# Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells

Authors: Jimin Xiong; Danijela Menicanin; Peter S. Zilm; Victor Marino; P. Mark Bartold; Stan Gronthos
Journal: Stem Cells International
 (2016)

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## Abstract

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein “spots” were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population.


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## Body

## 1. Introduction

Despite encouraging outcomes, therapeutic utilization of mesenchymal stem cells (MSC) is constrained by the lack of understanding and definition of their properties and developmental status followingex vivo expansion. Heterogeneity inherent within progenitor populations presents as one of the major limitations to their clinical application in regenerative medicine. The variability and inconsistencies in cellular properties allude to a hierarchical order within stem cell populations and result in the coexistence of subsets of distinct morphologies, phenotypes, proliferation rates, and biological functions [1–3]. Currently, there is a lack of individual or a set of markers that can distinguish different subsets within MSC-like populations of different origins from more differentiated fibroblastic cells in any tissue.Identification of stem/progenitor cells residing in the periodontium [4–6] has offered a potential novel therapeutic avenue for treating periodontal tissues damaged due to trauma, injury, and disease. Periodontal diseases are highly prevalent among all human populations and if untreated cause the destruction of periodontal supporting tissues and can potentially result in tooth loss. Predictable regeneration of periodontal tissues as a result of advanced periodontal diseases is beyond the scope of current technologies and, therefore, alternative strategies are being investigated.In addition to periodontal ligament stem cells (PDLSC), the periodontium contains multiple cell types including fibroblasts, endothelial cells, epithelial cell rests of Malassez (ERM), osteoblasts, and cementoblasts [7]. This array of specialised cell types is integrated into and cofunctions to provide the periodontium with its essential and unique structural and mechanical properties. This biological complexity and cellular heterogeneity highlights the need for identification of surface markers specific to each cell subset within the periodontium to enable identification and discriminant isolation of desired and required cell populations.It has been demonstrated that PDLSC share a phenotypic profile characteristic of bone marrow derived mesenchymal stem cells (BMSC) including expression of BMSC markers CD29, CD44, CD90, and CD105 [8]. Furthermore, PDLSC express the early BMSC and perivascular cell surface markers STRO-1 and CD146/MUC18 [4], with a subset of progenitors presenting with other antigens associated with perivascular tissues (alpha-smooth muscle actin and pericyte-associated antigen, 3G5) [9]. Together, these findings designate a possible perivascular origin of PDLSC, in accord with earlier findings by McCulloch and colleagues [10, 11]. In conjunction, comparative genomic analyses identified unique features exhibited by PDLSC when compared to BMSC and dental pulp stem cells (DPSC). These studies demonstrated increased levels of scleraxis (a tendon-specific transcription factor) [4] and PLAP-1 (periodontal ligament associated protein-1/asporin) expression in PDLSC [12]. A panel of markers, proposed for the current identification of PDLSC, includes alkaline phosphatase, type I collagen, periostin, runt-related transcription factor-2 (Runx2), and epithelial growth factor receptor, which are also expressed by BMSC, considering that both cell populations commonly hold the innate capacity for formation of mineralized matrix in the form of cementum and bone, respectively [13]. Since the cell surface markers described above are ubiquitously expressed by MSC-like populations derived from all dental tissues, specific cell surface antigens, capable of distinguishing between individual dental stem cell population subsets, are yet to be identified [14]. Therefore, our understanding of the cell surface phenotype of PDLSC falls short when considering the need to isolate and purify stem/progenitor cell subsets from the heterogeneous PDL population. This has driven the use of proteomics, the technology investigating global protein expression, to characterise the cell surface phenotype of PDLSC.Proteomic studies investigating dental tissues have been summarized by McCulloch [15]. While the majority of studies focused on protein expression by periodontal microbiota [16–18], a limited number of papers examined proteomic profiles of periodontal ligament cells and tissues [15]. In this study, we provide an insight into the cell surface proteome of PDLSC to identify potential discriminatory PDLSC markers not expressed by other cells residing in the periodontium.


## 2. Methods and Materials

### 2.1. Isolation of Human PDLSC and Gingival Fibroblasts

Human PDLSC and gingival fibroblasts (GF) were isolated from three donors and cultured as previously described (Human Research Ethics Committee of the University of Adelaide, Approval Number H-112-2008) [4, 19]. Briefly, gingival and periodontal ligament tissues were collected from excised gingiva and middle third of the root, respectively. The tissues were digested in equal volumes of collagenase type I (3 mg/mL; Worthington Biochemical, Lakewood, NJ) and dispase type II (4 mg/mL; Roche Diagnostics, Indianapolis, IN) for 2 hours at 37°C. Isolated cells were maintained and cultured in modified α-MEM media (α-MEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FCS (Thermo Electron, Melbourne, VIC, Australia), 50 U/mL and 50 μg/mL penicillin and streptomycin (Sigma-Aldrich), 1 mM sodium pyruvate, 2 mM L-glutamine (SAFC, Lenexa, KS, USA), and 100 μM L-ascorbate-2-phosphate (Novachem, Melbourne, VIC, Australia) at 37°C and 5% CO2 in a humidified environment, with a twice-weekly medium change. Cells were harvested and further expanded once upon reaching confluence. This process was repeated when cells reached 80% confluence until desired cell numbers were obtained.Human neonatal foreskins, collected from routine circumcisions, were used to isolate epithelial sheets after overnight incubation with 4 mg/mL dispase at 4°C, followed by trypsinization for 5 min at 37°C to obtain basal keratinocytes. Keratinocytes were cultured in DMEM containing 10% fetal calf serum, 20 ng/mL epidermal growth factor (Sigma-Aldrich), and 0.4μg/mL hydrocortisone (Sigma-Aldrich), at 37°C and 5% CO2 in a humidified environment, with a twice-weekly medium change.


### 2.2. Immunohistochemistry

Chamber slides (Nalge-Nunc Lab-Tek, Rochester, NY, USA) were seeded with 8 × 103 cells per cm2, in media with additives for 2 days. The slides were fixed with 4% paraformaldehyde (PFA) and endogenous peroxidase activity was inhibited using 0.5% H2O2 in methanol. The sections were incubated with primary antibodies or isotype control antibodies overnight at 4°C, secondary antibodies for 1 hour at room temperature, Vectastain ABC Reagents (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s recommendations, or horseradish-peroxidase-labelled streptavidin (Promega, Madison, WI, USA) at 1 in 1000 dilution and then developed with diaminobenzidine (Dako, Campbellfield, VIC, Australia). The slides were counterstained briefly with haematoxylin (ProSciTech, Thuringowa Central, QLD, Australia). Antibodies used in this study are 1B5, mouse IgG1 isotype control (1 : 25; Professor L. K. Ashman, University of Newcastle, NSW, Australia); mouse IgG1 anti-human CD73 (1 : 25; BD Pharmingen, Sparks, MD, USA); mouse IgG1 anti-human CD90 (1 : 25; BD Pharmingen); mouse IgG1 anti-human Annexin A2 (1 : 12.5; Invitrogen, Waltham, MA, USA); rabbit anti-human sphingosine kinase 1 (1 : 20; Cayman Chemical, Ann Arbor, MI, USA); normal rabbit Ig (1 : 20; Vector Laboratories, Burlingame, CA, USA); goat anti-mouse IgG biotin secondary antibody (1 : 200; Southern Biotechnology Associates, Birmingham, AL); and goat anti-rabbit IgG biotin secondary antibody (1 : 150; Vector Laboratories).


