5807.full Stem Cells

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SHED: Stem cells from human exfoliated deciduous teeth Masako Miura*, Stan Gronthos†, Mingrui Zhao‡, Bai Lu‡, Larry W. Fisher*, Pamela Gehron Robey*, and Songtao Shi*§ *Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892; †Mesenchymal Stem Cell Group, Division of Haematology, Institute of Medical and Veterinary Science, Frome Road, Adelaide 5000, South Australia, Australia; and  ‡ Section on Neural Development and Plasticity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 Edited by Anthony P. Mahowald, University of Chicago, Chicago, IL, and approved March 12, 2003 (received for review December 16, 2002)

To isolate high-quality human postnatal stem cells from accessible resources is an important goal for stem-cell research. In this study we found that exfoliated human deciduous tooth contains multipotent pote nt stem cells [stem cells from human exfoliated exfoliated decid deciduou uouss teeth (SHED)]. SHED were identified to be a population of highly prolifera prol iferative, tive, clono clonogeni genicc cells capab capable le of diff different erentiatin iating g into a variety of cell types including neural cells, adipocytes, and odonvivo o tra toblasts. tobl asts. Afte Afterr in viv transp nsplan lantat tation ion,, SHE SHED D wer were e fou found nd to be abl able e to induce bone formation, generate dentin, and survive in mouse brain along with expression of neural markers. Here we show that a naturally exfoliated human organ contains a population of stem cells that are completely different from previously identified stem cells. cel ls. SHE SHED D are no nott on only ly der derive ived d fro from m a ver very y acc access essibl ible e tis tissue sue resource but are also capable of providing enough cells for potential clinical application. Thus, exfoliated teeth may be an unexpected unique resource for stem-cell therapies including autologous stem-cell transplantation and tissue engineering. odontoblast    bone regeneration     neural differentiatio differentiation n     adipocyte    dental pulp stem cell

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ostnatal ostna tal ste stem m cel cells ls hav havee bee been n is isola olate ted d from a va varie riety ty of  tissues including including but not limited to bone marrow, brain, skin, hair follicles, skeletal muscle, and dental pulp (1–7). Recently, the extraordina extraordinary ry plas plastici ticity ty of post postnatal natal stem cel cells ls has been sugge sug geste sted, d, in whi which ch neu neuralstem ralstem ce cell llss maycontri maycontribut butee to blo blood od and skeletal muscle (8, 9), and bone marrow stem cells may contribute to muscle, liver, and neuronal tissue (10 –13). Recent emerging evidence suggests that cell-fusion events may account for somee of the som these se obs observa ervatio tions ns (14 (14,, 15) 15).. It is nec neces essa sary ry to gai gain n fur furthe therr insigh ins ightt int into o the cha charac racter terist istic icss of pos postna tnatal tal ste stem m ce cell llss and examine their full developmental potential  in vivo  (16). The transition from deciduous teeth to adult permanent teeth is a very unique and dynamic process in which thethe development and eruption of permanent teeth coordinate with resorption of the roots of deciduous teeth. It may take 7 years in humans to complete the ordered replacement of 20 deciduous teeth (17). In this study we isolated a distinctive population of multipotent stem cells from the remnant pulp of exfoliated deciduous teeth. The si signif gnific icanc ancee of thi thiss stu study dy is tha thatt it pro provid vides es evi eviden dence ce indicating that a naturally occurring exfoliated deciduous tooth is si simil milar ar in som somee way wayss to an umb umbil ilic ical al cord cord,, cont contain aining ing ste stem m ce cell llss that may offer a unique stem-cell resource for potential clinical applications. Materials and Methods Subjects Subje cts and Cell Culture.   Normal exfoliated human deciduous

incisors were colle incisors collected cted from 7- to 8-ye 8-year-o ar-old ld chil children dren under approved appr oved guidelines guidelines set by the Nat Nationa ionall Inst Institut itutes es of Hea Health lth Office of Human Subjects Research. The pulp was separated from a remnant crown and then digested in a solution of 3 mg ml collagenase type I (Worthington Biochem, Freehold, NJ) and 4 mgml dispase (Roche Molecular Biochemicals) Biochemicals) for 1 h at 37°C. Single-cell suspensions were cultured in a regular medium as reported (7). These techniques resulted in a population that we www.pnas.orgcgidoi10.1073 pnas.0937635100

have termed stem cells from human exfoliated deciduous teeth (SHED). Conditions for the induction of calcium accumulation were as reported (7), and recombinant human BMP-4 (R & D Systems)  was used to induce in duce osteogenic os teogenic differen differentiation. tiation. Calcium ac cumulation was detected by 2% Alizarin red S (pH 4.2) staining. The calcium concentration was measured by using a commercially avai av aila lable ble kit (ca (calc lcium ium kit 587 587-A -A,, Sig Sigma) ma).. The ind induct uction ion of  adipogenesis was performed as reported (18). For neural differentiation, ferentiat ion, Neurobasal A (GIBCOBRL), B27 suppl suppleme ement nt (GIBCO BRL) BRL),, 1% peni penicil cilli lin, n, 20 ngml epid epidermal ermal growth factor (BD Bioscience), and 40 ngml fibroblast growth factor (FGF) (BD Bioscience) were used to culture cells attached to 0.1% gelatin-coated dishes (StemCell Technologies, Vancou ver). For sphere-like cell-cluster formatio formation, n, 3% rat serum and B27 were added. Antibodies.   Rabbit antibodies included anti-HSP90 and basic

