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. 2021 Apr 20;11(3):379–385. doi: 10.1016/j.jobcr.2021.04.004

Differential expression of Notch related genes in dental pulp stem cells and stem cells isolated from apical papilla

Damrong Damrongsri a,d, Nunthawan Nowwarote b, Opor Sonpoung c, Suphalak Photichailert a, Thanaphum Osathanon a,d,∗
PMCID: PMC8093932  PMID: 33996433

Abstract

Although dental pulp and apical papilla are originated from neural crest cells, these tissues exhibit distinct characteristics. Notch signaling is one of the known signaling pathways regulating stemness and behaviors of stem cells. The aim of this study was to examine Notch signaling related gene expression profile comparing between coronal pulp tissues and apical pulp complex. Results demonstrated that coronal pulp tissue had higher expression levels of various genes in Notch pathway. However, NOTCH2, MAML2, DTX4, and NEDD4 mRNA levels were significantly lower in coronal pulp tissue than those of apical pulp complex. Furthermore, dental pulp stem cells (DPSCs) and stem cells isolated from apical papilla (SCAPs) were isolated and characterized. These two cell types exhibited similar mesenchymal stem cell surface markers. DPSCs expressed higher mRNA levels of NOTCH3, NOTCH4, DLL1, and HES1. In addition, SCAPs demonstrated higher colony formation and cell proliferation than DPSCs. In summary, cells and tissues from dental pulp and apical papilla exhibited the distinct gene expression profile of Notch related genes. This could be of one the signaling participated in control of DPSCs and SCAPs cells behaviors.

Keywords: Notch, Proliferation, Dental pulp stem cells, Stem cells isolated from apical papilla

1. Introduction

Dental pulp is a connective tissue resided in the pulp chamber and exhibits an important role in dentin-pulp homeostasis. Apical papilla is a soft tissue located at the apical of immature root during root development period.1,2 Although these two tissues originated from neural crest mesenchymal cells, they are different in both functional and structural aspects. Throughout tooth development period, stem cells in dental germ give rise to daughter cells that eventually differentiated into odontoblasts. The other daughter cells are mesenchymal stem cells and they continue to exist in pulp tissue after completed tooth development. These stem cells provide repair and regenerative capabilities throughout tooth lifespan. Dental pulp stem cells (DPSCs) and stem cells isolated from apical papilla (SCAPs) are stem cells isolated from dental pulp and apical papilla, respectively. These mesenchymal stem cells (MSCs) can be induced to differentiate into odontoblast-like cells, hence they are considered as potential sources in regenerative dentistry and tissue engineering approaches.1

Although DPSCs and SCAPs shares common characters, some distinct properties are also reported. Both cells express odonto/osteogenic marker genes in baseline and the expression levels are increased after treated with osteogenic induction medium.1,3 However, DPSCs and SCAPs showed different level of mineralization potential during osteoblast differentiation. DPSCs showed a higher level of dentin sialophosphoprotein (DSPP) and mineralization at early stage of differentiation when compared to SCAPs. DPSCs showed higher STRO-1 positive cells when compared with that of donor matched SCAPs.3

Notch pathway is one of several signaling pathways involving in control of stemness and differentiation of dental mesenchymal stem cells since they demonstrate crucial role in both odontogenesis and tooth regeneration.4,5 In mammals, canonical Notch pathway includes four transmembrane receptors (NOTCH 1–4) and five different ligands (DLL 1, 3, 4 and Jagged 1, 2). The activation of Notch signaling is initiated via direct cell-to-cell contact, resulting in binding of transmembrane receptor of one cell to membrane-bound ligand of another adjacent cell. The binding of receptors and ligands generates series of proteolytic cleavages of transmembrane receptor releasing Notch intracellular domain (NICD) that relocates to nucleus. NICD interacts with CSL/RPBj/CBF-1 transcription factors to modulate the expression of direct target genes, such as HES/HEY (hairy and enhancer of split/hairy and enhancer of split related with YRPW motif) families of transcription factors. Notch receptors and ligands are detected in both epithelium and mesenchyme in several stages of dental development.6 Moreover, Notch signaling is upregulated in mesenchymal cells in pulpal area of carious or injured adult teeth.5,7 Activation of Notch signaling leads to increase mineralization and suppress cell proliferation in dental pulp cells in vitro.8 Taken together, DPSCs and SCAPs exhibit the distinct characteristics and Notch signaling crucially regulates dental stem cell behaviors in tooth development, repair and homeostasis. In addition, Notch-mediated cell regulation and stimulation depend on differential expression of Notch receptors and ligands of local cells. Hence, the aim of the present study was to investigate the different expression patterns of Notch signaling genes in DSPCs and SCAPs.

