State-of-the-Art Differentiation Protocols for Patient-Derived Cardiac Pacemaker Cells

Abstract

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes raise the possibility of generating pluripotent stem cells from a wide range of human diseases. In the cardiology field, hiPSCs have been used to address the mechanistic bases of primary arrhythmias and in investigations of drug safety. These studies have been focused primarily on atrial and ventricular pathologies. Consequently, many hiPSC-based cardiac differentiation protocols have been developed to differentiate between atrial- or ventricular-like cardiomyocytes. Few protocols have successfully proposed ways to obtain hiPSC-derived cardiac pacemaker cells, despite the very limited availability of human tissues from the sinoatrial node. Providing an in vitro source of pacemaker-like cells would be of paramount importance in terms of furthering our understanding of the mechanisms underlying sinoatrial node pathophysiology and testing innovative clinical strategies against sinoatrial node dysfunction (i.e., biological pacemakers and genetic- and pharmacological- based therapy). Here, we summarize and detail the currently available protocols used to obtain patient-derived pacemaker-like cells.

Full text

Citation: Torre, E.; Mangoni, M.E.;
Lacampagne, A.; Meli, A.C.; Mesirca,
P. State-of-the-Art Differentiation
Protocols for Patient-Derived Cardiac
Pacemaker Cells. Int. J. Mol. Sci. 2024,
25, 3387. https://doi.org/10.3390/
ijms25063387
Academic Editor: Massimo Iacoviello
Received: 15 February 2024
Revised: 12 March 2024
Accepted: 14 March 2024
Published: 16 March 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Review
State-of-the-Art Differentiation Protocols for Patient-Derived
Cardiac Pacemaker Cells
Eleonora Torre
1,2
, Matteo E. Mangoni
1,2
, Alain Lacampagne
3
, Albano C. Meli
3,4
and Pietro Mesirca
1, 2,
*
1
Institut de Génomique Fonctionnelle, Universitéde Montpellier, CNRS, INSERM, 34090 Montpellier, France;
eleonora.torre@igf.cnrs.fr (E.T.); matteo.mangoni@igf.cnrs.fr (M.E.M.)
2LabEx Ion Channels Science and Therapeutics (ICST), 06560 Valbonne, France
3PhyMedExp, University of Montpellier, Inserm, CNRS, 371 Avenue du Doyen G. Giraud, CEDEX 5,
34295 Montpellier, France; alain.lacampagne@inserm.fr (A.L.); albano.meli@inserm.fr (A.C.M.)
4Montpellier Organoid Platform, Biocampus, University of Montpellier, CNRS, INSERM,
34090 Montpellier, France
*Correspondence: pietro.mesirca@igf.cnrs.fr
Abstract: Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes raise the possibility
of generating pluripotent stem cells from a wide range of human diseases. In the cardiology field,
hiPSCs have been used to address the mechanistic bases of primary arrhythmias and in investigations
of drug safety. These studies have been focused primarily on atrial and ventricular pathologies. Con-
sequently, many hiPSC-based cardiac differentiation protocols have been developed to differentiate
between atrial- or ventricular-like cardiomyocytes. Few protocols have successfully proposed ways to
obtain hiPSC-derived cardiac pacemaker cells, despite the very limited availability of human tissues
from the sinoatrial node. Providing an
in vitro
source of pacemaker-like cells would be of paramount
importance in terms of furthering our understanding of the mechanisms underlying sinoatrial node
pathophysiology and testing innovative clinical strategies against sinoatrial node dysfunction (i.e.,
biological pacemakers and genetic- and pharmacological- based therapy). Here, we summarize and
detail the currently available protocols used to obtain patient-derived pacemaker-like cells.
Keywords: sinoatrial node; hiPSC-derived cardiac pacemaker cells; protocols
1. Introduction
Heart automaticity is a fundamental physiological function that is reliant on the
presence of a highly specialized population of cardiomyocytes in the sinoatrial node (SAN).
These cells are referred to as pacemaker cells [
1
]. The spontaneous activity of pacemaker
cells is due to diastolic depolarization (DD), a slow depolarization phase that drives the
membrane potential from the end of an action potential (AP) to the threshold of a new
AP. SAN cells express a wide array of ion channels, which underlie the generation and
regulation of DD by the autonomic nervous system (ANS) [
1
]. Knowledge of the functional
role of ion channels in pacemaker activity mostly comes from small-animal models (i.e.,
rodents) [
2
–
4
]. Human biospecimens cannot fully replace animal models in cardiovascular
research because of the difficulty of performing the genetic manipulation of native human
myocytes. However, associations between data obtained in rodent models and insights
obtained from human-derived cells and cardiac tissue are important in terms of improving
the translational significance of preclinical research [
5
]. The availability of native human
cardiac tissue samples may be limited, especially tissue from individuals with congenital
heart diseases that do not require cardiac reparative surgery. Indeed, researchers commonly
obtain samples during surgical septal myectomy and cardiac catheterization from heart
transplants. However, the collection of SAN biopsies from patients for research purposes
is currently impossible because of the risk of irreversibly damaging automatic tissue, a
concern associated with surgical procedures. Consequently, living human SAN tissue
Int. J. Mol. Sci. 2024,25, 3387. https://doi.org/10.3390/ijms25063387 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2024,25, 3387 2 of 16
samples are not widely available and are mostly obtained from cardiac graft recipients’
diseased hearts. Searching for complementary sources of human-derived cardiomyocytes
is thus important. In this context, the differentiation of human-induced pluripotent stem
cells (hiPSCs) into cardiomyocytes (hiPSC-CMs) contributes to lessening the limitations in
the availability of
in vitro
human models and may constitute a paramount breakthrough
in the fields of disease modeling and personalized therapy [
6
,
7
]. Despite hiPSCs being
a different source of human cells than embryonic stem cells, some ethical limitations are
to be considered. First, the collection and harvesting of source cells in order to produce
hiPSCs requires patients’ written consent and ethical approval. This procedure implies
the confidential storage of information about the history of the patient’s disease and their
personal history. Another ethically challenging aspect is related to the usage of hiPSCs for
basic research and/or therapy. Indeed, most hiPSC-CM research is devoted to modeling
the idiopathic or inherited pathology of heart rhythm. This approach does not provide
immediate or even short-term benefits to the patient. Consequently, when providing
information to the patient to obtain written consent, the researcher needs to clearly state
that research using the patient’s cellular source will not affect or improve therapeutic
approaches to the pathology. This aspect brings substantial communication challenges
especially, when the given pathology may have a fatal outcome. In addition, access to
patients’ cellular sources may be limited by ethics in the case of secondary and acquired
arrhythmias and when studying potential risk factors because storing patients’ history
information that is not directly related to their primary pathology may be considered a
violation of privacy. Nevertheless, the study of several hiPSC lines derived from patients
has considerably advanced our knowledge of the mechanisms of primary arrhythmias.
While
in vitro
models of ventricular arrhythmias have been published [
8
,
9
], no hiPSC-
based model of primary sinoatrial node dysfunction is currently available. In addition,
while several efficient protocols exist for differentiating ventricular-like hiPSC-CMs [
9
,
10
],
protocols for differentiating pacemaker sinoatrial node-like cells (hiPSC-PMs) remain poorly
developed.
The purpose of this review article is to discuss the developmental and molecular
basis of the published protocols used to differentiate hiPSC-PMs. In this context, we
summarize the transcriptional cascade underlying SAN biogenesis in order to highlight the
key molecules that are important in promoting specification towards cardiac pacemaker
cells. We also discuss important electrophysiological hallmarks of the differentiation of
hiPSCs into hiPSC-PMs. Finally, we indicate potential research avenues via which to
improve differentiation into the hiPSC-PM phenotype.
2. Genesis of SAN: Molecular Determinants
2.1. Development of Cardiac Mesoderm
During differentiation, hiPSCs are treated using different molecules to promote cardiac
specification. Defining molecules involved in hiPSC differentiation is strictly dependent on
knowledge of the genesis of the heart.
In embryo development, gastrulation is a key event that begins with the generation
of a transient structure called a primitive streak, through which the three germ layers (en-
doderm, mesoderm, and ectoderm) are formed [
11
]. The primitive streak results from the
proliferation and the migration of the epiblast cells into the median plane of the embryonic
disc. As the cells invaginate, some of them displace the underlying hypoblast, forming the
embryonic endoderm. Some of the invaginated cells of the primitive streak are found be-
tween the epiblast and newly formed endoderm, generating the intra-embryonic mesoderm.
Later, the remaining cells of the epiblast give rise to the ectoderm (Figure 1)
[12–15]
. The
mammalian heart derives the lateral plate mesoderm from a specific region of mesoderm.
The formation of the mesoderm and its transition to a cardiac mesoderm occur due to
Wnt/
β
-catenin signaling [
16
]. Wnt/
β
-catenin signaling is activated when Wnt binds to its
receptor, which induces
β
-catenin stabilization.
β
-catenin migrates into the nucleus in order
to regulate target genes through its interaction with the TCF/LEF transcription factors.