FGF (bFGF) (Santa Cruz Biotechnology); anti-core-binding factor, runt domain,      subunit 1 (CBFA1) (Oncogene Research Products, Cambridge, MA); anti-endostatin, humanspecific speci fic mitoc mitochondri hondria, a, and glutami glutamicc acid deca decarboxylas rboxylasee (GAD)) (Che (GAD (Chemicon) micon);; and anti anti-al -alkali kaline ne phos phosphatas phatasee (ALP) (LF-47), (LF-4 7), bone sial sialoprote oprotein in (LF(LF-120), 120), matri matrixx extra extracell cellular ular phosphagl phosp haglycopro ycoprotein tein (MEP (MEPE) E) (LF(LF-155) 155),, and denti dentin n sia sialolophosphoprotein phosphoprotei n (DSPP) (LF-151) (National Institute of Dental and Craniofacial ResearchNational Institutes Institutes of Health). Goat antibodies included anti-MAP2 and Tau (Santa Cruz Biotechnology). Biotec hnology). Mouse antibod antibodies ies include included d anti-S anti-STRO-1 TRO-1 and CD14 CD146 6 (CC9 (CC9); ); glia gliall fibr fibrill illary ary aci acidic dic prot protein ein (GFAP), nestin, neurofilament neurofilament M (NFM), neuronal nuclei (NeuN), and 2  ,3  -cycli -cyclicc nucle nucleotide otide-3 -3  -phosphodiesterase (CNPase) (Chemicon (Che micon); ); and anti anti--IIIIII-tubul tubulin in (Pro (Promega mega). ). Rab Rabbit bit and murine isotype-matched negative control antibodie antibodiess were also used (Caltag Laboratories, Burlingame, CA).  Approximately imately 2.0  106 SHED were mixed with Transplantation. Approx 40 mg of hydroxyapatite tricalcium phosphate (HA TCP) ceramic powder (Zimmer, Warsaw, IN) and then transplante transplanted d s.c. into immuno immunocompromi compromised sed mice (NIH(NIH-bg-nubg-nu-xid, xid, Harla Harlan– n– Sprague–Dawley) as described (19). SHED were injected into the brain of immunocompromised mice according to specifications of an approved small-animal This paper was submitted directly (Track II) to the PNAS office. Abbreviations: SHED, stem cells from human exfoliated deciduous teeth; FGF, fibroblast growth grow th fact factor;bFGF, or;bFGF, basi basicc FGF;CBFA1,core-b FGF;CBFA1,core-bindin inding g fact factor,runt or,runt domai domain, n,  subun subunit it 1;GAD, glutamic acid decarboxylase; MEPE, matrix extracellula extracellularr phosphoglycoprotein; DSPP, dentinsialophosphop tinsialopho sphoprotei rotein; n; GFA GFAP,glial P,glial fibril fibrillaryacidicprotei laryacidicprotein; n; NFM NFM,, neuro neurofilam filamentM; entM; NeuN NeuN,, neuronal nuclei; CNPase, 2,3-cyclic nucleotide-3-phosphodiesterase; HATCP, hydroxyapatite phosphate; BMSSC, bone marrow stromal stem cell; DPSC, dental pulp tricalcium stem cell; ALP, alkaline phosphatase. §To

whom corresponden correspondence ce shoul should d be addre addressed ssed at: Nati National onal Institute Institute of Dent Dental al and CraniofacialResearchNati NationalInstit onalInstitutesof utesof Heal Health,Building30,Room th,Building30,Room 228,Conven 228,Conventt Driv Drive e MSC-4320, Bethesda, MD 20892. E-mail: [email protected] [email protected] gov.

