EMBRYONIC STEM CELLS/I NDUCED PLURIPOTENT STEM CELLS

A Small Synthetic Cripto Blocking Peptide Improves NeuralInduction,Dopaminergic Differentiation,and FunctionalIntegration of Mouse Embryonic Stem Cells in a Rat Modelof Parkinson’s DiseaseENZA LONARDO,a CLARE L. PARISH ,b,c,dSALVATORE PONTICELLI ,a DANIELA MARASCO,e DIOGO RIBEIRO ,bMENOTTI RUVO,f SANDRO DE FALCO ,a ERNEST ARENAS,b GABRIELLA M INCHIOTTI a

aStem Cell Fate Laboratory,Institute of Genetics and Biophysics,Naples,Italy,bLaboratory of MolecularNeurobiology,Department of Medical Biochemistry and Biophysics,Karolinska Institute,Stockholm,Sweden,cFlorey Neuroscience Institutes anddCentre for Neurosciences,The University of Melbourne,Parkville,Australia,eDepartment of Biological Science,University of Naples,Napoli,Italy,fInstitute of Biostructure and Bioimaging,CNR, Naples,Italy

Key Words.Embryonic stem cells• Neural differentiation• Cell transplantation• Cellular therapy• Parkinson’s disease

ABSTRACTCripto is a glycosylphosphatidylinositol-anchored corecep-tor that binds Nodal and the activin type I (ALK)-4 recep-tor, and is involved in cardiac differentiation ofmouseembryonic stem cells (mESCs).Interestingly,genetic abla-tion of cripto results in increased neuralization and mid-brain dopaminergic(DA) differentiation ofmESCs, aswellas improved DA cellreplacement therapy (CRT) in amodel of Parkinson’s disease (PD).In this study,we devel-oped a Cripto specific blocking toolthat would mimic thedeletion of cripto,but could be easily applied to embryonicstem cell(ESC) lines without the need of genetic manipu-lation.We thus screened a combinatorialpeptide libraryand identified a tetramerictripeptide,Cripto blocking

peptide (BP), which prevents Cripto/ALK-4 receptor inter-action and interfereswith Cripto signaling.Cripto BPtreatmentfavored neuroectoderm formationand pro-moted midbrain DA neuron differentiation ofmESCs invitro and in vivo.Remarkably,Cripto BP-treated ESCs,when transplanted into the striatum of PD rats,enhancedfunctionalrecovery and reduced tumor formation,mim-icking the effect of genetic ablation of cripto.We thereforesuggestthat specific blockers such as Cripto BP may beused to improvethe differentiation ofESC-derived DAneurons in vitro and their engraftmentin vivo, bringingus closer towards an application ofESCs in CRT. STEMCELLS 2010;28:1326–1337

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

Cripto is a glycosylphosphatidylinositol(GPI)-anchored core-ceptor thatbelongs to the EpidermalGrowth Factor-Cripto-1/FRL-1/Cryptic (EGF-CFC) protein family and plays importantroles during embryonic development,stem cellfunction,andcancerprogression [1,2]. Cripto bindsto ligandsof trans-forming growth factor beta (TGF-b) family such as Nodal and

Nodal-related growth differentiation factor-1 or -3,and inter-acts with the activin type I (ALK-4) receptor,thereby activat-ing a complex formed with Activin type IIB serine/threoninekinase receptor [3–5].Upon receptor activation,the intracellu-lar effectorsSmad2/Smad3 become phosphorylated and to-getherwith Smad4 translocate into the nucleusto regulategene expression [6].Cripto is thus a modulator of TGF-b sig-naling thatinhibits activin signaling [7,8] and the antiproli-ferativeeffectsof TGFb [9–11].Consistentwith that,we

Authors contributions:E.L.: conception and design,provision of study material,collection and/or assembly of data,data analysis andinterpretation,manuscriptwriting;C.L.P.:conception and design,provision of study material,collection and/or assembly of data,dataanalysisand interpretation,manuscriptwriting;S.P.: collection and/orassembly ofdata,dataanalysisand interpretation;D.M.:collection and/orassembly ofdata,data analysis and interpretation;D.R.: collection and/orassembly ofdata;M.R.: conception anddesign,collection and/or assembly of data,provision of study material,data analysis and interpretation;S.D.F.:conception and design,collection and/or assembly of data,data analysis and interpretation;E.A.: conception and design,financialsupport,provision of studymaterial,dataanalysisand interpretation,manuscriptwriting;G.M.: conception and design,financialsupport,provision ofstudymaterial,data analysis and interpretation,manuscriptwriting.E.L, C.L.P. equally contributed as firstauthor.E.A and G.M.equallycontributed as last author.Correspondence:Gabriella Minchiotti,Ph.D.,Stem CellFate Laboratory,Institute of Genetics and Biophysics,‘‘A. Buzzati-Traverso’’,CNR, Via Pietro Castellino 111,80,131 Naples,Italy.Telephone:39-081-6132357;Fax:39-081-6132595;e-mail:minchiotti@igb.cnr.itor ErnestArenas,MD., Ph.D.,Laboratory ofMolecularNeurobiology MBB,DBRM, KarolinskaInstitute,Scheelevag 1,17,177Stockholm,Sweden.Telephone:46-8-5248-7663;Fax: 46-8-34-1960;e-mail:Ernest.Arenas@ki.se;Received December22, 2009;accepted for publication May 27,2010; first published online in STEM CELLS EXPRESSJuly 16,2010.VC AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.458STEM CELLS 2010;28:1326–1337 www.StemCells.com

previously showed thatCripto acts through the Nodal/ALK-4pathway to negatively regulate neuraldifferentiation and per-mit the entry of embryonic stem cells (ESCs) into cardiac lin-eage [12].In support of these findings,disruption of cripto inmouse embryonic stem cells (mESCs) enhanced neurogenesisand midbrain dopaminergic (DA) differentiation in vitro [13,14].These resultssuggested thatCripto blocking could beused as a toolto improve the efficiency of neuraldifferentia-tion protocols for cell replacement therapy (CRT) in neurode-generative disorders such as Parkinson’s disease (PD) [15].

