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
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wileyonlinelib a y.com/jou nal/jcp
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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.
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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
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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
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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
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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
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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
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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.
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KIAMEHR ET AL.
|
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