PNAS      May 13, 2003      vol. 100      no. 10      5807–5812

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prot protocol ocol (Na (Nation tional al Inst Institut itutee of Denta Dentall and Crani Craniofac ofacial ial Research 01-185). Coordinates Coordinates for the target sites were determined by referencing a murine brain atlas (ref. 20; see Fig. 5 A). The anteropos ante roposteri terior, or, medi mediolat olateral eral,, and dorso dorsoventr ventral al coordin coordinate atess  were compute computed d relative to Bregma.   Ex vivo-expanded SHED (10,000 cells per ml) were infused to the dentate gyrus of the hippocampus (21, 22). Cells (0.5 ml per side) were infused to the coordinates (anteroposterior, (anteroposterior, mediola mediolateral, teral, and dorsoventra dorsoventral, l, respectively, 1.5 mm, 0.8 mm, and 2.0 mm) by using a 1-ml Hamilton Syringe. primers ers incl included uded pero peroxisome xisome prol prolifer iferator ator-RT-PCR.   The PCR prim activated recep activated receptortor- 2 s en en se se ( 5 -CTCCTATTGACCCAGAAAGC-3 ) (nucl (nucleoti eotides des 114 114– – 133 133)) and ant antise isense nse (5 GTAGAGCTGAGTCTTCTCAG-3) (nuc (nucleot leotide idess 441 441– – 460, GenBank accession no. XM  003059); 003059); lipoprotein lipase sense (5-ATGGAGAGCAAAGCCCTGCTC-3) (nuc (nucleot leotide idess 175 175– – 195) and antisense (5-GTTAGGTCCAGCTGGATCGAG-3) (nucleotides (nucleotid es 718 718– –738 738,, Ge GenBa nBank nk acc acces essio sion n no. XM  044682); 044682); CBFA1 CBFA 1 sens sensee (5-CAGTTCCCAAGCATTTCATCC-3) (nucleotide cle otidess 880 880– –900 900)) and ant antis isens ensee (5-TCAATATGGTCGCCAAACAG-3) (nuc (nucleot leotide idess 1304 1304– –1323 1323,, GenB GenBank ank acce accessio ssion n no. L40992); Osterix sense (5-GCAGCTAGAAGGGAGTGGTG-3) (nuc (nucleo leotide tidess 821 821– – 84 840) 0) an and d ant antis isen ense se (5-GCAGGCAGGTGAACTTCTTC-3) (nucl (nucleoti eotides des 1160 1160– –1179 1179,, GenBank Ba nk acc acces essi sion on no. XM  062600); 0 62600); Oste Osteoca ocalci lcin n sen sense se (5 CATGAGAGCCCTCACA-3  ) (nuc (nucleo leotid tides es 18 – 3 3) 3) a nd nd antisense antis ense (5-AGAGCGACACCCTAGAC-3 ) (nucle (nucleotide otidess 316 –332, GenBank accession no. X53698); and GAPDH sense (5-AGCCGCATCTTCTTTTGCGTC-3 ) (nucleotides 12– 12–32) and anti antisens sensee (5-TCATATTTGGCAGGTTTTTCT-3 ) (nucleotides 807– 807– 827, GenBank accession no. M33197). Total RNA  isolation, first-strand cDNA synthesis, and PCR processes were performed as described (23). In Situ  Hybridization.  Hybridization.  Human-specific alu and murine-specific pf1

sequences labeled with digoxigenin were used as probes for   in  situ  hybridization as described (7). Primers included human alu sense (5-TGGCTCACGCCTGTAATCC-3) (nucleotides 90– 90 – 108) and antisense (5 -TTTTTTGAGACGGAGTCTCGC-3 ) (nucleotides 344– 344 –364, GenBank accession no. AC004024) and murine mur ine pf1 (se (sense nse,, 5-CCGGGCAGTGGTGGCGCATGCCTTTAAATCCC-3) (nucleotides 170 –201) and antisense (5GTTTGGTTTTTGAGCAGGGTTCTCTGTGTAGC-3 ) (nucleotidess 275 cleotide 275– –306, GenBank accession no. X78319). weree sub subcul cultur tured ed int into o ei eight ght-Immunohistochemistry.   SHED wer 4

chamber slides (2     10 cells cells were fixed in 4% formaldehyde forper 15well) min (Nunc). and thenThe blocked and incubate incu bated d with prim primary ary anti antibodi bodies es (1:2 (1:200 00– –1:5 1:500 00 dil diluti ution) on) for 1 h, respect res pectivel ively. y. The samp samples les were subs subseque equently ntly incu incubate bated d with goat secondary antibodies of either IgG-rhodamine red or IgG-Cy2 (Jackson ImmunoResearch) for 45 min. For enzymatic immunohistochemical staining, the Zymed broad-spectrum immunopero pe roxida xidase se AE AEC C kit wa wass us used ed acco accordi rding ng to ma manuf nufac actu ture rerr protocol. Western Blot.  Primary antibodies were the same as those used in

immunohistoc immunohi stochemi hemical cal staini staining ng at dilu dilution tionss rang ranging ing from 1:20 1:200 0 to 1:1,000. Western blot was performed as reported (24). Fluorescence-Activated Fluorescence-Activ ated Cell Sorting.  SHED were collected from

culture and incubated with STRO-1 (IgM) antibodies or isotypematch ma tched ed neg negati ative ve cont controlanti rolantibod bodie iess for 1 h on ic ice. e. Fl Fluor uores escen cence ce-activated cell-sorting analysis was the same as described (23). Results Here we demonstrate that the remaining crown of exfoliated deciduous teeth contains a living pulp remnant comprised of a 5808      www.pnas.orgcgidoi10.1073 pnas.0937635100

