ORIGINAL ARTICLE
Ma u a ion o human plu ipo en s em cell de i ed
ca diomyocy es is imp o ed in ca dio ascula cons uc
Hanna Vuo enpa
¨a
¨.Ki si Pen inen .Tuula Heinonen .Ma i Pekkanen-Ma ila .
Je a-Riina Sa kanen .Timo Ylikomi .Ka iina Aal o-Se a
¨la
¨
Recei ed: 4 No embe 2016 / Accep ed: 17 Ma ch 2017 / Published online: 10 Ap il 2017
ÓThe Au ho (s) 2017. This a icle is an open access publica ion
Abs ac In o de o ansla e p eclinical da a in o
he clinical s udies, ele an in i o models wi h
s uc u e and key unc ional p ope ies simila o
na i e human issue should be used. In i o ca diac
models wi h ascula s uc u es mimic he highly
ascula ized myoca dium and p o ide in e ac ions
be ween endo helial cells, s omal cells and ca diomy-
ocy es. Cu en ly, human plu ipo en s em cell-
de i ed ca diomyocy es (hPSC-CMs) ha e been
shown o p esen imma u e mo phology and e al-like
elec ophysiological p ope ies ha may limi hei use
as physiological es pla o m. The aim o his s udy
was o de elop mul icellula in i o ca dio ascula
cons uc modeling human hea issue. In he ca dio-
ascula cons uc , hPSC-CMs we e cul u ed wi h a
ascula -like ne wo k o med by human o eskin
ib oblas s and human umbilical ein endo helial cells
ha se ed as a pla o m in he cons uc . Ca diomy-
ocy e o ien a ion, ma u a ion, elec ophysiological
p ope ies and d ug esponses o he ca dio ascula
cons uc we e cha ac e ized and compa ed o CM
monocul u e. hPSC-CMs in ca dio ascula cons uc
showed elonga ed mo phology and aligned wi h he
ascula -like ne wo k. Elec ophysiological p ope ies
and calcium me abolism o hPSC-CMs as well as
Elec onic supplemen a y ma e ial The online e sion o
his a icle (doi:10.1007/s10616-017-0088-1) con ains supple-
men a y ma e ial, which is a ailable o au ho ized use s.
Hanna Vuo enpa
¨a
¨and Ki si Pen inen ha e con ibu ed equally
o his wo k.
H. Vuo enpa
¨a
¨T. Heinonen T. Ylikomi
FICAM, Finnish Cen e o Al e na i e Me hods, Facul y
o Medicine and Li e Sciences, Uni e si y o Tampe e,
PL 100, 33014 Tampe e, Finland
e-mail: [email p o ec ed]
T. Heinonen
e-mail: [email p o ec ed]
T. Ylikomi
e-mail: [email p o ec ed]
K. Pen inen M. Pekkanen-Ma ila K. Aal o-Se a
¨la
¨
BioMediTech, Ins i u e o Biomedical Technology,
Facul y o Medicine and Li e Sciences, Uni e si y o
Tampe e, PL 100, 33014 Tampe e, Finland
e-mail: [email p o ec ed]
K. Pen inen (&)T. Ylikomi K. Aal o-Se a
¨la
¨
Facul y o Medicine and Li e Sciences, Uni e si y o
Tampe e, PL 100, 33014 Tampe e, Finland
e-mail: [email p o ec ed]
J.-R. Sa kanen T. Ylikomi
Depa men o Cell Biology, Facul y o Medicine and Li e
Sciences, Uni e si y o Tampe e, Tampe e, Finland
e-mail: [email p o ec ed]
K. Aal o-Se a
¨la
¨
Hea Hospi al, Tampe e Uni e si y Hospi al, Tampe e,
Finland
e-mail: [email p o ec ed]
123
Cy o echnology (2017) 69:785–800
DOI 10.1007/s10616-017-0088-1
esponse o E-4031 and ad enaline demons a ed
no mal physiological beha io . Inc eased exp ession
o ca diac s uc u al p o eins and ion channels in
ca dio ascula cons uc compa ed o CM monocul-
u e we e de ec ed. In conclusion, ascula -like ne -
wo k suppo s he s uc u al and unc ional ma u a ion
o hPSC-CMs. Ou esul s sugges ha ca dio ascula
cons uc p esen s mo e ma u e in i o ca diac model
compa ed o CM monocul u e and could he e o e
se e as an ad anced es sys em o ca diac sa e y and
e icacy assessmen as well as a model sys em o
biomedical esea ch.
Keywo ds Ca diomyocy es Vascula -like
ne wo k Ma u a ion
In oduc ion
In adul hea , he majo i y o ca diac cells a e
highly adap i e cells including ib oblas s, ascula
smoo h muscle cells and endo helial cells. 20–40%
o he ca diac cells a e ca diomyocy es (Soonpaa
and Field 1998; B u sae 2003). In ca diac mic oen-
i onmen , ca diomyocy es a e embedded in aligned
ex acellula ma ix (ECM) ha acili a es he coo -
dina ed con ac ile unc ion o he hea (Chien e al.
