ORIGINAL ARTICLE

Expression patterns of immune genes in long-term cultured dentalstem cells

Pukana Jayaraman1,2& Vijayendran Govindasamy1 & Nareshwaran Gnanasegaran1,3

&

Wijenthiran Kunasekaran1,2& Punitha Vasanthan1,2

& Sabri Musa2 &

Noor Hayaty Abu Kasim3

Received: 11 February 2015 /Accepted: 20 May 2015# Springer-Verlag Berlin Heidelberg 2015

AbstractBackground and objectives Long-term culture system is usedto prevent the impediment of insufficient cells and is good forlow starting materials such as dental pulp or periodontal liga-ment. In general, although cell viability and functionality arethe most common aspects taken into consideration in culturingcells for a long term, they may not truly represent the biolog-ical state of the cells. Hence, we explored the behaviour ofanother important aspect which is the immune properties inlong-term cultured cells.Methods Dental pulp stem cells from deciduous (SHED;n = 3) and permanent (DPSCs; n = 3) teeth as well as peri-odontal ligament stem cells (PDLSCs; n = 3) were culturedunder identical culture condition. The immune properties ofeach cell lines were profiled at passage 2 [P2] and passage 9[P9] as early and late passages, respectively. This was furthervalidated at the protein level using the Luminex platform.Results A major shift of genes was noticed at P9 with SHEDbeing the highest. SHED cultured at P9 displayed many genesrepresenting pathogen recognition (P < 0.001), immune sig-nalling (P < 0.001, pro-inflammatory (P < 0.001), anti-

inflammatory (P < 0.001) and immune-related growth andstimulation factor (P < 0.001) as compared to DPSCs andPDLSCs. Surprisingly, SHED also expressed many cytotox-icity genes (P < 0.001).Conclusions Communally, instabilities of immune genesfrom our findings suggest that long-term cultured cells maynot be feasible for transplantation purposes.Clinical relevance A complete biological characterizationcovering all major aspects including immune propertiesshould be made as prerequisite criteria prior to the use oflong-term cultured stem cells in clinical settings.

Keywords Immune properties . Gene expression profile .

Regenerativemedicine . Growth factors . Cytokines .

Chemokine

Introduction

Stem cell (SC) therapy has entered a new dimension followingthe establishment of many clinical trials. Among the common-ly used cells in clinical trials are mesenchymal stem cellsderived from the bone marrow (BM-MSCs). Recently, stemcells of dental origin such as the permanent teeth (DPSCs),exfoliated deciduous teeth (SHED), periodontal ligament(PDLSCs), dental follicle (DF) and dental apical papilla(SCAP) have emerged as another attractive source of cellsand have been geared to clinical trials especially in treatingneuro-ectoderm-related diseases [1–4]. Despite this, a contin-uous uninterrupted supply of stem cells is needed for success-ful completion of any clinical trials. This is carried out byculturing the cells in multiple subcultures to attain the requiredcell number for transplantation purpose. While the process iseasier by using BM-MSCs or umbilical cord stem cells, the

* Vijayendran Govindasamyvijay@hygieiainnovation.com; vijay07001@gmail.com

* Sabri Musasabrim@um.edu.my

1 GMP compliant Stem Cells Laboratory, Hygieia Innovation Sdn.Bhd, Federal Territory of Putrajaya, Lot 1G-2G, Lanai Complex,No.2, Persiaran Seri Perdana, Precinct 10, 62250 Putrajaya, Malaysia

2 Department of Paediatric Dentistry and Orthodontics, Faculty ofDentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia

3 Department of Restorative Dentistry, Faculty of Dentistry, Universityof Malaya, 50603 Kuala Lumpur, Malaysia

Clin Oral InvestDOI 10.1007/s00784-015-1497-1

main caveat surrounding the usage of dental stem cells in aclinical set up is the low cell yield.