PD has been identified as a priority target for CRT becausethe classical signs of the disease (tremor,rigidity,and hypoki-nesia) are caused by the progressive degeneration of a circum-scribed cellpopulation,the substantia nigra DA neurons.Theidea ofusing stem cellsfor CRT in PD patientshasbeenfueled by the finding thathuman ventralmidbrain fetaltissuegrafts,underspecific conditions,improve the symptoms andreduce the need of levodopa in these patients [16, 17]. Pluripo-tent stem cells have become an attractive cell source for CRTbecause they can be easily expanded and they can give raiseany cell type in an organism. Thus, much work has focused onthe development of methods for generating midbrain DA neu-rons from ESCs [18,19].However,a major remaining chal-lenge in the field is the need to improve the functional outcomeand the safety of ESC grafting [20, 21]. This has lead to the de-velopment of cell sorting strategies to eliminate unwanted cellsand/or selectthe desired celltypes [22,23],thus reducing oreliminating uncontrolled growth of ESCs and tumor formation[24–27].Other strategies have focused on reducing the pres-ence ofpluripotentcells and proliferating neuralprogenitorsby gaining furthercontrolovercriticaldevelopmentalproc-esses such as neural induction and neuronal differentiation.Inparticular,inhibition ofbone morphogenetic protein (BMP)signaling with Noggin [28,29],or a broaderinhibition ofLefty, activin and TGFb signaling,with Noggin andSB431542, have been reported to reduce the presence of pluri-potent cells and increase neural differentiation in ESC prepara-tions [28–30].Interestingly,transplantation of Noggin-treatedhumanEmbryonicStem (hES)-derivedDA cells lead toengraftmentof THþ cells and behavioralimprovementsinhemiparkinsonian rats,butthey also lead to tumor/outgrowthformation [28,29].Previous results from us and others haveshown that deletion of cripto in mESCs, increases DA differen-tiation [13, 14].Although transplantation of cripto/ mESCs-derived embryoid body (EBs) cells athigh density lead to tu-morformation [14],when grafted atlow density,they abol-ished tumor/outgrowth formation by ESCs in vivo,and pro-moted functionalrecovery in parkinsonian rats[13]. Thisfinding suggested thatstrategies aiming atachieving efficientCripto blocking,otherthan geneticmodification,could bebroadly used to improve the DA differentiation ofdifferentpluripotentcells.We therefore endeavored to develop a com-pound capable of inhibiting Cripto signaling in ESCs, with thegoalof using such a toolto enhance DA differentiation andimprove functionalrecovery aftertransplantation in animalmodels of PD.

For this purpose,we screened a random combinatorial tet-rameric tripeptide library builtwith non-naturalamino acidson a multimeric scaffold,which ensured enhanced recognitioncapabilitiesderiving from the large recognition surface andthe high flexibility of the finalcompounds [31].Peptide mix-turescomposing the library were utilized ascompetitorsofCripto/ALK-4 interaction in an enzyme-linked immunosorbentassay (ELISA)-based competition assay,following an iterativeprocess.Selected peptides were evaluated fortheirability toinhibitCripto-induced Smad2 phosphorylation in ESCsandpromote robustneuronaldifferentiation.These assays allowed

us to identify a tetrameric tripeptide,named Cripto blockingpeptide (Cripto BP).The action/activity ofthis peptide wasfurther tested on DA differentiation of mESCs in vitro.More-over,Cripto BP-treated mESCs were transplanted in parkinso-nian rats to examine their ability to promote functional recov-ery and reduce tissue overgrowth or tumor formation.

MATERIALS AND METHODS

Iterative Deconvolution of the TetramericTripeptide LibraryThe screening was performed by an ELISA assay (supportinginformation text). In the first step of the deconvolution process,the peptide pools were tested with a molar excess of 30,000-fold (calculated foreach single peptide)overCripto (8.6 10 10M; Fig. S1). Once identified an active pool(s), a dose-de-pendentinhibition using 200-,500-,1,000-,5,000-,12,500-and 30,000-fold excess was performed to confirm the inhibi-tory property (Fig.S2).The first active pool selected was thatlabeled as 4-23-X (X indicated random positions), where ‘‘23’’identified theamino acidL-cysteine(S-benzyl)[L-Cys(Bzl)](Fig. S2).This peptide poolwas resynthesized as 30 singlepeptides and submitted to the second screening using a molarexcess of 1,000-fold (calculated for each single peptide) overCripto;the screening allowed the identification of the peptide4-23-23 as the best performing in our test (Fig. S3).To finallyinvestigate the N-terminalposition,whereD-Glu was initiallyarbitrarily placed,30 single peptides labeled as B-23-23 weresynthesized and submitted to the final screening (Figs. S4, S5).The peptide indicated as 26-23-23,where ‘‘26’’ identified theamino acidL-methionine-sulfoxide [L-Met(O)],was the mole-cule showing the optimalinhibitory activity.The activity ofthis peptide was further confirmed using the high-performanceliquid chromatography (HPLC) purified molecule.

ESC Culture and DifferentiationMouse embryonic stem (ES)cell lines,both wild-type (R1[32]) and cripto/ [33],were used throughoutthe study andwere cultured as previously described [34].For in vitro car-diac differentiation as shown in Figure 3,ESCs were aggre-gated as EBs in 15% fetalbovine serum (FBS) as previouslydescribed [12,34].For the dopaminergic differentiation (Fig.4A), ESCs were seeded atlow density onto a stromalfeederlayerfor 14 days in the presence ofsequentialmorphogensand mitogens (sonic hedgehog [Shh],fibroblastgrowth factor2 [FGF2],FGF-8) as well as peptides (control peptide [CP] orCripto BP).See supporting information text for details.

Western Blotting and Smad2 Phosphorylation AssayTwo-day-oldcripto / mESC-derivedEBs were serumstarved for3 hours in Dulbecco’s modified Eagle’s medium(GIBCO, Invitrogen,Carlsbad,CA, http://www.invitrogen.com/site/us/en/home/brands/Gibco.html?CID=fl-gibco) withoutleukemiainhibitory factor(LIF) and in low serum (0.5%FBS). Followingstarvation,EBs were incubatedfor 20minutesat 37C with recombinantmouseCripto protein([12];0.5 lg/ml) either alone or in the presence of the indi-catedamountof peptides.Anti-Smad2and anti-phospho-Smad2 (Ser465/467)Antibodies (CellSignaling Technology,Denvers,MA, http://www.cellsignal.com/) were used,follow-ing the manufacturer’s instructions.

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RNA Preparation and Quantitative ReverseTranscription Polymerase Chain ReactionTotal RNAs was extracted with TRIzol kit (Life TechnologiesInc.,Carlsbad,CA, http://www.lifetechnologies.com/about-life-technologies/contact-us.html)accordingto manufacturer’sinstructions and quantitative polymerase chain reaction (QPCR)wasperformed using SYBR Green PCR mastermix (Euro-Clone,Milano,IT, http://www.euroclonegroup.it/),according tomanufacturer’s instructions.Primers are described in supportinginformation Table 2.

Determination of In Vitro Dopamine andDihydroxphenylacetic Acid LevelsDopamine release and turnover was determined in vitro usingreverse phase HPLC.DA turnover was expressed as the con-centrationof dihydroxphenylaceticacid (DOPAC) to DA[DOPAC/DA].Analysis was performed on triplicate wells onthree independent differentiation experiments.

6OHDA Lesioning and Transplantation of ESCsAll experiments were performed according to the guidelinesof the European Community and were approved by the localethicalcommittee.Sprague-Dawley rats(250–350 g;fromARC, Australia)were housed and treated according to theguidelines of the Australian and Howard Florey Institute ani-mal ethics committee (07-030).

