Resea ch A icle
Mu a ion-Speci ic Pheno ypes in hiPSC-De i ed
Ca diomyocy es Ca ying Ei he Myosin-Binding P o ein C O
𝛼-T opomyosin Mu a ion o Hype ophic Ca diomyopa hy
Ma isa Ojala,1Chand a P ajapa i,1Ris o-Pekka Pölönen,1K is iina Rajala,1
Ma i Pekkanen-Ma ila,1Jy ki Rasku,2Kim La sson,1and Ka iina Aal o-Se älä1,3,4
1BioMediTech, Uni e si y o Tampe e, 33014 Tampe e, Finland
2School o In o ma ion Sciences, Uni e si y o Tampe e, 33014 Tampe e, Finland
3Medical School, Uni e si y o Tampe e, 33014 Tampe e, Finland
4Hea Hospi al, Tampe e Uni e si y Hospi al, 33521 Tampe e, Finland
Co espondence should be add essed o Ka iina Aal o-Se ¨
al¨
a; ka [email p o ec ed]
Recei ed 21 May 2015; Re ised 22 July 2015; Accep ed 20 Sep embe 2015
Academic Edi o : Fa ah Sheikh
Copy igh © 2016 Ma isa Ojala e al. This is an open access a icle dis ibu ed unde he C ea i e Commons A ibu ion License,
which pe mi s un es ic ed 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.
Hype ophic ca diomyopa hy (HCM) is a gene ic ca diac disease, which a ec s he s uc u e o hea muscle issue. The clinical
symp oms include a hy hmias, p og essi e hea ailu e, and e en sudden ca diac dea h bu he mu a ion ca ie can also be o ally
asymp oma ic. To da e, o e 1400 mu a ions ha e been linked o HCM, mos ly in genes encoding o sa come ic p o eins. Howe e ,
he pa hophysiological mechanisms o he disease a e s ill la gely unknown. Two ounde mu a ions o HCM in Finland a e
loca ed in myosin-binding p o ein C (MYBPC3-Gln1061X)and𝛼- opomyosin (TPM1-Asp175Asn) genes. We s udied he p ope ies
o HCM ca diomyocy es (CMs) de i ed om pa ien -speci ic human induced plu ipo en s em cells (hiPSCs) ca ying ei he
MYBPC3-Gln1061X o TPM1-Asp175Asn mu a ion. Bo h ypes o HCM-CMs displayed pa hological pheno ype o HCM bu , mo e
impo an ly, we ound di e ences be ween CMs ca ying ei he MYBPC3-Gln1061X o TPM1-Asp175Asn gene mu a ion in hei
cellula size, Ca2+ handling, and elec ophysiological p ope ies, as well as hei gene exp ession p o iles. These indings sugges
ha e en hough he clinical pheno ypes o he pa ien s ca ying ei he MYBPC3-Gln1061X o TPM1-Asp175Asn gene mu a ion
a e simila , he gene ic backg ound as well as he unc ional p ope ies on he cellula le el migh be di e en , indica ing ha he
pa hophysiological mechanisms behind he wo mu a ions would be di e gen as well.
1. In oduc ion
Hype ophic ca diomyopa hy (HCM) is one o he mos
common gene ic ca diac diseases wi h wo ldwide p e alence
o 1 : 500, as well as he mos common cause o sudden
ca diac dea h (SCD) among young compe ing a hle es. HCM
isinhe i edinanau osomaldominan pa e n.Ne e heless,
a la ge clinical di e si y and age- ela ed pene ance a e ypical
o HCM. On he issue le el, HCM is cha ac e ized by he
disa ay o ca diomyocy es (CMs) and ib osis o ca diac
issue, as well as hickened in e en icula sep um o ee
le en icula wall. Clinical symp oms include a hy hmias,
p og essi ehea ailu e,ande enSCD,bu on heo he
hand hemu a ionca ie canbecomple elyasymp oma ic.
Al oge he mo e han 1400 mu a ions in 11 genes encoding
o he sa come ic p o eins ha e been iden i ied and ela ed
o HCM. The majo i y o he mu a ions a e ound ei he in
he 𝛽-myosin hea y chain (MYH7) o in he myosin-binding
p o ein C (MYBPC3) genes [1]. In Finland, wo ounde
mu a ions loca ed in MYBPC3 and 𝛼- opomyosin (TPM1)
genes and one common mu a ion in MYH7 gene oge he
accoun a ound 24% o all Finnish HCM cases [2, 3].
Al hough he gene ic in o ma ion ela ed o HCM has
been g owing in he ecen yea s due o he de elopmen
o sequencing echnologies, exac in o ma ion o he disease
mechanisms emains unclea . Thus, cu en medica ion o he
diseaseisdi ec ed owa d hesymp om elie and he eisno
speci ic he apy o p e en he onse o p og ession o he
Hindawi Publishing Co po a ion
S em Cells In e na ional
Volume 2016, A icle ID 1684792, 16 pages
h p://dx.doi.o g/10.1155/2016/1684792
2S em Cells In e na ional
disease [1]. Mos o he HCM s udies ha e been conduc ed
wi h model sys ems, mainly ei he wi h ansgenic mice o
by s udying human issues ob ained om su gical myec omy
om end-s age HCM pa ien s [4]. Howe e , animal models
ca y only he mu a ed gene lacking he es o he genome
and myec omy samples a e ob ained om pa ien s in he la e
s age o HCM de elopmen . The e o e, he disco e y o he
human induced plu ipo en s em cells (hiPSCs) has o e ed a
new aluable ool o model HCM and o he ca diac diseases
and os udy heunde lyingdiseasemechanisms[5].Toda e,
hiPSCs ha e al eady been used o model a a ie y o ca diac
diseases: elec ical de ec s, o example, long-QT synd ome
[6–8] and ca echolamine gic polymo phic en icula achy-
ca dia (CPVT) [9, 10] as well as ca diomyopa hies including
dila ed ca diomyopa hy (DCM) [11] and HCM [12–14].
