hiPSC-derived hepatocytes closely mimic the lipid profile of primary hepatocytes: A future personalised cell model for studying the lipid metabolism of the liver

Recei ed: 19 Ap il 2018 | Accep ed: 9 July 2018 DOI: 10.1002/jcp.27131 ORIGINAL RESEARCH ARTICLE hiPSC‐de i ed hepa ocy es closely mimic he lipid p o ile o p ima y hepa ocy es: A u u e pe sonalised cell model o s udying he lipid me abolism o he li e Mos a a Kiameh 1 | Anna Alexano a 1 | Leena E. Vii i 1 | Lau a Heiskanen 2 | Te hi Vihe aa a 2 | Dimple Kauhanen 2 | Kim Ek oos 5 | Reijo Laaksonen 1,2 | Reijo Käkelä 3 | Ka iina Aal o‐Se älä 1,4 1 Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland 2 Zo a Biosciences, Espoo, Finland 3 Facul y o Biology and En i onmen al Sciences, Uni e si y o Helsinki, Helsinki, Finland 4 Hea Hospi al, Tampe e Uni e si y Hospi al, Tampe e, Finland 5 Lipidomics Consul ing L d, Espoo, Finland Co espondence Mos a a Kiameh , Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland. Email: [email p o ec ed] Funding in o ma ion Finnish Ca dio ascula Founda ion; Ins umen a iumin Tiedesää iö; FP7 Heal h, G an /Awa d Numbe s: F2‐2013‐602222, 2012‐3057392 Abs ac Hepa ocy e‐like cells (HLCs) di e en ia ed om human‐induced plu ipo en s em cells o e an al e na i e pla o m o p ima y human hepa ocy es (PHHs) o s udying he lipid me abolism o he li e . Howe e , despi e hei g ea po en ial, he lipid p o ile o HLCs has no ye been cha ac e ized. He e, we comp ehensi ely s udied he lipid p o ile and a y acid (FA) me abolism o HLCs and compa ed hem wi h he cu en s anda d hepa ocy e models: HepG2 cells and PHHs. We di e en ia ed HLCs by i e commonly used me hods om h ee cell lines and ho oughly cha ac e ized hem by gene and p o ein exp ession. HLCs gene a ed by each me hod we e assessed o hei unc ionali y and he abili y o syn hesize, elonga e, and desa u a e FAs. In addi ion, lipid and FA p o iles o HLCs we e in es iga ed by bo h mass spec ome y and gas ch oma og aphy and hen compa ed wi h he p o iles o PHHs and HepG2 cells. HLCs esembled PHHs by exp essing hepa ic ma ke s: sec e ing albumin, lipop o ein pa icles, and u ea, and demons a ing simila i ies in hei lipid and FA p o ile. Unlike HepG2 cells, HLCs con ained low le els o lysophospholipids simila o he con en o PHHs. Fu he mo e, HLCs we e able o e icien ly use he exogenous FAs a ailable in hei medium and simul aneously modi y simple lipids in o mo e complex ones o ul ill hei needs. In addi ion, we p opose ha inc easing he polyunsa u a ed FA supply o he cul u e medium may posi i ely a ec he lipid p o ile and unc ionali y o HLCs. In conclusion, ou da a showed ha HLCs p o ide a unc ional and ele an model o in es iga e human lipid homeos asis a bo h molecula and cellula le els. © 2018 The Au ho s. Jou nal o Cellula Physiology Published by Wiley Pe iodicals, Inc. J Cell Physiol. 2019;234:3744–3761.3744 | wileyonlinelib a y.com/jou nal/jcp --------------------------------------------------------------------------------------------------------------------------- This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Abb e ia ions: APOA1, apolipop o ein A‐I gene; APOB, apolipop o ein B gene; ASGR, asialoglycop o ein ecep o ; BMP4, bone mo phogenic p o ein 4; CE, choles e yl es e ; Ce , ce amide; DAG, diacylglyce ol; DE, de ini i e endode m; EGF, epide mal g ow h ac o ; ELOVL, a y acid elongase; FA, a y acid; FADS, a y acid desa u ase; FASN, a y acid syn hase; Gb3, globo iaosylce amide; GCK, Glucokinase; Glc/GalCe , glucosyl/galac osylce amide; GSL, glycosphingolipid; HGF, hepa ocy e g ow h ac o ; hiPSC, human induced plu ipo en s em cell; HLC, hepa ocy e‐like cell; hLTR, Human Li e To al RNA; LacCe , lac osylce amide; LPC, lysophospha idylcholine; LPE, lysophospha idyle hanolamine; LPI, lysophospha idylinosi ol; LPL, lysophospholipid; M1, Me hod 1; M2, Me hod 2; M3, Me hod 3; M4, Me hod 4; M5, Me hod 5; MUFA, monounsa u a ed a y acid; PC, phospha idylcholine; PE, phospha idyle hanolamine; PHH, p ima y human hepa ocy e; PI, phospha idylinosi ol; PL, phospholipid; PUFA, polyunsa u a ed a y acid; SFA, sa u a ed a y acid; SL, sphingolipid; SM, sphingomyelin; TAG, iacylglyce ol; UGCG, UDP‐glucose ce amide glucosyl ans e ase. KEYWORDS a y acid (FA), gas ch oma og aphy, hepa ocy e‐like cell (HLC), HepG2, human‐induced plu ipo en s em cell (hiPSC), lipidomics, mass spec ome y (MS), p ima y human hepa ocy e (PHH) 1 | INTRODUCTION The li e plays an impo an ole in he egula ion o many physiological unc ions o he body, including lipid and ca bohyd a e me abolism, glycoly ic/u ea me abolism, plasma p o ein syn hesis, and he de oxi ica ion o a wide a ie y o molecules (Si‐Tayeb e al., 2010). Hepa ocy es, which comp ise abou 70% o he li e ’s mass, o igina e om he an e io po ion o he de ini i e endode m (DE), one o he h ee emb yonic laye s (Blouin, Bolende , & Weibel, 1977). Hepa ocy es handle many c ucial me abolic unc ions o he li e , including he syn hesis o lipop o eins, iacylglyce ols (TAGs), choles e ol, and phospholipids (PLs; Go dillo, E ans, & Gouon‐E ans, 2015). P ima y human hepa ocy es (PHHs) a e cu en ly conside ed he “gold s anda d”in cell modeling o s udying, o example, li e physiology, oxici y, and lipid homeos asis. Typically, PHHs a e ob ained om cada e ic dono s, bu hey a e sca ce and unc ionally he e ogeneous, and i is ha d o main ain hem in a cul u e. When cul u ed, PHHs loose unc ionali y ela i ely as , and hei li e ‐ speci ic ea u es p og essi ely de e io a e, which pa icula ly ham- pe s long‐ e m s udies (Elau e al., 2006; Godoy e al., 2013). To add ess hese limi a ions, a ious human hepa oma cell lines, including HepG2 and Huh7, ha e been used due o hei ease o handling, unlimi ed li e span, and s able pheno ype. Ne e heless, hey do no ai h ully mi o he me abolic ac i i ies o heal hy li e cells. In ac , he exp ession le els and p o iles o genes in ol ed in li e ‐speci ic unc ions a e poo ly p esen ed in hese sys ems (Olsa sky e al., 2007). As a esul , hepa oma cell lines ha e ailed o p edic he nume ous ad e se hepa o oxic side e ec s o new d ugs (Cas ell, Jo e , Ma ínez‐Jiménez, & Gómez‐Lechón, 2006). Al e na i ely, animal models, such as a s and mice, o animal p ima y hepa ocy es ha e been widely used o s udy lipid me abolism and lipop o ein p oduc ion in he li e (K ilek al, Lin, Cheng, & Abum ad, 1994). Howe e , he in o ma ion gained om mu ine cells is no ully ansla able o humans, and he e a e signi ican di e ences in lipid me abolism be ween he species. Animal models a e also expensi e and unsui able o la ge‐scale sc eening. Fu he mo e, due o g owing e hical conce ns, he e is an u gen need o educe he use o oden s and o he animal models in esea ch. Human‐induced plu ipo en s em cells (hiPSCs) p o ide an unlimi ed supply o issue‐speci ic