Comparative proteomic analysis of human embryonic stem cell-derived and primary human retinal pigment epithelium

1 Scien i ic RepoR s | 7: 6016 | DOI:10.1038/s41598-017-06233-9 www.na u e.com/scien i ic epo s Compa a i e p o eomic analysis o human emb yonic s em cell-de i ed and p ima y human e inal pigmen epi helium Heidi Hongis o1, An i Jylhä2, Janika Nä inen1,2, Jochen Rieck1, Tanja Ilma inen1, Zol án Ve éb3, Ulla Aapola2, Roge Beue man2,4, Go an Pe o ski3,5, Hannu Uusi alo2,6 & Heli Sko man1 Human emb yonic s em cell-de i ed e inal pigmen epi helial cells (hESC-RPE) p o ide an unlimi ed cell sou ce o e inal cell eplacemen he apies. Clinical ials using hESC-RPE o ea diseases such as age- ela ed macula degene a ion (AMD) a e cu en ly unde way. Human ESC-RPE cells ha e been ho oughly cha ac e ized a he gene le el bu hei p o ein exp ession p o ile has no been s udied a la ge scale. In his s udy, p o eomic analysis was used o compa e hESC-RPE cells di e en ia ed om wo independen hESC lines, o p ima y human RPE (hRPE) using Isoba ic ags o ela i e quan i a ion (iTRAQ). 1041 common p o eins we e p esen in bo h hESC-RPE cells and na i e hRPE wi h majo i y o he p o eins simila ly egula ed. The hESC-RPE p o eome e lec ed ha o no mal hRPE wi h a la ge numbe o me abolic, mi ochond ial, cy oskele al, and anspo p o eins exp essed. No signs o inc eased s ess, apop osis, immune esponse, p oli e a ion, o e inal degene a ion ela ed changes we e no ed in hESC-RPE, while impo an RPE speci ic p o eins in ol ed in key RPE unc ions such as isual cycle and phagocy osis, could be de ec ed in he hESC-RPE. O e all, he esul s indica ed ha he p o eome o he hESC-RPE cells closely esembled ha o hei na i e coun e pa s. The e inal pigmen epi helium (RPE) is a mul i unc ional, pola ized epi helial cell laye be ween he neu osen- so y e ina and he cho oid, which plays key oles in pho o ecep o unc ion and ision. The RPE cells anspo nu ien s, was e p oduc s, ions and luid be ween he cho oidal blood supply and he sub e inal space. RPE also phagocy izes shed pho o ecep o ou e segmen s (POS), abso bs sca e ed ligh , sec e es many impo an signal- ling molecules and unc ions in he e inoid isual cycle1. This highly me abolically ac i e cell ype is exposed o cons an ligh s imuli and high oxida i e s ess making i ulne able o oxida i e damage. Thus, abno mali ies in RPE cell unc ion may lead o e inal degene a ion and pho o ecep o cell dea h. The RPE is he ocal poin o many e inal degene a i e diseases such as age- ela ed macula degene a ion (AMD), he mos common cause o blindness in he elde ly in wes e n coun ies. AMD is a mul i ac o ial, age-associa ed disease cha ac e ized by accumula ion o insoluble d usen in he e ina, degene a ion o RPE and pho o ecep o s in he d y o m, and cho oidal neo ascula iza ion in he exuda i e, we o m o he disease2. T ea men op ions o he e inal degene a i e diseases such as AMD a e cu en ly e y limi ed and mos ly only delay disease p og ession. Cellula ansplan a ion o eplace he a ec ed RPE is conside ed as a p omis- ing he apeu ic s a egy o ea hese diseases. Macula ansloca ion and au ologous RPE ansplan a ion wi h pe iphe al RPE ha e demons a ed he easibili y and e ec i eness o au ologous RPE cell eplacemen he apy in AMD pa ien s, bu hese su gical p ocedu es ca y signi ican complica ions3. Many cell ypes ha e been es ed as a sou ce o RPE ansplan a ion issue including oe al RPE4 and RPE cell lines5, 6. Issues ela ed o sca ce issue 1BioMediTech Ins i u e, Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland. 2Depa men o Oph halmology, Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland. 3S em Cells and Eye Resea ch Labo a o y, Depa men o Oph halmology, Facul y o Medicine, Uni e si y o Szeged, Szeged, Hunga y. 4Singapo e Eye Resea ch Ins i u e and Duke-NUS School o Medicine, Singapo e, Singapo e. 5Cen e o Eye Resea ch, Depa men o Oph halmology, Oslo Uni e si y Hospi al and Uni e si y o Oslo, Oslo, No way. 6Tampe e Uni e si y Hospi al Eye Cen e , Uni e si y o Tampe e, Tampe e, Finland. Co espondence and eques s o ma e ials should be add essed o H.H. (email: heidi.m.hongis o@s a .u a. i) Recei ed: 3 Augus 2016 Accep ed: 12 June 2017 Published: xx xx xxxx OPEN www.na u e.com/scien i ic epo s/ 2 Scien i ic RepoR s | 7: 6016 | DOI:10.1038/s41598-017-06233-9 a ailabili y and cha ac e is ics o immo alized adul human cell lines, and he ac ha hey only weakly mimic some o he na i e RPE cha ac e is ics a e in i o passaging, make hese cells subop imal o ea men o he la ge popula ion o pa ien s7, 8. Human plu ipo en s em cells (hPSC), including bo h human emb yonic s em cells (hESC) and human induced plu ipo en s em cells (hiPSC) can be di e en ia ed o e inal cells, including pho o ecep o s and ma u e and unc ional RPE cells9. Thei high capaci y o sel - enew and wide di e en ia ion po en ial makes hem an excellen cell sou ce o bo h cellula models o esea ch pu poses as well as cell eplacemen he apy app oaches. Encou aging esul s ha e shown ha ansplan ed hESC-de