He e we ha e de i ed hiPSCs om pa ien s ca ying wo
o he Finnish HCM ounde mu a ions ei he in MYBPC3
(MYBPC3-Gln1061X)o inTPM1 (TPM1-Asp175Asn)gene.
We ha e di e en ia ed he pa ien -speci ic hiPSCs in o CMs
and compa ed he pheno ypes o he diseased and con ol
CMs.
2. Ma e ials and Me hods
2.1. E hical Issues. This s udy was conduc ed in acco dance
wi h he E hics Commi ee o Pi kanmaa Hospi al Dis ic
o es ablish, cul u e, and di e en ia e hiPSC lines (R08070).
Skin biopsies o hiPSC es ablishmen we e ecei ed om
he Hea Hospi al, Tampe e Uni e si y Hospi al, Tam-
pe e, Finland. Pa ien s dona ing skin biopsies signed an
in o med consen a e ecei ing bo h o al and w i en
desc ip ions o he s udy. The e a oma assay, desc ibed in
Sec ion 2.3.6, was app o ed by ELLA-Animal Expe imen
Boa d o Regional S a e Adminis a i e Agency o Sou he n
Finland (ESAVI/6543/04.10.03/2011).
2.2. Gene a ion and Cul u e o Pa ien -Speci ic hiPSC Lines.
hiPSC lines we e gene a ed om skin’s ib oblas s ei he wi h
Sendai ep og amming ec o s OCT4,KLF4,c-MYC,and
SOX2 using Cy oTune-iPS Rep og amming Ki (Li e Tech-
nologies L d., Paisley, UK) acco ding o he manu ac u e ’s
ins uc ions o by using pMX e o i al ec o s OCT4,KLF4,
c-MYC,andSOX2 wi h o wi hou C e-LoxP si e as desc ibed
ea lie [6, 15]. UTA.13602.HCMT, UTA.02912.HCMT, and
UTA.04511.WT hiPSC lines we e gene a ed by using Sendai
ec o s and UTA.07801.HCMM and UTA.06108.HCMM
by using pMX e o i al ec o s wi h C e-LoxP si e and
UTA.04602.WT was gene a ed by using pMX e o i al
ec o s wi hou C e-LoxP si e. In he p esen s udy, one
line o each pa ien was used. hiPSC lines we e de i ed and
cul u ed on mouse emb yonic ib oblas (MEF) eede cell
laye s (26000 cells/cm2, CellSys ems Bio echnologie Ve ieb
GmbH, T oisdo , Ge many) in human plu ipo en s em cell
(hPSC) cul u e medium consis ing o knockou -DMEM (ko-
DMEM,Gibco,Li eTechnologiesL d.)supplemen edwi h
20% knockou se um eplacemen (ko-SR, Gibco, Li e Tech-
nologies L d.), 1% nonessen ial amino acids (NEAA, Lonza
G oup L d., Basel, Swi ze land), 2 mM Glu aMax (Gibco, Li e
Technologies L d.), 50 U/mL penicillin/s ep omycin (Lonza
G oup L d.), 0.1 mM 2-me cap oe hanol (Gibco, Li e Tech-
nologies L d.), and 4 ng/mL basic ib oblas g ow h ac o
(bFGF, Pep oTech, Rocky Hill, NJ, USA).
2.3. Cha ac e iza ion o hiPSC Lines
2.3.1. Mu a ion Analysis by Geno yping. DNA samples om
hehiPSClineswe ep epa edwi hTaqManSample- o-
SNP Ki (Applied Biosys ems, Li e Technologies L d.) and
he p esence o MYBPC3-Gln1061X and TPM1-Asp175Asn
mu a ion in he pa ien -speci ic hiPSC lines was con i med
by cus om TaqMan SNP Geno yping Assays (Applied Biosys-
ems, Li e Technologies L d.) acco ding o he manu ac u e ’s
ins uc ions. In he geno yping assays, MYBPC3-gene as
well as TPM1-gene was ampli ied wi h speci ic p ime s.
Fu he mo e, he p esence o he mu a ions was assessed wi h
mu a ion-speci ic FAM labeled p obes. VIC labeled p obes
we e used o assess he p esence o he wild ype allele.
Sequences o hep ime sandp obesusedin heassaya e
lis ed in Supplemen a y Table 1 (see Supplemen a y Ma e ial
a ailable online a h p://dx.doi.o g/10.1155/2016/1684792).
2.3.2. The Exp ession o Mu an and Wild Type Alleles in
hiPSC-De i ed CMs. RNA samples we e collec ed and
ex ac ed om hiPSC-de i ed CMs (UTA.04511.WT,
UTA.02912.HCMT, UTA.07801.HCMM, and UTA.06108.
HCMM) wi h No gen’s To al RNA Pu i ica ion Plus Ki
(No gen Bio ek Co p., On a io, Canada) acco ding o manu-
ac u e ’s ins uc ions. 50–100 ng o RNA was ansc ibed o
cDNA by High-Capaci y cDNA Re e se T ansc ip ion Ki
(Applied Biosys ems, Li e Technologies L d.). The exp ession
o TPM1-Asp175Asn o MYBPC3-Gln1061X mu a ion on
mRNA le el in he hiPSC-de i ed CMs was s udied by
Cus om TaqMan SNP Geno yping Assays (Applied Bio-
sys ems, Li e Technologies L d.) simila ly as ha o
geno yping desc ibed abo e. Sequences o he p ime s and
p obesusedin heassaya elis edinSupplemen a yTable1.
