Title of Invention

A METHOD FOR TESTING AN AGENT FOR EFFECT ON HUMAN CARDIAC CELLS

Abstract Human embryonic stem cells form embryoid bodies in culture which contain differentiated human cells. Some of the human cells in embryoid bodies differentiate into cardiomyocytes. Here the biological and electrical characteristics of those car- diomyocytes arc described with reference to the use of cardiomyocytes derived from human embryonic stem cells in drag screening protocols for mechanisms of cardiac toxicity.
Full Text A METHOD FOR TESTING AN AGENT FOR EFFECT ON HUMAN CARDIAC CELLS
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority from U.S. provisional patent application S.N.
60/399,330 filed July 26, 2002.
BACKGROUND OF THE INVENTION
[0002] Human embryonic stem cells are human cells, that may be stably multiplied and
cultured in vitro, that are at least pluripotent and may be totipotent. By that it is meant that the
cells can differentiate into many different mature differentiated cell types of the human body and
may, in fact, be able to differentiate into all of the cell types of an adult human body. Human
embryonic stem cells are created from embryonic tissues and serially cultivated thereafter in an
in vitro culture.
[0003] In cultivation, human embryonic stem cells are normally maintained in an
undifferentiated state by culturing in conjunction with certain factors. Notably, the cultivation of
human embryonic stem cells upon fibroblasts feeder layers, or in the presence of factors derived
from fibroblasts, maintain the stem cells in an undifferentiated state. With the fibroblasts or the
factors from the fibroblasts removed, human embryonic stem cells can and will begin to
spontaneously differentiate into a variety of tissue types. Among the intermediate structures
formed by stem cells in the process of spontaneous differentiation into a variety of tissue types is
a structure known as an embryoid body, Embryoid bodies begin as aggregates formed in the
culture of embryonic stem cells. While culture conditions and cell line identity influence the rate
formation of embryoid bodies, under many conditions, embryoid bodies will both spontaneously
arise and spontaneously begin to differentiate into a variety of different tissue types.
[0004] Among the tissue types present in embryoid bodies are known to be
cardiomyocytes. These early cardiomyocytes are the precursors of human adult cardiac cells.
Adult cardiomyocytes permanently withdraw from the cell cycle and caimot regenerate. The fact
that cardiomyocytes were among the cells present in the embryoid bodies formed by stem cells
was evident by the fact that parts of the embryoid bodies will sometimes exhibit regular
heartbeat-like confractions. Thus it has been previously demonstrated that human embryonic
stem cells will differentiate into cells which have some of the fimctional properties of
cardiomyocytes. Exactly what form those cardiomyocytes take, and how mature they are in their
differentiation, was previously unknown. Also unknovm was what electromechanical
mechanisms are active in tiie cardiomyocytes present in embryoid bodies and wiiat sorts of analysis
of the behavior of those cardiomyocytes cells derived from stem cells can be performed.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING FIGURES
[0005] Fig. 1 is a graphical presentation of the amplitude of mechanical contractions
occurring in an embryoid body formed from human embryonic stem cells, measured over time to
determine the rate of contractions and the amplitude of contractions, gathered in the examples
below.
[0006] Fig. 2 is a graphical representation of the embryonic ventricular-type action
potential observed in cardiomyocytes derived from human embryonic stem cells.
[0007] Fig. 3 is a graphical representation of the embryonic atrial-type action potential
observed in cardiomyocytes derived from human embryonic stem cells.
[0008] Fig. 4 is a graphical representation of the nodal-type action potential observed in
cardiomyocytes derived from human embryonic stem cells.
[0009] Fig. 5 presented data from the ADP study referred to in the examples below.
DETAILED DESCRIPTION OF THE INVENTION
[00010] The instant invention is clearly directed to the uses of cardiomyocytes obtained
commercially from cultured cell lines and is not directed to the use of human embryos per se or
their destruction, for therapeutic purposes, that is, screening agents for potential effects on cardiac
cells. It is described here for the first time that cardiomyocytes derived from human embryonic
stem cells formed in embryoid bodies differentiate into all of the major cardiac muscle cell types,
including ventricular, atrial, and nodal cells. While not all embryoid bodies will contain
cardiomyocytes, those embryoid bodies which do contain cardiomyocytes will spontaneously beat.
It is also disclosed herein that the beat of such cardiomyocytes can be controlled and monitored,
which permits analysis of the m^nitude of such beats to measure the responsiveness of the cardiac
cells in culture to defined changes in environment and conditions. Monitoring the electrical
potentials of individual cells in an embryoid body can reveal the nature of individual
cardiomyocytes in the embryoid body and can be used to test the response of such cells to external
stimuli, such as potentially toxic or therapeutic agents. In particular, it has been found that it is
possible to evaluate the effect of chemicals on the HERG potassium channel of cardiac cells, and
thereby test in vitro the actual effect of drugs on human heart cells in a manner that has heretofore
not been possible.
[00011] The electrical activity of a cardiac cell is best characterized with reference to its
action potential. The action potential is a chart of the transmembrane electrical potential, from the
interior of the cardiomyocytes to the environment, which is measured over time. The action
potential of various types of mature and primitive cardiac cells are different from those of other

