Differentiation and Application of Human Pluripotent Stem Cells Derived Cardiovascular Cells for Treatment of Heart Diseases: Promises and Challenges

Abstract

Human pluripotent stem cells (hPSCs) are derived from human embryos (human embryonic stem cells) or reprogrammed from human somatic cells (human induced pluripotent stem cells). They can differentiate into cardiovascular cells, which have great potential as exogenous cell resources for restoring cardiac structure and function in patients with heart disease or heart failure. A variety of protocols have been developed to generate and expand cardiovascular cells derived from hPSCs in vitro. Precisely and spatiotemporally activating or inhibiting various pathways in hPSCs is required to obtain cardiovascular lineages with high differentiation efficiency. In this concise review, we summarize the protocols of differentiating hPSCs into cardiovascular cells, highlight their therapeutic application for treatment of cardiac diseases in large animal models, and discuss the challenges and limitations in the use of cardiac cells generated from hPSCs for a better clinical application of hPSC-based cardiac cell therapy.

Full text

fcell-09-658088 May 6, 2021 Time: 17:42 # 1
REVIEW
published: 12 May 2021
doi: 10.3389/fcell.2021.658088
Edited by:
Shijun Hu,
Soochow University, China
Reviewed by:
Meng Zhao,
Westlake University, China
Mingtao Zhao,
Nationwide Children’s Hospital,
United States
*Correspondence:
Jun Pu
pujun310@hotmail.com
Specialty section:
This article was submitted to
Stem Cell Research,
a section of the journal
Frontiers in Cell and Developmental
Biology
Received: 25 January 2021
Accepted: 25 March 2021
Published: 12 May 2021
Citation:
Gao Y and Pu J (2021)
Differentiation and Application
of Human Pluripotent Stem Cells
Derived Cardiovascular Cells
for Treatment of Heart Diseases:
Promises and Challenges.
Front. Cell Dev. Biol. 9:658088.
doi: 10.3389/fcell.2021.658088
Differentiation and Application of
Human Pluripotent Stem Cells
Derived Cardiovascular Cells for
Treatment of Heart Diseases:
Promises and Challenges
Yu Gao and Jun Pu*
Department of Cardiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Human pluripotent stem cells (hPSCs) are derived from human embryos (human
embryonic stem cells) or reprogrammed from human somatic cells (human induced
pluripotent stem cells). They can differentiate into cardiovascular cells, which have great
potential as exogenous cell resources for restoring cardiac structure and function in
patients with heart disease or heart failure. A variety of protocols have been developed
to generate and expand cardiovascular cells derived from hPSCs in vitro. Precisely and
spatiotemporally activating or inhibiting various pathways in hPSCs is required to obtain
cardiovascular lineages with high differentiation efficiency. In this concise review, we
summarize the protocols of differentiating hPSCs into cardiovascular cells, highlight their
therapeutic application for treatment of cardiac diseases in large animal models, and
discuss the challenges and limitations in the use of cardiac cells generated from hPSCs
for a better clinical application of hPSC-based cardiac cell therapy.
Keywords: human pluripotent stem cells (hPSCs), cardiovascular cells, differentiation, therapeutic application,
large animal
INTRODUCTION
Cardiovascular diseases are the leading causes of death in the world. It is estimated that more
than 5 million people die of myocardial infarction (MI) every year (Virani et al., 2020). Although
thrombolysis, coronary intervention, and coronary artery bypass graft have significantly improved
the prognosis, the high morbidity and mortality associated with MI indicate that current treatment
strategy is far from satisfactory.
Cell transfer therapy is being explored as a potential approach to repopulate damaged cardiac
tissue. In addition to skeletal myoblasts (Ye et al., 2007), bone marrow–derived cells (Assmus
et al., 2010), and mesenchymal stem cells (Ye et al., 2013a), pluripotent stem cells (PSCs)–derived
cardiovascular cells, including cardiovascular progenitor cells (CPCs), cardiomyocytes (CMs),
endothelial cells (ECs), and smooth muscle cells (SMCs), have been extensively studied.
