Isolation and characterization of human dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) – an in vitro study

INTRODUCTION

Stem cells are defined as clonogenic cells capable of both self-renewal and multi lineage differentiation.¹ Stem cells have been differentiated from variety of body parts. Stem cell therapy has shown great promise in the management of variety of diseases in medicine.

Recently stem cells have been isolated and grown from pulp tissue of permanent teeth (DPSCs), deciduous teeth (SHED), periodontal ligament, apical papilla from a immature tooth and it has been reported that they generated dentin like tissue both in vitro and in vivo studies in animals.^1,2,3 Transplanted skeletal or dental stem cells may one day be used to repair craniofacial bone or even repair or regenerate teeth.^2,3,4,5

The objectives of regenerative endodontic procedures are to regenerate pulp-like tissue, ideally, the pulp-dentin complex; regenerate damaged coronal dentin, such as following a carious exposure; and regenerate resorbed root, cervical or apical dentin.

If we have in hand populations of stem cells that reproducibly reform bone, cementum, dentin, and perhaps even periodontal ligament, it is possible to envision complete restoration of the hard tissues in the oral cavity using the patient’s own cells, thereby avoiding issues of his to compatability.⁶ This would be a more biological approach rather mere mechanical one.⁷

Recently it has been suggested that naturally occurring exfoliated teeth would be similar in some way to umbilical cord containing stem cells that may offer a unique stem cell resource for potential clinical applications. Gronthos et al in his study found that the deciduous tooth had multipotent stem cells which were highly proliferative and clonogenic capable of differentiating, into variety of cell types including neural cells, adipocytes and odontoblasts. After in vivo transplantation they were able to induce bone, generate dentin and survive in mouse brain along with expression of neural markers.²

Hence there is a need to gain clarity and further insight into specific properties of stem cells, derived from both, adult and deciduous tooth pulp, study their proliferation abilities, differentiation potential, and immunoreactivity profiles as these findings are likely to open up new horizons to make this concept a reality. Therefore methods to isolate and characterize the stem cell population are a preliminary step of any such research and are crucial for the development of novel therapies based on stem cell regeneration.

This study was undertaken to isolate and characterize dental pulp stem cellsobtained from adult(DPSCs) as well as deciduous teeth (SHED).

MATERIALS & METHODOLOGY

20 normal human permanent teeth from adults of age less than 25 years and 20 normal human deciduous teeth were collected for the study. Third molars, premolars and retained deciduous teeth indicated for extraction for orthodontic reasons were included in the study. Teeth with dental caries, pulpal, Periapical and periodontal disease, were excluded.

Preparation of culture media

Dulbecco’s Modified Eagle’s Media (DMEM) Knockout Media (GIBCO, Invitrogen) was supplemented with 10% Fetal Bovine Serum (HyClone USA), 5X Antibiotic-Antimycotic (Stock solution-100X) (GIBCO, Invitrogen Corporation) and 5mM L-glutamine. The enriched media was filtered through a 0.2 micron syringe filter (Millipore) and stored at 4˚C. Roswell Park Memorial Institute (RPMI) basal Medium (GIBCO, Invitrogen) was supplemented with 10% Fetal Bovine Serum (HyClone USA), 5X antibiotic-antimycotic and 5mM L-glutamine. The enriched media was filtered through a 0.2 micron syringe filter and stored at 4˚C.

Sample collection, storage and handling:

Normal human extracted permanent teeth and extracted/ exfoliated deciduous teeth were collected, cleaned and cut using sterilized dental burs to reveal the pulp chamber. Pulp was gently separated from the crown and roots using a small size broach and a blunt non cutting forceps. The extirpated pulp was stored in a falcon containing FBS (Fetal Bovine Serum) and transported to the laboratory, where further experiments were conducted.

Pulp tissue was digested in a solution of 3 mg/ml collagenase and 4 mg/ml dispase for 1 hour till the tissue digested. The cells were removed using a micropipette (Gilson’s Pipetman®) and were re-suspended in 5-10ml Dulbecco’s Phosphate Buffer Saline (DPBS) (GIBCO, invitrogen). It was then centrifuged at 1800 rpm for 5 minutes to obtain a pellet containing cells. The supernatant was discarded and the pellet was re-suspended in 5ml of DMEM Knockout media. Suspension was then plated onto a T25 Culture Flask and incubated in a carbon dioxide incubator which was maintained at 37˚C and 5% CO². Cells were incubated for 3 days for cell adhesion to occur, after which the media was replaced with fresh complete DMEM knockout media with 10%FBS. Subsequent media changes were carried out using the same media once in 2 days. Incubation was carried out till 80% confluence was observed under a phase contrast microscope (Nikon).

