Differentiation of dental pulp stem cells into islet-like aggregates

Hindawi Publishing CorporationStem Cells InternationalVolume 2013 Article ID 250740 10 pageshttpdxdoiorg1011552013250740

Research ArticleDifferentiation of Dental Pulp Stem Cells intoNeuron-Like Cells in Serum-Free Medium

Shahrul Hisham Zainal Ariffin1 Shabnam Kermani1

Intan Zarina Zainol Abidin1 Rohaya Megat Abdul Wahab2 Zulham Yamamoto1

Sahidan Senafi1 Zaidah Zainal Ariffin3 and Mohamad Abdul Razak4

1 School of Biosciences and Biotechnology Faculty of Science and Technology Universiti Kebangsaan Malaysia43600 Bangi Selangor Malaysia

2 Department of Orthodontics Faculty of Dentistry Universiti Kebangsaan Malaysia Jalan Raja Muda Abdul Aziz50300 Kuala Lumpur Malaysia

3 School of Biology Faculty of Applied Science Universiti Teknologi MARA 40450 Shah Alam Selangor Malaysia4Allianze University College of Medical Sciences Waziria Medical Square Jalan Bertam 2 13200 Kepala BatasPenang Malaysia

Correspondence should be addressed to Shahrul Hisham Zainal Ariffin shahroy8gmailcom

Received 13 December 2012 Revised 23 September 2013 Accepted 7 October 2013

Academic Editor Deepa Bhartiya

Copyright copy 2013 Shahrul Hisham Zainal Ariffin et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Dental pulp tissue contains dental pulp stem cells (DPSCs) Dental pulp cells (also known as dental pulp-derived mesenchymalstem cells) are capable of differentiating intomultilineage cells including neuron-like cellsThe aim of this study was to examine thecapability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors DPSCs were isolated fromteeth extracted from 6- to 8-week-oldmice andmaintained in complete mediumThe cells from the fourth passage were induced todifferentiate by culturing inmediumwithout serumor growth factors RT-PCRmolecular analysis showed characteristics ofCd146+Cd166+ and Cd31minus in DPSCs indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells After 5days of neuronal differentiation the cells showed neuron-like morphological changes and expressed MAP2 proteinThe activationof Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium whereasTub3was activated only after 5 days of neuronal differentiationThe proliferation of the differentiated cells decreased in comparisonto that of the control cells Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- andgrowth factor-free medium

1 Introduction

Dental pulp tissue contains many types of cells includingcommitted cells (eg endothelial cells) and uncommittedcells (ie DPSCs) DPSCs are of mesenchymal stem cells(MSCs) [1] In mice the majority of MSCs were isolated frombonemarrow [2] and peripheral blood [3 4]TheseMSCs canbe characterized by the expression of specific gene markerssuch as CD44 CD73 CD90 CD105 CD117 and CD166 [5 6]

DPSCs are capable of differentiating into multilineagecells [7ndash9] including neuron-like cells [10] Neuron-like cellsdifferentiated from MSCs derived from bone marrow cells

[11ndash13] and brain [14] However MSCs derived from dentalpulp that is DPSCs are also capable of differentiating intoneuron-like cells [10]The characteristics of MSCs from bonemarrow are similar to those cells derived from dental pulp[11] Both types of MSCs express Cd44 Cd106 Cd146 andCd166 [15ndash17]

Many factors are involved in neuronal differentiationincluding nestin [18] tubulin3 (Tub3) [19] and MAP2 [20]Nestin is involved in the radial growth of axons duringneuronal differentiation in vertebrate cells [19 21] ThereforeNestin is known as a neural marker and its presence canbe considered as a criterion for the ability to differentiate

2 Stem Cells International

Table 1 Primers involved in RT-PCR experiments

Geneaccession no Primers Sequences Expected product size (bp) Annealing temperature (∘C)

Cd146 (NM 023061) Forward 51015840-GGACCTTGAGTTTGAGTGG-31015840 479 60Reverse 51015840-CAGTGGTTTGGCTGGAGT-31015840

Cd166 (NM 009655) Forward 51015840-AACATGGCGGCTTCAACG-31015840 630 61Reverse 51015840-GACGACACCAGCAACGAG-31015840

Nestin (NM 016701) Forward 51015840-CGCTCGGGAGAGTCGCTT-31015840 215 64Reverse 51015840-CCAGTTGCTGCCCACCTTC-31015840

Gapdh (NM 008084) Forward 51015840-CAACGGCACAGTCAAGG-31015840 717 62Reverse 51015840-AAGGTGGAAGAGTGGGAG-31015840

Tub3 (NM 023279) Forward 51015840-ACGCATCTCGGAGCAGTT-31015840 125 61Reverse 51015840-CGGACACCAGGTCATTCA-31015840

Cd31 (NM 0010323781) Forward 51015840-GGTCTTGTCGCAGTATCAG-31015840 355 58Reverse 51015840-ATGGCAATTATCCGCTCT-31015840

into neurons [18 22] However Nestin has shown to beexpressed by other cell types such as hair follicle stem cells[23] pericytes [24] endothelial cells [25]myofibroblasts andpancreatic fibroblasts [26] Therefore analysis on expressionof other specific neuron markers such as Tub3 [27 28] andMAP2 [29 30] has been done concurrently for neuronalconfirmation Tub3 and MAP2 play a role in the stabilityof axons and neuronal cell bodies [20 31] Certain growthfactors such as epidermal growth factor basic fibroblastgrowth factor and retinoic acid were used for neuronalinduction [32ndash35] Dimethyl sulfoxide (DMSO) was alsoused to induce transformation of MSCs into neuron-likephenotypes in vitro [12 13]The objective of the present studywas to examine the directed differentiation of DPSCs intoneuron-like cells in the absence of chemical induction

2 Materials and Methods

21 Isolation ofDental Pulp Cells Incisor teethwere extractedfrom 6- to 8-week-old mice under sterile conditions andplaced in medium containing 1X PBS (Sigma USA) Theextracted dental pulp was washed with 1X PBS containing 1(vv) penicillin-streptomycin (Invitrogen USA)