Isolationof tionof SHED SHED.. ( A) The exfo exfoliate liated d prim primary ary inci incisor sor cont containe ained d dent dental al Fig. 1.   Isola Fig. pulp as shown (black triangles). The dashed line shows the occlusion edge of the incisor. (B  and  C ) Hematoxylineosin staining indicated dentin (D) and pulpof exfol exfoliateddecid iateddeciduousteeth.The uousteeth.The pulpcontai pulpcontainedodonto nedodontoblast blastss (arro (arrows), ws), blood vessels (open arrows), and connective tissues. The straight and curved dashed lines in  B  represent the occlusion and resorbed root surfaces, respectively. (D) Single colonies were formed after SHED were plated at low density and cultured for 2 weeks. (E ) SHED were capable of forming sphere-like clusters when cultured with the conditions described in  Materials and Methods. (F ) The sphere-like clusters could be dissociated by passage through needles and subsequently grew on 0.1% gelatin-coated dishes. (G) The proliferation rates of SHED, BMSSCs, and DPSCs were assessed by BrdUrd (BrdU) incorporation for 12 h. SHED showed a signi signifi ficantly higher proliferation rate in comparison with BMSSCs and DPSCs ( *, P   0.05, Student’ Student’s t  test).  test). (H) SHED were able to proliferate to   140 population doublings, which was signifi significantly higher (*,  P   0.05, Student’ Student’s  t  test) than BMSSCs and DPSCs.

normal dental pulp including connective tissue, blood vessels, and odontoblasts (Fig. 1  A –C). To isolate stem cells, single-cell suspensions were derived from the remnant pulp and placed at low density in liquid culture. Approximately 12– 12 –20 cells from each exfoliated incisor were capable of forming adherent colonies (Fig. 1 D), characteristic of other stromal stem-cell populations (7). Interestingly, when cultured either under a neuronaldiffe di fferen renti tiati ation on con condit ditio ion n or in 3% rat se serum rum wit with h B2 B27 7 supplement, these cells formed sphere-like clusters (Fig. 1 E) in  which highly proliferative cells aggregated together in clusters that either adhered to the culture dish or floated freely in the culture medium. After separating the sphere-like clusters, the cells were able to grow as individual fibroblastic cells (Fig. 1 F ). ). When compared with adult bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs), SHED showed a higher proliferation rate (Fig. 1 G) and a higher number of  population doublings (Fig. 1 H ). ). -expa xpande nded d SHE SHED D we were re fou found nd to exp expre ress ss the ce cell ll- Ex vivo-e surfacee mole surfac molecule culess STRO STRO-1 -1 and CD146 (MUC18), (MUC18), two earl earlyy mesenchymal stem-cell markers previously found to be present in BMSSCs and DPSCs (Fig. 2  D  and  E ). STRO-1- and CD146positive cells were found to be located around blood vessels of  the re remna mnant nt pul pulp p by imm immuno unohis histoc tochem hemica icall sta staini ining ng (Fi (Fig. g. 2 A and imply plying ing tha thatt SHE SHED D ma mayy hav havee ori origi ginat nated ed fro from m a per periva ivasc scula ularr  B), im microenvir micr oenvironme onment. nt. A mino minorr propo proportio rtion n (9%) of   ex viv vivo oexpanded SHED stained positive for the STRO-1 antibody with Miura  et al.

 