The cells were identified using a fluorescence microscope. OCT 4 and NANOG being the transcription factors were used to identify the stem cells and view under fluorescence microscope. Fluorescentstains DAPI and FITC were used to identify the stem cells as these cells will be stained.

Expansion and Subculturing

Once the culture reached 80% confluency, media was aspirated from the T25 flask. The flask was washed two times with DPBS for 5 minutes with gentle shaking to remove traces of FBS in media which contains trypsin inhibitors. 0.25% pre-warmed trypsin-EDTA was added to the flask, was agitated 3 minutes to re-suspend adherent cells. The flask was viewed under microscope to check for any remaining adherent cells. Media containing FBS (three times the volume of trypsin) or FBS (half the volume of trypsin) was added to neutralize trypsin and centrifugation was carried out at 1800 rpm for 5 min to obtain a pellet containing cells. Supernatant was discarded and pellet was re-suspended in 6ml of DMEM knockout media. To three fresh T25 flasks, 3ml of DMEM knockout media and 2ml of the cell suspension was added and incubated. Subsequent passages were carried out in T25 flasks or 35mm dishes. Aggregates of more than or equal to 50 cells were considered as colonies.

RNA Isolation

High quality intact RNA is essential for full length, high quality cDNA synthesis. Total RNA was prepared from collagenase/dispase digested cell suspensions of 6 week old DPSCs and SHED by using RNA STAT-60.

Materials Required

Confluent cultures, Trizol reagent (Inviitrogen). Chloroform (Merck), Isopropanol (Merck), 75% ethanol (Merck), RNase- and DNAse-free waiter (Sigma)

Protocol

• Cells were washed twice with Ix PBS and lysed by adding 1 ml Trizaol reagent in 35 mm Petri dish and passing the cells several times through the pipette.

• The lysate was transferred to 1.5 ml centrifuge tube and incubated for 5 minutes at room temperature.

• 200 µl of Chloroform was added to the tube, mixed by inverting 5 times and incubating for 2-3 minutes at room temperature.

• Centrifugation at 13000 rpm for 15 minutes at 4°C was done.

• Following centrifugation, the mixture separates into lower phenol chloroform red phase; an interphase and a colorless aqueous upper phase.

• Aqueous phase was transferred to a fresh tube.

• 0.5ml of isopropyl alcohol was added, mixed and incubated at room temperature for 10 minutes, to precipitate RNA,

• Centrifugation at 12000 rpm for 10 minutes at 4°C was done.

• The RNA pellet was washed with 75% ethanol twice.

• The RNA pellet was dried and dissolved in 30 µl of RNAse- and DNase-free water.

Reverse transcription – PCR

In molecular biology, reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique for amplifying a defined piece of a ribonucleic acid (RNA) molecule. First strand cDNA synthesis was perfomed by using a first strand cDNA synthesis kit and oligo dT Primer. It was diluted with MgCl2 and made ready for the PCR reactions. Characterization of cells

Characterization of cells

was done with the help of flow cytometry using surface markers like HLA-DR, CD73, CD44, CD106, CD34, CD10, CD123, CD7, CD31 following which RT-PCR was done using primers like Tdgf, Rex1 Oct4Sox2, hTert, Ncam1, b3 tubulin, Nestin, Handl, Brachury, Bmp 4, Gapdh.

RESULTS

Isolation and expansion of cells

Identification of cells was done with the help of a phase contrast microscope (NIKON ECLIPSE TE2000-U) at 10x magnification. The figures depict the cells at various stages of confluency. At the end of 5th day the cells reach about 90% confluency. After the cells reached confluence (5 days post plating) the cells were trypsinized at 0.25% concentration and replated. Subsequently immunoflouroscence was performed to check for the expression of pluripotency markers, oct4 and nanog. Examination of cells was done under fluorescence microscope (NIKON ECLIPSE TE2000-U) at 60x magnification. Oct4 and nanog being the stem cell specific markers were used to identify the stem cells within the heterogeneous cell adherent population. Cells were propagated to a minimum of 5 passages and characterized for mesenchymal markers by flow cytometry.