Dental pulp tissue was incubated for 1 hour in 4-unitcollagenase type I at 37∘C followed by several rounds ofenzymatic disaggregationThe cells were centrifuged at 1200 gfor 10 minutes at 25∘C and cultured in complete mediumconsisting of 120572-MEM (Invitrogen USA) supplemented with20 (vv) FBS (Biowest USA) and 1 (vv) penicillin-streptomycin The cells 1 times 105 cellsmL obtained were putin a T25 flask containing complete medium and cultured inan incubator with 5 CO

2atmosphere and 95 humidity at

37∘CAfter 24 hours the suspended cells were removed from

the medium and the flask was washed with 1X PBS solutionThe cells were grown in complete medium until 80 conflu-ency A solution of 025 (vv) trypsin-EDTA (Sigma USA)was used to detach the dental pulp cells from the flask surfacefor subculturing in another flask at 1 times 105 cellsmL Forcryopreservation and storage cells at the fourth passage were

placed in cryovials containing freezing medium consisting of120572-MEM 10 (vv) DMSO (Sigma USA) and 50 (vv) FBSand stored in liquid nitrogen In this study the cells usedwereat the fourth passage

22 Differentiation of Dental Pulp Stem Cells into NeuronalCells Approximately 1 times 105 cells were transferred into 24-well plates containing complete medium and were allowedto grow for 24 hours until adherence The medium wasdiscarded and the cells were washed with PBS The cellswere then cultured in serum- and growth factor free-mediumconsisting of 120572-MEM and 1 (vv) penicillin-streptomycinfor 5 days The medium was changed every 2-3 days duringthis 5-day period As a control the same number of cells wascultured in complete medium (consists of 120572-MEM 1 (vv)penicillin-streptomycin and 15 (vv) fetal bovine serum)for 5 days Cell morphology was monitored on days 2 and 5of neuronal differentiation using an Olympus phase-contrastmicroscope

23 Molecular Analysis Using RT-PCR Control and differen-tiated cells were detached using 025 (vv) trypsin-EDTAand centrifuged at 1400 g for 10 minutes Total RNA wasextracted using TRI-reagent (Sigma USA) according to themanufacturerrsquos protocol The purity of the total RNA wasassessed spectrophotometrically at 260 and 280 nm with anA260

A280

ratio of 18ndash20 considered acceptable Approxi-mately 300 ng of total RNA samplewas used for eachRT-PCRreactionwith theAccess Quick RT-PCR system kit (PromegaUSA) The primers were designed using the Primer Pre-mier 50 software program based on sequences obtainedfrom NCBI Information on the primers is summarized inTable 1

Primary cDNA synthesis was performed using AMVreverse transcriptase for 45minutes at 45∘C followed by deac-tivation for 2 minutes at 94∘C The amplification consistedof denaturation for 30 seconds at 94∘C annealing for 60seconds and extension for 60 seconds at 68∘C performedfor 40 cycles A final extension step was performed for 7minutes at 68∘CThe amplified products were separated using

Stem Cells International 3

1 (wv) agarose gel electrophoresis stained and analysedThe amplified products were subjected to DNA sequencingand verified using the BLASTN program from NCBI As forexpression level analysis a total of 1 120583g of total RNAwas usedfor each amplification and intensity was determined usingonline ImageJ 147 program (httprsbwebnihgovij)

24 Immunocytochemistry of MAP2 The induced neuronalcells were washed with 1X PBS and fixed at 4∘C with 4(vv) paraformaldehyde for 2 hours The cells were washed 3times with 005 PBS-Tween 20 followed by 2 PBS-Triton-X for 10 minutes Then the cells were washed again 3 timeswith 005 PBS-Tween 20 The cells were incubated at 37∘Cwith 10 goat serum diluted in 005 (vv) PBS-Tween 20plus 001mgmL BSA for 30 minutes prior to incubation ofprimary antibody This solution was removed and primaryantibody at 1 200 diluted with 005 (vv) PBS-Tween 20plus 3 (vv) goat serum and 1mgmL BSA were addedfor 24 hours at 4∘C Then the cells were washed 3 timeswith 005 (vv) PBS-Tween 20 followed by incubation ofsecondary antibody anti-mouse IgG-FITC for 30 minutesat room temperature diluted at 1 50 with Tween 20 plus3 (vv) goat serum and 1mgmL BSA Finally the cellswere washed 3 times with 005 (vv) PBS-Tween 20 priorto analysis using fluorescence microscope Negative controlused was undifferentiated cells without neuronal inductionthat is cultured in complete medium

25 3-(45-Dimethylthiazol-2-yl)-25-diphenyltetrazoliumBro-mide (MTT) Assay Approximately 1 times 104 cells were seededin 96-well plates and incubated for 24 hours at 37∘C Twogroups of cells were cultured one in complete medium(control) and the other in serum- and growth factor-freemedium After 24-hour incubation approximately 20120583LMTT (5mgmL) was added to each sample and the sampleswere incubated for another 4h at 37∘C The mixture wasslowly removed 200 120583L of DMSO was added to each welland the wells were measured by an ELISA Plate Reader at570 nm Each experiment was repeated in triplicate The cellswere subjected toMTT assay on the first third and fifth daysof neuronal induction The number of viable cells for eachanalysis was determined using a standard graph created priorto the experiment

26 Statistical Analysis Data from the differentiated and con-trol groups were compared using paired 119905-test in SPSS pro-gram version 1602 Differences with a 119875 value lt 005 wereconsidered statistically significant Data obtained were pre-sented as average (meanplusmn SD standard deviation) from threeindependent experiments (119899 = 3)