Human PSCs (hPSCs) include human embryonic stem cells (hESCs) and human induced
PSCs (hiPSCs). Theoretically, they can differentiate into all somatic cells found in the human
body and can be used as a disease model to explore the genetic mechanisms of diseases such
Frontiers in Cell and Developmental Biology | www.frontiersin.org 1May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 2
Gao and Pu Review of hPSCs-CVCs
as congenital heart defects (Lin et al., 2021) or test drugs (Protze
et al., 2019) or alternative cell sources for replacing diseased or
damaged tissue or disease models in vitro. A variety of protocols
have been developed to differentiate hPSC into cardiovascular
lineages in vitro. In this review, we focus on ongoing progress in
hPSC-based strategies for human cardiovascular cell derivation
and application. This review summarizes the available protocols
of differentiating hPSCs into cardiovascular cells, including
CMs, ECs, SMCs, and CPCs, and highlights their therapeutic
application for treatment of heart diseases in large animal models.
Finally, the review discusses the challenges and limitations in
the use of cardiac cells generated from hPSCs in the clinical
perspective for the treatment of cardiac disease.
EMBRYONIC HEART DEVELOPMENT
The differentiation of hPSCs into CMs is similar to the
process of the heart development and formation in vivo
(Figure 1). Detailed signaling and transcriptional networks in
heart development were described in a review by Bruneau
(2013). During the embryonic period, Nodal is expressed in
the epiblast and activates the distal visceral endoderm, which
moves toward the oval to form the anterior–posterior axis
(Perea-Gomez et al., 2002). Simultaneously, visceral endoderm
secretes Nodal antagonists, including Cerberus, Lefty1, and
Dickkopf-related protein 1, which make a gradient change
of Nodal and WNT signals in the front and rear directions
(Perea-Gomez et al., 2002). This promotes the development
of primitive streak, which indicates the start of gastrulation,
a process in which the inner cell mass is converted into the
trilaminar embryonic disk (Perea-Gomez et al., 2002). This disk
comprised the three germ layers: ectoderm, mesoderm, and
endoderm. The induction of cardiac mesoderm and distinct
populations of CPCs are primarily controlled by three families
of extracellular signaling molecules: wingless integrated (WNT),
fibroblast growth factor (FGF), and transforming growth factor
β(TGF-β), including WNT3a, bone morphogenetic protein
4 (BMP4), Nodal, and activin-A (Spater et al., 2014). These
signals induce the expressions of Brachyury and Eomes,
which are markers of early mesoderm formation (Lim and
Thiery, 2012). In the process of primitive streak migration,
cells temporarily activate the transcription factor mesoderm
posterior protein 1 (MESP1), which indicates entering the
stage of cardiac mesoderm development (Lim and Thiery,
2012). Later, mesodermal progenitors commit to cardiac cells
by WNT antagonist.
A subset of MESP1+cells begin to transcribe the
homeodomain transcription factors Nkx2.5, T-box 5 (Tbx5,
a marker of the first heart field), and islet1 (Isl1) genes
(a marker of the second heart field) (Evans et al., 2010).
These factors represent cardiac lineage markers in the early
developmental stages of the heart field. Nkx2.5 and Tbx5 are
typical markers of primitive heart tube cells involved in the
formation of the atria and left ventricular (LV) compartments,
whereas the secondary heart field is mainly related to the
development and formation of the right ventricle and outflow
tract (Evans et al., 2010). They are related to transcription factor
GATA4/5/6 and serum response factor (SRF). Subsequently,
genes related to the CMs are successively activated, such as
α-actinin, myosin light chain, myosin heavy chain (MHC),
and troponin, as well as myocyte enhancer factor-2 (MEF2)
that regulates heart structural genes (Evans et al., 2010).
These complexes process and lead to the proliferation and
maturation of CMs.
In summary, heart development can be roughly divided
into three stages: (1) gastrulation to cardiac specification
during which mesodermal progenitors are developed; (2) heart
development before beating during which cardiac progenitors are
developed; and (3) heart development at beating during which
myofibrillogenesis and trabeculation are developed.
DIFFERENTIATION OF hPSCs INTO CMs
Signaling Pathways Involved in
Differentiation of hPSCs Into CMs
The basic principle of the current method of inducing hPSCs
to differentiate into CMs in vitro is to simulate the heart
development in vivo. The same differentiation regulation
has been demonstrated in hESCs and hiPSCs. hPSCs are
differentiated into CMs based on three stages through spatial–
temporal modulation of signaling pathways, such as BMP,
activin-A, WNT, etc. (Filipczyk et al., 2007;Laflamme et al.,
2007;Kattman et al., 2011;Lian et al., 2012;Zhang et al., 2012;
Fonoudi et al., 2015).