Int. J. Mol. Sci. 2024,25, 3387 3 of 16
In the absence of Wnt signaling or the presence of an inhibitor such as the endoderm of
Dickkopf Wnt Signaling Pathway Inhibitor 1 (DKK1),
β
-catenin is degraded by a protein
complex, preventing target gene regulation.
Int. J. Mol. Sci. 2024, 25, 3387 3 of 17
(Figure 1) [12–15]. The mammalian heart derives the lateral plate mesoderm from a
specific region of mesoderm.
Figure 1. Generation of cardiac mesoderm during gastrulation and schematic representation of
cardiovascular lineage diversification. FHF, first heart field; SHF, second heart field; RA, retinoic
acid.
The formation of the mesoderm and its transition to a cardiac mesoderm occur due
to Wnt/β-catenin signaling [16]. Wnt/β-catenin signaling is activated when Wnt binds to
its receptor, which induces β-catenin stabilization. β-catenin migrates into the nucleus in
order to regulate target genes through its interaction with the TCF/LEF transcription
factors. In the absence of Wnt signaling or the presence of an inhibitor such as the
endoderm of Dickkopf Wnt Signaling Pathway Inhibitor 1 (DKK1), β-catenin is degraded
by a protein complex, preventing target gene regulation.
In particular, mesoderm specification is induced by the activation of Wnt/β-catenin
signaling (Figure 1). At post coitum embryonic (E) day 5.75 (E5.75) in developing mice
(corresponding roughly to week 2 of human gestation), activated Wnt/β-catenin signaling
induces the expression of mesendodermal markers, such as Brachyury (Bry) [17] and
Eomesodermin (EOMES) [18,19]. These directly activate the primary cardiac mesoderm
regulator, mesoderm posterior BHLH transcription factor 1 (Mesp1) [20,21], and the
family member Mesp2 [22]. This mechanism is reinforced by the activation of TGF-β
superfamily signaling by Nodal and BMP4 (bone morphogenetic protein 4) agonists
[23,24]. Together with Mesp1/2, Nodal and BMP4 induce the expression of the platelet-
derived growth factor receptor-α (Pdgfr-α), which can be used to effectively monitor the
emergence of cardiovascular mesoderm [25]. In the anterior part of the mesoderm,
Mesp1/2 activates the secretion of DKK1 from the endoderm (Figure 1). DKK1, which
Figure 1. Generation of cardiac mesoderm during gastrulation and schematic representation of
cardiovascular lineage diversification. FHF, first heart field; SHF, second heart field; RA, retinoic acid.
In particular, mesoderm specification is induced by the activation of Wnt/
β
-catenin
signaling (Figure 1). At post coitum embryonic (E) day 5.75 (E5.75) in developing mice
(corresponding roughly to week 2 of human gestation), activated Wnt/
β
-catenin signaling
induces the expression of mesendodermal markers, such as Brachyury (Bry) [
17
] and
Eomesodermin (EOMES) [
18
,
19
]. These directly activate the primary cardiac mesoderm
regulator, mesoderm posterior BHLH transcription factor 1 (Mesp1) [
20
,
21
], and the family
member Mesp2 [
22
]. This mechanism is reinforced by the activation of TGF-
β
superfamily
signaling by Nodal and BMP4 (bone morphogenetic protein 4) agonists [
23
,
24
]. Together
with Mesp1/2, Nodal and BMP4 induce the expression of the platelet-derived growth
factor receptor-
α
(Pdgfr-
α
), which can be used to effectively monitor the emergence of
cardiovascular mesoderm [
25
]. In the anterior part of the mesoderm, Mesp1/2 activates
the secretion of DKK1 from the endoderm (Figure 1). DKK1, which subsequently inhibits
Wnt/
β
-catenin signaling, converts the anterior mesoderm into a cardiogenic mesoderm
(Figure 1) [
20
,
21
,
26
]. Together with Wnt/
β
-catenin signaling inhibition, BMP4 and the
fibroblast growth factor (FGF) support cardiac mesoderm formation [
23
,
27
]. In the posterior
component, where Wnt/
β
-catenin signaling is still active and notochord (axial mesoderm)-
released Noggin inhibits BMP4, the lateral plate mesoderm becomes the hemangiogenic
mesoderm [28,29].