2008). Cells and ECM p o eins, mainly p oduced by
ib oblas s, a e connec ed ia cell–cell and cell–
ma ix in e ac ions o main ain he s uc u al o ga-
niza ion and unc ionali y o he hea ( an Sp eeu-
wel e al. 2014; P annkuche e al. 2010b). In ma u e
myoca dium, each ca diomyocy e has physical con-
ac wi h a leas one capilla y blood essel (Ga zoni
e al. 2009). The in e ac ions be ween ascula u e
and myoca dium a e bidi ec ional (Bha acha ya
e al. 2006), and ac i e h oughou he adul li e
a ec ing ca diac g ow h, unc ion and hy hm
(B u sae 2003). F om he onse o he ca diac
de elopmen , endo helial cells a e he p e equisi e
o myoca dial ma u a ion, physiological unc ion
and su i al (B u sae 2003). Endo helial cells and
ca diomyocy es in e ac wi h each o he wi h pa a-
c ine signals du ing hea de elopmen and ac i a e
he de elopmen o ca diac s uc u e. This in e ac-
ion is equi ed o p ope de elopmen o endo-
ca dium and myoca dium. Vascula endo helial cells
p oduce se e al compounds including angiopoie in 2
(Ang-2), whe eas angiopoie in 1 (Ang-1) and as-
cula endo helial g ow h ac o (VEGF) a e mainly
p oduced by ca diomyocy es (B u sae 2003;
Leucke e al. 2011; Hsieh e al. 2006). In addi ion,
ib oblas s sec e e ac o s wi h au oc ine and pa a-
c ine e ec s, such as VEGF, ib oblas g ow h
ac o s (FGFs) and ans o ming g ow h ac o be a
(TGF-b), ha gi e mechanical suppo and p omo e
he o ganiza ion o ca diomyocy es in o 3D s uc-
u es in collagen ma ices (P annkuche e al. 2010b;
Wong e al. 2007).
Ca dio oxici y is one o he leading causes o
ailu e o a new he apeu ic molecule p eclinical
de elopmen (B aam e al. 2010). P esen ly, pha ma-
ceu ical indus y elies upon animal es ing, al hough
he e a e undamen al di e ences in he elec ophys-
iological p ope ies o animal and human ca diomy-
ocy es (Fe ic and Radisic 2016). In pa icula , he
di e ences in ion channels and cu en s impac he
poo p edic i i y o d ug sc eening and oxici y s udies
om mu ine o humans (Polini e al. 2014). In addi ion
o animal models, ans ec ed non-ca diac cells ha e
been used in p eclinical es ing. Howe e , hese cells
exp ess usually only one ca diac ion channel and lack
o he cha ac e is ics o human ca diomyocy es. T ans-
ec ed non-ca diac cells lack a ca diac in acellula
en i onmen ha includes ca diomyocy e speci ic
accesso y p o eins, as well as, cell s uc u e (Ma in
e al. 2004). The e o e, he e is an unme need o
imp o ed human ca diomyocy e model o p eclinical
d ug sc eening especially o assessmen o ca -
dio oxic e ec s and o e alua ion o he e icacy o
new d ug candida es (Fe ic and Radisic 2016; Ke en-
ho en and Bohlen 2008). The cell model should mimic
e ec i ely human ca diomyocy es o example in
e ms o cell s uc u e, ca diac speci ic ion channels
and elec omechanical unc ion.
Func ional ca diomyocy es de i ed om human
plu ipo en s em cells could p o ide a signi ican
ad an age o e he p e iously used es sys ems.
Human plu ipo en s em cell de i ed ca diomyocy es
(hPSC-CMs) con ac spon aneously and espond
app op ia ely o ca dioac i e d ugs (Robe son e al.
2013). These cells could be use ul in ea ly e icacy and
oxici y sc eening, imp o ing he selec ion o lead
candida es and he educ ion o ad e se ou comes in
clinical s ages o d ug de elopmen (Da ila e al.
2004). Acco ding o hei mo phological and unc-
ional cha ac e is ics, hPSC-CM ha e been shown o
786 Cy o echnology (2017) 69:785–800
123
display p ope ies ypical o human e al ca diomy-
ocy es which may complica e hei u iliza ion and
in e p e a ion o he ob ained esul s (Sni e al. 2003;
Robe son e al. 2013). Compa ed o he human adul
ca diomyocy es he hPSC-CMs ha e been epo ed o
ha e lowe exp ession o he genes ela ed o he ion
anspo a ion, calcium handling and sa come e s uc-
u e (Yang e al. 2014). Fu he mo e, he mo phology
o he hPSC-CMs does no esemble he well-aligned
od-like na u e o adul human ca diomyocy es.
The e o e, he ma u a ion le el o hPSC-CMs can be
assessed by measu ing he exp ession le els o genes
encoding sa come ic p o eins such as ca diac T o-
ponin I and T as well as a- and bmyosin hea y chains.
In addi ion, he exp ession le els o he ca diac
speci ic ion channels and connexin 43 esponsible
o he in acellula communica ion can be used in
es ima ion o he ma u a ion s a e (Sa iani e al.
2007). In addi ion o he inc eased ime in cul u e,
elec ical s imula ion (Chan e al. 2013) and 3D
cul u e en i onmen (Schaa e al. 2011; an Sp eeu-
wel e al. 2014; Ga zoni e al. 2009; Vala ma hi e al.
