Clinical Research

Isolation and Characterization of Dental Pulp Stem Cellsfrom a Patient with Papillon–Lef�evre SyndromePakize Neslihan Taslı, MSc,* Sıdıka Tapsın, MSc,† Sezin Demirel, MSc,‡

Mehmet Emir Yalvac, PhD,† Serap Akyuz, PhD,§ Aysen Yarat, PhD,* and Fikrettin Sahin, PhD*

Abstract

Introduction: Papillon–Lef�evre syndrome (PLS) is a rareautosomal recessive disorder characterized by immunedysregulation because of a mutation in cathepsin cgene, resulting in hyperkeratosis of the palms, soles,elbows, and knees combined with premature loss ofthe primary and permanent dentitions. Periodontaltissue abnormalities in PLS patients were reported previ-ously. However, less is known about dental pulp tissuederived cells of PLS patients. This study aimed to showstem cell potential of PLS dental pulp stem cells (DPSCs)and provide new evidence regarding the pathophysi-ology of the disease. Methods: DPSCs were character-ized by using flow cytometry and immunocytochemistry.They were also induced to differentiate into adipogenic,osteogenic, chondrogenic, odontogenic, and myogeniccells. Results: The results revealed that PLS DPSCs arestained positive for mesenchymal stem cells surfacemarkers CD29, CD73, CD90, CD105, and CD166. PLSDPSCs were able to differentiate into adipogenic, oste-ogenic, chondrogenic, and odontogenic cell types prop-erly. PLS DPSCs expressed embryonic stem cell markersOct4, Sox2, cMYc, and Klf4 and showed similar prolifer-ation rate compared with DPSCs isolated from healthyyoung controls. Interestingly, it was found that unlikethe healthy DPSCs, PLS DPSCs are not able to form my-otubes with correct morphology. Conclusions: Thesedata are being reported for the first time; therefore,they might provide new insights to the pathology ofthe disease. Our results suggest that the PLS DPSCsmight be an autologous stem cell source for PLS patientsfor cellular therapy of alveolar bone defects and otherdental tissue abnormalities observed in PLS. (J Endod2013;39:31–38)

Key WordsDental pulp cells, differentiation, Papillon-Lef�evresyndrome, stem cells

From the *Department of Genetics and Bioengineering, FacultyDentistry, Faculty of Dentistry, Marmara University, Nisantasi CampuColumbus, Ohio; and §Department of Basic Medical Sciences-Bioche

Address requests for reprints to Dr Fikrettin Sahin, DepartmentA�gustos Campus, Kayisdagi cad, Kayisdagi, TR-34755 Istanbul, Turk0099-2399/$ - see front matter

Copyright ª 2013 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2012.09.024

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The Papillon–Lef�evre syndrome (PLS) is an autosomal recessive genetic disorderdescribed by Papillon and Lef�evre in 1924. PLS is shown to be associated with muta-

tions of the lysosomal cysteine protease cathepsin C gene (1), which is mainly expressedin the epithelial regions of palms, soles, knees, and elbows seen in palmoplantar hyper-keratosis and keratinized oral gingiva seen in premature loss of the primary and perma-nent teeth (2, 3). Lack of expression of cathepsin C in immune cells results ininactivation of neutrophil serine proteases (4), causing disruption in host immuneresponse tomicrobial infection in inflamed periodontal tissues or severe tissue destruc-tion in PLS (5). Current treatment of PLS basically aims to eliminate the causative viru-lent gram-negative anaerobic pathogen reservoir at the site of lesions by applyingphysical and chemical hygiene methods as well as systemic antibiotic treatment. Thesemethods are mostly infective, leaving antibiotic-resistant pathogens (6, 7). Not beingfully elucidated, it was reported that periodontal tissue malfunctions might haveplayed important roles in pathology of PLS (8, 9). On the other hand, there is noreport about the potential of PLS dental pulp stem cells (DPSCs) and theirmalfunctioning, which might be related to pathophysiology of the disease.