Int. J. Mol. Sci. 2024,25, 3387 4 of 16
2.2. First and Second Heart Fields
In mouse E8, corresponding to 3 weeks of human gestation, the specification of the
cardiac lineage begins when cardiac progenitor cells (CPCs) migrate anterolaterally from
cardiogenic mesoderm and form two different fields, known as the first and second heart
fields (FHF and SHF, respectively). At this point, cardiac transcription factors became
fundamental markers used to define the different populations.
The first pool of migrant cells becomes the FHF lineage: FHF cells are identified by
the expression of Nkx2-5 (NK2 transcription factor related, locus 5 [Drosophila]), Tbx5
(T-box transcription factor 5), HCN4 (hyperpolarization-activated cyclic nucleotide-gated
potassium channel 4), and Hand1 (Heart- and neural crest derivatives-expressed protein
1), among others markers [
30
]. FHF cells have been identified as contributing to the my-
ocardium of the left ventricle (LV), atrioventricular canal, and the proepicardium. Together
with cardiac mesoderm specification, FGF is also required in the maintenance of chamber
identity in developing ventricles, suppressing cardiomyocyte plasticity at this stage and
even in differentiated cardiomyocyte fields [31,32].
Later, SHF development depends on the use of instructive Wnts sourced from the al-
ready formed FHF [
33
]. In particular, the Wnt/
β
-catenin signaling regulates the expression
of the LIM (Lin11, Isl1, and Mec3) homeodomain transcription factor Islet1 (Isl1) in the SHF.
The SHF segregates into an anterior component (aSHF) and a posterior component (pSHF)
with different fates. aSHF cells give rise to the right ventricle (RV) and the outflow tract
(OFT), while cells of pSHF contribute to the formation of the atria first, followed by the
inflow tract (IFT) region or venous pole. From the IFT, the sinus horns and the primordial
SAN are formed. At day E10.5 in mice (day 32 in humans), the developing heart shows
well-defined chambers (Figure 1).
2.3. SAN Generates from Posterior SHF
SAN development is initiated in the presence of Isl1-expressing SHF-derived cells.
However, the imposition of the posterior limit of the SHF is, in part, controlled by retinoic
acid (RA) signaling, which restricts the border of aSHF while maintaining its posterior
component (Figure 1) [
34
]. Knockout mice deficient in retinaldehyde dehydrogenase 2
(Raldh2), which catalyzes the second oxidative step in RA biosynthesis, show alteration
in terms of SHF orientation [
35
]. aSFH expresses Hox genes as well as Tbx1, and these
antagonize RA signaling in this region. In the pSHF, RA is essential to restricting the
expression of Nkx2-5. This is suggested by the observation that RA deficiency leads to an
enlargement of the early domains expressing Nkx2-5 [35].
Moreover, pSHF itself is partitioned into right and left pSHF. In particular, Pitx2c
expression in the left pSHF contributes to atria development [
36
,
37
]. In the right side of
the pSHF, where Pitx2c is downregulated by Nodal [
38
], a negative regulation of Nkx2-5
is established, leading to the expression of Tbx3. Consequently, in the right pSHF, Tbx3
inhibits chamber myocardial differentiation, preventing the expression of Nppa (natriuretic
peptide precursor type A) and Cx40 (Connexin 40) proteins, which are specific atrial
chamber myocardial markers. Tbx3 not only represses genes required to differentiate
the working myocardium and limit the expression of atrial chamber markers, but also
stimulates genes required for pacemaker function, including HCN4, L-type Ca
2+
channel,
and slow gap junction Cx30.2, efficiently reprogramming atrial myocytes into functional
pacemaker cells [
37
,
39
]. Notably, the expression of HCN4 in FHF initially overlaps with
that of Nkx2-5, but subsequently becomes restricted to the newly recruited Nkx2-5-negative
venous pole components [
40
]. The population of cells co-expressing Tbx18, Tbx3, and
HCN4, but not expressing Cx40 and Nppa, constitutes the precursor population to SAN
specification [
41
]. The three-dimensional reconstruction of expression patterns show that,
during heart tube elongation, the Tbx18
+
progenitors remain spatially and temporally
separate from the Isl1
+
SHF. They only overlaps with the Isl1
+
domain at the right lateral
side of the IFT, where the SAN develops (Figure 1) [
42
]. Isl1 is regulated in a synergic way by
Tbx5 via Shox2 (short-stature homeobox protein 2) present in the IFT [
43
,
44
]. Biochemistry