2010; Pon es Soa es e al. 2012) ha e been u ilized in
he p oduc ion o mo e ma u e hPSC-CMs. Howe e ,
he use o sca olds o enable 3D en i onmen has been
associa ed wi h educed cell–cell con ac s, as well as
inco ec deposi ion and alignmen o ex acellula
ma ix in ca diac cons uc s (No o e e al. 2009).
In his s udy, a mul icellula in i o ca dio ascula
cons uc was de eloped. In he cons uc , hPSC-CMs
we e cul u ed wi h ascula -like ne wo k composed o
human ib oblas s and human umbilical ein endo he-
lial cells (HUVEC). Vascula -like ne wo k se es as a
suppo ing pla o m o c ea e na u al mic oen i on-
men , i.e. biological sca old, wi h cell–cell and cell–
ma ix in e ac ions. The e ec s o he ascula -like
ne wo k on ca diomyocy e mo phology, gene exp es-
sion and unc ionali y we e s udied. Resul s demon-
s a ed ha he ascula -like ne wo k enhanced he
s uc u al ma u a ion and inc eased he exp ession
le els o ca diac ion channels in hPSC-CMs in he
ca dio ascula cons uc . Ou s udy sugges s ha his
ca dio ascula cons uc p o ides a mo e ma u e
in i o ca diac model compa ed o CM monocul u e
and could he e o e se e as an ad anced es sys em
o ca diac sa e y and e icacy assessmen as well as a
model sys em in biomedical esea ch.
Ma e ials and me hods
E hics
This s udy con o ms o he p inciples ou lined in he
Decla a ion o Helsinki. The use o human umbilical
ein endo helial cells (HUVEC) and induced plu ipo-
en s em cells (iPSC) we e app o ed by E hics
Commi ee o he Pi kanmaa Hospi al Dis ic , Tam-
pe e, Finland (App o al Numbe s R08028 and
R08070, espec i ely) and a w i en in o med consen
was ob ained om all he pa icipan s.
Cell cul u e and di e en ia ion
Isola ion and cul u e o human umbilical ein
endo helial cells
HUVECs we e isola ed om human umbilical co d
ein by using enzyma ic p ocedu e as desc ibed
p e iously by us (Sa kanen e al. 2011). Cells we e
de ached om umbilical co ds wi h 0.05% collage-
nase I, cul u ed in EGM-2 medium (Lonza G oup L d,
Basel, Swi ze land, Table 1) and es ed o myco-
plasma con amina ion (MycoAle
Ò
Mycoplasma
De ec ion Ki , Lonza G oup L d) be o e expe imen al
use. We use HUVEC as a s anda d sou ce o
endo helial cells in cell cul u e s udies in ou labo a-
o y. The isola ion and cul u e p ocess o HUVECs has
been es ablished and alida ed p oducing cell lines
wi h low a iabili y (Sa kanen e al. 2011). Cells a e
ou inely s o ed in liquid ni ogen in passage 2 and
used o es ablishing cell cul u e models in passage 4.
Each HUVEC line is de i ed om a single dono and
quali y es ed ou inely o hei abili y o o m ubule
s uc u es in cell cul u e.
Cul u e o human o eskin ib oblas s
Human o eskin ib oblas s we e pu chased om
Ame ican Type Cul u e Collec ion (BJ, CRL-2522;
ATCC, Manassas, VA, USA). Cells we e cul u ed in
ib oblas medium (Table 1) consis ing o Minimum
Essen ial Medium wi h Ea le’s sal s, w/o L-glu amine
(Gibco, Van aa, Finland) supplemen ed wi h 10% FBS
(Gibco), 1% L-glu amine (Gibco) and 1% NEAA
(Gibco). Cells we e es ed o mycoplasma
Cy o echnology (2017) 69:785–800 787
123
con amina ion (MycoAle
Ò
Mycoplasma De ec ion
Ki , Lonza) be o e expe imen al use.
Gene a ion o pa ien -speci ic iPSC line and cell
cul u e o plu ipo en s em cells
In his s udy one comme cial human emb yonic s em
cell (hESC) line H7 pu chased om WiCell Resea ch
Ins i u e (Madison, WI, USA) and one iPSC line was
used o ca diomyocy e di e en ia ion. Pa ien -speci-
ic iPSC line UTA.04602.WT was es ablished om a
heal hy indi idual as desc ibed ea lie (Takahashi
e al. 2007). Sho ly, skin biopsy om he dono was
cul u ed in 0.2% gela in (Sigma-Ald ich, Espoo,
Finland) coa ed lask unde ib oblas cul u ing con-
di ions. iPSC line was es ablished using len i i us
in ec ion ollowed by e o i us in ec ion (Takahashi
e al. 2007). Cells, plasmids and eagen s used in his
p o ocol include: 293FT cells, Pla -E cells, pLen i6/
UbC/mSlc7a1- ec o (Addgene, Camb idge, MA,
USA), Vi aPowe
TM
Packaging Mix (Li e Technolo-
gies L d), Lipo ec amine
TM
2000 (Li e Technologies
L d, Van aa, Finland), Fugene 6 (Roche Diagnos ics,
Mannheim, Ge many), and pMX e o i al ec o s
(hOCT3/4, hSOX2, hKLF4 and hc-MYC, all om
Addgene). Resul s o he cha ac e iza ion o
UTA.04602.WT cell line ha e been desc ibed ea lie
(Lah i e al. 2012).