Thirty-seven animals received unilateralstereotaxic injec-tions of 6-Hydroxydopamine (OHD) into the substantia nigrapars compacta (SNpc) to create a complete lesion of the mid-brain DA neurons,as previously described [13].Lesioned ani-mals were selected for transplantation based on their responseto amphetamine-inducedrotationalbehavioras previouslydescribed [13].Animals making greater than six rotations/mi-nute (i.e., lesions greater than 80%) were selected for grafting.Two days prior to grafting animalsbegan cyclosporine-Aimmune suppression (10 mg/kg).Fourteen days postlesioningselected animals (based on behavioralrotations)were againanesthetized and stereotaxically injected with 1 ll of cell sus-pension ateach ofthe foursites.Stereotaxic coordinates offirstand second injection (from Bregma):anterior0.2 mm,lateral3.0 mm,ventral4.5 and 5.0 mm;third and fourthinjection,anterior 1.0 mm,lateral 2.5 mm,ventral 4.5 and 5.0mm; incisor bar 0 mm [35]).Wild-type mESCs were differen-tiated as outlined in Fig.S6a,including eitherCP or CriptoBP; media was also supplemented with Noggin (300 ng/ml,day 0–8,to enhance neuronalinduction)as well as Wnt5a(200 ng/ml,day 8–11),to promote DA neuron differentiation.Following in vitro differentiation,ESC coloniesweredis-sected away from the stromalfeederlayerand dissociatedusing collagenase/dispase with agitation (700 lg/ml,Roche,for 20 minutes,80 rpm).Cells were subsequently resuspendedata density of 3,000 cells/llin preparation for grafting.Fiverats received Sham injections (4 llN2 media,1 ll/site),16rats received a total of 12,000 mESCs-derived cells (4 1 llgrafts)pretreated with the CP and a further 16 rats receivedgrafts pretreated with the Cripto BP.

Animals were tested for functionalimprovements at2, 4,6, and 8 weeks post-transplantation by amphetamine-inducedrotationalbehavior.Eightweeks postgrafting,rats were killedby an overdose of sodium pentobarbital(100 mg/kg i.p.) andintracardially perfused aspreviously described [13].Brainswere removed,postfixed for 1 hour,and then left overnight at4 C in 20% sucrose in phosphate-buffered saline (PBS).Thefollowing day,20-lm-thick coronalsections were cutseriallythrough the striatum (12 series) and 50 lm through the SNpcand mounted directly onto chrom alum gelatinized slides.

Histological AnalysisImmunofluorescence analysiswere performed aspreviouslydescribed [12,13]. The following primary antibodieswereused:mouse anti-bIII-tubulin (Tuj1,1:1,000,Promega,Madi-son, WI, http://www.promega.com);rabbit anti-tyrosinehydroxylase(TH; 1:250, PelFreez,Rogers, AR, http://www.pelfreez-bio.com/);sheepanti-TH (1:250, PelFreez,Rogers,AR, http://www.pelfreez-bio.com/);Nurr-1 (1:500,SantaCruz, CA, USA, http://www.scbt.com/);rabbitanti-Pitx3 (1:200,a gift from P.Burbach,Rudolf Magnus Instituteof Neuroscience,Utrecht),rabbitanti-GIRK-2 (1:100,Chemi-con,Temecula,CA, http://www.millipore.com/company/cp1/redirect-ab),rabbitanti-GABA (1:500,Sigma-Aldrich,St.Louis, MO, http://www.sigmaaldrich.com/customer-service.html);rabbitanti-5HT (1:200,BiogenesisLtd, Dorset,UK,www.biogenesis.co.uk/);rabbitanti-NET (1:20,Chemicon;rabbitanti-cytokeratin (1:4,000,DAKO, Glostrup,DK, http://www.dako.com/);ratanti-CD29 (1:200,BD Pharmingen,SanJose, CA, http://www.bdbiosciences.com/),rabbitanti-Ki67(Neomarkers Thermofisher,CA, USA,http://www.thermofisher.com/global/en/about/home.asp),mouse anti-nestin(1:200,Chemicon,Temecula,CA, http://www.millipore.com/company/cp1/redirect-ab);mouseanti-Oct4 (1:100,SantaCruz, SantaCruz,CA, USA, http://www.scbt.com/),rabbitanti-glialfibril-lary acidicprotein,(GFAP, 1:200,DAKO, Glostrup,DK,http://www.dako.com/).Appropriatefluorophoreconjugatedsecondary antibodies: (Cyanin-2,Cy3,and Cy5,1:200,JacksonImmunoResearch,Suffolk, UK, http://www.jireurope.com/home/contact.asp)were used forvisualization.Labeling wasvisualized by light or fluorescent illumination using an invertedmicroscope(DMIRB, Leica Microsystems,Wetzelar,DE,http://www.leica-microsystems.com)or upright microscope(Leica DMLB2).Images were acquired on a DC 350 FX cam-era (Leica).

The grading analysis (Fig.3) was performed blinded bytwo investigators;to geta reliable unbiased analysis,all theEBs formed in suspension culture were individually plated in48 multiwellplates and used for morphological (Fig.3A, 3B)and immunofluorescence analysis (Fig.3C, 3D).The propor-tion ofneuron containing colonies (labeled using the imma-ture neuronalmarker bIII-tubulin,Tuj1) and TH neuron con-taining colonies were counted and expressed as a percentageof totalcolonies (Fig.4). The totalnumberof dopaminergicneurons/lm2 colony (identified using a marker for the precur-sor enzyme in DA synthesis,TH) were also counted (Fig.4).Experiments were performed in triplicate.

Semiquantitativescoring wasemployed to estimatethecell composition of the tumors,as previously described [14].Tumorswerestainedand scoredby blindedinvestigatorsusing the following scales:0 ¼ no cells (0%);1 ¼ few cells(<5%);2 ¼ moderate cells (5–20%);4 ¼ dominantcelltype(>50%).One seriesof slidescontaining grafted striata wascounterstained using 1% Neutralred to visualize the graft/tu-morsize and the gross morphology ofthe striatum.Area ofgraftsand tumorswas calculated using Stereo Investigatorsoftware (MicroBrightField, VT).

Statistical AnalysisValuesare expressed asmeansSEM or SD. The statisticalsignificance ofdifference wasdetermined by ANOVA fol-lowed by Tukey’s posthoc testor a Student’s t-test.Rankingof in vitro differentiation (Fig.3C) wasperformed using aWilcoxon rank sum test.StatisticalPackage for SocialScien-cessoftware package,version 11.0 SPSS,Chicago,IL andSigma Stat 3.0 software were used for statistical analysis withsignificance set at p < .05.

1328 Small Molecules for Stem Cell Therapies

RESULTS

Identification of a Novel Peptide that AntagonizesCripto/ALK-4 Receptor InteractionTo identify Cripto/ALK-4 antagonistscapable ofdisruptingdownstream signaling,we screened atetramerictripeptidelibrary synthesizedusing 29 non-naturalaminoacids toincrease peptide stability,plus Glycine,(supporting informa-tion Table 1).To increase the molecularsurface,the librarywas assembled as previously described [31] on a peptide scaf-fold composed ofa poly-lysine core [36].To facilitate thecharacterization of the peptide mixtures,and at the same timeto expedite the screening procedure,the first residue was arbi-trarily fixed.A negatively charged amino acid,theD-glutamicacid (D-Glu, denoted 4; Fig. 1A and supporting information Ta-ble 1),was thus initially chosen as the common,N-terminalresidue,to favor peptidesolubilization.The library wasarranged in 30 pools,each of them composed of 30 tetramerictripeptides, which differ for the amino acid (denoted B) on thesecond position (Fig.1A),while the residue in the third posi-tion (denoted X) was randomly added. The total complexity ofthe library was therefore of 302 ¼ 900 different peptides.Thescreening wascarried outby an ELISA-based competitionassay whereby the displacement of soluble recombinant Cripto