Int. J. Mol. Sci. 2024,25, 3387 5 of 16
experiments also demonstrate also that the Isl1
+
population requires Shox2 to repress the
Nkx2-5 [
45
,
46
] and positively regulates BMP4 for the subsequent SAN formation [
43
,
47
].
BMP4 seems to upregulate Tbx18 expression via Gata4 and supports the downregulation
of Nkx2-5 [48].
2.4. Map of SAN Markers
Defining critical markers involved in heart development and the differentiation of the
SAN could help to orientate the differentiation of hiPSCs into hiPSC-PMs.
While the SAN is generally considered to be a unique region of the heart, structural
studies have distinguished three different SAN sub-regions: the head, center, and tail [
49
].
These different subregions correspond in the adult SAN to a different spectrum of markers
that defines these territories. The transcriptional landscape of mouse SANs was revealed
via single-cell RNA sequencing (scRNA-seq) [
50
]. The SAN head is defined using the
principal markers that we listed in Section 2.3: Isl1
+
, Shox2
+
, Tbx3
+
, Tbx18
+
, Nppa
−
, Nkx2-
5
−
, and Cx40
−
. Few differences characterized the tail region (i.e., Tbx18
−
, Nkx2-5
+
) [
50
].
No information is available on the central part, which is supposed to be characterized by a
mixed spectrum of markers from the central and the tail regions.
3. Electrical Activity of the Native SAN
Along with markers of cardiac development, adult SAN is characterized by a specific
pattern of ion channels that ensures the generation of spontaneous activity (Figure 2).
The generation of automaticity in cardiac pacemaker cells is due to DD, a spontaneous,
slowly depolarizing phase of the AP cycle [
1
]. During this phase, the membrane potential
progressively becomes less negative until it reaches the threshold for triggering a new AP.
One of the key initiators of the DD is the hyperpolarization-activated “funny current” (I
f
),
an inward Na
+
/K
+
current predominantly carried by the HCN4 isoform in rodents [
3
]. In
humans, the expression of HCN isoform 1 (HCN1) is higher in the SAN than in HCN4 [
51
].
Consequently, HCN1 could be useful as a new specific molecular marker of the human SAN.
Int. J. Mol. Sci. 2024, 25, 3387 6 of 17
Figure 2. Main electrophysiological mechanisms of adult SAN cells are represented, including
signaling pathways involved in adrenergic and muscarinic regulation of pacemaker activity.
SERCA, sarco/endoplasmic reticulum Ca
2+
—ATPase; PLN, phospholamban.
The ANS has a decisive effect on HR acceleration and deceleration. In the adult heart,
the sympathetic branch of the ANS accelerates HR, while the parasympathetic branch
slows it down. SAN is enriched in adrenergic and muscarinic receptors [1]. The
sympathetic regulation of pacemaker activity is mediated by catecholamines (i.e.,
norepinephrine), activating the β-adrenergic receptors (β
1/2
-ARs). The activation of β-ARs
stimulates adenylyl cyclase (AC) activity, which converts ATP into cyclic AMP (cAMP).
Elevated cAMP directly stimulates I
f
via the cyclic nucleotide-binding domain (CNBD) of
the HCN protein and activates the protein kinase A (PKA). The catalytic subunit of PKA
enhances the activity of Ca
v
1.3 and Ca
v
1.2 channels via PKA-dependent phosphorylation
of the cardiac L-type channel regulatory partner protein Rad [57–59]. The regulation of T-
type Ca
2+
channels by PKA is still controversial; however, it is shown that Ca
v
3.1 channels
are stimulated by β-adrenergic agonists, probably via machinery similar to that used for
L-Type Ca
2+
channels [53]. The deceleration of HR is mediated by parasympathetic
regulation of cardiac automaticity via the activation of muscarinic receptors (M2Rs)
following the release of acetylcholine (ACh) from vagal nerve endings [1]. M2Rs are
negatively coupled with AC activity. Thus, the downregulation of cAMP and,
consequently, reductions in PKA phosphorylation reverse the signaling processes
involved in the sympathetic stimulation of HR, during which I
f
, Ca
v
1.3/Ca
v
1.2, and Ca
v
3.1
are positively stimulated. In addition, M2Rs directly activate the ACh-activated outward
K
+
current (I
KACh
), which induces the hyperpolarization of the membrane potential and
consequently reduces the pacemaker rate [60]. Two subunits of G-protein-activated
inwardly rectifying K
+
channels (GIRK1 and GIRK4) assemble as heterotetramers to form
cardiac I
KACh
channels [61].
The array of ion channels characterizing pacemaker cells can be considered valuable
instruments in efforts to define the population of hiPSC-PMs. Biophysical properties that
have been studied via single-cell recordings using patch clamp or planar lipid techniques
are still informative and accurate tools, disclosing the mechanisms underlying electrical
activity in hiPSC-PMs.
Figure 2. Main electrophysiological mechanisms of adult SAN cells are represented, including
signaling pathways involved in adrenergic and muscarinic regulation of pacemaker activity. SERCA,
sarco/endoplasmic reticulum Ca2+—ATPase; PLN, phospholamban.

