Effects of lysophosphatidic acid on human periodontal ligament stem cells from teeth extracted from dental patients

Byung Cheol Kim,Jae-In Song,Kyoung-Ha So,✉,Sang-Hwan Hyun,✉1 Laboratoryof Veterinary Embryologyand Biotechnology,Veterinary Medical Center and Collegeof Veterinary Medicine,Chungbuk National University,Seowon-gu,Cheongju 28644,Republic of Korea;

2 Instituteof Stem Cell&Regenerative Medicine,Chungbuk National University,Seowon-gu,Cheongju 28644,Republic of Korea.

Abstract Despitetheir potential applicationsin futureregenerativemedicine,periodontal ligament stem cells(PDLSCs)are dif f i cult to obtain in largeamountsfrom patients.Therefore,maintaining stemnesswhileexpanding thecell numbers for medical useisthekey to transitioning PDLSCsfrom thebench to theclinic.Lysophosphatidic acid(LPA),which is present in the human body and saliva,is a signaling molecule derived from phospholipids.In this study,we examined theeffectsof LPA on stemnessmaintenancein human PDLSCs.Several spindle-shaped and f i broblast-like periodontal ligament stem-like cell lines were established from PDLSC isolation.Among these cell lines,the most morphologically appropriate cell line was characterized.The expression levels of OCT4,NANOG(a stem cell marker),and CD90(a mesenchymal stem cell marker)were high.However,CD73(a negative marker of mesenchymal stem cells)expression was not observed.Notably,immuno f l uorescence analysis identi f i ed the expression of STRO-1,CD146(amesenchymal stem cell marker),and sex determining region Y-box 2 at theprotein level.In addition,lipid droplets were stained by Oil red O after the induction of adipogenesis for 21 days,and mineralized nodules were stained by Alizarin Red S after the induction of osteogenesis for 14 days.Alkaline phosphate staining also demonstrated the occurrence of osteogenesis.In summary,we established a human PDLSC line,which could be applied as a cell source for tissue regeneration in dental patients.However,further studies are needed to determine the detailed effects of LPA on PDLSCs.

Keywords:periodontal ligament stem cell,lysophosphatidic acid,stemness,primary cell culture

Introduction

Third molars or deciduous teeth are often extracted during dental procedures and then discarded.But from the extracted human teeth,stem cells may be obtained[1].Periodontal ligament stem cells(PDLSCs)havepotential applicationsin regenerativemedicineasa source for mesenchymal stem cells derived from neural crest cells.Postnatal stem cells can be found in various human tissues,such asthe bone marrow,umbilical cord blood,adipose tissue,and muscles[2-3].Among these stem cells,PDLSCs is more convenient and less invasive because they can be collected after a simple dental procedure;by contrast,bone marrow-derived stem cells require the patient to undergo a dif f i cult and painful procedure[4].PDLSCs are also associated with fewer immune-related problems because autologous transplantation is possible.Additionally,PDLSCs are free from the ethical problems unlike embryonic stem cells.

In dental research,PDLSCs have attracted attention for their potentials to regenerate and repair damaged dental structures[3,5].As shown in previous studies,PDLSCs can differentiate into neurogenic,cardiomyogenic,chondrogenic,and osteogenic lineages,as is needed to form periodontal ligaments,alveolar bone,cementum,peripheral nerves,and blood vessels[2,6-8].

Despite these advantages,PDLSCs can only be obtained in small amounts from a patient.The number of PDLSCsobtained from primary culture isonly 1,250 on average[9].However,for periodontal regeneration,at least 4×106cells are needed[10].Thus,primary cultured PDLSCs must proliferate for at least 12 doublings before clinical use[11].Therefore,maintaining stemness while expanding the cell numbers is essential for transitioning PDLSCs from the bench to the clinic.