2.3.3. Immunocy ochemis y. Undi e en ia ed hiPSC colo-
nies we e ixed wi h 4% pa a o maldehyde (PFA, Sigma-
Ald ich, Sain Louis, USA), s ained wi h p ima y an ibodies
o Nanog (R&D sys ems Inc., Minneapolis, MN, USA),
OCT4 (R&D sys ems Inc.), SOX2 (San a C uz Bio echnol-
ogy, San a C uz, CA, USA), TRA-1-60 (Millipo e, Bille ica,
MA, USA), and TRA-1-81 (Millipo e), and isualized wi h
seconda y an ibodies as desc ibed be o e [16]. Finally, he
cells we e moun ed wi h Vec ashield (Vec o Labo a o ies
Inc., Bu lingame, CA, USA) con aining 40,6-diamidino-2-
phenylindole (DAPI) o he nuclei s aining and imaged wi h
an Olympus IX51 phase con as mic oscope equipped wi h
luo escence op ics and Olympus DP30BW came a (Olympus
Co po a ion, Hambu g, Ge many).
2.3.4. RT-PCR. The RNA was ex ac ed om he hiPSC lines
by NucleoSpin RNA II Ki (Mache ey-Nagel GmbH & Co.,
D¨
u en, Ge many) and 500–1000 ng o RNA was ansc ibed
o cDNA by High-Capaci y cDNA Re e se T ansc ip ion Ki
S em Cells In e na ional 3
(Applied Biosys ems, Li e Technologies L d.). The p esence o
plu ipo ency genes Nanog,SOX2,REX1,OCT4,andc-MYC
and heabsenceo i allyimpo edexogenes(OCT4,SOX2,
c-MYC,andKLF4) we e con i med by RT-PCR. GAPDH
was used as an endogenous con ol. The p ime sequences
o plu ipo ency genes and i ally impo ed exogenes ha e
been published ea lie [5]. The p ime sequences used o
de ec ion o Sendai ansgenes a e desc ibed in Cy oTune-iPS
Rep og amming Ki ’s manual (Li e Technologies L d.).
2.3.5. Ka yo ype Analysis. The ka yo ypes o hiPSC lines we e
s udied by G-banding (Medix Labo a o ies, Espoo, Finland)
o by Ka yoLi e assay [17] (Tu ku Cen e o Bio echnology,
Uni e si y o Tu ku, Tu ku, Finland).
2.3.6. Plu ipo ency Analysis. The plu ipo ency o hiPSC lines
was con i med in i o by emb yoid body (EB) o ma ion
and in i o by e a oma assay. hiPSCs we e emo ed om
eede cell laye and cul u ed in suspension o o m EBs. The
EBs we e cul u ed in EB medium consis ing o ko-DMEM
supplemen ed wi h 20% e al bo ine se um (FBS, Biose a,
Boussens,F ance),1%NEAA(LonzaG oupL d.),2mM
Glu aMax (In i ogen, Li e Technologies L d.), and 50 U/mL
penicillin/s ep omycin (Lonza G oup L d) o 4–6 weeks
be o e RNA ex ac ion. 200 ng o RNA was ansc ibed o
cDNA o heRT-PCRanalysis.Thep esenceo all h eege m
laye s, endode m (AFP, SOX17), ec ode m (SOX1, NESTIN,
and Musashi), and mesode m (KDR, alpha ca diac ac in), was
s udied wi h RT-PCR.
Fo in i o plu ipo ency assay, hiPSCs we e injec ed
unde he es is capsule o nude mice and he o med
e a omas we e collec ed and ixed wi h 4% PFA 8 weeks a e
he injec ion. Te a omas we e embedded in pa a in, cu in
sec ions, and s ained wi h haema oxylin and eosin.
2.4. Di e en ia ion o Ca diomyocy es. hiPSCs we e di -
e en ia ed in o CMs by cocul u ing wi h mouse isce al
endode mal-like cells (END-2) (Hub ech Ins i u e, U ech ,
Ne he lands) as desc ibed be o e [18]. A e 15–30 days bea -
ing a eas we e cu om cocul u es and dissocia ed in o single
cells in EB medium by Collagenase A (Roche Diagnos ics,
Mannheim, Ge many) as desc ibed ea lie [18] and pla ed
o 0.1% gela in-coa ed co e slips o well pla es o u he
analysis.
2.5. Cha ac e iza ion o hiPSC-De i ed Ca diomyocy es
2.5.1. Immunocy ochemis y and Image Analysis. Dissocia ed
CMs we e ixed wi h 4% PFA and s ained wi h T oponin
T(cTnT,1:2000,ab64623,Abcam,Camb idge,MA,USA),
MYBPC (1 : 400, sc-166081, San a C uz Bio echnology), and
TPM1 (1 : 200, sc-73225, San a C uz Bio echnology) p ima y
an ibodies, ollowed by labeling wi h seconda y an ibodies.
Images we e ob ained wi h Olympus IX51 phase con as
mic oscope equipped wi h luo escence op ics and Olympus
DP308W came a (Olympus Co po a ion) o wi h Zeiss Axio-
Scope A1 luo escen mic oscope and Zeiss AxioCam MRc5
came a (Ca l Zeiss, Jena, Ge many). Size o he T oponin T
s ainedCMswasanalyzed om46 o50CMsineachcellline
by in-house made so wa e (unpublished me hod). CMs we e
analyzed om pic u es ob ained wi h Olympus IX51 phase
con as mic oscope. The p opo ion o mul inuclea ed CMs
was de e mined om he same images (46–50 CMs/cell line).
