This article provides a stepwise guide to establish a primary culture of dental pulps stem cells using the explant culture method and characterization of these cells based on ICSCRT guidelines. The cells isolated by this protocol can be considered as mesenchymal stem cells for further applications.
The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an extracted tooth. The neural crest-derived ecto-mesenchymal origin of dental pulp stem cells (DPSCs) bestows a high degree of plasticity that owes to its multifaceted benefits in tissue repair and regeneration. There are various practical ways of harvesting, maintaining, and proliferating adult stem cells being investigated for their use in regenerative medicine. In this work, we demonstrate the establishment of a primary mesenchymal stem cell culture from dental tissue by the explant culture method. The isolated cells were spindle-shaped and adhered to the plastic surface of the culture plate. The phenotypic characterization of these stem cells showed positive expression of the international society of cell therapy (ISCT)-recommended cell surface markers for MSC, such as CD90, CD73, and CD105. Further, negligible expression of hematopoietic (CD45) and endothelial markers (CD34), and less than 2% expression of HLA-DR markers, confirmed the homogeneity and purity of the DPSC cultures. We further illustrated their multipotency based on differentiation to adipogenic, osteogenic, and chondrogenic lineages. We also induced these cells to differentiate into hepatic-like and neuronal-like cells by adding corresponding stimulation media. This optimized protocol will aid in the cultivation of a highly expandable population of mesenchymal stem cells to be utilized in the laboratory or for preclinical studies. Similar protocols can be incorporated into clinical setups for practicing DPSC-based treatments.
Adult stem cells have transpired into a powerful therapeutic tool for cell-directed treatments and therapies due to their plasticity, paracrine mechanisms, and immunomodulatory properties1,2,3. The encouraging data from stem cell-based preclinical studies have inspired researchers to work for the bench to-bedside translation. The type of stem cells used for stem cell therapy plays a significant role in successful outcomes. In preclinical and clinical studies, the most widely reported source for mesenchymal stem cells (MSCs) remain bone marrow4,5. However, major drawbacks to using bone marrow-derived stem cells (BMSCs) include their rare population, highly invasive procedures for isolation, and their limited ability to expand. Therefore, alternative sources of MSCs are being explored. In this regard, dental tissues, with their ease of accessibility, enormous plasticity, high regenerative potential, and high proliferative ability, have now been deemed as a rich and potential alternative source of stem cells6,7,8,9,10.
Dental pulp stem cells (DPSCs) were the first type of dental stem cells to be isolated and characterized by Gronthos in 200011. DPSCs have grabbed the attention for tissue engineering applications because of their high proliferation rate, significant differentiation potential, ease of accessibility with effortless culturing, and, most importantly, their ability to be obtained from a discarded tooth without any ethical concern12. The limitations posed by other stem cell sources, such as BMSCs and adipose-derived stem cells (ADSCs), in their isolation and inadequate self-renewal capacities are circumvented by DPSCs13. Human DPSCs can be obtained from human primary teeth, permanent teeth, wisdom teeth, exfoliated deciduous teeth (SHEDs), and apical papillae. Moreover, DPSCs can also be isolated from supernumerary teeth, which are generally discarded14. DPSCs express neural crest-associated markers and have the potential to differentiate into neuronal cells both in vitro and in vivo15. In addition to their neurogenic potential, DPSCs can differentiate into other cell lineages, such as osteogenic, chondrogenic, adipogenic, hepatic, and myogenic, when given specific differentiation conditions13. Thus, these multipotent cells hold great potential for cell-based therapy and can be employed for the regeneration of various tissues. Studies have also reported the potential role of DPSCs in the reconstruction of the cornea16, repair of myocardial infarction17, and their potential therapeutic role in diseases like limb ischemia18, Alzheimer's 19, Parkinson's 20, and aging21. Therefore, dental tissue-derived stem cells can be used not only for dental regeneration, but also for the repair and regeneration of non-dental organs like eyes16, hearts17, livers22, bones23 etc.
There are two particular methods for the isolation of an MSC population from pulp tissue - enzymatic digestion and explant culture24,25. Successful establishment of primary cultures without any significant difference in the quantity and properties of DPSCs have been reported by both these methods26. In this study, we have focused on the isolation of DPSCs by the explant method27, since this method generates DPSCs without contamination of hematopoietic and endothelial cells, as compared to enzymatic digestion which can result in fibroblast contamination28.