from coated ALK-4 by the peptide pools was evaluated.Theiterative procedure consisted of three screening rounds,whichled to the selection of the 26-23-23 tetramer formed byL-me-thionine-sulfoxide [L-Met(O)](denoted as 26)in position 1,and S-benzylatedL-cysteine [Cys(Bzl)] (denoted as 23) in posi-tions 2 and 3 of the tripeptide (Fig.1A), (for details see sup-porting information Textand Figs.1–4).The selected tetra-meric tripeptide (26-23-23,supporting information Fig.5) wasthereafter named Cripto BP.This peptide blocked the interac-tion of Cripto and ALK-4 with an estimated IC50 of 400 nM,while the control tetrameric Peptide (CP),D-Ser-Ala-Cha,anda dimeric peptide (DP) variant of the 26-23-23 peptide had noeffect (Fig.1B).We thus performed ELISA assays to evaluatewhetherCripto BP-or CP-coated to microtiterplates couldbind recombinantCripto and/orALK-4 receptor.Cripto butnot ALK-4 specifically recognized the immobilized Cripto BP,in a dose-dependentmanner(Fig. 1C). NeitherCripto norALK-4 recognized the CP, confirming the specificity of CriptoBP for Cripto binding.Cripto BP Antagonizes Cripto Signaling,ImpairingCardiomyogenesis,and Promoting Neurogenesisin mESCsGiven the neutralizing activity ofthe peptide,we firstaskedwhether Cripto BP was able to block Smad2-dependent Cripto

Figure 1. (A): Schematic representation of the iterative screening strategy.(1) NH2-D-Glu (D-glutamic acid,also denoted as ‘‘4’’,see supportinginformation Table 1) is the common N-terminal residue,(B) identifies the fixed residue,which denotes 1 of 30 amino acids (aa1-30,supporting in-formation Table 1) and X indicates random positions (4-B(1–30)-X). The totalcomplexity of the firstlibrary was therefore of 302 ¼ 900 differentpeptides. In the first screening round (1), 4.23.X identifies the first active pool, where ‘‘23’’ indicatesL-cysteine(S-benzyl) [L-Cys(Bzl)]. In the secondround of screening (2) the pool 4-23-X was re-synthesized in 30 single peptides (4-23-B(1–30)), and the residue ‘‘23’’ was identified in the third posi-tion (4-23-23).Finally,30 single peptides (B(1–30)-23-23) were submitted to the third screening round (3),which identified the residue ‘‘26,’’ where‘‘26’’ indicatesL-methionine-sulfoxide [L-Met(O)].The 26-23-23 peptide is also referred to as Cripto BP.(B) Dose-dependentinhibition of Cripto/ALK-4 interaction exerted by Cripto BP,CP (i.e., tetrameric tripeptideD-Ser-Ala-Cha) and dimeric peptide (DP; i.e., dimeric tripeptide 26-23-23) inELISA-based competition assays (n ¼ 3).(C): Dose-dependentinteraction of Cripto or ALK-4 to Cripto BP.The indicated peptides (BP and CP)were coated (50 lM) on the plates and increasing amounts of either Cripto or ALK-4 receptor were used in binding, ranging from 50 to 200 ng/ml.Cripto but not ALK-4 bound Cripto BP; whereas,both Cripto and ALK-4 failed to bind the CP. Mean 6 SD,*, p < .0005. Abbreviations: ALK-4,activin type I receptor; BP, blocking peptide; CP, control peptide; CRIPTO, Cripto; ELISA, enzyme-linked immunosorbent assay.

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signaling in mESCs.We thus evaluated whether Cripto BP wasable to prevent Cripto-induced Smad2 phosphorylation of serumstarved 2-day-old cripto/ EBs. Recombinantsoluble Criptoprotein stimulated Smad2 phosphorylation, as assessed by West-ern blot analysis ([12] and Fig.2); this effect was not modifiedby the presence ofincreasing amounts ofeitherCripto DP orCP (2.5 lM and 25 lM; Fig. 2). On the contrary,Cripto-induced Smad2 phosphorylation wasinhibited by Cripto BPand completely blocked at 25 lM (125 molar excess; Fig.2).We then assessed whether Cripto BP was able to antagonize theendogenous Cripto signaling and thus mimicking the effectofgenetic ablation of cripto in a mESC differentiation assay,thatis, blocking cardiomyogenesis and inducing neuronaldifferen-tiation,underthe well-established conditions,which promotecardiac differentiation ofmESCs [12,37].Two day-old wild-type EBs were treated with increasing amounts of either CP orCripto BP, ranging from 5.0–25 lM, and the percentage of EBscontaining beating areas was scored from day 8 to 12 of the dif-ferentiation (n ¼ 60 EBs/group; Fig.3B). Given that the pep-tideswere dissolved in dimethylsulfoxide (DMSO),controluntreated EBs were compared with DMSO-treated EBs.Inter-estingly,neitherDMSO,the tetrameric CP (Fig.3B),northeDP (data notshown)affected the percentage ofbeating EBs.Instead,treatmentof wild-type EBs with Cripto BP,induced adose-dependent inhibition of cardiomyogenesis, as shown by theprogressive reduction ofrhythmically contracting EBs(at 25lM, BP 3.7% 6 3.5 vs.CP 82.9% 6 11.17,**, p < .0001;Fig. 3B).

We previously showed thatcripto / ESCs spontaneouslydifferentiate into neurons during the time window when wild-type ESCs are primed forcardiomyogenesis by Cripto [12].Thus,if specific and effective,the Cripto BP should be ableto redirect ESC fate and to promote neural induction and neu-rogenesis in mESCs,even if they are cultivated in conditions(i.e.,EBs in 15% FBS) that favor cardiac differentiation [34].

To evaluate the peptide(s)activity,2-day-old wild-type EBswere treated with either CP,DP, or BP and both morphologi-cal and immunofluorescence analysis were performed on 13-day-old EBs(Fig. 3A). To betterdefine the activity ofthepeptides,we used anti-bIII tubulin antibodies to identify neu-rons in culture and arbitrarily defined four grades of neuronaldifferentiation (Fig.3C) ranging from the absence of neurons(Grade 0)to full neuronaldifferentiation (Grade 3),thatis,presenceof a densenetwork ofbIII-tubulin-positivecells.The presence of either few isolated neurons or smallareas ofbIII-tubulin positivecells defined intermediatephenotypes,grade 1 and grade 2,respectively (Fig.3C).Our data clearlyindicated that Cripto BP but not DP or CP treatment promotesrobustneuronaldifferentiation,characterized by a high per-centage of grade 3 (full neuronal differentiation, Fig.3C) phe-notype (BP 45.6% 6 5.2 vs.CP 6.9% 6 0.6,Mean 6 SD *,p .03; Fig. 3D).Accordingly,upon treatmentwith controlpeptides (CP or DP),the majority of the EBs scored showeda grade 0 (CP 75.9% 6 3.05 and DP 77.2% 6 7,vs. BP17.4% 6 1.15,Mean 6 SD,*, p .03; Fig. 3D).To supportthesefindings,we performedthe molecularanalysisandexamined theexpression profileof neuronaldifferentiationmarkersin the samecardiacdifferentiation conditions,byquantitativereversetranscription polymerasechain reaction(QRT-PCR).In line with ourprevious findings in cripto/

ESCs [12],expression of the pan neuronalmarker NFM wassignificantly induced in 13-day-old EBs upon treatmentwithCripto BP.Concomitantly,expression ofthe cardiac specificmarker myosin light chain-2 ventricular as well as the mesen-doderm marker alpha-fetoprotein were strongly reduced (Fig.3E). Thus,our resultssuggesta fate switch in ESCsfrommesoderm/cardiomyocytesto neurons.Accordingly,expres-sion of the panmesodermalmarkerBrachyury/T wasalsostrongly reduced in 2-day-old EBs upon treatment with CriptoBP (data notshown).Moreover,Cripto BP-treated wild-typeEBs also showed induction of markers typical of DA develop-mentand differentiation such as,Pitx3 [38],Shh [39],Wnt5a[40],TH (the rate limiting enzyme in DA synthesis) and do-pamine transporter (DAT; Fig.3E),in line with previous find-ingsin cripto / ESCs [12].Thus,our resultssuggestthatCripto BP can enhance the neural differentiation of ES cells.