DPSCs were isolated from primary teeth and permanent teeth previously (10–13).DPSCs were reported to have an extensive proliferation and differentiation capacity(14), which makes them an important source of stem cells for regenerative medicine(11, 15, 16). It was reported that DPSCs can be directly implanted into the pulpchamber of a severely injured tooth to regenerate the pulp tissue in the presence ofproper growth factors (17). It was also shown that DPSCs might be used in cellulartherapy of some neurologic diseases via their secretion of neuroprotective moleculessuch as fibroblast growth factor and vascular endothelial growth factor (18, 19).Recent studies also showed that dental pulp derived from mesenchymal stem cells(MSCs) can be cryopreserved for long-termwithout losing their proliferation and differ-entiation capacity (20). Immune regulatory functions of DPSCs include directing thetrafficking of immune cells and regulating the inflammatory responses (21). Therefore,on characterization of PLS DPSCs it remains to elucidate the roles of these cells in immu-nopathology of PLS.

MethodsIsolation of PLS DPSCs

Four vital human primary teeth, 2 primary canines and 2 primary molars, beforethe onset of physiological root resorption were extracted from a 6-year-old patient withPLS caused by severe periodontal destruction. The patient had no systemic disease.Written informed consents were obtained from the PLS patient and from 8-year-oldhealthy donor and their parents after receiving approval by the Institutional Ethics

of Engineering and Architecture, Yeditepe University, Istanbul, Turkey; †Department of Pediatrics, Istanbul, Turkey; ‡Center for Gene Therapy, Research Institute at Nationwide Children’s Hospital,mistry, Faculty of Dentistry, Marmara University, Nisantasi Campus, Istanbul, Turkey.of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, 26ey. E-mail address: fsahin@yeditepe.edu.tr

Stem Cell Potential of PLS Dental Pulp Stem Cells 31

TABLE 1. List of Primers Used in Real-time PCR Analysis

Marker Sequences Reference

Oct4 50-CTTGAATCCCGAATGGAAAGG-30 2550-CCTTCCCAAATAGAACCCCCA-30

Sox2 50-CCCCCGGCGGCAATAGCA-30 2550-TCGGCGCCGGGGAGATACAT-30

c-Myc 50-CCTTGCAGCTGCTTAGACGC-30 2550-TCTGCTGCTGCTGCTGGTAG-30

Klf4 50-ATTAATGAGGCAGCCACCTG-30 2550-GGTCTCTCTCCGAGGTAGGG-30

nanog 50-CTAAGAGGTGGCAGAAAAACA-30 2650-CTGGTGGTAGGAAGAGTAAAGG-30

hTERT 50-ACCCTAACTGAGAAGGGCGTAG-30 2350-GTTTGCTCTAGAATGAACGGTG-30

GAPDH 50-TGGTATCGTGGAAGGACTCA-30 2450-GCAGGGATGATGTTCTGGA-30

cMyc, v-myc myelocytomatosis viral oncogene homolog; hTERT, human telomerase reverse tran-

scriptase; Klf4, Krueppel-like factor4; Oct4, octamer-binding transcription factor 4; Sox2, SRY

(sex determining region Y)-box 2.

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Committee of Yeditepe University, Turkey. Extractions of the teeth wereconducted under local anesthesia. The extracted teeth were immedi-ately placed in sterile growth medium containing Dulbecco modifiedessential medium (DMEM) (Sigma-Aldrich, St Louis, MO) supple-mented with 10% fetal bovine serum (FBS) (Biological Industries,Beit Haemek, Israel) and 1% of penicillin, streptomycin, and amphoter-icin solution (Biological Industries) and transferred to the laboratorywithin 2 hours. DPSCs from a healthy donor were isolated as previouslydescribed by our group (22).