types. Here it is demonstrated that all three main classes of action potentials, nodal-like,
embryonic atrial-like, and embryonic ventricular-like, can be observed in cardiomyocytes formed
by human embryonic stem cells. Impaling individual beating outgrowths revealed reproducible
action potential morphologies recorded from cells suggesting that each outgrowth, or embryoid
body, is composed of a predominant cell type.
[00012] It has been demonstrated previously that one can find cardiomyocytes in embryoid
bodies formed from h\iman embryonic stem cells. The differentiation of human embryonic stem
cells into a variety of tissue types within the body is most commonly done through the formation
of what are known as embryoid bodies, referred to here as EBs. EBs are aggregations of cells
which begins as irregular clumps found in the cultivated cultures of embryonic stem cells that
begin to exhibit differentiated tissue types within their structure. Some embryoid bodies will
spontaneously beat, suggesting the presence of cardiomyocyte-type behavior. Previous studies
using immunostaining techniques on fixed cells showed the presence of cardiac-specific proteins
in EBs from human embryonic stem cells. Extracellular recordings of electrical activity from
aggregates of cells have supported the notion that spontaneously electrically active hearts cells
are present in the EBs, but these recordings cannot provide information as to the types of
cardiomyocytes present in the EBs, as the measures potentials were an average of Imany cells
present in the area of the extracellular electrode. There are a variety of techniques which can
give rise to embryoid body formation, and tlie method used can result in a greater or lesser
percentage of embryoid bodies which do contain cardiomyocytes and the types of
cardiomyocytes formed has been previously uncharacterized. Thus while it has been previously
shown that cardiomyocytes are formed in EBs, the type of cardiomyocytes, the capabilities of
these cells, and accessibility of these cells to intracellular elecfrophysiological recordings have
previously not been demonsfrated. It is described here that the major cell types of mature heart
muscle, including ventricular, atrial, and nodal cells can all be found within embryoid bodies
formed from embryonic stem cells.
[00013] The cultivation of cardiomyocytes from human embryonic stem cells permits
simple studies of human cardiac cell characteristics to be conducted in vitro. At the simplest
level, it is possible to culture an EB that contains cardiomyocytes in a vessel or culture container
and observe the pulsation or beating of that embryoid body. While not all embryoid bodies will
form cardiomyocytes and exhibit pulsing or beating behavior, those embryoid bodies that do
rhythmically pulse will be found to contain cardiomyocyte cells within them. At a more
sophisticated level, described in greater detail below, the various embryonic cardiac cell types

can separately have their action potentials taken and then the variations in action potentials can
be observed following various chemical, electrical or physical perturbations of the cells.
[00014] The examples described below used cardiomyocytes derived from human
embryonic stem cells which produced embryoid bodies maiotained in culture 40-95 days. This is
the stage at which we found heterogeneity of action potential morphologies. While action
potentials with characteristics of atrial and ventricular myocytes were observed, the relatively
positive MDP (-50 to -60 mV) and the slow action potential upstroke (5-30 V/sec) contrasts with
neonatal and adult human atrial and ventricular cardiomyocytes which have resting membrane
potentials in the range of-80 mV and dV/dtmax ranging from 150 to 350 V/s.l8 The stem cell-
derived cardiomyocytes likely correlate with the "intermediate" stage described for the murine
stem cell system. The limited data available describing the action potential in human embryonic
and fetal hearts suggests that by 7 to 8 weeks of development the resting membrane potential and
dV/dtmax of atrial and ventricular myocytes reaches that of adult cells. Thus we referred to the
atrial and ventricular action potentials observed in this study as embryonic because they have
properties of the action potentials anticipated in human embryos prior to 7 weeks of
development. The nodal type action potentials observed were simply described as nodal because
this action potential morphology shows little change during development. This strikingly slow in
vitro development of action potential properties compared to the mouse system is likely related to
the markedly different gestational periods comparing mice and man.
[00015] It has been assiimed that since enzymatic dissociation of a collection of embryoid
body outgrowths has yielded diverse cardiomyocyte cell types, that each outgrowth is composed
of a heterogeneous mix of cardiomyocytes, perhaps in part mimicking the heterogeneous
collection of myocytes in the developing heart. However, the current infracellular recording of
action potentials with sharp microelectrodes were unique in that repeated distinct cellular
measurements were made from individual outgrowths, and we found that each outgrowth is
populated by a predominant cell type. Thus we postulate that each outgrowth responds to its
unique nucroenvironment resulting in differentiation and proliferation of one predominant type
of cardiac cell.
[00016] One characteristic property of the intact heart or certain cardiomyocyte cell types
is an intrinsic or spontaneous beating rate. Cells that set the beating rate are sometimes referred
to as pacemaker cells. The cardiomyocytes present in EBs exhibit a spontaneous beating or
contraction characterized by particular patterns of activity (episodic as opposed to continuous) as
well as a frequency of beating. By either measuring directly action potentials as above or by
measuring time or amplitude of cell contractions, it is possible to characterize the effect of drugs