Int. J. Mol. Sci. 2024,25, 3387 6 of 16
Moreover, SAN myocytes constitute a unique type of cardiac cell as they co-express
two functionally distinct isoforms of L-type Ca
2+
channels: the cardiac Ca
v
1.2 (Cacna1C)
isoform, which couples excitation to contraction in the working myocardium [
52
], and
the Ca
v
1.3 (Cacna1D) isoform, which contributes to SAN DD [
2
]. Together with L-type
Ca
2+
channels, the T-type Ca
2+
channel Ca
v
3.1 contributes to DD [
53
]. The AP upstroke of
pacemaker cells is mainly driven by Ca
2+
rather than Na
+
channels (I
Na
) [
54
]. In the rabbit
SAN, I
Na
does not participate in the generation of automaticity per se (in the central SAN),
but it can influence heart rate (HR) by contributing to impulse propagation in the SAN and
from the SAN to the atrium [55]. The activation of Cav1.3 triggers sarcoplasmic reticulum
(SR) Ca
2+
release via ryanodine receptor 2 (RyR2) [
56
]. Ca
2+
released from SR stimulates
the activity of the Na
+
/Ca
2+
exchanger (NCX1). This permits the restoration of diastolic
Ca2+ content, generating an inward Na+current.
The ANS has a decisive effect on HR acceleration and deceleration. In the adult heart,
the sympathetic branch of the ANS accelerates HR, while the parasympathetic branch slows
it down. SAN is enriched in adrenergic and muscarinic receptors [
1
]. The sympathetic
regulation of pacemaker activity is mediated by catecholamines (i.e., norepinephrine),
activating the
β
-adrenergic receptors (
β1/2
-ARs). The activation of
β
-ARs stimulates
adenylyl cyclase (AC) activity, which converts ATP into cyclic AMP (cAMP). Elevated
cAMP directly stimulates I
f
via the cyclic nucleotide-binding domain (CNBD) of the HCN
protein and activates the protein kinase A (PKA). The catalytic subunit of PKA enhances
the activity of Ca
v
1.3 and Ca
v
1.2 channels via PKA-dependent phosphorylation of the
cardiac L-type channel regulatory partner protein Rad [
57
–
59
]. The regulation of T-type
Ca
2+
channels by PKA is still controversial; however, it is shown that Ca
v
3.1 channels are
stimulated by
β
-adrenergic agonists, probably via machinery similar to that used for L-Type
Ca
2+
channels [
53
]. The deceleration of HR is mediated by parasympathetic regulation
of cardiac automaticity via the activation of muscarinic receptors (M2Rs) following the
release of acetylcholine (ACh) from vagal nerve endings [
1
]. M2Rs are negatively coupled
with AC activity. Thus, the downregulation of cAMP and, consequently, reductions in PKA
phosphorylation reverse the signaling processes involved in the sympathetic stimulation
of HR, during which I
f
, Ca
v
1.3/Ca
v
1.2, and Ca
v
3.1 are positively stimulated. In addition,
M2Rs directly activate the ACh-activated outward K
+
current (I
KACh
), which induces the
hyperpolarization of the membrane potential and consequently reduces the pacemaker
rate [
60
]. Two subunits of G-protein-activated inwardly rectifying K
+
channels (GIRK1 and
GIRK4) assemble as heterotetramers to form cardiac IKACh channels [61].
The array of ion channels characterizing pacemaker cells can be considered valuable
instruments in efforts to define the population of hiPSC-PMs. Biophysical properties that
have been studied via single-cell recordings using patch clamp or planar lipid techniques
are still informative and accurate tools, disclosing the mechanisms underlying electrical
activity in hiPSC-PMs.
4. Protocols to Differentiate hiPSC-PMs
We used critical checkpoints in heart development to establish several protocols for
the
in vitro
differentiation of hiPSC-PMs. Previously listed markers are used to validate
the quality of the hiPSC-PM population. Electrophysiological properties are tested later to
evaluate the phenotypes of these cells.
After elucidating the complexity of SAN development in terms of signaling and
molecular markers, it became clear that the manipulation of single transcriptional factors
alone could not be sufficient to induce the
in vitro
differentiation of hiPSC-PMs. Using
this approach, two protocols were proposed by increasing the expression of Tbx3 [
62
] or
Shox2 [
63
]. However, Tbx3 and Shox2 remain two downstream factors in heart develop-
ment. Upstream events (i.e., cardiac mesoderm induction, FHF and SHF segregation) are
important commitment steps to generate, as much as possible, a significant population of
hiPSC-PMs in mixed cultures. In particular, it was the study by Zhu et al. [
64
] that first
considered the regulation of an appropriate signaling pathway as an approach to enriching

Int. J. Mol. Sci. 2024,25, 3387 7 of 16
human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Neuregulin (NRG)-1β,
along with its receptor tyrosine kinase, ErbB, is involved in cardiac specification in the
differentiation of hESC-CMs. Their role suggests that manipulating NRG-1
β
/ErbB signal-
ing affects the ratio of Nodal- to working-type cells in differentiating hESC-CM cultures.
However, reducing hESC-CM heterogeneity via the manipulation of this pathway did not
consider several other pathways involved in SAN specification (i.e., Wnt/
β
-catenin and
RA signaling), which are fundamental to imposing the presence or absence of specific
tissue markers.
4.1. Transgene-Dependent Methods to Obtain hiPSC-PMs
Later, several groups tried to obtain hiPSC-PMs, combining the manipulation of
pathways involved in heart development with cell sorting experiments to purify the
population of interest (Figure 3).
The first strategy was proposed by the research group led by Christine Mummery [
65
].
These authors recognized the importance of precisely modulating the transition from
CPCs to differentiated cardiomyocytes in improving cardiac lineage specification. In
particular, they took advantage of the increasing expression of Pdgfr-
α
in CPCs to follow
cardiac progression and the progression of podoplanin (PDPN), whose expression is largely
restricted to the SAN [
66
]. In addition, they used hESCs, in which Nkx2-5 was associated
with eGFP (Nkx2-5-eGFP), together with a doxycycline (dox)-inducible MYC transgene.
They tried to restrain Nkx2-5 enhancement in hESCs, activating MYC transgene by dox
at day 4.75 to obtain SAN-like cells. In order to reinforce Nkx2-5 downregulation, the
Nodal pathway was inhibited (Figure 3). The authors selected a Nkx2-5-eGFP
−
Pdgfr-
α+
PDPN
+
population. However, when maintaining Nkx2-5-eGFP
−
Pdgfr-
α+
PDPN
+
cells in the self-renewal stage, the use of dox combined with Nodal inhibition was not
sufficient. To amplify the Nkx2-5-eGFP
−
Pdgfr-
α+
PDPN
+
population, mitogenic activators
were added (i.e., insulin-like growth factor-1, IGF-1; hedgehog agonist, Hh). On day 12,
MYC transgene was inactivated by removing dox in order to put Nkx2-5-eGFP
−
Pdgfr-
α+
PDPN
+
cells through cardiac differentiation. During the cardiac differentiation stage,
IGF-1 and Hh stimulation were maintained, as was Nodal pathway inhibition; moreover,
the Wnt pathway was inhibited. In this way, Nkx2-5-eGFP
−
Tbx3
+
Shox2
+
HCN4
+
cells
were obtained (Figure 3). Although this strategy produces a large number of cells, the
overexpression of MYC may generate pro-oncogenic cell types that cannot be used
in vivo
to generate biological pacemakers.
Nkx2-5 was also used to monitor hiPSC differentiation in a protocol proposed by
Gordon Keller’s group [
67
]. They started with two different cell lines: the HES3Nkx2-
5
GFP/w
cell line, similar to the Nkx2-5-eGFP hESC line, and a non-genetically modified
hiPSC line. A previously described stepwise development protocol [
25
] was used to obtain
a mixed population of hiPSC-CMs (Figure 3). Briefly, Bry
+
hiPSCs were selected and
cultured as embryonic bodies (EBs). EBs were maintained in culture in a low-oxygen
environment until day 12. During this period, cells were first treated with FGF, BMP4, and
Nodal to obtain Pdgfr-
α+
Mesp1
+
EBs. On day 3, Nodal and FGF pathways were inhibited
while the BMP4 route remained active and supported by RA stimulation. On day 20, cells
were sorted using Nkx2-5, SIRPA (pan-cardiac myocyte marker), and CD90 (mesenchymal
marker). The Nkx2-5
−
SIRPA
+
CD90
−
population expressed considerably higher levels
of genes associated with SAN compared to the Nkx2-5
+
population, including Isl1, Tbx3,
Tbx18, Shox2, and HCN isoforms 1 and 4. The advantage of this protocol comes from the
possibility of generating hiPSC-PMs for the development of clinically compliant biological
pacemakers without the need for the genetic manipulation of hiPSCs.