3 Results

31 Identification of Mesenchymal Stem Cells in Dental PulpTissue The identity of dissociated cells isolated from dentalpulp tissue using collagenase was confirmed by their capacityto form adherent colonies consisting of sphere-like clustersof cells (Figure 1) Averages of 68 times 104 cellscm2 were found

capable to obtain colonies after 24 hours cultured in thecomplete medium Then the suspended cells were discardedand only adherent cells were expanded in the mediumThe suspended cells may have been cells that were unableto survive in the medium The colonies began to changetheir shape during the second passage The cells assumed afibroblast-like morphology with a long thin body during thefourth passage and became confluent after 2 to 3 days of invitro culture in complete medium

Molecular analysis was performed to validate the types ofcells in the fourth passageThe total RNA was extracted fromthe fourth passage of dental pulp cells and was subjected toRT-PCR analysis (Figure 2) This analysis showed Cd146 andCd166 amplicons in these cells whereas activation of Cd31was not observedThe amplicon ofGapdhwas found in dentalpulp cells both before and after differentiation Analysis ofexpression level for Cd146 and Cd166 was shown to produce1180plusmn168 and 775plusmn143 respectively when comparedto Gapdh (100) which was used to normalize the cellularmRNA level

32 Morphological Changes into Neuron-Like Cells andExpression of MAP2 Protein After 5 days of culture most ofthe cells showed a morphological change to a long thin bodyshape (Figure 3) The cytoplasm was contracted toward thenucleus and assumed a multipolar shape The cells displayedsmall spherical and contracted bodies and a conical cyto-plasm with branches resembling the neuronal perikaryonaxon and dendrite The perikaryon dendrite and axon ofa neuronal cell are indicated respectively by a white arrowan open arrowhead and a black arrow Approximately 60ndash70 of the cell population differentiate into neuron-likecells an indication that differentiation occurred due to theabsence of serum and growth factors but not because ofspontaneous differentiation DPSCs expressed the neuron-specific protein marker MAP2 after 5 days of culture inserum- and growth factor-free medium (Figure 3) HoweverMAP2 was not detected in DPSCs or undifferentiated cellswithout neuronal induction that is cultured in completemedium (negative control) (Figure 3)

33 Activation of NeuronalMarkers Thecells showed expres-sion of Cd146+ Cd166+ and Cd31- before differentiationRT-PCR analysis showed the presence of a Nestin amplicon(sim215 bp) in the cells both before and after differentiationHowever the intensity of Nestin activation was significantlyhigher after differentiation compared with before differen-tiation (Figure 4(a)) An amplicon of Tub3 (sim125 bp) wasobserved after day 5 of neuronal differentiation (Figure 4(b))Tub3 was shown to be activated after 5 days of neuronaldifferentiation Gapdh was used as a positive control bothbefore and after differentiation A Gapdh amplicon (sim717 bp)was found in cells both before and after differentiationindicating thatGapdh remains activated in both types of cells(Figure 4(c))

34 Proliferation of Dental Pulp Stem Cells during Differ-entiation Cell viability studies were performed to assess


times100

(a)

times100

(b)

times400

(c)

times400

(d)

times400

(e)

Figure 1 Characteristics of isolated and in vitromouse dental pulp stem cells Colonies derived from dental pulp at the first passage (a) andafter 24 hours of culture (b) Colonies began to show changes in shape after the second passage (c) a fibroblastic cell shape after the fourthpassage (d) and confluence after 2-3 days of culture in complete medium (e)

the proliferation capacity of both differentiated and undiffer-entiated cells during neuronal differentiationThenumbers ofcontrol and differentiated cells were significantly (119875 lt 005)increased upon differentiation compared with day 0 of cul-ture (Figure 5)However the proliferation capacity of the cellsbegan to change gradually after 24 hours of culture until day

5 of neuronal differentiation with the number of control cellsremaining higher Both types of cellsmaintained their growthrate during the initial 24 hours of culture However thedifferentiated cells showed a reduction of growth rate after 24hours whereas the undifferentiated (control) cells maintainedtheir growth rate resulting in an increased number of viable


(i) (ii)Cd146

500bp400bp sim479bp

(i) Undifferentiated cells(ii) Differentiated cells

(a)

(i) Cd166 (ii)

700bp600bp

sim630bp


(b)

(i) (ii)Cd31


(c)

(i) (ii)

sim717bp sim717bp

Gapdh

800bp700bp


(d)

Figure 2 Activation of mesenchymal stem cell markers The activation of Cd146 (sim479 bp) (a) and Cd166 (sim630 bp) (b) was observedonly in cells before differentiation indicating that the cells were mesenchymal stem cells Cd31 was inactivated before and after neuronaldifferentiation (c) Gapdh (sim717 bp) a housekeeping gene was expressed before and after differentiation (d) Panel (i) is representingundifferentiated cells that is cells before neuronal differentiation while panel (ii) is representing differentiated cells that is cells after neuronaldifferentiation

cells Statistical analysis showed a significant difference (119875 lt005) in cell number between the two types of cells at days 3and 5 of culture (Figure 5)

4 Discussion

The cells formed a fibroblast-like morphology during thefourth passage Molecular analysis showingCd146 andCd166amplicon but not Cd31 validated that the fibroblast-like cellswere MSCs rather than hematopoietic stem cells Cd146 andCd166 are mesenchymal stem cell markers [36 37] Cd146is an early mesenchymal stem cell marker expressed withindental pulp tissues [36] Cd166 is a cell adhesion moleculethat plays important roles in tight cell-to-cell interactionand in the regulation of MSCs differentiation [38] WhileCd166 is expressed in a wide variety of tissues it is usually

restricted to subsets of cells involved in processes of dynamicgrowth andor migration including neural development andimmune response [39] The amplicon of Gapdh found inDPSC both before and after differentiation indicated thatGapdh is expressed in both types of cells

To confirm the differentiation of DPSCs to a neuronalphenotype and to demonstrate that this differentiation wasnot an artefact three analyses were performed morphologi-cal changes expression of MAP2 and activation of neuronalmarkers After 2 days of culture most of the cells resembledmultipolar neuron However some fibroblast-like cells withspread-outmorphologywere still observed in the populationWe suggest that DPSCs changed gradually and differentiatedinto neuron-like cells after 5 days when cultured in serum-and growth factor-free medium DPSCs from various tis-sues differentiated into neuronal cells by displaying neuronmorphology [40 41] similar to our observations Thus


times400

(a)

times400

(b)

times400

(c)

times400

(d)

times400

(e)

times400

(f)

times400

(g)