### 2.3. Immunophenotypic Profiling

Single cell suspensions of 2 × 105 cells were blocked with 5% FCS, 1% bovine serum albumin (BSA, SAFC), 50 U/mL penicillin, 50 μg/mL streptomycin, and 5% normal human serum (Red Cross, SA, Australia) in HBSS on ice. Cells were treated with primary or isotype control antibodies (CD73, CD90, and Annexin A2) at a concentration of 20 μg/mL, followed by incubation with phycoerythrin (PE) conjugated goat anti-mouse IgG1 (1 : 50; Southern Biotechnology Associates). Samples were fixed in PBS with 0.1% formalin and 20 mg/mL glucose. Analysis was performed on a fluorescence-activated cell sorter fitted with 250 MW argon laser (Beckman Coulter Cytomics FC500, using CXP Cytometry List Mode Data Acquisition and Analysis Software version 2.2; Beckman Coulter, Miami, FL, USA).


### 2.4. Proteomic Analysis

All equipment and reagents were purchased from Bio-Rad Laboratories (Hercules, CA, USA) unless stated otherwise. CyDye DIGE Fluor minimal dye was purchased from GE Healthcare (Buckinghamshire, UK).


### 2.5. Cell Surface Labelling Using CyDye DIGE Fluor Minimal Dye

CyDye fluorescent cell surface protein labelling was performed as previously reported [4, 20]. Briefly, approximately 20 million subconfluent PDLSC or GF were detached with either 1 mM PUCK’s EDTA or 3 mg/mL type I collagenase and aliquoted into ~5 million cells per tube. Cells were washed in ice cold HBSS (pH 7.4) followed by ice cold HBSS (pH 8.5) and centrifuged at 800 ×g for 2 minutes. The cell pellets were resuspended in 200 μL labelling buffer containing HBSS (pH 8.5) and 1 M urea. Cells were then labelled with 600 pmol of either Cy2, Cy3, or Cy5 or CyDye DIGE Fluor minimal dyes on ice in the dark for 20 minutes. Staining was quenched by adding 20 μL lysine (10 mM) for 10 minutes. Surface-labelled cells were pelleted by centrifugation and resuspended in 202 μL HBSS (pH 7.4). An aliquot (2 μL) was taken prior to and after labelling to check for labelling efficiency using flow cytometry.


### 2.6. Membrane Protein Enrichment

Proteins were isolated and fractionated using a phase separation kit (Mem-PER, Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Briefly, 150μL reagent A containing 1 μL protease inhibitor (Sigma) was added to cell pellets containing PDLSC or GF. Following 10-minute incubation, 450 μL mixture of reagents B and C was added to cell lysates and tubes were kept on ice for 30 minutes. The preparation was centrifuged at 10,000 ×g for 3 minutes, at 4°C, and the supernatant was incubated at 37°C for 20 minutes. Following centrifugation at 10,000 ×g and phase separation, the hydrophobic fraction containing membrane proteins was carefully removed and purified using ReadyPrep 2-D Cleanup Kit. Membrane protein enrichment efficiency was assessed and cells were subjected to up to three membrane fractionation steps.


### 2.7. Membrane Protein Separation by Two-Dimensional Gel Electrophoresis (2DE)

Membrane proteins were solubilised with ReadyPrep reagent 3 buffer for 1 hour at room temperature. The protein was solubilised by gentle aspiration through a fine-gauge needle, as previously described by Zilm et al. [21]. The protein concentration was determined using RC/DC Protein Assay Kit according to the manufacturer’s instructions. Proteins were separated in the first dimension using 11 cm immobilised pH gradient (IPG) strips (pH 3–10) which had been passively rehydrated for 24 hours in 330 μL rehydration/extraction buffer #3, containing 0.2% (w/v) pH 3–10 ampholytes and 1.2% (v/v) De-Streak Reagent (GE Healthcare, Buckinghamshire, UK). IEF was performed using a Protean IEF cell. Briefly, membrane protein preparations containing 150 μg protein were cup-loaded onto the anode end of the IPG strip. The IEF cycle consisted of 8 steps outlined in Table 1, with a 50 μA/strip current limit, and the temperature was maintained at 20°C. Duplicate IPG strips were run concurrently. Following IEF, the IPG strips were equilibrated as previously described [22]. Polyacrylamide gels (18
×
18 cm) containing 8% T, 3.3% C, 0.1% SDS, and 375 mM Tris/HCl (pH 8.8) were cast without stacking gels using a Protean II XL casting chamber. Proteins were separated in the second dimension using a Protean II XL Multicell (Bio-Rad Laboratories) in tris-glycine tank buffer (25 mM Tris, 192 mM glycine, and 0.1% (w/v) SDS) and resolved at 7 mA/gel.Table 1
IEF conditions for 11 cm IPG (3–10).
Step number
Voltage
Voltage ramping mode
Time
Step  1
150 V
Linear
1 hour
Step  2
300 V
Linear
2.30 hours
Step  3
600 V
Linear
1.50 hours
Step  4
1200 V
Linear
1.50 hours
Step  5
4000 V
Slow
1.50 hours
Step  6
8000 V
Slow
1 hour
Step  7
8000 V
Linear
30000 volt-hours
Step  8
500 V
Slow
0.15 hours


### 2.8. Gel Visualisation

Gels were scanned using a Typhoon Trio Variable Mode Imager (Molecular Dynamics Inc., Sunnyvale, CA) with a pixel resolution of 100μm at the CyDye excitation and emission wavelengths described by the manufacturer. Image analysis was performed using PD-Quest software (version 7.2, Bio-Rad Laboratories). Replicate groups, each containing four gels, were used for analysis. Protein spots were automatically detected and manually edited. Gel staining was normalized using the total density in gels.


### 2.9. Flamingo Fluorescent Staining

To visualise all proteins, gels were fixed in 40% ethanol (v/v)/10% acetic acid (v/v) in Milli-Q water and stained with Flamingo Fluorescent Stain (Bio-Rad Laboratories) according to the manufacturer’s instructions. Gels were destained in 0.1% (v/v) Tween 20 in Milli-Q water for 10 minutes prior to imaging. Gels were scanned using a Typhoon Trio Variable Mode Imager using a green laser (532 nm) excitation source and610
±
30 nm bandpass emission filter.


### 2.10. Automated Spot Picking

Gel images were scanned using a Typhoon Trio Variable Mode Imager and imported into DeCyder software (version 6.5, GE Healthcare) and spots were detected using the automated method. Spots of interest were selected to generate a pick-list. The pick-list was exported from DeCyder and imported into Spot Picker software (version 1.2, GE Healthcare). Spots were excised using the Ettan Spot Cutting Robot (GE Healthcare) according to the manufacturer’s instructions. Gel plugs were washed twice with 0.1 M ammonium bicarbonate buffer (NH4HCO3), followed by Milli-Q water, then dehydrated in acetonitrile (ACN), and dried.


### 2.11. Protein Identification by Liquid Chromatography-Electrospray Ionisation-Ion Trap (LC-ESI-IT) Mass Spectrometry (MS)

Each gel plug was digested with 10μL of 5 mM ammonium bicarbonate with 10% ACN containing 100 ng trypsin (Promega) for 16 hours at 37°C. Peptides were extracted sequentially with 1% formic acid (FA), 50% ACN/0.1% FA, and ACN, and the combined extracts were concentrated by centrifugal evaporation and diluted in 6 μL 3% ACN/0.1% FA. Vacuum concentrated samples were resuspended in 0.1% FA in 2% ACN to a total volume of ~8 μL. LC-ESI-IT MS/MS was performed using an online 1100 series HPLC system (Agilent Technologies) and HCT Ultra 3D-Ion Trap mass spectrometer (Bruker Daltonics). The LC system was interfaced to the MS using an Agilent Technologies Chip Cube operating with a ProtID-Chip-150 (II), which integrates the enrichment column (Zorbax 300SB-C18, 4 mm, 40 nL), analytical column (Zorbax 300 SB-C18, 150 mm × 75 μm), and nanospray emitter. 5 μL samples were loaded onto the enrichment column set at a flow rate of 4 μL/min in Mobile Phase A (0.1% FA in 2% v/v ACN) and resolved with 1–30% gradient of Mobile Phase B (0.1% FA in 98% w/v ACN) over 32 minutes at 300 nL/min. Ionizable species (300 <m/z< 3,000) were trapped and the two most intense ions eluting at the time were fragmented by collision-induced dissociation. Active exclusion was used to exclude a precursor ion for 30 seconds following the acquisition of two spectra.