or other interventions on the spontaneous beating rate and patterns of the cardiomyocytes in
culture. Agents that increase or decrease the rate of beating in the EBs may be predicted to have
a similar effect on intact human hearts. Likewise, agents which alter the pattern of beating by
increasing the pause duration in episodic beating, may be predicted to have propensity to produce
heart block in patients.
[00017] One problem in the testing of cardiomyocytes in EBs is that the rate of
spontaneous beating is variable can be dependent on a variety of factors. Since an objective is to
obtain controlled data demonstrating the effect of exogenous substances on cardiomyocyte
behavior in EBs, one way to provide a controlled baseline of activity is to artificially regulate the
beating of the cardiomyocytes in culture. This can be readily done by applying an electrical field
stimulation to the EB. This is done most simply by applying electrodes to opposite sides of the
culture container in which the EB is contained. If periodic exciting voltages are applied between
those electrodes (e.g. 40 volt DC pulses of 10 microsecond duration at 1 hertz) the EBs will
exhibit regular pulsing or beating at the firequency of the electrode stimulation. It is necessary as
a part of this process to maintain the EB culture at a constant temperature, since temperature
changes can also effect the amplitude of EB beats. It is possible to optically scan the EBs during
such beating, using imaging processing observing the EB with video microscopy, to determine
the amplitude of the beats which occur in such an EB. It then becomes possible to stimulate the
EB with a chemical or other stimulus to observe what effect the stimulation has on the magnitude
of the beat produced by the EB. Agents which antagonize electromechanical activity in cardiac
cells will reduce the amplitude of such beats and agents which agonize such electricomechanical
activity will increase the magnitude of such beats. Additionally other properties of the beat can
be monitored such as the rate of contraction and relaxation of the embryoid body providing
additional mechanistic information.
[00018] Shown in Figure 1 is data obtained firom such a study. In Figure 1, an EB is
contained within a temperature controlled small culture vessel that has been electrically
stimulated at 1 hertz and therefore exhibits a basal amplitude of pulses or beats, the amplitude of
which, judged optically by physical displacement of the edge of the EB, is arbitrarily defined to
have a control or basal level of 100. Then, a stimulus, such as a test compound, is added to the
medium in which the EB is resting which can affect the electrical and mechanical characteristics
of a cardiac cell. The chemical stimulant or test compound in this study was the addition of 1
micromolar isoproteranol, which is a known agonist for /3-adrenergic receptors in heart cells that
can activate an increase in the rate and magnitude of heart contractions. Isoproteranol is known
to be a mimic of the "fight or flight" response in adult heart cells. Since the beating rate of the

EB is controlled by the rate of the artificially applied field stimulation, the rate of pulsing of the
EB does not change, but as shown in Figure 1, the amplitude of the beat or contraction of the EB
increases dramatically upon the application of the chemical stimulus. This demonstrates that
chemicals having an effect upon heart contraction can be modeled using cardiomyocytes in
culture contained within EBs firom embryonic stem cells.
[00019] It is also possible to probe the electric action potential characteristics of individual
cardiomyocytes in an EB. This is done by creating a very fine microelectrode and physically
directing that microelectrode into the EB. While it is not possible to select which cell is probed
by that electrode, it is possible to measure the electrical signal experienced by the electrode and
determine into what type of cardiomyocyte the probe has been extended based upon the electrical
signal created by the cell. This is possible because the different types of heart cells have
distinctive electrophysiological properties due to the expression of a unique set of ion channels
and other proteins.
[00020] Thus an effort was undertaken to characterize the action potentials of cells
occurring in beating EBs. These studies were conducted in intact EB outgrowths to avoid the
possible alterations in electrical behavior which might arise from cell isolation or replating of
isolated cardiac cells. Because the focus of this effort was, in part, to determine if multiple types
of cardiomyocytes can be obtained from human embryonic stem cells, the study was conducted
on cells in a time window of 40 to 95 days of differentiation of EBs, a time period selected to
provide adequate time for distinct cell types to resolve.
[00021] Shovm in Figures 2,3 and 4 are electrical signals obtained firom such a probe
when placed into different cells in embryoid bodies arising in human stem cell cultures. In
Figure 2, an embryonic ventricular cell type of action potential has been detected. The waveform
is characteristic of ventricular cells, as determined by the magnitude and shape of the potentials
generated by the cell. In Figure 3, an embryonic atrial cell type action potential is illustrated. In
Figure 4 a nodal type electrical characteristic is charted. These signals, all taken from actual
cardiomyocytes in EBs in culture, are diagnostic of cell type to those knowledgeable in the field
of cardiac electrophysiology. These action potentials demonstrate that three major cell types of a
mature heart are present among the cardiomyocytes in an EB in m vitro culture. Again, it was
observed that various beating EBs would have differing predominant cell types, and all three
main cell types can be observed.
[00022] While it has been proposed before that embryonic stem cell-derived
cardiomyocytes might be usefiil for some forms of drug screening, of particular interest is the
effect the potential drugs might have on repolarization (return to baseline) of the action potential.