i ed RPE cells (hESC-RPE) can media e unc ional pho o ecep o escue in he Royal College o Su geons (RCS) a model o e inal degene a ion10–12. Mo eo e , ongoing phase I/II clinical s udies ha e ecen ly demons a ed ha i is possible o sa ely implan hESC-RPE o end s age pa ien s wi h AMD and o he e inal degene a i e diseases13. Simila s udies wi h au ologous hiPSC-RPE ha e also been ini ia ed in Japan14, al hough suspended la e o one o he wo pa ien s due o sa e y conce ns ega ding genomic s abili y o he pa ien ’s hiPSCs15. Ou esea ch g oup, along wi h many o he s, has shown ha he hESC-RPE s uc u e, unc ion, and physi- ology closely esembles ha o hei na i e coun e pa s wi h a high a e o pigmen a ion, polygonal, cuboidal epi helial cell mo phology, cellula ine s uc u e, and exp ession o many RPE signa u e genes and p o eins16–22. In addi ion he cells show epi helial in eg i y and unc ionali y wi h he abili y o phagocy ose POS and sec e ion o g ow h ac o s18. Howe e , la ge scale compa a i e s udies o he p o eome, he o al p o ein complemen o a genome, o he hESC-RPE cells a e lacking, while he p o eome o hRPE, especially ela ed o AMD has been ex ensi ely s udied7, 23–25. S ingen cha ac e iza ion o hPSC-RPE using s anda dized and quan i iable me hods is impo an o gain con idence ha hey a e sa e and adequa e eplacemen s o diseased RPE i hey a e u ilised in clinical se ings. In his s udy, we ha e pe o med a compa a i e p o eomic analysis o in i o di e en ia ed hESC-RPE cells o p ima y hRPE using Isoba ic ags o ela i e quan i a ion (iTRAQ). iTRAQ quan i ica ion is based on inco po- a ion o s able iso opes in o pep ides ollowed by liquid ch oma og aphy ac iona ion and andem mass spec- ome y analysis. iTRAQ-based p o eomics has p e iously been used o example o s udy ea ly s age hESC di e en ia ion26 and changes in isual cycle p o eins in mice RPE in conjunc ion wi h RPE65 gene deli e y27. We ha e also p e iously success ully applied he echnique o cha ac e iza ion o co neal epi helial cells de i ed om hPSC28. In his s udy, he esul s showed ha he o e all spec um o p o ein exp ession o he hESC-RPE cells and he p ima y RPE we e simila . Resul s A la ge numbe o common p o eins we e exp essed and egula ed a simila le el in hESC-RPE and hRPE. iTRAQ p o eomics was pe o med o compa e p o ein exp ession o hESC-RPE o p ima y hRPE. The expe imen al se up and wo k low o he s udy is shown in Fig.1a. Th ee eplica e samples o wo hESC lines Regea08/017 and Regea08/023 di e en ia ed o RPE cells ( e e ed o as 08/017 and 08/023 hESC-RPE), we e compa ed o hRPE samples om h ee di e en cada e ic human dono eyes. Diges ed pep ides we e labelled wi h iTRAQ eagen s as shown in Fig.1a, inco po a ing one biological sample o 08/017, one sample o 08/023, and one sample o hRPE o each o he analysis eac ions. The labelled, combined samples we e subjec ed o h ee LC-MS/MS analyses, which we e conduc ed in wo echnical eplica es each. Fo da a analysis he eplica e esul s we e combined as shown o allow ela i e quan i a i e compa ison o he p o ein exp ession in hESC-RPE and hRPE. The iTRAQ p o eomics allows he iden i ica ion and quan i ica ion o p o eins ha a e exp essed in all sam- ples included in each labelling (Analysis 1.1–3.2 in Fig.1). 2643 unique p o eins (Unip o Accession) exp essed by bo h he hESC-RPE and hRPE we e iden i ied in a leas one echnical eplica e sample (Fig.1b). The numbe o p o eins p esen a e s ingen da a p ocessing and il e ing o ensu e da a quali y a e shown in Fig.1b. 1041 unique p o eins exp essed in a leas wo biological eplica e samples o bo h 08/017 and 08/023 hESC-RPE, and in he hRPE dono sample we e included o inal da a analysis. A comple e lis o he p o eins is p o ided in Supplemen a y TableS1. Log2 scale was used o analysing he da a. As p o ein exp ession o he hESC-RPE we e compa ed o he p o ein exp ession in hRPE, o e wo- old di e ences in p o ein exp ession (co esponding o old change alues >1 o <−1 on log2 scale) we e conside ed signi ican o e exp ession (up egula ion) o unde exp ession (down egula ion). Majo i y o he p o eins we e exp essed a simila le els in hESC-RPE and hRPE. 82% o he p o eins we e simila ly exp essed in 08/017 hESC-RPE and 81% in 08/023 hESC-RPE, espec- i ely, when compa ed o hRPE (Fig.2a and c). Only 7% o he p o eins we e up egula ed in 08/017 and 8% in 08/023 hESC-RPE cells, while 11% we e down egula ed in bo h cell lines compa ing o hRPE. Pa hway analysis o he o e - and unde exp essed p o eins in he hESC-RPE compa ed o hRPE was conduc ed o bo h 08/017 and 08/023 o inspec i en iched ac i e pa hways a ise om he di e en ially exp essed se o p o eins. IPA (Ingenui y Pa hway Analysis, Qiagen) co e analysis o he canonical pa hways indica ed ha p o eins associa ed wi h mi ochond ial dys unc ion, oxida i e phospho yla ion, and pho o ansduc ion pa hways we e down egu- la ed in hESC-RPE (Fig.2b and d). The log2 old change alues o he di e en ially exp essed p o eins o bo h 08/017 and 08/023 a e p esen ed in Supplemen a y TableS2, and isualised in