BMP-4 commits hPSCs into mesodermal lineage cells alone
or in combination with activin-A. BMP signaling controls the
expressions of GATA4, SRF, and MEF2C transcription factors
(Klaus et al., 2012). Combination of activin-A and BMP4
induces KDR+PDGFRα+cardiogenic mesoderm in hPSCs,
which expressed MESP1 between days 3 and 4 and Nkx2.5
by day 8 of differentiation (Kattman et al., 2011). Combining
activin-A and BMP-4 with Matrigel-generated high purity (up to
98%) and yield (up to 11 CMs/input PSC) of CMs from hPSCs
(Zhang et al., 2012).
WNT plays a bidirectional role in differentiation, depending
on the time point of differentiation. At stage 1, both classical
activation, which suppresses the catenin/GSK3 pathway, and
non-classical signal transduction, which involves the C protein
kinase C/C-Jun N-terminal kinase, have been shown to induce
mesodermal lineage from hPSCs (Cohen et al., 2008). At stage
2, WNT antagonist, such as DKK1 and IWP, directs mesodermal
progenitors to cardiac progenitors (Willems et al., 2011). Nkx2.5,
Isl1, and Baf60c are controlled by WNT/β-catenin signaling
(Klaus et al., 2012).
Although either WNT activation/GSK3 inhibition (Ye et al.,
2013b;Tan et al., 2019;Tao et al., 2020) or BMP4/activin-A
(Laflamme et al., 2007;Hudson et al., 2012;Zhang et al., 2012;Ye
et al., 2013b) has been shown to induce mesodermal lineage from
hPSCs, WNT activator alone, such as CHIR99021, has gained
popularity because of its cheap price and reproducible results
(Lian et al., 2012;Su et al., 2018;Tan et al., 2019;Tao et al., 2020).
Frontiers in Cell and Developmental Biology | www.frontiersin.org 2May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 3
Gao and Pu Review of hPSCs-CVCs
FIGURE 1 | Schematic diagram of the development of heart cells in vivo.(A) Mutual regulation between epiblast and distal visceral endoderm (DVE) through Nodal,
Cerberus, Lefty1, and DRP1 signals leads to a gradient distribution of the concentrations of Nodal and WNT, which results in the formation of primitive streak. During
primitive streak migration, a small number of cells express mesoderm posterior protein 1 (MESP1), marking the beginning of heart development. MESP1+cells finally
differentiate into various cells that form the heart, such as endothelium, smooth muscle, and myocardium. (B) The migration of the primitive streak from posterior to
anterior also marks the beginning of gastrulation, a crucial event in embryonic development. During this period, the embryo becomes a trilaminar embryonic disk,
and the heart develops from the mesoderm.
Three-Dimensional Environment for CM
Differentiation
To mimic in vivo cardiac cell development, hPSCs were cultured
in embryoid bodies (EBs) or spheroids (Itskovitz-Eldor et al.,
2000;Kehat et al., 2001;Xu et al., 2002;He et al., 2003;Zhang
et al., 2009;Kattman et al., 2011;Fonoudi et al., 2015;Kempf
et al., 2015) or in suspended microcarriers (Ting et al., 2014) to
differentiate into CMs. Differentiation of hPSCs in EBs results
in three embryonic germ layer formation (Itskovitz-Eldor et al.,
2000). Early studies showed that the cells in EBs generated
spontaneous contraction and contained mixed cell populations
of nodal-, atrial-, and ventricular-like cells, and the efficiency was
quite low (Kehat et al., 2001;He et al., 2003;Zhang et al., 2009).
The size of EBs seems to be a critical factor that affects
differentiation efficiency. Centrifugation (Ng et al., 2005;
Burridge et al., 2007), engineered microwells (Mohr et al., 2006,
2010), and micropatterning (Bauwens et al., 2008) have been
employed to produce more homogenously sized EBs, which
is helpful for maximizing mesoderm formation and cardiac
induction (Bauwens et al., 2008). More recently, spatial–temporal
modulation of WNT signaling and activation of sonic hedgehog
signaling in hPSCs, cultured in stirred suspension bioreactors,
led to the generation of approximately 100% beating EBs
containing highly pure (∼90%) CMs in 10 days (Fonoudi et al.,
2015). Using bioreactors, 4 ×107to 5 ×107CMs can be
generated per differentiation batch at >80% purity in 24 days
(Kempf et al., 2015).