Agents which prolong repolarization, and hence increase action potential duration, have the
possibility Of causing drug-induced long QT syndrome, winch is associated with potentially
lethal ventricular arrhythmias. The name QT syndrome is not an abbreviation, it refers to the
time interval between points of the action potential chart which are ai'bitrarily named Q and T.
This syndrome represents one of the major forms of toxicity seen across multiple classes of
pharmaceutical agents. While modulation of a variety of ion channels can prolong the action
potential, in humans the potassium channel known as the HERG channel is particularly
susceptible to blockade by drugs, leading to prolongation of the action potential and QT interval
on the surface electrocardiogram. Currently no in vitro tectmique is available for screexung for
action potential prolongation and HERG channel block in human cardiac myocytes. While a
variety of screening approaches are under use to evaluate candidate drugs using animal heart
cells and expression of HERG channels in non-cardiac cells, these methods have failed on
multiple occasions. In fact, recent history reveals several important pharmaceuticals, notably
terfenadine (sold as Seldane'^'^) and astemizole (sold as HismanaF**) which were approved by
FDA for marketing but which were subsequently removed from the market when they were
foimd to have adverse effect upon HERG channel activity in hxmian heart cells, because they lead
to rare but lethal cardiac arrhythmias in humans. When and if human ES-derived
cardiomyocytes would express the HERG charmel genes was unknown before the work described
here.
[00023] The applicants here have determined that the HERG chaimel response of
cardiomyocytes in culture created from human embryonic stem cells can be tested and that they
do have the same responsive characteristics as mature heart cells in adult humans. Shown in
Figure 5 is an experiment conducted on an atrial type cell, which is a cardiomyocyte in an EB in
culture. Note tliat the electrical profile of the potential generated by that cell, labeled basal in
Figure 5, is characteristic of an embryonic atrial type action potential. To the culture in which
that basal signal was being observed, 500 nanomolar E4031 was added. E4031 is a known
highly specific HERG channel blocker which blocks the rapid delayed rectifying potassium
current (Ikt) in adult human heart cells. The addition of that chemical to the medium in which the
EB was cultured led to the modification of the action potential generated by that atrial type cell,
as illustrated by the curve marked "500 nM E4031" in Figure 5. The action potential was
prolonged due to delayed return to a resting state. This is precisely the effect that this molecule,
E4031, is known to have on adult heart cells and precisely tlie effect that one would predict from
the blockage of the HERG channel. Accordingly, this test demonstrates that it is possible to test

molecules for their effect on the HERG channel in adult human cardiac cells by testing
cardiomyocytes derived from human embryonic stem cells in culture.
[00024] The applicants have determined that the electrical activity of cardiomyocytes in
EBs can also display electrical activity known as delayed after depolarizations (DADs). This
electrical property is found in diseased human heart muscle or heart muscle treated with agents
which cause calcium overload. These DADs serve as the basic mechanisms leading to triggered
arrhythmias including some forms of potentially lethal ventricular tachycardia. The ability of
EBs to demonstrate the complex cellular environment needed for generation of DADs makes
them a suitable model for testing interventions including drugs to modulate DAD formation and
potentially derive new pharmacological therapies for heart arrhythmias.
[00025] Following here are methods and materials and several examples describing the
work conducted with cardiomyocytes derived from himian embryonic stem cells. In particular,
techniques are described which enable the isolation of large quantities of ventricle type, atrial
type or nodal type cells from EBs generated from human embryonic stem cells. This makes
possible the collection and culturing of large numbers of such cells for drug screening or other
toxicity testing purposes.
EXAMPLES
[00026] £B formation and cardiac differentiation
[00027] The hES cell lines HI, H7, H9 and H14 were derived and maintained as
previously described. For EB formation, ES cell colonies were dispersed into cell aggregates
containing approximately 500-800 cells using Img/ml dispase. The cell aggregates were then
cultured in suspension in cell culture flasks (BD Bioscience) with ES cell medium without basic
fibroblast growth factor for 6 days with media changed daily. To promote cardiac differentiation,
6-day old EBs were fransferred to the 6 well plates coated with 0.1% gelatin in media consisting
of DMEM supplemented with 15% FBS (selected for cardiac differentiation), 2 mmol/L L-
glutamine, and 1% nonessential amino acids. During differentiation, the media was changed
daily. Spontaneously contracting cells appeared as clusters in outgrowths from the EBs. These
beating EBs were maintained in long-term cultures for up to 95 days.
[00028] Immunostaining
[00029] Beating foci were isolated with Pasteur pipettes and digested with 0.05 % trypsin
for 20 min with intermittent vortexing. After cells were centrifuged and resuspended in DMEM
mediirai containing 20% FCS and 0.5% chicken embryo extracts (GIBCO/BRL), cells were