All the procedures described in the study have been approved by the Institute Ethics Committee (IEC# 9195/PG-12 ITRG/2571-72) of PGIMER, Chandigarh. All the cell culture related experiments need to be performed in a Class II biological safety cabinet (BSC) following aseptic technique. Dental pulp was obtained from healthy teeth of three (F/14, M/14, and M/20) patients undergoing third molar extractions for orthodontic reasons. Before the sample collection, written informed consent was obtained from the patient/guardian in accordance with the guidelines provided by the ethics committee of PGIMER, Chandigarh.
1. Establishment of primary culture of dental pulp stem cells (DPSCs) from human dental tissue
NOTE: Any decayed teeth should not be used.
2. Removal of pulp tissue from the tooth and cell culture of DPSCs (time: 60-120 min)
NOTE: All the steps after tooth transportation have been performed in the cell and tissue culture lab inside a biosafety cabinet level 2.
3. DPSC expansion
4. Identification of stem cell phenotypic markers (time: 90 – 120 min)
NOTE: For the characterization of cells harvested from the pulp tissue, use cells between the third and fifth passage.
5. DPSC differentiation into multiple lineages
NOTE: Use 75%-80% confluent cultures of DPSCs at the third to fifth passage for the assessment of multipotency. A control cell group containing α-MEM should be used for all types of differentiations described below.
6. Adipogenic differentiation of DPSCs, oil red O staining, and quantification
NOTE: The initial steps of seeding are the same as above (i.e., osteogenic differentiation step 5.1).
7. Chondrogenic differentiation of DPSCs, Alcian blue staining, and quantification
NOTE: Chondrogenic differentiation was induced in the monolayer culture of DPSCs. The initial steps of seeding are the same as above (i.e., osteogenic differentiation step 5.1).
8. Differentiation of DPSCs toward hepatic-like lineage and characterization
9. Neural differentiation of DPSCs toward neuronal-like lineage and characterization
Here, we describe how researchers can establish a pure culture of DPSCs via the explant method6,7,8,9,10 and induce them toward multiple lineages to establish the purity of culture for downstream applications.
We established a primary culture of DPSCs from the small tissue of pulp extracted from the third molar tooth of patients, as shown in Figure 1A. Initially, very few rounded cells were observed in the immediate vicinity of explants or tissue on the fourth and fifth day post-explant seeding. However, by the 12th day, the count of cells that emerged out of the explants increased, and by the end of second week (14th-16th day), most of the explants got surrounded by many adherent cells with spindle-shaped morphology. At this time, the explant could be removed from the plates and the cells were allowed to grow in the presence of complete media. After reaching 70%-75% confluence (third week), the cells were sub-cultured and expanded for further experimental setup. Through the explant method, we obtained a uniform culture of stem cells with positivity of MSC CD markers CD90, CD105, CD73, and an almost negligible cell population of another cell type, endothelial cell marker CD34 and hematopoietic cell marker CD45 (Figure 1B).
We also differentiated DPSCs successfully into cells of osteogenic lineage, as evident by positive alizarin red stain uptake in calcified granules in osteocytes, adipogenic lineage, as demonstrated by the uptake of oil red O staining by lipid droplets in differentiated adipocytes, and chondrogenic lineage, as shown by positive Alcian blue staining (Figure 2). DPSCs also showed efficient differentiation toward hepatic-like lineage, as evident by the typical cuboidal morphology and uptake of LDL-550 by live cell (Figure 3A) staining. The differentiated hepatocyte-like cells were fixed and processed for staining with the LDL receptor antibody. Figure 3B shows the positive expression of the LDL receptor in differentiated hepatocyte- like cells along with LDL-550. DPSCs also demonstrated a marked ability to differentiate into neuronal-like cells, as shown by the positive staining of MAP-2 and NFM, typical neuronal proteins (Figure 4). In a nutshell, our study shows that DPSCs can be isolated and cultured as a pure population, with a >95% purity for stem cell markers, and differentiated into various lineages for further downstream applications like cell therapy, drug testing, and disease modeling.
Figure 1: Primary culture of DPSCs. (A). Differential interference contrast (DIC) images showing various stages in development of DPSC primary culture: 1) explant at the initial budding stage with rounded bubble type cells coming out of dental tissue on the second day of seeding; 2) establishment of a mess-like network of cells coming out of tissue on the fourth day; 3) cells with full spindle-shaped morphology at confluence on the 13th day and cells covering the entire surface of the culture dish after the removal of tissue. Scale bar = 100 µm, (B). Stem cell marker characterization by flow cytometry with histograms and a bar graph showing the positive expression of stemness markers and negative expression of non-stem cell markers in DPSCs (n = 3). Each experiment was repeated in triplicate at least three times. Please click here to view a larger version of this figure.