Supplemen a y Fig.S1. O e all, mos ly homogenous da a wi h simila exp ession pa e ns ac oss he mass spec ome y analyses ( h ee analyses wi h wo echnical eplica es each) was p oduced, as shown in Supplemen a y Fig.S2. Va ia ions in he exp ession le els be ween he h ee analyses we e hough o esul om he h ee indi idual hRPE dono samples used o compa ison in each analysis. A compa ison o a pooled sample om he h ee hRPE dono s o a single dono (Dono 1) was done o e alua e in e -dono a iabili y in p o ein exp ession. The esul s showed mos ly simila exp ession wi h only 2% o he p o eins di e en ially exp essed be ween pooled dono sample and Dono 1, indi- ca ing ha he di e ences be ween he dono s we e sub le (Supplemen a y Fig.S3). www.na u e.com/scien i ic epo s/ 3 Scien i ic RepoR s | 7: 6016 | DOI:10.1038/s41598-017-06233-9 hESC-RPE showed a no mal RPE p o ein exp ession p o ile. The PANTHER (P o ein ANalysis Th ough E olu iona y Rela ionships) Classi ica ion Sys em and Gene On ology (GO) e m mapping we e used o examine speci ic p o ein g oups o in e es . PANTHER classi ica ion o he exp essed p o eins acco ding o biological p ocess, classi ied he p o eins in o 13 g oups as shown in Fig.3. The la ges g oup o p o eins was me - abolic p ocesses which included 407 p o eins. Mos o hese p o eins (369) ell unde he subclass o p ima y me - abolic p ocesses. Cellula p ocesses included 380 p o eins and con ained p o eins in ol ed in cell communica ion Figu e 1. Wo k low o he s udy. (a) S udy design o he iTRAQ p o eomics. Human ESC-RPE samples om h ee independen di e en ia ion expe imen s consis ed o h ee eplica e samples each. Human RPE samples we e collec ed om h ee cada e ic dono eyes. To al p o ein was ex ac ed and diges ed wi h ypsin. Pep ides we e labeled wi h iTRAQ 4-plex labels 114–117 shown in colo s, and analyzed as echnical duplica es wi h Nano-RPLC- T iple TOF ins umen a ion. Fo inal da a analyses he esul s we e pooled oge he om all six LC-MS/MS analyses sepa a ely o 08/017 nad 08/023 hESC-RPE. (b) A Venn diag am illus a ing he da a p ocessing be o e da a analyses om o al numbe o p o ein hi s o 1041 p o eins accep ed o inal da a analyses. www.na u e.com/scien i ic epo s/ 4 Scien i ic RepoR s | 7: 6016 | DOI:10.1038/s41598-017-06233-9 (76), cell cycle (47), cellula componen mo emen (19), cy okinesis (11) and g ow h/p oli e a ion (1). Cellula componen o ganisa ion and biogenesis was he hi d la ges g oup wi h 154 p o eins. Localisa ion wi h 123 p o- eins included p o eins in ol ed in anspo . A subclass esponse o s ess, he majo subclass unde esponse o s imulus, included 34 p o eins. Immune sys em p ocesses included 23 p o eins. To analyse he speci ic p o ein g oups mo e ca e ully, GO e m mapping was used o iden i y p o eins con- nec ed o speci ic GO e ms (Supplemen a y TableS3). The p o ein exp ession p o ile o hESC-RPE cells was ound o closely esemble ha o na i e RPE. A la ge numbe o mi ochond ial p o eins we e exp essed a simila le el (85% o 08/017 and 86% o 08/023) o sligh ly down egula ed, in he hESC-RPE cells compa ed o p ima y hRPE (Fig.4a). All oge he 139 p o eins in ol ed in cellula s ess we e iden i ied (Fig.4b). O hese 78% we e simila ly egula ed in 08/017 hESC-RPE and 79% in 08/023 hESC-RPE compa ed o hRPE. P o eins ha ell in o he ca ego y cellula s ess included many p o eins in ol ed in immune esponses, such as majo his ocompa i- bili y complex MHC class I an igen (F agmen , HLA-A) and CD59 an igen ha we e simila ly egula ed. O he p o eins o in e es in he class included galec in-3-binding p o ein (LGALS3BP), clus e in (CLU), annexin A1 (ANXA1) and −5 (ANXA5), apolipop o eins A (APOA) and -E (APOE), c ys allin alpha A (CRYAA), c ys allin alpha B (CRYAB), cys a in C, ca alase (CAT), galec in-1, supe oxide dismu ase 1 (SOD1) and −2 (SOD2), and p o ein S100. A la ge numbe o cy oskele al p o eins (189) we e also mos ly simila ly egula ed in he hESC-RPE and hRPE (83% o 08/017 and 80% o 08/023) (Fig.4c). Cy oskele al ke a ins: −8, −10, −18, and −19 could be de ec ed and o hese −8, 10, −18 we e up egula ed in bo h hESC-RPE lines as we e ez in (EZR), ib illin-1 Figu e 2. P o ein exp ession di e ences be ween hESC-RPE and hRPE. The p opo ions o up egula ed (g een) and down egula ed ( ed) p o eins acco ding o ela i e exp ession le els o he p o eins compa ed o hRPE o (a) 08/017 hESC-RPE and (c) 08/023 hESC-RPE. Fo he majo i y o p o eins in bo h RPE lines, he e we e no di e ences in exp ession le el be ween hESC-RPE cells and hRPE (g ey). Numbe o p o eins included = 1041. De aul se ings o IPA we e used o conduc co e pa hway analysis o he o e - and unde exp essed p o eins (188 p o eins o 08/017 and 195 p o eins o 08/023). Ten en iched pa hways wi h lowes p- alues o he di e en ially exp essed p o eins in (b) 08/017 and (d) 08/023 hESC-RPE compa ed o hRPE. To al numbe o genes/p o eins in each pa hway shown on op o each ba and pe cen age p esen in he da a analysed on he y-axis (le ). Up- and down egula ion o he p o eins in he da a is shown in ed and g