plated onto gelatin (0.3%) coated coverglasses and incubated in 10% FCS medium for two days.
Immunostaining was done as described elsewhere.
[00030] Intracellular electrophysiology
[00031] A single beating, microdissected EB outgrowth was cultured on a glass coverslip
for 1-10 days. The coverslip was then attached to the bottom of an experimental chamber
mounted on an inverted microscope (Nikon Diaphot 200). The EBs were perfused with Tyrodes
solution consisting of (mmol/L): UONaCl, 1 MgC12,10 HEPES, 10 Glucose, 1.8 CaC12, pH 7.4
with NaOH at 37° C. Contractions were measured using video edge detection. For intracellular
electrophysiology experiments, sharp glass microelectrodes were fabricated with resistances of
30-100 MQ when filled with 3 mol/L KCl. Spontaneously beating EBs were impaled with the
microelectrodes and pipette capacitance was nulled. Litracellular recordings of membrane
potential were made using an Axoclamp-2A ampHfier in Bridge Mode (Axon Instruments, Foster
City, CA), and recordings which showed a stable maximum diastoHc potential (MDP) for at least
5 minutes were included in data analysis. In some experiments, the preparation underwent
electrical field stimulation at rates from 1 to 3 Hz. Data were digitized at 20 kHz and filtered at 2
kHz. APs were analyzed using pClamp8.02 (Axon Instruments, Foster City, CA) and Origin 6.0
software (Microcal Inc, Northampton, MA) to determine AP duration at 50% and 90% of
repolarization (APD50 and APD90), AP amplitude (APA), maximimi diastohc potential (MDP),
and the maximum rate of rise of the AP upstroke (dV/dtmax).
[00032] Contraction Measurements
[00033] Contractions were measured using video edge detection. A single beating
embryoid body (EB) outgrowth cultured on a glass coverslip was attached to the bottom of an
experimental chamber mounted on an inverted microscope (Nikon Diaphot 200). The
preparation was continuously perfused with Tyrodes solution containing (mmol/L): 140 NaCl, 1
MgCl2,10 HEPES, 10 Glucose, 1.8 CaCl2, pH 7.4 with NaOH with additional drugs as indicated.
Electrical field stimulation with Grass SD-9 stimulator (Quincy,MA) was carried out with two
platinum electrodes along opposite walls of the 200-^1 experimental chamber (Warner
Instrument Corp). The stimulation protocol was from 1 to 3 Hz, 10-ms duration, and 30 to 50 V
at 37°C. Individual beating EBs were monitored with Video Edge Detector VED 105 (Crescent
Electronics) through CCD BW Camera NL-2332 (National Electronic) and Sony BW Video
Monitor PVM-97 (Sony Coip). The twitch responses at sharp edge of beating EB outgrowth
were recorded at 1 kHz through DigiData 1200 A/D converter with pClamp 8.2 acquisition software
(both from Axon Instrument, Foster City, CA). The contractile responses are normalized to basal

levels. The experimental chamber temperature was controlled at 37+0.5'C by Dual Automatic
Temperature Controller TC-344B (Warner Instrument Corp).
[00034] Cardiac differentiation in EBs
[00035] Our initial studies showed that HI, H7, H9 and H14 ES cell lines can form EBs
with spontaneously contracting outgrowths. Beating EBs are first observed approximately 10
days into differentiation and after 30 days approximately 10-25% of EBs show spontaneous
contractions. With daily gentle media changes and low EB density, the EBs continued to
contract in culture for a period of observation of up to 95 days of differentiation. The remainder
of the experiments then focused on EBs derived from H9 and H14 cell lines, and results from
these two cell lines were indistinguishable.
[00036] Immunostaining was performed to confirm the presence of CMs in the beating EB
outgrowlhs and to examine contractile/sarcomeric protein organization. Beating foci were
digested and plated as a monolayer for immunostaining using antibodies against a-actinin,
sarcomeric myosin heavy chain (MHC), and cardiac Troponin I (cTnl). Cells isolated from
beating foci resumed spontaneous beating after 6-48 hrs plating on coverglasses.
[00037] Staining with anti a-actinin antibodies showed varying cytoplasmic patterns
ranging from unorganized myofilaments to well organized sarcomeric myofilaments with Z-
lines. Sarcomeric MHC staining showed an abundant signal distributed throughout cytoplasm,
which is a typical staining pattern with this antibody.
[00038] Immunostaining of cTnl showed well-organized parallel myofilament and a
striated pattern of I bands in some cells. These observations clearly indicated that cardiac
myocytes are present in differentiating EBs and some CMs show significant sarcomeric
organization. Although cells were from beating foci, there are non-CMs indicated by nuclear
staining but lack of cardiac specific protein immunostaining. The percentage of CMs isolated
from beating foci varied widely, ranging from 2 % to 70 %.
[00039] Positive inotropic response to p-adrenergic stimulation
[00040] An increase in confractility of cardiac muscle in response to p-adrenergic
stimulation requires appropriate surface membrane receptors coupled to a signaling pathway that
stimulates a variety of ion channels, membrane transporters and myofilament proteins. However,
the responsiveness of cardiac contractility to P-adrenergic stimulation changes over the course of
development with the earliest embryonic cardiac myocytes being unresponsive to p-adrenergic
agonists. Therefore, we sought to determine if the beating EB outgrowths showed a change in
contractile properties in response to the p-adrenergic agonist isoproterenol (Iso). Contractions of
the EB outgrowths were measured usmg video edge-detection techniques during electrical field
-10-