di e en ia ed cell ypes o disease modeling and cell he apy. Hepa ocy es di e en ia ed om plu ipo en s em cells ci cum en he p oblem o he limi ed a ailabili y o cells aced when wo king wi h PHHs. They show e y simila cha ac e is ics o PHHs; o ins ance, hey sec e e albumin as well as u ea, and o some ex en exp ess d ug anspo e s and cy och ome P450 enzymes (Kia e al., 2012). Unlike oden hepa ocy es and some human hepa oma cells, hese hepa ocy e‐like cells (HLCs) a e sensi i e o hepa i is C i us in ec ion and suppo i al eplica ion simila o PHHs (Schwa z e al., 2012). Pa ien ‐speci ic iPSC lines p o ide us wi h an ad an ageous sys em o HLCs om pa ien s o di e en gene ic backg ounds, a p e equisi e o he de elopmen o pe sonalised medicine. The gene a ion o hiPSC‐de i ed HLCs (hiPSC‐HLCs) in la ge quan i ies enables hei use in modeling inbo n e o s o hepa ic me abolism, unde s anding he molecula basis o li e cell di e - en ia ion, s udying disease mechanisms, and acili a ing d ug disco e y and sa e y. HLCs ha e al eady p o en o be o g ea alue in de eloping no el he apeu ics (Medine e al., 2013; Szkolnicka e al., 2014) and iden i ying he noncoding mic o‐RNAs egula ing human li e damage (Szkolnicka e al., 2016; Yang e al., 2016). HLCs ha e also been success ully used as in i o cell cul u e sys ems, o example, o ecapi ula e he pa hophysiology o amilial hype cho- les e olemia (Cayo e al., 2012) and human choles e ol homeos asis (K uege e al., 2013). Lipid de ec s a e cen al o he pa hogenesis o many common diseases, such as a he oscle osis (Meikle e al., 2011; S übige e al., 2012) and nonalcoholic a y li e disease (Ruhanen e al., 2017; Younossi e al., 2016). The e o e, hiPSC‐HLCs o e a g ea pla o m o in es iga ing he basic mechanisms o lipid me abolism and i s dys egula ion in a pa ien ‐speci ic manne . Howe e , o da e, no de ailed s udies ha e ye been pe o med on he lipid p o ile and a y acid (FA) me abolism o HLCs. To be e use HLCs as a cell model o s udy he ole o molecula lipids in li e diseases, i is essen ial o know hei lipid p o ile in ela ion o ac ual human adul li e issue and o he cu en ly used cell models, namely PHHs and hepa oma cell lines. Hepa ic di e en ia ion me hods ha e g ea ly imp o ed o e he las decade, enabling he e icien gene a ion o high quali y HLCs om plu ipo en s em cells (Came on e al., 2015; Hannan, Sege i z, Touboul, & Vallie , 2013; Hay e al., 2008; Kajiwa a e al., 2012; Si‐Tayeb e al., 2010). Ne e heless, he expe imen al de ails and some di e en ia ion ac o s di e be ween he cu en p o ocols, which migh a ec he pheno ype o he HLCs. To ep oduce he physiological condi ions o he human li e , i is c ucial o gene a e unc ional HLCs ha a e as simila as possible o PHHs. In his s udy, we desc ibe i e p o ocols o gene a ing HLCs om hiPSCs and comp ehensi ely compa e he mo phology, gene ics, biochemis y, and unc ional ai s o he HLCs p o- duced. In addi ion, we compa e he HLCs wi h he wo mos common cell models cu en ly used—PHHs and HepG2 cells—as well as human li e issue. Mos impo an ly, he lipid p o iles o HLCs,PHHs,andHepG2cellsa eanalyzedbybo hmass spec ome y (MS) and gas ch oma og aphy, and he simila i ies and main di e ences be ween he cell ypes a e discussed in ligh o selec ed key genes in ol ed in FA me abolism. Finally, he lipid p o ile o HLCs is ully cha ac e ized, and he po en ial and KIAMEHR ET AL. | 3745 limi a ions o HLCs as a model o s udying lipid me abolism a e e alua ed. 2 | MATERIAL AND METHODS 2.1 | E hical issues The s udy and pa ien ec ui men ha e been app o ed by he E hics Commi ee o Tampe e Uni e si y Hospi al (app o al numbe : R12123). All pa icipan s p o iding skin biopsies we e adul s mo e han 18 yea s old who had signed an in o med consen o m a e ecei ing bo h o al and w i en desc ip ions o he s udy. 2.2 | hiPSC ep og amming and cell cul u e Th ee hiPSC lines (UTA.10100.EURCAs, UTA.11104.EURCAs, and UTA.11304.EURCCs) we e gene a ed di ec ly om he ib oblas s o h ee indi iduals. Plu ipo ency was induced wi h he Sendai ep og amming ki (OCT4, SOX2, KLF4, C‐MYC; Cy oTune; Li e Technologies, USA) based on he p o ocol desc ibed by Ohnuki, Takahashi, Yamanaka, (2009) and Takahashi & Yamanaka, (2006). hiPSCs we e hen main ained as desc ibed be o e (Kiameh e al., 2017). De ails o he hiPSC cell cul u e a e also desc ibed in he Suppo ing In o ma ion. 2.3 | Hepa ic di e en ia ion In all me hods excep Me hod 4 (M4), hiPSCs we e ans e ed om mouse emb yonic ib oblas s o Gel ex TM (USA; Ca : A14133‐ 01, 1:50 dilu ion), kep in mTeSR1 TM medium, and adap ed o he changed cul u e condi ions o a ew passages be o e commencing he di e en ia ion. In M4, hiPSCs we e adap ed on Laminin 521 (BioLamina, Lo : 80104) i s , and he di e en ia ion was comple ed on Laminin mix 111/521‐coa ed pla es (3:1 a io, 10 µg/ml). The hiPSCs used in all me hods we e a passage 20 o highe be o e commencing he di e en ia ion. Th ee lines we e di e en ia ed using each me hod, excep Me hod 5 (M5), which was applied o wo cell lines. Figu e 1 shows a schema ic iew o all i e me hods used in he s udy. De ails o he i e di e en ia ion me hods a e desc ibed in de ail in he Suppo ing In o ma ion. 2.4 | PHHs and HepG2 cells C yop ese ed PHHs (Ca . No. HMCPIS, Lo . HU8210, USA) we e pu chased om Gibco ® , and hepa ocellula ca cinoma cells (HepG2, ATCC‐HB‐8065, Lo . No. 59947519) we e pu chased om ATCC ™ . Bo h cells we e pla ed acco ding o he manu ac u e ’s ins uc ions. PHHs we e cul u ed in William’s E medium (A1217601, Gibco, USA) supplemen ed wi h cock ail B (Gibco, CM 4000) and dexame hasone, whe eas he HepG2 cells we e cul u ed in Dulbecco’s modi ied Eagle medium supplemen ed wi h 10% FBS. 2.5 | RT‐polyme ase chain eac ion RNA ex ac ion and polyme ase chain eac ion (PCR) o plu ipo- ency ma ke s (OCT4,NANOG,SOX2, and SSEA4) we e pe o med as published be o e (Manzini, Vii i, Ma ila, & Aal o‐Se älä, 2015). 2.6 | Quan i a i e PCR (qPCR) analysis RNA samples we e collec ed a he hiPSC, DE, and HLC s ages, and RNA was ex ac ed using an RNeasy ki (Qiagen, Ge many, Ca . No. 74106). Complemen a y DNA (cDNA) was gene a ed using a high capaci y cDNA Re e se T ansc ip ion ki (Applied Biosys ems) acco ding o he manu ac u e ’s ins uc ions in he p esence o an RNase inhibi o . cDNA was mul iplied ei he by he Powe SYBR G een PCR Mas e Mix (Li e Technology, Ca . No. 1408470, Aus in, TX) and gene‐speci ic p ime s (OCT4,SOX17,FOXA2,AFP,ALB)o by he TaqMan Uni e sal Mas e Mix (Applied Biosys ems, 4304437, Aus in, TX) and gene‐speci ic TaqMan p obes (APOA1,APOB,FADS1, FADS2,ELOVL2,ELOVL5,FASN) using he BioRad CFX384 Real‐Time PCR De ec ion Sys em. Values we e no malized o GAPDH, which was used as an endogenous con ol, and ela i e quan i ica ion was FIGURE 1 A schema ic ep esen a ion o he i e hepa ic di e en ia ion p o ocols used o gene a ing hepa ocy e‐like cells (HLCs). Di e en ia ion in Me hod 1 and Me hod 2 (M1 and M2) we e s a ed wi h hiPSC colonies, in Me hod 3 and Me hod 4 (M3 and M4) wi h dissocia ed iPSC single cells, and in Me hod 5 (M5) wi h 24 h s pos ‐cul u ed hiPSCs. Pla es in Me hod 4 (M4) we e coa ed wi h a mix o Laminin 111/521 (3:1 a io) ins ead o he Gel ex TM used in he emainde o he me hods. hiPSC: human‐induced plu ipo en s em cell [Colo igu e can be iewed a wileyonlinelib a y.com] 3746 | KIAMEHR ET AL. calcula ed by he ΔΔCT me hod (Li ak & Schmi gen, 2001). PHH was used as he e e ence sample. Fi s Choice ® Human Li e To al RNA (hLTR, Ca . No. AM7960), pu chased om Ambion®, was used as an ex a con ol. The esul s om qPCR we e compa ed o he HLCs and he h ee e e ence samples (PHHs, hLTR, and HepG2 cells). 