Flow Cytometry AnalysisThe surface antigens of PLS DPSCs (passages 3–4) were charac-

terized by flow cytometry analysis as described earlier (22). Briefly,first, the cells were incubated with primary antibodies prepared inphosphate-buffered saline (PBS) (Invitrogen, Gibco, UK; pH 7.4) for1 hour. Primary antibodies against CD29 (catalog #BD556049),CD34 (catalog #SC-51540), CD45 (catalog #SC-70686), CD90 (catalog#SC-53456), CD105 (catalog #SC-71043), CD133 (catalog #SC-65278), CD166 (catalog #SC-53551) (Santa Cruz Biotechnology Inc,Santa Cruz, CA; 100 tests in 2 mL), and CD73 (catalog #BD 550256)(Zymed, San Francisco, CA) were used with 1:100 dilution. Then thecells were washed with PBS to remove excess primary antibodies, fol-lowed by incubating with fluorescein isothiocyanate–conjugated(FITC) chicken anti-mouse secondary antibodies (catalog #SC-2989;200 mg/0.5 mL) at 4�C for 1 hour, except for CD29, against whichphycoerythrin, conjugated monoclonal antibody was used. The flow cy-tometry analysis of the cells was completed by using Becton DickinsonFACS Calibur Flow cytometry system (Becton Dickinson, San Jose, CA;model no. 342975). Five thousand events were counted for eachsample.

Cell Proliferation AssayPLS DPSCs and DPSCs (passages 3–4) were seeded at a concentra-

tion of 4000 cells/well on 96-well plates (BIOFIL; Filmar, Switzerland).The cells were counted for next 11 days in 2-day intervals.

Real-time Polymerase Chain Reaction AnalysisTotal RNA isolation was performed by using the High Pure RNA

isolation kit (Roche, Indianapolis, IN) in accordance with the manufac-turer’s instructions. Complementary DNA (cDNA) cDNA synthesis fromRNA samples was done by using the High Fidelity cDNA synthesis kit(Roche) in accordance with the manufacturer’s instructions. For thedetermination of gene levels, real-time polymerase chain reaction(PCR) was performed by using Maxima SYBR Green/ROX. SynthesizedcDNA was used as a template. Ten microliters of Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, EU), 0.3 mmol/L of forward primer,0.3mmol/L of reverse primer, 200 ng template and dH2O were mixed ina final concentration of 20 mL. Serial dilution of PLS DPSCs and DPSCscDNA was used for relative quantitation of the expression of Oct4, Klf4,Sox2, c-Myc, and nanog genes. cDNAs of PLS DPSCs on passages 3, 15,and 30 were used for relative quantitation of expression of human telo-merase reverse transcriptase (hTERT) (23) gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (24) gene was used in this studyas a housekeeping gene for normalization of data. iCycler RT-PCR detec-tion system (Bio-Rad, Berkeley, CA) was used for real-time PCR. Primersequences for Oct4, Klf4, Sox2, c-Myc (25), nanog (26), and hTERTgenes are shown in Table 1.

Immunocytochemistry AnalysisPLS DPSCs (passage 3–4) grown on 48-well plates were fixed with

2% of paraformaldehyde and permeabilized by incubating with 0.1%

32 Taslı et al.

Triton-X-100/PBS pH 7.4 for 5 minutes. Nonspecific binding of anti-bodies was blocked by incubating with 2% goat serum (diluted inPBS) for 20 minutes. Samples were incubated with primary antibodiesovernight at 4�C. Each sample was washed twice for 5 minutes with PBSto remove unbound primary antibodies (COL1A #ab292, SMA #ab5694;abcam, a-Actin #sc-58670, DMP1 #sc-73633, BMP2 #sc-73743, OCN#sc-30044, COL2A #sc-28887, DSP #sc-33586; Santa Cruz Biotech-nology). After washing, secondary antibodies (goat polyclonalanti-rabbit IgG-Alexa 488, goat polyclonal anti-mouse IgG-Alexa 488conjugate #A21121, goat polyclonal anti-mouse IgG-Alexa 488#A11008; Invitrogen, Carlsbad, CA) were added and incubated for 1hour. DAPI (40, 6-diamidino-2-phenylindole) (AppliChem, Darmstadt,Germany) was used to stain the nuclei of the cells by incubating for20 minutes at 4�C, followed by rinsing with PBS 3 times. Sampleswere observed under fluorescence microscope (Nikon EclipseTE200; Nikon, Minnesota, MN).