Stimulation to control the beating rate. The magnitude of deflection of the edge of the outgrowth
with each stimulated contraction gives a measure of contractility. Fig. 2 demonstrates the
coivtractile pattern of an EB stimulated at I Hz uiider basal conditions and then after superfusion
with 1 [j-mol/L Iso. A clear increase in the magnitude of the contraction is observed, and on
average l^imol/L Iso resulted in a 33 plus or minus 27% increase in the contraction magnitude
(n=5, p=0.05). This measurement showed significant variability from EB to EB in part due to the
distinct and complex geometry of each beating outgrowth. These results demonstrate that P-
adrenergic receptors are present in hmnan embryonic stem cell-derived cardiomyocytes and
stimulation of these receptors produces a positive inotropic response.
[00041] Patterns of spontaneous electrical activity
{00042] Observations of beating EBs in culture revealed at least two distinct patterns of
beating, continuous beating or episodic beating. To investigate this beating pattern fiirther, we
made intracellular recordings of action potentials witli sharp microelectrodes in twenty
spontaneously contracting EBs. Continuous electrical activity was documented in 12/20 EBs.
EBs with continuous electrical activity had spontaneous action potential rates that were relatively
constant throughout the recording period and ranged between 38 and 106 bpm. In 8/20 EBs,
episodic activity was observed, and a clear periodicity of activity was evident. Each burst of
activity is characterized by action potentials resuming at a relatively rapid rate that then tapers,
followed by another pause. For episodic activity, tlie duration of active periods and pauses
varied from EB to EB, and there was a rough parallel in the duration of spontaneous electrical
activity and pauses for each EB.
[00043] Multiple types of action potentials
[00044] To characterize the types of cardiomyocytes m the EBs, we examined the shape
and properties of action potentials from 105 stable impalements of 20 different EBs. At the time
window of differentiation that we studied (40-95 days), there was clear heterogeneity in the
morphology of the action potentials; however, the action potentials could be classified into 3
major types: nodal-like, embryonic atrial-like, and embryonic ventricular-like (Fig. ). This
classification was based on the properties of the action potential as measured by the maximum
rate of rise of the action potential (dV/dtmax), the action potential duration (APD), action
potential amplitude (APA), and prominence of phase 4 depolarization as summarized in the
Table. Nodal-like action potentials (Fig. ) were characterized by prominent phase-4
depolarization, slow upstroke (dV/dtmax), and a smaller APA. Embryom'c ventricular-like actioi
potentials could be distinguished by the presence of a significant plateau phase of the action
potential resulting in a significantly longer duration compared to the more triangular shaped
-11-

embryonic-atrial action potentials. In addition, embryonic ventricular-like action potentials
generally showed a trend for slower spontaneous rates of activity the longer the EBs were
maintained in culture from 40 to 95 days.
[00045] These latter two classes of action potentials are referred to as embryonic, because
they have properties more reminiscent of embryonic hearts, which are quite distinct from
neonatal and adult cardiac muscle. In particular, the embryonic action potentials are
characterized by more depolarized maximum diastolic potentials (MDP) and "slow" type action
potentials based on low dV/dtmax (~5-30 V/sec)
[00046] To compare action potentials, and hence cardiac cell types in a given EB
outgrowth, we made multiple separate impalements with up to 14 separate recordings per
outgrowth. Our findings were that multiple intracellular recordings from a single EB are
characterized by a predominant action potential phenotype. To provide a quantitative
comparison of all of the action potentials recorded from each impalement of a single EB, we
plotted the measured APD90s grouped per EB. In general, the APD90s clustered closely
together for a given EB but showed variabihty from EB to EB studied. These results suggest that
for any given beating EB outgrowth, spontaneous differentiation favors a predominant cardiac
myocyte cell type based on the reproducible action potential morphology observed.
[00047] Rate adaptation of action potentials
[00048] A ftindamental property of cardiomyocytes is the ability to adapt to an increase in
heart rate with a decrease in APD. Rate adaptation is present in atrial and ventricular muscle,
and it can be impaired in certain disease states. Shortening of APD with rate has also been
observed in embryonic (7-12 wk) human ventricular muscle. Therefore, we sought to determine
if the embryonic ventricular-like action potentials exhibited appropriate rate adaptation. Isolated
EB outgrowths were subjected to electrical field stimulation at three different rates, and steady
state action potentials were then recorded and analyzed. An increase in stimulation frequency
from 1 to 2 Hz resulted in APD50 and APD90 shortening on average approximately 20% (Fig
5C), and there was an additional small decrease in APD as the rate was increased to 3 Hz.
However, there were no changes in APA or upstroke of the action potential evident at the
different stimulation rates tested. These results demonsfrate that embryonic ventricular-like
cardiomyocytes present in beating EBs have the necessary ion channels and regulatory properties
to exhibit rate adaptation. Similar results were also observed for embryonic atrial-like myocytes.
[00049] Human stem cell-derived cardiomyocytes have significant Ikt
[00050] Repolarization of the cardiac action potential is due to multiple ionic currents witli
an important role played by voltage gated K+ chaimels; however, there is significant species