Two-Dimensional Environment for CM
Differentiation
Directed differentiation of hPSCs in monolayer is a more
convenient method as compared with cardiac differentiation in
three-dimensional (3D) environment. Two most efficient and
popular CM differentiation methods are the GiWi small molecule
differentiation protocol by Lian et al. (2012) and the matrix
sandwich method by Zhang et al. (2012) (Figure 2). Lian et al.
(2012) showed that temporal modulation of WNT signaling is
essential and sufficient for efficient cardiac lineage induction in
hPSCs under defined and growth factor–free conditions. WNT
activation at the initial stage of hPSC differentiation enhanced
CM generation, whereas shRNA knockdown of β-catenin during
this stage fully blocked CM specification. Sequential treatment
of hPSCs with GSK3, such as CHIR99021, followed by chemical
inhibitors of WNT (IWP2) signaling in the later stage produced
a high yield (up to 98%) and functional CMs from multiple
hPSC lines. Zhang et al. (2012) demonstrated that extracellular
matrix also plays an important role in hPSC differentiation.
The Matrigel, an extracellular matrix, promotes an epithelial-to-
mesenchymal transition, combined with activin-A, BMP4, and
basic FGF (bFGF) generated high yield (up to 11 CMs/input PSC)
and pure (up to 98%) CMs from hPSCs.
Other factors that affect the differentiation efficiency of
hPSCs have been documented, including cell density, cell culture
matrices, vascular endothelial growth factor 165 (VEGF165),
heparin, and insulin. A complete confluence of hiPSCs is required
Frontiers in Cell and Developmental Biology | www.frontiersin.org 3May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 4
Gao and Pu Review of hPSCs-CVCs
FIGURE 2 | Schematic diagram of differentiation of hPSCs into cardiomyocytes. (A) Schematic diagram of matrix sandwich protocol. Extracellular matrix application
promoted epithelial–mesenchymal transition of human PSCs. (B) Schematic diagram of GiWi small molecule differentiation protocol, which proved that timing
regulation of Wnt signal was critical. (C) Schematic diagram of activin-A/BMP-4/VEGF protocol, which efficiently differentiated cardiomyocytes from both integrated
and non-integrated hiPSCs. (D) Schematic diagram of flexible Wnt signal suppression protocol, which indicated that the differentiation of various cell types can be
flexibly changed.
during the differentiation process and increases the yield of CMs
(Zhang et al., 2015). In addition to Matrigel, other extracellular
matrices, such as recombinant human cadherin, vitronectin,
laminin-521 and laminin-511, fibronectin, and a fibronectin
mimetic, support efficient CM differentiation of hPSCs (Burridge
et al., 2014). A low concentration of VEGF-A at differentiation
stage 2 efficiently differentiated hiPSCs into CMs, especially the
one reprogrammed from blood mononuclear cells (Ye et al.,
2013b). Heparin can act as a WNT modulator to promote CM
production condition (Lin et al., 2017). Insulin can redirect
differentiation from cardiogenic mesoderm and endoderm to
neuroectoderm in differentiating hESCs (Freund et al., 2008).
Xeno-Free and Chemically Defined
Systems for CM Differentiation From
hiPSCs
To be safe for clinical application, a xeno-free and chemically
defined differentiation system is required. E8 medium and
StemMACS iPS-Brew XF medium have been developed as
xeno-free media for maintenance and expansion of hPSCs.
Vitronectin XF and human recombinant laminin are coating
matrix for maintaining hPSC growth and differentiation of
hPSCs into cardiac cells (Hayashi and Furue, 2016;Yap et al.,
2019). Transferrin has been used to replace B27 in a chemically
defined medium for CM differentiation from hiPSCs (Zhang
et al., 2020). hiPSC-CMs derived from transferrin-supplemented
medium have similar transcriptome and the maturation level
compared to those generated in B27 minus insulin medium.
High CM differentiation efficiency using xeno-free and
chemically defied system have been reported (Burridge et al.,
2014;Tan et al., 2018). Burridge et al. (2014) obtained contractile
cell sheets of up to 95% cardiac troponin T (cTNT) CMs
using RPMI 1640 medium supplemented with L-ascorbic acid 2-
phosphate, recombinant human albumin, and small molecules.