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possessed stem-cell stem-cell characteristics. characteristics. ( A–E ) The remnant pulp showed STRO-1 (open arrows in  A ) and CD146 (open arrows in  B ) immunoposit immunopositive ive Fig. 2.   SHED possessed vivo o-expa staining stain ing for cell cellss in peri perivasc vascular ular area areas. s. Fluo Fluoresce rescence-a nce-activa ctivated ted cell cell-sort -sorting ing anal analysis ysis show showed ed that ex viv -expandedSHED ndedSHED cont contained ained 9% STRO STRO-1-po -1-positiv sitive e cells( C ). ). SHEDexpressedSTRO-1( SHEDexpres sedSTRO-1( D) an and d CD CD14 146( 6( E ) (arro (arrows).( ws).( F –I ) SHEDexpress SHEDexpressed ed theosteoge theosteogenicand nicand angi angiogen ogenic ic marke markers rs ALP,MEPE, bFGF bFGF,, andendostat andendostatin. in. ( J and K ) SHE SHED D wereeitherculturedwith wereeithercultur edwith regul regular ar medi medium( um( J ) or L-asco -ascorbate rbate-2-ph -2-phosph osphate,dexame ate,dexamethas thasone,and one,and inorg inorganicphosph anicphosphatefor atefor 4 week weekss (K ). ). Aliz Alizarinred arinred stai stainingshowed ningshowed mineralized nodule formation in the induction (K ). ). (L) Western blot analysis showed an up-regulated expression of CBFA1, ALP, MEPE, bone sialoprotein (BSP), andDSPP andDSP P aft afterthe erthe ind induct uctionas ionas des descri cribedabo bedabove.HSP9 ve.HSP90 0 wa wass use used d toasses toassesss theamou theamountof ntof pro protei tein n loa loadedpersamp dedpersample.( le.( M ) Huma Human n reco recombin mbinantBMP-4 antBMP-4 (300ngml, 24 h) was added to induce a signifi significant up-regulation of CBFA1, Osterix, and Osteocalcin (OC) in SHED as detected by semiquantitative PCR.

f luoresc luorescence-act ence-activated ivated cell-sorting analysis (Fig. 2C). Further immunohistotypic analysis demonstrated that cultured SHED

structure (Fig. 3  A  and  B ). Importantly, the regenerated dentin  was immunoreactive immunor eactive to dentin-specific den tin-specific DSPP D SPP antibody antibod y (Fig. 3 C).

expressed stromal- and vascular-related markers ALP, MEPE, bFGF, and endostatin (Fig. 2   F – I ). ). To investigate the potential of SHED to differentiate into mineralized tissue, established secondary SHED cultures were supplemented supplemente d w ith   L -ascorbate-2-phosphate -ascorbate-2-phosphate,, dexametha dexamethasone, sone, and inorganic phosphate as described (7). Alizarin red-positive nodules formed in the SHED cultures after 4 weeks of induction (Fig. (Fi g. 2   J   and   K ), ), indi indicati cating ng cal calcium cium accum accumulat ulation ion   in vit vitro ro.  Accordingly,  Accor dingly, Western blot analysis revealed that various bone markers mark ers CBFA1, ALP, MEPE MEPE,, and bone sialoprote sialoprotein in were up-regulated under the induction (Fig. 2 L). In addition, DSPP  was induced by the mineralizing induction (Fig. 2 L). Furthermore, mor e, BM BMPP-4 4 tre treat atmen mentt wa wass ca capab pable le of ind induci ucing ng an upup-reg regula ulated ted expression of CBFA1, Osterix, and Osteocalcin by semiquantitative RT-PCR (Fig. 2 M ). ). The These se data indi indicate cated d that SHED possess poss essed ed the abil ability ity to diffe different rentiate iate into func function tional al odont odontobla oblaststlike cells  in vitro. To validate the capacity of SHED to form odontoblasts,   ex  vivo-expanded SHED were transplanted into immunocompromised mise d mice (7, 23). The tran transpla splants nts yiel yielded ded huma human-sp n-speci ecific fic alu-positive odontoblasts directly associated with a dentin-like

These findings indicated that human SHED satisfie satisfiess one important stem-cell attribute: the ability to differentiate into odontoblasts   in vivo. However, SHED were unable to regenerate a complete dentin– dentin–pulp-like complex as do DPSCs  in vivo  (Fig. 3 recipient  A and  E ). In addition, SHED were capable of inducing recipient murine cells to differentiate into bone-forming cells as noted by murine-specific pf1   in situ   hybridization (Fig. 3 L) and lacked DSPP expression (Fig. 3 D). Importantly, skin fibroblasts were never capable of inducing bone formation after   in vivo   transplantation (data not shown). These data indicated that SHED are distinctively different from DPSCs in respect to the odontogenic differentiation and osteogenic induction. We next examined the characteristics of clonal cell strains, each originating from a single cell of deciduous pulp. When 12 single-colony-derived SHED clones were transplanted into immunocompromised mice, only one fourth (3 of 12) of the clones demonstrated a potential to generate ectopic dentin-like tissue on the HA TCP carrier equivalent to that generated by multicolony-de colon y-derive rived d SHED (Fig (Fig.. 3 E and G). SHE SHED D fromeith fromeither er si singl nglee or multiple colonies were found to form dentin-like tissue (Fig. 3 F ) and to survive in the fibrous tissue within the transplants

Miura  et al.

