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ORIGINAL ARTICLE
Year : 2016  |  Volume : 8  |  Issue : 1  |  Page : 16-21

OCT4 and SOX2 are reliable markers in detecting stem cells in odontogenic lesions


Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Institute, Bangalore, Karnataka, India

Date of Web Publication6-May-2016

Correspondence Address:
Dr. Venkatesh Vishwanath Kamath
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Institute, Munnekolala, Marathalli, Bangalore - 560 037, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0975-8844.181920

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  Abstract 

Context (Background): Stem cells are a unique subpopulation of cells in the human body with a capacity to initiate differentiation into various cell lines. Tumor stem cells (TSCs) are a unique subpopulation of cells that possess the ability to initiate a neoplasm and sustain self-renewal. Epithelial stem cell (ESC) markers such as octamer-binding transcription factor 4 (OCT4) and sex-determining region Y (SRY)-box 2 (SOX2) are capable of identifying these stem cells expressed during the early stages of tooth development. Aims: To detect the expression of the stem cell markers OCT4 and SOX2 in the normal odontogenic tissues and the odontogenic cysts and tumors. Materials and Methods: Paraffin sections of follicular tissue, radicular cyst, dentigerous cyst, odontogenic keratocyst, ameloblastoma, adenomatoid odontogenic tumor, and ameloblastic carcinoma were obtained from the archives. The sections were subjected to immunohistochemical assay by the use of mouse monoclonal antibodies to OCT4 and SOX2. Statistical Analysis: The results were evaluated by descriptive analysis. Results: The results show the presence of stem cells in the normal and lesional tissues with these stem cell identifying markers. SOX2 was found to be more consistent and reliable in the detection of stem cells. Conclusion: The stem cell expressions are maintained in the tumor transformation of tissue and probably suggest that there is no phenotypic change of stem cells in progression from normal embryonic state to its tumor component. The quantification and localization reveals interesting trends that indicate the probable role of the cells in the pathogenesis of the lesions.

Keywords: Octamer-binding transcription factor 4 (OCT4), odontogenic lesions, sex-determining region Y (SRY)-box 2 (SOX2), stem cells


How to cite this article:
Banerjee A, Kamath VV, Sundaram L, Krishnamurthy SS. OCT4 and SOX2 are reliable markers in detecting stem cells in odontogenic lesions. J Orofac Sci 2016;8:16-21

How to cite this URL:
Banerjee A, Kamath VV, Sundaram L, Krishnamurthy SS. OCT4 and SOX2 are reliable markers in detecting stem cells in odontogenic lesions. J Orofac Sci [serial online] 2016 [cited 2018 Dec 16];8:16-21. Available from: http://www.jofs.in/text.asp?2016/8/1/16/181920


  Introduction Top


Odontogenic lesions are one the most commonly encountered pathological entities in the head and neck region. The molecular pathogenesis and the mechanisms involved in their aggressive behavior are mostly unknown. A probable explanation for these could be the presence of stem cells, hypothesizing that odontogenic lesions contain a small population of the stem cells, which has three important imbibed properties such as self-renewal, colony formation, and pluripotency. [1],[2],[3] Stem cells are unspecialized cells defined as clonogenic cells that have the capacity for self-renewal and the potential to differentiate into one or more mature cellular lineages. [4],[5] They support in the histiogenesis and organogenesis during development, maintaining a balance in the cell turnover process and also have a regenerative capacity in the adult tissues. [6] It is generally a well-agreed fact that the embryo is a potential source of pluripotent progenitor cells, which divides and gives rise to different types of cells, specialized in forming different tissues.

There are two initial concepts, which support the stem cell model - first, the Caenorhabditis elegans that is a small worm the entire lineage map of which has been described and second, the hematopoietic cell lineage, which has a more emphasized representation of the diverging pathways of cell lineage. [7] There are numerous factors, which control the molecular and cellular patterns of expression of the stem cells. Human stem cells are broadly of two types - the somatic type and the germline type. [8] Dental stem cells are mesenchymal stem cells, which are derived from the somatic variety. [8] Based on the sources of the dental stem cells, it is further classified in to dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), dental follicle precursor cells (DFPCs), and stem cells of apical papilla (SCAPs). [8] The derivation of various sources of stem cells has been shown in [Figure 1]. There are few cancer stem cell (CSC) markers such as CD133, CD44, and ABCG2, which have shown positive expression in ameloblastomas and have pointed to the role of resident stem cells in tumor progression. [9],[10],[11]
Figure 1: The lineage of stem cells in the human

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There are four important cell specific factors, which are expressed in the cells with pluripotent capacity, i.e., octamer-binding transcription factor 4 (OCT4), sex-determining region Y (SRY)-box 2 (SOX2), NANOG, and c-Myc. [4],[12] These factors also get combined together and show their expression. OCT4 and SOX2 are the two major transcription factors, which are required to maintain the pluripotent and self-renewal capacity. [6] ESC markers such as OCT4 and SOX2 are capable of identifying these stem cells expressed during the early stages of tooth development (dental papilla and dental lamina cells). [13],[14] This study is focused on the identification of stem cells in the selected odontogenic lesions by the two potential markers, OCT4 and SOX2.


