Stem cell-derived cardiomyocytes (CMs) are often electrophysiologically immature and heterogeneous, which represents a major barrier to their in vitro and in vivo application. Therefore, the purpose of this study was to examine whether Neuregulin-1β (NRG-1β) treatment could enhance in vitro generation of mature “working-type” CMs from induced pluripotent stem (iPS) cells and assess the regenerative effects of these CMs on cardiac tissue after acute myocardial infarction (AMI). With that purpose, adult mouse fibroblast-derived iPS from α-MHC-GFP mice were derived and differentiated into CMs through NRG-1β and/or dimethyl sulfoxide (DMSO) treatment. Cardiac specification and maturation of the iPS was analyzed by gene expression array, quantitative real-time polymerase chain reaction, immunofluorescence, electron microscopy, and patch-clamp techniques. In vivo, the iPS-derived CMs or culture medium control were injected into the peri-infarct region of hearts after coronary artery ligation, and functional and histology changes were assessed from 1 to 8 weeks post-transplantation. On differentiation, the iPS displayed early and robust in vitro cardiogenesis, expressing cardiac-specific genes and proteins. More importantly, electrophysiological studies demonstrated that a more mature ventricular-like cardiac phenotype was achieved when cells were treated with NRG-1β and DMSO compared with DMSO alone. Furthermore, in vivo studies demonstrated that iPS-derived CMs were able to engraft and electromechanically couple to heart tissue, ultimately preserving cardiac function and inducing adequate heart tissue remodeling. In conclusion, we have demonstrated that combined treatment with NRG-1β and DMSO leads to efficient differentiation of iPS into ventricular-like cardiac cells with a higher degree of maturation, which are capable of preserving cardiac function and tissue viability when transplanted into a mouse model of AMI.
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References
1.
PassierR, van LaakeLW and MummeryCL. (2008). Stem-cell-based therapy and lessons from the heart. Nature, 453:322–329.
2.
BeltramiAP, BarlucchiL, TorellaD, BakerM, LimanaF, ChimentiS, KasaharaH, RotaM, MussoE, et al. (2003). Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell, 114:763–776.
3.
WinterEM, van OorschotAA, HogersB, van der GraafLM, DoevendansPA, PoelmannRE, AtsmaDE, Gittenberger-de GrootAC and GoumansMJ. (2009). A new direction for cardiac regeneration therapy: application of synergistically acting epicardium-derived cells and cardiomyocyte progenitor cells. Circ Heart Fail, 2:643–653.
4.
van LaakeLW, PassierR, Monshouwer-KlootsJ, VerkleijAJ, LipsDJ, FreundC, den OudenK, Ward-van OostwaardD, KorvingJ, et al. (2007). Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction. Stem Cell Res, 1:9–24.
5.
LaflammeMA, ChenKY, NaumovaAV, MuskheliV, FugateJA, DuprasSK, ReineckeH, XuC, HassanipourM, et al. (2007). Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol, 25:1015–1024.
6.
TakahashiK and YamanakaS. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126:663–676.
7.
Iglesias-GarciaO, PelachoB and ProsperF. (2013). Induced pluripotent stem cells as a new strategy for cardiac regeneration and disease modeling. J Mol Cell Cardiol, 62:43–50.
8.
MauritzC, SchwankeK, ReppelM, NeefS, KatsirntakiK, MaierLS, NguemoF, MenkeS, HausteinM, et al. (2008). Generation of functional murine cardiac myocytes from induced pluripotent stem cells. Circulation, 118:507–517.
9.
ZhangJ, WilsonGF, SoerensAG, KoonceCH, YuJ, PalecekSP, ThomsonJA and KampTJ. (2009). Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ Res, 104:e30–e41.
10.
NelsonTJ, Martinez-FernandezA, YamadaS, Perez-TerzicC, IkedaY and TerzicA. (2009). Repair of acute myocardial infarction by human stemness factors induced pluripotent stem cells. Circulation, 120:408–416.
11.
KawamuraM, MiyagawaS, MikiK, SaitoA, FukushimaS, HiguchiT, KawamuraT, KurataniT, DaimonT, et al. (2012). Feasibility, safety, and therapeutic efficacy of human induced pluripotent stem cell-derived cardiomyocyte sheets in a porcine ischemic cardiomyopathy model. Circulation, 126:S29–S37.
12.
XiJ, KhalilM, ShishechianN, HannesT, PfannkucheK, LiangH, FatimaA, HausteinM, SuhrF, et al. (2010). Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells. FASEB J, 24:2739–2751.
13.
OdieteO, HillMF and SawyerDB. (2012). Neuregulin in cardiovascular development and disease. Circ Res, 111:1376–1385.
ZhaoYY, SawyerDR, BaligaRR, OpelDJ, HanX, MarchionniMA and KellyRA. (1998). Neuregulins promote survival and growth of cardiac myocytes. Persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular myocytes. J Biol Chem, 273:10261–10269.
16.