 

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Fig.3.   Tran Transpla splantedSHED ntedSHED intoimmuno intoimmunocomp compromi romisedmice. sedmice. ( A and B) After8 week weekss of tran transplan splantatio tation, n, SHEDwere ableto diffe differenti rentiateinto ateinto odon odontobla toblasts(open sts(open arrows) that were responsible for the dentin-like structure (D) formation on the surfaces of HA ( A). The same fi same  field eld is shown for human-specifi human-speci fic alu  in situ hybridization, indicating indicating the human origin of odontoblasts (open arrows, B). The black dashed line represents interface between newly formed dentin (D) and HATCP(HA).(C ) Immu Immunohis nohistoch tochemica emicall stai stainingof ningof antianti-DSPPantibo DSPPantibody dy show showss a posi positivestainin tivestaining g on theregener theregenerateddentin(blackarrows) ateddentin(blackarrows).. (D) Incont Incontras rastt toDPSC E ) Of 12 sele tran transplan splants,newly ts,newly generatedbone(B) ratedbone(B) by hostcells in thesame SHEDtransplantshows SHEDtranspl no reac reactivit tivity y to theDSPP dantib antibody.( ody.( ctedsingle-colon y-derived rived SHED SHE D strain str ains, s, onl only y 3gene (25%) (25 %) were we re cap capabl able e of genera gen eratin ting g den dentin tin in vivo viv o. New Newly lyantshows formeddent for meddentin in (ar (arrow rows) s) wasfoun wasfound to be adjace adj acent nt to selectedsinglethesurfacesof thesurfa cesof colony-de HATCP carrier carr ier situ u hybri (HA)and asso associate ciated d withconnecti withconnective ve tissu tissue e (CT) (CT).. (F ) Huma Human-sp n-speci ecifi fic al alu u in sit hybridizat dization ion show showed ed that huma human n cells(open arrow arrows) s) were asso associate ciated d withdentin formatio form ation n (D) andwere resid residingwithin ingwithin theconnecti theconnective-ti ve-tissuecompar ssuecompartmen tmentt (CT).( G) Theremaini Theremaining ng 75%(9 of 12)single-c 12)single-colon olony-der y-derivedSHED ivedSHED strai strains ns wereunable to generate dentin  in vivo. (H)  In situ  hybridization demonstrated that alu-positive human cells survived in the connective-tissue compartment (CT) in the transplants in which there was no odontogenesis. Human cells were also found to surround the blood vessels (arrows). ( I ) Seven of 12 (58.4%) single-colonyderived SHED lines induced a very limited amount of bone formation (B) on the surface of HA TCP (HA). ( J ) Of 12 single-colony-derived SHED lines, 5 (41.6%) were able to induce a signifi signi ficant amount of bone formation (B) on the surfaces of HA TCP (HA). (K ) The alu  in situ  hybridization showed human cells (arrows) attached to the surfaces of HATCP (HA) at the initial site of bone formation (B). The black dashed lines represent the interface between newly formed bone (B) and HATCP (HA). (L)  In situ  hybridization studies showed the murine-speci murine-specifi fic pf1 DNA probe reacting with osteoblasts and osteocytes (arrows) associated with the new bone formation (B).

(Fig. 3 H ) as demonstrated by human-specific alu  in situ  hybridization. These results infer that SHED may contain subpopulations of cells, either differentiating differentiating into odontoblast odontoblastss or residi residing ng in the connective-tissue compartments. Surprisingly, all transplanted plan ted sing singlele-colon colony-de y-deriv rived ed SHED clon clones es were capa capable ble of  inducing indu cing bone forma formation tion in immu immunocomp nocompromi romised sed mic mice. e. Appr Approxoximate im ately ly 40% of the clonal clonal cell strains strains (5 of 12) induce induced d a significant amount of new bone, whereas the remaining 60% (7 of 12) induced a limited amount of bone (Fig. 3  I  and  and  J ). ). SHED  were found to be located on the sur faces of H A TCP but did not participate participa te in bone formation as indicate indicated d by human-spec human-specific ific alu in si situ tu hybridization (Fig. 3 K ). ). In contrast, murine host cells were found to differentiate into osteoblasts and osteocytes as shown by reac reactivity tivity to muri murine-s ne-speci pecific fic pf1 in si hybridiza dization tion (Fig (Fig.. 3 L). situ tu hybri Next we studied the potential of SHED to develop into other cell lineages such as neural cells and adipocytes. To elucidate the neuralneur al-diffe different rentiati iation on pote potentia ntiall of SHED SHED,, we exa examine mined d the expression of neural markers in SHED. We found that cultured SHED expressed expressed a var variety iety of neur neural al cell markers markers incl includin udingg nestin,   III-tubuli III-tubulin, n, GAD, NeuN, GFAP, NFM, and CNPase as measured by immunocytochemical staining (Fig. 4   A– H ) and Western blot analysis (Fig. 4 I ). ). After 4 weeks of neural inductive culture, expression levels of neuronal markers including   IIItubulin, GAD, and NeuN were increased, whereas the levels of  nesti ne stin, n, GFAP GFAP,, NF NFM, M, and CN CNPas Pasee rem remai ained ned unc unchan hanged ged (Fi (Fig. g. 4 I ). ). When cultured under these conditions, SHED lost their fibroblastic morphology and developed multicytoplasmic processes 5810      www.pnas.orgcgidoi10.1073 pnas.0937635100