### 2.12. Protein Identification Using Web-Based Bioinformatics Tools

MS and MS/MS spectra were subjected to peak detection and deconvolution using Data Analysis (version 3.4, Bruker Daltonics, Billerica, MA, USA). Compound lists were exported into BioTools (version 3.1, Bruker Daltonics) and then submitted to Mascot (version 2.2, Boston, MA, USA) using the following parameters: fixed modification = carbamidomethyl (C), variable modification = oxidation (M), MS mass tolerance = 1.5 Da, MS/MS mass tolerance = 0.8 Da, peptide charge = 1+, 2+, or 3+, and missed cleavages = 3. Data were matched to the Swiss-Prot protein database.


## 2.1. Isolation of Human PDLSC and Gingival Fibroblasts

Human PDLSC and gingival fibroblasts (GF) were isolated from three donors and cultured as previously described (Human Research Ethics Committee of the University of Adelaide, Approval Number H-112-2008) [4, 19]. Briefly, gingival and periodontal ligament tissues were collected from excised gingiva and middle third of the root, respectively. The tissues were digested in equal volumes of collagenase type I (3 mg/mL; Worthington Biochemical, Lakewood, NJ) and dispase type II (4 mg/mL; Roche Diagnostics, Indianapolis, IN) for 2 hours at 37°C. Isolated cells were maintained and cultured in modified α-MEM media (α-MEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FCS (Thermo Electron, Melbourne, VIC, Australia), 50 U/mL and 50 μg/mL penicillin and streptomycin (Sigma-Aldrich), 1 mM sodium pyruvate, 2 mM L-glutamine (SAFC, Lenexa, KS, USA), and 100 μM L-ascorbate-2-phosphate (Novachem, Melbourne, VIC, Australia) at 37°C and 5% CO2 in a humidified environment, with a twice-weekly medium change. Cells were harvested and further expanded once upon reaching confluence. This process was repeated when cells reached 80% confluence until desired cell numbers were obtained.Human neonatal foreskins, collected from routine circumcisions, were used to isolate epithelial sheets after overnight incubation with 4 mg/mL dispase at 4°C, followed by trypsinization for 5 min at 37°C to obtain basal keratinocytes. Keratinocytes were cultured in DMEM containing 10% fetal calf serum, 20 ng/mL epidermal growth factor (Sigma-Aldrich), and 0.4μg/mL hydrocortisone (Sigma-Aldrich), at 37°C and 5% CO2 in a humidified environment, with a twice-weekly medium change.


## 2.2. Immunohistochemistry

Chamber slides (Nalge-Nunc Lab-Tek, Rochester, NY, USA) were seeded with 8 × 103 cells per cm2, in media with additives for 2 days. The slides were fixed with 4% paraformaldehyde (PFA) and endogenous peroxidase activity was inhibited using 0.5% H2O2 in methanol. The sections were incubated with primary antibodies or isotype control antibodies overnight at 4°C, secondary antibodies for 1 hour at room temperature, Vectastain ABC Reagents (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s recommendations, or horseradish-peroxidase-labelled streptavidin (Promega, Madison, WI, USA) at 1 in 1000 dilution and then developed with diaminobenzidine (Dako, Campbellfield, VIC, Australia). The slides were counterstained briefly with haematoxylin (ProSciTech, Thuringowa Central, QLD, Australia). Antibodies used in this study are 1B5, mouse IgG1 isotype control (1 : 25; Professor L. K. Ashman, University of Newcastle, NSW, Australia); mouse IgG1 anti-human CD73 (1 : 25; BD Pharmingen, Sparks, MD, USA); mouse IgG1 anti-human CD90 (1 : 25; BD Pharmingen); mouse IgG1 anti-human Annexin A2 (1 : 12.5; Invitrogen, Waltham, MA, USA); rabbit anti-human sphingosine kinase 1 (1 : 20; Cayman Chemical, Ann Arbor, MI, USA); normal rabbit Ig (1 : 20; Vector Laboratories, Burlingame, CA, USA); goat anti-mouse IgG biotin secondary antibody (1 : 200; Southern Biotechnology Associates, Birmingham, AL); and goat anti-rabbit IgG biotin secondary antibody (1 : 150; Vector Laboratories).


## 2.3. Immunophenotypic Profiling

Single cell suspensions of 2 × 105 cells were blocked with 5% FCS, 1% bovine serum albumin (BSA, SAFC), 50 U/mL penicillin, 50 μg/mL streptomycin, and 5% normal human serum (Red Cross, SA, Australia) in HBSS on ice. Cells were treated with primary or isotype control antibodies (CD73, CD90, and Annexin A2) at a concentration of 20 μg/mL, followed by incubation with phycoerythrin (PE) conjugated goat anti-mouse IgG1 (1 : 50; Southern Biotechnology Associates). Samples were fixed in PBS with 0.1% formalin and 20 mg/mL glucose. Analysis was performed on a fluorescence-activated cell sorter fitted with 250 MW argon laser (Beckman Coulter Cytomics FC500, using CXP Cytometry List Mode Data Acquisition and Analysis Software version 2.2; Beckman Coulter, Miami, FL, USA).


## 2.4. Proteomic Analysis

All equipment and reagents were purchased from Bio-Rad Laboratories (Hercules, CA, USA) unless stated otherwise. CyDye DIGE Fluor minimal dye was purchased from GE Healthcare (Buckinghamshire, UK).


## 2.5. Cell Surface Labelling Using CyDye DIGE Fluor Minimal Dye

CyDye fluorescent cell surface protein labelling was performed as previously reported [4, 20]. Briefly, approximately 20 million subconfluent PDLSC or GF were detached with either 1 mM PUCK’s EDTA or 3 mg/mL type I collagenase and aliquoted into ~5 million cells per tube. Cells were washed in ice cold HBSS (pH 7.4) followed by ice cold HBSS (pH 8.5) and centrifuged at 800 ×g for 2 minutes. The cell pellets were resuspended in 200 μL labelling buffer containing HBSS (pH 8.5) and 1 M urea. Cells were then labelled with 600 pmol of either Cy2, Cy3, or Cy5 or CyDye DIGE Fluor minimal dyes on ice in the dark for 20 minutes. Staining was quenched by adding 20 μL lysine (10 mM) for 10 minutes. Surface-labelled cells were pelleted by centrifugation and resuspended in 202 μL HBSS (pH 7.4). An aliquot (2 μL) was taken prior to and after labelling to check for labelling efficiency using flow cytometry.


## 2.6. Membrane Protein Enrichment

Proteins were isolated and fractionated using a phase separation kit (Mem-PER, Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Briefly, 150μL reagent A containing 1 μL protease inhibitor (Sigma) was added to cell pellets containing PDLSC or GF. Following 10-minute incubation, 450 μL mixture of reagents B and C was added to cell lysates and tubes were kept on ice for 30 minutes. The preparation was centrifuged at 10,000 ×g for 3 minutes, at 4°C, and the supernatant was incubated at 37°C for 20 minutes. Following centrifugation at 10,000 ×g and phase separation, the hydrophobic fraction containing membrane proteins was carefully removed and purified using ReadyPrep 2-D Cleanup Kit. Membrane protein enrichment efficiency was assessed and cells were subjected to up to three membrane fractionation steps.