2. Materials and methods

2.1. Histological analysis

Teeth scheduled for extraction in accordance with patient’s treatment plan were obtained for histological analysis and cell isolation. For histological evaluation, teeth were fixed with 4% formaldehyde for 24 ​h and subsequently decalcified for 2 weeks. Specimens were then dehydrated in ethanol and embedded in paraffin. Histological sections were obtained at 8 ​μm in thickness using microtome. Specimens were stained with hematoxylin and eosin staining as well as Masson’s trichrome staining.

For immunofluorescence staining, specimens were deparaffinized, rehydrated, and subsequently incubated with primary antibody at 4 ​°C overnight. Rabbit anti-Hes1 antibody (cat. No. ab108937; Abcam, USA) has been used as primary antibody. Subsequently, sections were washed with phosphate buffer saline and incubated with biotinylated goat anti rabbit IgG and subsequently with Step-FITC (S3762; Sigma) for 2 ​h at room temperature for visualizing protein expression. DAPI was used for nuclei counterstaining.

2.2. Cell isolation and culture

Tissues were gently removed from extracted teeth and minced into small pieces. Cell explantation was performed to isolate cells from both dental pulp and apical papilla. The isolated cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM Gibco BRL, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% l-glutamine, 100 U/ml penicillin, and 100 ​μg/mL streptomycin (Gibco) and maintained in 5% Co2 with humidified atmosphere at 37 ​°C.

2.3. Flow cytometry analysis

Expression of surface protein marker was determined using flow cytometry analysis. After trypsinization, single cell suspension were obtained and then stained with fluorescence conjugated primary antibody in 1% horse serum solution. Protein expression analysis was achieved using a FACSCalibur Flow cytometer (BD Bioscience, CA, USA). The antibodies were FITC conjugated anti-human CD44 (BD Bioscience Pharmingen, NJ, USA), PerCP-CyTM5.5-conjugated anti-human CD90 (BD Bioscience Pharmingen), PE-conjugated anti-human CD105 (Immuno Tools, Friesoythe, Germany), and PerCP-conjugated anti-CD45 (Immuno Tools).

2.4. Colony forming unit assay

Cells were seeded at density of 500 ​cells/dish in 60 ​mm tissue culture dish and kept for 14 days in growth medium. Culture medium was changed every 2 days. Samples were fixed with 10% buffered formalin and washed twice using phosphate buffered saline. Coomassie blue dye was used for colony staining and visualization.

2.5. Proliferation assay

Cell viability was employed to determine the increase of cell number at different time points using MTT assay. Briefly, cells (12,500 ​cells) were seeded in 24 wells-plate and maintain in growth medium. At selected time points, cells were incubated for 30 ​min with 0.5 ​mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (USB Corporation), allowing formation of formazan crystal. The crystals were then solubilized in dimethylsulfoxide and glycine buffer. The absorbance was quantified at 570 ​nm using microplate reader (ELx800; BIO-TEK®).

2.6. Real-time polymerase chain reaction (PCR)

Total RNA was isolated using Trizol reagent (RiboEx™, GeneAll® Seoul, Korea). RNA quality and quantity were assessed using NanoDrop™. RNA samples at 1 ​μg each were converted to cDNA using a reverse transcriptase (Promega, WI, USA). PCR reaction was examined using FastStart® Essential DNA Green Master in CFX connect Real-Time PCR (Bio-Rad, Singapore). Product specificity was assessed using melting curve analysis. The expression levels of target genes were normalized to the expression levels of 18S and subsequently normalized to the control condition. The oligonucleotide sequences used were 18S forward: ​5’-GGCGTCCCCCAACTTCTTA-3’, ​reverse: ​5’-GGGCATCACAGACCTGTTATT-3’, NOTCH3 forward 5′-TCTCAGACTGGTCCGAATCCAC-3’; reverse 5′-ACACTTGCCTCTTGGGGGTAAC-3′, NOTCH4 forward 5′-ATGCGAGGAAGATACGGAGTGG-3’; reverse 5′-TCGGAATGTTGGAGGCAGAAC-3′, DLL1 forward 5′-TGTGACGAGTGTATCCGCTATCC-3’; reverse 5′-AGGGCTTATGGTGTGTGCAGTAG-3′, HES1 forward 5′-AGGCGGACATTCTGGAAATG-3’; reverse 5′-CGGTACTTCCCCAGCACACTT-3’.9, 10, 11

