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The handle http://hdl.handle.net/1887/42928 holds various files of this Leiden University dissertation
Author: Devalla, Harsha D.
Title: Don’t skip a beat : studying cardiac rhythm in human pluripotent stem cell-derived cardiomyocytes
Issue Date: 2016-09-14
Chapter 1
General Introduction
Cardiac development and disease
The heart forms from the mesodermal germ layer of the embryo and is one of the first functional organs. Development of the heart begins with bilateral fields of cardiac precursors fusing to form a crescent shaped structure at 17-19 days of gestation in humans (Sylva et al 2014).
This is followed by the formation of a linear heart tube, which undergoes rightward looping.
The first heartbeat is detectable around 22 days of human development. The vertebrate heart then divides into four chambers – two ventricles and two atria, which are separated by fibrous skeletal structures, derived from the endocardial cushions. Simultaneously, the blood vessels that connect the heart to pulmonary and systemic circulation begin to develop within the pharyngeal arches of the embryo (Moore et al, 2015). Electrical activity of the heart is coordinated by a specialized conduction system (CS) that allows contraction of the heart essential for its role as a mechanical pump. The right side of the heart receives deoxygenated blood from the body, which is sent to the lungs while the left side supplies oxygenated blood coming from the lungs to the rest of the body.
Heart disease, both congenital and acquired, is responsible for millions of deaths each year. Chromosomal abnormalities (e.g. Trisomy21, DiGeorge syndrome) and mutations in cardiogenic transcription factors such as GATA4, TBX5 have been reported to cause congenital heart disease (CHD) (Fahed et al, 2013). Septal defects (atrial/ventricular/atrioventricular) are the most commonly occurring forms of CHD. Acquired cardiac disease on the other hand maybe multifactorial (e.g. coronary heart disease) although genetic predisposition that increases the risk of an individual for heart attack or sudden cardiac death (SCD). For example, heritable mutations in ion channel genes such as KCNQ1 cause long QT syndrome, the symptoms of which manifest in childhood or adolescence and patients suffering from this disease are prone to SCD (Modell & Lehmann 2006).
Research performed on experimental animal models has been essential for understanding the genetic and molecular pathways involved in physiology and pathophysiology of the heart.
In particular, mouse experimental models have been widely used due to ease of genetic manipulation and maintenance (Fox et al, 2007). However, there are noteworthy differences between a human and mouse heart including variations in distribution of potassium channels and heart rate (Hamlin & Altschuld, 2011). These biological distinctions underline the importance of relevant models to study human cardiac function.
Human pluripotent stem cell models of cardiac development and disease
Human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have proven to be valuable for studying cardiac differentiation and disease in vitro. Isolation of ESCs from blastocysts of human embryos in 1998 marked the beginning of a new era in experimental medicine (Thomson et al, 1998).
hESCs were shown to be capable of unlimited proliferation in culture and were also able to differentiate to derivatives of all three germ layers. The availability of hESC lines provided a tool for emulating differentiation of human lineages in vitro and protocols were developed for generation of cardiomyocytes (CMs) from hESCs (Kehat et al, 2001; Mummery et al, 2002).
Differentiation of hESCs to CMs gave insight into early steps of cardiovascular specification
and established that this model mimics cardiac development in vivo (Beqqali et al 2006; Cao
10 Chapter 1
et al, 2008).
With the realization that mammalian heart has limited regenerative capacity, research into alternative sources of CMs for cell repair gained momentum. hESC-derived CMs were viewed as an attractive option for transplantation into damaged heart. Several studies demonstrated that hESC-CMs engraft with the host tissue when injected into injured rodent heart and may improve function, although beneficial effects were not maintained in long-term studies (Caspi et al, 2007; van Laake et al 2007, Laflamme et al, 2007). However, heterogeneity of CM preparations posed risk of teratoma formation and arrhythmias. Moreover, the possibility of immunologic rejection of graft tissue and poor survival of implanted CMs had to be addressed.
In addition, application of hESC-derived CMs for safety pharmacology also attracted interest.
Figure 1. Human pluripotent stem cells (hPSCs). Human embryonic stem cells (hESCs) were derived from inner cell mass of blastocyst stage of human embryos in 1998 and human induced pluripotent stem cells (hiPSCs) were generated by reprogramming human adult somatic cells in 2007. hESCs and hiPSCs are characterized by prolonged self-renewal in culture and the ability to differentiate to cell types representing all three germ layers such as brain and skin (ectoderm derivatives); gut and liver (endoderm derivatives), heart and blood vessels (mesoderm derivatives).