2.5.2. Ca2+ Imaging. The clus e s o CMs we e cu , dissoci-
a ed, pla ed on 0.1% gela in-coa ed co e slips, and cul u ed
o 1,3,and6weeks.Tos udy heCa
2+ handling p ope ies o
hiPSC-de i ed CMs, cells we e loaded wi h 4 𝜇M Fu a-2 AM
(Molecula P obes, Li e Technologies L d.) o 30 minu es
in pe usa e medium. The pe usa e medium consis ed o
(in mM) 137 NaCl, 5 KCl, 0.44 KH2PO4, 20 HEPES, 4.2
NaHCO3, 5 D-glucose, 2 CaCl2,1.2MgCl
2,and1Na-py u a e
dissol edinH
2O. pH o he pe usa e medium was adjus ed
o 7.4 wi h NaOH. The co e slip, con aining he dissocia ed
hiPSC-de i ed CMs, was moun ed o an RC-25 eco ding
chambe and con inuously pe used wi h pe usa e medium
p ehea ed o 35-36∘C by an SH-27B inline-hea e con olled
byaTC-324Buni (all omWa ne Ins umen sInc.,
Hamden, USA). The pe usion was con olled by a g a i y
d i en VC38 applica ion sys em (ALA Scien i ic Ins umen s
Inc., NY, USA). Co e slip was pe used o 15 minu es o
Fu a-2 AM dees e i ica ion be o e expe imen al eco dings.
Ca2+ handling o spon aneously bea ing CMs was imaged
wi h an in e ed IX70 mic oscope using UApo/340 x20 ai
objec i e (Olympus Co po a ion) and ANDOR iXon 885
CCD came a (Ando Technology, Bel as , No he n I eland)
synch onized wi h a Polych ome V ligh sou ce by a eal ime
DPScon oluni .TILL isIONo Li eAcquisi ionso wa e
(TILL Pho onics, Munich, Ge many) was used o eco ding.
Fu a-2 AM was exci ed a 340 nm and 380 nm ligh and he
emission was eco ded o 10–30 seconds a 505 nm.
Fo Ca2+ imaging analysis, single bea ing CMs we e
selec ed as egions o in e es s and backg ound noise,
eco ded om a cell- ee a ea in he same co e slip, was
sub ac ed be o e u he p ocessing. Da a is p esen ed as
a ios o 340/380 nm (F340/F380). The spon aneously bea ing
CMs we e di ided in o i e di e en hy hm ca ego ies based
on he abno mali ies obse ed in hei Ca2+signals: no mal
bea ing wi h egula peaks (no mal); mo e han h ee peaks
which do no e u n o he baseline (oscilla ion); signals wi h
small o middle sized ampli ude e en s in he beginning, in
heend,o inbe ween woCa
2+ spikes (low/middle peaks);
wo o h ee peaks which do no e u n o he baseline
(double peaks); Ca2+ spikes wi h p olonged ise o decay ime
(pla eau abno mali y). In he low/middle peaks ca ego y, he
small ampli ude was a leas 10% om he p eceding Ca2+
spike ampli ude. Full-leng h, 10–30 seconds long eco dings
we e analyzed, while mos o he analyzed eco dings we e 12
seconds long. The dis ibu ion o CMs in di e en ca ego ies
is p esen ed o each cell lines sepa a ely.
2.5.3. Elec ophysiological Measu emen s: Reco ding and Anal-
ysis o Ac ion Po en ials. The ac ion po en ials (APs) we e
eco ded by con en ional pa ch clamp [19] in pe o a ed
pa ch con igu a ion using Ampho e icin B [20] in inal
concen a ion o 0.24 mg/mL [8]. Da a acquisi ion was
4S em Cells In e na ional
Table1:TaqManassaysusedinqRT-PCRexpe imen s.
Gene Desc ip ion/alias Func ion TaqMan assay ID
EEF1A1 Euka yo ic ansla ion elonga ion ac o 1 alpha 1 Housekeeping gene Hs00265885 g1
GAPDH Glyce aldehyde-3-phospha e dehyd ogenase Housekeeping gene Hs02758991 g1
TNNT2 T oponin T Sa come ic gene Hs00165960 m1
MYH6 Myosin hea y chain 6 Sa come ic gene Hs01101425 m1
ACTN2 𝛼-ac inin 2 Sa come ic gene Hs00153809 m1
TPM1 𝛼- opomyosin Sa come ic gene Hs00165966 m1
MYBPC3 Myosin-binding p o ein C Sa come ic gene Hs00165232 m1
TTN Ti in Sa come ic gene Hs00399225 m1
TNNC1 T oponin C ype 1 Sa come ic gene Hs00896999 g1
MYL9 Myosin, ligh chain 2/MLC2 Sa come ic gene Hs00697086 m1
MYL7 Myosin, ligh chain 7 Sa come ic gene Hs01085598 g1
NPPA Na iu e ic pep ide A Hype ophy ma ke Hs01081097 m1
NPPB Na iu e ic pep ide B Hype ophy ma ke Hs01057466 g1
HCN4 Hype pola iza ion ac i a ed cyclic nucleo ide-ga ed po assium channel 4 Po assium channel Hs00975492 m1
KCNQ1 Vol age-ga ed po assium channel, KQT-like sub amily, membe 1 Po assium channel Hs00923522 m1
CACNA1C Vol age-dependen calcium channel, L ype, alpha 1C subuni /CaCNA1.2 Calcium channel Hs00167681 m1
SCN5A Vol age-ga ed sodium channel, V ype, alpha subuni Sodium channel Hs00165693 m1
SLC8A1 Solu e ca ie amily 8, membe 1/NCX1 Na+/Ca2+ exchange Hs01062258 m1
PLN Phospholamban/PLB P o ein kinase subs a e Hs01848144 s1
ATP2A2 ATPase, Ca2+ anspo ing, ca diac muscle, slow wi ch 2/SERCA2a Ca2+-ATPase Hs00544877 m1
CASQ2 Calseques in Ca2+ binding p o ein in SR Hs00154286 m1
ITPR2 Inosi ol 1,4,5- isphospha e ecep o , ype 2/IP3R2 Ca2+ ecep o Hs00181916 m1
RYR2 Ryanodine ecep o 2 (ca diac) Ryanodine ecep o Hs00892883 m1
conduc ed using Axon Se ies 200B pa ch-clamp ampli ie
connec ed o Digida a 1440a AD/DA con e e d i en by
pCLAMP 10.2 so wa e (all om Molecula de ices LLC).