UTA.04602.WT cells and H7 hESCs we e cul u ed
on mi omycin C inac i a ed mouse emb yonic ib ob-
las s (MEF) in KSR medium which consis ed o
DMEM/F-12 (In i ogen) supplemen ed wi h 20%
KnockOu se um eplacemen (In i ogen), 1% non-
essen ial amino acids (Lonza), 2 mM Glu amax
(In i ogen), 50 U/ml penicillin/s ep omycin
(Lonza), 0.1 mM be a me cap oe hanol (In i ogen)
and 7.8 ng/ml basic ib oblas g ow h ac o (R&D
Sys ems). The medium was e eshed daily, and he
s em cell colonies we e passaged on o a new MEF
laye once a week using 1 mg/ml collagenase IV
(In i ogen).
Di e en ia ion o ca diomyocy es
Di e en ia ion o plu ipo en s em cells in o ca -
diomyocy es was ca ied ou wi h ei he by co-
cul u ing hESC o iPSC wi h mu ine isce al endo-
de m-like (END-2) cells (Humb ech Ins i u e,
U ech , The Ne he lands) as desc ibed ea lie (Mum-
me y e al. 2003) o wi h small molecule di e en i-
a ion me hod ia empo al modula ion o canonical
Wn signaling (Lian e al. 2013). B ie ly, in END-2
me hod undi e en ia ed hPSC colonies we e dissec ed
mechanically in o agg ega es and pla ed on op o
mi omycin C (Toc is) ea ed END-2 cells in he hPSC
medium. The cul u e medium was supplemen ed wi h
2.92 mg/ml o asco bic acid (Sigma-Ald ich). The
medium was e eshed on days 5, 8 and 12. On day 14,
10% se um eplacemen was added o he medium.
B ie ly, o small molecule di e en ia ion hPSCs
main ained on a Gel ex-coa ed su ace in mTeSR1
(S emcell Technologies, Cologne, Ge many) we e
dissocia ed in o single cells wi h Accu ase (Li e
Technologies) a 37 °C o 5 min and hen seeded
on o a Gel ex-coa ed cell cul u e dish a 50,000 cell/
cm
2
in mTeSR1 supplemen ed wi h 5 lM ROCK
inhibi o (Toc is, Minneapolis, MN, USA) o 24 h.
Cells we e hen cul u ed in mTeSR1, changed daily.
On di e en ia ion day 0, medium was exchanged wi h
RPMI medium (Li e Technologies) wi h 19B27
Ò
Table 1 Cul u e media used o de elopmen o cell models
Ac onym Basal
medium
Se um G ow h ac o s Supplemen a ion
Fib oblas medium MEM 10% FBS – 1% L-glu amine, 1% NEAA
EGM-2 medium EBM-2 2% FBS VEGF, FGF-2,
IGF, EGF
Hyd oco isone, asco bic
acid, hepa in
Angiogenic s imula ion medium EBM-2 2% FBS 10 ng/ml VEGF,
1 ng/ml FGF-2
1% L-glu amine
EB 5% DMEM/F12 5% FBS – 1% NEAA,
1% Glu amax,
0.5% Pen/S ep
VEGF ascula endo helial g ow h ac o , FGF-2 ib oblas g ow h ac o 2, EGF epide mal g ow h ac o
788 Cy o echnology (2017) 69:785–800
123
Supplemen minus insulin (Li e Technologies) wi h
10 lM CHIR99021 (S emgen , Lexing on, MA,
USA). 24 h la e , medium was exchanged wi h
RPMI/B27 wi hou insulin. On day 3, medium was
exchanged wi h RPMI/B27 wi hou insulin supple-
men ed wi h 5 lM IWP4 (Mil enyi Bio ech, Be gisch
Gladbach, Ge many). On day 5, medium was
exchanged wi h RPMI/B27 wi hou insulin. On day
7 and e e y 3 days ollowing, medium was exchanged
wi h RPMI/B27.
Bo h di e en ia ion me hods o med bea ing ca -
diomyocy e agg ega es. These we e mechanically
excised and ea ed wi h collagenase A (Roche
Diagnos ics) o dissocia e bea ing agg ega es o single
cell le el (Mumme y e al. 2003). In each expe imen ,
ca diomyocy es pla ed on he monocul u e and on op
o he ascula -like ne wo k o igina ed om he same
di e en ia ion and dissocia ion ba ch.
De elopmen o cell models
Human ca diomyocy e monocul u e
A e dissocia ion, human hPSC-CMs we e seeded in
EB 5% medium (Table 1) a densi y o
0.01–0.04 910
6
cells/cm
2
in 0.1% gela in ype A
(Sigma-Ald ich) coa ed 48-well pla es o immuno-
cy ochemical and qRT-PCR analyses and in mic o-
elec ode a ay (MEA) pla o ms and gela in coa ed
12 mm co e slips o unc ional analyses. EB 5%
medium was changed 1–2 days a e cell seeding and
he ea e wice a week. hPSC-CM monocul u e in EB
5% medium was used as a con ol h oughou he
s udy.