  Materials and Methods Top


This study aimed at evaluation of OCT4 and SOX2 expression in odontogenic tissues and odontogenic lesions. The study was approved by the institutional review board (IRB). The study involved the use of formalin fixed paraffin embedded tissues of previously diagnosed cases of follicular cysts, radicular cysts, dentigerous cysts, adenomatoid odontogenic tumor, ameloblastoma, and ameloblastic carcinoma from our department. Relevant features such as age, sex, and site were obtained from the records of the patients. Seminoma and glioma were used as the positive controls of OCT4 and SOX2 markers, respectively.

A total of two cases of follicular tissues, four cases of radicular cysts, two cases of dentigerous cysts, two cases of odontogenic keratocysts, two cases of adenomatoid odontogenic tumors, four cases of ameloblastomas, one case of ameloblastic carcinoma, one case of seminoma (for OCT4), and one case of glioma (SOX2) were assessed. The following chemicals were used: Anti-rabbit monoclonal secondary antibody, anti-OCT4 rabbit monoclonal primary antibody, anti-SOX2 rabbit monoclonal primary antibody (PathnSitu labs), 3,3'-diaminobenzidine (DAB) chromogen, hydrogen peroxide (0.3%), bovine serum albumin (BSA), phosphate buffer saline (PBS), sodium citrate buffer (pH 6.0), xylene, alcohol (50%, 70%, and 100%), distilled water, Harris' hematoxylin, mounting medium (DPX). Serial sections of 4-um thicknesses were made onto silane-coated slides for immunohistochemistry. The tissue sections were deparaffinized and were then rehydrated. Endogenous hydrogen peroxide in tissue sections was blocked by immersing the slides in PBS containing 0.3% hydrogen peroxide in a staining bath for 30 min at room temperature and then the antigen was retrieved by using the microwave method (three cycles). After antigen retrieval, the staining bath was placed at room temperature for 20 min. The tissue sections were incubated with a mixture of 3% BSA (blocking agent) and 1% PBS in a humid chamber at room temperature for 30 min. After 30 min, the excess BSA from tissue sections was removed. The slides were washed by immersing in PBS for 5 min at room temperature. The tissue sections were incubated with rabbit monoclonal primary antibody in a humidifying chamber for 45 min. The slides were washed by immersing in PBS for 5 min at room temperature. The tissue sections were incubated with rabbit monoclonal secondary antibody at 1:300 dilution in 2% BSA-TBS for 35 min at room temperature. The slides were washed by immersing in PBS for 5 min at room temperature (repeated twice). The DAB substrate was prepared just prior to use. The slides were immersed in DAB substrate solution in a staining bath at room temperature for 5 min. The tissue sections were counterstained by immersing the slides in hematoxylin in a staining bath for 2 min. The slides were washed in running tap water for 5 min. The tissue sections were dehydrated by immersing the slides in increasing concentrations of alcohol (50%, 70%, 100%) in a staining bath for 3 min each at room temperature. The slides were immersed in xylene in a staining bath for 6 min at room temperature (repeated twice). The slides were mounted in permanent mounting medium and air-dried. The staining was observed under a light microscope. Anti-OCT4 and anti-SOX2 antibodies stained brown against light blue background. The distribution pattern of the markers was evaluated and analyzed for each type of lesion. Statistical analysis was performed by Statistical Package for the Social Sciences (SPSS) version 18, IBM corporation.


  Results Top


The immunoreactivity of OCT4 and SOX2 in follicular tissue and odontogenic lesions

There was a diffuse cytoplasmic expression of OCT4 in the follicular tissue interspersed in between the inflammatory cells, whereas the odontogenic epithelium showed nuclear positivity for SOX2 marker in the basal and the suprabasal layers, along with a few inflammatory cells [Figure 2]a-c. In the case of the dentigerous cysts, SOX2 showed a distinct nuclear positivity in the basal cells [Figure 3]a and b, whereas there was a negative immunoreactivity by OCT4 marker except for a few scattered red blood cells (RBCs) and inflammatory cells that expressed cytoplasmic and nuclear positivity. In the case of odontogenic keratocyst (OKC), the odontogenic epithelium showed positive expression for SOX2 marker [Figure 4]a and c and was negative for OCT4 marker. Few inflammatory cells in the OKC showed intense focal positive OCT4 staining [Figure 4]b. In the case of radicular cyst OCT4 and SOX2 proteins was expressed by the chronic inflammatory cells and very few suprabasal cells of odontogenic epithelium expressed OCT4 positivity Adenomatoid odontogenic tumor showed positive expression for both the markers in its tumor component and ameloblastoma showed negative OCT4 and SOX2 staining. OCT4 staining showed mild and was seen in only few tumor cells in ameloblastic carcinoma interspersed between the stromal cells [Figure 5]a and b. SOX2 staining showed intense positive nuclear immunoexpression in the tumor cells of ameloblastic carcinoma [Figure 5]c. The percentage of expression showed by the cases is enlisted in [Table 1], [Table 2], [Table 3] and the intensity of expression is enlisted in [Table 4], [Figure 6].
Figure 2: (a) Follicular tissue — hemotxylin-eosin stained stained section 10× (b) Expression of OCT4 in follicular tissue (10×); note –– positivity is also seen focally other than the epithelium depicting the cluster of ectomesenchymal cells (c) Expression of SOX2 in follicular tissue (10×)