Figure 2: Multilineage differentiation of DPSCs. Bright field microscopic images showing alizarin red staining after osteogenic differentiation, oil red O staining post-adipogenic differentiation, and Alcian blue staining after chondrogenic differentiation in DPSCs. Scale bar = 100 µm (n = 3). Each experiment was repeated in triplicate at least three times. Please click here to view a larger version of this figure.
Figure 3: Differentiation of DPSCs toward hepatocyte-like cells and uptake of LDL. (A). Light microscopic images for control and hepatic-like cells differentiated from DPSCs; the last image on the extreme right shows LDL uptake in the live differentiated cells. (B). Fluorescent microscopic images showing the expression of the LDL receptor (green) and uptake of LDL-550 (red) (fixed cells) in DPSCs post-hepatic differentiation. DAPI was used as a nuclear stain. Scale bar = 200 µm (n = 3). Each experiment was repeated in triplicate at least three times. Please click here to view a larger version of this figure.
Figure 4: Differentiation of DPSCs to neuronal lineage. Fluorescent microscopic images for DPSCs after 41 days of neural differentiation, showing a positive expression of neural lineage-specific antibodies' neurofilament and MAP-2. PI was used as a nuclear stain. Scale bar = 20 µm (n = 3). Each experiment was repeated in triplicate at least three times. Please click here to view a larger version of this figure.
Stem cells have pinned the hopes of curing numerous diseases, owing to their plasticity, robustness, immunomodulatory properties, paracrine mechanisms, and homing efficiencies. Dental pulp tissue is considered the most potent and valuable source of stem cells, with eminent plasticity and a regenerative capability. Here, we demonstrate the isolation of DPSCs, utilizing the widely adopted explant culture method, in which the cells migrate from pieces of pulp tissue or explants to grow into a homogenous cell culture that morphologically resembles spindle-shaped fibroblast-like cells. The explant method yields more uniform cultures of DPSCs, devoid of other cell types such as endothelial cells and pericytes, which remain during the enzymatic procedure for DPSC culture establishment34. We showed that DPSCs derived by the explant method express ISCT-recommended mesenchymal cell surface CD markers, such as CD90, CD73, and CD105. They are also devoid of CD45, CD34, and HLA-DR marker-expressing cells, which indicate the homogeneity and purity of DPSC culture. The lack of HLA-DR also makes them suitable for transplantation purposes, with minimum rejection possibilities. There are a few critical steps in the protocol to ensure the efficiency and purity of DPSC culture. The explants should be washed at least four times with PBS before being put in the culture plates to avoid any possible contamination. Also, the explants should adhere well to the culture well surface, as detached explants would not yield any cells. Hence, utmost care should be taken while handling explants during media change. The media should be added carefully, dropwise along the corners of the well, to not disturb the explants. Once the cells grow in the plate wells, tissue pieces should be removed carefully using the tip, in order not to damage any surrounding cells. During the retrieval of DPSCs at the first passage from plate wells, there might be very few cells, making it impossible to see any visible cell pellet in the tube after centrifugation. Some cells might also stick to the walls of the centrifuge tube, making it hard to get any cells during the upscaling process. Care should be taken to rinse the culture plate and 15 mL tube well with PBS to retrieve as many cells as possible. All cell-based differentiation experiments should be performed in DPSCs below passage number eight, as higher passages might lead to the accumulation of genetic aberrations and spontaneous differentiation, hence affecting the results.
There are also a few limitations of the protocol. The explant culture is a slow protocol and yields enough cells after 15-20 days of culture only. In between, there is a risk of contamination, and the explant (dental pulp pieces) can get detached from the plate surface, which would not yield any cells later. Hence, utmost care should be taken while changing the media. Also, repeated PBS washings are important before seeding the explants, as teeth are rich in oral micro flora and there are very high chances of contamination in the culture if proper washing is not performed. Human DPSCs are ectodermal in origin and during tooth development; they migrate from the neural crest and later differentiate into mesenchymal cells. This confers special properties of both mesodermal and ectodermal lineage in them34. Arthur et al. postulated that the neural crest-derived cells are predisposed to neural differentiation35; the spontaneous neural differentiation of DPSCs was also reported by Kim et al.36. Another study has indicated the potential role of the distal C-terminus L-type voltage-gated calcium channel in directing DPSCs to attain a neuronal phenotype37. Previously, our lab demonstrated the cryopreservation efficiency of these DPSCs by preserving them at -80 °C for 5 years10. These cells were able to maintain stemness characteristics, proliferation, and differentiation ability, as well as a stable karyotype without any structural or numerical abnormalities. In addition, in a comparative analysis, our lab also demonstrated the superior tendency of DPSCs toward neurogenic7 and osteogenic8 lineages, as compared to BMSCs. Their superior competence and inherit tendency toward osteogenic and neural lineage make these cells the best-suited candidate for stem cell therapies in bone disorders and neurodegenerative diseases.