een colou s espec i ely, and p- alue (o ange line) is shown in -log scale, y-axis ( igh ). Figu es in (b,c) c ea ed wi h IPA (Qiagen) so wa e. www.na u e.com/scien i ic epo s/ 5 Scien i ic RepoR s | 7: 6016 | DOI:10.1038/s41598-017-06233-9 (FBN1), ib illin-2 (FBN2), and ilamin A (FLNA). P oli e a ion and cell cycle p o eins also showed mos ly sim- ila egula ion in hESC-RPE and hRPE (Fig.4d). Impo an p o eins o RPE unc ion and cha ac e is ics we e exp essed in hESC-RPE cells. P o eins in ol ed in he pe cep ion o ligh s imulus we e iden i ied and 13 o hese 23 p o eins we e down- egula ed in he 08/017 hESC-RPE and 11 p o eins in he 08/023 hESC-RPE compa ed o he p ima y RPE, while he es we e simila ly exp essed (Fig.5a). Impo an RPE-speci ic isual cycle p o eins iden i ied included o example e inal-speci ic ATP-binding casse e anspo e (ABCA4), e inoid isome ohyd olase (RPE65), Figu e 3. Dis ibu ion o p o eins. A pie cha showing dis ibu ion o he p o eins acco ding o hei biological p ocess using Pan he Classi ica ion Sys em. The p o eins we e classi ied by hei co esponding gene names. 852 o he 1041 p o eins we e classi ied in o one o mo e o he 13 g oups. Figu e 4. P o eins in ol ed in mi ochond ial unc ions, s ess, cy oskele on, and p oli e a ion. Rela i e exp ession o (a) mi ochond ial, (b) cellula s ess- ela ed, (c) cy oskele al, and (d) p oli e a ion and cell cycle p o eins in bo h hESC-RPE cell lines compa ed o hRPE. Each do ep esen s a single p o ein, p esen ed as mean old changes on a log2 scale, whe e y = 0 deno es equal p o ein exp ession. Red dashed lines ma k he bo de s o o e and unde exp ession (2- old di e ence in exp ession). Fo p o ein names and exac exp ession alues in each class see Supplemen a y TablesS4–S7. www.na u e.com/scien i ic epo s/ 6 Scien i ic RepoR s | 7: 6016 | DOI:10.1038/s41598-017-06233-9 e inaldehyde-binding p o ein 1 (RLBP1), and e inol dehyd ogenase 11 (RDH11). The e we e 46 p o eins in ol ed in ex acellula ma ix (ECM) and cell adhesion. Among hose we e in eg in subuni s αV (ITGAV) and h ee be a chains −1 (ITGB1), −4 (ITGB4) and −8 (ITGB8), o which he in eg in ß-4 was up egula ed in bo h hESC-RPE lines. In eg in binding alin-1 (TLN1) was sligh ly up egula ed as was in e cellula adhesion molecule 1 (ICAM-1) and galec in-3 binding p o ein (LGALS3BP). Neu al cell adhesion molecule 1, (NCAM- 1) was down egula ed in he hESC-RPE, while i onec in (VTN) was among he simila ly exp essed p o eins (Fig.5b). P o eins in ol ed in melanogenesis included pigmen epi helium-de i ed ac o (SERPINF1) ha was simila ly exp essed in hESC-RPE and hRPE, while y osinase- ela ed p o ein 1 (TYRP1), was up egula ed in 08/017 hESC-RPE. A o al o 98 p o eins in ol ed in phagocy osis, p o ein deg ada ion and au ophagy we e iden i ied. These included p o easome subuni s and 26 S p o easome p o eins. In e es ing p o eins in ol ed in phagocy osis included in eg in alpha V, pa o he αVβ5 in eg in, and i s ligands e aspanin (CD81, ag- men ), and lac adhe in (MFG-E8), and hese p o eins we e exp essed a simila le el as compa ed o hRPE. Also, CD44, CD36 (sca enge ecep o class B, membe 2), and ca hepsin-D we e exp essed a simila le el in he hESC-RPE and hRPE. T anspo e s and ion channels (90 p o eins in o al) exp essed included chlo ide in acel- lula channel p o eins 1 and −6 (CLIC1, and CLIC6). Na+K+- anspo ing ATPase subuni s alpha-1 and be a-1 (ATP1A1, ATP1B1) we e simila ly egula ed. Aquapo in 1 ( agmen ) (AQP1) was up egula ed in hESC-RPE. Vol age-dependen anion-selec i e channel p o eins 1 (VDAC1) and −2 (VDAC2) we e down egula ed in hESC-RPE while VDAC3 was exp essed a simila le el. Amino acid anspo e (SLC1A3) was down egula ed in hESC-RPE, while la ge neu al amino acids anspo e small subuni 1 (SLC7A5) was up egula ed (Fig.5b). Discussion Ul ima ely, p o eins exp essed by a cell de e mine he pheno ypic exp ession o genomic in o ma ion29. The p o- eome is a dynamic en i y cha ac e izing he cell ype and changing wi h he physiological s a us o he cell. Pionee ing p o eomic p o iling s udies ha e e ealed he p o ein exp essions in human RPE/cho oid complex Figu e 5. P o eins in ol ed in impo an RPE unc ions. (a) Rela i e mean exp ession le els o he 23 p o eins in ol ed in pe cep ion o ligh s imulus and (b) o selec ed impo an RPE p o eins. P o ein exp ession le els shown on log2 scale. Red dashed lines ma k he bo de s o o e exp ession and unde exp ession (o e 2- old di e ence in exp ession). E o ba s deno e s anda d de ia ion. Fo he ull p o ein names and exp ession alues see Supplemen a y TableS8. *Quan i ica ion based on less han wo pep ides. www.na u e.com/scien i ic epo s/ 7 Scien i ic RepoR s | 7: 6016 | DOI:10.1038/s41598-017-06233-9 as well as in i o cul u ed RPE cells7, 25, 30–32, changes in RPE p o eome associa ed wi h he p og essi e s ages o AMD23, 24, 33, 34, and in esponse o oxida i e s ess35. The p o ein con en o d usen and lipo uscin has been elu- cida ed wi h p o eomic app oaches36. To ou bes knowledge, la ge scale analyses o hPSC-RPE p o