Cardiotoxicity is the major cause for withdrawal of pharmaceutical compounds during
preclinical development (Chi et al, 2013). Drugs inducing prolongation of QT interval (a
parameter measuring the electrical depolarization and repolarization of the ventricles) on
the electrocardiogram (ECG) may lead to ventricular arrhythmias and as a consequence,
11 Introduction
may cause SCD. By measuring electrical properties of hESC-CMs in response to drugs, cardiotoxicity could be predicted in vitro (Chaudhary et al 2006; Braam et al 2010). Despite the tremendous scope for application of hESC-derived CMs in basic and preclinical research, they raise ethical concerns in some countries. hESCs are isolated from human pre-implantation embryos formed by in vitro fertilization and since the embryo is destroyed during stem cell isolation, the use of these cells for research has been under scrutiny.
A major breakthrough was achieved in 2007 with the discovery of hiPSCs, generated by reprogramming somatic cells (Takahashi et al, 2007). hiPSCs resembled hESCs in morphological and molecular characteristics. Most importantly, they were also capable of unlimited self-renewal and had the ability to differentiate to derivatives of all three germ layers.
The use of somatic cells for conversion into stem cells also circumvents some of the ethical barriers associated with hESCs. Introduction of hiPSCs opened up new avenues in stem cell biology and for the first time, stem cells generated from patients were being used for cardiac disease modeling in vitro (Moretti et al, 2010; Itzhaki et al, 2011). Furthermore, patient-specific hiPSC-derived cell types would be immunocompatible for cell replacement therapies.
In the recent years, excellent protocols have been developed for guided differentiation of CMs from hPSCs. Recapitulating embryonic cardiovascular development during hPSC differentiation using human recombinant growth factors promoted efficient differentiation (Kattmann et al, 2011). Further optimization of differentiation strategies to include small molecules (Lian et al, 2012; Burridge et al, 2014) and exclude any xenogenic components (Lian et al, 2015) greatly enhanced efficiency of CM differentiation. Identification of cell surface markers, such as SIRPA and VCAM1 has also made it possible to separate CMs from other cell types in culture (Elliott et al, 2011; Dubois et al, 2011).
Combined with other novel technologies in the fields of genetic medicine, tissue engineering,
and genome editing, hPSC-CM models will be valuable for 1) understanding lineage decisions
determining CM specification, 2) unraveling molecular basis of disease, 3) translational
applications such as target/drug discovery, diagnostic medicine and developing effective
treatment strategies.
12 Chapter 1
Outline of this thesis
In this dissertation, we describe research performed with hPSCs in order to study cardiac differentiation and disease.
In chapter 2, we reviewed our current understanding of cardiac lineage specification in vivo, with the aim of using this knowledge to guide differentiation to specific cardiac subtypes in vitro.
We discussed the importance of generating cardiac subtypes from hPSCs for applications in disease modeling, tissue engineering and regenerative medicine.
In chapter 3, we described the generation of atrial-like CMs from hESCs and demonstrate their value in preclinical research for testing selectivity of drugs being developed for atrial fibrillation. In addition, we also described a role for orphan nuclear transcription factors, COUP-TFI and COUP-TFII in hESC-atrial CMs.
In chapter 4, we studied the association of a splice site mutation in TECRL to catecholaminergic polymorphic ventricular tachycardia (CPVT). CMs generated from patient-specific hiPSCs were valuable to study the electrophysiological features of TECRL mutant CMs and test the effect of Flecainide, a class Ic antiarrhythmic drug found to be effective in other forms of CPVT.
In chapter 5, we performed further characterization of TECRL mutant hiPSC-CMs in order to identify dysregulated gene and signaling pathways. Functional analyses demonstrated altered mitochondrial function in TECRL mutant CMs. This work provides a framework for future studies aiming to elucidate the link between TECRL and inherited cardiac arrhythmias.
In chapter 6, we investigated the molecular signature of the developing cardiac conduction system (CCS) in human fetal hearts. A panel of genes implicated in CCS development was evaluated by immunohistochemistry. This work serves as a reference for gene expression in the human cardiac CS during early stages of fetal development.
In chapter 7, we discussed the significance of findings presented in this thesis with respect
to cardiac development and their clinical impact. Scope for future work in the field is also
proposed.
13 Introduction
References
Beqqali A, Kloots J, Ward-van Oostwaard D, Mummery C, Passier R (2006). Genome-Wide Transcriptional Profiling of Human Embryonic Stem Cells Differentiating to Cardiomyocytes. Stem Cells 24:1956–1967
Braam SR, Tertoolen L, van de Stolpe A, Meyer T, Passier R, Mummery CL (2010). Prediction of drug-induced cardiotoxicity using human embryonic stem cell-derived cardiomyocytes. Stem Cell Res 4:107–116
Burridge PW, Matsa E, Shukla P, Lin ZC, Churko JM, Ebert AD, Lan F, Diecke S, Huber B, Mordwinkin NM et al (2014).