Lysophosphatidic acid(LPA),a serum-derived growth factor present in the human body,is a signaling molecule derived from phospholipids.LPA can stimulate cell proliferation,differentiation,migration,and survival[12-15]and appears to havepro-/anti-in f l ammatory and pro-/anti-apoptotic properties[16].For example,LPA enhances the survival of human umbilical cord stem cellsin ischemic conditions[17].In thedental f i eld,LPA is present in normal human saliva[18-19]and plasmaand may affect theperiodontal ligament in vivo.Cheng et al.have showed that LPA enhances the repair of human dental pulp cellsby controlling cell adhesion,migration,and differentiation[20-21].Furthermore,LPA rescueshuman dental pulp cellsfrom ischemia-induced apoptosis by accelerating the repair of injured or damaged tissues[22].LPA also controls regenerative responses of human gingival and periodontal ligament f i broblasts and helps in oral wound healing[23].In addition,a recent study in our laboratory showed positive effects of LPA in porcine female genital cells.A 30μmol/L LPA treatment during in vitro maturation improved the developmental potential of parthenogenic embryos and in vitro fertilized embryos in swine.

The Hippo pathway is a newly recognized key regulator of stem cells,organ growth,and tissue repair,and its dysregulation is associated with cancer development[24].Yes-associated protein(YAP),an effector inhibited by the Hippo pathway,is upregulated in mouse stem and progenitor cells[25].A study of human induced pluripotent stem cells showed that the Hippo pathway blocked reprogramming pluripotency,indicating YAPmightregulatepluripotency in pluripotentstem cells[26-27].LPA can induce overexpression of YAP,thereby promoting and maintaining naïve pluripotency by suppressing differentiation-inducing effects[28].

Based on these f i ndings,we hypothesized that LPA migh tenhancethestemnessof PDLSCs.However,LPA also has negative effects,such as promoting tumor growth and bone destruction[29].Therefore,in this study,we examined the effects of LPA on stemness maintenance in human PDLSCs.

Materials and methods

Chemicals

All chemicalsused in thisstudy werepurchased from Sigma-Aldrich Chemical Company(St.Louis,MO,USA),unlessotherwiseindicated.LPAwasdissolved in 0.1%(w/v)BSA in PBSand frozen at-20℃before addition to culture medium.

Samples and cell culture

This study was approved and supervised by the Institute of Review Board of Chungbuk National University(CBNU-201608-BMBRETC-322-01).Only healthy adults(18 to 30 years old)who provided informed consent for participation were enrolled in this study.Plaque on the teeth was removed with a curette,and the teeth were disinfected with chlorhexidine solution and washed with salinetwice.Thenon-decayed third molars were then extracted by a dentist and stored in Dulbecco’s PBS with 1%antimycotic/antibiotic solution(Gibco,Carlsbad,CA,USA).Periodontal ligaments were separated gently from the mid-third of the root,minced into tiny pieces and immersed in a mixture of 1:1 type I collagenase(Worthington Biochem,Freehold,NJ,USA)/dispase II solution.Cells were incubated at 37℃for 60-90 minutes with gentle shaking and isolated to a single-cell suspension using a 70-μm cell strainer(SPL Life Science,Seoul,Republic of Korea).PDLSCs were cultured in Dulbecco’s modi f i ed Eagle’s medium(DMEM;Gibco)containing 10%FBS(Gibco)and 1%nonessential amino acids(Gibco),hereafter referred to as growth medium,under humidi f i ed conditions of 5%CO2in air at 37℃.