This is because, compared to other tissues, the starting ma-terial is extremely low and requires additional subcultures toachieve an adequate cell number. For example, a single um-bilical cord is able to generate more than 1 × 108 cells within asubculture [5], while the use of dental tissues to generate asimilar cell number requires an additional of four to five sub-cultures and that too in a pooled sample situation [6]. Despitethis, using stem cells for transplantation has proven to be safeupon long-term expansion [7, 8]. Recently, it was reported thatDPSCs cultured at long-term expansion are able to maintaincell function, proliferation and viability thus making themsuitable for therapeutic usage [9]. While these results lookpromising, most of them focused in the plasticity, stemnessand immunophenotyping However, another vital aspect worthconsidering in long-term cell culture systems is the immuneproperties. In general, stem cells are regarded as immuno-privileged cells with convincing evidence showing that theyaffect innate and adaptive immune cells in two possible steps:they decrease T- and B-cell proliferation by cell-cell contactand release a wide range of paracrine factors [10]. Similarmechanisms were also reported in stem cells of dental origins[11–13].

There are several methods to evaluate the immune proper-ties of stem cells, and one of them is by looking at their genelevel. In this context, gene expression profiling is a reliablemethod and has been commonly used in understanding thebiology and the ontogeny of the cells [14, 15]. Here, we haveinvestigated for the first time the immune properties of dental-derived stem cells in long-term expansion. It is believed thatby understanding the pattern of immune properties of the den-tal stem cells especially at late culture will eventually addvalue to the existing knowledge on the safety of celltransplantation.

Materials and methods

Sample collection

Our experiments were conducted with institutional ethic ap-proval (Medical Ethics Clearance Number DF CO1107/0066[L]), and the study subjects provided informed consentprior to the commencement of the study.

Isolation and expansion of cells

We have established dental pulp stem cells from deciduousteeth (SHED) [n = 3; ages 7–11 years] and permanent teeth(DPSCs) [n = 3; ages 24–35] as previously described [16]whereas periodontal ligament stem cells (PDLSCs) [n = 3;ages 7–11 years] were isolated and cultured up to passage 8

(P8) according to previously reported protocols with slightmodification [17]. In this study, we have regarded cells at P2and P9 as early and late passages, respectively.

TaqMan® Array Human Immune Gene Array

We have isolated total cellular RNA from all cell lines at P2and P9 with TRIzol® RNA isolation reagent (Invitrogen). Thecomplementary DNAs (cDNAs) were synthesized by using a1-μg RNA sample through a reverse transcriptase enzyme kit(Invitrogen). We profiled the immune-related genes usingTaqMan® Human Immune Gene Array. Briefly, we loadedthe cDNAs on the microfluidic cards for thermal cycling onan ABI PRISM 7900HT Sequence Detection System (FisherScientific), and expression values for target genes were nor-malized to the expression of 18srRNA. For estimation of thefold change by TLDA when the initial transcript levels wereundetectable, the initial cycle threshold (CT) value wasassigned to be 35, which would lead to a possible underesti-mation of the actual fold change.

Validation of immune genes by using Procarta® cytokineprotein array

This study validated the secretion of immune genes randomlyselected by using a customized human cytokine protein array(Panomics, Redwood City, CA). Briefly, we have collectedconditioned mediums from all cell lines at P2 and P9, andan assay was performed according to the manufacturer’s in-struction. The results were analysed using Luminex platform.

Statistical analysis

We have conducted an experiment independently to ensure thereproducibility of the result. The descriptive statistical analy-ses were performed using the software SPSS for Windows(Version 11.0; SPSS Predictive Analytics, Chicago, IL) andthe data analysed by using two-way analysis of variance(ANOVA) with the significance level set at P = 0.05. Tukeypost hoc multiple comparison tests were carried out to deter-mine the differences between the groups.

Results

Gene expression variability between SHED, DPSCsand PDLSCs at early and late passages

To understand the immunological behaviour between thesecell lines during their transition period from P2 (early passage)to P9 (late passage), we looked at the expression patterns ofimmuno-regulated genes. The genes were defined as being 2-fold up- or downregulated in each sample at P2 and P9 when