2.7. Bioinformatics analyses

Raw data of microarray expression profiles of coronal pulp and apical pulp complexes gene expression analyses were downloaded from NCBI Gene Expression Omnibus (GSE71048).12 Data was processed in Excel to obtain text format for bioinformatics analyses. Genes in Notch were listed according to KEGG database. Bioinformatics analyses were performed using NetworkAnalyst online program.13 Differential gene expression was determined using built-in statistical analyses. The significant difference was determined at FDR <0.05. Heat map of differential gene expression was generated using Heatmapper.14

2.8. Statistical analyses

Cell culture experiments were performed in biological quadruplicate from different donors. Data were presented as mean ​± ​standard error of mean (SEM). Mann Whitney U test was utilized for two group comparison and the statistically significant difference was considered when p ​< ​0.05. The statistical analyses were conducted using Prism 8 (GraphPad Software, CA, USA).

3. Results

3.1. Characteristics of dental pulp and apical papilla tissues and the isolated cells

Dental pulp tissues exhibited denser connective tissues and blood vessels than those of apical papilla (Fig. 1). Higher cellular density was observed in apical papilla compared with that of dental pulp tissues. Both DPSCs and SCAPs exhibited the similar spindle shape morphology. These cells were confirmed as mesenchymal cells by exhibiting mesenchymal marker CD44, CD90, and CD105 but lacking of hematopoietic surface marker CD45 expression (Fig. 2A and B). However, no statistically significant difference in percentage of positive cells in each marker between DPSCs and SCAPs was observed (Fig. 2C).

Fig. 1.

Fig. 1

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Representative histological images of dental pulp (A and B) and apical papilla (C and D). A and C were stained with hematoxylin and eosin staining. C and D were stained with Masson’s trichrome staining.

Fig. 2.

Fig. 2

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Mesenchymal stem cell surface marker expression. (A and B) Histogram of fluorescence intensity of fluorescence staining of hematopoietic surface marker (CD45) and mesenchymal stem cell surface marker (CD44, CD90, and CD105). (C) Graph demonstrated the percentage of cell expressing each marker.

3.2. Bioinformatics analyses of gene expression profiles of dental pulp tissues and apical pulp complex

Previous report performed microarray analysis to evaluate the profiles of gene expression in dental pulp tissues and apical pulp complex.12 In present study, raw dataset was re-analyzed using bioinformatics tools to identify the differential gene expressions related to Notch pathways (Fig. 3A). Results demonstrated that coronal pulp tissues had lesser mRNA levels of NOTCH2, MAML2, DTX4, NEDD4 compared with those of apical pulp complex. In contrast, fifteen genes were expressed higher in coronal pulp than those of apical pulp complex, namely NOTCH3, NOTCH4, DLL1, JAG2, MAML3, FURIN, ADAM17, HES1, HES2, HES4, HEY2, HEYL, MIB1, CNTN6, and PCSK5.

Fig. 3.

Fig. 3

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(A) Heatmap diagram demonstrated the differential expression of Notch signaling related genes between apical pulp complex and coronal pulp tissues. (B–E) Differential expression of selected genes between DPSCs and SCAPs was validated using real-time polymerase chain reaction. Asterisks indicate statistically significant difference. Immunofluorescence staining of HES1 was examined in coronal pulp tissue (F) and apical papilla area (G).