Reverse transcription polymerase chain reaction(RT-PCR)

To determine the mRNA levels of stem cell markers,total mRNA was extracted with TRIzol reagent(Invitrogen Corp.,Carlsbad,CA,USA)from cultured PDLSCs at passage 3 according to the manufacturer’s instructions.The total RNA concentration was determined after measuring the absorbance at 260 nm.Complementary DNA(cDNA)was prepared by subjecting 1μg of total RNA to reverse transcription using Moloney murine leukemia virus(MMLV)reverse transcriptase(Invitrogen Corp.)and random primers(9-mers;Takara Bio,Inc.,Otsu,Shiga,Japan).Next,for RT-PCR,the cDNA from hPDLSCs was ampli f i ed in a f i nal volume of 20μL containing 10 pmol of forward and reverseprimers,2 Uof Taq polymerase,and 5 pmol of dNTP mixture(iNtRON Biotechnology,SungNam,Republic of Korea).PCR was then performed for 40 cycles under the following conditions:denaturation for 30 seconds at 95℃,annealing for 30 seconds at 57℃,and extension for 30 seconds at 72℃.The reaction products were analyzed on a 1.5% agarose gel prestained with ethidium bromide.The gels were scanned using a Lumino graphⅡ(ATTO,Japan),and PCR products were compared with a 100-bp DNA marker(iNtRON Biotechnology).

Immuno f l uorescence(IF)analysis

For IF analysis of protein levels in PDLSCs,f i xed cells were washed with PBS and permeabilized with 0.2%Triton X-100 for 5 minutes.The cells were blocked with 10%normal goat serum in PBS and incubated with primary antibodies overnight at 4℃.The primary antibodies were anti-STRO-1(MAB4315;Millipore,Temecula,CA;1:200 dilution),anti-melanoma cell adhesion molecule(MCAM)/CD146(ABT1488;Millipore;1:200),anti-sex determining region Y-box 2(SOX2;AVB5603;Millipore;1:200).Secondary antibodies were applied and nuclei were stained with Hoechst 33342.The stained cells were examined using a confocal microscope and ZEN 2009 Light Edition software(Carl Zeiss).

Mesenchymal differentiation and staining

To determinethe in vitro mesenchymal differentiation potential of PDLSCs,weperformed invitro osteogenesis and adipogenesis.After osteogenic and adipogenic differentiation,cells were stained and analyzed for expression of adipogenic and osteogenic marker genes by RT-PCR.

For osteogenesis analysis,14-day osteogenic induction was conducted using a StemPro Osteogenesis Differentiation kit according to the manufacturer’s protocol.After the 14-day osteogenic induction,cells were f i xed with 10%formaldehyde for 10 minutes and stained with 2%Alizarin Red Ssolution for 20 minutes at room temperature.For alkaline phosphatase staining,cells were f i xed with 4%paraformaldehyde for 10 minutes and stained with 50:1 Tris Cl,NBT/BCIP solution(Roche,Basel,Switzerland)for 20 minutes at room temperature.For adipogenesis analysis,21-day adipogenic induction was conducted using a StemPro Adipogenesis Differentiation Kit according to the manufacturer’s protocol.After 21 days of cell differentiation,adipocyte-like cells were f i xed with 4%paraformaldehyde for 10 minutes,incubated with Oil red O working solution for 10 minutes,and observed under alight microscope.After measurement,Oil red O dye was eluted by adding 100%isopropanol and incubating for 10 minutes with gentle shaking.Solutions were transferred to 96-well plates,and the optical density(OD)was measured at 500 nm with 100%isopropanol as a blank using an enzyme-linked immunosorbent assay(ELISA)reader(Epoch,BioTek,VT,USA)[30].

Cell proliferation assays

To evaluate the effects of LPA on PDLSC proliferation,cell proliferation assays were conducted.PDLSCs were seeded at a density of 3×103cells/well in 96-well plates(Becton Dickinson Labware,Franklin Lakes,NJ,USA)in a humidi f i ed atmosphere of 5%CO2at 37℃.Cells were incubated with cell culture medium for 24 hours,treated with different concentrations of LPA(0 or 10μmol/L),and incubated for 1,3,or 7 days.Cell viability was detected following the addition of Ezcytox solution(Dogen Bio,Seoul,Republic of Korea).Ez-cytox solution(10μL)wasadded to each well of the 96-well plate,and the plates were incubated for 1 hour at 37℃.OD was measured at 450 nm using an ELISA reader(Epoch,BioTek,VT,USA)and the number of viable cells was calculated.