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comparing to SHED at P2 which acted as a control with anaccompanying P value of 0.05. Since huge fold variationswere noticed among all the samples, they were tentativelyclassified into five distinct categories based on their fold ex-pression value as follows: upregulated genes consisting ofthree categories, namely highly upregulated (≥50-fold), medi-um upregulated (≥10- to 49.99-fold) and low upregulated (≥2-to 9.99-fold) followed by unchanged genes (≥0.5- to 1.99-fold) and downregulated (≤0.499-fold) categories. In this re-gard, in P2, it was noticed that in the highly upregulated cat-egory, there was no gene percentage expressed in SHEDwhereas there were no significant differences of gene expres-sion percentage between DPSCs and PDLSCs. On the otherhand, in the medium upregulated category, no significant dif-ferences of gene expression percentage were found among all

the cell lines. PDLSCs showed a significantly lower percent-age (P < 0.05) of genes in the low upregulated category ascompared to SHED and DPSCs. Surprisingly, in P9, a tremen-dous augmentation of gene percentage was seen in all upreg-ulated categories within all cell lines (P < 0.05). In terms ofunchanged category, a significant swift (P < 0.05) was ob-served in SHED, DPSCs and PDLSCs from P2 to P9 wherethe genes were decreased from 78 to 6 %, 57 to 9 % and 60 to9 % respectively. In contrast, a significant increment(P < 0.05) of gene percentages ranging from 8 to 17 % wasobserved in all the cell lines between the same passages(Fig. 1). To better understand the role played by eachimmuno-related gene within the cell lines and passages, wefurther categorized the genes according to their similar biolog-ical functions.

Fig. 1 Distribution of genes ofSHED, DPSCs and PDLSCs atpassage 2 (P2) and passage 9(P9), respectively. A total of fivecategories, namely highlyupregulated (fold change ≥50),medium upregulated (fold changeof 10–49.9), low upregulated(fold change of 2–9.9),unchanged (fold change of 0.5–1.9) and downregulated (foldchange of ≤0.49), were employedin this study to describe their geneexpressions

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SHED cultured at P9 displayed many genes representingpathogen recognition as well as immune signallingand transduction

Among the 15 genes grouped under the category of pathogenrecognition aswell as signalling and transcription, 7were highlysignificant (HLA-DRB1, CD80, CD34, TNFRSF18, PTPRC,SELE and SELP; P < 0.001) in SHED cultured at P9 as com-pared to SHED cultured at P2. A similar observation with lessersignificant values (P < 0.01; P < 0.05) was observed for DPSCsand PDLSCs cultured in P9 as compared to P2 (Table 1).

Pro- and anti-inflammatory gene profiling of SHED,DPSCs and PDLSCs at P2 and P9

The transition of SHED from P2 to P9 resulted in the incre-ment of seven pro-inflammatory genes namely TNF, ILIB,IFN- , LTA, IL-12B, IL-1A and IL-15 (P < 0.001).However, except for IFN- , not much significance was noticedfor the same group of genes in DPSCs and PDLSCs during thetransition period. On the other hand, tremendous increments(P < 0.001) of anti-inflammatory genes such as IL-4, IL-5, IL-7, IL-9, IL-10 and IL-13 were observed in SHED cultured atP9 as compared to P2. Surprisingly, we did not observe asimilar expression in DPSCs and PDLSCs indicating SHED

experiencing immune-related gene turbulence when culturedfor a prolonged period of time (Table 2).

SHED cultured at P9 displayed many genes representingmany growth and stimulation factors as wellas chemokines

It is worth noting that growth and other stimulation factorsalso play an integral part in immune systems. Accordingly,we noticed that TGF-β1 and other stimulation factors suchas CSF3 and IL2RAwere highly expressed in SHED culturedat P9 as compared to P2 (P < 0.001), and again these geneswere stagnant in DPSCs and PDLSCs in similar culture con-ditions. In terms of chemokine gene expression, only thosegenes related to the CXC group such as CCL5, CXCL11,CCR2, CCR4, CCR5 and CCR7 were noticeable in all celllines at P9 (Table 3).

DPSCs and PDLSCs cultured at P9 expressed lessercellular toxicity and apoptosis-related genes as comparedto SHED cultured at P9

We postulated that the agitations of most of the immune genesin SHED cultured at P9 were most likely contributed by theelevated expression (P < 0.001) of cellular toxicity and

Table 1 Fold changes of genes related to pathogen recognition and signalling and transcription

*P value < 0.05, **P value < 0.005, ***P value < 0.001

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Table 2 Fold change of genes related to cytokines

*P value < 0.05, **P value < 0.005, ***P value < 0.001

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apoptosis such as GNLY, GZMB, PRF1, CD3E, CD8A,CD19, CD28, CD38, CD40LG and CTLA4.