2.7 | Immunos aining Cells we e ixed, s ained, and isualized as desc ibed be o e (Kiameh e al., 2017). De ails a e also p o ided in he Suppo ing In o ma ion. The pe cen age o ALB‐posi i e cells and binuclea HLCs we e calcula ed manually by coun ing HLCs in 3–5 s ained a eas and calcula ing he a e age. 2.8 | low‐densi y lipop o ein up ake The abili y o he cells o up ake low‐densi y lipop o ein (LDL) was e alua ed by incuba ing he HLCs wi h labeled LDL (Cell‐based assay ki , Cayman, USA, Ca . No. 10011125) o 4 h , a e which he cells we e imaged by luo escen mic oscopy. 2.9 | FACS analysis To analyze he numbe o CXCR4‐posi i e cells, endode mal cells we e de ached wi h Ve sene (Gibco ® , UK), suspended in 3%–5% bo ine se um albumin bu e , s ained wi h a PE‐conjuga ed CXCR4 an ibody (R&D Sys ems FAB173P, Minneapolis, 10 µl o 10 6 cells) o 15 min a RT, washed h ee imes, and analyzed using he Accu i TM C6 de ice (BD Biosciences). 2.10 | Albumin, u ea, and TAG sec e ion A he la e s age o hepa ic di e en ia ion, HLCs we e e alua ed o hei unc ionali y. The albumin, u ea, and TAG con en o he condi ioned medium we e de e mined, espec i ely, wi h he Human Albumin ELISA Quan i a ion ki (Be hyl Labo a o y), he Quan i- Ch om TM U ea Assay Ki (BioAssay Sys ems, USA), and he T iglyce ide Quan i ica ion Ki (BioVision Inc., Ca . No. K622‐100, USA) acco ding o he manu ac u e s’ins uc ions. The alues we e no malized o cell numbe s. The esul s we e hen compa ed wi h he da a om he PHHs and HepG2 cells, which we e cul u ed in pa allel wi h he HLCs. 2.11 | Lipid mass spec ome y 2.11.1 |Lipid sample p epa a ion and ex ac ion Lipids (sphingolipids [SL], choles e ol, glyce olipids, and glyce opho- spholipids) we e ex ac ed om he HLCs by Hamil on Robo ics AB and s udied by sho gun lipidomics. The de ailed p ocedu e is desc ibed by Kiameh e al., (2017). 2.11.2 |Mass spec ome ic analyses and da a p ocessing In he sho gun lipidomics (choles e yl es e [CE], diacylglyce ol [DAG], sphingomyelin [SM], lysophospha idylcholine [LPC], lysopho- spha idyle hanolamine [LPE], lysophospha idylse ine, lysophospha i- dylglyce ol, and lysophospha idylinosi ol [LPI]), lipid ex ac s we e analyzed on a hyb id iple quad upole/linea ion ap mass spec o- me e (QTRAP 5500) equipped wi h a obo ic nano low ion sou ce (NanoMa e, Ad ion Biosciences Inc., I haca, NJ) as desc ibed by Heiskanen, Suoniemi, Ta, Ta aso , & Ek oos, (2013). Molecula lipids we e analyzed in posi i e ion mode using lipid class‐speci ic p ecu so ion o neu al loss scans (Ek oos, Che nushe ich, Simons, & She chenko, 2002; Ek oos e al., 2003). Sphingolipids (ce amide [Ce ], glucosyl/galac osylce amide [Glc/ GalCe ], lac osylce amide [LacCe ], and globo iaosylce amide [Gb3]) and molecula PLs (phospha idylcholines [PC], phospha idyle hano- lamines [PE], and phospha idylinosi ols [PI]) we e analyzed wi h a a ge ed app oach using ul a‐high‐p essu e liquid ch oma og aphy‐ mass spec ome y (UHPLC‐MS; Me ill, Sulla ds, Allegood, Kelly, & Wang, 2005). An analy ical Acqui y BEH C18, 2.1 × 50 mm column wi h a pa icle size o 1.7 µm (Wa e s, Mil o d, MA) hea ed o 60°C was used. Mobile phases consis ed o 10 mM ammonium ace a e in wa e wi h 0.1% o mic acid (sol en A) and 10 mM ammonium ace a e in ace oni ile:isop opanol (4:3, / ) con aining 0.1% o mic acid (sol en B). The low a e was se o 500 µl/min. Sphingolipids we e sepa a ed wi h a 15 min linea g adien om 75% B o 100% B, while molecula PLs we e analyzed using a 10 min g adien om 75% B o 80% B. Bo h sphingolipids and PLs we e analyzed on a hyb id iple quad upole/linea ion ap mass spec ome e (5500 QTRAP) equipped wi h an UHPLC sys em (CTC HTC PAL au osample and Rheos Alleg o pump o Shimadzu Nexe a X2) using a mul iple eac ion moni o ing based me hod in posi i e ion mode o sphingolipids and nega i e ion mode o molecula PLs. Cu ain gas was se a 25; he ion sp ay ol age was se a 5000 V in posi i e ion mode and −4500 V in nega i e ion mode, and he ion sou ce was hea ed o 400°C in posi i e mode and o 300°C in nega i e ion mode. The collision ene gy was op imized o each lipid class. Iden i ied lipids we e quan i ied by no malizing agains hei espec i e in e nal s anda d (Ejsing e al., 2006) and o al p o ein concen a ions in he cell sample. To al p o ein concen a ions we e de e mined using he Mic o BCA ™ P o ein Assay Ki (The mo Scien i ic Pie ce P o ein Resea ch P oduc s) acco ding o he manu ac u e ’s ins uc ions. Da a p ocessing was pe o med by Mul iQuan , LipidView (AB Sciex) so wa e and SAS. 2.12 | FA gas ch oma og aphy To con i m ou obse a ions om he MS analysis o molecula lipids and o in es iga e he e ec o he medium on he FA p o ile o he cells, he FA composi ion o HLCs, PHHs, and HepG2 cells and hei media was analyzed by gas ch oma og aphy as desc ibed in de ail by Kiameh e al., (2017). B ie ly, he acyl chains in he cell pelle KIAMEHR ET AL. | 3747 lipids o he lipid esidues o he ni ogen‐d ied media we e con e ed o FA me hyl es e s (FAMEs) in a anses e i ica ion eac ion wi h 1% me hanolic H 2 SO 4 . The quan i a i e analysis o he FAMEs, which we e ex ac ed in o hexane, was pe o med using a Shimadzu GC‐2010 Plus gas ch oma og aph wi h a lame‐ioniza ion de ec o , and he FAME s uc u es we e iden i ied by Shimadzu GCMSQP2010 Ul a wi h a mass selec i e de ec o . In bo h sys ems, he componen s o he FAME mix u es we e sepa a ed in ZB‐wax capilla y columns (30 m, 0.25 mm ID, 0.25 μm ilm; Phenomenex). The calcula ions o he FA composi ions and concen a ions ollowed s anda d p ocedu es (Kiameh e al., 2017), and he FAs we e ma ked by using he abb e ia ions: [ca bon numbe ]:[numbe o double bonds] n‐[posi ion o he i s double bond calcula ed om he me hyl end] (e.g., 22:6n‐3). 2.13 | S a is ical analysis G aphPad P ism e sion 5.02 so wa e was used o he da a analysis. Da a a e p esen ed as means ± s anda d de ia ion wi h n ep esen ing he numbe o independen expe imen s. The esul s we e compa ed using one‐way analysis o a iance, ollowed by Bon e oni’s mul iple‐compa ison es . A p alue < 0.05 was con- side ed s a is ically signi ican . 