Quantitative RT-PCR(qRT-PCR)

For gene expression analysis,total mRNA was extracted with TRIzol reagent(Invitrogen Corp.)from PDLSCs cultured for 7 days with or without 10μmol/L LPA according to the manufacturer’s instructions.To determine the conditions for logarithmic-phase PCR ampli f i cation of target mRNA,1-μg aliquots were ampli f i ed using differing numbers of cycles.Glyceraldehyde 3-phosphate dehydrogenase(GAPDH)was ampli f i ed by PCR to rule out the possibility of RNA degradation and control for variations in mRNA concentrations in the RT reaction.Quantitative realtime PCR was performed with SYBR Premix Ex Taq(Takara Bio,Inc.)for 40 cycles using the following cycling parameters:denaturation at 95℃for 15 seconds,annealing at 57℃for 15 seconds,and extension at 72℃for 15 seconds.All oligonucleotide primer sequences are presented in Table 1.The f l uorescence intensity was measured at the end of the extension phase of each cycle and the threshold value for the f l uorescence intensity of all samples was set manually.Thereaction cycleat which the PCRproduct exceeded this f l uorescence intensity threshold was deemed the threshold cycle(Ct)in the exponential phase of PCR ampli f i cation.The expression of each target genewasquanti f i ed relativeto that of theinternal control gene(GAPDH).The relativequanti f i cation was based on a comparison of Ct values at a constant f l uorescenceintensity.Theamount of transcript present wasinversely related to the observed Ct.For each twofold dilution in transcript amount,the Ct was expected to increase by one.The relative expression(R)was calculated using the equation R=2-[ΔCtsample-ΔCtcontrol].Gene expression values were normalized to those of GAPDH and the experiments were repeated at least three times.

Results

Morphological analysis

From PDLSC isolation,several spindle-shaped and f i broblast-like periodontal ligament stem-like cell lines were established(Fig.1).These cells could adhere to plastic and had colony-forming ability.Among these cell lines,themostmorphologically appropriatecell line was characterized.Cells were passaged for morphological observation,and the cells over passage 7 showed severe loss of initial morphology.As the passage number increased,the degree of morphological change also increased.Moreover,further passaging of the cells slowed theproliferation rateand increased the cell size.

RT-PCR revealed highly expressed markers of undifferentiated,self-renewing,stem cells,such as POU5F1(OCT4)and NANOG in PDLSC(Fig.2).CD90,a mesenchymal stem cell marker,also showed high expression.However,CD73,a negative marker of mesenchymal stem cells,was not expressed.LL24,a human lung f i broblast,was used as a negative control.

IF was used to evaluate the expression of key mesenchymal stem cell surface markers,including STRO-1 and MCAM/CD146,and the stem cell marker SOX2(Fig.3).Compared with the other markers,STRO-1 showed very high expression and was expressed throughout the cell(Fig.3A-C).Moreover,MCAM/CD146 and SOX2 showed high expression in and near the nucleus rather than in the periphery or margin of the cell(Fig.3D-I).

Alkalinephosphatestaining indicated early osteogenesis as purple-colored cells(Fig.4A and B).Addition-ally,mineralized nodules were stained by Alizarin Red S after 14 days of incubation in osteogenic induction medium(Fig.4C and D).Mineralized nodules were stained a reddish color.The expression of osteocalcin(OCN),a key marker of osteogenesis,was slightly increased in theosteogenic group compared with that in thecontrol group(Fig.4E).Lipid droplets were stained by Oil red O after incubation for 21 days in adipogenic induction medium(Fig.4F-I).Stained Oil red O was dissolved with 70%isopropanol,and the OD was evaluated.There was a signi f i cant(P＜0.05)difference between the control group and the 21-day adipogenesis group(Fig.4J).Finally,the expression of peroxisome proliferator-activated receptorγ,an adipogenic marker,was slightly increased in the adipogenesis group compared with that in the control group(Fig.4K).Therefore,these f i ndings con f i rmed the multiple differentiation potential of PDLSCs.