Vascula -like ne wo k om co-cul u e o HUVEC
and ib oblas
The co-cul u e was es ablished as desc ibed ea lie
(Sa kanen e al. 2011). B ie ly, ib oblas s (p 6–7),
we e seeded a 20,000 cells/cm
2
in ib oblas medium
(Table 1) in 48-well pla es o immunocy ochemical
and qRT-PCR analyses and in MEA-pla o ms and
12 mm diame e co e slips o unc ional analyses
and g own o 2–3 days o con luency. HUVEC we e
seeded on op o con luen ib oblas cul u es a
4000 cells/cm
2
in EGM-2 medium (Table 1). The
day a e cell seeding, angiogenic s imula ion medium
con aining EBM-2 (Lonza), 2% FBS, 1 mM L-glu-
amine, 10 ng/ml ascula endo helial g ow h ac o
(VEGF, Sigma) and 1 ng/ml ib oblas g ow h ac o 2
(FGF-2, Sigma) (Table 1) was applied o cells. The
angiogenic s imula ion medium was changed wice
du ing he 6 day co-cul u e p io o CM seeding.
Ca dio ascula cons uc
EB 5% medium (Table 1) was changed o
HUVEC ? ib oblas co-cul u e be o e seeding
hPSC-CMs. Dissocia ed hiPSC- o hESC-de i ed
ca diomyocy es in EB 5% medium (Fig. 1) we e
seeded on op o he HUVEC ? ib oblas co-cul u e
a day 6, when he ascula -like ne wo k was al eady
o med, a a densi y o 0.01–0.04 910
6
cells/cm
2
in
48-well pla es o immunocy ochemical and qRT-
PCR analyses and in mic oelec ode a ay (MEA)
pla o ms and 12 mm diame e co e slips o unc-
ional analyses. 1–2 days a e ca diomyocy e seeding
i s EB medium change was pe o med and he ea e
Fig. 1 Es ablishmen o
ca dio ascula cons uc
Cy o echnology (2017) 69:785–800 789
123

h ee imes in a week. The iabili y o ca dio ascula
cons uc s was e alua ed isually unde mic oscope
by assessing he con ac ion o he ca diomyocy es
and ascula -like ne wo k o ma ion o he HUVECs
and ib oblas s a leas h ee imes in a week.
Medium de elopmen
In ca dio ascula cons uc , medium was designed o
p ima ily suppo ca diomyocy e unc ionali y and
seconda ily o induce ascula -like ne wo k o ma-
ion. EB medium (Table 1) wi h di e en se um
concen a ions including 0, 2, 5, 10, and 20% FBS
(Biose a-Immuno Diagnos ic Oy, Ha
¨meenlinna, Fin-
land) we e es ed wi h duplica e wells o dissocia ed
ca diomyocy es due o ou objec i e o ha e low-
se um o se um- ee cul u e condi ions, o a oid
exogenous bias in ou cell cul u e sys em and o
de elop in i o model ha mimics closely he in i o
si ua ion in o de o imp o e he p edic i e alue o
he es sys em. In ha , i is impo an o p o ide
physiologically ele an en i onmen wi h low/no-
se um cul u e condi ions. Cells we e cul u ed o
9 days and ixed wi h 4% PFA (Sigma-Ald ich) o
immunocy ochemical s ainings wi h goa -an i-ca diac
oponin-T (an i-Tn , 1:1500, Abcam, Camb idge,
U.K.). Cells we e moun ed wi h Vec ashiel (Vec o
Labo a o ies, Bu lingame, CA, USA) con aining
DAPI o s aining nuclei. The esul s o he se um
concen a ion es s can be ound in he supplemen al
da a (Fig. S1).
Immunocy ochemis y
Ca diomyocy es in CM monocul u e and in ca dio-
ascula cons uc we e s ained ei he wi h goa an i-
ca diac- oponin-T (an i-Tn , 1:1500, Abcam) o wi h
mouse an i-Tn (1:500, Abcam) and he ascula -like
o ma ion was isualized wi h basemen memb ane
ma ke mouse collagen IV (an i-ColIV, 1:500, Sigma)
o 1 h a RT o in ?4°C o/n. Polyclonal IgG Alexa
Fluo 568 (Abcam) o goa an i-Tn and polyclonal
IgG FITC (1:100, Sigma) o an i-ColIV and mouse
an i-Tn we e used as seconda y an ibodies o 30 min
in RT. Fluo escence was isualized wi h Nikon
Eclipse Ti-S mic oscope (Nikon, O awa a, Tochigi,
Japan) o wi h con ocal lase scanning mic oscope
Zeiss LSM780 Lase Scanning Con ocal Mic oscope
(ZEISS, Obe kochen, Ge many). The images we e
p ocessed wi h Zen2009 (con ocal images, Ca l Zeiss)
and wi h Adobe Pho oshop so wa e 7.0 (Adobe
Sys ems). The esul s a e om a leas 4 indi idual
expe imen s pe o med in duplica es.
