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Electrophysiological Profiling of Cardiomyocytes in Embryonic
Bodies Derived From Human Embryonic Stem Cells
Therapeutic Implications
Rachel D. Vanderlaan, Gavin Y. Oudit, Peter H. Backx
H
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observed variability may reflect differences in plating density, culture medium composition, or period of gestation.13
Another factor that may influence cardiomyocyte differentiation is the inductive interactions between anterior visceral
endoderm-like cells and cardiac progenitor cells.14
He and colleagues used an array of tools to phenotype hES
cell– derived cardiomyocytes. Immunostaining and ultrastructural analysis established that beating EBs displayed a sarcomeric pattern and Z-lines as reported previously by others.8,13,14 Previous reports have also demonstrated that beating
EBs express thin- (cardiac troponin I and T) and thickfilament (myosin heavy and light chains) proteins, creatine
kinase-MB, cardiac transcription factors such as GATA-4,
Nkx2.5, and MEF-2, and the cardiac-specific marker
ANF.8,13,14 Collectively, these findings suggest that EBs
contain cardiomyocytes. Consistent with this suggestion, in
situ action potentials (APs) within a beating outgrowth
recorded using microelectrode impalements (intracellular recordings) revealed typical cardiac-like depolarization patterns
with AP heterogeneity resembling AP profiles observed in
nodal, atrial, and ventricular cardiomyocytes. These observations build on recent findings showing similar electrical
heterogeneity in hES cells cultured with visceral endodermlike cells.14 Electrical heterogeneity was also observed as two
distinct types of spontaneous electrical activity (ie, continuous and episodic) in EBs,11 which may depend on intercellular connexin-containing gap junction expression.12 The
authors speculate that the episodic pattern could be due to
conduction block related to tissue geometry, impaired cellto-cell coupling, or reduced cellular excitability, although the
role of altered intracellular Ca2⫹ cycling due to an immature
Ca2⫹ regulatory system cannot be ruled out. The phenotyping
and segregation of hES cell– derived cardiomyocytes into
different subpopulations may allow highly specific cell therapies to be developed thereby minimizing possible side
effects. For example, nodal cells may be used for pacemaker
support in sick sinus syndrome and atrioventricular block,
whereas the use of “pure” ventricular cells might allow
selective therapy of myocardial diseases without the creation
of a nidus of proarrhythmogenic pacemaker/nodal cells. How
undifferentiated hES cells transform into different types of
cardiac myocytes will clearly be an important area of future
research. Indeed, the ability of the vascular cytokine, endothelin, to transform avian embryonic heart muscle cells into
impulse-conducting Purkinje fibers provides evidence that
humoral factors can induce electrically distinct cell populations in the heart.16
uman embryonic stem (hES) cells are derived from
the inner mass cells of developing blastocytes and
have the ability to generate cells from three embryonic germ layers. Since their initial culturing in 1981, ES
cells have revolutionized the mouse genetics field1,2 by
allowing the creation of mouse models of disease as well as
the molecular study of the differentiation of pluripotent cells
into various somatic cell types including cardiomyocytes and
vascular smooth cells.3–5 Some key features of ES cells that
have made them particularly useful in research include their
ability for self-renewal, diploid karyotype stability, and continuous telomerase activity.6 –9 Since human fetuses are not
generally available for scientific study, hES cells represent an
in vitro model for embryonic differentiation, with the ability
to understand more about cell lineage commitment and the
process of differentiation. Importantly, they also represent a
potential source of cells for therapeutic uses such as the
regeneration of functional myocardium and conducting tissue. As a consequence of these capabilities and their derivation from human embryos, hES cells have been a subject of
numerous ethical and moral debates.10
In this issue of Circulation Research, He and coworkers
characterize the electrophysiological and contractile properties of cardiomyocytes derived from hES cells.11 This work,
together with previous studies by Gepstein’s group8,12 and
other recent reports,13,14 expands our understanding of cardiac
differentiation of hES cells, building a foundation for future
research and for new therapeutic strategies using hES cells. In
agreement with other hES studies,8 He et al found that about
20% of the embryoid bodies (EBs) (three-dimensional cell
aggregates of hES cells in culture) remained as spontaneous
beating “cardiac-like” cell clusters after 45 days in culture.