3 | RESULTS 3.1 | Cell mo phology du ing hepa ic di e en ia ion In all me hods, d ama ic mo phological changes we e obse ed, pa icula ly du ing he i s ew days o DE di e en ia ion. In all me hods excep M5, mig a ing DE cells possessed a spiky mo phol- ogy, whe eas in M5, mig a ing cells we e ins ead mo e ound o squa e‐shaped (Suppo ing In o ma ion Figu e S1a). In addi ion, he amoun o cell dea h in M5 was conside ably lowe han in he o he me hods. No mo phological di e ences we e obse ed be ween he DE cells ea ed wi h CHIR 99021 (Me hod 1, M1) o Wn 3 (Me hods 2–4, M2–4). Ini ia ing di e en ia ion wi h single cells in M3 and M4 did no yield a highe e iciency o DE o ma ion compa ed wi h M1 and M2, which we e s a ed wi h colonies (Suppo ing In o ma ion Figu es S1a and S3). Howe e , we did obse e cells wi h a DE mo phology appea ing one o e en wo days ea lie in me hods ini ia ed wi h single cells compa ed wi h me hods ini ia ed wi h colonies (Suppo ing In o ma ion Figu e S1a). A he hepa ic speci ica ion s age, cells ea ed wi h basic ib oblas g ow h ac o , bone mo phogenic p o ein 4 (BMP4), and hepa ocy e g ow h ac o (HGF; M1) clea ly had a di e en mo phology compa ed wi h he cells ea ed wi h DMSO (M2–5; Suppo ing In o ma ion Figu e S1b), which migh imply di e en pa hways owa d hepa oblas s in hose p o ocols. Binuclea ion is a ea u e o adul hepa ocy es and gene ally conside ed a sign o e minal di e en ia ion (Miyaoka & Miyajima, 2013). We ound 29% o he PHHs and on a e age 10% o he HLCs o be binuclea ( Figu es 1b and 2a). The HLCs di e en ia ed by M4 showed he closes binuclea i y (16%) o he PHHs. No indi idual cell line was shown o be mo e po en in gene a ing binuclea cells. 3.2 | Cha ac e iza ion o DE and HLCs a he p o ein le el hiPSCs exp essed OCT4 p o ein, which was los du ing he DE s age, while he exp ession o DE ma ke s SOX17 and FOXA2 was up egula ed (Suppo ing In o ma ion Figu e S2a). The e iciency o he DE di e en ia ion, es ima ed by measu ing he CXCR4 exp es- sion by low cy ome y, did no di e ac oss M1, M2, and M3 (Suppo ing In o ma ion Figu e S2b). In M4 and M5, he amoun o CXCR4‐posi i e cells was lowe han in he o he me hods. The imma u e hepa ic ma ke AFP was exp essed in hepa ic p ogeni o cells and emained exp essed un il he la e s ages in all i e me hods (Figu e 2a). In addi ion, ALB, LDL ecep o (LDL‐R), and asialoglycop o ein ecep o (ASGR) we e all exp essed in ma u e HLCs (Figu e 2a and Suppo ing In o ma ion Figu e S5). The a e age o he ALB‐posi i e cells in M1 o M5 was 9.8%, 16.6%, 20.8%, 37.7%, and 31.5%, espec i ely, and he pe cen age in M4 was signi ican ly highe han in M1, M2, and M3, bu no in M5 (Figu e 2b). Mo e han 90% o M2‐ o M5‐HLCs we e posi i e o ASGR (da a no shown). 3.3 | Gene egula ion As expec ed, OCT4 was highly exp essed a he iPSC s age, whe eas a he DE s age, OCT4 was d ama ically down egula ed in mos o he cell lines, and SOX17 and FOXA2 we e highly exp essed (Suppo ing In oma ion Figu e S6). Fu he di e en ia ion owa d HLCs esul ed in signi ican down egula ion o SOX17, whe eas FOXA2 emained up egula ed du ing he es o he di e en ia ion and ma u a ion o he HLCs (Figu e 2c and Suppo ing In oma ion Figu e S7). The HLCs exp essed SOX17 a he same le els as hLTR. The le el o FOXA2 in he HLCs was compa able o hose in he e e ence samples PHH, hLTR, and HepG2. AFP was up egula ed du ing he ea ly and la e hepa ic di e en ia ion s ages, indica ing he imma u e cha ac e is ic o he HLCs. The exp ession o AFP in he M2‐HLCs was s a is ically signi ican ly highe han in he M1‐HLCs. ALB was d ama ically up egula ed in ma u e HLCs, up o 2 × 10 5 ‐ old compa ed wi h ha in he hiPSCs, and i s le els emained close bu below hose ound in he PHHs. The exp ession o ALB in he M5‐HLCs was signi ican ly highe han in he M1‐HLCs (p< 0.01) and M3‐HLCs (p< 0.05). The le els o ALB exp ession we e compa able be ween he HepG2 and he PHHs; howe e , ALB exp ession was abou 19‐ old highe in he hLTR when compa ed wi h he PHHs. 3.4 | Hepa ic ma u a ion and unc ionali y The li e is esponsible o p oducing se um albumin. The e o e, we e alua ed he abili y o HLCs o sec e e albumin in o he condi ioned medium. All HLCs we e able o sec e e albumin (Figu e 2d). Albumin sec e ion by UTA.11304 di e en ia ed by M3 was abou ou old 3748 | KIAMEHR ET AL. FIGURE 2 Con inued. KIAMEHR ET AL. | 3749 highe compa ed wi h he same cell line di e en ia ed by M1 o M2, and wo old highe compa ed wi h M4 (da a no shown), bu no o he s a is ically signi ican di e ences we e obse ed be ween he di e en ia ion p o ocols. Howe e , PHHs sec e ed signi ican ly la ge amoun s o albumin han he HLCs o e en he HepG2 cells. The le el o sec e ed u ea by he cell lines di e en ia ed by M3 and M4 was conside ably highe compa ed wi h same lines di e en ia ed by M1, M2, and M5 and ela i ely close o he amoun o u ea sec e ed by he PHHs (Figu e 2d). In ac , he le el o u ea sec e ed by he HLCs di e en ia ed by M1, M2, and M5 was compa able o he HepG2 cells, which sec e ed 6.5‐ old less u ea han he PHHs. TAG sec e ion in he cell lines di e en ia ed by M3, M4, and M5 was a simila le els as in he PHHs (Figu e 2d). Howe e , cell lines di e en ia ed by M1 and M2 sec e ed, on a e age, 5.9‐and 3.8‐ old mo e TAG han he PHHs. We we e no able o de ec sec e ed TAG in he HepG2 cul u e medium due o medium in e e ence. Apolipop o ein A‐I(APOA1) encodes o apoA, which is he main p o ein componen o high‐densi y lipop o eins (HDL). The p oduc o apolipop o ein B (APOB) is he main p o ein componen o e y‐low‐densi y lipop o ein (VLDL) and LDL. The exp ession o bo h APOA1 and APOB in he HLCs was compa able o he HepG2 cells, PHHs, and hLTR, which u he indica ed he unc ionali y o he HLCs (Figu e 2c). In e es ingly, bo h APOA1 and APOB we e exp essed almos 5.5‐ old mo e in hLTR compa ed wi h he PHHs. All he cell lines we e able o up ake he labeled LDL om he cul u e medium (Suppo ing In o ma ion Figu e S5). This was con i med by s aining he LDL‐R by monoclonal an ibody (Suppo ing In o ma ion Figu e S4). 