On he day o use, he co e slips con aining dissocia ed
hiPSC-de i ed CMs we e ans e ed o RC-24N eco ding
chambe (Wa ne Ins umen s Inc.) and moun ed on an
in e ed Olympus IX71 mic oscope (Olympus Co po a ion).
The pa ch elec odes had ip esis ance o 3.0–3.5 MΩand
con ained he ollowing in acellula solu ion (in mM): 132
KMeSO4,20KCl,1MgCl
2, and 1 CaCl2(pH was adjus ed o
7.2 wi h KOH). The ex acellula solu ion con ained (in mM)
143 NaCl, 4.8 KCl, 1.8 CaCl2, 1.2 MgCl2,5glucose,and10
HEPES (pH was adjus ed o 7.4 wi h NaOH). The p ehea ed
ex acellula solu ion was con inuously pe used wi h simila
se upcompa ed owha isp esen edinSec ion2.5.2.Pa ch
pipe es (Ha a d Appa a us L d., Hollis on, MA, USA)
we e eshly p epa ed using PC-10 mic opipe e pulle and
hen lame-polished wi h MF-830 mic o o ge (bo h om
Na ishige In ., Tokyo, Japan).
APs we e eco ded in he gap- ee mode in he cu en
clamp om he spon aneously bea ing hiPSC-de i ed CMs.
Cu en -clamp eco dings we e digi ally sampled a 20 kHz
and il e ed a 2 kHz using low pass Bessel il e on eco ding
ampli ie . Bea s pe minu e (BPM), AP du a ion (APD50
and APD90), AP ampli ude (APA), and maximum dias olic
po en ial (MDP) we e analyzed om he eco ded APs by
using O igin 9.1 (O iginLab Co p., No hamp on, USA).
Only en icula -like wa e o ms a e p esen ed he e o a oid
any biasness among di e en hiPSC lines. The en icula -
like CMs we e cha ac e ized by APD90/APD50 <1.3 and APA
>90 mV.
2.5.4. Real-Time qRT-PCR Analysis. A e one week o cul-
u e, dissocia ed CMs we e collec ed in o a lysis solu ion
bu e o CellsDi ec One-S ep qRT-PCR Ki (Li e Technolo-
gies L d.) acco ding o he manu ac u e ’s ins uc ions. Two
eplica e samples we e collec ed and s o ed a −70∘Cun il he
DNase I diges ion and e e se ansc ip ion-speci ic a ge
ampli ica ion (RT-STA) by using CellsDi ec One-S ep qRT-
PCR Ki . Real-Time qPCR was pe o med wi h Bioma k HD
sys em (Fluidigm Co p., San F ancisco, USA) acco ding o
he manu ac u e ’s ins uc ions. The TaqMan assays (Li e
TechnologiesL d.)usedin heqRT-PCRa ecollec edin
Table1.Allsampleswe eanalyzedinduplica eand he old
changeswe ecalcula edby he2−ΔΔCT me hod [21]. EEF1A1
and GAPDH genes we e used as endogenous con ol genes
and UTA.04511.WT cell line was used as a calib a o .
2.5.5. Wes e n Blo . hiPSC-de i ed CMs we e lysed in M-
PER p o ein ex ac ion eagen (The mo Scien i ic, Li e
Technologies L d.), supplemen ed wi h comple e p o ease
inhibi o cock ail (Roche Diagnos ics). The p o ein concen-
a ion was quan i ied wi h BCA p o ein assay ki (The mo
Scien i ic, Li e Technologies L d.). 10 𝜇go p o einwas un
o 4–15% mini-PROTEAN TGX p ecas polyac ylamide gel
S em Cells In e na ional 5
(Bio-Rad, He cules, CA, USA) and ans e ed o PVDF
memb ane (Ame sham Hybond-P, GE Heal hca e, Li le
Chal on , UK). Memb anes we e blocked wi h 5% milk o
1 h a RT and p o eins we e s ained wi h MYBPC (1 : 1500, sc-
166081, San a C uz Bio echnology), cTnT (1 : 2000, ab64623,
Abcam), TPM1 (1 : 200, sc-73225, San a C uz Bio echnology),
o 𝛽-ac in (1 : 1000, sc-47778, San a C uz Bio echnology) p i-
ma y an ibodies o e nigh a +4∘C. Ho se adish pe oxidase-
(HRP-) conjuga ed polyclonal abbi an i-mouse (DAKO,
P0260) and abbi an i-goa IgG (San a C uz Bio echnology,
sc-2922) we e used as seconda y an ibodies. S ained p o eins
we e de ec ed by using Cla i y ECL subs a e (Bio-Rad) and
isualized by Molecula Image ChemiDOc XRS+ (Bio-Rad).