Int. J. Mol. Sci. 2024,25, 3387 8 of 16
Int. J. Mol. Sci. 2024, 25, 3387 8 of 17
embryonic bodies (EBs). EBs were maintained in culture in a low-oxygen environment
until day 12. During this period, cells were first treated with FGF, BMP4, and Nodal to
obtain Pdgfr-α
+
Mesp1
+
EBs. On day 3, Nodal and FGF pathways were inhibited while the
BMP4 route remained active and supported by RA stimulation. On day 20, cells were
sorted using Nkx2-5, SIRPA (pan-cardiac myocyte marker), and CD90 (mesenchymal
marker). The Nkx2-5
−
SIRPA
+
CD90
−
population expressed considerably higher levels of
genes associated with SAN compared to the Nkx2-5
+
population, including Isl1, Tbx3,
Tbx18, Shox2, and HCN isoforms 1 and 4. The advantage of this protocol comes from the
possibility of generating hiPSC-PMs for the development of clinically compliant biological
pacemakers without the need for the genetic manipulation of hiPSCs.
Figure 3. Summary of experimental designs used to induce hiPSC-PMs through transgene-based
methods combined with cardiac development-based pharmacological manipulation [65,67,68].
Finally, Ren et al. [68] designed a pacemaker-like differentiation protocol based on
the modulation of the Wnt pathway (Figure 3). Indeed, they showed that the re-activation
of Wnt/β-catenin signaling was sufficient to enforce the cellular identity of the pacemaker
phenotype. They validated this finding in a TNTT2-GFP hiPSC line. To this end, they
induced mesoderm formation via the activation of the Wnt/β-catenin signaling from day
0 to day 1. The Wnt/β-catenin signal was inhibited from day 3 until day 5 to force cardiac
mesoderm generation. Because they observed that the Wnt5b isoform was able to silence
Nkx2-5 and upregulate SAN transcription factors (i.e., Isl1 and Tbx18) after cardiac
mesoderm induction in zebrafish, they re-activated the Wnt/β-catenin signaling from day
5 until day 7. On day 15, the TNTT2-GFP
+
cell population exhibited an increase in SAN
markers (i.e., Isl1, Tbx18, Tbx3, Shox2, BMP4, and HCN4) and a reduction in Nkx2-5
expression with the re-activation of Wnt/β-catenin signaling compared to TNTT2-GFP
+
cells population, where Wnt/β-catenin signaling was not re-activated.
4.2. Transgene-Free Methods to Obtain hiPSC-PMs
While these pioneering strategies were fundamental to providing a rich source of
hiPSC-PMs, the high complexity of protocols that are reliant on genetic manipulation may
reduce the chances of reproducibility, driving several groups to propose differentiation
Figure 3. Summary of experimental designs used to induce hiPSC-PMs through transgene-based
methods combined with cardiac development-based pharmacological manipulation [65,67,68].
Finally, Ren et al. [
68
] designed a pacemaker-like differentiation protocol based on the
modulation of the Wnt pathway (Figure 3). Indeed, they showed that the re-activation of
Wnt/
β
-catenin signaling was sufficient to enforce the cellular identity of the pacemaker
phenotype. They validated this finding in a TNTT2-GFP hiPSC line. To this end, they
induced mesoderm formation via the activation of the Wnt/
β
-catenin signaling from
day 0 to day 1. The Wnt/
β
-catenin signal was inhibited from day 3 until day 5 to force
cardiac mesoderm generation. Because they observed that the Wnt5b isoform was able to
silence Nkx2-5 and upregulate SAN transcription factors (i.e., Isl1 and Tbx18) after cardiac
mesoderm induction in zebrafish, they re-activated the Wnt/
β
-catenin signaling from
day 5 until day 7. On day 15, the TNTT2-GFP
+
cell population exhibited an increase in
SAN markers (i.e., Isl1, Tbx18, Tbx3, Shox2, BMP4, and HCN4) and a reduction in Nkx2-5
expression with the re-activation of Wnt/
β
-catenin signaling compared to TNTT2-GFP
+
cells population, where Wnt/β-catenin signaling was not re-activated.
4.2. Transgene-Free Methods to Obtain hiPSC-PMs
While these pioneering strategies were fundamental to providing a rich source of
hiPSC-PMs, the high complexity of protocols that are reliant on genetic manipulation may
reduce the chances of reproducibility, driving several groups to propose differentiation pro-
tocols based mainly on the pharmacological modulation of cardiac development pathways
in order to obtain hiPSC-PMs (Figure 4).
Liu et al. [
69
] started with a cardiac differentiation protocol similar to the one of Ren
et al. [
68
] (Figure 4): between day 0 and day 1, Wnt/
β
-catenin signaling was activated,
being switched off from day 3 until day 5. On day 5, hiPSCs were treated with a BMP4
activator, FGF inhibitor, and a potent RAr
β
(RA receptor
β
) agonist that acts as an antagonist
against RAr
α
and RAr
γ
. In particular, RArs are expressed widely [
70
,
71
]: RAr
α
shows
ubiquitous expression in the developing heart [
72
]. RAr
β
expression is restricted in the OFT
mesenchyme and is found through the developing myocardium [
72
,
73
]. RAr
γ
transcripts
are specifically detected in the endocardial cushion tissue and large developing vessels [
70
].
Moreover, RAr
α
activation seems to improve the atrial differentiation of hiPSCs [
74
]. In