3.5 | Lipid p o iles o HLCs di e en ia ed by di e en me hods The HLCs di e en ia ed by M3, M4, and M5 showed supe io unc ionali y as o TAG and u ea sec e ion when compa ed wi h M1 and M2. In addi ion, he M5‐HLCs exp essed highe ALB compa ed wi h he HLCs di e en ia ed by M1 and M3. The e o e, we selec ed he HLCs di e en ia ed by M3, M4, and M5, analyzed hei lipid p o ile by MS, and compa ed hei lipid p o ile. In addi ion, he lipid con en s o hei uncondi ioned media we e s udied o in es iga e he in luence o cul u e media lipids and hei FAs on he cells. O e all, 15 majo lipid classes—including CE, DAG, PC, LPC, PI, LPI, PE, LPE, SM, Ce , LacCe , Glc/GalCe , and Gb3—we e in es iga ed, and al oge he mo e han 150 molecula species we e de ec ed and s udied (Suppo ing In o ma ion Tables S2 and S3). The lipid p o ile o he HLCs di e en ia ed by M3, M4, and M5 closely esembled each o he ( Figu es ). In ac , only he le els o h ee PC species (PC 16:1–20:4, PC 17:0–18:1, and PC 17:0–20:4) we e sligh ly, bu s a is ically signi ican ly, lowe in M4 (Figu e 3, ma ked by blue a ows) compa ed wi h he o he me hods. No o he signi ican di e ences we e ound in any o he molecula species be ween he me hods. When compa ing he o al le els o lipid classes, only Ce was ound a signi ican ly lowe concen a ions in he cells p oduced by M5 compa ed wi h hose p oduced by M4 (Figu e 5b). Howe e , a he molecula species le el, none o he Ce species di e ed signi ican ly be ween M4 and M5 (Figu e 5a and Suppo ing In o ma ion Table S2). 3.6 | Lipid p o iles o HLCs, PHHs, and HepG2 cells Nex , we compa e he lipidomes o he HLCs wi h he PHHs and HepG2 cells and desc ibe ou indings o each lipid class sepa a ely. In addi ion, we highligh he di e ences and simila i ies be ween he PHHs and HepG2 cells. 3.6.1 |Phospholipids In he HLCs, he molecula species o PC, PE, and PI con aining sa u a ed FAs (SFAs) and monounsa u a ed FAs (MUFAs) we e mos ly simila o hose in he PHHs, wi h he excep ions o PC 18:1/ 18:1 (p< 0.05), PC 17:0–18:1 (p< 0.001), and PE 18:0–18:1 (p< 0.05), which we e s a is ically signi ican ly highe in he HLCs (Figu e 3, ma ked by ed a ows). When he PHHs we e compa ed wi h he HepG2 cells, a la ge numbe o species showed s a is ically signi ican di e ences, pa icula ly he species con aining 14:0, 16:0, and 18:1 FAs. In PC and PE, he molecula species con aining polyunsa u a ed FAs (PUFAs) we e p esen in signi ican ly highe concen a ions in he PHHs compa ed wi h bo h he HLCs and HepG2 cells. This di e ence was especially p onounced o he species con aining an SFA coupled wi h FA 18:2 o i s de i a i e 20:4 (e.g., 16:0–18:2, 18:0–18:2, 16:0–18:2, and 18:0–20:4). The HepG2 cells, howe e , con ained mo e o he species whe e an MUFA was FIGURE 2 Cha ac e iza ion and unc ionali y o hiPSC‐de i ed hepa ocy e‐like cells (hiPSC‐HLCs) di e en ia ed om h ee cell lines by i e me hods and hei compa ison o p ima y human hepa ocy es (PHHs), HepG2 cells, and human li e o al RNA (hLTR). (a) Immunos aining o cells o AFP ( ed) and ALB (g een). Nuclei a e s ained wi h DAPI (blue). The ed inse on he lowe igh shows he compa ison o he mo phology and binuclea i y ( ed a ows) o M5‐HLCs and PHHs. The scale ba ep esen s 200 µm o he HLCs and 100 µm o he PHHs and HepG2 cells. (b) The g aph on he le shows he a e age pe cen age o binuclea HLCs in each me hod and hei compa ison o PHHs. The g aph on he igh shows he a e age pe cen age o ALB‐posi i e HLCs in each me hod. Each ba ep esen s he mean ± SD o he manual coun s om he immunos aining image analysis o a leas i e a eas. (c) Real‐ ime qPCR analysis o he SOX17,FOXA2,AFP,ALB,APOA1, and APOB genes a he hiPSC and hepa ic s age and hei compa ison o he e e ence samples. Each sample was un in iplica e and he ba s ep esen he mean ± SD o h ee biological eplica es om h ee indi idual cell lines. The gene exp ession da a we e no malized o he housekeeping gene GAPDH and a e p esen ed ela i e o he PHHs. (d) Biochemical analysis o he condi ioned media om he HLCs o albumin, u ea, and iacylglyce ol. Values a e no malized as 1 million cells pe 24 h . The ba s ep esen he mean ± SD o h ee biological eplica es o he h ee cell lines. *p< 0.05, **p< 0.01, ***p< 0.001. DAPI: 6‐diamidino‐2‐phenylindole; n.s.: no signi ican ; qPCR: quan i a i e PCR; SD: s anda d de ia ion [Colo igu e can be iewed a wileyonlinelib a y.com] 3750 | KIAMEHR ET AL. coupled o a PUFA (o ano he MUFA). The o al le els o PC and PI classes we e simila among all h ee compa ed cell ypes. To al PE was, howe e , de ec ed a lowe le els in he HLCs compa ed wi h he PHHs (s a is ically signi ican in M4 and M5 s he PHHs). This was mos ly due o he highe le els o PE 18:0–18:2 and PE 18:0– 20:4 in he PHHs. The concen a ion o PC, PE, and PI in he HLC and PHH media was ei he ze o o negligible, while he HepG2 medium con ained high amoun s o PC (12.9 µM) bu only minimal amoun s o PE and PI (Suppo ing In o ma ion Figu e S7 and Table S3). The p o ile and concen a ion o molecula lysophospholipids (LPLs)— such as LPC, LPE, and LPI—in he HLCs we e e y close o hose o he PHHs (Figu e 3a). Consequen ly, he o al LPL le els o he HLCs and PHHs we e also simila (Figu e 3b). The HepG2 cells, howe e , con ained conside ably highe le els o LPL as o al le els and s ikingly high le els o he molecula lyso‐species, wi h he 18:1 acyl esidue in each LPL class (Figu e 3 and Suppo ing In o ma ion Table S2). 3.6.2 |Neu al lipids CE concen a ion was he highes in heHepG2cells,in e media ein he HLCs, and he lowes in he PHHs. The CE species p o ile o he HLCs and HepG2 cells was di e en because 16 CE species showed s a is ically signi ican ly highe concen a ions in he HepG2 cells FIGURE 3 Lipidomic analysis o phospholipids (PLs) and lysophospholipids (LPLs) in he HLCs di e en ia ed by M3, M4, and M5, and hei compa ison o PHH and HepG2 cell lipids. (a) P o ein‐no malized concen a ion o molecula species de ec ed in each class o PLs (Phospha idylcholine [PC], phospha idylinosi ol [PI], and phospha idyle hanolamine [PE]) as well as LPLs (lysophospha idylcholine [LPC], lysophospha idylinosi ol [LPI], and lysophospha idyle hanolamine [LPE]). The a ows e e o species ha we e ound o be s a is ically signi ican ly di e en be ween HLCs in M3, M4, and M5 (blue a ows) o be ween he HLCs and PHHs ( ed a ows) by one‐way analysis o a iance. (b) To al concen a ions o PLs and LPLs calcula ed om he sum o all he molecula species in hose speci ic classes. Each sample was un in iplica e and he ba s ep esen he mean ± s anda d de ia ion o he s udied cell lines [Colo igu e can be iewed a wileyonlinelib a y.com] KIAMEHR ET AL. | 3751 (Figu e 4). When in e p e ing hese di e ences, he e ec o medium CE concen a ion was conside ed. The HepG2 medium con ained 154 µM o CE, whe eas he CE concen a ions we e negligible in he HLC and PHH media (Suppo ing In o ma ion Figu e S7 and Table S3). Simila o PLs, he HLCs con ained highe le els o DAG species wi h FA 18:1 when compa ed wi h he PHHs. Howe e , he le el o majo DAG species 16:0–18:1 de ec ed in he HLCs was clea ly close o ha de ec ed in he PHHs compa ed wi h wha was ound in he HepG2 cells. The PHHs, on he o he hand, con ained highe le els o DAG species wi h FA 18:2 ( he mino species DAG 18: 1–18:2 being an excep ion wi h i s equally low le els in he HLCs, PHHs, and HepG2 cells). As also obse ed in PC, he DAG species wi h ela i ely sho chain FAs (e.g., 14:0, 16:0, and 16:1) we e de ec ed a highe concen a ions in he HepG2 cells han in he HLCs and PHHs (Figu e 4A). 