ImageJ so wa e (Na ional Ins i u es o Heal h, USA) was
used o compa e he exp ession o MYBPC, cTnT, and TPM1
wi h he 𝛽-ac in exp ession om he same cell line.
2.6. S a is ical Analysis. Fo s a is ical analysis, con ol cell
lines and cell lines in each mu a ion we e combined in g oups:
UTA.04602.WT and UTA.04511.WT hiPSC lines in WT-CM
g oup, UTA.02912.HCMT and UTA.13602.HCMT in HCMT-
CM g oup, and UTA.07801.HCMM and UTA.06108.HCMM
in HCMM-CM g oup. Mann-Whi ney 𝑈 es wi h Bon e -
oni’s co ec ion was used o analyze he di e ences be ween
WT-, HCMT-, and HCMM-CMs in cell size analysis, p opo -
ion o mul inuclea ed CMs, and Ca2+ imaging expe imen s
as well as in gene exp ession analysis. Fo he s a is ical
compa ison be ween he h ee g oups, one-way ANOVA
ollowed by Tukey es was used o he pa ch-clamp esul
analysis. 𝑝 < 0.05 was conside ed s a is ically signi ican . All
e o ba s a e p esen ed as s anda d e o o he mean (SEM).
3. Resul s
3.1. hiPSCs We e De i ed om HCM Pa ien s wi h Di e en
Backg ounds. We de i ed hiPSCs om ou pa ien s ca ying
a HCM causing mu a ion ei he in TPM1 (TPM1-Asp175Asn)
o in MYBPC3 (MYBPC3-Gln1061X). UTA.13602.HCMT and
UTA.02912.HCMTs ca y TPM1-Asp175Asn and UTA.07801.
HCMM and UTA.06108.HCMM MYBPC3-Gln1061X mu a-
ion. The hiPSC lines and hei mu a ions and abb e ia ions,
used below, a e p esen ed in Table 2. UTA.13602.HCMT (46,
XX) is de i ed om a 48-yea -old emale, whose mo he
died suddenly a he age o 51. Ou pa ien has had one
collapse a he age o 20 wi h no mal hea s uc u e, bu
la e sligh hickening o sep um (16 mm) has been obse ed.
Cu en ly she is no on medica ion due o low blood p essu e.
UTA.02912.HCMT (46, XY) is de i ed om a 33-yea -old
male whose amily membe has died suddenly a he age
o 21. The maximal sep al hickness o ou pa ien has been
measu ed o be 26 mm on echoca diog am. The pa ien has
been asymp oma ic bu is cu en ly on 𝛽-blocke medica ion.
UTA.07801.HCMM (46, XY) is de i ed om a 61-yea -old
male wi h no SCDs in he amily. On echoca diog am, his
myoca dial sep um has been obse ed o be 25 mm. He has
a ial ib illa ion and he is on 𝛽-blocke medica ion. Due o
b adyca dia and equen nonsus ained en icula achyca -
dia episodes, an implan able ca dio e e de ib illa o (ICD)
Table 2: The hiPSC lines and hei mu a ions and abb e ia ions used
in he s udy.
G oup Cell line Mu a ion Name in
Figu e 2( )
WT UTA.04602.WT — WT1
UTA.04511.WT — WT2
HCMT UTA.02912.HCMT TPM1-Asp175Asn HCMT1
UTA.13602.HCMT TPM1-Asp175Asn HCMT2
HCMM UTA.07801.HCMM MYBPC3-Gln1061X HCMM1
UTA.06108.HCMM MYBPC3-Gln1061X HCMM2
has been implan ed. UTA.06108.HCMM (46, XY) is de i ed
om a 55-yea -old male whose a he died suddenly a he
age o 36 and uncle a he age o 38. Ou pa ien has been
asymp oma ic wi h maximal sep al hickness o 22 mm on
echoca diog am. He is no on medica ion due o low blood
p essu e. Con ol hiPSC lines we e de i ed om heal hy
indi iduals: UTA.04602.WT (46, XX) om a 56-yea -old
emale and UTA.04511.WT (46, XY) om a 34-yea -old male.
The plu ipo en cha ac e is ics o he hiPSC-lines used
we e assessed (Figu e 1 and Supplemen a y Figu es 1–5).
UTA.04602.WT cell line has been cha ac e ized ea lie [22].
All he lines o med colonies, which exp essed p o eins and
genes ypical o hPSCs. The i ally ans e ed exogenous
genes we e silenced and ka yo ypes o he hiPSC lines we e
no mal. The plu ipo ency o hiPSC lines was p o en in
i o by EB o ma ion o in i o by e a oma o ma ion.
The p esence o TPM1-Asp175Asn and MYBPC3-Gln1061X
mu a ions in he pa ien -speci ic hiPSC lines was con i med
by cus om TaqMan SNP Geno yping Assays (Supplemen a y
Figu e 6).
3.2. Mu a ion-Speci ic HCM Pheno ypes We e Obse ed in
hiPSC-De i ed Ca diomyocy es. All he cell lines used in he
p esen s udy di e en ia ed in o ca diomyocy es simila ly.