Figure 3 shows the Flow cytometry analysis of dental pulp derived stem cells using a panel of cell surface markers. Figure (A) shows the characterization of SHED. Figure (B) and (C) show the characterization of DPSCs. In the present study, the cells were strongly positive for lymphocyte differentiation marker CD73, early adhesion and hyaluronan marker CD 44, and leukocytic cell marker CD 10. Cultures of DPSC and SHED failed to react with endothelial cell marker CD106, immune cell marker HLA-DR, and were consistently negative for CD34 (marker for early hematopoietic stem cells). They were also negative for T cell marker CD 7 and endothelial cell marker CD 31 .The cells were dimly positive for hematopoietic stem cell marker CD123.

Figure 4 shows RT-PCR analysis of the expression of various pluripotency markers and derm markers in dental pulp derived stem cells at different passages. The cells showed positive expression for pluripotent markers, ectodermal markers and mesodermal markers. The cells were found to express oct4 at both the mRNA and protein levels. They expressed ectodermal markers like ncam1, β3 tubulin and nestin. They also expressed mesodermal markers like hand1 , bmp4 and gapdh. SHED and DPSCs showed similar results. The cells however did not express endodermal markers.

DISCUSSION

This study along with the other previous studies provides evidence that remnant dental pulp derived from DPSCs and SHED contains a multipotent stem-cell population. These stem cells can be isolated and expanded, thereby providing a unique and accessible population of stem cells from an easily available tissue resource.

This study was conducted using the pulp tissue from deciduous and permanent teeth of patients below the age of 25 years. Extracted teeth from young patients were selected because of the presence of more cells and less fiber tissue in the pulp, aiding the motive of our study. The pulp tissue was transported to the laboratory within an hour.

The media used in the study was knockout DMEM (Dulbecco’s Modified Eagle’s Media containing antibiotic and antimycotic) [Invitrogen]. Knockout™ D-MEM is a basal medium optimized for growth of undifferentiated Embryonic Stem cells that has fewer components than the other Medias, enriched for more self-renewing population.⁸

A flow cytometry analysis of dental pulp derived stem cells using a panel of cell surface markers revealed a similar expression pattern for a variety of markers for both DPSCs and SHED. In the present study, the cells were strongly positive for lymphocyte differentiation marker CD73, early adhesion and hyaluronan marker CD 44, and leukocytic cell marker CD 10.

Cultures of DPSC and SHED failed to react with endothelial cell marker CD106, immune cell to express oct4 at both the mRNA and protein levels. They expressed ectodermal markers like ncam1, β3 tubulin and nestin. They also expressed mesodermal markers like hand1 , bmp4 and gapdh. SHED and DPSCs showed similar results. The cells however did not express endodermal markers.

marker HLA-DR, consistently negative for CD34 (marker for early hematopoietic stem cells). They were also negative for T cell marker CD 7 and endothelial cell marker CD 31(PECAM-1). The cells were dimly positive for hematopoietic stem cell marker CD123. These results show that these cells are not hematopoietic in origin and that they are pure mesenchymal stem cells. Since single marker can be expressed by a variety of cells, positive and negative expression of multiple markers were sought after, for the characterization of cells.

The present study goes along with the study of Gronthos, who showed that profiles for both cell populations of DPSC and BMSC (bone marrow stem cells) failed to react with the hematopoietic markers CD14 (monocyte/macrophage), CD45 (common leukocyte antigen), CD34 (hematopoietic stem/progenitor cells/endothelium).