Int. J. Mol. Sci. 2024,25, 3387 9 of 16
this way, FGF signaling, which is known to sustain ventricular development, was inhibited.
BMP4 was added to support SAN differentiation, and RAr
β
-selective activation prevented
atrial, endocardial, and vessel specification, promoting SAN differentiation. Cells treated
with this method showed higher levels of expression of Shox2, Tbx3, Tbx18, HCN4, and
TNNT2 in comparison to untreated ones.
A simplified version of this protocol was proposed by Yechikov [
75
] (Figure 4). After
the activation of Wnt/
β
-catenin signaling between days 0 and 1, inhibition of Wnt/
β
-
catenin signaling was accompanied by Nodal signaling inactivation between days 3 and 5.
Yechikov based this protocol on the concept that Nodal is an upstream effector of Pitx2c and
that the inhibition of Pitx2c via Nodal inactivation could promote hiPSC-PM differentiation.
In addition to the simplicity of this protocol, treated cells exhibited increased transcript and
protein expression of Tbx3, Tbx18, Shox2, and Isl1.
Wiesinger et al. [
76
] used a modified Protze-based protocol [
67
] to generate a roadmap
of transcriptional changes during hiPSC-PM differentiation. Briefly, hiPSCs were initially
directed towards a mesodermal lineage by supplementing the medium with BMP4, Wnt
and Nodal activators; then hiPSCs were further guided towards a cardiac fate by inhibiting
the Wnt/
β
-catenin signaling pathway (Figure 4). At day 4, alongside inhibiting Wnt/
β
-
catenin signaling, the addition of BMP4 and RA in combination with Nodal and FGF
inhibition, drove cardiac mesoderm to cardiomyocytes with a pacemaker-like profile. The
obtained hiPSC-PMs exhibited higher expression levels of Shox2, Tbx3, Tbx18, HCN4,
HCN1, Cacna1d, Cacna1g, and TNNT2 compared to the ventricular-like counterpart.
An exception to these methods was the protocol proposed by Schweizer [
77
] (Figure 4).
They provided a transgene-free approach by co-culturing hiPSCs with mouse visceral
endoderm-like (END-2) cells till day 12 in serum-free medium. After, beating clusters
were dissected and transferred to dishes with serum-enriched medium. They observed a
decrease in Tbx5 and Nkx2-5 expression with an increase in Shox2 expression. However,
co-culturing hiPSC-PMs with END-2 cells has a high xenogeneic risk if we consider the
possible medical applications. Moreover, interaction mechanisms between END-2 cells
and hiPSCs are still unknown and it is difficult to predict how changes in END-2 cell
culture can influence the hiPSCs differentiation. For these reasons, recently Schweizer
group established four END2-cell independent protocols to obtain hiPSC-PMs [
78
]. Here,
three protocols proposed combine Wnt/
β
-catenin signaling activation/inactivation, Nodal
inactivation, and RA administration at different time points or combined in different ways
(Figure 4). The fourth protocol (STEMCELL protocol) consists of a STEMdiff™ Atrial
Cardiomyocyte Differentiation Kit from STEMCELL Technologies (STEMCELL protocol).
Protocols are compared to the END-2 cells dependent one. SAN marker expression was
higher using the STEMCELL protocol.
More recent findings bring the role of cadherin-5 protein (CDH5 or VE-Cadherin) on
the differentiation of hiPSC-PMs [
79
]. CHD5 is a cell adhesion glycoprotein expressed in
vascular endothelial cells [
80
]. Treatment with CDH5 during cardiac differentiation led
to an increased proportion of hiPSC-PMs [
79
] (Figure 4). In particular, CDH5 seems to
synergically support Wnt/
β
-catenin signaling to enhance TCF activity [
81
] when added
between days 5 and 7. However, the mechanism remains to be clarified because CDH5 may
inhibit Wnt/
β
-catenin signaling sequestering
β
-catenin into cadherin complex at the cell
surface and preventing TCF activation [82].

Int. J. Mol. Sci. 2024,25, 3387 10 of 16
Int. J. Mol. Sci. 2024, 25, 3387 10 of 17
between days 5 and 7. However, the mechanism remains to be clarified because CDH5
may inhibit Wnt/β-catenin signaling sequestering β-catenin into cadherin complex at the
cell surface and preventing TCF activation [82].
Figure 4. Summary of experimental designs to induce hiPSC-PMs through transgene-free methods
[69,75–79].
Given that transgene-dependent or independent protocols, deciphering and combing
the complex interplay between BMP4, RA, Nodal, and Wnt/ β-catenin signaling pathways
Figure 4. Summary of experimental designs to induce hiPSC-PMs through transgene-free meth-
ods [69,75–79].
Given that transgene-dependent or independent protocols, deciphering and combing
the complex interplay between BMP4, RA, Nodal, and Wnt/
β
-catenin signaling pathways
in SAN development holds the key to unable improved methods to obtain differentiated
hiPSC-CM cultures containing high percentages of hiPSC-PMs [23,80,83].