Differentiation TestsPLS DPSCs with low passage number (3 or 4) were induced to

differentiate into osteogenic, odontogenic, chondrogenic, and adipo-genic cells on the basis of protocols below.

Adipogenic Differentiation. PLS DPSCs (passages 3–4) werecultured in 6-well plates at a concentration of 3000 cells/cm�2 ingrowth medium. After 24 hours, the medium was replaced withDMEM supplemented with 10% FBS, 1 mmol/L dexamethasone, 5mg/mL�1 insulin, 0.5 mmol/L iso-butyl-methyl-xanthine, and 60mmol/L indomethacin (Sigma-Aldrich) for 2 weeks. Intracellular lipidvesicles were observed under a light microscope (Nikon TS100; Nikon)Oil red O staining was performed to visualize lipid vesicles in the cells.Oil red O staining solution was prepared by dissolving 0.5 g oil red O(Sigma-Aldrich) in 100 mL isopropanol. The cells were fixed with2% paraformaldehyde for 30 minutes; then they were rinsed withPBS and stained with oil red O diluted (6:4) in PBS for 1 hour. Afterstaining, the cells were washed with PBS and observed under lightmicroscope.

Osteogenic Differentiation. The cells (passages 3–4) werecounted and cultured in 6-well plates at a concentration of 3000cells/cm�2 in growth medium. After 48 hours, their medium was re-placed with osteogenic medium (DMEM supplemented with 10%FBS, 0.1 mmol/L�1 dexamethasone, 10 mmol/L b-glycerol-phosphate,50 mmol/L ascorbate) (Sigma-Aldrich). The cells were incubated inosteogenic medium for 10 days, and the medium was changed every

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Figure 1. Flow cytometry analysis. PLS DPSCs are positive for MSC markers and negative for hematopoietic stem cell markers. NC, negative control (non-stainedPLS DPSCs).

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other day. At day 10, alkaline phosphatase (ALP) activity assay, vonKossa staining, and immunocytochemistry analysis were performed toconfirm osteogenic differentiation.

Odontogenic Differentiation. The cells (passages 3–4) werecounted and cultured in 6-well plates at a concentration of 3000cells/cm�2 in growth medium. After 48 hours, the medium was re-placed with odontogenic medium (DMEM supplemented with 10%FBS, 0.01 mmol/L�1 dexamethasone, 50 nmol/L b-glycerol-phosphate,50mmol/L ascorbate. The cells were incubated in odontogenic mediumfor 10 days, and the solution was changed every other day. At day 10,ALP activity assay, von Kossa staining, and immunocytochemistry exper-iments were conducted to confirm odontogenic differentiation.

ALP Activity Assay. After osteogenic differentiation and odonto-genic differentiation, the cells were trypsinized and centrifuged at

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1000 rpm for 5 minutes. The pellets were resuspended in 500-mLcell lysis buffer containing 0.2% Triton-X-100 diluted in PBS, incubatedfor 30 minutes at room temperature, shaking at 850 rpm. After lysingthe cells, 25 mL of protein lysate and 75 mL of Randox reagent (ALPligand) (Randox ALP detection kit-RANDOX) were mixed in 96-wellplate and incubated for 15 minutes, followed by measuring absorbanceat 405 nm by using an enzyme-linked immunosorbent assay platereader to detect enzyme activity.

von Kossa Staining. After 10 days of incubation with osteogenic orodontogenic medium in 24-well plates, the cells were fixed with 2% ofparaformaldehyde at 4�C for 30 minutes. After fixation, the cells werestained by using the von Kossa method (Bio-optica, Milan, Italy), andcalcium depositions were observed using light microscope (NikonTS100; Nikon).