Table 1 Primer sequences used for gene expression analysis

Cells in the group treated with 10μmol/L LPA showed signi f i cantly(P＜0.05)higher proliferation rates than untreated (control)cells at day 1(1.12±0.003)and day 7(4.32±0.005)(Fig.5),and those treated with 10μmol/L LPA for 3 days showed a tendency toward increased proliferation.

Notably,there were no signi f i cant differences in the expression of stemness-related genes such as POU5F1,NANOG,and SOX2 after 7 days of culture in the presence of 10μmol/L LPA(Fig.6).Unexpectedly,YAP expression was low in the 10μmol/L LPA-treated group.

Fig.1 Morphology of human periodontal ligament stem cells(hPDLSCs).The morphology of PDLSCs was evaluated under light microscopy at passage number 3.Spindle-shaped and f i broblast-like periodontal ligament stem-like cell lines were established.Scale bars,400μm(A)and 100μm(B).

Fig.2 Expression of mesenchymal stem cell markersin periodontal ligament stem cells.Expression of POU5F1,NANOG,CD90,and CD34 mRNAs was analyzed by RT-PCR.LL24:human lung f i broblast was used as a negative control.

Discussion

PDLSCs are human dental stem cells that contain a population of multipotent postnatal stem cells with biological characteristics similar to bone marrowderived undifferentiated stem cells.PDLSCs can differentiate into periodontal ligament f i broblasts,osteoblasts,and cementoblasts and repair allogenic bone defects without immunological rejection[31].

In the present study,we successfully established and characterized PDLSCs isolated from extracted teeth from dental patients.The established cell lines showed expression of POU5F1 and NANOG,the pluripotent markers expressed on PDLSCs[6],as well as CD90,a mesenchymal stem cell marker.Additionally,the absence of CD73 expression demonstrated that these cellswerenotderived fromhematopoietic stemcells.At theprotein level,although not PDLSC-speci f i c,wealso observed expression of the mesenchymal stem cell surface markers STRO-1 and CD146,the valuable indicatorsof immaturemesenchymal stem cells[11].The expression of SOX2 has been found in PDLSCs as well[6].Taken together,these mRNA and protein analyses demonstrated that the cells were PDLSCs.The ef f i ciency of differentiation potential into multiple lineages in vitro was also veri f i ed by IF staining and marker gene expression analysis.

Fig.3 Immuno f l uorescenceanalysisof human PDLSCs.A:Expressionof STRO-1.B:Expressionof CD146.C:Expressionof SOX2.D-F:Nucleiwerestained with Hoechst.G-I:Merged images.Expression wasevaluated by confocal laser scanning microscopy at 200×(scalebar=100μm).

Lysophospholipids are bioactive signaling molecules that act through the binding of their speci f i c G-proteincoupled receptors to exert pleiotropic effects on a wide range of cells.The most widely studied signaling lysophospholipids are LPA and sphingosine-1-phosphate(S1P).LPA and S1P can exert developmental,physiological,and pathological effects on the central nervous system and induce biological effects in various types of stem cells[32].Moreover,LPA has been identi f i ed as a strong mitogenic factor.Saunders et al.reported that LPA increased cell growth and inhibited apoptosis via the redox-dependent activation of extracellular signal-regulated serine/threonine kinase-,Akt-,and nuclear factor(NF)-k B-dependent signaling pathways in ovarian cancer cells[33].

Furthermore,an association between LPA signaling and reactive oxygen species-mediated signaling has been suggested in other cell types[34-36].LPA stimulates the extracellular signal regulated/mitogen-activated protein kinase(ERK/MAPK)cascade,and is thought to be important for proliferation.LPA increases the proliferation and migration of ASCs via Nox4-induced reactive oxygen species(ROS)generation.miR-210 expression is associated with LPA-induced stimulation,and Serpine1 mediates the LPA-induced migration of ASCs.Therefore,phospholipid derivatives,such as LPA and S1P,may be used to stimulate ASCs during stem cell expansion[37].