## 2.7. Membrane Protein Separation by Two-Dimensional Gel Electrophoresis (2DE)

Membrane proteins were solubilised with ReadyPrep reagent 3 buffer for 1 hour at room temperature. The protein was solubilised by gentle aspiration through a fine-gauge needle, as previously described by Zilm et al. [21]. The protein concentration was determined using RC/DC Protein Assay Kit according to the manufacturer’s instructions. Proteins were separated in the first dimension using 11 cm immobilised pH gradient (IPG) strips (pH 3–10) which had been passively rehydrated for 24 hours in 330 μL rehydration/extraction buffer #3, containing 0.2% (w/v) pH 3–10 ampholytes and 1.2% (v/v) De-Streak Reagent (GE Healthcare, Buckinghamshire, UK). IEF was performed using a Protean IEF cell. Briefly, membrane protein preparations containing 150 μg protein were cup-loaded onto the anode end of the IPG strip. The IEF cycle consisted of 8 steps outlined in Table 1, with a 50 μA/strip current limit, and the temperature was maintained at 20°C. Duplicate IPG strips were run concurrently. Following IEF, the IPG strips were equilibrated as previously described [22]. Polyacrylamide gels (18
×
18 cm) containing 8% T, 3.3% C, 0.1% SDS, and 375 mM Tris/HCl (pH 8.8) were cast without stacking gels using a Protean II XL casting chamber. Proteins were separated in the second dimension using a Protean II XL Multicell (Bio-Rad Laboratories) in tris-glycine tank buffer (25 mM Tris, 192 mM glycine, and 0.1% (w/v) SDS) and resolved at 7 mA/gel.Table 1
IEF conditions for 11 cm IPG (3–10).
Step number
Voltage
Voltage ramping mode
Time
Step  1
150 V
Linear
1 hour
Step  2
300 V
Linear
2.30 hours
Step  3
600 V
Linear
1.50 hours
Step  4
1200 V
Linear
1.50 hours
Step  5
4000 V
Slow
1.50 hours
Step  6
8000 V
Slow
1 hour
Step  7
8000 V
Linear
30000 volt-hours
Step  8
500 V
Slow
0.15 hours


## 2.8. Gel Visualisation

Gels were scanned using a Typhoon Trio Variable Mode Imager (Molecular Dynamics Inc., Sunnyvale, CA) with a pixel resolution of 100μm at the CyDye excitation and emission wavelengths described by the manufacturer. Image analysis was performed using PD-Quest software (version 7.2, Bio-Rad Laboratories). Replicate groups, each containing four gels, were used for analysis. Protein spots were automatically detected and manually edited. Gel staining was normalized using the total density in gels.


## 2.9. Flamingo Fluorescent Staining

To visualise all proteins, gels were fixed in 40% ethanol (v/v)/10% acetic acid (v/v) in Milli-Q water and stained with Flamingo Fluorescent Stain (Bio-Rad Laboratories) according to the manufacturer’s instructions. Gels were destained in 0.1% (v/v) Tween 20 in Milli-Q water for 10 minutes prior to imaging. Gels were scanned using a Typhoon Trio Variable Mode Imager using a green laser (532 nm) excitation source and610
±
30 nm bandpass emission filter.


## 2.10. Automated Spot Picking

Gel images were scanned using a Typhoon Trio Variable Mode Imager and imported into DeCyder software (version 6.5, GE Healthcare) and spots were detected using the automated method. Spots of interest were selected to generate a pick-list. The pick-list was exported from DeCyder and imported into Spot Picker software (version 1.2, GE Healthcare). Spots were excised using the Ettan Spot Cutting Robot (GE Healthcare) according to the manufacturer’s instructions. Gel plugs were washed twice with 0.1 M ammonium bicarbonate buffer (NH4HCO3), followed by Milli-Q water, then dehydrated in acetonitrile (ACN), and dried.


## 2.11. Protein Identification by Liquid Chromatography-Electrospray Ionisation-Ion Trap (LC-ESI-IT) Mass Spectrometry (MS)

Each gel plug was digested with 10μL of 5 mM ammonium bicarbonate with 10% ACN containing 100 ng trypsin (Promega) for 16 hours at 37°C. Peptides were extracted sequentially with 1% formic acid (FA), 50% ACN/0.1% FA, and ACN, and the combined extracts were concentrated by centrifugal evaporation and diluted in 6 μL 3% ACN/0.1% FA. Vacuum concentrated samples were resuspended in 0.1% FA in 2% ACN to a total volume of ~8 μL. LC-ESI-IT MS/MS was performed using an online 1100 series HPLC system (Agilent Technologies) and HCT Ultra 3D-Ion Trap mass spectrometer (Bruker Daltonics). The LC system was interfaced to the MS using an Agilent Technologies Chip Cube operating with a ProtID-Chip-150 (II), which integrates the enrichment column (Zorbax 300SB-C18, 4 mm, 40 nL), analytical column (Zorbax 300 SB-C18, 150 mm × 75 μm), and nanospray emitter. 5 μL samples were loaded onto the enrichment column set at a flow rate of 4 μL/min in Mobile Phase A (0.1% FA in 2% v/v ACN) and resolved with 1–30% gradient of Mobile Phase B (0.1% FA in 98% w/v ACN) over 32 minutes at 300 nL/min. Ionizable species (300 <m/z< 3,000) were trapped and the two most intense ions eluting at the time were fragmented by collision-induced dissociation. Active exclusion was used to exclude a precursor ion for 30 seconds following the acquisition of two spectra.


## 2.12. Protein Identification Using Web-Based Bioinformatics Tools

MS and MS/MS spectra were subjected to peak detection and deconvolution using Data Analysis (version 3.4, Bruker Daltonics, Billerica, MA, USA). Compound lists were exported into BioTools (version 3.1, Bruker Daltonics) and then submitted to Mascot (version 2.2, Boston, MA, USA) using the following parameters: fixed modification = carbamidomethyl (C), variable modification = oxidation (M), MS mass tolerance = 1.5 Da, MS/MS mass tolerance = 0.8 Da, peptide charge = 1+, 2+, or 3+, and missed cleavages = 3. Data were matched to the Swiss-Prot protein database.


## 3. Results

### 3.1. Membrane Protein Expression ofEx Vivo Expanded Human PDLSC

CyDye-tagged membrane-associated proteins derived from human PDLSC followingex vivo expansion were separated by 2-dimensional electrophoresis. Based on the CyDye imaging, a total of 80 well-resolved proteins spots with a molecular weight range of 10–110 kDa were detected after automatic exclusion of pseudospots and the locations of the identified protein spots on the representative raw image are shown in Figure 1.Figure 1
Representative raw 2DE gel of CyDye labelled proteins ofex vivo expanded human periodontal ligament stem cells and the location of proteins identified on the raw 2DE image. Following cell surface labelling with CyDye and membrane protein enrichment, proteins were separated by 2DE using a pI range of pH 3–10 and a molecular weight range of 10–110 kDa. Following image analysis, 80 well-resolved protein spots were detected. A total of 32 membrane-associated protein spots were consistently found on replicate gels.


