Journal of Conservative Dentistry
Home About us Editorial Board Instructions Submission Subscribe Advertise Contact e-Alerts Login 
Users Online: 237
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size
 


 
Table of Contents   
ORIGINAL ARTICLE  
Year : 2013  |  Volume : 16  |  Issue : 5  |  Page : 423-428
Differentiation of isolated and characterized human dental pulp stem cells and stem cells from human exfoliated deciduous teeth: An in vitro study


1 Ahmedabad Municipal Corporation Dental College, Ahmedabad, Gujarat, India
2 Department of Conservative Dentistry and Endodontics, Dayananda Sagar College of Dental Sciences, Shavige Malleshwara hills, Kumarswamy Layout, Bangalore, Karnataka, India
3 Former CEO, Stempeutics India Limited, Bangalore, Karnataka, India
4 Adult Mesenchymal Stem Cell Group, Manipal University of Regenerative Medicine, Bangalore, Karnataka, India

Click here for correspondence address and email

Date of Submission17-Apr-2013
Date of Decision07-Jun-2013
Date of Acceptance17-Jul-2013
Date of Web Publication3-Sep-2013
 

   Abstract 

Aims and objectives: Isolation, characterization and differentiation of dental pulp stem cells (DPSCs) and stem cells from exfoliated human deciduous teeth (SHED).
Methods: The pulp tissue was digested in collagenase and cultured in DMEM Dulbecco's Modified Eagle's Media). The stem cells were identified and isolated. Surface characterization of cells was done with flow cytometer using surface markers. An immuno cytochemistry analysis was done. Differentiation potential was analyzed using various differentiation markers.
Results: Flow cytometry analyses for various CD markers showed similar results for both DPSCs and SHED. The cells showed positive expression for pluripotent, ectodermal and mesodermal markers. Cells differentiated into osteoblasts and adipocytes.
Conclusion: The study demonstrated that stem cells existed in deciduous and permanent pulp tissue. The stem cells present in pulp tissue can be isolated, cultivated and expanded in vitro. Both DPSCs and SHED show almost a similar expression pattern profile for variety of antigens tested.

Keywords: Dental pulp stem cell; dentin regeneration; pulp regeneration; stem cell; tissue engineering; tooth regeneration

How to cite this article:
Vishwanath VR, Nadig RR, Nadig R, Prasanna JS, Karthik J, Pai VS. Differentiation of isolated and characterized human dental pulp stem cells and stem cells from human exfoliated deciduous teeth: An in vitro study. J Conserv Dent 2013;16:423-8

How to cite this URL:
Vishwanath VR, Nadig RR, Nadig R, Prasanna JS, Karthik J, Pai VS. Differentiation of isolated and characterized human dental pulp stem cells and stem cells from human exfoliated deciduous teeth: An in vitro study. J Conserv Dent [serial online] 2013 [cited 2019 Jul 24];16:423-8. Available from: http://www.jcd.org.in/text.asp?2013/16/5/423/117509

   Introduction Top


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 and have 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 human dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), and periodontal ligament apical papilla of immature teeth. It has been reported that the stem cells obtained from the above said sources can generate dentin like tissue both in vitro and in vivo studies in animals. [1],[2],[3] Transplanted skeletal or dental stem cells have the potential to "rephrase" repair craniofacial defects and repair/regenerate teeth. [2],[3],[4],[5]

The objectives of regenerative endodontic procedures are to: a) Regenerate pulp-like tissue, b) repair damaged coronal dentin following a carious exposure of pulp tissue, c) regenerate resorbed root, and d) induce apicogenisis.

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 histocompatability. [6] This would be a more biological approach rather mere mechanical one. [7]

It has been suggested that 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, 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 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 cells obtained from adult (DPSCs) as well as deciduous teeth (SHED).


   Materials and Methods Top


Twenty normal human permanent teeth from adults of age less than 25 years and 20 normal human deciduous teeth were collected. 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 (FBS; HyClone USA), 5× antibiotic-antimycotic (stock solution-100×) (GIBCO, Invitrogen Corporation), and 5 mM L-glutamine. The enriched media was filtered through a 0.2 μm syringe filter (Millipore) and stored at 4°C.