Using a bovine serum albumin–free and chemically defined
system, Tan et al. (2018) were able to differentiate hPSCs
into clinical-grade CMs, which generated greater than 80%
cTNT +CMs.
Purification of hPSC-Derived CMs
Although self-beating immature CMs can be obtained through
the above methods, the differentiation efficiency is cell line
dependent. There are still many unknown non-CMs, such
as undifferentiated hPSCs or cell differentiation into other
directions. In order to make the hPSC-CM have therapeutic
value, non-CMs need to be removed. A detailed description of
strategies for purification of hPSC-CM can be found in review by
Ban et al. (2017).
Genetic Modification
Genetic method to enrich CM was first developed (Anderson
et al., 2007;Xu et al., 2008;Kita-Matsuo et al., 2009;
Ma et al., 2011). Cardiac-specific promoter, such as αMHC
promoter, with puromycin or neomycin selection gene, was
introduced into hESCs to generate stable transgenic cell
lines (Xu et al., 2008;Kita-Matsuo et al., 2009). The drug
selected CMs were 96% pure and could be cultured for over
4 months. Anderson et al. developed two genetic selection
systems: (1) negative selection of proliferating cells with the
herpes simplex virus thymidine kinase/ganciclovir gene system
and (2) positive selection of CMs expressing a bicistronic
reporter: αMHC promoter driven green fluorescent protein
(GFP) with puromycin-resistance gene (Anderson et al., 2007).
However, only the puromycin method enriched CMs up to
91.5% purity, which was about 2.7-fold that of the negative
selection method.
Nkx2.5 is expressed in early cardiac mesoderm cells
throughout the left ventricle and atrial chambers during
embryogenesis (Evans et al., 2010). GFP was engineered to
Frontiers in Cell and Developmental Biology | www.frontiersin.org 4May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 5
Gao and Pu Review of hPSCs-CVCs
the Nkx2.5 locus of hESC to facilitate the monitoring CM
differentiation (Elliott et al., 2011). Den Hartogh et al. (2016)
generated a dual fluorescent reporter MESP1 (mCherry)/Nkx2.5
(GFP) line in hPSC. This enabled the visualization of precardiac
MESP1 +mesoderm and their further commitment toward
the cardiac lineage through activation of Nkx2.5 (Den Hartogh
et al., 2016). Jung et al. (2014) used nodal cell inducer TBX3,
coupled with MHC6 promoter–based antibiotic selection, which
can obtain 80% of functional sinus pacemaker cells. Although
genetic modification seems to improve the purity of hPSC-CMs,
it may be more useful for monitoring CM differentiation rather
than for purifying hPSC-CMs.
Cell Surface Markers
Efforts have been made to identify cell surface markers
on CMs. Dubois et al. (2011) screened 370 known CD
antibodies and found that signal-regulatory protein α(SIRPα)
is a marker specifically expressed on hPSC-CMs. Cell sorting
targeting SIRPαcan enrich cardiac precursors and hPSC-
CMs up to 98% purity. In addition, vascular cell (VC)
adhesion molecule 1 has been identified as a cell surface
marker for cTnT expressing CMs from 242 antibodies by
Uosaki et al. (2011).
Lin et al. (2012) developed a protocol to select CPCs based
on cell surface markers during differentiation stages. hPSCs were
cultured in EBs and dissociated. The low-KDR/c-Kit−CPCs were
isolated by fluorescence activated cell sorting. After culture with
VEGF/DKK1, cells were further isolated based on CD166. The
CD166+cells were differentiated into CMs, and CD166−cells
were differentiated into SMCs.
Physical or Chemical Methods
Purification of CMs using a Percoll gradient or metabolic
selection has been established. Early study using EBs for hPSC-
CM differentiation results in cells from three embryonic germ
layers (Itskovitz-Eldor et al., 2000). Percoll density centrifugation
can enrich CMs reaching 70% (Xu et al., 2002). Because of
the special metabolic mode of CMs, a medium containing
lactate without glucose has been used to inhibit the growth
of non-CMs, so that only CMs can survive, which increases
CM purity up to 99% (Tohyama et al., 2013). Fluorescent
molecular beacons targeting the mRNA of MHC6/7 in CMs
have been developed to enrich cTnT+CMs up to 97%
(Ban et al., 2013).