### 3.2. Identification of Proteins Expressed by Human PDLSC

Following spot excision and analysis by mass spectrometry, a total of 32 protein spots were identified as membrane-associated proteins (Figure1). Table 2 outlines the details of the membrane-associated proteins, including the protein name, spot number, predicted molecular weight and pI values, ID/total queries, combined ion scores, and coverage. Some proteins were identified in multiple spots (e.g., 5′-nucleotidase, Annexin A2, and sphingosine kinase 1) suggesting the presence of isoforms, possibly as a result of posttranslational modifications. Differences in the observed molecular weight/pI and the expected values were observed in some proteins (e.g., sphingosine kinase 1), possibly due to posttranslational modifications, proteolysis, or protein aggregation. Importantly, this approach was validated by the identification of MSC-associated stem cell surface proteins, 5′-nucleotidase (CD73) and Thy-1 membrane glycoprotein (CD90), previously shown to be expressed by PDLSC [8]. Furthermore, MS identified other membrane-associated markers, such as Annexin A2 and sphingosine kinase 1, the expression of which had not previously been reported by human PDLSC. All four proteins were chosen for further confirmatory analyses.Table 2
Membrane-associated proteins on human PDLSC.
Swiss-Prot 57.7Accession number
Protein name
Spot number
Predicted MW (kDa)/pI
ID/total queries
Combined ion scores
Coverage (%)
1A01_HUMAN
HLA class I histocompatibility antigen, A-1 alpha chain
243
41.1/6.1
2/612
66
10
1C06_HUMAN
HLA class I histocompatibility antigen, Cw-6 alpha chain
239
41.4/5.7
4/638
175
18
5NTD_HUMAN
5′-Nucleotidase
78
63.9/6.6
13/518
287
17
80
63.9/6.6
8/513
206
11
90
63.9/6.6
41/546
872
32
99
63.9/6.6
13/535
191
15
101
63.9/6.6
39/517
945
38
103
63.9/6.6
42/539
933
33
109
63.9/6.6
25/536
606
22
111
63.9/6.6
2/558
34
6
326
63.9/6.6
53/544
961
35
86
63.9/6.6
25/356
521
31
97
63.9/6.6
45/414
1044
39
AMPB_HUMAN
Aminopeptidase B
94
73.2/5.5
5/559
172
10
ANXA2_HUMAN
Annexin A2
285
38.8/7.6
36/581
489
47
288
38.8/7.6
87/585
1907
67
289
38.8/7.6
56/600
950
55
292
38.8/7.6
3/594
76
11
304
38.8/7.6
14/536
241
28
CAP2_HUMAN
Adenylyl cyclase-associated protein 2
154
53.1/6.0
2/576
67
6
156
53.1/6.0
4/557
102
8
165
53.1/6.0
3/619
102
11
CO1A1_HUMAN
Collagen alpha-1(I) chain
292
139.9/5.6
7/594
103
4
CO6A3_HUMAN
Collagen alpha-3(VI) chain
20
345.2/6.3
9/568
159
3
DNJA1_HUMAN
DnaJ homolog subfamily A member 1
208
45.6/6.6
4/521
95
12
EHD3_HUMAN
EH domain-containing protein 3
139
62.0/6.1
4/600
71
8
EZRI_HUMAN
Ezrin
65
69.5/5.9
28/558
455
26
FLNC_HUMAN
Filamin-C
29
293.4/5.6
4/492
49
2
78
293.4/5.6
3/518
65
1
139
293.4/5.6
14/600
363
6
147
293.4/5.6
13/525
237
4
191
293.4/5.6
4/586
147
2
GELS_HUMAN
Gelsolin
46
86.0/5.9
46/636
1011
35
K2C1_HUMAN
Keratin, type II cytoskeletal 1
25
66.2/8.2
5/578
128
9
46
66.2/8.2
11/636
456
17
47
66.2/8.2
7/491
170
7
80
66.2/8.2
11/513
227
13
91
66.2/8.2
3/666
94
12
94
66.2/8.2
4/559
146
8
98
66.2/8.2
2/639
57
7
104
66.2/8.2
2/520
80
3
111
66.2/8.2
5/558
79
10
130
66.2/8.2
3/614
60
8
147
66.2/8.2
20/525
256
13
153
66.2/8.2
2/615
126
7
154
66.2/8.2
3/576
107
8
228
66.2/8.2
10/664
466
22
289
66.2/8.2
14/600
386
15
292
66.2/8.2
2/594
55
3
324
66.2/8.2
5/570
127
10
KAP2_HUMAN
cAMP-dependent protein kinase type II-alpha regulatory subunit
178
45.8/5.0
13/621
360
22
NOMO2_HUMAN
Nodal modulator 2
19
140.4/5.5
9/599
244
8
20
140.4/5.5
10/568
189
12
25
140.4/5.5
10/578
226
8
27
140.4/5.5
11/583
224
10
28
140.4/5.5
11/436
244
12
NUCB2_HUMAN
Nucleobindin-2
178
50.3/5.0
3/621
100
5
PDIA6_HUMAN
Protein disulfide-isomerase A6
175
48.5/5.0
6/592
209
12
178
48.5/5.0
7/621
278
10
RUVB2_HUMAN
RuvB-like 2
191
51.3/5.5
25/586
614
34
SBP1_HUMAN
Selenium-binding protein 1
165
52.9/5.9
7/619
126
8
SNX4_HUMAN
Sorting nexin-4
155
52.2/5.7
5/637
157
14
SPHK1_HUMAN
Sphingosine kinase 1
19
42.9/6.6
12/599
253
18
20
42.9/6.6
5/568
188
16
21
42.9/6.6
4/518
138
11
34
42.9/6.6
3/517
200
11
STML2_HUMAN
Stomatin-like protein 2
239
38.6/6.9
13/638
584
37
240
38.6/6.9
6/656
266
27
STXB3_HUMAN
Syntaxin-binding protein 3
111
68.6/8.0
5/558
97
7
118
68.6/8.0
6/479
103
7
SWP70_HUMAN
Switch-associated protein 70
94
69.4/5.7
3/559
80
5
99
69.4/5.7
6/535
136
7
THY1_HUMAN
Thy-1 membrane glycoprotein
304
18.2/9.0
3/536
55
16
UBP14_HUMAN
Ubiquitin carboxyl-terminal hydrolase 14
130
56.5/5.2
6/614
176
15
132
56.5/5.2
8/611
184
18
133
56.5/5.2
3/635
100
5
136
56.5/5.2
15/642
372
24
ULA1_HUMAN
NEDD8-activating enzyme E1 regulatory subunit
133
60.7/5.2
6/635
127
8
VATB2_HUMAN
V-type proton ATPase subunit B, brain isoform
153
56.8/5.6
4/615
191
12
154
56.8/5.6
9/576
252
14
155
56.8/5.6
7/637
300
16
VDAC1_HUMAN
Voltage-dependent anion-selective channel protein 1
300
30.9/8.6
7/601
128
18
304
30.9/8.6
34/536
902
59
305
30.9/8.6
19/524
319
38
VIME_HUMAN
Vimentin
130
53.7/5.1
2/614
68
6
161
53.7/5.1
28/626
710
46
175
53.7/5.1
44/592
1026
63
178
53.7/5.1
46/621
1306
58
222
53.7/5.1
3/630
87
6
228
53.7/5.1
5/664
139
9
239
53.7/5.1
4/638
139
9
240
53.7/5.1
5/656
169
12
VINC_HUMAN
Vinculin
25
124.3/5.5
14/578
226
16
27
124.3/5.5
30/583
643
25


### 3.3. Validation of the Expression of 5′-Nucleotidase, Thy-1 Membrane Glycoprotein, Annexin A2, and Sphingosine Kinase 1