Figure 3 Characteristics of differentiated cells Neuron-like cells appeared among the dental pulp stem cells ((a) (b)) 5 days after neuronalinduction (c)Theperikaryon dendrites and axons of neurons are indicated respectively bywhite arrows open arrowheads and black arrows(d) Immunofluorescence staining for neuron markers MAP2 was performed after 5 days of neuronal induction ((e) (f)) MAP2 marker wasnot detected in DPSCs without neuronal induction that is negative control (g)

morphological analysis indicated that DPSCs isolated fromdental pulp tissue differentiated into neurons

RT-PCR analysis showed the presence of a Nestin ampli-conNestin is a neuron marker in adult rat and human brains[42] Both Nestin and Tub3 were also used as markers toinvestigate neuronal differentiation in the hippocampus ofmice after DPSC implantation [43] The expression of bothNestin andTub3 after differentiation indicated that theDPSCsdifferentiated into neuronal cells Nestin is one of the inter-mediate filaments found in the cytoskeleton of vertebrate cells[19 44]Nestin expression was used to track the proliferationmigration and differentiation of neuronal stem cells Tub3 isexpressed in all eukaryotic cells It contributes tomicrotubulestability in neuronal cell and plays a role in axonal transportIn the present study Tub3 was activated after 5 days ofneuronal differentiationGapdhwas used as a positive controlboth before and after differentiationGapdh is a housekeepinggene which has always been activated by all mammalian cellswhether by undifferentiated or differentiated cells [45] It wasused to determine the RNA quality of isolated DPSCs and tonormalize the levels ofmRNAsAGapdh ampliconwas foundin cells both before and after differentiation indicating thatGapdh remains activated in both types of cells

Nestin activation was increased during neuronal differ-entiation Nestin is known to be expressed within fibrousdental pulp tissue however its expression continued tobe detected by the majority of DPSCs following neuronalinduction that is expression of Nestin was increased when

cells differentiated into neuron [28] MAP2 and Tub3 areexpressed only after neuronal differentiation and are there-fore utilized as markers of mature neuronal cells during thefinal stages of growth [46ndash48] MAP2-positive neuronal cellshave been shown to be adult neuronal cells produced byneuronal induction Varied expression of Nestin in severalcell types such as hair follicle stem cells pericytes endothelialcells myofibroblasts and pancreatic fibroblasts makes Nestinnot specific to neuron [23ndash26] However combination ofNestin expression togetherwith the expression of other neuralmarkers that is Tub3 and MAP2 can support neuronaldifferentiation In addition characterization of the neuron-like cell by morphological analysis also showed a positiveresult

While growth factors such as FGF and retinoic acid havebeen employed in previous studies neuronal induction wasinduced in the present study solely by excluding serum andgrowth factors from the complete medium Our observationson Tub3 andNestin activation neuron-like cells morphologyand MAP2 expression provide evidence for the directeddifferentiation of mouse dental pulp stem cells into neuron-like cells in serum- and growth factor-free medium

The proliferation of the differentiated cells continuedalthough their growth rate was lower than that of the controlgroup Theoretically proliferation and differentiation of cellscannot occur simultaneouslyThe signalling cues that coordi-nate these two processes are largely unknown However celldifferentiation and proliferation are regulated simultaneously


(i) (ii)Nestin

sim215bpsim215bp400bp

200bp

(i) Undifferentiated cells

(ii) Differentiated cells

(a)

(i) (ii)Tub3

sim125bp200bp

100bp



(b)

Gapdh(i) (ii)

sim717bpsim717bp



800bp

700bp

(c)

Figure 4 Activation of specific neuronal markersThe activation of Nestin (sim215 bp) (a) and Tub3 (sim125 bp) (b) indicated that the cells haddifferentiated into neurons Gapdh (sim717 bp) a housekeeping gene was activated before and after differentiation (c) Panel (i) is representingundifferentiated cells that is cells before neuronal differentiation while panel (ii) is representing differentiated cells that is cells after neuronaldifferentiation

but independently that cells often start differentiating longbefore they stop dividing and that the initiation of differ-entiation is not restricted to any particular segment of thecell cycle [49 50] In the present study differentiated cellsbegan to divide slowly after 24 hours of culture and continueduntil the end of the differentiation period suggesting thatthe cells undergo differentiation immediately after neuronaldifferentiation has been induced Although the number ofcells increased upon the induction of differentiation the cellssubsequently showed a growth rate reduction and focused onthe differentiation process

Capabilities of adult stem cells to differentiate into cellsfrom different germ layers or cell lineages are known as

transdifferentiation Stem cells from bone marrow whichoriginated from mesoderm were shown to be able to differ-entiate into liver lung gastrointestinal tract and skin cellswhich derived from endoderm and mesoderm [51] Periph-eral blood stem cells (hematopoietic stem cells) also werefound able to differentiate into mature cells which werenot originated from hematopoietic cells such as liver skinepithelial and gastrointestinal tract cells [52] Changes thatoccur to cell microenvironments such as addition of certaingrowth or differentiation factors during in vitro cell culturewere found to be able to induce transdifferentiation [53]Deletion of serum also contributed to this transdifferentia-tion since addition of serum in the medium allowed growth


0 1 3 5Days of differentiation

lowast

lowast9876543210

Cel

ls vi

abili

ty (times

104)

ControlDifferentiate to neuron-like cells

Figure 5 Viability of cells during directed differentiation The via-bility of control (undifferentiated cells cultured in complete me-dium) versus differentiated cells cultured in serum- and growthfactor-free mediumThe numbers of both control and differentiatedcells were significantly increased upon differentiation comparedwith day 0 of cultureThe results are summarized as the mean plusmn SDStatistical significance was determined using SPSS program version1602 lowastStatistical analysis showed significant differences (119875 lt 005)of viable cells at days 3 and 5 of culture as compared to control

andmaintenance of cells and prevented embryonic stem cellsdifferentiation into neuronal cells [54] In this study theabsence of serum and growth factors during culture may leadto DPSCs transdifferentiation into neuron-like cells