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Figure 3: (a) Dentigerous cyst — hemotxylin-eosin stained section 10×) (b) SOX2 expression in the basal and suprabasal layers of the odontogenic epithelium (10×). OCT4 showed no expression those areas


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Figure 4: (a) Odontogenic keratocyst (hemotxylin-eosin stained section 10×) (b) OCT4 positively expressed in the inflammatory cells and few basal cells of the odontogenic epithelium in the odontogenic keratocyst (10×) (c) Expression of SOX2 is shown focally in the basal and suprabasal layers of the odontogenic epithelium in the odontogenic keratocyst (10×)


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Figure 5: (a) Ameloblastic carcinoma (hemotxylin-eosin stained section 10×) (b) Nuclear OCT4 expression in the tumor cells of ameloblastic carcinoma (40×) (c) Intense nuclear positivity in the tumor cells of ameloblastic carcinoma by SOX2 marker (10×)


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Figure 6: Graph showing the expression percentages of the markers


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Table 1: Represents the percentage of expression in the various cases of the markers OCT4 and SOX2 (*in percentages)


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Table 2: Expression of OCT4


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Table 3: Expression of SOX2


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Table 4: Degree of expression of the markers


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  Discussion Top


OCT4 (also known as OCT3) or octamer-binding protein transcription factor 4 belongs to the Pit-Oct-Unc (POU) transcription factor family. [13],[15] The POU family of transcription can activate the expression of their target genes through binding a structural sequence motif of an AGTCAAAT consensus sequence. During the early stages of mammalian development, OCT4 is generally expressed in all the blastomeres; gradually, the expression gets restricted to only the inner cell mass and gets downregulated in the trophoectoderm and the endoderm. Researches targeting the OCT4 for disruption has shown the loss of pluripotency. [13],[16]

SOX2 family of transcription factors or sex-determining region (SRY) carries a characteristic high-mobility group (HMG) domain that binds DNA in a sequence-specific manner and those proteins, which contain a HMG domain with 50% or higher amino acids in similarity to the HMG domain of SRY are referred to as SOX proteins. SOX factors have a very important role to play in the development, tissue homeostasis, reprogramming, and cancer. Studies have shown that the zygotic deletion of the SOX2 have resulted in failure to form the pluripotent epiblast. [17] SOX2 is also required to form the two differentiated structures, i.e., the trophectoderm and the inner cell mass. [6]

OCT4 expression was seen in both the epithelial and mesenchymal components in the developing tooth bud [18] and also in carcinomas; [19] similar expressions were seen in our study in cases of follicular tissue, dentigerous cyst, radicular cyst, and ameloblastic carcinoma depicting the presence of ESCs and its role in the pathogenesis. There is a possibility of blast cell population in the region of inflammatory cells and RBCs, which might have shown positivity in the inflamed connective tissue component of dentigerous cyst. The dedifferentiating potential of the cells in the basal and the suprabasal layer of the epithelium in cases of dentigerous cyst and odontogenic cyst may be a reason for the expression of SOX2 protein, which was in consistence with previous studies. [20] In case of ameloblastoma, only a mild cytoplasmic positivity was appreciated by SOX2 marker and hence, pointed toward the scanty stem cell population; our study is very similar to the previous reports where no positivity was seen with SOX2 marker. [21] Qiao et al. found the expression of SOX2 to be proportional to the progression of the disease in their study conducted on transforming oral mucosa. [22] In our study, we got a similar expression in the odontogenic lesions in respect to (i.r.t) follicular tissue and dentigerous cyst. The same progression was not found in ameloblastoma and ameloblastic carcinoma. This strongly suggests that ameloblastic carcinomas very well arise from direct transformation of preexisting odontogenic rests rather than from preexisting ameloblastoma.


  Conclusion Top


SOX2 and OCT4 served as reliable markers for identifying the stem cell population. SOX2 was more intensely expressed in odontogenic tissues as well as in lesions. This study depicted that stem expression was also maintained in the tumor transformation of tissues.

Note: This study was presented as a research paper in the 23 rd National Conference of the Indian Association of Oral and Maxillofacial Pathologists, Bengaluru, Karnataka, India.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]


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