Stem Cell Potential of PLS Dental Pulp Stem Cells 33

Figure 2. (A) Cell proliferation of PLS DPSCs and DPSCs for 11 days; (B)mRNA levels of hTERT gene in PLS DPSCs at passages 3, 15, and 30; (C)comparison of expression of ESC markers in PLS DPSCs and DPSCs. Valuesare given as means � standard deviations.

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Chondrogenic Differentiation. For chondrogenic differentia-tion a micromass culture system was applied as previously described(27). Briefly, 20� 104 cells (passages 3–4) in 12.5 mL medium drop-lets were placed in the center of each well of 24-well plate. To provide

34 Taslı et al.

cell attachment, the plates were put in incubator for 2 hours, followedby addition of 500 mL chondrogenic differentiation medium (DMEMsupplemented with 1% insulin-transferrin-selenium-G, 1.0 mg/mL�1

human serum albumin, 10 ng/mL�1 transforming growth factor b1,14 mL/mL�1 ascorbate acid, 0.1 mmol/L dexamethasone, 1% strepto-mycin/penicillin, 5 mg/mL�1 linoleic acid), and incubated for 10days, changing differentiation medium every other day. At day 10, alcianblue staining and immunocytochemistry analysis were conducted toconfirm chondrogenic differentiation. Alcian blue staining solutionwas prepared by dissolving 1 g alcian blue dye (Sigma-Aldrich) in100 mL 3% acetic acid. The cells were first fixed with 2% paraformal-dehde for 30 minutes and washed with PBS 3 times, followed by incu-bation with alcian blue staining solution for 30 minutes. Afterincubation, staining solution was removed, and the cells were washedwith PBS 3 times. Samples were observed under light microscope.

Myogenic Differentiation. The cells (passages 3–4) werecounted and cultured in 6-well plates at a concentration of 3000cell/cm2 in growth medium. After 48 hours, the medium was replacedwith myogenic medium (Ham’s F-12/DMEM supplemented with 10%FBS, 0.4mg/mL dexamethasone, 1% donor horse serum, 1 ng/mL basicfibroblast growth factor, 1% L-glutane) (Sigma-Aldrich). The cells wereincubated with myogenic medium for 10 days, and the medium waschanged every other day. At day 10, immunocytochemistry analysiswas performed to confirm myogenic differentiation.

Statistical AnalysisFor statistical analysis, the Student’s t test was used. A P value of

<.05 was accepted as statistically significant. Results were expressedas mean � standard deviation. For these procedures GraphPad Prism5 statistics software (La Jolla, CA) was used.

ResultsCharacterization of PLS DPSCs

Flow cytometry analysis revealed that PLS DPSCs were stained posi-tively for MSC markers such as CD29, CD105, CD90, CD73, and CD166.However, they were stained negatively for hematopoietic markers suchas CD34, CD45, CD44, and CD14 (Fig. 1).

Cell Proliferation Assay and Real-timePCR Analysis Results

PLS DPSCs and DPSCs were counted every other day for 11 days.PLS DPSCs and DPSCs showed very similar patterns of proliferation(Fig. 2A). PLS DPSCs reached 30th passage showing reduced prolifer-ation rate, which was confirmed by real-time PCR analysis on the basisof the fact that hTERT is expressed in stem cells and immortalized celllines (28). It was shown that hTERT expression is gradually decreasingas the passage number increases in PLS DPSCs (Fig. 2B). When theexpression of embryonic stem cell (ESC) markers was compared inPLS DPSCs (passage 3) and DPSCs (passage 3), it was found that thereis no significant difference in the expression of these markers in bothcell types (Fig. 2C).