Quan i a i e eal ime-PCR
Gene exp ession in CM monocul u e, ascula -like
ne wo k and ca dio ascula cons uc we e analyzed.
The o al RNA was ex ac ed a day 1, 6, 7 and 18
(Fig. 1) using Pu eLink RNA Mini Ki (Li e Tech-
nologies) ollowing he manu ac u e ’s p o ocol.
Concen a ion and pu i y o RNA was assessed using
spec opho ome y wi h mic opla e eade in Va -
ioskan Flash Spec opho ome e (The moScien i ic,
Espoo, Finland) be o e u he use. Re e se ansc ip-
ion o he o al RNA o cDNA was pe o med using
iSc ip cDNA syn hesis ki (Bio-Rad, Helsinki, Fin-
land) ollowing manu ac u e ’s ins uc ions. qRT-
PCR was pe o med acco ding o s anda d p o ocols
on Abi P ism 7300 ins umen (Applied Biosys ems,
Espoo, Finland) o on Bio- ad CFX96 Real Time
Sys em (BioRad). Fo each ime poin wo biological
eplica e samples we e collec ed and samples we e
analyzed as iplica es. The exp ession o angiogenesis
ela ed genes including VEGF-A, FGF-2, PDGF-b,
TGF-b1, Angiopoie in-1, Angiopoie in-2and ca diac
ela ed genes including CACNA1C, TNNT2, KCNJ2,
CX43, MYH6, MYH7 we e s udied wi h SYBR chem-
is y. In addi ion, he exp ession o ca diac ela ed
genes ADRB1 and SCN5a as well as e e ence gene
GAPDH we e s udied using Taqman chemis y wi h
Taqman Uni e sal PCR Mas e Mix (Applied Biosys-
ems). The ollowing Taqman assays (209) we e used:
Hs02330048_s1 o ADRB1, Hs00165693_m1 o
SCN5A and Hs02758991_g1 o GAPDH (Applied
Biosys ems). SYBR p ime sequences can be ound in
supplemen al da a (Supplemen a y Table 1). The
ela i e exp ession le els we e de e mined by using
he compa a i e me hod (DDC ) and GAPDH was
used as an endogenous con ol (Li ak and Schmi gen
2001, 402–408).
Func ional analyses
Mic oelec ode a ay (MEA) measu emen s
The abili y o ca dio ascula cons uc o conduc
elec ical signal was analyzed using he MEA sys em
790 Cy o echnology (2017) 69:785–800
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(Mul i Channel Sys ems MCS GmbH, Reu lingen,
Ge many). The MEA pla o ms (8 98 s anda d
MEAs o 6-well MEAs) we e i s hyd ophilized wi h
FBS and hen coa ed wi h 0.1% gela in ype A (Sigma-
Ald ich).
Field po en ials we e eco ded a day 10 o 18
(Fig. 1)a 37°C, and signals we e eco ded o 2 min.
The sampling equency was 20 kHz. Field po en ials
we e eco ded du ing spon aneous baseline bea ing,
and wi h 1 lM ad enaline (Sigma-Ald ich) o 300 nM
E-4031 (Sigma-Ald ich), which we e incuba ed 2 min
be o e measu emen s. D ugs we e dilu ed and mea-
su emen s pe o med in EB 5% medium. The ield
po en ials we e eco ded wi h MC_Rack .4.5.7
so wa e (Mul i Channel Sys ems MCS GmbH).
Signals we e analyzed wi h Ca diomyocy e MEA
Da a Analysis (Ca dioMDA) so wa e (P adhapan
e al. 2013).
Analysis o Calcium
2?
cycling
Ca dio ascula cons uc s we e loaded wi h 4 lmol/L
Fu a-2 AM (In i ogen, Molecula P obes) o 30 min
in HEPES based medium. Measu emen s we e
assessed in 37 °C and he ex acellula solu ion
consis ed o (in mmol/L): 137 NaCl, 5 KCl, 0.44
KH
2
PO
4
, 20 HEPES, 4.2 NaHCO
3
,5D-glucose, 2
CaCl
2
, 1.2 MgCl
2
and 1 Na-py u a e (pH was adjus ed
o 7.4 wi h NaOH).
Ca
2?
measu emen s we e eco ded a day 10 o 11
(Fig. 1) and conduc ed on an in e ed IX70 mic o-
scope (Olympus Co po a ion, Van aa, Finland) wi h a
UApo/340 209ai objec i e (Olympus). Images we e
acqui ed wi h an ANDOR iXon 885 CCD came a
(Ando Technology, Bel as , U.K.) synch onized wi h
a Polych ome V ligh sou ce by a eal ime DSP
con ol uni and TILL isION so wa e (TILL Pho on-
ics, G a
¨ el ing, Ge many). Fu a-2 in ca diomyocy es
was exci ed a 340 and 380 nm ligh and he emission
was eco ded a 505 nm. Fo Ca
2?
analysis, egions o
in e es s we e selec ed o spon aneously bea ing cells
and backg ound noise was sub ac ed be o e u he
p ocessing. The Ca
2?
le els a e p esen ed as a io-
me ic alues o F340/F380. The changes in Ca
2?
we e eco ded du ing spon aneous baseline bea ing
and spon aneous bea ing a e 2-min 1 lM ad enaline
(Sigma-Ald ich) pe usion.
