Maturation of human pluripotent stem cell derived cardiomyocytes is improved in cardiovascular construct

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) 69:785–800 123 (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 . 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