Roswell Park Memorial Institute (RPMI) basal Medium (GIBCO, Invitrogen) was supplemented with 10% FBS (HyClone USA), 5× antibiotic-antimycotic, and 5 mM L-glutamine. The enriched media was filtered through a 0.2 μm syringe filter and stored at 4°C.

Sample collection, storage, and handling

Teeth surfaces were cleaned and cut using sterilized dental burs to reveal the pulp chamber, pulp was gently separated using a small size broach, and a blunt non-cutting forceps stored in a falcon containing FBS and transported to the laboratory.

Pulp tissue was digested in a solution of 3 mg/ml collagenase and 4 mg/ml dispase for 1 h till the tissue digested. The cells were removed using a micropipette (Gilson's Pipetman®), resuspended in 5-10 ml Dulbecco's PBS (DPBS) (GIBCO, invitrogen) and centrifuged at 1,800 rpm for 5 min to obtain a pellet containing cells. The supernatant was discarded and the pellet was resuspended in 5 ml of DMEM knockout media. Suspension was then plated onto a T25 culture flask and incubated for 3 days in a carbon dioxide incubator maintained at 37°C and 5% CO 2 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).

Oct4 and Nanog being the transcription factors were used to identify the stem cells and view under fluorescence microscope. Fluorescent stains diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate (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 twice with DPBS for 5 min to remove traces of FBS. 0.25% pre-warmed trypsin-ethylenediaminetetraacetic acid was added to the flask, was agitated 3 min to resuspend adherent cells, and viewed under microscope to check for any remaining adherent cells. Media containing FBS was added to neutralize trypsin and centrifugation was carried out at 1,800 rpm for 5 min to obtain a pellet containing cells. Supernatant was discarded and pellet was resuspended in 6 ml of DMEM knockout media. To fresh T25 flasks, 3 ml of DMEM knockout media and 2 ml of the cell suspension was added, incubated, and subsequent passages were carried out. 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.

Reverse transcription-polymerase chain reaction

First strand cDNA synthesis was performed by using a first strand cDNA synthesis kit and oligo dT primer, diluted with MgCl 2 and made ready for PCR reactions.

Characterization of cells

Done with the help of flow cytometry using surface markers like HLA-DR, CD73, CD44, CD106, CD34, CD10, CD123, CD7, CD31 following which Reverse transcription-polymerase chain reaction (RT-PCR) was done using primers like Tdgf, Rex1 Oct4 Sox2, hTert, Ncam1, b3 tubulin, Nestin, Handl, Brachury, Bmp4, and Gapdh.

Osteoblastic differentiation


After exposure to conditioned medium, cells were washed with PBS, fixed with 10% formalin, and washed again three times with PBS. Extracellular alkaline phosphatase (ALP) activity was examined histochemically using Fast Violet B salt (Sigma). After ALP staining, the samples were washed with PBS and stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma). Images were taken at random locations.

Mineralization

Medium was removed, cells washed with PBS, and evaluated for calcium production by staining with 10% alizarin Red and Von Kossa as these dyes bind to calcium salts.

Adipocytic differentiation

Cells were plated in 10% DMEM and incubated at 37°C in 10% CO2. Preadipocytes were grown to confluency in DMEM. A distinct change in the morphology of the cells was observed in the next 2 days. The media was changed to insulin media and then to 10% FBS. Cells were fed with 10% FBS every 2 days and visualized using Oil O Red stain after full differentiation.