Int. J. Mol. Sci. 2024,25, 3387 11 of 16
5. Electrophysiological Properties of hiPSC-PMs
Besides cardiac development markers, ion channels confer electrophysiological proper-
ties that can be studied by the patch clamp technique to affirm the cellular identity expected
from hiPSC-PMs. Native SAN has a unique characteristic of spontaneously beating rapidly.
However, one of the main issues with hiPSC-CMs is that almost all of the cardiac subtypes
(i.e., ventricular-, atrial- and hiPSC-PMs) have spontaneous activity [
10
,
76
,
84
]. The best way
to analyze the functional characteristics of the supposed hiPSC-PMs is to deeply delineate
the ion channels profile by combining information obtained from APs recordings (Table 1).
Table 1. Summary of electrophysiological parameters evaluated in the different protocols.
Birket, Nat.
Biotechnol.,
2015 [65]
Protze, Nat.
Biotechnol.,
2017 [67]
Schweizer,
Stem Cell
Research and
Therapy,
2017 [77]
Ren, Devel-
opmental
Cell,
2019 [68]
Liu, Stem
Cell
Research and
Therapy,
2020 [69]
Yechikov,
Stem Cell
Research,
2020 [75]
Wiesinger,
eLife,
2022 [76]
Zhang, J.
Cell.
Physiol.,
2023 [79]
AP rate ++++++++
dV/dtmax ++ +++
If+ + + (indirectly) + (indirectly) +
ICaL
ICaT +
INa +
IKACh +
Adrenergic
response + +
Muscarinic
response + +
In general, hiPSC-PMs have faster AP rate and slower maximum upstroke veloc-
ity (dV/dt
max
) [
65
,
67
–
69
,
75
–
77
,
79
] compared to atrial- and ventricular-like hiPSC-CMs.
As mentioned above, HCN1 and HCN4 contribute to the generation of I
f
in the human
SAN. Expression of I
f
is considered as the electrophysiological hallmark to be tested in
hiPSC-PM differentiation protocols [
65
,
67
,
76
,
77
,
79
]. I
f
is normally recorded with regular
voltage-clamp protocols [
65
,
67
,
79
], but sometimes I
f
is assessed indirectly by treating cells
with a selective I
f
inhibitor (Ivabradine) and measuring the AP rate reduction
[76,77]
. How-
ever, the expression of I
f
alone is not sufficient to define a pacemaker-like cell because
automaticity relies also on the expression of voltage-gated L- and T-type Ca
2+
channels
(i.e., Ca
v
1.3 and Ca
v
3.1). To date, the functional expression of voltage-gated Ca
2+
channels
isoforms is poorly defined in hiPSC-PMs. Despite different groups showing the expression
of different Ca
2+
channel isoforms, patch clamp recordings of I
Ca
are still focused on total
Ca
2+
current, without making a distinction between the two L-type isoforms Ca
v
1.2 and
Ca
v
1.3 [
67
]. Notably, Ca
v
1.3 channels activate at more negative potentials compared to
Ca
V
1.2 and are responsible for a large fraction of the total L-type Ca
2+
current (I
CaL
) in
the SAN [
1
]. In the native SAN, both Ca
v
1.3 and Ca
v
1.2 are expressed, whereas Ca
v
1.2 is
the only isoform of the adult working myocardium. The fact that the expression of I
CaL
activatesat negative voltages (
−
50,
−
40 mV), just as in the native SAN [
2
], is suggestive
of a more pacemaker-like profile in hiPSC-PMs compared to ventricular- and atrial-like
hiPSCs [
67
]. Recently, the black mamba toxin calciseptine [
85
] was found to be a selective
inhibitor of Ca
v
1.2 vs. Ca
v
1.3 isoform and it could be a useful pharmacological tool to
use when studying the relative expression of Ca
v
1.3 in hiPSC-CMs. Consistent with a
SAN-like phenotype, hiPSC-PMs are characterized by a reduced I
Na
current in comparison
with ventricular-like hiPSCs [
67
]. This is in line with a reduction in the SAN dV/dt
max
,
which is mainly dependent on Ca
2+
channels [
1
,
54
]. In addition, the expression of I
KAch
is
also evaluated and increases in hiPSC-PMs compared to ventricular-like ones [
67
]. Finally,
because adrenergic and muscarinic receptors are critical for the modulation of the response

Int. J. Mol. Sci. 2024,25, 3387 12 of 16
rate in the SAN [
1
], pharmacological regulation with Isoproterenol (
β
-AR stimulator) or
M2R activators may be useful for assessing the proper functioning of positive–negative
chronotropic machinery
[67,77,78]
. In summary, even if no single marker or electrophysio-
logical hallmark has been reported as sharply defining the pacemaker-like differentiation
fate of hiPSC-CMs, the available data suggest an association between action potential
waveform, the steepness of diastolic depolarization, and expression of sinoatrial node ionic
currents (i.e., I
KACh
, I
f
, I
CaL
, I
CaT
), providing a reliable criterion for defining this pathway.
Further research will be required to define more discriminatory marker(s).
6. Conclusions
Nowadays, hiPSC-CMs are widely used as tool for disease modeling and represent
valid platforms for the pharmacological screening of molecules and drugs. However,
hiPSC-CMs have been mostly limited to model-inherited cardiopathies, which primarily
affect ventricular cardiomyocytes. Hence, the current knowledge in the field mainly
focuses on differentiating ventricular-like hiPSC-CMs. It is important to develop an
in vitro
multiscale model of the human SAN based on hiPSC-CM and hiPSC-PM differentiation
protocols. From this perspective, the recent development of multi-chamber cardioids
derived from hiPSC-CMs [
86
] may constitute an important step forward in producing 3D
models in which pacemaker-like myocytes, derived, for example, from patients carrying
sinoatrial node dysfunction, are fused with atrial and ventricular chamber-like tissue. This
would be of paramount importance in terms of further understanding the mechanisms
underlying sinus node dysfunction and testing innovative pharmacologic or molecular
strategies translatable to clinics. In addition, the ability to generate hiPSC-PMs offers the
potential to create biological pacemakers and specialized conduction tissues for treating
patients with conduction system failures. Variability in the differentiation of hiPSC-CM
subpopulations and the degree of cellular maturation may constitute a limitation. In this
regard, the manipulation of cardiac development pathways appears a reliable method
to drive hiPSCs towards an SAN phenotype capable of reducing variability. From this
perspective, this review highlights the significant strides made in the past decade in
generating hiPSC-PMs and emphasizes the critical contribution of insights gained from
developmental biology to these achievements. The regulation of Wnt/
β
-catenin signaling,
Nodal, RA, and BMP4 pathways seems to be fundamental to determining cardiac cells’
fates. However, many authors do not directly report data from differentiation time points
to quantify the relative degree of efficiency of a given protocol to differentiate pacemaker-
like cardiomyocytes in a mixed population of hiPSCs-CMs. Indeed, authors evaluate the
efficiency of differentiation as the relative increase in expression of molecular markers of the
sinoatrial node in hiPSCs-PMs compared to differentiated hiPSCs-CMs in parallel cultures,
rendering comparison between different protocols difficult. Future work will thus require
a coupled molecular and functional analysis of hiPSC-PM to ensure that they faithfully
relate to the phenotypes of donor patients. This analysis will be essential in advancing
the potential clinical applications of hiPSC-PMs and their relevance in modeling human
cardiac conditions and diseases.
Author Contributions: Writing and original draft preparation, E.T.; figures preparation, E.T.; review
and editing, M.E.M., A.L., A.C.M. and P.M. All authors have read and agreed to the published version
of the manuscript.
Funding: This work is supported by the Fondation Leducq (TNE 19CV03) and the Agence Nationale
de la Recherche (ANR-22-CE17-0012-02). E.T. is supported by Lefoulon Delalande postdoctoral
fellowship.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.

Int. J. Mol. Sci. 2024,25, 3387 13 of 16
Acknowledgments: All the Figures are created with Biorender.com, accessed on 1 December 2019.
We thank Stefan Dubel for scientific editing.
Conflicts of Interest: The authors declare no conflicts of interest.
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