Bea ingagg ega eswe e o med14–20daysa e heini ia ion
o cocul u ing wi h END-2 cells and he e was no di e ence
be ween he lines when he bea ing a eas appea ed. A e
ca diac di e en ia ion, bea ing clus e s we e dissocia ed in o
single cells and cul u ed o 1, 3, and 6 weeks. The di e ences
in he cell sizes and Ca2+ handling p ope ies be ween
wo di e en mu a ions and con ol cells we e analyzed in
each ime poin . When compa ing di e en ypes o CMs,
HCMM-CMs we e signi ican ly la ge han HCMT-CMs and
WT-CMs in all h ee ime poin s (Figu es 2(a) and 2(b),
𝑛=96–100, 𝑝 < 0.005 in all cases). The enla gemen
o HCMT-CMs was de ec ed a e h ee weeks o cul u e
when hey we e signi ican ly la ge han WT-CMs (𝑝<
0.005). Gene ally, he size o he hiPSC-de i ed CMs in all
g oups inc eased when he cells cul u ed o h ee weeks.
Wi hin each g oup, he e we e no di e ences in cell sizes
be ween h ee and six weeks, excep wi h HCMT-CMs, which
seemed o be smalle in size 6-week ime poin . Howe e ,
du ing 6 weeks o cul u e, cell ypes o he han CMs had
he endency o o e g ow he CM cul u e, which migh
ha e a ec ed he cellula enla gemen . This phenomenon was

6S em Cells In e na ional
OCT4 TRA-1-60
Nanog SOX2 TRA-1-81
(a)
500
250
500
250
Exogenous
Endogenous
OCT4
OCT4
KLF4 SOX2
SOX2
c-MYC
c-MYC
++++
GAPDH
Nanog
H2O
H2O
H2O
REX1
(b)
123 45
6 7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22 XY
(c)
Mesode m Endode m Ec ode m
(d)
Figu e 1: Cha ac e iza ion o UTA.13602.HCMT cell line. (a) The hiPSCs o med colonies exp essing Nanog, OCT4, SOX2, TRA-1-60, and
TRA-1-81.Scaleba s:200𝜇m. (b) The i ally ans e ed Sendai exogenes, exo-OCT4 (483 bp), exo-KLF4 (410 bp), exo-SOX2 (451 bp), and exo-
c-MYC (532 bp), we e silenced in he hiPSCs. + indica es posi i e con ols, o which RNA was ex ac ed om cells 1 week a e ansduc ion.
hiPSCs exp essed endogenous Nanog (287 bp), OCT4 (144 bp), REX1 (306 bp), SOX2 (151 bp), and c-MYC (328 bp). GAPDH (302 bp) was
used as a housekeeping con ol. (c) The hiPSC line was ka yo ypically no mal, 46 XX. (d) The plu ipo ency o hiPSCs was con i med by in
i o e a oma assay, in which hiPSCs o med all h ee ge m laye s (mesode m, endode m, and ec ode m).
S em Cells In e na ional 7
WT HCMT HCMM
(a)
12
10
8
6
4
2
×104
(pixels)
n = 100
n = 100
n = 100
n = 100
n = 100
n=96
n = 100
n = 100
n = 100
∗∗ ##
WT 1w
WT 3w
WT 6w
HCMT 1w
HCMT 3w
HCMT 6w
HCMM 1w
HCMM 3w
HCMM 6w
$$
&&
(b)
∗∗
50
40
30
20
10
(%)
WT 1w
WT 3w
WT 6w
HCMT 1w
HCMT 3w
HCMT 6w
HCMM 1w
HCMM 3w
HCMM 6w
(c)
∗∗
50
40
30
20
10
(%)
WT 1w
WT 3w
WT 6w
HCMT 1w
HCMT 3w
HCMT 6w
HCMM 1w
HCMM 3w
HCMM 6w
(d)
No mal
246810
Time (s)
F340/F380
Oscilla ion
246810
Time (s)
F340/F380
Low/middle peaks
246810
Time (s)
F340/F380
Pla eau abno mali y
246810
Time (s)
F340/F380
Double peaks
246810
Time (s)
F340/F380
(e)
Figu e 2: Con inued.
8S em Cells In e na ional
50
60
70
80
90
40
30
20
10
(%)
n=
WT11w
WT13w
WT16w
WT21w
WT23w
WT26w
HCMT11w
HCMT13w
HCMT16w
HCMT21w
HCMT23w
HCMT26w
HCMM11w
HCMM13w
HCMM16w
HCMM21w
HCMM23w
HCMM26w
89 24 20 99 66 50 99 104 19 145 24 97 78 141 83 90 29 42
( )
Figu e 2: The cell size and Ca2+ handling o hiPSC-de i ed CMs a e 1-, 3-, and 6-week cul u e as single cells. (a) Rep esen a i e images o
WT-CMs (WT), HCMT-CMs (HCMT), and HCMM-CMs (HCMM) s ained wi h an ibodies o cTnT ( ed) and MYBPC (g een) p o eins.
Scale ba s a e 100 𝜇m. (b) The size o he HCMM-CMs was signi ican ly la ge in all h ee ime poin s when compa ed o WT- and HCMT-
CMs ($$𝑝< 0.005 when compa ed o WT-CMs o HCMT-CMs in he 1-week ime poin , ∗∗𝑝< 0.005 when compa ed o WT-CMs o
HCMT-CMs in 3-week ime poin , and ##𝑝< 0.005 when compa ed o WT-CMs o HCMT-CMs in 6-week ime poin ). HCMT-CMs
we e signi ican ly la ge han WT-CMs in 3-week ime poin (&&𝑝< 0.005 when compa ed o WT-CMs. 𝑛 = 100, excep in HCMT 6 w
𝑛=96.) (c) The p opo ion o he mul inuclea ed CMs was signi ican ly highe in HCMT-CMs han in WT-CMs and HCMM-CMs when
bo h cell lines and all ime poin s we e combined o each g oup (in s a is ical analysis 𝑛=6,∗𝑝< 0.05). The a e ages o mul inuclea ed
CMs we e de e mined om he same cells, whose sizes and 𝑛numbe s a e p esen ed in (b). (d) Signi ican ly mo e CMs wi h Ca2+ handling
abno mali ies we e obse ed in HCMT-CMs han in WT-CMs and HCMM-CMs when bo h cell lines and all ime poin s we e combined
o each g oup (in s a is ical analysis 𝑛=6,∗𝑝< 0.05). The p opo ions o CMs wi h abno mali ies in hei Ca2+ handling we e de e mined
om he same Ca2+ imaging esul s p esen ed in ( ). The o al 𝑛numbe s o he analyzed CMs a e p esen ed in ( ). (e) Rep esen a i e
images o Ca2+ hy hm ca ego ies. ( ) Dis ibu ions o hiPSC-de i ed CMs in di e en Ca2+ hy hm ca ego ies (e) in each ime poin . WT1 =
UTA.04602.WT, WT2 = UTA.04511.WT, HCMT1 = UTA.02912.HCMT, HCMT2 = UTA.13602.HCMT, HCMM1 = UTA.07801.HCMM, and
HCMM2 = UTA.06108.HCMM.