To confirm the expression of selected proteins including CD73, CD90, Annexin A2, and sphingosine kinase 1 (SPK1), additional studies were performed to investigate their expression in human PDLSC, GF, and keratinocytes (epithelial cell population). Flow cytometric analysis demonstrated high surface expression of CD73 and CD90 and low cell surface levels of Annexin A2 expression in human PDLSC and GF populations (Figure2). In contrast, human keratinocytes showed a lack of cell surface expression for CD73, CD90, and Annexin A2 (Figure 2). Table 3 summarizes levels of surface expression of these four antigens on assessed cell types. In summary, CD73 and CD90 were expressed by human PDLSC and GF, but not by human keratinocytes, confirming that they are MSC-associated markers. Annexin A2 was demonstrated to be expressed at low levels by human PDLSC (1.92–7.83%) and human GF (2.41–4.66%), while human keratinocytes were largely negative for Annexin A2 expression (0.88–1.64%). Previous studies have shown that SPK1 can translocate to the plasma membrane upon cell stimulation by cytokines [23–29]. No positive expression was detected with the anti-SPK1 antibody to the human cell types by flow cytometric analysis (data not shown), most likely because the available antibody reagent did not react with the extracellular domain of SPK1.Table 3
Flow cytometric analysis of cell surface expression of CD73, CD90, and Annexin A2. Data represent median% (range);n
=
3 replicate experiments.
Antigen
PDLSC
GF
Keratinocytes
CD73
99.90
99.97
0.82
(99.4–99.9)
(99.9–100)
(0.78–0.84)
CD90
99.98
99.98
1.58
(99.8–100)
(99.9–100)
(1.55–1.62)
Annexin A2
4.48
2.92
0.86
(1.92–7.83)
(2.41–4.66)
(0.86–1.64)Figure 2
Validation of surface expression of CD73, CD90, and Annexin A2 using flow cytometric analysis in human (a) periodontal ligament stem cells, (b) gingival fibroblasts, and (c) skin keratinocytes.
(a)
(b)
(c)Additional studies were performed to investigate the expression of Annexin A2 and SPK1 in human PDLSC, GF, and keratinocytes, using immunocytochemistry. All cell types studied were positive to anti-Annexin A2 and anti-SPK1 antibodies (Figure3). Of note, no reactivity was observed with the anti-CD73 or anti-CD90 antibodies to all cell types examined (data not shown), indicating that the specific epitopes identified by these antibodies were compromised following processing for immunocytochemical analysis.Figure 3
The expression of Annexin A2 and sphingosine kinase 1 (SPK1) in fixed and permeabilized (a) human periodontal ligament stem cells (PDLSC), (b) gingival fibroblasts (GF), and (c) keratinocytes using immunohistochemistry. All cell types studied were positive to anti-Annexin A2 and anti-SPK1 antibodies. Scale bar = 50μm.
(a)
(b)
(c)


## 3.1. Membrane Protein Expression ofEx Vivo Expanded Human PDLSC

CyDye-tagged membrane-associated proteins derived from human PDLSC followingex vivo expansion were separated by 2-dimensional electrophoresis. Based on the CyDye imaging, a total of 80 well-resolved proteins spots with a molecular weight range of 10–110 kDa were detected after automatic exclusion of pseudospots and the locations of the identified protein spots on the representative raw image are shown in Figure 1.Figure 1
Representative raw 2DE gel of CyDye labelled proteins ofex vivo expanded human periodontal ligament stem cells and the location of proteins identified on the raw 2DE image. Following cell surface labelling with CyDye and membrane protein enrichment, proteins were separated by 2DE using a pI range of pH 3–10 and a molecular weight range of 10–110 kDa. Following image analysis, 80 well-resolved protein spots were detected. A total of 32 membrane-associated protein spots were consistently found on replicate gels.


## 3.2. Identification of Proteins Expressed by Human PDLSC

Following spot excision and analysis by mass spectrometry, a total of 32 protein spots were identified as membrane-associated proteins (Figure1). Table 2 outlines the details of the membrane-associated proteins, including the protein name, spot number, predicted molecular weight and pI values, ID/total queries, combined ion scores, and coverage. Some proteins were identified in multiple spots (e.g., 5′-nucleotidase, Annexin A2, and sphingosine kinase 1) suggesting the presence of isoforms, possibly as a result of posttranslational modifications. Differences in the observed molecular weight/pI and the expected values were observed in some proteins (e.g., sphingosine kinase 1), possibly due to posttranslational modifications, proteolysis, or protein aggregation. Importantly, this approach was validated by the identification of MSC-associated stem cell surface proteins, 5′-nucleotidase (CD73) and Thy-1 membrane glycoprotein (CD90), previously shown to be expressed by PDLSC [8]. Furthermore, MS identified other membrane-associated markers, such as Annexin A2 and sphingosine kinase 1, the expression of which had not previously been reported by human PDLSC. All four proteins were chosen for further confirmatory analyses.Table 2
Membrane-associated proteins on human PDLSC.
Swiss-Prot 57.7Accession number
Protein name
Spot number
Predicted MW (kDa)/pI
ID/total queries
Combined ion scores
Coverage (%)
1A01_HUMAN
HLA class I histocompatibility antigen, A-1 alpha chain
243
41.1/6.1
2/612
66
10
1C06_HUMAN
HLA class I histocompatibility antigen, Cw-6 alpha chain
239
41.4/5.7
4/638
175
18
5NTD_HUMAN
5′-Nucleotidase
78
63.9/6.6
13/518
287
17
80
63.9/6.6
8/513
206
11
90
63.9/6.6
41/546
872
32
99
63.9/6.6
13/535
191
15
101
63.9/6.6
39/517
945
38
103
63.9/6.6
42/539
933
33
109
63.9/6.6
25/536
606
22
111
63.9/6.6
2/558
34
6
326
63.9/6.6
53/544
961
35
86
63.9/6.6
25/356
521
31
97
63.9/6.6
45/414
1044
39
AMPB_HUMAN
Aminopeptidase B
94
73.2/5.5
5/559
172
10
ANXA2_HUMAN
Annexin A2
285
38.8/7.6
36/581
489
47
288
38.8/7.6
87/585
1907
67
289
38.8/7.6
56/600
950
55
292
38.8/7.6
3/594
76
11
304
38.8/7.6
14/536
241
28
CAP2_HUMAN
Adenylyl cyclase-associated protein 2
154
53.1/6.0
2/576
67
6
156
53.1/6.0
4/557
102
8
165
53.1/6.0
3/619
102
11
CO1A1_HUMAN
Collagen alpha-1(I) chain
292
139.9/5.6
7/594
103
4
CO6A3_HUMAN
Collagen alpha-3(VI) chain
20
345.2/6.3
9/568
159
3
DNJA1_HUMAN
DnaJ homolog subfamily A member 1
208
45.6/6.6
4/521
95
12
EHD3_HUMAN
EH domain-containing protein 3
139
62.0/6.1
4/600
71
8
EZRI_HUMAN
Ezrin
65
69.5/5.9
28/558
455
26
FLNC_HUMAN
Filamin-C
29
293.4/5.6
4/492
49
2
78
293.4/5.6
3/518
65
1
139
293.4/5.6
14/600
363
6
147
293.4/5.6
13/525
237
4
191
293.4/5.6
4/586
147
2
GELS_HUMAN
Gelsolin
46
86.0/5.9
46/636
1011
35
K2C1_HUMAN
Keratin, type II cytoskeletal 1
25
66.2/8.2
5/578
128
9
46
66.2/8.2
11/636
456
17
47
66.2/8.2
7/491
170
7
80
66.2/8.2
11/513
227
13
91
66.2/8.2
3/666
94
12
94
66.2/8.2
4/559
146
8
98
66.2/8.2
2/639
57
7
104
66.2/8.2
2/520
80
3
111
66.2/8.2
5/558
79
10
130
66.2/8.2
3/614
60
8
147
66.2/8.2
20/525
256
13
153
66.2/8.2
2/615
126
7
154
66.2/8.2
3/576
107
8
228
66.2/8.2
10/664
466
22
289
66.2/8.2
14/600
386
15
292
66.2/8.2
2/594
55
3
324
66.2/8.2
5/570
127
10
KAP2_HUMAN
cAMP-dependent protein kinase type II-alpha regulatory subunit
178
45.8/5.0
13/621
360
22
NOMO2_HUMAN
Nodal modulator 2
19
140.4/5.5
9/599
244
8
20
140.4/5.5
10/568
189
12
25
140.4/5.5
10/578
226
8
27
140.4/5.5
11/583
224
10
28
140.4/5.5
11/436
244
12
NUCB2_HUMAN
Nucleobindin-2
178
50.3/5.0
3/621
100
5
PDIA6_HUMAN
Protein disulfide-isomerase A6
175
48.5/5.0
6/592
209
12
178
48.5/5.0
7/621
278
10
RUVB2_HUMAN
RuvB-like 2
191
51.3/5.5
25/586
614
34
SBP1_HUMAN
Selenium-binding protein 1
165
52.9/5.9
7/619
126
8
SNX4_HUMAN
Sorting nexin-4
155
52.2/5.7
5/637
157
14
SPHK1_HUMAN
Sphingosine kinase 1
19
42.9/6.6
12/599
253
18
20
42.9/6.6
5/568
188
16
21
42.9/6.6
4/518
138
11
34
42.9/6.6
3/517
200
11
STML2_HUMAN
Stomatin-like protein 2
239
38.6/6.9
13/638
584
37
240
38.6/6.9
6/656
266
27
STXB3_HUMAN
Syntaxin-binding protein 3
111
68.6/8.0
5/558
97
7
118
68.6/8.0
6/479
103
7
SWP70_HUMAN
Switch-associated protein 70
94
69.4/5.7
3/559
80
5
99
69.4/5.7
6/535
136
7
THY1_HUMAN
Thy-1 membrane glycoprotein
304
18.2/9.0
3/536
55
16
UBP14_HUMAN
Ubiquitin carboxyl-terminal hydrolase 14
130
56.5/5.2
6/614
176
15
132
56.5/5.2
8/611
184
18
133
56.5/5.2
3/635
100
5
136
56.5/5.2
15/642
372
24
ULA1_HUMAN
NEDD8-activating enzyme E1 regulatory subunit
133
60.7/5.2
6/635
127
8
VATB2_HUMAN
V-type proton ATPase subunit B, brain isoform
153
56.8/5.6
4/615
191
12
154
56.8/5.6
9/576
252
14
155
56.8/5.6
7/637
300
16
VDAC1_HUMAN
Voltage-dependent anion-selective channel protein 1
300
30.9/8.6
7/601
128
18
304
30.9/8.6
34/536
902
59
305
30.9/8.6
19/524
319
38
VIME_HUMAN
Vimentin
130
53.7/5.1
2/614
68
6
161
53.7/5.1
28/626
710
46
175
53.7/5.1
44/592
1026
63
178
53.7/5.1
46/621
1306
58
222
53.7/5.1
3/630
87
6
228
53.7/5.1
5/664
139
9
239
53.7/5.1
4/638
139
9
240
53.7/5.1
5/656
169
12
VINC_HUMAN
Vinculin
25
124.3/5.5
14/578
226
16
27
124.3/5.5
30/583
643
25