To further validate the differential gene expression, real-time polymerase chain reaction was conducted. Similar to bioinformatic results, DPSCs expressed significant higher NOTCH3, NOTCH4, DLL1, and HES1 mRNA levels than SCAPs (Fig. 3B–E). Furthermore, HES1 protein expression was examined using immunofluorescence staining. Coronal dental pulp tissues exhibited a low expression level of HES1 in stromal area (Fig. 3F) while a high HES1 level was observed in association with blood vessels in apical area (Fig. 3G).

3.3. SCAPs exhibited higher proliferative ability than DPSCs

Our previous study reported that trigger of Notch signaling in human dental pulp cells generated the suppression of cell cycle progression and eventually inhibited cell proliferation.8 Hence, we hypothesized that the higher Notch component expression in DPSCs could affect cell proliferation compared with that of SCAPs. In colony forming unit assay, both cell types were able to form colonies when cells were seeded in low density (Fig. 4A). However, it is noted that the DPSCs colony number and density were lesser than those of SCAPs. Correspondingly, DPSCs had lower percentage of cell number at day 7 compared with that of SCAPs (Fig. 4B).

Fig. 4.

Fig. 4

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(A) Colony formation assay and (B) proliferation assay of DPSCs and SCAPs. Asterisks indicate statistically significant difference compared with day 1. Bar indicates statistically significant difference between groups.

4. Discussion

Throughout tooth development, dental papilla develops from the proliferation and condensation of ectomesenchymal cells during cap stage.15 In late bell stage, the appearance of epithelial root sheath (HERS) induces root formation while dental papilla elongates and moves apically at the same time.15,16 When reaching the root apex, the dental papilla becomes the apical papilla.15 Dental papilla and apical papilla finally convert to dental pulp tissue at crown and root apex of the teeth respectively and both area are separated by dense apical cell rich zone which becomes diaphragm of developing root.1,15,17 Despite the fact that dental pulp and apical papilla originate from similar cell population, their characteristics are different. Moreover, stem cells derived from dental pulp and apical papilla exhibited distinct behaviors. These dental stem cells provide numerous potentials and benefits for tooth repair and regeneration. However, better understanding and establishment of appropriate cells are important for improving ability and application in repair and regeneration of dental tissue.

Previous study in human immature teeth showed different patterns of gene expression profiles between coronal and apical pulp complex using a microarray technique.12 Results demonstrated that genes responsible for mineralization of dentin are strongly expressed in coronal pulp complex. In the present study, we utilized this public gene expression repository to identify raw data of interest and further employed for extended bioinformatics analyses on our research objective focusing on Notch signaling as it plays crucial roles in tooth development, repair and regeneration. In this research, bioinformatics analyses and real time PCR indicated the upregulation of Notch receptors, ligand and target transcription factor mRNA in DPSCs compared to SCAPs.

Many evidences suggested that Notch receptors and their signaling pathways played crucial roles in control of cell proliferation and differentiation in various cell lineages including skeletal and tooth development both embryonic and post-natal stages.18, 19, 20 Importantly, the consequences of Notch signaling are cell-context dependent, which depending on differentiation stage and type of target cells. The present findings that DPSCs express higher NOTCH3 and NOTCH4 mRNA than SCAPs are corresponding to the idea that Notch signaling pathway involved in differentiation and mineralization of DPSCs. NOTCH3 mRNA was detected in primary dental pulp cells isolated from rat.21 In addition, NOTCH3 gene expression was upregulated in response to pulp tissue injury in rat.5,22 NOTCH3 protein has been expressed in co-localization with CD146 and RGS5, which are used as markers for pericytes.23,24 Moreover, CD 146-positive DPSCs have been shown to improve formation of dentin-pulp like structure.25 NOTCH4 and NOTCH4 intracellular domain (N4ICD) were increased throughout osteogenic differentiation of human mesenchymal stem cells (hMSCs).26 Moreover, NOTCH4 expression was increased in both primary human osteoblast and SaOS-2 human osteosarcoma cell line during osteogenic differentiation.27 NOTCH4 gene expression was also upregulated during differentiation of vascular progenitor cells (VPCs) from embryonic stem cell (ESCs).28

It has been shown that NOTCH3 was expressed in the area associated with blood vessels.23 Only 7% of SCAPs were positively stained of NOTCH3 as examined by flow cytometry analysis.23 When SCAPs were differentiated into osteoblast cells, the expression levels of NOTCH3 were reduced.23 Similar to the present study, Notch signaling target, HES1 protein, was detected in area nearby blood vessels in apical papilla. These data implied the significant of Notch signaling of pericyte function regarding repair and regeneration of dentin-pulp complex. However, further investigation is necessitated to confirm this hypothesis.