3.6.3 |Sphingolipids Despi e highe o al SM concen a ion, HLCs mimicked he SM p o ile o PHHs and HepG2 cells, excep ha SM d18:1/15:0 and SM d18:1/16:1 we e p esen a s a is ically signi ican ly highe concen- a ions in he HepG2 cells (Figu e 5a). Bo h he HLCs and HepG2 cells con ained signi ican ly highe le els o SM d18:1/16:0 compa ed wi h he PHHs. The le el o SM in he HLC and PHH media was unde ec able, whe eas he HepG2 medium con ained 6.5 µM o SM (Suppo ing In o ma ion Figu e S7 and Table S3). In e ms o he o e all SL p o ile, he HepG2 cells si ua ed be ween he PHHs and HLCs. In ac , lipid class da a showed ha he HLCs con ained lowe le els o Ce bu highe le els o LacCe , Glc/ GalCe , and Gb3 (membe s o glycosphingolipid [GSL] amily) compa ed wi h he PHHs (Figu e 5b). Close examina ion showed ha Ce s and, pa icula ly, he sa u a ed species wi h long‐and e y‐ long‐chain FAs (e.g., Ce d18:0/22:0 and Ce d18:1/24:0) we e highe in he PHHs (Figu e 5a), in ac , he Ce species p o ile o he HLCs esembled mo e ha o he HepG2 cells. On he o he hand, he HLCs con ained mo e GSLs—especially he species d18:1/16:0, d18:1/24:0, and d18:1/24:1—and he di e ences we e he mos p onounced in he Glc/GalCe class. The HepG2 medium con ained ace amoun s o SLs, and he concen a ion o SLs in he HLC and PHH media was ei he negligible o unde ec able (Suppo ing In o ma ion Figu e S7 and Table S3). 3.7 | FA analysis The HLCs con ained 15 mol% PUFAs e sus 29 mol% and 10.5 mol % in he PHHs and HepG2 cells, espec i ely (Figu e 6a). The FA FIGURE 4 Lipidomic analysis o choles e yl es e (CE) and diacylglyce ol (DAG) in he HLCs di e en ia ed by M3, M4, and M5, and hei compa ison o PHH and HepG2 cell lipids. (a) P o ein‐no malized concen a ion o molecula species de ec ed o he CE and DAG lipid class. (b) To al concen a ions o CE and DAG calcula ed om he sum o all he molecula species in hose speci ic classes. Each sample was un in iplica e and he ba s ep esen he mean ± s anda d de ia ion o he s udied cell lines [Colo igu e can be iewed a wileyonlinelib a y.com] 3752 | KIAMEHR ET AL. The au ho s g a e ully acknowledge he Tampe e acili y o iPS Cells and Flow Cy ome y o hei se ices. CONFLICTS OF INTEREST The au ho s decla e no con lic s o in e es in his s udy. ORCID Mos a a Kiameh h p://o cid.o g/0000-0003-1894-3237 REFERENCES Ana skaya, O. V., & Vinog ado , A. E. (2007). Genome mul iplica ion as adap a ion o issue su i al: E idence om gene exp ession in mammalian hea and li e . Genomics,89(1), 70–80. h p://doi.o g/10. 1016/j.ygeno.2006.08.014 Behbahan, I. S., Duan, Y., Lam, A., Khoobya i, S., Ma, X., Ahuja, T. P., & Ze n, M. A. (2011). New app oaches in he di e en ia ion o human emb yonic s em cells and induced plu ipo en s em cells owa d hepa ocy es. S em Cell Re iews,7(3), 748–759. h ps://doi.o g/10. 1007/s12015‐010‐9216‐4 Biebe ich, E. (2012). I ‘s a lipid’s wo ld: Bioac i e lipid me abolism and signaling in neu al s em cell di e en ia ion. Neu ochemical Resea ch, 37, 1208–1229. h ps://doi.o g/10.1007/s11064‐011‐0698‐5 Blouin, A., Bolende , R. P., & Weibel, E. R. (1977). Dis ibu ion o o ganelles and memb anes be ween hepa ocy es and nonhepa ocy es in he a li e pa enchyma. A s e eological s udy. The Jou nal o Cell Biology,72(2), 441–455. Came on, K., Tan, R., Schmid ‐Heck, W., Campos, G., Lyall, M. J., Wang, Y., …Hay, D. C. (2015). Recombinan laminins d i e he di e en ia ion and sel ‐o ganiza ion o hESC‐de i ed hepa ocy es. S em Cell Repo s, 5(6), 1250–1262. Cas ell, J. V., Jo e , R., Ma ínez‐Jiménez, C. P., & Gómez‐Lechón, M. J. (2006). Hepa ocy e cell lines: Thei use, scope and limi a ions in d ug me abolism s udies. Expe Opinion on D ug Me abolism & Toxicology, 2(2), 183–212. h ps://doi.o g/10.1517/17425255.2.2.183 Cayo, M. A., Cai, J., Dela o es , A., No o, F. K., Nagaoka, M., Cla k, B. S., … Duncan, S. A. (2012). JD induced plu ipo en s em cell‐de i ed hepa ocy es ai h ully ecapi ula e he pa hophysiology o amilial hype choles e olemia. Hepa ology,56(6), 2163–2171. h ps://doi.o g/ 10.1002/hep.25871 Chen, Y. F., Tseng, C. Y., Wang, H. W., Kuo, H. C., Yang, V. W., & Lee, O. K. (2012). Rapid gene a ion o ma u e hepa ocy e‐like cells om human induced plu ipo en s em cells by an e icien h ee‐s ep p o ocol. Hepa ology,55(4), 1193–1203. h ps://doi.o g/10.1002/hep.24790 Choi, S. C., Choi, J. H., Cui, L. H., Seo, H. R., Kim, J. H., Pa k, C. Y., …Lim, D. S. (2015). Mixl1 and Flk1 a e key playe s o Wn /TGF‐βsignaling du ing DMSO‐induced mesode mal speci ica ion in P19 cells. Jou nal o Cellula Physiology,230(8), 1807–1821. Cingolani, F., Fu e man, A. H., & Casas, J. (2016). Ce amide syn hases in biomedical esea ch. Chemis y and Physics o Lipids,197,25–32. h ps://doi.o g/10.1016/j.chemphyslip.2015.07.026 Czysz, K., Minge , S., & Thomas, N. (2015). DMSO e icien ly down egula es plu ipo ency genes in human emb yonic s em cells du ing de ini i e endode m de i a ion and inc eases he p o iciency o hepa ic di e en ia ion. PLoS One,10(2), e0117689. h ps://doi.o g/10. 1371/jou nal.pone.0117689 Diasc o, D. D., Vogel, R. L., Johnson, T. E., Wi he up, K. M., Pi zenbe ge , S. M., Ru ledge, S. J., …Schmid , A. (1998). High a y acid con en in abbi se um is esponsible o he di e en ia ion o os eoblas s in o adipocy e‐like cells. Jou nal o Bone and Mine al Resea ch,13(1), 96– 106. h ps://doi.o g/10.1359/jbm .1998.13.1.96 Ejsing, C. S., Duchosla , E., Sampaio, J., Simons, K., Bonne , R., Thiele, C., … She chenko, A. (2006). Au oma ed iden i ica ion and quan i ica ion o glyce ophospholipid molecula species by mul iple p ecu so ion scanning. Analy ical Chemis y,78(17), 6202–6214. h ps://doi.o g/ 10.1021/ac060545x Ek oos, K., Che nushe ich, I. V., Simons, K., & She chenko, A. (2002). Quan i a i e p o iling o phospholipids by mul iple p ecu so ion scanning on a hyb id quad upole ime‐o ‐ ligh mass spec ome e . Analy ical Chemis y,74(5), 941–949. h ps://doi.o g/10.1021/ ac015655c Ek oos, K., Ejsing, C. S., Bah , U., Ka as, M., Simons, K., & She chenko, A. (2003). Cha ing molecula composi ion o phospha idylcholines by a y acid scanning and ion ap MS3 agmen a ion. The Jou nal o Lipid Resea ch,44(11), 2181–2192. h p://doi.o g/10.1194/jl . D300020‐JLR200 Elau , G., Henkens, T., Papeleu, P., Snyke s, S., Vinken, M., Vanhaecke, T., & Rogie s, V. (2006). Molecula mechanisms unde lying he dedi e - en ia ion p ocess o isola ed hepa ocy es and hei cul u es. Cu en D ug Me abolism,7(6), 629–660. h p://doi.o g/10.2174/138920006 778017759 Gaul , C. R., Obeid, L. M., & Hannun, Y. A. (2010). An o e iew o sphingolipid me abolism: F om syn hesis o b eakdown. Ad ances in Expe imen al Medicine and Biology,1–23. h ps://doi.o g/10.1007/978‐ 1‐4419‐6741‐1_1 Ge bal‐Chaloin, S., Funakoshi, N., Caillaud, A., Gondeau, C., Champon, B., &Si‐Tayeb, K. (2014). Human induced plu ipo en s em cells in hepa ology: Beyond he p oo o concep . The Ame ican Jou nal o Pa hology,184(2), 332–347. h ps://doi.o g/10.1016/j.ajpa h.2013. 