correlating with either   III-tubulin GAD or   III-tubulin NFM expression (Fig. 4   J –O). The long cellular processes could be  viewed best after toluidine blue stain staining ing and were immunoreactiv ac tivee to MAP2 and Ta Tau u ant antibo ibodi dies es (Fi (Fig. g. 4 P –S). Aft After er the neu neural ral inductive culture, SHED continued to express glial cell makers such as nestin, nestin, CNPa CNPase, se, GFAP, and NFM (Fig. 4  T –W ). ). Neural developmental developmental potential was studied further in vivo  by injecting SHED into the dentate gyrus of the hippocampus of  immunocompromised mice (Fig. 5 A). Histologic Histological al examinat examination ion showe sho wed d tha thatt SHE SHED D survi survive ved d for 10 day dayss in insid sidee the mou mouse se bra brain in microenvir micr oenvironme onment nt as note noted d by huma human-sp n-specif ecific ic anti antimito mitochonchondrial antibody staining and c ontinued to express neural markers such as NFM (Fig. 5 B).  After 5 weeks of culture cult ure with wit h an adipogenic adipog enic inductive indu ctive mixture, 5% of cultured SHED were found to possess the potential to develop into Oil red O-positive lipid-laden fat cells (Fig. 6 A). This correlated with an up-regulation in the expression of two adipocyte-specific adipocyte-specif ic transcripts transcripts,, peroxisome proliferat proliferator-activa or-activated ted receptor-  2 and lipoprotein lipase, as detected by semiquantitative RT-PCR (Fig. 6 B). Discussion Here we provide evidence that remnant dental pulp derived from exfo exfolia liated ted deci deciduous duous teet teeth h contain containss a mult multipot ipotent ent stem stem-ce -cell ll population. These stem cells can be isolated and expanded   ex  vivo, thereby providing a unique and accessible population of  stem cells from an unexpected tissue resource. Previous experMiura  et al.

 

      Y       G       O       L       O       I       B       L       L       E       C

Neural al diffe differenti rentiationof ationof SHED SHED.. ( A–H) Immu Immunocy nocytoch tochemica emicall stai staining ning depi depicts cts SHEDexpressi SHEDexpressing ng nest nestin, in, GFAP GFAP,, NFM, CNPa CNPase, se, III-t III-tubul ubulin, in, GAD, and NeuN NeuN.. Fig. 4.   Neur Fig. (I ) Western blot analysis confi confirmed that SHED expressed neural markers as described above. After 4 weeks of culture in the presence of B27 supplement, bFGF (40 ngml), and epidermal growth factor (20 ngml) (Neural Diff. ), expression levels of III-tubulin, GAD, and NeuN were up-regulated when compared with regular culture conditions as described in  Materials and Methods  (Neural Diff. ). However, expression levels of nestin, GFAP, CNPase, and NFM remained the same after the treatment. ( J –O) SHED may coexpress neuronal markers including   III-tubulin (green)GAD (red) and   III-tubulin (green)NFM (red). The morpholo morp hology gy of SHEDshowed elon elongate gated d cellcell-cytop cytoplasmi lasmicc proce processesthat ssesthat some sometime timess coex coexpressneural pressneural mark markers ers (tria (triangle ngles) s) or onlyexpress indi individu vidual al neur neural al mark marker er (open arrows). (P –S ) Toluidine blue (0.1%) staining depicting the altered morphology of SHED after induction with neural culture medium ( P  and  and  Q , arrows). Immunopositive staining for anti-MAP2 and anti-Tau antibodies on dendrites and axon ( R and  S , arrows), respectively. Double-staining Double-staining experiments showing III-tubulin-positive cells were also detected in the same  fi  field eld (R, triangle, green). (T –W ) SHED continued to express glial cell markers including nestin (red), CNPase (red), GFAP (red), and NFM (green) by immunocytostaining.

iments have shown that dental pulp tissue of adult teeth contains a population of DPSCs that are capable of differentiating into odontoblasts and adipocytes as well as expressing nestin and GFAP and form a dentinpulp-lik pulp-likee c omplex after  in vivo  transplantation (23). Deciduous teeth are significantly different from

sphere-like cell-c sphere-like cell-cluster luster for mation, osteoinductive capacity   in failure to re recons constit titute ute a den denti tin n–pulp-lik pulp-likee complex.  vivo, and failure SHED apparently represent a population of multipotent stem cells that are perhaps more immature than previously examined postnatal stromal stem-cell populations.

permanent teeth with regards to their developmental processes, processes, tissue structure, and function. Therefore, it is not a surprise to find that SHED are distinct from DPSCs with respect to their higher proliferation rate, increased cell-population doublings,

The mechanisms controlling the growth and replacement of  teeth are largely unknown (17), in particular with respect to how craniofac crani ofacial ial compon components ents including including bone and soft tiss tissues ues surrounding teeth participate in the process of tooth development.