S a is ics
qPCR da a a e exp essed as mean alues ±SD.
S a is ical analysis o he qPCR da a was pe o med
by IBM SPPS S a is ics 22-so wa e and by using
Mann-Whi ney U- es o independen samples. Bon-
e oni-co ec ion was included when mo e han wo
g oups we e analyzed. A p alue less han 0.05 was
conside ed s a is ically signi ican .
In MEA and Ca
2?
imaging da a, he signi icance o
di e ences be ween he wo g oups (CM monocul u e
and ca dio ascula cons uc ) was e alua ed wi h he
unpai ed S uden ’s es . The signi icance o changes
wi hin a g oup was e alua ed wi h he pai ed S uden ’s
es . MEA and Ca
2?
imaging da a a e exp essed as
a e age ±SEM. In Ca
2?
imaging da a n e e s o he
numbe o cells and in MEA da a n e e s o numbe o
MEA wells. A p alue less han 0.05 was conside ed
s a is ically signi ican .
Resul s
Vascula -like ne wo k se es as a suppo ing
and in e ac i e pla o m o ca dio ascula
cons uc
Exp ession le els o angiogenesis ela ed genes we e
analyzed a days 1, 6 and 18 in ascula -like ne wo k
o assess he p oduc ion o g ow h ac o s and
ma u a ion s a e o ascula s uc u es (Fig. 2). Du ing
he i s six days he exp ession o ib oblas g ow h
ac o 2 (FGF-2), ans o ming g ow h ac o b(TGF-
b)and angiopoie in-1(Ang-1) had an inc easing end
whe eas VEGF exp ession emained cons an . S a is-
ically signi ican inc ease was obse ed in angiopoi-
e in 2 (Ang-2) and pla ele de i ed g ow h ac o b
(PDGF-b) exp ession. Du ing he days 6–18, he
exp ession o a o emen ioned genes emained con-
s an excep o Ang-2, which had signi ican ly
inc eased exp ession le el.
The exp ession le els o he angiogenesis ela ed
genes we e also assessed om he ca dio ascula
cons uc a day 6 and 18 (Fig. 2). The exp ession
le els o FGF-2,TGF-band angiopoie in-1 emained
qui e cons an du ing cul u e, whe eas Ang-2exp es-
sion was dec eased in ca dio ascula cons uc
Cy o echnology (2017) 69:785–800 791
123
Fig. 2 Rela i e gene exp ession le els o angiogenesis ela ed
genes including ascula endo helial g ow h ac o (VEGF),
ib oblas g ow h ac o 2 (FGF-2), ans o ming g ow h ac o
be a (TGF-b), angiopoie in 1 (Ang-1), angiopoie in 2 (Ang-2)
and pla ele de i ed g ow h ac o be a (PDGF-b) in ca dio as-
cula cons uc and in ascula -like ne wo k a ime poin s 1, 6
and 18. The GAPDH was used as a endogenous con ol.
*p 0.05
Fig. 3 Mo phology o human plu ipo en s em cell de i ed
ca diomyocy es in amonocul u e and in bca dio ascula
cons uc a day 14 in 10% FBS. O ien a ion o human
plu ipo en s em cell de i ed ca diomyocy es ( oponin T, ed)
wi h ascula -like ne wo k (collagen IV, g een)inc,
dca dio ascula cons uc a day 7 in 5% FBS. CCon ocal
3D z-s ack p ojec ion ha ing he scale ba s included in he
image. The e ical heigh is 100 lm and he ho izon al leng h
be ween wo majo ick in e als is also 100 lm. In a,band
d he scale ba s ep esen 50 lm. (Colo igu e online)
792 Cy o echnology (2017) 69:785–800
123
compa ed o he ascula -like ne wo k. Howe e , he
exp ession le els o VEGF and PDGF-bwe e signi -
ican ly highe in he ca dio ascula cons uc .
Vascula -like ne wo k enhances ma u e ca diac
pheno ype and o ien a ion o plu ipo en s em cell
de i ed ca diomyocy es in ca dio ascula
cons uc
Compa ed o he ound mo phology and he andom
o ien a ion o ca diomyocy es in monocul u e
(Fig. 3a; S1), mo e ma u e mo phology wi h elon-
ga ed ca diomyocy es was de ec ed in he p esence o
ascula -like ne wo k (Fig. 3b). Mo eo e , 2D and 3D
p ojec ions showed ha ca diomyocy es co-localized
longi udinally and pa allel wi h ubula s uc u es
(Fig. 3c, d). On he con a y, ca diomyocy es in
monocul u e emained ounded, less o ganized and
he o ien a ion o he cells along he ubule s uc u es
could no be de ec ed.