09.026 Glück, T., Rupp, H., & Al e , P. (2016). Mechanisms inc easing n‐3highly unsa u a ed a y acids in he hea . Canadian Jou nal o Physiology and Pha macology,94(3), 309–323. h ps://doi.o g/10.1139/cjpp‐2015‐0300 Godoy, P., Hewi , N. J., Alb ech , U., Ande sen, M. E., Ansa i, N., Bha acha ya, S., …Hengs le , J. G. (2013). Recen ad ances in 2D and 3D in i o sys ems using p ima y hepa ocy es, al e na i e hepa ocy e sou ces and non‐pa enchymal li e cells and hei use in in es iga ing mechanisms o hepa o oxici y, cell signaling and ADME. . A chi es o Toxicology,87, 1315–1530. h ps://doi.o g/10.1007/s00204‐013‐ 1078‐5 Godoy, P., Schmid ‐Heck, W., Na a ajan, K., Lucendo‐Villa in, B., Szkol- nicka, D., Asplund, A., …Hengs le , J. G. (2015). Gene ne wo ks and ansc ip ion ac o mo i s de ining he di e en ia ion o s em cells in o hepa ocy e‐like cells. Jou nal o Hepa ology,63(4), 934–942. h ps://doi.o g/10.1016/j.jhep.2015.05.013 Go dillo, M., E ans, T., & Gouon‐E ans, V. (2015). O ches a ing li e de elopmen . De elopmen ,142(12), 2094–2108. h ps://doi.o g/10. 1242/de .114215 Hannan, N. R. F., Sege i z, C.‐P., Touboul, T., & Vallie , L. (2013). P oduc ion o hepa ocy e‐like cells om human plu ipo en s em cells. Na u e P o ocols,8(2), 430–437. Hay, D. C., Fle che , J., Payne, C., Te ace, J. D., Gallaghe , R. C. J., Snoeys, J., …I edale, J. P. (2008). Highly e icien di e en ia ion o hESCs o unc ional hepa ic endode m equi es Ac i inA and Wn 3a signaling. P oceedings o he Na ional Academy o Sciences,105(34), 12301– 12306. h ps://doi.o g/10.1073/pnas.0806522105 Heiskanen, L. A., Suoniemi, M., Ta, H. X., Ta aso , K., & Ek oos, K. (2013). Long‐ e m pe o mance and s abili y o molecula sho gun lipidomic analysis o human plasma samples. Analy ical Chemis y,85(18), 8757– 8763. h ps://doi.o g/10.1021/ac401857a Hodson, L., & Fielding, B. A. (2013). S ea oyl‐CoA desa u ase: Rogue o innocen bys ande ? P og ess in Lipid Resea ch,52,15–42. h ps://doi. o g/10.1016/j.plip es.2012.08.002 KIAMEHR ET AL. | 3759 Hu, C., & Li, L. (2015). In i o cul u e o isola ed p ima y hepa ocy es and s em cell‐de i ed hepa ocy e‐like cells o li e egene a ion. P o ein & Cell,6(8), 562–574. h ps://doi.o g/10.1007/s13238‐015‐0180‐2 Huang, H. ‐C., & Lin, J. ‐K. (2012). Pu‐e h ea, g een ea, and black ea supp esses hype lipidemia, hype lep inemia and a y acid syn hase h ough ac i a ing AMPK in a s ed a high‐ uc ose die . Food & Func ion,3(2), 170–177. h ps://doi.o g/10.1039/C1FO10157A Jensen‐U s ad, A. P. L., & Semenko ich, C. F. (2012). Fa y acid syn hase and li e iglyce ide me abolism: Housekeepe o messenge ? Biochimica e Biophysica Ac a,1821, 747–753. h ps://doi.o g/10. 1016/j.bbalip.2011.09.017 Kajiwa a, M., Aoi, T., Oki a, K., Takahashi, R., Inoue, H., Takayama, N., … Yamanaka, S. (2012). Dono ‐dependen a ia ions in hepa ic di e - en ia ion om human‐induced plu ipo en s em cells. P oceedings o he Na ional Academy o Sciences,109(31), 12538–12543. h ps://doi. o g/10.1073/pnas.1209979109 Kamada, N., Kawashima, H., Saku adani, E., Akimo o, K., Ogawa, J., & Shimizu, S. (1999). P oduc ion o 8,11‐cis‐eicosadienoic acid by a del a 5 and del a 12 desa u ase‐de ec i e mu an de i ed om he a achidonic acid‐p oducing ungus Mo ie ella alpina 1S‐4. Jou nal o he Ame ican Oil Chemis s Socie y,76(11), 1269–1274. h ps://doi.o g/ 10.1007/S11746‐999‐0138‐8 Ka aku a, M., Hashimo o, M., Okui, T., Shahda , H. M., Ma suzaki, K., & Shido, O. (2013). Omega‐3 polyunsa u a ed a y acids enhance neu onal di e en ia ion in cul u ed a neu al s em cells. S em Cells In e na ional,2013,1–9. h ps://doi.o g/10.1155/2013/490476 Kia, R., Sison, R. L. C., Heslop, J., Ki e ingham, N. R., Hanley, N., Mills, J. S., …Gold ing, C. E. P. (2012). S em cell‐de i ed hepa ocy es as a p edic i e model o d ug‐induced li e inju y: A e we he e ye ? B i ish Jou nal o Clinical Pha macology,75(4), 885–896. h ps://doi. o g/10.1111/j.1365‐2125.2012.04360.x Kiameh , M., Vii i, L. E., Vihe aa a, T., Kois inen, K. M., Hil o, M., Ek oos, K., …Aal o‐Se älä, K. (2017). Lipidomic p o iling o pa ien ‐speci ic induced plu ipo en s em cell‐de i ed hepa ocy e‐like cells. Disease Models & Mechanisms, 030841. h ps://doi.o g/10.1242/dmm.030841 Kim, M. H., Kim, M. O., Kim, Y. H., Kim, J. S., & Han, H. J. (2009). Linoleic acid induces mouse emb yonic s em cell p oli e a ion ia Ca 2+/PKC, PI3K/Ak , and MAPKs. Cellula Physiology and Biochemis y,23(1–3), 53–64. h ps://doi.o g/10.1159/000204090 K uege , W. H., Tanasije ic, B., Ba be , V., Flamie , A., Gu, X., Manau ou, J., & Rasmussen, T. P. (2013). Choles e ol‐sec e ing and s a in‐ espon- si e hepa ocy es om human ES and iPS cells o model hepa ic in ol emen in ca dio ascula heal h. PLoS One,8(7), e67296. K ilek al, K., Lin, J., Cheng, W., & Abum ad, N. (1994). Fa y acids as de e minan s o iglyce ide and choles e yl es e syn hesis by isola ed hepa ocy es: Kine ics as a unc ion o a ious a y acids. Jou nal o Lipid Resea ch,35(10), 1786–1794. Li ak, K. J., & Schmi gen, T. D. (2001). Analysis o ela i e gene exp ession da a using eal‐ ime quan i a i e PCR and he 2−ΔΔCT me hod. Me hods,25(4), 402–408. Mallanna, S. K., & Duncan, S. A. (2013). Di e en ia ion o hepa ocy es om plu ipo en s em cells. Cu en P o ocols in S em Cell Biology,1 (Suppl.26), 1G.4.1–1G.4.13. h ps://doi.o g/10.1002/97804701518 08.sc01g04s26 Manzini, S., Vii i, L. E., Ma ila, S., & Aal o‐Se älä, K. (2015). A compa a i e iew on easy o deploy non‐in eg a ing me hods o pa ien ‐speci ic iPSC p oduc ion. S em Cell Re iews and Repo s,11(6), 900–908. h ps://doi.o g/10.1007/s12015‐015‐9619‐3 Massa, M. L., Gaglia dino, J. J., & F ancini, F. (2011). Li e glucokinase: An o e iew on he egula o ymechanisms o i s ac i i y. IUBMB Li e,63, 1–6. h ps://doi.o g/10.1002/iub.411 Medine, C. N., Lucendo‐Villa in, B., S o ck, C., Wang, F., Szkolnicka, D., Khan, F., …Hay, D. C. (2013). De eloping high‐ ideli y hepa o oxici y models om plu ipo en s em cells. S em Cells T ansla ional Medicine,2 (7), 505–509. h ps://doi.o g/10.5966/sc m.2012‐0138 Meikle, P. J., Wong, G., Tso o es, D., Ba low, C. K., Wei , J. M., Ch is ophe , M. J., …Kingwell, B. A. (2011). Plasma lipidomic analysis o s able and uns able co ona y a e y disease. A e ioscle osis, Th ombosis, and Vascula Biology,31(11), 2723–2732. h ps://doi.o g/10.1161/ ATVBAHA.111.234096 Me ill , A. H., J ., Sulla ds, M. C., Allegood, J. C., Kelly, S., & Wang, E. (2005). Sphingolipidomics: High‐ h oughpu , s uc u e‐speci ic, and quan i a i e analysis o sphingolipids by liquid ch oma og aphy andem mass spec ome y. Me hods,36(2), 207–224. h ps://doi. o g/10.1016/j.yme h.2005.01.009 