induction of Differentiation of DPSCs into Mineralized Cells & Quantified Alizarin Red S Assay
Alizarin Red S standard were prepared by first diluting the dilution buffer & used for making 2-fold serial dilutions based on the table.
High Range concentration of dye | Low Range concentration of dye | ||||||||||||
2 mM | 1 mM | 500 μM | 250 μM | 125 μM | 62.5 μM | 31.3 μM | 15.6 μM | 7.8 μM | 3.9 μM | 1.9 μM | 0.9 μM | 0.4 μM | Blank |
(Notes: A paired t-test analysis was used to determine the differences in the amount of mineralized matrix produced ED and OG groups after induction of differentiation. The results were expressed as mean ±SEM. (significance assumed for P < 0.05)
(Note: The QPCR data were analyzed using the comparative CT method. A paired t-test analysis was used to determine the differences in gene expression between ED & OG groups before and after the differentiation, and also between differentiated cells of two groups. The results were expressed as mean ±SEM. (Significance assumed for P < 0.05) (Triplicate technical replications)
Figure 1. Enzymatic dissociated DPSC (DPSC-ED) at day 10, 15 & 18. Magnification bar =200 μm.
Figure 2. Outgrown DPSCs (DPSC-OG) on day 5, 10, 13 and 18. Magnification bar =200 μm.
Figure 3. Outgrown DPSCs (DPSC-OG) & enzymatic digested DPSCs (DPSC-ED) Morphologies in passage 3. Magnification bar =200 μm. Click here to view larger figure.
Figure 4. Overlaid Histogram of immunophenotyping results in DPSC-ED (dark red) and DPSC-OG (Blue). (Yellow lines represent isotypes). Click here to view larger figure.
Figure 5. Alizarin Red S staining results in differentiated DPSC-OG (c), and differentiated DPSC-ED (d).(a) & (b) referred to the controls. Quantification of Alizarin Red S assay show more dye absorption in differentiated DPSC-ED compared to the differentiated OG group (e). Click here to view larger figure.
Figure 6. QPCR results of the expression of odontoblast and mineralized-related genes (DSPP) and (MEPE, ALP) respectively, in differentiated DPSC-ED & DPSC-OG. The expression of MEPE, ALP, & DSPP in differentiated cells compare to the control confirmed the differentiation of both groups into odontoblast (a-c). These results indicated the significant up-regulation of ALP & MEPE after the differentiation of ED and OG groups (a & b). Meanwhile, ALP and MEPE expressions were significantly higher in differentiated DPSC-ED compared to the differentiated DPSC-OG (a & b). However, there is no difference in the expression of DSPP in differentiated cells of both groups (c) (Housekeeping gene: Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein; YWHAZ). Click here to view larger figure.
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 needle29, Gracey curette30, dental fissure burs4 , etc. Those methods are time-consuming, while considering low level of overheating of pulp by adding water continuously. However, in current technique, dental diamond disc is used for the isolation of pulp tissue, which is a rapid way to cut tooth with minimum contamination in one-step process, and minimizes the overheating of pulp during a cutting process. Moreover, hDPSCs are finally differentiated into odontoblasts that have been never compared in the case of human permanent teeth by discussed aim. Comparative assessment of differentiated hDPSCs (ED & OG groups) by quantitative Alizarin Red S assay indicated the higher mineralization potential of hDPSC-ED in comparison with OG group. These results also confirmed by QPCR. DSPP, ALP and MEPE as the mineralization markers up-regulated after the odontoblast differentiation in both groups. On the other hand, the expressions of MEPE & ALP were higher in differentiated hDPSC-ED compared to the control. In agreement with previous study about deciduous teeth27, stem cells derived from permanent teeth by using ED or OG isolation methods also exhibit similar differentiation behaviors under the same conditions. Moreover, for better comparative evaluation, in this study, both hDPSC-ED & hDPSC-OG were isolated from the same individuals.
Expressions of MSC markers and the absence of heamatopietic/endothelial markers identify both types of hDPSCs as mesenchymal stem cells. On the other hand, CD 105 /CD 146 were higher in DPSC-OG compared to the ED groups. Despite the existing evidence according to the presence of STRO-1 in DPSCs31, our results showed no expression of STRO-1 in both groups. However, the significant diversity in surface marker expression in different studies might be due to the either cultivation conditions such as passage number, composition of media, etc. or interindividual variations between different donors. The absence of STRO-1 in both groups may indicate that the expression of this marker might not directly affect odontoblast differentiation potential (not shown in video).
Different hDPSCs isolation condition might bring different differentiation capacity. These differences might be due to the existence of different populations in the pulp tissue. Thus, selecting the isolation methods might be useful for targeted-tissue specific repair & regeneration for future clinical approaches. Our results indicated the higher mineralization capacity of enzymatic digested DPSC compared to the outgrown ones. However, additional studies, such as the assessment of the formation of tubular structure in vivo & in vitro, are required to determine which of these two procedures is applicable for functional dentin/pulp tissue regeneration therapies.
The authors have nothing to disclose.
We gratefully acknowledge Dr. Leila Shakeri & Dr. Aref Dournaei for their critical discus and Mr. Mohammad Reza Khadem Sharif for his technical supports.
Name of the reagent | Company | Catalogue or Lot. number | Comments (optional) |
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 |