In our previous studies,we showed that treatment with 30μmol/L LPA during in vitro maturation improved the developmental potential of parthenogenic embryos and in vitro fertilized embryos in swine.Moreover,LPA enhances the survival of human hematopoietic stem/progenitor cells under ischemic conditions[17].It is also crucial for periodontal wound healing and gingival in f l ammation[38].LPA enhances the proliferation of human corneal endothelial cells induced by LPA through phosphatidylinositol 3-kinase and Rho-associated protein kinase pathways[39].

Fig.4 In vitro differentiation of PDLSCs.A and B:Alkalinephosphatasestaining 14 daysafter osteogenic induction.Imageswerecollected after 24 hoursof staining.Cand D:Alizarin Red Sstaining.Mineralized noduleswerestained areddish color(C:×100,scalebar=50μm,D:×200,scalebar=100μm).E:mRNA expression of PDLSCmarkers,analyzed by RT-PCR,beforeand 14 daysafter osteogenic induction.F-I:Oil red Ostaining 21 daysafter adipogenic induction.Lipid dropletswerestained asreddish beads(×400,scalebar=20μm).J:ODvaluesat 500 nm wereevaluated after Oil red Ostaining(P＜0.05).K:mRNA expression of PDLSCmarkers,analyzed by RT-PCR,beforeand 21 days after adipogenic induction.

Fig.5 Proliferation rates of PDLSCs treated with 10μmol/L LPA for 1,3,and 7 days.*P＜0.05.

Fig.6 Gene expression changes in PDLSCs after 7 days of culture in the presence of 10µmol/L LPA,as analyzed by RTqPCR.

In astudy of embryonic stem cells,Han et al.showed that LPA could substitute for YAP to generate transgene-free human naïve pluripotent stem cells[28].Additionally,Cuizhu et al.demonstrated that YAP regulated the apoptosis and proliferation of hPDLSCs;speci f i cally,knockdown of the YAP gene by small interfering RNA inhibited proliferation,induced apoptosis,and altered the cell cycle of PDLSCs[40].

For the application in the dental clinic,expansion of PDLSCs without loss of stemness is essential.In our study,10μmol/L LPA promoted the proliferation of PDLSCs.However,there were no signi f i cant differences in the expression of stemness-related genes,although YAP mRNA tended to bedownregulated rather than upregulated.Collectively,our f i ndings demonstrated that LPA enhanced PDLSCs proliferation,but a signi f i cant connection between LPA and stemnessrelated genes in PDLSCs was not identi f i ed in this study.Further studies on the mechanisms of LPAinduced PDLSCproliferation and proteinexpressionare needed to clarify the effects of LPA on PDLSCs.

In conclusion,LPA treatmentcan enhance PDLSCs proliferation via ROS mediated signaling andpromote cell survival through reversible activation or inhibition of pro-proliferative and regulatory signaling proteins.Thus,LPA treatment of PDLSCs culture may be useful for reprogramming human induced pluripotent stem cells and producing cells for clinical studies of therapeutic regeneration.

Acknowledgments

This work wassupported,in part,by agrant from the“Korea Research Fellowship(KRF)Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Science and ICT(NRF-2015H1D3A1066175)”,the“NRFGrant funded by the Ministry of Science and ICT(NRF-2016R1D1A1B-03933191,NRF-2017R1A2B4002546)”,the“Global Research and Development Center(GRDC)Program through the NRFfunded by the Ministry of Scienceand ICT(NRF-2017K1A4A3014959)”,and“Korea Institute of Planning and Evaluation for Technology in Food,Agriculture,Forestry and Fisheries(IPET)through Agri-Bio industry Technology Development Program,funded by Ministry of Agriculture,Food and Rural Affairs(MAFRA)(318016-5)”,Republic of Korea.