The number of cardiomyocytes within these EB outgrowths
varied widely (between 2% to 70%). Others have previously
reported that up to 70% of human EBs can be induced to beat
spontaneously after 16 days in culture13 whereas approximately 90% of mouse EBs beat after differentiation.15 This
The opinions expressed in this editorial are not necessarily those of the
editors or of the American Heart Association.
From the Departments of Medicine and Physiology, Heart and Stroke/
Richard Lewar Centre of Excellence, University of Toronto, Ontario,
Canada.
Correspondence to Dr Peter H. Backx, DVM, PhD, Heart and
Stroke/Richard Lewar Centre of Excellence, 150 College St, Room 68,
Fitzgerald Building, Toronto, Ontario, M5S 3E2. E-mail
[email protected]
(Circ Res. 2003;93:1-3.)
© 2003 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000082767.38055.03
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Circulation Research
July 11, 2003
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Functional aspects of hES cell– derived cardiomyocytes
were determined by He and colleagues using two different
approaches. First, by increasing the EB outgrowth pacing
rates, these authors observed AP shortening adaptation in
ventricular-like cardiomyocytes; this physiological response
leads to systolic shortening at high heart rates thereby
maintaining diastolic time for ventricular filling as shown in
ventricular myocardium of human embryos.17 Second, the
impact of the specific IKr blocker, E-4031,18 on AP profile and
triggered arrhythmias (early [EAD] and delayed [DAD]
afterdepolarizations) was examined. Human cardiac APs
represent complex interactions between depolarizing (such as
Na⫹ and Ca2⫹ currents) and repolarizing currents (such as
transient outward [Ito] and delayed rectifier [IKr and IKs] K⫹
currents).18,19 The application of E-4031 led to prolongation
of atrial- and ventricular-like APs thereby providing pharmacological evidence that IKr contributes to repolarization in
these myocytes. These data complement recent studies in hES
cells showing expression of KvLQT1 (pore-forming subunit
of IKs) and Kv4.3 (pore-forming subunit of Ito).14 Future
studies will undoubtedly unravel the temporal and spatial
expression patterns of pore-forming and accessory subunits
contributing to cardiac ion channels in hES cell– derived
cardiomyocytes, which are known to be developmentally
regulated.19,20 In addition, although EBs are only 4 to 10
layers thick, it will be of great interest to determine whether
regional “transmural” variations in AP profile occur within
EBs, thus providing important new approaches for studying
the regulatory factors governing the three-dimensional organization of AP profile and cardiac electrophysiology.
The application of E-4031 and the associated AP prolongation was also associated with EADs and DADs, suggesting
that these myocytes have the capacity for arrhythmogenesis.