   Results Top


Isolation and expansion of cells

Identification of cells was done with the help of a phase contrast microscope at ×10 magnification. [Figure 1] depicts the cells at various stages of confluency. At the end of 5 th day, the cells reach about 90% confluency after which they are trypsinized at 0.25% concentration and replated. Subsequently immunoflouroscence was performed to check for the expression of pluripotency markers, Oct4 and Nanog.
Figure 1: Mesenchymal stem cells derived from the dental pulp (a) at 24 h, (b) at 48 h, (c) after 3 days, and (d) after 5 days post plating

Click here to view


Examination of cells was done under fluorescence microscope at ×60 magnification [Figure 2]. 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 five passages and characterized for mesenchymal markers by flow cytometry.
Figure 2: Expression of self-renewal markers by DPSCs. Immunoflourescence picture depicting the expression of Oct4 and Nanog in DPSCs. DAPI staining had also been performed to detect all the cells in the field

Click here to view


In the present study [Figure 3], the cells were strongly positive for lymphocyte differentiation marker CD73, early adhesion and hyaluronan marker CD 44, and leukocytic cell marker CD 10; 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), T cell marker CD 7, and endothelial cell marker CD 31. The cells were dimly positive for hematopoietic stem cell marker CD123, found to express Oct4 at both the mRNA and protein levels.
Figure 3: Immunophenotyping of dental pulp derived stem cells (a) depicts the stem cells from deciduous teeth and (b and c) depicts stem cells from permanent teeth

Click here to view


[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.
Figure 4: mRNA analysis of pluripotency markers and derm markers in dental pulp derived stem cells at different passages (a) showing the analysis of pluripotency markers, (b) depicting analysis of ectodermal markers, and (c) mesodermal markers. Positive control indicates embryonic stem cell control and negative control depicts the cDNA control

Click here to view


SHED and DPSCs showed similar results. The cells however did not express endodermal markers.

Differentiation of cells

Differentiated cells were visualized using Alizarin Red and Von Kossa stain for mineralization. Oil Red O stain was used to look for adipocytes.

Differentiation of stem cells

[Figure 2] shows the differentiation of DPSCs into osteoblasts and adipocytes with use of Von Kossa stain and Oil Red O stain, respectively. [Figure 3] shows the osteoblastic differentiation at higher magnification at 15 th and 21 st day. [Figure 4] shows the adipocytes differentiated from DPSCs at higher magnification.


   Discussion Top


This study was conducted using the pulp tissue from deciduous and permanent teeth of patients below the age of 25 years because of the presence of more cells and less fiber tissue, aiding the motive of our study.

The media used in the study was knockout DMEM which optimized the 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 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. They failed to react with endothelial cell marker CD106, immune cell marker HLA-DR, consistently negative for CD34 (marker for early hematopoietic stem cells), negative for T cell marker CD 7, endothelial cell marker CD 31(PECAM-1), 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 bone marrow stem cells (BMSCs) failed to react with the hematopoietic markers CD14, CD45, and CD34.

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

The study by Miura et al., showed that ex vivo-expanded 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 BMSCs and DPSCs. Pierdomenico demonstrated that the cells expressed CD29, CD166; while CD45, CD34, and CD14 proved negative which is again in confluence with our study. [9] 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 Sonoyama, stem cells from the apical papilla (SCAP) 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 BMSCs 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 BMSCs. [11]

Cheng did experiments on chimpanzee teeth 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 for CD29, CD44, CD59, CD73, CD90, CD105, CD150, and CD166. The present study also got similar results. [12]

In the present study, RT-PCR analysis of the expression of various pluripotency markers and derm markers showed positive expression for pluripotent markers, ectodermal markers, and mesodermal markers expressing Oct4 at both the mRNA and protein levels. They expressed ectodermal markers like ncam1, β3 tubulin and nestin, and mesodermal markers like hand1, bmp4, and gapdh. Both SHED and DPSCs showed similar results indicating that these cells might have the capacity to differentiate into ectoderm and mesodermal organs. [13] The cells 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 dentin sialophosphoprotein (DSPP), bone sialoprotein (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. [5] Another crucial study, suggests that nestin plays a potential role in odontoblast differentiation and that bone morphogenic protein-4 is involved in nestin upregulation. [8]

mRNA isolated from chimpanzee DPSCs (ChDPSCs) was used for RT-PCR analysis by Cheng, the expression of stem cell (Nanog, Rex 1, and Oct4) and differentiation (osteonectin, OC, and osteopontin) markers was detected. BSP was not detected in ChDPSCs. [12]