5 Conclusions

Dental pulp stem cells are induced to differentiate into neu-ronal cells when cultured in serum- and growth factor-freemedium

Authorsrsquo Contribution

Shahrul Hisham Zainal Ariffin is head of this project Hedesigned the experiment and characterized the cell linesShabnam Kermani performed the experiments Intan ZarinaZainol Abidin analyzed the data and corrected and rewrotethe paper Rohaya Megat Abdul Wahab designed the exper-iment and analyzed the data Zulham Yamamoto wrote thepaper Sahidan Senafi and Zaidah Zainal Ariffin analyzedthe data and corrected the paper Mohamad Abdul Razakdesigned the experiment and the research approaches Allauthors have read and approved the paper

Acknowledgments

This research project was funded by Grants FRGS12011SGUKM0213 and ERGS12012SKK11UKM025 from theMinistry of Higher Education of Malaysia and UKM-DLP-2012-025 UKM-DLP-2012-001 andDPP-2013-024 fromUni-versiti Kebangsaan Malaysia

References

[1] I Kerkis A Kerkis D Dozortsev et al ldquoIsolation and charac-terization of a population of immature dental pulp stem cellsexpressing OCT-4 and other embryonic stem cell markersrdquoCells Tissues Organs vol 184 no 3-4 pp 105ndash116 2007

[2] E Javazon J Tebbets and K Beggs ldquoIsolation expansionand characterization of murine adult bone marrow derivedmesenchymal stem cellsrdquo Blood vol 102 pp 180Bndash181B 2003

[3] S H Zainal Ariffin I Z Zainol Abidin M D Yazid and RMegat Abdul Wahab ldquoDifferentiation analyses of adult suspen-sion mononucleated peripheral blood cells of Mus musculusrdquoCell Communication and Signaling vol 8 article 29 2010

[4] M D Yazid S H Zainal Ariffin S Senafi Z Zainal Ariffinand R Megat Abdul Wahab ldquoStem Cell Heterogeneity ofmononucleated cells from murine peripheral blood molecularanalysisrdquo TheScientificWorldJOURNAL vol 11 pp 2150ndash21592011

[5] M D Yazid S H Z Ariffin S Senafi M A Razak and R MA Wahab ldquoDetermination of the differentiation capacities ofmurinesrsquo primary mononucleated cells and MC3T3-E1 cellsrdquoCancer Cell International vol 10 article 42 2010

[6] T Hermida-Gomez I Fuentes-Boquete M J Gimeno-Longaset al ldquoQuantification of cells expressing mesenchymal stemcell markers in healthy and osteoarthritic synovial membranesrdquoJournal of Rheumatology vol 38 no 2 pp 339ndash349 2011

[7] Y Sumita S Tsuchiya I Asahina H Kagami andM J HondaldquoThe location and characteristics of two populations of dentalpulp cells affect tooth developmentrdquo European Journal of OralSciences vol 117 no 2 pp 113ndash121 2009

[8] C Gandia A N A Arminan J M Garcıa-Verdugo et alldquoHuman dental pulp stem cells improve left ventricular func-tion induce angiogenesis and reduce infarct size in rats withacute myocardial infarctionrdquo Stem Cells vol 26 no 3 pp 638ndash645 2008

[9] A Graziano R Drsquoaquino M G Cusella-De Angelis et alldquoScaffoldrsquos surface geometry significantly affects human stemcell bone tissue engineeringrdquo Journal of Cellular Physiology vol214 no 1 pp 166ndash172 2008

[10] S Gronthos M Mankani J Brahim P G Robey and S ShildquoPostnatal human dental pulp stem cells (DPSCs) In Vitro andIn Vivordquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 25 pp 13625ndash13630 2000

[11] S Shi P G Robey and S Gronthos ldquoComparison of humandental pulp and bone marrow stromal stem cells by cDNAmicroarray analysisrdquo Bone vol 29 no 6 pp 532ndash539 2001

[12] B Neuhuber G Gallo L Howard L Kostura A Mackay andI Fischer ldquoReevaluation of In Vitro differentiation protocolsfor bone marrow stromal cells disruption of actin cytoskeletoninduces rapid morphological changes and mimics neuronalphenotyperdquo Journal of Neuroscience Research vol 77 no 2 pp192ndash204 2004

[13] G Munoz-Elıas D Woodbury and I B Black ldquoMarrowstromal cells mitosis and neuronal differentiation stem celland precursor functionsrdquo Stem Cells vol 21 no 4 pp 437ndash4482003

[14] T R Brazelton F M V Rossi G I Keshet and H M BlauldquoFrom marrow to brain expression of neuronal phenotypes inadult micerdquo Science vol 290 no 5497 pp 1775ndash1779 2000

[15] A J Sloan and R J Waddington ldquoDental pulp stem cells whatwhere howrdquo International Journal of Paediatric Dentistry vol19 no 1 pp 61ndash70 2009


[16] A C W Zannettino S Paton A Arthur et al ldquoMultipotentialhuman adipose-derived stromal stem cells exhibit a perivascu-lar phenotype in vitro and in vivordquo Journal of Cellular Physiologyvol 214 no 2 pp 413ndash421 2008

[17] S Gronthos J Brahim W Li et al ldquoStem cell properties ofhuman dental pulp stem cellsrdquo Journal of Dental Research vol81 no 8 pp 531ndash535 2002

[18] C A Messam J Hou and E O Major ldquoCo-expression ofnestin in neural and glial cells in the developing human CNSdefined by a human specific anti-nestin antibodyrdquo ExperimentalNeurology vol 161 no 2 pp 585ndash596 2000

[19] D Guerette P A Khan P E Savard and M Vincent ldquoMolec-ular evolution of type VI intermediate filament proteinsrdquo BMCEvolutionary Biology vol 7 article 164 2007