Although this may be related to spontaneous Ca2⫹ release,19 it
could also be at least partially related to myocyte injury after
microelectrode impalement. In either case, relative high
expression/activity of the Na⫹-Ca2⫹ exchanger observed in
fetal/neonatal myocytes is likely to play an important role in
the formation of these arrhythmias.21,22 Activation of G
protein– coupled receptors, including ␤-adrenergic (␤1- and
␤2-AR), ␣-adrenergic, and muscarinic (M2) receptors, is
known to strongly influence contractile properties and beating rates of cardiomyocytes derived from hES.13,14 Consistent
with this, He and coworkers showed that the acute application
of the ␤-adrenergic agonist, isoproterenol, leads to a marked
positive inotropic effect in hES cell– derived EB outgrowths.11 These effects could be related to activity of protein
kinase A on L-type Ca2⫹ channel (␣1c) and phospholamban,
both of which are expressed in these preparations14 and are
likely to elevate peak systolic intracellular Ca2⫹ concentration
recorded previously in these preparations.8 In addition, the
L-type Ca2⫹ channel blockers, diltiazem and verapamil, have
profound negative chronotropic effects on hES cell– derived
cardiomyocytes13,14 as expected from the observation that
pacemaker activity is critically dependent on L-type Ca2⫹
channel current.23
Overall, this study by He et al,11 together with several other
recent reports, provides a solid phenotypic analysis of the
electrophysiological and contractile features of EB-derived
cardiomyocytes and underscores the complex machinery that
exists within these cells. The observation that fetal myocyte
transplantation can lead to stable intracardiac grafts and
improvement of cardiac function24 has stimulated interest in
the use of ES cells, with their self-renewal and pluripotent
characteristics, as a potential therapeutic tool for cardiovascular diseases. Because heart disease and failure are often
characterized by a loss of functioning cardiomyocytes, which
are terminally differentiated and show a very limited capacity
for regeneration,25 transplantation of hES cells or cardiac
grafts may be a means of reversing the functional changes
observed in cardiac patients. In particular, the study by He et
al suggests that it might be possible to introduce preselected
nodal, atrial, or ventricular myocytes to appropriately affected regions, thus tailoring therapy for heart disease patients. While the idea has conjured much interest, many
questions regarding the feasibility of cell-based therapy still
exist. Questions concerning proarrhythmogenic integration,
functional coupling with host tissues in context of the
extracellular matrix, the role of epigenetic factors (eg, hemodynamics), and rejection and vascularization of the graft (eg,
concomitant use of endothelial precursor cells) will all need
to be addressed.9,24,26 Undoubtedly, a complete understanding
of the electrophysiology and excitation-contraction coupling
in hES cell– derived cardiomyocytes will provide insights
into human embryonic cardiomyocyte development, signaling pathways, and transcriptional events while fostering the
potential therapeutic applications of hES cells.
References
1. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells
from mouse embryos. Nature. 1981;292:154 –156.
2. Martin GR. Isolation of a pluripotent cell line from early mouse embryos
cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl
Acad Sci U S A. 1981;78:7634 –7638.
3. Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitro
development of blastocyst-derived embryonic stem cell lines: formation
of visceral yolk sac, blood islands and myocardium. J Embryol Exp
Morphol. 1985;87:27– 45.
4. Maltsev VA, Wobus AM, Rohwedel J, Bader M, Hescheler J. Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents. Circ Res.
1994;75:233–244.
5. Drab M, Haller H, Bychkov R, Erdmann B, Lindschau C, Haase H,
Morano I, Luft FC, Wobus AM. From totipotent embryonic stem cells to
spontaneously contracting smooth muscle cells: a retinoic acid and
db-cAMP in vitro differentiation model. FASEB J. 1997;11:905–915.
6. Carpenter MK, Rosler E, Rao MS. Characterization and differentiation of
human embryonic stem cells. Cloning Stem Cells. 2003;5:79 – 88.
7. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ,
Marshall VS, Jones JM. Embryonic stem cell lines derived from human
blastocysts. Science. 1998;282:1145–1147.
8. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A,
Livne E, Binah O, Itskovitz-Eldor J, Gepstein L. Human embryonic stem
cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest. 2001;108:407– 414.
9. Gepstein L. Derivation and potential applications of human embryonic
stem cells. Circ Res. 2002;91:866 – 876.
10. Robertson JA. Human embryonic stem cell research: ethical and legal
issues. Nat Rev Genet. 2001;2:74 –78.
11. He JQ, Ma Y, Lee Y, Thomson JA, Kamp TJ. Human embryonic stem
cells develop into multiple types of cardiac myocytes: action potential
characterization. Circ Res. 2003;93:32–39.
12. Kehat I, Gepstein A, Spira A, Itskovitz-Eldor J, Gepstein L. Highresolution electrophysiological assessment of human embryonic stem
cell-derived cardiomyocytes: a novel in vitro model for the study of
conduction. Circ Res. 2002;91:659 – 61.