Differentiation Analysis ResultsOdontogenic Differentiation Results. After 14 days of incuba-tion with odontogenic differentiationmedium, PLS DPSCs were analyzedfor odontogenic differentiation markers. ALP activity increased whenthe cells were induced to differentiate (Fig. 3A). Immunocytochemistryanalysis revealed that differentiated cells were expressing odontogenicmarkers such as collagen type 1 (COL1A), dentin matrix protein 1

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Figure 3. ALP activity in odontogenic and osteogenic differentiation. (A) Comparison of ALP activity in odontogenic differentiation. (B) Comparison of ALP activityin osteogenic differentiation. Student’s t test was performed, and values are given as means � standard deviations. *P < .05.

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(DMP1), bone morphogenetic protein 2 (BMP2), and dentin sialo-phosphoprotein (DSPP) (Fig. 4).

Adipogenic Differentiation Results. After adipogenic differen-tiation, PLS DPSCs formed lipid vesicles that were visualized by oil red Ostaining (Fig. 5A).

Osteogenic Differentiation Results. On osteogenic differenti-ation, PLS DPSCs were analyzed for ALP activity. The results indicate thatALP activity was significantly higher in PLS DPSCs induced to differen-tiate compared with undifferentiated PLS DPSCs (Fig. 3B). Immunocy-tochemistry results demonstrated that osteogenic PLS DPSCs wereexpressing high amount of osteocalcin (OCN) and COL1A (Fig. 4B).Von Kossa staining data indicate that on osteogenic differentiation,PLS DPSCs have higher Ca+2 deposits than undifferentiated PLS DPSCs(Fig. 5B).

Chondrogenic Differentiation. On chondrogenic differentiation,PLS DPSCs were shown to express chondrogenic marker COL2A deter-mined by immunocytochemistry. COL2A was highly expressed inchondrogenic differentiated cells compared with undifferentiated PLSDPSC MSCs (Fig. 4C). Alcian blue staining of acid mucosubstancesand acetic mucins confirmed chondrogenic differentiation of PLS DPSCs(Fig. 5C).

Myogenic Differentiation. On myogenic differentiation, PLSDPSCs were shown to express myogenic markers smooth muscle actin(SMA) and alpha-actin (a-actin) determined by immunocytochemistry.SMA was highly expressed in myogenic differentiated PLS DPSCs andDPSCs (Fig. 4D). Interestingly, it was found that although PLS DPSCswere expressing both SMA and a-actin, they were not able to form my-otubes with correct morphology and lacked correct parallel arrange-ments of actin filaments. On the other hand, DPSCs were shown tohave regular myotube morphology with parallel localization of actinfilaments.

DiscussionBone marrow has been shown to be the main source of MSCs

with therapeutic properties (29). In addition to bone marrow,MSCs can be obtained from several tissues such as nerve, liver, syno-vium, muscle, skin, and cartilage (30). Recent studies have shown thatdental tissues such as dental pulp and dental follicle might be an alter-native source of MSCs. DPSCs were reported to differentiate into adi-pogenic, osteogenic, chondrogenic, odontogenic, neurogenic, andendogenic cells (31). Therefore, DPSCs might be used as an autolo-

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gous stem cell source for cellular therapy and tissue engineeringapplications.