[20] C Janke and M Kneussel ldquoTubulin post-translational mod-ifications encoding functions on the neuronal microtubulecytoskeletonrdquo Trends in Neurosciences vol 33 no 8 pp 362ndash372 2010

[21] L Chang and R D Goldman ldquoIntermediate filaments mediatecytoskeletal crosstalkrdquo Nature Reviews Molecular Cell Biologyvol 5 no 8 pp 601ndash613 2004

[22] T Tohyama V M Lee L B Rorke M Marvin R D G McKayand J Q Trojanowski ldquoNestin expression in embryonic humanneuroepithelium and in human neuroepithelial tumor cellsrdquoLaboratory Investigation vol 66 no 3 pp 303ndash313 1992

[23] R M Hoffman ldquoThe potential of nestin-expressing hair folliclestem cells in regenerative medicinerdquo Expert Opinion on Biolog-ical Therapy vol 7 no 3 pp 289ndash291 2007

[24] J P Levesque ldquoA niche in a dish pericytes support HSCrdquo Bloodvol 121 pp 2816ndash2818 2013

[25] S Suzuki J Namiki S Shibata Y Mastuzaki and H OkanoldquoThe neural stemprogenitor cell marker nestin is expressed inproliferative endothelial cells but not in mature vasculaturerdquoJournal of Histochemistry and Cytochemistry vol 58 no 8 pp721ndash730 2010

[26] Y Kishaba D Matsubara and T Niki ldquoHeterogeneous expres-sion of nestin in myofibroblasts of various human tissuesrdquoPathology International vol 60 no 5 pp 378ndash385 2010

[27] M K Lee J B Tuttle L I Rebhun D W Cleveland andA Frankfurter ldquoThe expression and posttranslational modifi-cation of a neuron-specific beta-tubulin isotype during chickembryogenesisrdquo Cell Motility and the Cytoskeleton vol 17 no2 pp 118ndash132 1990

[28] A Arthur G Rychkov S Shi S A Koblar and S GronthoseldquoAdult human dental pulp stem cells differentiate toward func-tionally active neurons under appropriate environmental cuesrdquoStem Cells vol 26 no 7 pp 1787ndash1795 2008

[29] N Kalcheva J Albala K OrsquoGuin H Rubino C Garner andB Shafit-Zagardo ldquoGenomic structure of human microtubule-associated protein 2 (MAP-2) and characterization of additionalMAP-2 isoformsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 92 no 24 pp10894ndash10898 1995

[30] S Kermani K Karbalaie S H Madani et al ldquoEffect of lead onproliferation and neural differentiation ofmouse bonemarrow-mesenchymal stem cellsrdquo Toxicology In Vitro vol 22 no 4 pp995ndash1001 2008

[31] R W L Lim and S Halpain ldquoRegulated association ofmicrotubule-associated protein 2 (MAP2) with Src and Grb2evidence for MAP2 as a scaffolding proteinrdquo Journal of Biologi-cal Chemistry vol 275 no 27 pp 20578ndash20587 2000

[32] T Doniach ldquoBasic FGF as an inducer of anteroposterior neuralpatternrdquo Cell vol 83 no 7 pp 1067ndash1070 1995

[33] D P Hill and K A Robertson ldquoCharacterization of the cholin-ergic neuronal differentiation of the human neuroblastoma cellline LA-N-5 after treatment with retinoic acidrdquo DevelopmentalBrain Research vol 102 no 1 pp 53ndash67 1997

[34] V Tropepe M Sibilia B G Ciruna J Rossant E F Wagnerand D Van Der Kooy ldquoDistinct neural stem cells proliferatein response to EGF and FGF in the developing mouse telen-cephalonrdquo Developmental Biology vol 208 no 1 pp 166ndash1881999

[35] K Guan H Chang A Rolletschek and A MWobus ldquoEmbry-onic stem cell-derived neurogenesis retinoic acid inductionand lineage selection of neuronal cellsrdquoCell and Tissue Researchvol 305 no 2 pp 171ndash176 2001

[36] M Miura S Gronthos M Zhao et al ldquoSHED stem cells fromhuman exfoliated deciduous teethrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no10 pp 5807ndash5812 2003

[37] A P Croft and S A Przyborski ldquoMesenchymal stem cells fromthe bone marrow stroma basic biology and potential for celltherapyrdquo Current Anaesthesia and Critical Care vol 15 no 6pp 410ndash417 2004

[38] H Fujiwara K Tatsumi K Kosaka et al ldquoHuman blastocystsand endometrial epithelial cells express activated leukocytecell adhesion molecule (ALCAMCD166)rdquo Journal of ClinicalEndocrinology and Metabolism vol 88 no 7 pp 3437ndash34432003

[39] G W M Swart ldquoActivated leukocyte cell adhesion molecule(CD166ALCAM) developmental and mechanistic aspects ofcell clustering and cell migrationrdquo European Journal of CellBiology vol 81 no 6 pp 313ndash321 2002

[40] D R Martin N R Cox T L Hathcock G P Niemeyer and HJ Baker ldquoIsolation and characterization of multipotential mes-enchymal stem cells from feline bone marrowrdquo ExperimentalHematology vol 30 no 8 pp 879ndash886 2002

[41] L Qian and W M Saltzman ldquoImproving the expansion andneuronal differentiation of mesenchymal stem cells throughculture surface modificationrdquo Biomaterials vol 25 no 7-8 pp1331ndash1337 2004

[42] M L Hendrickson A J Rao O N A Demerdash and R EKalil ldquoExpression of nestin by neural cells in the adult rat andhuman brainrdquo PLoS ONE vol 6 no 4 Article ID e18535 2011

[43] A HC Huang B R Snyder PH Cheng and A W S ChanldquoPutative dental pulp-derived stemstromal cells promote pro-liferation and differentiation of endogenous neural cells in thehippocampus ofmicerdquo StemCells vol 26 no 10 pp 2654ndash26632008