eome ha e no been p e iously pe o med. The objec i e o his s udy was o compa e he p o ein exp ession p o ile o RPE cells di e en ia ed om hESCs o he na i e p ima y hRPE p o eome. We chose a sample se up wi h a di e en biological eplica e o bo h hESC-RPE and hRPE included in each analysis, in o de o obse e ue biological di e ences be ween he samples. In he quan i a i e 4-plex iTRAQ analyses, 1041 common p o eins we e p esen in bo h hESC-RPE cell lines and p ima y hRPE samples. Impo an ly, majo i y o hese p o eins (>80%) we e simila ly egula ed in he hESC-RPE and na i e hRPE, showing ha he hESC-RPE cells esembled hei na i e coun e pa s. The exp ession pa e ns o he wo hESC-RPE lines we e also simila , showing ha ou di e en ia- ion p o ocol yielded homogenous RPE cells wi h low cell line speci ic a ia ion in p o ein exp ession. The p o ein p o ile o he hESC-RPE cells esembled ha o na i e p ima y hRPE. A la ge numbe o me a- bolic and espi a o y p o eins we e exp essed e lec ing he highly me abolically ac i e na u e o he RPE. The pa hway analysis o he di e en ially exp essed p o eins indica ed mi ochond ial dys unc ion and down egula ed oxida i e phospho yla ion in he hESC-RPE, e lec ing educed ene gy equi emen s in he in i o cul u ed hESC-RPE cells compa ed o na i e cells. This is consis en wi h some p e ious published esul s on he p o- eome o in i o cul u ed RPE cells7. Apa om single up egula ed p o eins, no signs o inc eased s ess, apop- osis, immune esponse, p oli e a ion, o e inal degene a ion- ela ed changes we e obse ed in he hESC-RPE. Cellula s ess ma ke s and many p o eins conside ed as bioma ke s o AMD we e simila ly egula ed: galec in-3-binding p o ein (LGALS3BP) which was sligh ly up egula ed in hESC-RPE has been implica ed as AMD ma ke , bu also in ol ed in a achmen and sp eading o RPE cells37. Clus e in (CLU) and apolipop o ein E (APOE) a e majo cons i uen s o d usen36, and we e simila ly exp essed in hESC-RPE and hRPE. C ys allin alpha A (CRYAA), down egula ed in hESC-RPE and c ys allin alpha B (CRYAB) ha was simila ly egula ed, a e p o ec i e o oxida i e inju y and bo h d usen cons i uen s as well as AMD bioma ke s34, 38. Cys a in-C (CST3) is abundan ly sec e ed by he RPE39, while no much is known abou he unc ion o cys a in-B (CSTB) in he RPE. Annexins (ANXA1, ANXA5) in ol ed in many p ocesses including in lamma ion and apop osis40, we e also simila ly egula ed as we e me abolic p o eins such as p ohibi in (PHB), supe oxide dismu ases 1 and −2 (SOD1, SOD2), and glu a hione pe oxidase (GPX1) ha ha e been implica ed as oxida i e s ess bioma ke s41–43. While in lamma ion- ela ed p o ein in e cellula adhesion molecule 1 (ICAM-1) was up egula ed, MHC class I an igen ( agmen , HLA-A) was simila ly egula ed in bo h hESC-RPE lines and MHC class II an igen was no de ec ed. Ou unpublished esul s, in acco dance wi h published da a by o he s44, show ha in he s anda d cul u e condi ions, ou hESC-RPE cells exp ess MHC class I bu no MHC class II an igens wi hou exposu e o p oin lamma o y cy okines. The memb ane complemen egula o y p o ein CD59 was also simila ly egula ed. P o ein S100, and mac ophage mig a ion inhibi o y ac o (MIF) ha e p e iously been shown o pa o macula and pe iphe al RPE p o eome25. As highly di e en ia ed monolaye o pola ized cells, he RPE cells main ain hemsel es ou side he cell cycle. Du ing in i o cell cul u e and ce ain pa hologies, RPE cells unde go dedi e en ia ion (epi helial- o-mesenchyme ansi ion, EMT) o become ac i ely p oli e a ing cells acqui ing a ib oblas ic mo phology. Dedi e en ia ed RPE cells exp ess cell cycle ma ke s and cy oke a ins such as cy oke a in −8, −18, and −1945. Cy okele al c osslinke s ac in binding ilamin-A (FLNA) and ez in (EZR), which is localised o apical mic o illi in he RPE, we e up eg- ula ed in he hESC-RPE. Ez in has a ole in POS phagocy osis bu i s o e exp ession has also been linked EMT46. Fib illins −1 and −2 a e s uc u al p o eins o he ECM mic o ib ils, in ol ed in a a ie y o unc ions including eye de elopmen and mo phogenesis47. O e exp ession o he cy oskele al emodelle s and cy oke a ins -8 and -18 in bo h hESC-RPE lines and cy oke a in-19 in 08/023 hESC-RPE, mos p obably e lec s he in i o na u e o he cells. The in i o hPSC-RPE di e en ia ion in ol es se ial passaging a e which he cells p oli e a e o colonize he cell cul u e su ace and hen egain hei epi helial pheno ype. The hRPE cells used o compa ison in his s udy we e no in i o cul u ed o a oid he cul u e induced changes o hei cellula pheno ype and p o eome. No signs o ans e o mo e ac i e s a us o cell cycle o p oli e a ion was de ec ed in he hESC-RPE cells. Impo an ly, highly RPE-speci ic p o eins such as he isual cycle p o eins we e iden i ied in he hESC-RPE. The RPE laye is he p inciple si e o 11-cis e inal egene a ion in he isual cycle. RLBP1 and RPE65 a e impo - an biochemical RPE ma ke s p esen in hESC-RPE and hiPSC-RPE18. The down egula ion o exp ession o hese p o eins is expec ed o he in i o di e en ia ed and cul u ed