In this study for the first time, DPSCs from PLS patient were isolatedand characterized. The results revealed that PLS DPSCs were positive forMSC surface markers and negative for hematopoietic cell markers. Itwas shown that like regular DPSCs isolated from healthy individuals,PLS DPSCs were expressing ESCmarkers and having a high proliferationrate in in vitro culture. As the passage number increased, PLS DPSCsshowed decreasing expression of hTERT, which might indicate agingof cells. We have also demonstrated that PLS DPSCs have capacity todifferentiate into odontogenic, adipogenic, osteogenic, and chondro-genic cells. Odontogenic differentiation of cells was confirmed byshowing expression of odontogenic markers such as ALP (32),DMP1 (33), BMP2 (34), DSPP (35), and COL1A (36). Oil red O stain-ing is a standard method to visualize lipid vesicles inside the cells (37).Adipogenic differentiation was proved by staining lipid vesicles by oilred O staining on differentiation. To determine osteogenic differentia-tion capacity of PLS DPSCs, expressions of osteogenic markers suchas ALP, OCN (38), and COL1A (39) were detected. Calcium depositionsformed by osteogenic cells were demonstrated by using von Kossa stain-ing method. This study revealed that PLS DPSCs were forming cartilage-like structures that were stained blue by using alcian blue staining.Chondrogenic marker COL2A (40) was also shown to be expressedby PLS DPSCs on chondrogenic differentiation.

Interestingly, our data demonstrated that on myogenic differenti-ation, PLS DPSCs were expressing myogenic differentiation markersSMA and a-actin, but the alignment of a-actin protein was found tobe different from the alignment of this protein in myotubes formed bydifferentiation of healthy DPSCs. These data might suggest that PLSDPSCs are not able to differentiate into smooth muscle cells completely.This might result in 2 important consequences that can be associated tolosing of teeth in these patients. One consequence, inadequate forma-tion of capillary vessels because of improper organization of smoothmuscles, may result in food and oxygen deprivation of dental pulp ordental follicle tissue, resulting in loss of tooth. The second is abnormallocalization of a-actin filaments, which may be linked to formation ofabnormal periodontal ligament sheets, giving rise to loss of tooth. Exactmechanism of tooth loss in PLS still remains to be elucidated, but thesedata showed that role of progenitor cells in dental pulp might play rolein the pathogenesis of the syndrome.

The fact that DPSCs have immune regulatory functions (21) mightbring new insight to the pathology of PLS. Establishing PLS DPSC cell

Stem Cell Potential of PLS Dental Pulp Stem Cells 35

Figure 4. Immunocytochemistry analysis. (A) Odontogenic PLS DPSCs and PLS DPSCs were analyzed for COL1A, DMP1, BMP2, and DSPP. (B) Osteogenic PLSDPSCs and PLS DPSCs were analyzed for OCN and COL1A. (C) Chondrogenic PLS DPSCs and PLS DPSCs were analyzed for COL2A. (D) Myogenic PLS DPSCs andDPSCs were analyzed for myogenic differentiation markers SMA and a-actin on myogenic differentiation Scale bars = 100 mm.

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36 Taslı et al. JOE — Volume 39, Number 1, January 2013

Figure 5. (A and B) Oil red O staining of adipogenic PLS DPSCs. Arrows indicate stained lipid vesicles. (C and D) von Kossa staining results. Ca+2 deposits inosteogenic differentiation, formed by osteogenic PLS DPSCs (C) and PLS DPSCs (D). (E and F) Alcian blue staining results. (E) Chondrogenic PLS DPSCs stainedblue after staining. (F) PLS DPSCs remained colorless. Scale bars = 100 mm.

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lines might provide a model system to research the role of these cells inimmunopathology of PLS. We expect that PLS DPSCs might handle theimmunological changes in PLS less efficiently than DPSCs in normalconditions, but this still remains to be elucidated.

The results obtained in this study revealed that PLS DPSCsmight be potential sources of stem cell in dental tissue engineeringapplications such as alveolar bone and pulp regeneration for PLSpatient. On the other hand, PLS DPSCs might be used as a tool tostudy the immunopathologic events leading to loss of teeth in PLSpatients.

AcknowledgmentsThe authors thank Burcin Keskin for her help in flow cytometry

analysis.The authors deny any conflicts of interest related to this study.

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JOE — Volume 39, Number 1, January 2013