variability of the exact type of K+ channels present. In human heart, current through HERG
potassium channels (KCNH2), Ikt, plays a major role in repolarization of the action potential.
HERG channels are also important in drug development as they represent a promiscuous target
for drug block that can result in action potential prolongation and the potentially lethal
ventricular arrhythmias torsades de pointes. Therefore, we examined the contribution of Ikt to
repolarization of action potentials in human embryonic stem cell-derived cardiomyocytes
utilizing the HERG specific channel blocker E-4031. Application of 500 nM E-4031 resulted in
action potential prolongation in both embryonic atrial and embryonic ventricular-like
cardiomyocytes. Prolongation of the AP was most evident for temiinal repolarization ^hase 3)
where HERG current is maximal. In embryonic atrial-like cardiomycytes, APD90 but not
APD50 was significantly prolonged, and in embryonic ventricular-like cardiomyocytes
significant prolongation of both APD50 and APD90 was produced by E-4031 with a larger effect
onAPD90. There were not statistically significant effects by E-4031 onAPAorMDP. These
results suggest that HERG channels are expressed in both embryonic atrial-like and embryonic
ventricular-like cardiomyocytes and that Ikt contributes significantly to repolarization of the
action potentials in these cell t>pes.
[00051] Provoked early and delayed after depolarizations
[000521 A major mechanism underlying certain types of cardiac arrhythmias is triggered
activity, which results from after depolarizations. These can be divided into early
afterdepolarizations (EADs) which occur during the repolarization of the action potential or
delayed after depolarizations (DADs) which occur after fiiU repolarization. EADs and DADs
result firom different cellular mechanisms, but both require a specific and complex set of
interacting ion chaimels and Ca2+ cycling proteins present in cardiac myocytes. Therefore, we
examined embryonic ventricular-like cardiomyocytes for the ability to develop EADs and DADs.
EADs typically occur in the setting of a prolonged action potential. EADs were defined as
depolarizations occurring near the action potential plateau and were observed in 3/5 embryonic
ventricular-like CMs treated with E-4031. EADs were never observed in the absence of E-4031.
DADs typically occur during Ca2+ overload such as produced by injury or digoxin toxicity.
DADs were observed to occur spontaneously in a small number of cells immediately following
microelectrode impalement presumably due to injury associated with impalement and associated
Ca2+ overload. These cells were not used for characterization of action potential properties, but
they demonstrate the abihty of the human embryonic stem cell-derived cardiomyocytes to exhibit
DADs.
WE CLAIM :
1. A method for testing an agent for effect on human cardiac cells comprising the steps
of
cuituring aggregates of approximately 500-800 undifferentiated human embryonic stem
cells to produce embryoid bodies;
differentiating the embryoid bodies in in vitro culture for between 40 and 95 days to
derive atrial-, ventricular- and nodal cardiomyocyte cell types;
piercing a single cardiomyocyte with an electrode so that the transmembrane action
membrane of that cardiomyocyte can be electrically measured;
measuring the transmembrane action potential of the single cardiomyocyte;
assessing the transmembrane action potential of the cardiomyocyte to characterize
the cardiomyocyte as to the cell type of the human heart that the action potential most
resembles among the cell types selected from the group consisting of ventricular, atrial
and nodal cell types;
exposing the cardiomyocyte to the agent; and
observing whether the action potential of the cardiomyocyte changes after the exposure
to the agent.
2. The method as claimed in claim 1 wherein the deriving is conducted by permitting
the human embryonic stem cells to form embryoid bodies and wherein the measuring includes
impaling the single cardiomyocyte within an embryoid body with the electrode.
3. A method for testing an agent for its effect on the electrical properties of the HERG
channel in human cardiac cells comprising the steps of
cuituring aggregates of approximately 500-800 undifferentiated human embryonic stem
cells to produce embryoid bodies;
differentiating the embryoid bodies in in vitro culture for between 40 and 95 days to
derive atrial-, ventricular- and nodal cardiomyocyte cell types;
inserting an electrode into the interior of a single cardiomyocyte in culture in order to be
able to measure the transmembrane action potential of the cardiomyocyte;
measuring the duration of the transmembrane action potential of the cardiomyocyte;
assessing the transmembrane action potential of the cardiomyocyte to characterize the
cardiomyocyte as to the cell type of the human heart that the action potential most resembles
among the cell types selected from the group consisting of ventricular, atrial and nodal cell
types;
exposing the cardiomyocyte to the agent; and
observing whether the action potential duration is changed by the agent, as would be the
case if the HERG channel is altered.
4. The method as claimed in claim 3 wherein the culturing is conducted by permitting
the human embryonic stem cells to form embryoid bodies and wherein the measuring includes
impaling an embryoid body with an electrode.
5. A method for testing an agent for its likelihood of triggering delayed after
depolarization events in human cardiac cells comprising the steps of
culturing aggregates of approximately 500-800 undifferentiated human embryonic stem
cells to produce embryoid bodies;
differentiating the embryoid bodies in in vitro culture for between 40 and 95 days to
derive atrial-, ventricular- and nodal cardiomyocyte cell types;
inserting an electrode into the interior of a single cardiomyocyte in culture in order to be
able to measure the transmembrane action potential of the cardiomyocyte;
obtaining a chart of the transmembrane action potential of the cardiomyocyte over time;
assessing the transmembrane action potential of the cardiomyocyte to characterize the
cardiomyocyte as to the cell type of the human heart that the action potential most resembles
among the cell types selected from the group consisting of ventricular, atrial and nodal cell
types;
exposing the cardiomyocyte to the agent; and
observing whether a delayed after polarization event is triggered by the agent.
6. The method as claimed in claim 5 wherein the culturing is conducted by permitting
the human embryonic stem cells to form embryoid bodies and wherein the measuring includes
impaling an embryoid body with an electrode.
7. A method for testing an agent for its likelihood of triggering long QT syndrome in
patients by testing human Cardiac cells comprising the steps of
culturing aggregates of approximately 500-800 undifferentiated human embryonic stem
cells to produce embryoid bodies;
differentiating the embryoid bodies in in vitro culture for between 40 and 95 days to
derive atrial-, ventricular- and nodal cardiomyocyte cell types;
separately inserting an electrode into the interior of several single cardiomyocytes in the
culture in order to be able to measure the transmembrane action potential of the cardiomyocytes;
obtaining a chart of the transmembrane action potential of a plurality of the
cardiomyocytes over time;
assessing the transmembrane action potential of the cardiomyocytes to characterize the
cardiomyocytes as to the cell type of the human heart that the action potential most resembles
among the cell types selected from the group consisting of ventricular, atrial and nodal cell
types;
exposing the cardiomyocytes to the agent; and
observing whether action potential duration is prolonged, as an indicator of the risk of
long QT syndrome by the agent in any of the cardiomyocytes.
8. The method as claimed in claim 7 wherein the culturing is conducted by permitting
the human embryonic stem cells to form embryoid bodies and wherein the measuring includes
impaling embryoid bodies with an electrode.
9. A method for testing an agent for effect on human cardiac cells comprising the steps
of
culturing aggregates of approximately 500-800 undifferentiated human embryonic stem
cells by in vitro culture to produce embryoid bodies;
selecting amongst the embryoid bodies for embryoid bodies which demonstrate the
presence of atrial-, ventricular- and nodal cardiomyocyte cell types;
piercing the embryoid body to place a fine electrode inside a single cardiomyocyte
within the embryoid body so that the transmembrane action membrane of that cardiomyocyte
can be electrically measured;
measuring the transmembrane action potential of the single cardiomyocyte;
assessing the transmembrane action potential of the cardiomyocyte to characterize the
single cardiomyocyte as to the cell type of the human heart that the action potential most
resembles among the cell types selected from the group consisting of ventricular, atrial and
nodal cell types;
exposing the cardiomyocyte to the agent; and
observing whether the action potential of the cardiomyocyte changes after the exposure
to the agent.