Chemically defined generation of human cardiomyocytes. Nat Meth 2014 11:855–860
Cao F, Wagner RA, Wilson KD, Xie X, Fu J-D, Drukker M, Lee A, Li RA, Gambhir SS, Weissman IL et al (2008).
Transcriptional and Functional Profiling of Human Embryonic Stem Cell-Derived Cardiomyocytes. PLoS ONE. 3:e3474 Caspi O, Huber I, Kehat I, Habib M, Arbel G, Gepstein A, Yankelson L, Aronson D, Beyar R, Gepstein L (2007).
Transplantation of Human Embryonic Stem Cell-Derived Cardiomyocytes Improves Myocardial Performance in Infarcted Rat Hearts. J Am Coll Cardiol 50:1884–1893
Chaudhary KW, Barrezueta NX, Bauchmann MB, Milici AJ, Beckius G, Stedman DB, Hambor JE, Blake WL, McNeish JD, Bahinski A et al (2006). Embryonic Stem Cells in Predictive Cardiotoxicity: Laser Capture Microscopy Enables Assay Development. Toxicol Sci. 90:149–158
Chi KR. Revolution dawning in cardiotoxicity testing. Nat Rev Drug Discov. 2013;12:565–567.
Dubois NC, Craft AM, Sharma P, Elliott DA, Stanley EG, Elefanty AG, Gramolini A, Keller G (2011). SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat Biotechnol 29:1011-1018 Elliott DA, Braam SR, Koutsis K, Ng ES, Jenny R, Lagerqvist EL, Biben C, Hatzistavrou T, Hirst CE, Yu QC et al (2011) NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat Meth 8:1037–1040 Fahed AC, Gelb BD, Seidman JG, Seidman CE (2013). Genetics of congenital heart disease. Circ Res. 112: 707-720 Fox J, Barthold S, Davison M, Newcomer C, Quimby F, Smith A, eds (2007). The Mouse in Biomedical Research. Volume 1–4, Second Edition (American College of Laboratory Animal Medicine). Burlington, MA: Elsevier.
Hamlin RL, Altschuld RA (2011). Extrapolation from mouse to man. Circ Cardiovasc Imaging 4:2-4
Itzhaki I, Maizels L, Huber I, Zwi-Dantsis L, Caspi O, Winterstern A, Feldman O, Gepstein A, Arbel G, Hammerman H et al (2011). Modelling the long QT syndrome with induced pluripotent stem cells. Nature 471:225-229
Kattman SJ, Witty AD, Gagliardi M, Dubois NC, Niapour M, Hotta A, Ellis J, Keller G (2011). Stage-Specific Optimization of Activin/Nodal and BMP Signaling Promotes Cardiac Differentiation of Mouse and Human Pluripotent Stem Cell Lines.
Cell Stem Cell 8:228–240
Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, Livne E, Binah O, Itskovitz-Eldor J, Gepstein L (2001). Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 108:407–414
Laflamme MA, Chen KY, Naumova AV, Muskheli V, Fugate JA, Dupras SK, Reinecke H, Xu C, Hassanipour M, Police S 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
Lian X, Zhang J, Azarin SM, Zhu K, Hazeltine LB, Bao X, Hsiao C, Kamp TJ, Palecek SP (2012). Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions.
Nat Protoc 8:162–175.
Lian X, Bao X, Zilberter M, Westman M, Fisahn A, Hsiao C, Hazeltine LB, Dunn KK, Kamp TJ, Palecek SP (2015).
Chemically defined, albumin-free human cardiomyocyte generation. Nat Meth 12:595–596
Modell SM, Lehmann MH (2006). The long QT syndrome family of cardiac ion channelopathies: A HuGE review. Genet Med 8(3):143–155.
Moore KL, Persaud TVN, Torchia MG (2015). Cardiovascular system. In The Developing Human: Clinically Oriented Embryology (10th ed.) pp 283-334. Philadelphia:Saunders
Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flügel L, Dorn T, Goedel A, Höhnke C, Hofmann F (2010). Patient- Specific Induced Pluripotent Stem-Cell Models for Long-QT Syndrome. N Engl J Med 363:1397–1409
Mummery C, Ward D, Van Den Brink CE, Bird SD, Doevendans PA, Opthof T, La Riviere De AB, Tertoolen L, Van Der Heyden M, Pera M (2002). Cardiomyocyte differentiation of mouse and human embryonic stem cells. J Anat 200:233–242
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Sylva M, van den Hoff MJ, Moorman AF (2014). Development of the human heart. Am J Med Genet A 164A(6): 1347-71 Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007). Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 131:861–872
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998). Embryonic Stem Cell Lines Derived from Human Blastocysts. Science 282:1145–1147
van Laake LW, Passier R, Monshouwer-Kloots J, Verkleij AJ, Lips DJ, Freund C, Ouden den K, Ward-van Oostwaard D, Korving J, Tertoolen LG (2010). 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
15 Introduction