In our experiment, increased level of Notch ligand DLL1 expression was observed in DPSCs in correlation with decreased level of CFU ability and cell proliferation compared to SCAPs. Although the differentiation was not directly evaluated in both DPSCs and SCAPs, but the decrease of colony forming unit ability and proliferation in DPSCs suggest more potential of differentiation in DPSCs. Previous reports have been shown that DDL1 was associated with stem cell differentiation in dental pulp tissue. Activation of Notch signaling by DLL1 favored odontoblastic differentiation of DPSCs indicated by increased both level of DSPP expression and the number of calcified nodule formation.29 Moreover, DLL1 was upregulated during odontoblastic differentiation of dental pulp cells30 and bone regeneration.31 Surface immobilization of DLL1 enhanced osteogenic differentiation of stem cells isolated from human exfoliated deciduous teeth (SHEDs).32 In addition, DLL1 promoted osteogenic differentiation in mouse embryonic fibroblasts (MEFs) by regulating BMP signaling.33

HES family genes are group of key Notch target genes of Notch signaling. In this study, our finding of increased HES1 expression in DPSCs indicates downstream activation of Notch signaling pathway. Additionally, DPSCs had both lower level of colony forming units and cell proliferation rate when compared to those of SCAPs which correspond to more differentiated developing tissue of DPSCs. In previous study, dramatic increase of HES1 mRNA expression in human dental pup cells was correlated with decreases of colony forming unit ability, cell proliferation, and gene expression associated with DNA replication and regulation of cell cycle.8 Moreover, HES1 mRNA expression was also increased in hMSCs that cultured in osteogenic medium.26 DPSCs also showed significantly higher odontoblast differentiation related cytokine expressions whereas strong proliferation related cytokine expressions were detected in SCAPs.34 Although both coronal and apical pulp stem cells have abilities to differentiate and form hard tissue in vivo when subcutaneously transplanted in mice using carrier, DPSCs can be more useful because of their homogenous lineage and differentiation potential.35 Similar to previous reports, SCAPs exhibit higher cell proliferation and colony forming unit capability than those of DPSCs.1,3 The higher proliferation in SCAPs corresponds to markedly higher telomerase enzyme activity in SCAPs than DPSCs.36 Taking these evidences together, it implicates that endogenous Notch signaling could involve and participate in the control of cell growth and differentiation in these mesenchymal stem cells. However, further in-depth investigation is certainly required to confirm this observation.

In conclusion, coronal and apical pulp tissue complex demonstrated distinct Notch signaling related gene expression pattern. Although the influence of this differentially expressed genes is not yet functionally proven, we speculate that different Notch expression pattern may involve in control of cell differentiation and proliferation in DPSCs and SCAPs. Further study on identifying the differentially Notch signaling expression in these cells and its relation to cell function is indeed required.

Ethical consideration

All experiments and protocol were approved by the Human Research Ethics Committee, Faculty of Dentistry, Chulalongkorn University (study code HREC-DCU 2020-091 ).

Authorship declaration

D.D. contributed to data interpretation, data analysis, and manuscript preparation. N.N. contributed in cell isolation and characterization. O.S. performed real-time polymerase chain reaction assay. S.P. performed proliferation assay. T.O. contributed in study conceptualization, data interpretation and analysis. All authors critically revised the manuscript and gave final approval for publication.

Declaration of competing interest

All authors declare no competing interests.

Acknowledgement

This study was supported by the Faculty of Dentistry Research Fund (028/2020) to D.D. and in part by the Dental Research Fund, Dental Research Project 3200502#2/2017 of the Faculty of Dentistry, Chulalongkorn University and Thailand Research Fund (RSA6180019) to T.O. The authors thank Dr. Katekunya Sasiprapakul, Dr. Phichamon Rungsirikunnan, and Dr. Teena Dilokwanich for their assistance in raw data of microarray preparation.

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