hESC-RPE as compa ed o na i e issue, since he hESC-RPE cells ha e no been in con ac wi h he neu al e ina o e inoids. I has been p e iously shown ha hiPSC-RPE cells possess he machine y o p ocess e inoids and can exhibi a unc ional isual cycle in i o and in i o. Human iPSC-RPE cells main ain RPE65 p o ein exp ession du ing cul u e in con as o p ima y cells and a e incuba ion wi h all- ans e inol, hiPSC-RPE elease 11-cis e inaldehyde in o he cul u e media48. ECM and cell adhesion- ela ed p o eins iden i ied included in eg in subuni s αV and h ee be a chains: 1, 4, and 8. In eg in ß-4, pa o he p ima y laminin ecep o in eg in α6ß4, was up egula ed in bo h hESC-RPE lines. Laminin is an impo an componen o B uch’s memb ane and he epi helial cell basemen memb ane. In eg in α6ß4 p omo es a achmen o RPE o B uch’s memb ane49 and is hus impo an o in eg a ion o hESC-RPE cell ansplan s. Abso p ion o s ay ligh by he abundan melanin g anules is ano he impo an unc ion o he RPE. P o eins in ol ed in melanin biosyn hesis such as y osinase- ela ed p o ein 1 (TYRP1) was de ec ed as being up egula ed in he hESC-RPE. High pigmen a ion s a us and sec e ion o e ina p o ec i e pigmen epi helium-de i ed ac o , PEDF (SERPINF1) by ou hESC-RPE cells has been shown p e iously18. RPE cells a e among he mos ac i e phagocy es in he body. Ci cadian shedding o POS ac i a es a synch o- nized phagocy ic esponse by RPE cells e e y mo ning50. Many phagocy osis ela ed p o eins we e de ec ed in hESC-RPE. The in eg in α β5 is in ac equi ed o POS binding o he RPE51. In eg in α β5 complexes wi h he e aspanin CD81, an in e ac ion ha p omo es pa icle binding. Engul men ecep o CD36 a he apical, www.na u e.com/scien i ic epo s/ 8 Scien i ic RepoR s | 7: 6016 | DOI:10.1038/s41598-017-06233-9 phagocy ic su ace o RPE cells and lac adhe in (Milk a globule-EGF ac o 8) a e also in ol ed in POS ecog- ni ion. Ca hepsin-D (CTSD) is a majo p o eoly ic enzyme in phagocy ic cells ha diges s opsin. Ez in is known o bind o lysosomal-associa ed memb ane p o ein 1 (LAMP-1) and CD44 du ing he o ma ion o phagocy ic acuoles50, 52. The RPE also unc ions as a ba ie be ween he neu al e ina and he enes a ed cho oid pe o ming a a ie y o ec o ial anspo unc ions (wa e , ions, me aboli es, nu ien s and was e p oduc s) be ween he wo compa - men s1. Impo an anspo e s and ion channels we e iden i ied in he hESC-RPE such as Na+K+- anspo ing ATPase subuni s alpha-1, and be a-1 and −2 (ATP1A1, ATP1B1) localized a he apical su ace o he RPE as shown p e iously o ou hESC-RPE18. Aquapo in 1 ( agmen ) (AQP1), channel p o ein ha media es wa e anspo , is apically exp essed bo h in human oe al RPE p ima y cul u es and in hESC-RPE cells19. P o eomic analyses o he plasma memb ane a e subcellula ac iona ion could p o ide u he insides in o exp ession o anspo e and channel p o eins by he hPSCs. A speci ic no mal RPE gene exp ession signa u e o 154 genes has been p oposed o alida ion o RPE-like cells de i ed om hPSC53. In ha s udy, only eigh p o eins we e analysed a p o ein le el. The changes a he mRNA le el o en do no lead o co esponding changes a he p o ein le el and la ge scale, p o ein le el s udies o he hPSC-RPE a e he e o e also impo an o alida ion. The iTRAQ me hod is limi ed o he de ec ion o only hose p o eins ha a e p esen in all samples o each iTRAQ labelling. Thus p o eins exp essed in he hESC-RPE only, o solely in he p ima y hRPE sample could no be de ec ed wi h his me hod. In addi ion, a 30 kDa il e was used in he expe imen al se up o educe noise and ion supp ession caused by he small molecules in he samples. P o eins unde he limi such as leci hin e inol acyl ans e ase (LRAT), a 25 kDa isual cycle p o ein and an impo an RPE ma ke , could no be de ec ed. In he iTRAQ, quan i ica ion o low abundancy p o eins can cause miss quan i ica ion a ising om backg ound ions54, and he e o e ca e ul da a checking is impo an espe- cially o p o eins wi h ex eme old change alues. Da a dependen analysis (DDA) mode is equi ed o iTRAQ label in ensi y based quan i ica ion and causes missing alue gene a ion in he da a as numbe o eplica e uns inc ease. This is a well-documen ed phenomenon in ITRAQ analyses55 and was also obse ed in ou da a wi h 661 p o eins de ec ed in only one analysis un i.e. one biological eplica e (Supplemen a y Table S1, p. 12–19). Despi e o hese limi a ions o he me hod, iTRAQ enables mul iplexing, ha is compa ing ou di e en samples in a single mass spec ome y expe imen . Highly eliable compa a i e, quan i a i e da a can hus be p oduced especially wi h samples ha ha e simila p o ein exp ession pa e ns56. Al hough he 1041 p o eins de ec ed only p esen s a ac ion o he o al RPE p o eome o o e 4000 indi idual p o eins de ec ed om RPE-cho oid sam- ples in o he s udies25, he da a p esen ed p o ides new, impo an , and quan i a i e in o ma ion on exp ession o o e 1000 common p o eins be ween hESC-RPE and hRPE. The idea o his s udy was o quan i a i ely compa e he p o eome o hESC-RPE o hRPE, no o accumula e he whole hESC-RPE p o