More recently, it has been shown that synergy between
CHIR99021 and concurrent removal of cell–cell contact
can massively expand hiPSC-CMs in vitro (i.e., 100- to
250-fold) (Buikema et al., 2020). The lymphoid enhancer
binding factor/T-cell–specific transcription factor activity
and AKT phosphorylation are underlying mechanisms
for a synergistic effect. The differentiated hPSC-CMs are
often a mixture of several CM subtypes, such as atrial-,
ventricular-, and pacemaker-like CMs, which cannot meet
the requirements of precision medicine. A comprehensive
description of how chamber-specific CMs are produced
during development and how atrial-, ventricular-, and
pacemaker-like CMs are induced in vitro, can be found in
review of Zhao et al. (2020).
DIFFERENTIATION OF hPSCs INTO ECs
Endothelial cells are a thin layer of specialized cells that
directly contact with the blood flow, the circulatory system,
and blood throughout the body. Therefore, the function of ECs
involves multiple fields of vascular biology, such as nxutrient
exchange, immune cell adhesion and migration, and intercellular
communication (Lerman and Zeiher, 2005). If ECs are damaged
or dysfunctional, it is easy to cause atherosclerosis and other
common cardiovascular diseases (Lerman and Zeiher, 2005).
Embryonic Origins of ECs
The development of blood vessels in the embryo is slightly
different from CMs. The initial embryonic blood vessels come
from the extraembryonic mesoderm of the yolk sac (Goldie
et al., 2008). The progenitor cells differentiate to form a solid
cell mass called “blood island,” which will fuse to form a
primitive network of tubules known as a vascular plexus. The
outer layer of cells gradually becomes flattened to become
the most primitive ECs, whereas the inner cells form the
primitive hematopoietic stem cells (Goldie et al., 2008). These
differentiated blood islands continue to fuse to form the
vascular plexus, which is further remodeled to form arteries
or veins. In addition, the endothelium of cardiac coronary
arteries originates from the sinus venosus through VEGF-C–
stimulated angiogenesis during the development of the heart
(Chen et al., 2014). The coronary endothelium of interventricular
septum is differentiated from the endocardium progenitor cells
(Harris and Black, 2010).
Signaling Pathways in EC Differentiation
hPSCs need to be differentiated into mesodermal progenitor
cells by regulating WNT signaling pathway followed by
commitment to endothelial lineage principally by VEGF
signaling (Su et al., 2018;Wang K. et al., 2020). VEGF
is a key growth factor in EC differentiation from hPSCs
(Olsson et al., 2006;Nourse et al., 2010). VEGF/VEGF
receptor (VEGFR) signaling promotes vascular endothelial
differentiation by up-regulating ETV2 expression (Liu et al.,
2015). Synergistically using BMP4, FGF2, and VEGF up-
regulate the mitogen-activated protein kinase (MAPK) and
PI3K pathways to induce early vascular progenitors from
hiPSC-derived mesodermal progenitors through regulation of
the ETS family transcription factors, ETV2, ERG, and FLI1
(Harding et al., 2017).
ETV2 is a dispensable regulator for vascular EC development.
It is expressed in hematopoietic and endothelial progenitors
in the yolk sac (Koyano-Nakagawa et al., 2012). ETV2
acts downstream of BMP, Notch, and WNT signaling to
regulate blood and vessel progenitor specification. Chromatin
immunoprecipitation assay by Liu et al. (2015) showed
that ETV2 can bind not only to promoters or enhancers
of Flk1 and Cdh5, but also to other genes that perform
Frontiers in Cell and Developmental Biology | www.frontiersin.org 5May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 6
Gao and Pu Review of hPSCs-CVCs
critical roles in vascular endothelial or hematopoietic
cells, including GATA2, Meis1, Dll4, Notch1, Nrp1/2, Flt4,
Fli1, RhoJ, and MAPK.
3D Environment for EC Differentiation
Similar to CM, 3D and two-dimensional (2D) culture
systems have been applied to differentiate hPSCs into ECs
(Levenberg et al., 2002;Li et al., 2011;Adams et al., 2013;
Sahara et al., 2014;Zhang et al., 2014, 2017;Patsch et al., 2015;
Sivarapatna et al., 2015;Gil et al., 2016;Liu et al., 2016;Harding
et al., 2017;Su et al., 2018;Wang K. et al., 2020). 3D system
includes EB formation or patch-mediated EC differentiation
(Levenberg et al., 2002;Li et al., 2011;Adams et al., 2013;Zhang
et al., 2014;Sivarapatna et al., 2015;Su et al., 2018).