FukazawaR, MillerTA, KuramochiY, FrantzS, KimYD, MarchionniMA, KellyRA and SawyerDB. (2003). Neuregulin-1 protects ventricular myocytes from anthracycline-induced apoptosis via erbB4-dependent activation of PI3-kinase/Akt. J Mol Cell Cardiol, 35:1473–1479.
17.
SeguchiO, TakashimaS, YamazakiS, AsakuraM, AsanoY, ShintaniY, WakenoM, MinaminoT, KondoH, et al. (2007). A cardiac myosin light chain kinase regulates sarcomere assembly in the vertebrate heart. J Clin Invest, 117:2812–2824.
18.
IivanainenE, PaateroI, HeikkinenSM, JunttilaTT, CaoR, KlintP, JaakkolaPM, CaoY and EleniusK. (2007). Intra- and extracellular signaling by endothelial neuregulin-1. Exp Cell Res, 313:2896–2909.
19.
XuG, WatanabeT, IsoY, KobaS, SakaiT, NagashimaM, AritaS, HongoS, OtaH, et al. (2009). Preventive effects of heregulin-beta1 on macrophage foam cell formation and atherosclerosis. Circ Res, 105:500–510.
20.
LiuX, GuX, LiZ, LiX, LiH, ChangJ, ChenP, JinJ, XiB, et al. (2006). Neuregulin-1/erbB-activation improves cardiac function and survival in models of ischemic, dilated, and viral cardiomyopathy. J Am Coll Cardiol, 48:1438–1447.
21.
GuX, LiuX, XuD, LiX, YanM, QiY, YanW, WangW, PanJ, et al. (2010). Cardiac functional improvement in rats with myocardial infarction by up-regulating cardiac myosin light chain kinase with neuregulin. Cardiovasc Res, 88:334–343.
22.
RentschlerS, ZanderJ, MeyersK, FranceD, LevineR, PorterG, RivkeesSA, MorleyGE and FishmanGI. (2002). Neuregulin-1 promotes formation of the murine cardiac conduction system. Proc Natl Acad Sci U S A, 99:10464–10469.
23.
ZhuWZ, XieY, MoyesKW, GoldJD, AskariB and LaflammeMA. (2010). Neuregulin/ErbB signaling regulates cardiac subtype specification in differentiating human embryonic stem cells. Circ Res, 107:776–786.
24.
IrizarryRA, BolstadBM, CollinF, CopeLM, HobbsB and SpeedTP. (2003). Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res, 31:e15.
25.
GentlemanR, CareyV, DudoitS, IrizarryR and HuberW. (2005). Bioinformatics and Computational Biology Solutions Using R and Bioconductor. Springer, NY.
PelachoB, NakamuraY, ZhangJ, RossJ, HeremansY, Nelson-HolteM, LemkeB, HagenbrockJ, JiangY, et al. (2007). Multipotent adult progenitor cell transplantation increases vascularity and improves left ventricular function after myocardial infarction. J Tissue Eng Regen Med, 1:51–59.
28.
Benavides-VallveC, CorbachoD, Iglesias-GarciaO, PelachoB, AlbiasuE, CastanoS, Munoz-BarrutiaA, ProsperF and Ortiz-de-SolorzanoC. (2012). New strategies for echocardiographic evaluation of left ventricular function in a mouse model of long-term myocardial infarction. PLoS One, 7:e41691.
29.
BehfarA, Perez-TerzicC, FaustinoRS, ArrellDK, HodgsonDM, YamadaS, PuceatM, NiederlanderN, AlekseevAE, ZingmanLV and TerzicA. (2007). Cardiopoietic programming of embryonic stem cells for tumor-free heart repair. J Exp Med, 204:405–420.
ZwiL, CaspiO, ArbelG, HuberI, GepsteinA, ParkIH and GepsteinL. (2009). Cardiomyocyte differentiation of human induced pluripotent stem cells. Circulation, 120:1513–1523.
32.
UosakiH, FukushimaH, TakeuchiA, MatsuokaS, NakatsujiN, YamanakaS and YamashitaJK. (2011). Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression. PLoS One, 6:e23657.
33.
WangZ, XuG, WuY, GuanY, CuiL, LeiX, ZhangJ, MouL, SunB and DaiQ. (2009). Neuregulin-1 enhances differentiation of cardiomyocytes from embryonic stem cells. Med Biol Eng Comput, 47:41–48.
34.
SunM, YanX, BianY, CaggianoAO and MorganJP. (2011). Improving murine embryonic stem cell differentiation into cardiomyocytes with neuregulin-1: differential expression of microRNA. Am J Physiol Cell Physiol, 301:C21–C30.
35.
KimHS, ChoJW, HidakaK and MorisakiT. (2007). Activation of MEK-ERK by heregulin-beta1 promotes the development of cardiomyocytes derived from ES cells. Biochem Biophys Res Commun, 361:732–738.
36.
HaoJ, GalindoCL, TranTL and SawyerDB. (2014). Neuregulin-1beta induces embryonic stem cell cardiomyogenesis via ErbB3/ErbB2 receptors. Biochem J, 458:335–341.
ChenHS, KimC and MercolaM. (2009). Electrophysiological challenges of cell-based myocardial repair. Circulation, 120:2496–2508.