Increased Dopaminergic Differentiation of ESCsTreated with Cripto BPWe nextexamined whether Cripto BP treatmentwas able tofurtherenhance DA differentiation ofmESC,using the stro-malcell-based protocoldescribed by Barberietal. [41],withminor modifications (Fig.4A).In brief,mESCs were plated atlow density onto stromalcells (PA6) for a period of 5 days,to promote neuralinduction.At day 5 the media was replen-ished and supplemented with Shh and FGF-8 (for ventral mid-brain patterning).From day 8 to 11,cultures where changedinto neuronal(N2) media in the presence of Shh and FGF-8as wellas basic FGF (to expand the poolof DA precursors).Finally (day 11–14),the cells were exposed to the survivalfactorsbrain-derivedneurotrophicfactor, glial cell line-derived neurotrophic factor (GDNF),and ascorbic acid.In allcultures, the CP, or Cripto BP was added to the differentiatingESCs from day 1 to day 11 (Fig.4A).

We first examined whetherimmatureand proliferatingcellsremained in the culturesat the end ofdifferentiation,day 14.Interestingly,Oct4þ cells (a pluripotentstem cellmarker) as wellas Ki67þ cells (a nuclear cellcycle marker)were presentboth in CP-and BP-treated cultures.However,while extremely few/rare Oct4þ cells were identified in CP(notshown)and BP (Fig.4B), Ki67þ cells were relatively

Figure 2. Cripto BP prevents Cripto-induced Smad2 phosphoryla-tion. Two-day-oldcripto / mouseembryonicstem cells-derivedembryoid bodieswerestimulated with recombinantCripto protein(0.5 lg/ml) either alone or in the presence of increasing amount(2.5lM and 25 lM) of the peptides:DP (dimeric 26-23-23),CP (controltetramericD-Ser-Ala-Cha)and BP (tertrameric26-23-23),or leftuntreated as control.Cripto BP,butnotCP or DP inhibits Cripto-induced Smad2 phosphorylation,as shown by Western blot,usinganti-phospho-Smad2 antibodies.Levelsof total Smad2werealsocompared.Smad2 phosphorylation was expressed as ratio between ar-bitrary densitometric units of P-Smad2 and Smad2.Data are mean 6SEM, n 3. Abbreviations:BP, blocking peptide;CP, controlpep-tide; DP,dimeric peptide.

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abundantboth in CP (notshown)and BP cultures (Fig.4C,BP). To furthercharacterize the ESC cultures,we thus per-formed immunocytochemistry againstnestin (neuroectoderm),cytokeratin (ectoderm/epidermis)and CD29 (mesoderm andectoderm).We found an increase in the presence ofneuralrosettes and nestin staining in BP cultures (Fig.4D,4E) com-pared with CP cultures(Fig. 4E0). Although cytokeratinþcells were detected in both CP- and BP-differentiated cultures(Fig.4F,BP), few CD29þ cells were present only in CP cul-tures(Fig. 4G), suggesting thatCripto BP prevented meso-derm differentiation to promote neural induction,as describedfor cripto/ ESCs [13,14].The efficacy of the neural induc-tion was also evidentby the abundanceof both neurons(Tuj1) and glial cells (GFAP) in the BP cultures (Fig.4H).

We subsequently assessed the effectof BP on neuronaland DA differentiation in these culture conditions.First,wefound thatwhile treatmentof cultures with CP had no effecton neuralinduction orTH differentiation,a clear increasewas detected with BP (Fig.4I–4K).Following differentiationin the presence ofthe Cripto BP,the majority ofcolonieswithin the cultureswere immunoreactivefor bIII-tubulin

(91% comparedwith 66% in CP, Fig. 4I). Furthermore,Cripto BP was capable of significantly increasing the propor-tion of coloniescontaining TH-irDA neurons(62%)com-pared with 34% in CP (Fig. 4J). Additionally, these TH-ir col-oniescontained significantly more DA neurons (THþ cells/lm colony,286%,Fig. 4K–4M).Interestingly,we found thatDA differentiation of Cripto BP-treated ESCs could be furtherenhanced by addition of Noggin [28,29] and Wnt5a [40,42](supporting information Fig.6).Many of the TH-labeled neu-rons (in CP- and Cripto BP-treated cultures) showed a bipolarmorphology(Fig. 4N). Immunohistochemistryfor Nurr1,Pitx3,and G protein-activated inwardly rectifying potassiumchanneltwo (GIRK2; the latter,a markerpreferentiallyexpressed in SNpc DA neurons[43]),confirmed thatTH-ircells within the cultures (CP orBP treated)were ofa mid-brain DA phenotype (Fig.4O–4Q) and,at least a notable pro-portion possessed a SNpc phenotype (Fig.4Q).Interestingly,few cells within the cultures were immunoreactive forotherneuronalsubpopulationsmarkersincludingc-aminobutyricacid (GABA-ir) and serotonin (5HT-ir) (Fig.4R–4S).Further-more,we analyzed the ability of the dopamine cells generated

Figure 3. Cripto BP prevents cardiomyogenesis and induces neurogenesis in wild-type ESCs.(A): In vitro cardiac differentiation scheme of wild-type ESCs.(B): Cripto BP (butnotCP or DMSO) prevented cardiomyogenesis.Peptides were added to 2-day-old wild-type mouse ESCs-derivedEBs; the percentage of EBs with rhythmically contracting areas was scored on day 13 (n ¼ 60 EBs/group; data are mean 6 SD; *, p < .001, **, p< .0001). (C): Grades of neuronal differentiation of wild-type EBs, as arbitrarily defined. Grade 0: absence of neurons; Grade 1: few, isolated neuronsimmunoreactive to b-III tubulin antibodies; Grade 2: small areas of b-III tubulin-ir cells; Grade 3: dense network of b-III tubulin-ir cells. Nuclei werevisualized by 40,6-diamidino-2-phenylindole (DAPI) counterstaining.Scale bar (A–C) ¼ 100 lM,(D) ¼ 75 lM in.(D): BP (but not CP or DP) pro-moted robust neuronal differentiation in wild-type EBs.Two-day-old wild-type EBs were treated with 25 lM of the indicated peptides and immuno-fluorescence analysis was performed on day 13 (n ¼ 60 EBs/group,data are mean 6 SD;**, *, p .03). The degree of neuronal differentiationwas scored according to panel (C). (E): Gene expression analysis of mesoderm and neuronal markers. Two-day-old wild-type EBs were treated eitherwith the indicated peptides at25 lM (CP; BP) or vehicle (DMSO) and the expression profile of mesoderm/cardiac and neuronalmarkers was ana-lyzed on day 13,by quantitative reverse transcription polymerase chain reaction.mRNA was normalized to glyceraldehyde 3-phosphate dehydrogen-ase (GAPDH) and presented as fold change in gene expression relative to the control (DMSO ¼ 1). Data are mean 6 SD, n 3; *, p < .05 **, p <.01.Abbreviations:AFP, alpha-fetoprotein;BP, blocking peptide;CP, controlpeptide;DAT, dopamine transporter;DMSO,dimethylsulfoxide;DP,dimeric peptide; EB, embryoid body; ESC, embryonic stem cell; NFM, neurofilament medium polypeptide; TH, tyrosine hydroxylase.