Validation of immune genes using cytokine array

We randomly selected few genes that showed expression pat-terns in good agreement with the PCR array data (Fig. 2).

Discussion

The findings of this study have prompted a revisit of some ofthe key aspects of stem cells for clinical usage. One of it is thesuitability of using pro-long cultured cells. Generally, cellscultured for a long term are considered to be safe [18] al-though a few studies reported that they undergo genomic in-stabilities [19, 20]. More recently, studies have shown thatDPSCs cultured up to P14 too have maintained high mitogen-ic and functionality thus making them a safe source for ther-apeutic usage [9]. Nevertheless, the latter study as well asothers in the past [21, 22] mainly focused on proliferationand functionality and may not truly represent the state of thecells. This is true in our situation that despite maintainingproliferation and functionality (data not shown), a significantshift was observed in the immunomodulatory aspect especial-ly at long-term cultured cells. Interestingly, this shift happenedacross all tested cell lines with SHED being the highest.

There is a sharp increment of immune gene expressionrelated to T-lymphocyte activation such as HLA-DR andCD80 across all cell lines at P9. We postulated that thepresence of these molecules indicates that the cells arelosing their stemness and transforming into terminally dif-ferentiated cells [23] and potential of creating a graft-versus-host disease scenario if transplanted in allogeneicsettings. Further, we noticed a significant increase in thenumber of pro-inflammatory genes during the transitionof the cell lines from P2 to P9. In vivo condition, thesegenes are expressed at large during the early inflammationphase to eradicate infection, and the overexpression ofthese genes especially TNF, IL1B, IFN- , LTA, IL12B,IL1A and IL2 could potentially harm the host tissues[24–26]. Likewise, we also noticed an increment of sev-eral of anti-inflammatory genes at P9. In an ideal situa-tion, the expressions of pro-inflammatory and anti-inflammatory genes complement to each other, for exam-ple, the pattern of TNF is inversely proportional to IL-10[27].In contrast, we found that both groups of genes werehighly expressed in P9 indicating a disturbance in thegenetic makeup though this needs further investigations.It was not surprising to see the elevation of TGF-β ex-pression at P9. The pivotal function of TGF-β in the im-mune system is to maintain tolerance via the regulation oflymphocyte proliferation, differentiation, and survival[28] by suppressing pro-inflammatory cytokines. CCL5,CXCL11, CCR2, CCR4, CCR5 and CCR7 were also

Table 3 Fold change of genes related to cellular cytotoxicity, surface molecules and apoptosis

*P value < 0.05, **P value < 0.005, ***P value < 0.001

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upregulated in all cell lines at P9. All these molecules aremembers of the CXC chemokine family and, apart fromplaying a role in immunogenicity, they are potent pro-moters of angiogenesis and mediate their angiogenicproperties [29]. Perhaps this might contribute to the con-tinuous growth of the cells. It was surprising to notice thatdespite the cells being cultured in the same culture condi-tions, SHED surpassed other cell lines in expressing un-wanted immune genes at a late passage. This perhaps isattributed to the high expression of cellular toxicitymarkers such as GNLY and GZMB in SHED as comparedto DPSCs and PDLSCs. These genes are known to beinvolved as intracellular effectors of target cell death, withrecent data suggesting that GNLY may have a role in thepropagation of immune signals [30].

Collectively, occurrences of immune gene turbulence fromour findings suggest that long-term culture conditions of stemcells may not be feasible for use in transplantation. Havingsaid that, what is the remedy for using cells having a lowstarting material such as dental pulp or periodontal ligamenttissue? To overcome this issue, large expansions of cells usinga 3D bioreactor [31] are possibly made to dental stem cells atthe early passage without triggering any side effects [32].Apart from that, pooled allogeneic samples are another choice,but further characterization needs to be done to maintain their

functionality. In conclusion, we proposed a comprehensivebiological characterization which includes the immune prop-erties before the stem cells cultured at a later passage arebrought into clinical application.

Acknowledgments The work is part of an ongoing project betweenHygieia Innovation and the Faculty of Dentistry, University Malaya,and supported by a University of Malaya-Ministry of Education highimpact research grant (UM.C/HIR/MOHE/DENT/02).

Conflict of interest The authors declare that they have no competinginterests.

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