Although isolation and 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 Top


  • The study demonstrated that stem cells existed in human deciduous and permanent pulp tissue that can be isolated, cultivated, and expanded in vitro
  • These stem cells can be successfully differentiated into adipocytes and osteoblasts
  • Further studies should include analysis of diverse cell populations to elucidate their potential to differentiate into various other cell types followed by in vivo studies in animals and humans
  • 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. [14]


 
   References Top

1.Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem cell properties of human dental pulp stem cells. J Dent Res 2002;81:531-5.  Back to cited text no. 1
[PUBMED]    
2.Gronthos S, Mankani M, Brahmin J, Robey PG, Shi S. Postnatal human dental pulp cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 2000;97:13625-30.  Back to cited text no. 2
    
3.Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, et al. SHED: Stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A 2003;100:5807-12.  Back to cited text no. 3
[PUBMED]    
4.Robey PG. Stem cells near the century mark. J Clin Invest 2000;105:1489-91.  Back to cited text no. 4
[PUBMED]    
5.Wang X, Thibodeau B, Trope M, Lin LM, Huang GT. Histologic characterization of regenerated tissues in canal space after the revitalization/revascularization procedure of immature dog teeth with apical periodontitis. J Endod 2010;36:56-63.  Back to cited text no. 5
[PUBMED]    
6.Sedgley CM, Botero TM. Dental stem cells and their sources. Dent Clin North Am 2012;56:549-61.  Back to cited text no. 6
[PUBMED]    
7.Shetty S, Farooq U, Chandra SB, Nanjappa S. Molecular biology and preservation of tooth vitality current implication. Endodontol 2006;2:12.  Back to cited text no. 7
    
8.Ward CM, Barrow K, Woods AM, Stern PL. The 5T4 oncofoetal antigen is an early differentiation marker of mouse ES cells and its absence is a useful means to assess pluripotency. J Cell Sci 2003;116:4533-42.  Back to cited text no. 8
[PUBMED]    
9.Pierdomenico L, Bonsi L, Calvitti M, Rondelli D, Arpinati M, Chirumbolo G, et al. Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation 2005;80:836-42.  Back to cited text no. 9
[PUBMED]    
10.Kerkis I, Kerkis A, Dozortsev D, Stukart-Parsons GC, Gomes Massironi SM, Pereira LV, et al. Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cell markers. Cells Tissues Organs 2006;184:105-16.  Back to cited text no. 10
[PUBMED]    
11.Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C, et al. Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLos One 2006;1:e79.  Back to cited text no. 11
[PUBMED]    
12.Cheng PH, Snyder B, Fillos D, Ibegbu CC, Huang AH, Chan AW. Postnatal stem/progenitor cells derived from the dental pulp of adult chimpanzee. BMC Cell Biol 2008;9:20.  Back to cited text no. 12
[PUBMED]    
13.Shekar R, Ranganathan K. Phenotypic and growth characterization of human mesenchymal stem cells cultured from permanent and deciduous teeth. Indian J Dent Res 2012;23:838-9.  Back to cited text no. 13
[PUBMED]  Medknow Journal  
14.Brar GS, Toor RS. Dental stem cells: Dentinogenic, osteogenic, and neurogenic differentiation and its clinical cell based therapies. Indian J Dent Res 2012;23:393-7.  Back to cited text no. 14
[PUBMED]  Medknow Journal  

Top
Correspondence Address:
Veena S Pai
224/8/1, Mahamaya, 2nd Main, 1st Cross, Byrappa Block. T. R Nagar, Bangalore - 560 028, Karnataka
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-0707.117509

Rights and Permissions


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

This article has been cited by
1 Stem Cell Therapy for Osteoporosis
Ben Antebi,Gadi Pelled,Dan Gazit
Current Osteoporosis Reports. 2014;
[Pubmed] | [DOI]



 

Top
 
 
 
  Search
 
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  
 


    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
   Conclusion
    References
    Article Figures

 Article Access Statistics
    Viewed3571    
    Printed73    
    Emailed2    
    PDF Downloaded246    
    Comments [Add]    
    Cited by others 1    

Recommend this journal