[45] R D Barber D W Harmer R A Coleman and B J ClarkldquoGAPDH as a housekeeping gene analysis of GAPDH mRNAexpression in a panel of 72 human tissuesrdquo PhysiologicalGenomics vol 21 no 3 pp 389ndash395 2005

[46] M Cristofanilli SThanos J Brosius S Kindler and H TiedgeldquoNeuronal MAP2 mRNA species-dependent differential den-dritic targeting competencerdquo Journal of Molecular Biology vol341 no 4 pp 927ndash934 2004

[47] C B Johansson S Momma D L Clarke M Risling ULendahl and J Frisen ldquoIdentification of a neural stem cell in


the adult mammalian central nervous systemrdquo Cell vol 96 no1 pp 25ndash34 1999

[48] E Karaoz B N Dogan A Aksoy et al ldquoIsolation and in vitrocharacterisation of dental pulp stem cells from natal teethrdquoHistochemistry and Cell Biology vol 133 pp 95ndash112 2010

[49] G Brown P J Hughes and R H Michell ldquoCell differentiationand proliferationmdashsimultaneous but independentrdquo Experi-mental Cell Research vol 291 no 2 pp 282ndash288 2003

[50] H Geng R Lan G Wang et al ldquoInhibition of autoregulatedTGFbeta signaling simultaneously enhances proliferation anddifferentiation of kidney epithelium and promotes repair fol-lowing renal ischemiardquo American Journal of Pathology vol 174no 4 pp 1291ndash1308 2009

[51] D S Krause N D Theise M I Collector et al ldquoMulti-organmulti-lineage engraftment by a single bone marrow-derivedstem cellrdquo Cell vol 105 no 3 pp 369ndash377 2001

[52] M Korbling R L Katz A Khanna et al ldquoHepatocytes andepithelial cells of donor origin in recipients of peripheral-bloodstem cellsrdquo New England Journal of Medicine vol 346 no 10pp 738ndash746 2002

[53] L Yang S Li H Hatch et al ldquoIn vitro trans-differentiation ofadult hepatic stem cells into pancreatic endocrine hormone-producing cellsrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 99 no 12 pp 8078ndash80832002

[54] M Darmon J Bottenstein and G Sato ldquoNeural differentiationfollowing culture of embryonal carcinoma cells in a serum-freedefinedmediumrdquoDevelopmental Biology vol 85 no 2 pp 463ndash473 1981

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Primers involved in RT-PCR experiments

Geneaccession no Primers Sequences Expected product size (bp) Annealing temperature (∘C)

Cd146 (NM 023061) Forward 51015840-GGACCTTGAGTTTGAGTGG-31015840 479 60Reverse 51015840-CAGTGGTTTGGCTGGAGT-31015840

Cd166 (NM 009655) Forward 51015840-AACATGGCGGCTTCAACG-31015840 630 61Reverse 51015840-GACGACACCAGCAACGAG-31015840

Nestin (NM 016701) Forward 51015840-CGCTCGGGAGAGTCGCTT-31015840 215 64Reverse 51015840-CCAGTTGCTGCCCACCTTC-31015840

Gapdh (NM 008084) Forward 51015840-CAACGGCACAGTCAAGG-31015840 717 62Reverse 51015840-AAGGTGGAAGAGTGGGAG-31015840

Tub3 (NM 023279) Forward 51015840-ACGCATCTCGGAGCAGTT-31015840 125 61Reverse 51015840-CGGACACCAGGTCATTCA-31015840

Cd31 (NM 0010323781) Forward 51015840-GGTCTTGTCGCAGTATCAG-31015840 355 58Reverse 51015840-ATGGCAATTATCCGCTCT-31015840

into neurons [18 22] However Nestin has shown to beexpressed by other cell types such as hair follicle stem cells[23] pericytes [24] endothelial cells [25]myofibroblasts andpancreatic fibroblasts [26] Therefore analysis on expressionof other specific neuron markers such as Tub3 [27 28] andMAP2 [29 30] has been done concurrently for neuronalconfirmation Tub3 and MAP2 play a role in the stabilityof axons and neuronal cell bodies [20 31] Certain growthfactors such as epidermal growth factor basic fibroblastgrowth factor and retinoic acid were used for neuronalinduction [32ndash35] Dimethyl sulfoxide (DMSO) was alsoused to induce transformation of MSCs into neuron-likephenotypes in vitro [12 13]The objective of the present studywas to examine the directed differentiation of DPSCs intoneuron-like cells in the absence of chemical induction

2 Materials and Methods

21 Isolation ofDental Pulp Cells Incisor teethwere extractedfrom 6- to 8-week-old mice under sterile conditions andplaced in medium containing 1X PBS (Sigma USA) Theextracted dental pulp was washed with 1X PBS containing 1(vv) penicillin-streptomycin (Invitrogen USA)

Dental pulp tissue was incubated for 1 hour in 4-unitcollagenase type I at 37∘C followed by several rounds ofenzymatic disaggregationThe cells were centrifuged at 1200 gfor 10 minutes at 25∘C and cultured in complete mediumconsisting of 120572-MEM (Invitrogen USA) supplemented with20 (vv) FBS (Biowest USA) and 1 (vv) penicillin-streptomycin The cells 1 times 105 cellsmL obtained were putin a T25 flask containing complete medium and cultured inan incubator with 5 CO

2atmosphere and 95 humidity at

37∘CAfter 24 hours the suspended cells were removed from

the medium and the flask was washed with 1X PBS solutionThe cells were grown in complete medium until 80 conflu-ency A solution of 025 (vv) trypsin-EDTA (Sigma USA)was used to detach the dental pulp cells from the flask surfacefor subculturing in another flask at 1 times 105 cellsmL Forcryopreservation and storage cells at the fourth passage were

placed in cryovials containing freezing medium consisting of120572-MEM 10 (vv) DMSO (Sigma USA) and 50 (vv) FBSand stored in liquid nitrogen In this study the cells usedwereat the fourth passage