39.
EdwardsMK, HarrisJF and McBurneyMW. (1983). Induced muscle differentiation in an embryonal carcinoma cell line. Mol Cell Biol, 3:2280–2286.
40.
HalbachM, PfannkucheK, PillekampF, ZiomkaA, HannesT, ReppelM, HeschelerJ and Muller-EhmsenJ. (2007). Electrophysiological maturation and integration of murine fetal cardiomyocytes after transplantation. Circ Res, 101:484–492.
41.
CarpenterL, CarrC, YangCT, StuckeyDJ, ClarkeK and WattSM. (2011). Efficient differentiation of human induced pluripotent stem cells generates cardiac cells that provide protection following myocardial infarction in the rat. Stem Cells Dev, 21:977–986.
42.
ReineckeH, ZhangM, BartosekT and MurryCE. (1999). Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts. Circulation, 100:193–202.
43.
HalbachM, BaumgartnerS, SahitoRG, KrausgrillB, MaassM, PeinkoferG, LadageD, HeschelerJ and Muller-EhmsenJ. (2014). Cell persistence and electrical integration of transplanted fetal cardiomyocytes from different developmental stages. Int J Cardiol, 171:e122–e124.
44.
MauritzC, MartensA, RojasSV, SchnickT, RathertC, ScheckerN, MenkeS, GlageS, ZweigerdtR, et al. (2011). Induced pluripotent stem cell (iPSC)-derived Flk-1 progenitor cells engraft, differentiate, and improve heart function in a mouse model of acute myocardial infarction. Eur Heart J, 32:2634–2641.
45.
HalbachM, PeinkoferG, BaumgartnerS, MaassM, WiedeyM, NeefK, KrausgrillB, LadageD, FatimaA, et al. (2013). Electrophysiological integration and action potential properties of transplanted cardiomyocytes derived from induced pluripotent stem cells. Cardiovasc Res, 100:432–440.
46.
ShibaY, FernandesS, ZhuWZ, FiliceD, MuskheliV, KimJ, PalpantNJ, GantzJ, MoyesKW, et al. (2012). Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature, 489:322–325.
47.
MirotsouM, JayawardenaTM, SchmeckpeperJ, GnecchiM and DzauVJ. (2010). Paracrine mechanisms of stem cell reparative and regenerative actions in the heart. J Mol Cell Cardiol, 50:280–289.
48.
SasakiH, RayPS, ZhuL, GalangN and MaulikN. (2000). Oxidative stress due to hypoxia/reoxygenation induces angiogenic factor VEGF in adult rat myocardium: possible role of NFkappaB. Toxicology, 155:27–35.
49.
SekoY, TakahashiN, TobeK, KadowakiT and YazakiY. (1999). Pulsatile stretch activates mitogen-activated protein kinase (MAPK) family members and focal adhesion kinase (p125(FAK)) in cultured rat cardiac myocytes. Biochem Biophys Res Commun, 259:8–14.
50.
ZentilinL, PuligaddaU, LionettiV, ZacchignaS, CollesiC, PattariniL, RuoziG, CamporesiS, SinagraG, et al. (2009). Cardiomyocyte VEGFR-1 activation by VEGF-B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction. FASEB J, 24:1467–1478.
51.
StevensKR, KreutzigerKL, DuprasSK, KorteFS, RegnierM, MuskheliV, NourseMB, BendixenK, ReineckeH and MurryCE. (2009). Physiological function and transplantation of scaffold-free and vascularized human cardiac muscle tissue. Proc Natl Acad Sci U S A, 106:16568–16573.
52.
TemplinC, ZweigerdtR, SchwankeK, OlmerR, GhadriJR, EmmertMY, MullerE, KuestSM, CohrsS, et al. (2012). Transplantation and tracking of human-induced pluripotent stem cells in a pig model of myocardial infarction: assessment of cell survival, engraftment, and distribution by hybrid single photon emission computed tomography/computed tomography of sodium iodide symporter transgene expression. Circulation, 126:430–439.
53.
TullochNL, MuskheliV, RazumovaMV, KorteFS, RegnierM, HauchKD, PabonL, ReineckeH and MurryCE. (2011). Growth of engineered human myocardium with mechanical loading and vascular coculture. Circ Res, 109:47–59.
54.
XiongQ, HillKL, LiQ, SuntharalingamP, MansoorA, WangX, JameelMN, ZhangP, SwingenC, KaufmanDS and ZhangJ. (2011). A fibrin patch-based enhanced delivery of human embryonic stem cell-derived vascular cell transplantation in a porcine model of postinfarction left ventricular remodeling. Stem Cells, 29:367–375.
55.
FormigaFR, PelachoB, GarbayoE, ImbuluzquetaI, Diaz-HerraezP, AbizandaG, GaviraJJ, Simon-YarzaT, AlbiasuE, et al. (2014). Controlled delivery of fibroblast growth factor-1 and neuregulin-1 from biodegradable microparticles promotes cardiac repair in a rat myocardial infarction model through activation of endogenous regeneration. J Control Release, 173:132–139.
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