## 3.3. Validation of the Expression of 5′-Nucleotidase, Thy-1 Membrane Glycoprotein, Annexin A2, and Sphingosine Kinase 1

To confirm the expression of selected proteins including CD73, CD90, Annexin A2, and sphingosine kinase 1 (SPK1), additional studies were performed to investigate their expression in human PDLSC, GF, and keratinocytes (epithelial cell population). Flow cytometric analysis demonstrated high surface expression of CD73 and CD90 and low cell surface levels of Annexin A2 expression in human PDLSC and GF populations (Figure2). In contrast, human keratinocytes showed a lack of cell surface expression for CD73, CD90, and Annexin A2 (Figure 2). Table 3 summarizes levels of surface expression of these four antigens on assessed cell types. In summary, CD73 and CD90 were expressed by human PDLSC and GF, but not by human keratinocytes, confirming that they are MSC-associated markers. Annexin A2 was demonstrated to be expressed at low levels by human PDLSC (1.92–7.83%) and human GF (2.41–4.66%), while human keratinocytes were largely negative for Annexin A2 expression (0.88–1.64%). Previous studies have shown that SPK1 can translocate to the plasma membrane upon cell stimulation by cytokines [23–29]. No positive expression was detected with the anti-SPK1 antibody to the human cell types by flow cytometric analysis (data not shown), most likely because the available antibody reagent did not react with the extracellular domain of SPK1.Table 3
Flow cytometric analysis of cell surface expression of CD73, CD90, and Annexin A2. Data represent median% (range);n
=
3 replicate experiments.
Antigen
PDLSC
GF
Keratinocytes
CD73
99.90
99.97
0.82
(99.4–99.9)
(99.9–100)
(0.78–0.84)
CD90
99.98
99.98
1.58
(99.8–100)
(99.9–100)
(1.55–1.62)
Annexin A2
4.48
2.92
0.86
(1.92–7.83)
(2.41–4.66)
(0.86–1.64)Figure 2
Validation of surface expression of CD73, CD90, and Annexin A2 using flow cytometric analysis in human (a) periodontal ligament stem cells, (b) gingival fibroblasts, and (c) skin keratinocytes.
(a)
(b)
(c)Additional studies were performed to investigate the expression of Annexin A2 and SPK1 in human PDLSC, GF, and keratinocytes, using immunocytochemistry. All cell types studied were positive to anti-Annexin A2 and anti-SPK1 antibodies (Figure3). Of note, no reactivity was observed with the anti-CD73 or anti-CD90 antibodies to all cell types examined (data not shown), indicating that the specific epitopes identified by these antibodies were compromised following processing for immunocytochemical analysis.Figure 3
The expression of Annexin A2 and sphingosine kinase 1 (SPK1) in fixed and permeabilized (a) human periodontal ligament stem cells (PDLSC), (b) gingival fibroblasts (GF), and (c) keratinocytes using immunohistochemistry. All cell types studied were positive to anti-Annexin A2 and anti-SPK1 antibodies. Scale bar = 50μm.
(a)
(b)
(c)