Mesicek, J., Lee, H., Feldman, T., Jiang, X., Skobele a, A., Be dyshe , E. V., …Kolesnick, R. (2010). Ce amide syn hases 2, 5, and 6 con e dis inc oles in adia ion‐induced apop osis in HeLa cells. Cellula Signalling, 22(9), 1300–1307. h ps://doi.o g/10.1016/j.cellsig.2010.04.006 Michalopoulos, G. K., Bowen, W. C., Mulè, K., & Luo, J. (2003). HGF‐, EGF‐, and dexame hasone‐induced gene exp ession pa e ns du ing o ma- ion o issue in hepa ic o ganoid cul u es. Gene Exp ession,11(2), 55– 75. h ps://doi.o g/10.3727/000000003108748964 Miyaoka, Y., & Miyajima, A. (2013). To di ide o no o di ide: Re isi ing li e egene a ion. Cell Di ision,8, 8. h ps://doi.o g/10.1186/1747‐ 1028‐8‐8 Moss, A. J. (1991). Choles e ol and a he oscle osis: Diagnosis and ea men . Jou nal o he Ame ican Medical Associa ion,266(20), 2910–2911. N ambi, J. M. (1999). Regula ion o s ea oyl‐CoA desa u ase by polyunsa u a ed a y acids and choles e ol. Jou nal o Lipid Resea ch, 40(9), 1549–1558. Re ie ed om. h p://www.ncbi.nlm.nih.go / pubmed/10484602 Ohnuki, M., Takahashi, K., & Yamanaka, S. (2009). Gene a ion and cha ac e iza ion o human induced plu ipo en s em cells. In Schlaege , T. (Ed.), Cu en p o ocols in s em cell biology. John Wiley & Sons, Inc. h ps://doi.o g/10.1002/9780470151808 Olsa sky, K. M., Page, J. L., Johnson, M. C., Za bl, H., S om, S. C., & Omiecinski, C. J. (2007). Gene exp ession p o iling and di e en ia ion assessmen in p ima y human hepa ocy e cul u es, es ablished hepa oma cell lines, and human li e issues. Toxicology and Applied Pha macology,222(1), 42–56. h ps://doi.o g/10.1016/j. aap.2007. 03.032 Rasmussen, T. P. (2015). Genomic medicine and lipid me abolism: LDL a ge s and s em cell esea ch app oaches, T ansla ional Ca diome a- bolic Genomic Medicine (99–118). London UK: Academic P ess. h ps:// doi.o g/10.1016/B978‐0‐12‐799961‐6.00005‐6 Ruhanen, H., Nidhina Ha idas, P. A., Eskelinen, E. L., E iksson, O., Olkkonen, V. M., & Käkelä, R. (2017). Deple ion o TM6SF2 dis u bs memb ane lipid composi ion and dynamics in HuH7 hepa oma cells. Biochimica e Biophysica Ac a,1862(7), 676–685. h ps://doi.o g/10. 1016/j.bbalip.2017.04.004 Rui, L. (2014). Ene gy me abolism in he li e . Comp ehensi e Physiology,4 (1), 177–197. h ps://doi.o g/10.1002/cphy.c130024 San os, N. C., Figuei a‐Coelho, J., Ma ins‐Sil a, J., & Saldanha, C. (2003). Mul idisciplina y u iliza ion o dime hyl sul oxide: Pha ma- cological, cellula , and molecula aspec s. Biochemical Pha macology, 65, 1035–1041. h ps://doi.o g/10.1016/S0006‐2952(03)00002‐9 Schwa z, R. E., Fleming, H. E., Khe ani, S. R., & Bha ia, S. N. (2014). Plu ipo en s em cell‐de i ed hepa ocy e‐like cells. Bio echnology Ad ances,32, 504–513. h ps://doi.o g/10.1016/j.bio echad .2014. 01.003 Schwa z, R. E., T ehan, K., And us, L., Sheahan, T. P., Ploss, A., Duncan, S. A., …Bha ia, S. N. (2012). Modeling hepa i is C i us in ec ion using human induced plu ipo en s em cells. P oceedings o he Na ional Academy o Sciences o he Uni ed S a es o Ame ica,109, 2544–2548. h ps://doi.o g/10.1073/pnas.1121400109/‐/DCSupplemen al.www. pnas.o g/cgi//10.1073/pnas.1121400109 Si‐Tayeb, K., No o, F. K., Nagaoka, M., Li, J., Ba le, M. A., Du is, C., … Duncan, S. A. (2010). Highly e icien gene a ion o human hepa o- 3760 | KIAMEHR ET AL. cy e–like cells om induced plu ipo en s em cells. Hepa ology,51(1), 297–305. Sp eche , H. (2000). Me abolism o highly unsa u a ed n‐3 and n‐6 a y acids. Biochimica e Biophysica Ac a,1486, 219–231. h ps://doi.o g/ 10.1016/S1388‐1981(00)00077‐9 S übige , G., Aldo e ‐Macasae , E., Bicke , W., Sobal, G., Will o ‐Eh inge , A., Pock, K., …Belgacem, O. (2012). Ta ge ed p o iling o a he ogenic phospholipids in human plasma and lipop o eins o hype lipidemic pa ien s using MALDI‐QIT‐TOF‐MS/MS. A he oscle osis,224(1), 177– 186. h ps://doi.o g/10.1016/j.a he oscle osis.2012.06.010 Sulli an, G. J., Hay, D. C., Pa k, I. ‐H., Fle che , J., Hannoun, Z., Payne, C. M., …Wilmu , I. (2010). Gene a ion o unc ional human hepa ic endode m om human induced plu ipo en s em cells. Hepa ology, 51(1), 329–335. h ps://doi.o g/10.1002/hep.23335 Szkolnicka, D., Fa nwo h, S. L., Lucendo‐Villa in, B., S o ck, C., Zhou, W., I edale, J. P., …Hay, D. C. (2014). Accu a e p edic ion o d ug‐induced li e inju y using s em cell‐de i ed popula ions. S em Cells T ansla ional Medicine, 3(2), 141–148. h ps://doi.o g/10.5966/sc m.2013‐0146 Szkolnicka, D., Lucendo‐Villa in,B.,Moo e,J.K.,Simpson,K.J.,Fo bes, S. J., & Hay, D. C. (2016). Reducing hepa ocy e inju y and nec osis in esponse o pa ace amol using noncoding RNAs. S em Cells T ansla ional Medicine,5(6), 764–772. h ps://doi.o g/10.5966/sc m.2015‐0117 Takahashi, K., & Yamanaka, S. (2006). Induc ion o plu ipo en s em cells om mouse emb yonic and adul ib oblas cul u es by de ined ac o s. Cell,126(4), 663–676. Takayama, K., Inamu a, M., Kawaba a, K., Sugawa a, M., Kikuchi, K., Higuchi, M., …Mizuguchi, H. (2012). Gene a ion o me abolically unc ioning hepa ocy es om human plu ipo en s em cells by FOXA2 and HNF1α ansduc ion. Jou nal o Hepa ology,57(3), 628–636. h ps://doi.o g/10.1016/j.jhep.2012.04.038 Tokuda, N., Numa a, S., Li, X., Nomu a, T., Takizawa, M., Kondo, Y., … Fu ukawa, K. (2013). β4GalT6 is in ol ed in he syn hesis o lac osylce amide wi h less in ensi y han β4GalT5. Glycobiology,23 (10), 1175–1183. h ps://doi.o g/10.1093/glycob/cw 054 Wiśniewski, J. R., Vildhede, A., No én, A., & A u sson, P. (2016). In‐dep h quan i a i e analysis and compa ison o he human hepa ocy e and hepa oma cell line HepG2 p o eomes. Jou nal o P o eomics,136, 234– 247. h ps://doi.o g/10.1016/j.jp o .2016.01.016 Yang,D.,Yuan,Q.,Balak ishnan,A.,Ban el,H.,Klusmann,J.,Manns,M.P.,… Sha ma,A.D.(2016).Mic oRNA‐125b‐5p mimic inhibi s acu e li e ailu e. Na u e Communica ions,7,1–11. h ps://doi.o g/10.1038/ ncomms11916 Younossi, Z. M., Koenig, A. B., Abdela i , D., Fazel, Y., Hen y, L., & Wyme , M. (2016). Global epidemiology o non‐alcoholic a y li e disease‐ Me a‐analy ic assessmen o p e alence, incidence and ou comes. Hepa ology,64(1), 73–84. h ps://doi.o g/10.1002/hep.28431 Zhang, J. Y., Ko hapalli, K. S. D., & B enna, J. T. (2016). Desa u ase and elongase‐limi ing endogenous long‐chain polyunsa u a ed a y acid biosyn hesis. Cu en Opinion in Clinical Nu i ion and Me abolic Ca e,19(2), 103–110. h ps://doi.o g/10.1097/MCO. 0000000000000254 SUPPORTING INFORMATION Addi ional suppo ing in o ma ion may be ound online in he Suppo ing In o ma ion sec ion a he end o he a icle. How o ci e his a icle: Kiameh M, Alexano a A, Vii i LE, e al. hiPSC‐de i ed hepa ocy es closely mimic he lipid p o ile o p ima y hepa ocy es: A u u e pe sonalised cell model o s udying he lipid me abolism o he li e . J Cell Physiol. 2019;234:3744–3761. h ps://doi.o g/10.1002/jcp.27131 KIAMEHR ET AL. | 3761