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in vitro to synthesize,release and utilize dopamine.Althoughboth Cripto BP and CP-treated cultures were able to utilizeDA (as reflected by dopamine turnover;ratio ofDOPAC toDA, Fig.4T0 0 0), we noted that Cripto BP treatmentof mESCs

significantly increasesthe KCl-evoked release ofdopamine(Fig.4T0) and DOPAC (Fig.4T00) within the cultures,reflec-tive ofenhanced DA differentiation.Thus,our results showthatCripto BP,in addition to promoting neuralinduction (as

Figure 4.

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shown in EB cultures,Fig.3),was also capable of enhancingthe numberof DA neuronsby 2.8-foldsin a neuralmedia(Fig.4).

Enhanced Functional Recovery in ParkinsonianRats Receiving ESC Grafts Treated with Cripto BPWe nextexamined the ability ofmESC-derived DA neuronsto survive,integrate and induce functional recovery in an ani-malmodelof PD. Previous studies on the function of cripto

in the DA differentiation ofESCs in vivo have been per-formed using cripto/ ESCs grown for4 days as EBs [13,14].Given the high yield of DA neurons in wt mESCs treatedwith Cripto BP,cells differentiated as described in supportinginformation Figure 6,in the presence ofeither Cripto BP orCP, were used for transplantationin 6-hydroxydopaminelesioned rats,an animalmodelof PD. The validity ofthemodel was assessedby amphetamine-inducedrotationalbehaviorand histologicalexamination,which confirmed thatall animals had near complete unilaterallesions of SNpc DA

Figure 5. Cripto BP reduces the incidence and size of tumors following mouse embryonic stem cells transplantation.(A): Pretreatment of em-bryonic stem cells(ESCs)with BP reduced tumorincidence upon transplantation compared with CP treatment.Neuralred-stained sectionsshowed that upon tumor occurrence, animals receiving Cripto BP-pretreated ESCs (C) showed significantly reduced tumor volume by comparisonto CP treatment(B). (D): Semiquantitative analysis of graftcomponents within tumors (refer to ‘‘Materialand Methods’’ section for scoring).(E): Tumors contained abundant proliferating cells (Ki67þ; [E, G]) and undifferentiated neural cells (Nestinþ; [F, G]) within the graft. However,in all cases,these tumors also contained cells from other germ layer cells including mesoderm (CD29þ,[H]),endothelial cells (CD29þ,[I]), andectoderm/epiderm cells (cytokeratinþ,[J]) as wellas the rare presence of undifferentiated ESCs (Oct4þ,[K]), indicating thatthe tumors wereteratomas.Student’s t-test,* p < .05.Abbreviations:BP, Cripto blocking peptide;CP, controlpeptide;CPu,caudate putamen;Ctx,cortex;Lv,lateral ventricle.

Figure 4. Cripto BP enhances neuronalinduction and dopaminergic differentiation.(A): In vitro differentiation scheme.ESCs were seeded atlow density onto a stromalfeeder layer for 14 days in the presence of sequentialmorphogens and mitogens (Shh,FGF2,FGF-8) as wellas thepresence orabsence ofthe CP orCripto BP (12.5 lM).Differentiated cultures rarely contained pluripotentESCs (Oct4,[B]), butcontainednumerous proliferating cells (Ki67þ,[C]).No difference in Oct4þ or Ki67þ cells was detected between BP and CP,however BP cultures con-tained many more neuralstem cells (rosettes,[D],and nestinþ cells,[E]) compared with CP (E0). Both CP and BP cultures contained ectoder-mal/epiderm cells (cytokeratinþ cells,[F]), however only CP cultures expressed mesodermalcells (CD29þ cells,[G]). The efficacy of neuralinduction in BP cultures was reflected by abundance of neuralrosettes (D),and the dense neuronaland glialstaining (Tujþ and GFAP,respec-tively,[H]. (I): In vitro differentiation ofESCs in the presence ofBP significantly increased the percentage ofcolonies containing neurons(TUJ1).(J): Cripto BP significantly increased the proportion of colonies containing THþ dopaminergic neurons,and (K) the number of DA neu-rons/colony.(I–K): Mean 6 SD, **, p < .01,***, p < .0001.Photomicrographs illustrating THþ neurons within neuronal-enriched ESC colo-nies following (L) CP administration,and (M–N) Cripto BP treatment.THþ cells within the cultures acquired the expression of markers typicalof midbrain DA neurons such as Nurr1 (O),Pitx3 (P),and GIRK2 (Q),confirming the DA phenotype of the THþ neurons.Few cells with otherneuronalphenotypes such as GABA (R) or 5HT (S) were present.No TH/GABA colocalization was detected.(T): Cripto BP-treated ESC cul-tures showed significantly increased dopamine and DOPAC release in vitro following KCl evoked release.Data represents mean 6 SEM,n ¼ 3,*, p < .05.Scale bar (B-H and L,M) ¼ 200 lm,(N–S) ¼ 50 lm.Abbreviations:5HT,5-hydroxitriptamine;AA, ascorbic acid;BDNF,brain-derived neurotrophic factor; BP,Cripto blocking peptide; CP,control peptide; DA,midbrain dopaminergic; DOPAC,dihydroxyphenylacetic acid;ESC,embryonic stem cell; FGF,fibroblast growth factor; GABA,c-aminobutyric acid; GDNF,Glial cell line-derived neurotrophic factor; GFAP,glial fibrillary acidic protein; PA6,mouse stromal cells; SRM,serum replacement medium; TH,tyrosine hydroxylase.

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neurons(cell loss>90% and >6 rotations/minute;data notshown).When the viability ofthe grafts was examined,wefound that97% of the animals (31 of32 rats)transplantedwith 12,000 mESC-derived cellsshowed viable graftsat 8weeks.The majority ofthe ratsreceiving ESCspretreatedwith CP showed the presence oftumors/overgrowths withinthe grafts (Fig.5A, 5B). Interestingly,tumorformation wasreduced by more than half,but not abolished, in rats receivingESCs pretreated with Cripto BP (Fig.5A, 5C).To determinethe nature of these tumors (i.e.,neuralovergrowths or terato-mas),we examined the grafts by immunohistochemistry andscored the celltypes(Fig. 5E–5K, see ‘‘Methods’’section).

The majority ofthese tumors(all CP-pretreated and 3/4 ofBP-pretreatedESCs) containedectodermal-epidermalandmesodermalcells,indicating thatthey were teratomas.Thiswas confirmed by the presence of scarce Oct4þ cells in thesegrafts(Fig. 5K). Thus, while mesodermalcells werenotobservedafter Cripto BP treatmentin vitro, thesecellsappeared in vivo,indicating thatthe levelof antagonism maynot be sufficient to prevent mesoderm formation from the fewremaining ESCs in vivo [14].