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Fig. 6.   Adipogenic differentiation differentiation of SHED. ( A  A) Cultured SHED formed Oil red O-positive lipid clusters after 5 weeks of induction in the presence of 0.5 mM isobutylmethylxanthine, 0.5  M hydrocortisone, and 60  M indomethacin. (B) A signifi significant up-regulation of peroxisome proliferator-activated receptor- 2 (PPAR 2) 2) and lipoprotein lipase (LPL) was observed in the group induced with the adipogenic mixture (Adip) as compared with the control group (Cont) by RT-PCR. Fig. 5.   Trans Transplant plantation ation of SHED SHED into the brain. ( A) Diagram indicating injection of SHED into the dentate gyrus of the hippocampus. ( B) SHED were cultured in the neural-differentiation medium as described in  Materials and  Methods for1 we week,afterwhic ek,afterwhich h 5,0 5,000 00 cel cells ls in 0.5 l of PBSwereinjec PBSwereinjectedinto tedinto thedentategyrusof thehippoca thehippocampusof mpusof immu immunoco nocompro mpromise mised d mice mice.. Afte Afterr 10 days, day s, thebrai thebrain n was wasfi fixed and prep preparedfor aredfor immu immuno nofl fluore uorescen scence ce stain staining ing with NFM and human-specifi human-specific anti-mitochondrial antibody. The anti-mitochondrial antibody immunostaining showed human SHED (arrows, green) in the dentat den tate e gyr gyrus us of thehipp thehippoca ocampu mpuss wit with h coe coexpr xpress essionof ionof NFM(arro NFM(arrows,red) ws,red).. In merged images, coexpression of human mitochondria and NFM showed colocalization of antigen expression as indicated by yellow  fl  fluorescence uorescence (arrows). (Magnifi (Magnification, 20.)

SHED D dem demons onstra trated ted a str strong ong cap capaci acity ty to ind induce uce rec recipi ipient ent SHE cell-mediated bone formation  in vivo. According to our investigations, SHED could not differentiate directly directly into osteoblasts but did ind induce uce new bon bonee for format mation ion by form forming ing an ost osteoi eoindu nducti ctive ve template to recruit murine host osteogenic cells. These data imply im ply tha thatt dec decid iduou uouss tee teeth th ma mayy not onl onlyy pro provideguida videguidancefor ncefor the eruption of permanent teeth, as generall generallyy assumed, but may also be involved in inducing bone formation during the eruption of  permanent teeth. It is notable that SHED expressed neuronal and glial cell markers, which may be related to the neural crest-cell origin of  the dental pulp (25). Neural crest cells play a pivotal role in

embryonicc dev embryoni devel elopm opment ent,, givi giving ng ris risee to a var varie iety ty of ce cell ll type typess suc such h as neural cells, pigment cells, smooth muscle, craniofacial cartilage til age,, and bon bonee (26 (26). ). Pre Previou viouss stu studie diess dem demons onstra trated ted tha thatt BMSSCs were also capable of differentiating into neural-like cells after   in vivo   transplantation (27). Dental pulp cells are known to produce neurotrophic factors and even rescue motoneurons after spinal cord injury (28). Moreover, neural progenitors genit ors were identified identified recently recently in mamm mammali alian an derma dermall skin layers (6). These evidences support the notion that stem cells of  nonneural tissue may be capable of differentiating into neural cells. Our study provides evidence that SHED represent a population tio n of pos postna tnatal tal ste stem m cel cells ls cap capab able le of ext extens ensiv ivee pro proli life ferat ratio ion n and multipot mult ipotenti ential al differ differenti entiatio ation. n. Deci Deciduous duous teet teeth h ther therefor eforee may be an ideal resource of stem cells to repair damaged tooth structures, induce bone regeneration, and possibly to treat neural tissue injury or degenerative diseases. However, the biological signif si gnific icanc ancee of the exis existen tence ce of SHE SHED D re remai mains ns to be det determ ermine ined. d. This study provi provides des a des descrip cription tion of a stem stem-ce -cell ll popul populati ation on residing in exfoliated human deciduous teeth and establishes the foundation for further studies to determine the efficacy of using SHED in cellular-based therapies. This work was supp This supporte orted d by the Division Division of Int Intramu ramural ral Research, Research, National Nation al Institute of Dental and Craniofacial Research of the National Institutes of Health, and Department of Health and Human Service.

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