eome, which would equi e a much la ge sample size and a di e en MS me hodology. New me hods like sequen ial window acquisi ion o all heo e ical agmen ion spec a (SWATH), migh enable analysis up o 4000 p o eins om RPE samples. Building a whole p o eome lib a y o he hPSC-RPE is a a ge o ou u u e s udies. To conclude, we we e able o de ec 1041 common p o eins be ween he wo hESC-RPE samples and p ima y hRPE and demons a ed ha he hESC-RPE di e en ia ion yields high quali y RPE cells. The hESC-RPE cells di - e en ia ed in i o, in se um- ee condi ions and in he absence o con ac wi h hei na u al niche, s ill esembled he hRPE exp essing RPE speci ic unc ional p o ein machine y. No p o ein abno mali ies indica i e o pa hological beha iou o he hESC-RPE was de ec ed which is impo an conside ing he u u e clinical applica ions. P o eomic app oaches enabling absolu e, label- ee quan i ica ion such as SWATH echnique could be used in he u u e o e en mo e p ecisely compa e he hPSC-RPE p o eome o na i e issue. As he hPSC-RPE also p o ide unlimi ed oppo uni ies o model e inal degene a i e diseases in i o, quan i a i e, compa a i e p o eomics opens possibil- i ies o unde s and p o ein le el disease mechanisms and disco e new he apeu ic a ge s o de as a ing diseases such as AMD, and also aid in alida ion o hPSC-RPE o clinical applica ions. Me hods Human emb yonic s em cell-de i ed RPE cells. The wo hESC lines p e iously de i ed a ou labo- a o y a Uni e si y o Tampe e we e used in his s udy: Regea08/017 (46;XX) and Regea08/023 (46;XY)57. The undi e en ia ed hESCs we e cul u e in hESC cul u e medium consis ing o KnockOu DMEM supplemen ed wi h 20% KnockOu Se um Replacemen (ko-SR, The mo Fishe Scien i ic), 2 mM Glu amax, 0.1 mM 2-me cap- oe hanol, 1% Non-essen ial amino acids, 50 U/ml penicillin/s ep omycin, and 8 ng/ml human basic ib oblas g ow h ac o (bFGF) on inac i a ed human o eskin ib oblas (CRL-2429; Ame ican Type Cul u e Collec ion) eede cells. Undi e en ia ed s em cell colonies we e passaged wi h T ypLE Selec on o esh eede cell laye e e y 10 days. Bo h cell lines a e ou inely ka yo yped and cha ac e ized o hei sel - enewal and di e en ia ion capaci ies as well as absence o mycoplasma. The hESCs we e di e en ia ed o RPE as p e iously desc ibed18. B ie ly, hESCs we e manually cu o suspen- sion and cul u ed as loa ing cell agg ega es in RPE basic medium consis ing o same eagen s as hESC medium excep o 15% ko-SR and no bFGF. Fo en ichmen , pigmen ed cells we e isola ed and seeded o human placen al collagen IV (5 µg/cm2). Fo u he ma u a ion he cells we e seeded o collagen IV (5 µg/cm2) coa ed PET cell cul u e inse s (Millipo e) and cul u ed o 129 days (Regea08/017) o 139 days (Regea08/023). Regea08/017 a passage le els 33, 34 and 70, and Regea08/023 a passage le els 44, 61, and 70 we e used o gene a ing RPE cells. Cells om h ee independen di e en ia ion expe imen s ( eplica es 1–3) we e used o he s udy. Each biological eplica e consis ed o h ee cell cul u e inse s. The hESC-RPE cells we e collec ed wi h T ypLE Selec , washed wice wi h PBS, pelle ed by cen i uga ion and s o ed as d y pelle s a −80 °C un il p o ein ex ac ion. Human RPE cells. P ima y hRPE om h ee di e en dono s we e used. The dono eyes had no mac- oscopical o mic oscopical signs o e inal degene a ion. Fo human e inal pigmen epi helium isola ion, www.na u e.com/scien i ic epo s/ 9 Scien i ic RepoR s | 7: 6016 | DOI:10.1038/s41598-017-06233-9 ollowing enuclea ion, he eye bulb was washed wi h 5% po idone iodine (Be adine), he an e io segmen was cu app oxima ely 3.5 mm pos e io o he limbus and he an e io segmen , he i eous and neu al e ina we e emo ed. The eyecup was hen illed wi h RPE cell cul u e medium (DMEM/F12, supplemen ed wi h 10% FCS, 1% an ibio ics-an imyco ics and 1% L-Glu amin) and he cells collec ed by a special glass cell sc ape o gen ly emo e he RPE om B uch’s memb ane while con inuously ba hed in hei own cell cul u e medium. The RPE was collec ed as a pelle by cen i uga ion (1000 pm, 10 min). The RPE pelle was washed wice wi h PBS, ol- lowed by cen i uga ion a e each wash, and shipped o u he analysis a −20 °C. P o ein ex ac ion. Human ECS-RPE pelle s we e suspended in 200 µl and hRPE pelle s in 400 µl o RIPA lysis bu e , supplemen ed wi h 0.1% HALT p o ein inhibi o cock ail, and dissocia ed using a VWR disposable pes le. The samples we e hen o exed o 2 min, incuba ed in an ul asonic ba h o 5 min, and on ice o 25 min. Dissocia ed samples we e cen i uged a 14 800 pm o 15 min a 4 °C, supe na an s ans e ed o clean ubes and hei p o ein concen a ions de e mined using he DC P o ein Assay Ki II (Bio-Rad Labo a o ies) acco ding o he manu ac u e ’s ins uc ions. Bo ine se um albumin (BSA; Bio-Rad Labo a o ies) was used as a s anda d, and all samples analysed in duplica es. 15 µg o o al p o ein/hESC-RPE inse we e used and pooled oge he o each biological hESC-RPE eplica e (1–3) o bo h cell lines (45 µg in o al). Equal amoun (45 µg) was used o each hRPE dono sample (1–3) (Fig.1). Addi ional pooled hRPE sample was p epa ed by pooling 15 µg o p o ein om each hRPE dono (45 µg o p o ein in o al). P o eins we e p ecipi a ed by adding six olumes o cold ace one, ollowed by quick o exing and o e nigh incuba ion a −20 °C. The ollowing day, samples we e cen i uged a 14 800 pm o 15 min a 4 °C. The ace one was disca ded and e apo a ed o 10 min, and he pelle s subjec ed o p o ein diges ion. P o ein diges ion, iTRAQ labelling, and mass spec ome y analysis. P o ein diges ion, label- ling and mass spec ome y was done as desc ibed in de ail p e iously28. 