Interestingly,despite more nestinþ cells were detected inBP compared with CP-pretreated ES cells (Fig.5D),we didnot detectareaswith very high cell density/parenchymal

Figure 6. Cripto BP induces behavioral and anatomical improvements in embryonic stem cells (ESC)-grafted parkinsonian rats.(A): Treatmentof ESCs with Cripto BP (red line) significantly improves amphetamine-induced rotationalbehavior of grafted animals compared with CP-treatedgrafts (blue line) and sham grafts (green).(B): An increase in the number of TH-ir cells/lm3 was detected within the graft following transplanta-tion of ESCs treated with Cripto BP compared with CP.(C): Correlation of THþ cells and functional improvement in grafted animals.No statis-ticaldifference was detected between CP (blue) and Cripto BP (red).Bold line represents regressions and faintlines,95% confidence intervals.(D): Photomicrographs illustrating TH-ir neurons within the striatum following grafting of ESCs treated with CP and (E) Cripto BP.(F): Numer-ous hypertrophied THþ fibers seen outside the graftsite,indicative of good graftintegration.(G): High power image of a TH cellwithin thegraft showing the bipolar morphology.(H): THþ/Nurr1þ,(I) THþ/DATþ, (J) THþ/Pitx3þ, (K) THþ/GIRK2þ (fill arrowhead) staining withinthe graft,confirming the midbrain dopaminergic phenotype of the TH neurons in Cripto BP grafts.(L): Norepinephrine (labeled with NET) fiberscould be seen throughout the graft but did not colocalize with TH.TH neurons within the grafts did not colocalize with (M) GABA or,serotonin(5HT (5-hydroxitriptamine),not shown).Data are mean 6 SD.*, p < .05,**, p < .01,***, p < .001.Scale bar (Di,Ei) ¼ 400 lm,(Dii, Diii,Eii, G) ¼ 100 lm,(F, H, I–M) ¼ 50 lm.Abbreviations:BP, Cripto blocking peptide; CP,controlpeptide; DAT,dopamine trasporter; GABA,c-aminobutyric acid; NET,norepinephrine transporter; TH,tyrosine hydroxylase; GIRK2,G protein-activated inwardly rectifying potassium chan-nel two; Nurr1, nuclear receptor related one; Pitx3,paired-like homeodomain transcription factor three.

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distortion containing nestinþ cells and Ki67þ cells in the ab-sence ofmesodermaland endodermalcells,indicating thatthere were no cases ofisolated neuralovergrowth (data notshown).Remarkably,in the cases where tumorswereobserved,animals receiving ESCs pretreated with Cripto BPshowed a significantreduction in tumor volume (0.94 1010

lm3 6 0.27 10 10) compared with CP (2.22 1010 lm3 60. 44 10 10, Mean 6 SEM,*, p < .05),as assessed in neu-ral red-stained sections (Fig.5B–5C).Thus,ourresults indi-cate thatCripto BP reduces the incidence and size of mESC-derived teratomas by 60%.

We nextexamined the impactof ESC transplantation onamphetamine-induced circling behaviorand dopamine neuronengraftmentin ratsthatdid notpresentany tumor(n ¼ 11Cripto BP grafts and n ¼ 4 CP grafts,15 BP grafts and 16 CPgrafts of the total).Functionally,the motor behavior of parkin-sonian rats receiving ESCs grafts significantly improved by 8weeks compared with sham-treated animals (Fig.6A).Graftingof 12,000 ESCs pretreated with CP showed modestlevels offunctionalimprovementcompared with shamsfrom 4 to 8weeks.Most remarkably,animals receiving the same number ofESCs treated with the Cripto BP showed significantreductionsin amphetamine-induced rotationalbehavior as early as week 2postgrafting.Moreover,the reduction in motor asymmetry pro-gressively (and significantly) improved from 6 weeks postgraft-ing compared with CP grafts (Fig. 6A).

Subsequently, we examined whether the functional improve-mentobserved in Cripto BP-treated animals was related to anincrease in TH-ir neuron counts within the grafts of tumor-freeanimals.Importantly,the volume of the grafts was notsignifi-cantly different between treatments (CP ¼ 8.79 108 6 1.20 108 and BP ¼ 9.43 108 6 0.88 108, p ¼ .687), and no THneuronswere found within the striatum ofanimalsreceivingsham grafts (data not shown). When we examined the number ofTH cells,we found that animals grafted with Cripto BP-treatedESCs showed a consistent increase in THþ cells/lm3 comparedwith CP-treated ESCs (p ¼ .046,Fig. 6B–6E).However,wefound no statistically significant difference between CP and BP-pretreated ESC grafts when examining the correlation betweenbehavioralimprovementand THþ neuron number(Fig. 6C),indicating thatthe improvementis mainly contributed by anincrease in THþ neurons.We observed thatmany of the THþcells within the grafts displayed a bipolar morphology (Fig. 6G)as well as an extensivenetworkof hypertrophiedneuritesthroughoutthe striatum (Fig.6F). Immunohistochemistryrevealed that many of the TH-ir cells within the grafts also coex-pressed Nurr1, DAT, Pitx3, and GIRK2, verifying their midbrainDA identity (Fig.6H–6K).Interestingly,Pitx3/TH and GIRK2/TH colabeling illustrated thatseveralTHþ neurons within thegrafts were SNpc midbrain-like DA neurons, both in CP and BPgrafts,(filled arrowheads in Fig.6K). Although norepinephrinefibers (labeled with norepinephrine transporter,NET) could beseen within the grafts (Fig.6L),no TH-ir cell bodies within thegraft colocalized with other neurotransmitters,including norepi-nephrine (Fig. 6L), GABA (Fig. 6J), and serotonin (not shown).

In summary,our resultsillustrate thata synthetic smallpeptide,Cripto BP,improves the generation of midbrain DAneurons from ESCs in vitro and in vivo, reduces the incidenceof teratomas and results in a behavioralimprovementin par-kinsonian rats.

DISCUSSION

In this study,we reportthe identification (by a combinatorialscreening) of a novelsmall synthetic peptide (Cripto BP) that

antagonizesthe onco-developmentalsignalingmolecule,Cripto [2].Although Cripto blocking antibodieshave beenused in the pastto suppress tumor cellgrowth [44],syntheticmolecules/peptides offernumerous advantages overbiothera-peutics as they are easier and cheaper to produce, more stable,and generally free of contaminants of biologicalorigin.Com-binatorialscreenings have been successfully used in the lasttwo decades to identify smallmolecules/peptides foruse indrug discovery and cellbiology [45].Recently,cell-basedscreeningshave identified synthetic moleculesthatregulatestem celldifferentiation/proliferation,which have been usedas probes to study the underlying biology of stem cells [46–49].Although many basic questionsregarding identificationof targets and mechanisms of action of the molecules identi-fied in cell-based screenings stillremain to be answered,ithas become evidentthatsuch synthetic molecules could leadto significantadvances in stem cellbiology.In our study weused a reverse screening approach using a defined mecha-nism/target,the Cripto/ALK-4 receptorcomplex,and there-fore screened a synthetic peptide library to identify a CriptoBP. The choice ofa synthetic peptide library was made aspeptides are known to be optimalprobes to targetthe extrac-ellular domains of membrane receptors,provided their typicalhigh affinity and selectivity,minimaldrug-drug interactions,low accumulation capacity,and low toxicity [31].Moreover,tetrameric peptides are extremely flexible molecules,have ahigh-recognition surface,are highly resistantto proteases andcan thus be applied in functionalassays and in a broad rangeof applications both in vitro and in vivo.