In general, DPSCs and BMSCs(Bone marrow stem cells) exhibited a similar expression pattern for a variety of markers associated with endothelium vascular cell adhesion molecule.¹ smooth muscle (a-smooth muscle actin), bone (alkaline phosphatase, type I collagen, osteonectin, osteopontin, and osteocalcin), and fibroblasts (type III collagen and fibroblast growth factor.²

Masako Miura in his study showed that ex vivoexpanded SHED were found to express the cell surface molecules STRO-1 and CD146, the two early mesenchymal stem-cell markers previously found to be present in BMSSCs and DPSCs. Laura Pierdomenico demonstrated that the cells expressed CD29, CD166, while CD45, CD34, CD14 proved negative which is again in confluence with our study.⁹ Irina Kerkis showed in her study that immature dental pulp stem cells are positive for CD13 and CD31 and negative for CD34, CD43, and CD45.¹⁰

According to Wataru Sonoyama, SCAP(stem cells from the apical papilla) at passage 1 expressed many surface markers including STRO-1, ALP, CD24, CD29, CD73, CD90, CD105, CD106, CD146, CD166 and ALP but were negative for CD34, CD45, CD18 and CD150. STRO-1 and CD146 have been identified as early Mesenchymal stem cell markers present on both BMMSCs and DPSCs. According to the researcher, CD24 appears to be a specific marker for SCAP, not detectable in other mesenchymal stem cells including DPSCs and BMMSCs.¹¹

Pei-Hsun Cheng, did experiments on chimpanzee teeth. There was 98% similarity of their genomes, making the chimpanzee the closest living relative to humans; and found that Both chimpanzee DPSCs and human BMSCs share identical expression profiles on common cell surface antigens. They were all negative for hematopoietic cell surface markers: CD14, CD18, CD24, CD34, and CD45; and positive CD29, CD44, CD59, CD73, CD90, CD105, CD150 and CD166. The present study also got similar results.¹

In the present study, RT-PCR analysis of the expression of various pluripotency markers and derm markers was done at different passages. The cells showed positive expression for pluripotent markers, ectodermal markers and mesodermal markers. The cells were found to express oct4 at both the mRNA and protein levels. They expressed ectodermal markers like ncam1, β3 tubulin and nestin. They also expressed mesodermal markers like hand1, bmp4 and gapdh. SHED and DPSCs showed similar results. This indicates that these cells might have the capacity to differentiate into ectoderm and mesodermalorgans. The cells however did not express endodermal markers.

Oct-4 and Nanog, markers of cells were expressed which indicates the pluripotent behavior of the cells. However the expression pattern needs to be confirmed at a protein level to emphasize if there is any physical significance.

These results were similar to the results obtained by Gronthos, in which transcripts for DSPP, BSP, OC, and glyceraldehyde-3- phosphate dehydrogenase were detected. Even Masuka moura showed that SHED expressed a variety of neural cell markers including nestin, III-tubulin, as measured by immunocytochemical staining.5Another crucial study, suggests that nestin plays a potential role in odontoblast differentiation and that bone morphogenic protein-4 is involved in nestin up-regulation.⁸

mRNA isolated from chimpanzee DPSCs (ChDPSCs) was used for RT-PCR analysis by Pei-Hsun Cheng,. The expression of stem cell (Nanog, Rex-1, Oct-4) and differentiation (Osteonectin, Osteocalcin, Osteopontin) markers was detected. Bone sialoprotein was not detected in ChDPSCs.¹²

In our study, out of 20 samples used, few of them could not be successfully processed and expanded in vitro suggesting the complexity and technique sensitivity of the procedure involved. Furthermore, the risk of contamination, viability of the cells, addition of ideal factors, inherent nature of the cells and the requirement of a perfect environment cannot be overlooked in performing this study. Identifying and rectifying these limitations is important to complement the technique of isolation, expansion and maintenance of these multilineage cell lines.

Although isolation, characterization is a preliminary step in stem cell research, it is certainly a crucial step and the data obtained will aid in conducting further research on efficacy of ex vivo expanded stem cells for various cell based therapies in dentistry.

Extensive studies in vivo, on animals with suitable combination of growth factors and scaffold materials, is essential before resorting to human trails.

CONCLUSION

1. The study demonstrated that stem cells existed in human deciduous and permanent pulp tissue.

2. The stem cells present in deciduous and permanent teeth pulp tissue can be isolated, cultivated and expanded in vitro.

3. Further studies should include analysis of diverse cell populations to elucidate their potential to differentiate into various cell types followed by in vivo studies in animals and humans.

4. Our study together with the work of other studies indicates the potential for using DPSCs and SHED as a source of pluripotent stem cells for future cellular based therapies in medicine and dentistry.