Human embryonic stem cells form embryoid bodies in culture which contain differentiated human cells. Some of
the human cells in embryoid bodies differentiate into cardiomyocytes. Here the biological and electrical characteristics of those car-
diomyocytes arc described with reference to the use of cardiomyocytes derived from human embryonic stem cells in drag screening
protocols for mechanisms of cardiac toxicity.

Documents:

1949-KOLNP-2004-ABSTRACT 1.1.pdf

1949-kolnp-2004-abstract.pdf

1949-kolnp-2004-assignment.pdf

1949-KOLNP-2004-CLAIMS 1.1.pdf

1949-kolnp-2004-claims.pdf

1949-KOLNP-2004-CORRESPONDENCE.pdf

1949-kolnp-2004-description (complete).pdf

1949-KOLNP-2004-DESCRIPTION (COMPLETED) 1.1.pdf

1949-KOLNP-2004-DRAWINGS 1.1.pdf

1949-kolnp-2004-drawings.pdf

1949-kolnp-2004-examination report.pdf

1949-KOLNP-2004-FORM 1.1.1.pdf

1949-kolnp-2004-form 1.pdf

1949-kolnp-2004-form 13.pdf

1949-kolnp-2004-form 18.pdf

1949-kolnp-2004-form 3.pdf

1949-kolnp-2004-form 5.pdf

1949-KOLNP-2004-FORM-27.pdf

1949-kolnp-2004-gpa.pdf

1949-kolnp-2004-granted-abstract.pdf

1949-kolnp-2004-granted-assignment.pdf

1949-kolnp-2004-granted-claims.pdf

1949-kolnp-2004-granted-correspondence.pdf

1949-kolnp-2004-granted-description (complete).pdf

1949-kolnp-2004-granted-drawings.pdf

1949-kolnp-2004-granted-examination report.pdf

1949-kolnp-2004-granted-form 1.pdf

1949-kolnp-2004-granted-form 13.pdf

1949-kolnp-2004-granted-form 18.pdf

1949-kolnp-2004-granted-form 3.pdf

1949-kolnp-2004-granted-form 5.pdf

1949-kolnp-2004-granted-gpa.pdf

1949-kolnp-2004-granted-reply to examination report.pdf

1949-kolnp-2004-granted-specification.pdf

1949-KOLNP-2004-INTENATIONAL PUBLICATION.pdf

1949-KOLNP-2004-OTHERS.pdf

1949-kolnp-2004-reply to examination report.pdf

1949-kolnp-2004-specification.pdf


Patent Number 239500
Indian Patent Application Number 1949/KOLNP/2004
PG Journal Number 13/2010
Publication Date 26-Mar-2010
Grant Date 24-Mar-2010
Date of Filing 17-Dec-2004
Name of Patentee WISCONSIN ALUMNI RESEARCH FOUNDATION
Applicant Address 614 WALNUT STREET, P.O.BOX 7365, MADISON, WI
Inventors:
# Inventor's Name Inventor's Address
1 THOMSON JAMES A 1807 REGENT STREET, MADISON, WI 53705
2 MA YUE 2350 CHALET GARDENS ROAD, APT 15, FITCHBURG WI 53711
3 HE,JIA QIANG 506 EAGLE HEIGHTS, APT, E. MADISON, WI 53705
4 KAMP TIMOTHY J 4203 WANETAH TR, MADISON, WI 53711
PCT International Classification Number C12N
PCT International Application Number PCT/US2003/023174
PCT International Filing date 2003-07-25
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/399,330 2002-07-26 U.S.A.