obse ed especially wi h UTA.13602.HCMT cell line (da a
no shown). When all ime poin s in each g oup (WT-CM,
HCMT-CM, and HCMM-CM) we e combined, he numbe
o mul inuclea ed CMs was signi ican ly highe in HCMT-
CMs han in WT-CMs and HCMM-CMs (𝑛=6in s a is ical
analysis, 𝑝 < 0.05, Figu e 2(c)).
The Ca2+handling p ope ies o hiPSC-de i ed CMs we e
s udied by Ca2+ imaging. HCMT-CMs had signi ican ly mo e
abno mali ies han WT-CMs and HCMM-CMs, when all
ime poin s in each g oup we e combined (Figu e 2(d), 𝑛=
6in s a is ical analysis, 𝑝 < 0.05). Spon aneously bea ing
CMs we e di ided in o i e di e en hy hm ca ego ies
(no mal, oscilla ion, low/middle peaks, double peaks, and
pla eau abno mali y) based on he abno mali ies obse ed in
hei Ca2+ signals (Figu es 2(e) and 2( )). HCMT-CMs had
signi ican ly highe numbe o double peaks han HCMM-
CMs (𝑝 < 0.05,𝑛=6in s a is ical analysis) when bo h cell
lines and all ime poin s we e combined o each g oup.
3.3. Ac ion Po en ial Cha ac e is ics o WT and HCM hiPSC-
De i ed Ca diomyocy es. The spon aneous ac ion po en ials
we e eco ded om he bea ing hiPSC-de i ed CMs o
es ablish he elec ophysiological baselines. Mos o he cells
(>80%) we e en icula -like CMs in all he hiPSC lines
s udied. Fo his eason, only en icula -like wa e o ms
a e p esen ed he e. We i s analyzed he pe cen age o
he a hy hmias in each cell line (Figu es 3(a)–3( )) and
ound simila pe cen age in bo h cell lines wi hin he
g oups (UTA.04602.WT (13%) e sus UTA.04511.WT (15%),
UTA.02912.HCMT (42%) e sus UTA.13602.HCMT (47%),
and UTA.07801.HCMM (50%) e sus UTA.06108.HCMM
(50%)). Based on he pe cen age o he a hy hmias, we com-
bined hiPSC-de i ed CMs in o g oups (WT-CM, HCMT-
CM, and HCMM-CM) o u he analysis.
Bo h HCMT-CMs and HCMM-CMs had mo e
a hy hmic e en s including delayed a e depola iza ions
(DADs) and ea ly a e depola iza ions (EADs) when
compa ed o he WT-CMs (WT-CM (14%), HCMT-
CM (45%), and HCMM-CM (50%)). We quan i ied he
occu ence o DADs in hiPSC-de i ed CMs as a a e
(DADs/min) calcula ed as o al numbe o DADs/ o al
numbe o APs.We ound ha heDAD a einHCMM-
CMs was signi ican ly highe han in WT-CMs (Figu e 3(g),
𝑝 < 0.005).
S em Cells In e na ional 9
WT
(a)
HCMT
(b)
HCMM
(c)
HCMT
(d)
HCMM
(e)
n = 23 n = 20 n = 24 n = 47 n = 32 n = 22
80
60
40
20
(%)
DADs and EADs
EADs
DADs
No mal
UTA.04602.WT
UTA.04511.WT
UTA.02912.HCMT
UTA.13602.HCMT
UTA.07801.HCMM
UTA.06108.HCMM
( )
2
4
6
8
DADs/min
n=43
WT
n=71
HCMT
n=54
HCMM
∗∗
(g)
Figu e 3: A hy hmogenic e en s (DADs and EADs) we e obse ed in HCM-CMs. (a)–(e) Rep esen a i e eco dings o con ol hiPSC-
de i ed CMs (WT) and hiPSC-de i ed CMs ca ying TPM1-Asp175Asn (HCMT) o MYBPC3-Gln1061X (HCMM) mu a ions. Typical DADs
(a ows) a e p esen ed in (b) and (c) and EADs (a ows) in (d) and (e) o HCMT-CMs and HCMM-CMs, espec i ely. Scale ba s ep esen
40 m and 5 seconds, espec i ely. Scale ba s in (a) a e ep esen a i e o (b) and (c), and scale ba s in (d) a e ep esen a i e o (e). ( )
Dis ibu ion o CMs exhibi ing a hy hmogenic e en s in each cell line. (g) DAD a e was signi ican ly highe in HCMM-CMs han in WT-
CMs (∗∗𝑝< 0.005).
16 S em Cells In e na ional
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