Altogether, we describe here an efficient protocol to obtain a pure population of DPSCs by the explant method, as evident by their stem cell marker expression and multilineage differentiation capability.
The authors have nothing to disclose.
We acknowledge the funding support to AK from the Department of Health Research (DHR), ICMR, Govt. of India (DHR-NRI Grant # R.12015/01/2022-HR). SR has received funding from ICMR, Govt. of India (Grant # 2020-7593/SCR-BMS) and PS has received fellowship from CSIR, Govt. of India. We are also thankful to Ms. Sandhya Tokhi and Ms. Bhupinder Kaur for assistance in flow cytometry, and central sophisticated instrumentation core (CSIC) and PGIMER, Chandigarh for providing infrastructural support.
6 well cell culture plate | Costar | 3516 | For cell culture |
Alcian blue stain | EZstain chondrocyte staining kit, HiMedia | CCK029 | |
alizarin red S stain | Sigma-Aldrich | TMS-008 | Osteogenic stain |
Antibiotic cocktail | Himedia | A002-5X50ML | To prevent culture contamination |
Ascorbic Acid | Himedia | TC094-25G | Chondrogenic induction |
B27 supplement | Gibco | 17504044 | For neural induction |
bFGF ( basic Fibroblast Growth Factor) | Gibco | PHG0024 | For neural induction |
CD 105 | BD-Pharmingen | 560839 | |
CD 35 | Biolegend | 343604 | |
CD 45 | Biolegend | 304006 | |
CD 73 | Biolegend | 344016 | |
CD 90 | Biolegend | 328107 | Characterization |
cetyl pyridinium chloride (CPC) | Sigma-Aldrich | 1104006 | For Alizarin Red extraction |
Dexamethasone 21-phosphate disodium | Sigma-Aldrich | D1159-100MG | |
Dulbecco's Phosphate Buffered Saline | Himedia | TS1006-5L | For washing purpose |
EGF (Epidermal Growth Factor) | Gibco | PHG0311 | For hepatic and neural induction |
EVOS LED microscope | Invitrogen | For fluorescence imaging | |
EZ stain Chondrocyte staining kit | Himedia | CCK029-1KT | Chondro stain Kit |
FACS Canto flow cytometer | BD Biosciences | For cell characterization | |
Fetal Bovine Serum | Gibco | 16000044 | For primary culture |
Fetal Bovine Serum | Sigma-Aldrich | F2442 | For cell culture |
G5 supplement | Gibco | 17503012 | For neural induction |
HGF( Hepatocyte Growth Factor) | Sigma-Aldrich | H1404 | For hepatic Induction |
HLA-DR | Biolegend | 307605 | |
Human TGF-β3 | Peprotech | #100-36E-10U | |
Insulin-Transferrin-Selenous acid premix | Sigma-Aldrich | I3146 | For hepatic Induction |
ITS premix | Corning | 354350 | |
LDL Uptake Assay kit | Abcam | ab133127 | For hepatic characterization |
Low glucose DMEM | Gibco | 11885-084 | For hepatic induction |
MAP2 antibody | Sigma-Aldrich | M4403 | For neural characterization |
N2 supplement | Gibco | 17502048 | For neural induction |
Neural Basal Media | Gibco | 21103049 | For neural induction |
NFM antibody | Sigma-Aldrich | N4142 | For neural characterization |
Nikon Elipse TS100 microscope | Nikon | For fluorescence imaging | |
Oil Red O | Sigma-Aldrich | 01391-250Ml | Adipogenic stain |
Oncostatin M | R&D Systems | 295-OM-010/CF | For hepatic Induction |
Petridish | Tarson | 460090-90MM | For tissue cutting |
Potassium phosphate monobasic | Sigma-Aldrich | 15655-100G | Osteogenic induction |
Propan-2-ol | Thermo Fisher | Q13827 | For Oil Red O extraction |
Sodium pyruvate solution | Sigma life sciences | S8636-100ML | |
Trypsin-EDTA | Sigma-Aldrich | T4049 | For cell passaging |
Whatman filter paper | merck | WHA1001325 | filter paper |
α- Minimum Essential Media (α-MEM) | Sigma-Aldrich | M0643-10X 1L | Media for primary culture |
β-glycerophosphate disodium salt hydrate | Sigma-Aldrich | G9422-50G |