Vanderlaan et al
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
13. Xu C, Police S, Rao N, Carpenter MK. Characterization and enrichment
of cardiomyocytes derived from human embryonic stem cells. Circ Res.
2002;91:501– 8.
14. Mummery C, Ward-Van Oostwaard D, Doevendans P, Spijker R, Van
Den Brink S, Hassink R, Van Der Heyden M, Opthof T, Pera M, De La
Riviere AB, Passier R, Tertoolen L. Differentiation of human embryonic
stem cells to cardiomyocytes: role of coculture with visceral
endoderm-like cells. Circulation. 2003;107:2733–2740.
15. Fijnvandraat AC, van Ginneken AC, de Boer PA, Ruijter JM, Christoffels
VM, Moorman AF, Lekanne Deprez RH. Cardiomyocytes derived from
embryonic stem cells resemble cardiomyocytes of the embryonic heart
tube. Cardiovasc Res. 2003;58:399 – 409.
16. Gourdie RG, Wei Y, Kim D, Klatt SC, Mikawa T. Endothelin-induced
conversion of embryonic heart muscle cells into impulse-conducting
Purkinje fibers. Proc Natl Acad Sci U S A. 1998;95:6815– 6818.
17. Jezek K, Pucelik P, Sauer J, Bartak F. Basic electrophysiological parameters and frequency sensitivity of the ventricular myocardium of human
embryos. Physiol Bohemoslov. 1982;31:11–19.
18. Tristani-Firouzi M, Sanguinetti MC. Structural determinants and biophysical properties of HERG and KCNQ1 channel gating. J Mol Cell
Cardiol. 2003;35:27–35.
19. Oudit GY, Kassiri Z, Sah R, Ramirez RJ, Zobel C, Backx PH. The
molecular physiology of the cardiac transient outward potassium current
(Ito) in normal and diseased myocardium. J Mol Cell Cardiol. 2001;33:
851– 872.
Cardiac Development in Human Embryonic Bodies
3
20. Franco D, Demolombe S, Kupershmidt S, Dumaine R, Dominguez JN,
Roden D, Antzelevitch C, Escande D, Moorman AF. Divergent
expression of delayed rectifier K⫹ channel subunits during mouse heart
development. Cardiovasc Res. 2001;52:65–75.
21. Haddock PS, Coetzee WA, Artman M. Na⫹/Ca2⫹ exchange current and
contractions measured under Cl⫺-free conditions in developing rabbit
hearts. Am J Physiol. 1997;273:H837–H846.
22. Balaguru D, Haddock PS, Puglisi JL, Bers DM, Coetzee WA, Artman M.
Role of the sarcoplasmic reticulum in contraction and relaxation of
immature rabbit ventricular myocytes. J Mol Cell Cardiol. 1997;29:
2747–2757.
23. Lipsius SL, Huser J, Blatter LA. Intracellular Ca2⫹ release sparks atrial
pacemaker activity. News Physiol Sci. 2001;16:101–106.
24. Dowell JD, Rubart M, Pasumarthi KB, Soonpaa MH, Field LJ. Myocyte
and myogenic stem cell transplantation in the heart. Cardiovasc Res.
2003;58:336 –350.
25. Nadal-Ginard B, Kajstura J, Leri A, Anversa P. Myocyte death, growth,
and regeneration in cardiac hypertrophy and failure. Circ Res. 2003;92:
139 –150.
26. Nir SG, David R, Zaruba M, Franz WM, Itskovitz-Eldor J. Human
embryonic stem cells for cardiovascular repair. Cardiovasc Res. 2003;
58:313–323.
KEY WORDS: embryonic stem cells 䡲 embryonic bodies
䡲 cardiomyocytes 䡲 action potentials
䡲
electrophysiology
Electrophysiological Profiling of Cardiomyocytes in Embryonic Bodies Derived From
Human Embryonic Stem Cells: Therapeutic Implications
Rachel D. Vanderlaan, Gavin Y. Oudit and Peter H. Backx
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Circ Res. 2003;93:1-3
doi: 10.1161/01.RES.0000082767.38055.03
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