Based on these considerations,we designed an assay toidentify competitorpeptidesof the Cripto/ALK-4 receptorcomplex thatcould be used to controlESC fate.Consistentwith this idea,we found thatCripto BP prevented Cripto-de-pendentSmad2 phosphorylation,blocked cardiomyogenesisand favored neurogenesis in ESCs,mimicking the phenotypeof cripto/ ESCs [12,13].Thus,these data and our in vitroresults showing thatCripto BP selectively binds Cripto andprevent its in vitro binding to ALK-4 receptor, support the ideathat,despite the function of ALK-4 as a receptor for multipleTGFb-related ligands [50],the neutralizing activities thatwedetect are the result of specific targeting of the Cripto protein.

Interestingly,the BMPs antagonist Noggin was previouslyshown to induceneuroectodermalcell developmentandenhanceproduction ofDA neuronsfrom hESCs[28, 29].Moreover,it has been recently reported thata combination ofNoggin and thesmallmoleculeSB431542,which inhibitsALK-4, -5,and -7 receptors [51] and blocks SMAD signaling,efficientlyinduceneuralconversionof humanpluripotentstem cells and allow their subsequent DA differentiation [30].Our protocoldiffers from previous studies in thatwe devel-oped and used a selective inhibitor(Cripto BP),instead ofbroader inhibitors such as Noggin and particularly SB431542,which blocks signaling by several TGF-b superfamily ligands.Ourwork indicates thata selective blocking with Cripto BPis sufficientto improve neuraldifferentiation ofmESCsinvitro. This improvementwas not at the expenseof anincreased differentiation ofOct4þ cells,as theirnumber didnot change by Cripto BP,but was rather due to a switch frommesodermalto neuraldifferentiation.We thussuggestthatadditionalcell sortingor BMP inhibitorscould furtherimprove this protocol.In agreementwith this,we found thatNoggin and Wnt5a enhanced the production ofDA neuronsby Cripto BP in vitro.These findings indicate thatCripto BPcan indeed be used in combination with other factors to fur-ther improve DA differentiation.

With regard to celltransplantation in animalmodelsofPD, Chiba et al. [29], previouslyfoundthatgraftingof

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hemiparkinsonian rats with hESCs-derived DA neuronspre-treated with Noggin lead to significantengraftmentof DAneurons and behavioralimprovement,butall animals showedcell overgrowths.These results differ from the study by Sonn-tag etal. [28],who showed teratomas in about25% oftheanimalsgraftedwith Noggin-treatedcells and behavioralimprovementthatcorrelated with THþ cellnumbers in onlytwo of the grafted animals.In our study,we found that CriptoBP treatment,led to grafts containing many more THþ cells(9,304 6 850 forBP and 5,841 6 1,939 forCP), and to aclear behavioralimprovement in hemiparkinsonian rats.How-ever,ourapproach did notentirely eliminate tumors.CriptoBP significantly reduced the incidence of teratoma formation(by 58%)and the size oftumors,but teratomaswere stillobserved in as many as 27% of the grafted rats,even in thepresence ofadditionalfactors,thatis, Wnt5a and Noggin.Previous studies have shown thatwhile transplantation ofasfew as 20,00 cripto/ ES-derived EB cells resulted in func-tional recoveryand no tumor/overgrowthformationESCs[13],50,000 cripto/ ES-derived EB cells did not prevent tu-mor/overgrowth formation,but lead to smaller grafts that con-tained more neurons [14].We thus explored whether differentcell grafting densities ofmESCs,differentiated as describedin Figure 4A,could reduce tumor/outgrowth formation whilemaintaining behavioralimprovement,butfound thatcellden-sity playsa seemingly insignificantrole in predifferentiatedmESCs in the presence of Cripto BP (supporting informationFig. 7). We also explored whetherthe efficiency ofCriptoblocking with Cripto BP may have decreased in vivo,butfound thatinfusion of Cripto BP peptide in vivo did notfur-therenhance the effectof Cripto BP pretreatmentin vitro(data not shown). Thus,our results suggest that additional fac-torspresentduring DA differentiation and/oraftergraftingmay play an additionalrole in tumor/outgrowth formation.These data are thus in agreementwith the results by Sonntaget al.,who suggested thatactivation ofotherpathways,in acell autonomous or nonautonomous manner,may also contrib-ute to cell overgrowth in vivo [14].Finally,it should be notedthatfew previous studies have reported robustengraftmentofmES cell-derived DA neurons in animalmodels of PD in theabsence oftumorformation by eitherusing transgenes[52,53], feeders[41] or knockoutES cells [13] (see[20] forreview).Our differentiation protocol,while aiming atavoid-ing genetic modification,requires the use of PA6 feeders.Inour protocol,we introduced some modifications to the proto-col by Barberi,which togetherwith some variability intro-duced by PA6 cocultivation,may have contributed to incom-plete specification ofthe ESCs and tumorformation.In thefuture,it will thus be importantto determine whetheraddi-tional instructive/differentiation factors,inhibitors of pathwaysinvolved in tumorformation and cellseparation techniques,can be used to improvethe functionalintegration ofES-

derived DA neurons in models of PD,making feeder-free andtransgene-free DA differentiation protocols safer,as wellasmore reproducible and robust.

CONCLUSION

In conclusion,our strategy providesevidence thata smallsynthetic peptide obtained from an in vitro screen can notonly targetspecific proteins and signaling pathways in vitro,butcan also enhance neuralinduction in mESCs,midbrainDA neuron differentiation and DA release.More importantly,Cripto BP can improve the number and the functional integra-tion of ESC-derivedDA neurons, leadingto behavioralimprovements in animalmodels of PD.This is the firsttimethata reverse screening approach has been used in stem cellbiology to improve stem celltransplantation.However,thepresence ofteratomas in a significantnumberof the graftedanimals remains as an importantlimiting factor.Hence,fur-ther efforts are required to elucidate the mechanisms responsi-ble for tumorformation and optimize CRT.Future studieswill examine whether hESCs respond to Cripto BP in a simi-lar manner as mESCs and whether the screening and targetingof additionalsignaling pathways may improve the therapeuticapplication of stem cells in CRT for PD.

ACKNOWLEDGMENTS

We are grateful to the members of the Stem Cell Fate Laboratoryand the Arenas laboratory forhelpfuldiscussion.This workwassupported by the Telethon Foundation to G.M.(grantsGGP05112, GGP08120) and S.D.F. (GGP08062), AssociazioneItaliana Ricerca sul Cancro (AIRC) to G.M. and S.D.F.; and bygrants from the European Union (Eurostemcell),the SwedishResearch Council (VR2008), Norwegian Research Council andKarolinska Institutet, to E.A. M.R. is funded by a FIRB (project,n RBNE03PX83_005). C.L.P. was supported by a Human Fron-tiers Science Program Long-term Fellowship and a NationalHealth and Medical Research Council, Australia Career Devel-opment Award. D.R. was supported by a by a grant form the Por-tuguese Praxis XXI program of the Fundac¸ao para a Ciencia e aTecnologia.

DISCLOSURE OF POTENTIAL CONFLICTSOF INTEREST

The authors indicate no potential conflicts of interest.

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