The exp ession le els o ca diac ela ed genes
we e analyzed in he ca dio ascula cons uc and in
he CM monocul u e a one day a e ca diomyocy e
seeding (day 7) and a day 18 (Fig. 4). Resul s
showed ha he exp ession o ca diac muscle
myosin ansc ip s MYH6 and MYH7 as well as
ca diac oponin T inc eased be ween day 7 and 18
(p 0.05) whe eas he exp ession emained con-
s an o dec eased in he CM monocul u e. The
exp ession le el o gap junc ion ma ke connexin 43
was also shown o inc ease signi ican ly in ca dio-
ascula cons uc (p=0.002) be ween day 7–18
while emaining low in he CM monocul u e. A day
18 he exp ession le els o MYH6, MYH7, oponin
Tand connexin 43 we e all a highe le el in he
ca dio ascula cons uc when compa ed o he CM
monocul u e (p 0.05) (Fig. 4).
In he ca dio ascula cons uc , he exp ession
le els o ion channels CACNA1C, KCNJ2 and SCN5A
did no inc ease signi ican ly be ween days 7–18
(Fig. 4). Howe e , simila ly o s uc u al genes, he e
was s a is ically signi ican di e ence when exp es-
sion le els we e compa ed be ween ca dio ascula
cons uc and CM monocul u e a day 18. The
Fig. 4 Rela i e gene exp ession le els o ca diac ela ed genes
oponin T (TNNT2), ca diac muscle myosins (MYH6,MYH 7),
connexin 43 (Cx43), calcium channel (CACNA1C), po assium
channel (KCNJ2), sodium channel (SCN5A) and be a-1-
ad ene gic ecep o (ADBR1) a ime poin s day 7 and day 18
in ca diomyocy e monocul u e and in ca dio ascula cons uc .
The GAPDH was used as an endogenous con ol. *p 0.05
Cy o echnology (2017) 69:785–800 793
123
Sa iani L, Be iol E, S illi ano F, Mugelli A, Ce bai E, Jaconi
ME (2007) De elopmen al changes in ca diomyocy es
di e en ia ed om human emb yonic s em cells: a
molecula and elec ophysiological app oach. S em Cells
(Day on, Ohio) 25:1136–1144
Schaa S, Shibamiya A, Mewe M, Ede A, S oh A, Hi MN,
Rau T, Zimme mann WH, Con adi L, Eschenhagen T,
Hansen A (2011) Human enginee ed hea issue as a
e sa ile ool in basic esea ch and p eclinical oxicology.
PLoS ONE 6:e26397
Sekine H, Shimizu T, Hobo K, Sekiya S, Yang J, Yama o M,
Ku osawa H, Kobayashi E, Okano T (2008) Endo helial
cell cocul u e wi hin issue-enginee ed ca diomyocy e
shee s enhances neo ascula iza ion and imp o es ca diac
unc ion o ischemic hea s. Ci cula ion 118:S145–S152
Sni M, Keha I, Geps ein A, Coleman R, I sko i z-Eldo J,
Li ne E, Geps ein L (2003) Assessmen o he ul as uc-
u al and p oli e a i e p ope ies o human emb yonic s em
cell-de i ed ca diomyocy es. Am J Physiol Hea Ci c
Physiol 285:H2355–H2363
Soonpaa MH, Field LJ (1998) Su ey o s udies examining
mammalian ca diomyocy e DNA syn hesis. Ci c Res
83:15–26
Synne g en J, Giesle TL, Adak S, Tandon R, Noaksson K,
Lindahl A, Nilsson P, Nelson D, Olsson B, Englund MC,
Abbo S, Sa ipy P (2007) Di e en ia ing human
emb yonic s em cells exp ess a unique housekeeping gene
signa u e. S em Cells 25:473–480
Takahashi K, Tanabe K, Ohnuki M, Na i a M, Ichisaka T,
Tomoda K, Yamanaka S (2007) Induc ion o plu ipo en
s em cells om adul human ib oblas s by de ined ac o s.
Cell 131:861–872
Vala ma hi MT, Goodwin RL, Fusele JW, Da is JM, Yos MJ,
Po s JD (2010) A 3-D ca diac muscle cons uc o
explo ing adul ma ow s em cell based myoca dial
egene a ion. Bioma e ials 31:3185–3200
an Sp eeuwel AC, Bax NA, Bas iaens AJ, Foolen J, Loe akke
S, Bo ochin M, an de Scha DW, Chen CS, Baaijens FP,
Bou en CV (2014) The in luence o ma ix (an)iso opy on
ca diomyocy e con ac ion in enginee ed ca diac mic o-
issues. In eg Biol Quan Biosci Nano Mac o 6:422–429
Vuo enpaa H, Ikonen L, Kujala K, Hu ala O, Sa kanen JR,
Ylikomi T, Aal o-Se ala K, Heinonen T (2014) No el
in i o ca dio ascula cons uc s composed o ascula -
like ne wo ks and ca diomyocy es. In Vi o Cell De Biol
Anim 50:275–286
Wong T, McG a h JA, Na sa ia H (2007) The ole o ib oblas s
in issue enginee ing and egene a ion. B J De ma ol
156:1149–1155
Yang X, Pabon L, Mu y CE (2014) Enginee ing adolescence:
ma u a ion o human plu ipo en s em cell-de i ed ca -
diomyocy es. Ci c Res 114:511–523
800 Cy o echnology (2017) 69:785–800
123