## 4. Discussion

Initially, the first proteomic reference map of undifferentiated periodontal ligament fibroblasts identified 117 proteins, consistently expressed across three clones, which included a variety of expected cytoskeleton- and metabolism-related proteins [30]. This comparative analysis of the proteome revealed that the percentage of total cytoskeleton-related proteins identified in periodontal ligament fibroblasts (26.5%) was higher than that in dermal fibroblasts (15%). It was proposed that this difference is assigned to mechanical loading and rapid remodelling associated with periodontal ligament tissue [30].Assessment of protein expression during differentiation of PDLSC identified 29 proteins, differentially expressed during early cementoblastic/osteogenic differentiation [31], and demonstrated a reduction in expression of cytoskeletal proteins and their binding partners, potentially attributed to cytoskeletal rearrangements during differentiation processes [32]. Interestingly, higher expression of the calcium-binding protein Annexin A4 was noted following osteogenic differentiation. Annexins are thought to play an important role in osteogenic development including Annexin A2 and Annexin A5 which are highly expressed in skeletal tissues and upregulated in osteogenic cultures of MSC [33, 34].A direct comparison of protein expression profiles between ovine PDLSC, DPSC, and BMSC identified 58 differentially expressed proteins between at least two MSC populations, with the expression of 6 proteins upregulated in PDLSC relative to both DPSC and BMSC, 5 proteins upregulated in DPSC relative to both PDLSC and BMSC, and 1 protein upregulated in BMSC relative to both PDLSC and DPSC [35]. An increase in PDLSC expression of heat-shock protein beta 1, Annexin A3, and Annexin A4 compared to DPSC and BMSC was thought to relate to high turnover of periodontal tissues.The aim of the present study was to determine the surface expression profile of human PDLSC and to compare the expression of prospective cell surface markers in human PDLSC, GF, and keratinocytes (as a source of epithelial cells). Our findings identified 80 proteins expressed on the surface of human PDLSC, 32 of which were membrane associated and four of which were selected for further validation due to their known association with other MSC-like populations as a proof-of-principal analysis. These include CD73 and CD90, well known MSC-associated markers, and Annexin A2 and SPK1. Annexin A2 is calcium dependent [36–42] and has been reported to be associated with the stem cell niche [43–49] and SPK1 has recently been demonstrated to be associated with the progenitor phenotype of endothelial cells [50]. CD73 and CD90 were highly expressed by human PDLSC and GF but not by human keratinocytes, indicating that these antigens could be used as potential markers for distinguishing mesenchymal from epithelial cell populations. Annexin A2 was demonstratively expressed at the cell surface at low copy number by human PDLSC and GF, using flow cytometric analysis, while human keratinocytes lacked any cell surface expression of Annexin A2. Expression of SPK1 was detected in all analysed cell types using immunocytochemical analysis.CD73, originally defined as a lymphocyte differentiation antigen, functions as a cosignalling molecule on T lymphocytes and is required for lymphocyte binding to endothelium [51]. Expression of CD73 has been demonstrated on various cell types including lymphocytes, endothelial cells, and MSC. It is thought to play physiological roles in epithelial ion and fluid transport, maintaining barrier functions, mediating endothelial permeability, adapting to hypoxia, and contributing to microbial responses [52]. CD73 is an extracellular enzyme that catalyzes the formation of immunosuppressive adenosine by converting adenosine 5′-monophosphate (AMP) to its bioactive intermediate, adenosine, which in turn activates adenosine receptors, when released into the extracellular space, and regulates various physiological functions [52, 53]. Adenosine signalling, modulated by CD39 and CD73 expression, has been highlighted as a novel modulator in the immunosuppression of T-cell proliferation by MSC [54, 55]. As such, this may be contributory to immunomodulatory properties of PDLSC [8] and may highlight an avenue for anti-inflammatory therapy in periodontal disease [4, 56].The expression of CD90 on PDLSC has been well documented [56–59]; however, its role in PDLSC function remains largely unknown. CD90, also known as Thy-1 (thymocyte differentiation antigen-1), is found to be expressed in various cell types such as hematopoietic stem/progenitor cells [60], hepatic stem cells in human fetal liver [61], liver cancer stem cells [62], neurons, fibroblasts, vascular pericytes, and MSC [63]. Its expression is developmentally regulated [64] and remains one of the minimal criteria for defining human MSC, proposed by the committee for the International Society for Cellular Therapy (ISCT) [63, 65]. While the biological role of CD90 is unclear, a number of associated immunological and nonimmunological functions have been previously addressed [64]. In addition to its involvement in T-cell activation [64], it is believed to be associated with many cellular processes and pathological conditions in a context-dependent manner [63], including cell-cell and cell-matrix interactions, cell motility, and thymocyte adhesion to epithelium [64]. Moreover, CD90 expression is also associated with fibroblast phenotypes relevant to wound healing and fibrosis. The differential CD90 expression is associated with cell-extracellular matrix interactions and cell migration and, as such, is correlated with distinct cellular morphology [64].Annexin A2, firstly identified as an intracellular protein, has since been found extracellularly both in secreted and in membrane bound form [66]. While Annexin A2 monomer is largely present in the cytoplasm, the formation of the heterotetramer allows its binding to the plasma membrane [67–70]. Potential roles of extracellular Annexin A2 include plasminogen activation, cell-cell adhesion, and immunoglobulin transport [66]. Increasing evidence has highlighted the roles of the Annexin family of calcium-dependent, phopholipid-binding proteins in the mineralization process [36–42] and found them to be highly expressed in calcifying cartilage and bone and to serve to initiate mineralization of extracellular matrix [71]. Previously, it has been suggested that Annexin members also have the capacity to function in a compensatory manner of each other during skeletal development [72]. In a study investigating intracellular processes involved in mineralization, the overexpression of Annexin A2 has been shown to increase ALP activity and cartilage and bone formation, while diminished Annexin A2 expression resulted in decreased mineralization [38]. Collectively, previous findings related to Annexins in dental tissues are consistent with their roles in support of osteogenic differentiation and formation of minerals [31, 33, 35, 73, 74].We identified Annexin A2 as one of the cell surface proteins expressed by human PDLSC. Further flow cytometric analysis showed the surface expression of Annexin A2 at low copy number by human GF. As a member of the Annexin family which plays important roles in the mineralization process [36–42], the low expression of Annexin A2 in human GF may be correlated to the fact that human GF demonstrated limited osteogenic potential when cultured in osteogenic conditions (data not shown). A recent study [75] suggested that Annexin A2 regulates adhesion, homing, and engraftment within stem cell niches at endosteal [43–47] and vascular [48, 49] sites; hence, we propose that it may be a potential marker of the PDLSC niche in periodontal tissues.SPK1, the more characterised of the two SPK isoforms, enhances cell growth and proliferation and is involved in immune regulation and tumorigenesis [76]. This highly conserved lipid kinase catalyzes the phosphorylation of proapoptotic sphingosine to form antiapoptotic sphingosine-1-phosphate (S1P) [77] and, as such, is an important cell fate determinant [78]. S1P is a key sphingolipid metabolite that regulates various physiological and pathological processes such as cell proliferation, differentiation, apoptosis, migration, invasion, and angiogenesis [78] and is thought to promote cell growth and proliferation and suppresses apoptosis [79]. In addition to their roles in regulating cell proliferation and apoptosis, SPK-S1P-S1P receptors have been shown to be involved in immune regulation such as immune cell trafficking, activation, and T-cell differentiation [77].Multiple studies have demonstrated that SPK and S1P play important roles in the maintenance of stem cells [76] including endothelial progenitor cells, rate of endothelial progenitor cell differentiation [50, 80, 81], neural progenitors [82], human embryonic stem cells [76, 83], hematopoietic stem cell [76], and muscle progenitors [84]. Furthermore, SPK1 is a maker of oncogenic potential, tumour progression, and cancer prognosis in numerous tissue types [85, 86]. It is predominantly a cytosolic enzyme, which lacks an obvious membrane anchoring sequence. However, considerable evidence has suggested that SPK1 can be translocated to the plasma membrane upon cell stimulation by growth factors and cytokines [23–25, 27–29, 83]. The present study initially identified SPK1 in human PDLSC by proteomic analysis and SPK1 expression was demonstrated in all cell types examined by immunocytochemical analysis. However, we were unable to demonstrate the cell surface expression of this enzyme, limited by the availability of an SPK1 antibody suitable for flow cytometry.


## 5. Conclusion

In summary, this study is the first, to date, to investigate the cell surface proteome ofex vivo expanded human PDLSC. In addition to the expression of recognised MSC-associated cell surface antigens CD73 and CD90, PDLSC were also found to express two novel cell surface proteins, Annexin A2 and sphingosine kinase 1. Interestingly, previous studies have implicated CD73, CD90, Annexin A2, and sphingosine kinase 1 expression in the maintenance of various stem cell populations. Importantly, this study found that human skin epithelial cells lacked the expression of CD73, CD90, and Annexin A2. These proteomic findings provide the platform to further define the cell surface protein expression profile of PDLSC in order to further characterise this cell population and support development of novel isolation and purification strategies.



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