22 Differentiation of Dental Pulp Stem Cells into NeuronalCells Approximately 1 times 105 cells were transferred into 24-well plates containing complete medium and were allowedto grow for 24 hours until adherence The medium wasdiscarded and the cells were washed with PBS The cellswere then cultured in serum- and growth factor free-mediumconsisting of 120572-MEM and 1 (vv) penicillin-streptomycinfor 5 days The medium was changed every 2-3 days duringthis 5-day period As a control the same number of cells wascultured in complete medium (consists of 120572-MEM 1 (vv)penicillin-streptomycin and 15 (vv) fetal bovine serum)for 5 days Cell morphology was monitored on days 2 and 5of neuronal differentiation using an Olympus phase-contrastmicroscope

23 Molecular Analysis Using RT-PCR Control and differen-tiated cells were detached using 025 (vv) trypsin-EDTAand centrifuged at 1400 g for 10 minutes Total RNA wasextracted using TRI-reagent (Sigma USA) according to themanufacturerrsquos protocol The purity of the total RNA wasassessed spectrophotometrically at 260 and 280 nm with anA260

A280

ratio of 18ndash20 considered acceptable Approxi-mately 300 ng of total RNA samplewas used for eachRT-PCRreactionwith theAccess Quick RT-PCR system kit (PromegaUSA) The primers were designed using the Primer Pre-mier 50 software program based on sequences obtainedfrom NCBI Information on the primers is summarized inTable 1

Primary cDNA synthesis was performed using AMVreverse transcriptase for 45minutes at 45∘C followed by deac-tivation for 2 minutes at 94∘C The amplification consistedof denaturation for 30 seconds at 94∘C annealing for 60seconds and extension for 60 seconds at 68∘C performedfor 40 cycles A final extension step was performed for 7minutes at 68∘CThe amplified products were separated using






3 Results








times100

(a)

times100

(b)

times400

(c)

times400

(d)

times400

(e)





(i) (ii)Cd146

500bp400bp sim479bp


(a)

(i) Cd166 (ii)

700bp600bp

sim630bp


(b)

(i) (ii)Cd31


(c)

(i) (ii)

sim717bp sim717bp

Gapdh

800bp700bp


(d)



4 Discussion





times400

(a)

times400

(b)

times400

(c)

times400

(d)

times400

(e)

times400

(f)

times400

(g)









(i) (ii)Nestin


200bp



(a)

(i) (ii)Tub3

sim125bp200bp

100bp



(b)

Gapdh(i) (ii)

sim717bpsim717bp



800bp

700bp

(c)







lowast

lowast9876543210

Cel

ls vi

abili

ty (times

104)




5 Conclusions




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






3 Results








times100

(a)

times100

(b)

times400

(c)

times400

(d)

times400

(e)





(i) (ii)Cd146

500bp400bp sim479bp


(a)

(i) Cd166 (ii)

700bp600bp

sim630bp


(b)

(i) (ii)Cd31


(c)

(i) (ii)

sim717bp sim717bp

Gapdh

800bp700bp


(d)



4 Discussion





times400

(a)

times400

(b)

times400

(c)

times400

(d)

times400

(e)

times400

(f)

times400

(g)









(i) (ii)Nestin


200bp



(a)

(i) (ii)Tub3

sim125bp200bp

100bp



(b)

Gapdh(i) (ii)

sim717bpsim717bp



800bp

700bp

(c)







lowast

lowast9876543210

Cel

ls vi

abili

ty (times

104)




5 Conclusions




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


times100

(a)

times100

(b)

times400

(c)

times400

(d)

times400

(e)





(i) (ii)Cd146

500bp400bp sim479bp


(a)

(i) Cd166 (ii)

700bp600bp

sim630bp


(b)

(i) (ii)Cd31


(c)

(i) (ii)

sim717bp sim717bp

Gapdh

800bp700bp


(d)



4 Discussion





times400

(a)

times400

(b)

times400

(c)

times400

(d)

times400

(e)

times400

(f)

times400

(g)









(i) (ii)Nestin


200bp



(a)

(i) (ii)Tub3

sim125bp200bp

100bp



(b)

Gapdh(i) (ii)

sim717bpsim717bp



800bp

700bp

(c)







lowast

lowast9876543210

Cel

ls vi

abili

ty (times

104)




5 Conclusions




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(i) (ii)Cd146

500bp400bp sim479bp


(a)

(i) Cd166 (ii)

700bp600bp

sim630bp


(b)

(i) (ii)Cd31


(c)

(i) (ii)

sim717bp sim717bp

Gapdh

800bp700bp


(d)



4 Discussion





times400

(a)

times400

(b)

times400

(c)

times400

(d)

times400

(e)

times400

(f)

times400

(g)









(i) (ii)Nestin


200bp



(a)

(i) (ii)Tub3

sim125bp200bp

100bp



(b)

Gapdh(i) (ii)

sim717bpsim717bp



800bp

700bp

(c)







lowast

lowast9876543210

Cel

ls vi

abili

ty (times

104)




5 Conclusions




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


times400

(a)

times400

(b)

times400

(c)

times400

(d)

times400

(e)

times400

(f)

times400

(g)









(i) (ii)Nestin


200bp



(a)

(i) (ii)Tub3

sim125bp200bp

100bp



(b)

Gapdh(i) (ii)

sim717bpsim717bp



800bp

700bp

(c)







lowast

lowast9876543210

Cel

ls vi

abili

ty (times

104)




5 Conclusions




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(i) (ii)Nestin


200bp



(a)

(i) (ii)Tub3

sim125bp200bp

100bp



(b)

Gapdh(i) (ii)

sim717bpsim717bp



800bp

700bp

(c)







lowast

lowast9876543210

Cel

ls vi

abili

ty (times

104)




5 Conclusions




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



lowast

lowast9876543210

Cel

ls vi

abili

ty (times

104)




5 Conclusions




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Differentiation of dental pulp stem cells into islet-like aggregates

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Transcript of Differentiation of dental pulp stem cells into islet-like aggregates