30 kDa size exclusion il e s (Pall Co po a ion) we e used in sample p epa a ion. Samples we e labelled wi h he isoba ic iTRAQ ags as shown in Fig.1 and pooled oge he o he mass spec ome y analysis. Labelled samples we e analysed by Nano-RPLC- T ipleTOF ins umen a ion using Eksigen ekspe ™ 425 NanoLC coupled o high speed T ipleTOF™ mass spec ome e (Sciex 5600+). A capilla y RP-LC column (cHiPLC® Ch omXP C18-CL, 3 µm pa icle size, 120 Å, 75 µm i.d × 15 cm; Eksigen , Conco d, Canada) was used o LC sepa a ion o pep ides. Da a p ocessing. P o ein pilo so wa e e sion 4.0.8085 (Ab Sciex) was used o analyse MS/MS da a sea ched agains he UniP o /Swiss-P o p o ein da abase o p o ein iden i ica ion. Some impo an se ings in he Pa agon sea ch algo i hm in p o ein pilo we e con igu ed as ollows. Sample ype: iTRAQ 4plex (pep ide labelled), Cys-alkyla ion: MMTS, Diges ion: T ypsin, Ins umen : T ipleTOF 5600+, Sea ch e o : ho ough ID. False disco e y a e (FDR) analysis was pe o med in he P o ein pilo and FDR <1% was se o p o ein iden i i- ca ion. Only pep ides wi h 99% con i ma ion we e included in iden i ica ion and quan i ica ion. Sha ed pep ides we e excluded om quan i ica ion ha was pe o med using P o einPilo so wa e wi h s aigh a e age o pep- ides (p o eins wi h dis inc i e pep ides, n = 1964, Fig.1b). Da a p ocessing included log2- ans o ma ion and cen al endency no maliza ion. G aphical p esen a ion o he da a no maliza ion is shown in Supplemen a y Fig.S4. Ranking o p o eins (measu e o quali y) was c ea ed by aking he mean o he ankings in di e en uns. Duplica e p o eins wi h he same gene names we e me ged so ha only one p o ein name was kep . I bo h SwissP o and T embl duplica e p o eins we e p esen , SwissP o was chosen (numbe o p o eins a e duplica e check, n = 1729, Fig.1b). The spec al peaks o p o eins wi h o e 4- old di e ence (log2 old change alue >2 o <−2) in exp ession in ei he hESC-RPE line compa ing o hRPE we e manually checked, and mis- quan i ied p o eins we e omi ed. These included pho o ansduc ion ela ed p o eins such as opsin, eco e in, hodopsin, od ou e segmen memb ane p o ein 1, and pe iphe in-2 ha we e p esen in high abundance in he human dono samples bu misquan i ied due o noise ions in he hESC-RPE. The manual checking con i med ha hese pho o ecep o speci ic p o eins we e no p esen in he hESC-RPE (da a no shown). The da a was il e ed so, ha only p o eins which we e iden i ied in a leas wo biological eplica e samples o bo h hESC-RPE lines and human RPE (i.e. in wo o he h ee analysis uns), we e conside ed eliable and included in da a anal- yses. P o eins based on de ec ion in only one biological eplica es (one analysis un, n = 661) we e il e ed ou . Finally, p o eins wi h ei he echnical o biological CV >100 (n = 27), we e excluded om he inal da a analysis. The p o eins il e ed ou a e shown in Supplemen a y TableS1. P- alues, which a e shown in he Supplemen a y TableS1 and Supplemen a y Fig.S1, we e ob ained using a one sample - es , whe e he means o echnical ep- lica es we e aken p io o es ing. Unadjus ed p- alues we e p esen ed (as no signi ican changes we e seen o 08/017 hESC-RPE o hRPE, and only se en p o eins (Unip o IDs: O15537, B7ZB41, J3KPF3, Q6FHV6, Q76LA1, V9HWH2, X5DNM4) eached signi icance in old change o 08/023 wi h adjus ed he p- alues). Classi ica ion in o speci ic g oups o in e es was done by GO mapping wi h he GO e ms connec ed o each p o ein’s UniP o accession using UniP o .ws package in R (Ma c Ca lson. UniP o .ws: R In e ace o UniP o Web Se ices. R package e sion 2.10.2.). The GO e ms o in e es as well as hei o sp ing e ms, i.e. mo e speci ic e ms, we e included in he GO e m mapping p ocess due o he loose hie a chical s uc u e o GO anno a ion. The GO e ms used a e lis ed in Supplemen a y TableS3. P ocessing o he da a, GO mapping, box- plo s, and olcano plo s o isualiza ion we e c ea ed using R so wa e e sion 3.2.3 (R Co e Team, Founda ion o S a is ical Compu ing, Vienna, Aus ia, h ps://www.Rp ojec .o g/). QIAGEN’s Ingenui y® Pa hway Analysis (IPA®, QIAGEN Redwood Ci y, www.qiagen.com/ingenui y) was used o conduc canonical pa hway analyses using he di e en ially exp essed (log2 old change >1 o <−1) p o eins in 08/017 and 08/023 hESC-RPE com- pa ed o hRPE. The PANTHER Classi ica ion Sys em Ve sion 11.0 ( elease da e July 15, 2016) was used o classi y he p o eins acco ding o hei in ol emen in biological p ocesses (h p://pan he db.o g/). Cha s we e c ea ed in Mic oso Excel 2013.