Early studies cultured hESCs in EBs to facilitate formation
of the three embryonic germ layers and purified differentiated
ECs by cell sorting based on CD31 (Levenberg et al., 2002;
Li et al., 2011;Adams et al., 2013;Gil et al., 2016). ECs
can be differentiated from EBs of hESCs under hemangioblast
differentiation conditions in two stages (Gil et al., 2016). EBs were
cultured with BMP4 for 2 days and dissociated into single cells
and cultured with BMP4, VEGF, stem cell factor, thrombopoietin,
Flt-3 ligand, and bFGF for another 2 days to obtain ECs.
Approximately 37% of hESCs differentiated into ECs as assessed
by flow cytometry.
Adams et al. (2013) differentiated hiPSCs in EBs to get
ECs. Although only 18% of cells differentiated into ECs, which
have biological function to react to proinflammatory factors,
such as interleukin 1β(IL-1β), tumor necrosis factor α, and
lipopolysaccharide.
To monitor EC differentiation, an hESC cell line was
engineered with VE-cadherin promoter-driven GFP EBs (Sahara
et al., 2015). BMP4 and a GSK3βinhibitor were applied in
an early phase and followed by treatment with VEGF-A and
inhibition of the Notch signaling pathway in a later phase for
EC differentiation in EBs (Sahara et al., 2015). This resulted in
differentiation efficiency up to 50% within 6 days.
Recently, ECs were more efficiently generated from EBs
based on the modulation of signaling pathways involved in
mesodermal progenitor cells in the early stage and endothelial
specification at a later stage (Sivarapatna et al., 2015). Human EBs
were first differentiated into mesoderm using BMP-4 followed
by dissociation and cultured as monolayer and further treated
with VEGF to specify EC fate (Sivarapatna et al., 2015). The
differentiation protocol improved EC differentiation efficiency by
greater than 50%.
It was found that 3D environment promoted hiPSC
differentiation into ECs when hiPSCs were seeded into
thrombin–fibrinogen patch (Zhang et al., 2014). 3D environment
enhanced EC differentiation through up-regulation of p38MAPK
and extracellular signal–regulated kinase 1/2 (ERK1/2) signaling
pathways (Su et al., 2018). Synergistically using CHIR99021
with U-46619, a prostaglandin H2 analog that activates ERK1/2
and p38MAPK signaling, not only more efficiently induces
mesodermal progenitors in early stage, but also enhances ETV2
transcription factor expression at later stage, which leads to >85%
hiPSCs converted to EC fate (Su et al., 2018).
2D Environment for EC Differentiation
With the understanding of signaling pathways required
in EC differentiation from hPSCs, 2D monolayer for EC
differentiation gains popular. hPSCs are differentiated into
intermediate mesodermal progenitor cells at early stage followed
by commitment to endothelial specification at later stage by
VEGF (Patsch et al., 2015;Sahara et al., 2015;Sivarapatna et al.,
2015;Gil et al., 2016;Zhang et al., 2017;Su et al., 2018;Rosa et al.,
2019;Wang K. et al., 2020) (Figure 3).
Although a combination of BMP4 and bFGF commits hPSCs
into mesodermal lineage (Ikuno et al., 2017;Rosa et al., 2019),
synergically using GSK3 inhibitor with BMP4 and/or activin-
A has been shown to more efficiently and rapidly commit
hPSCs to a mesodermal fate, and subsequent exposure to
VEGF-A resulted in efficient differentiation of hPSCs into ECs
(Patsch et al., 2015;Sahara et al., 2015;Zhang et al., 2017).
CHIR99021 alone has been used for induction of mesoderm
at stage 1 of EC differentiation (Liu et al., 2016). At a later
stage, VEGF alone or combined with other factors induces
EC differentiation from mesodermal progenitors (Patsch et al.,
2015). Combining cyclic adenosine monophosphate has been
shown to efficiently induced EC differentiation as it increases
the expression of VEGFR2 and another VEGFR, neuropilin1,
through protein kinase A activation (Yamamizu et al., 2009;
Ikuno et al., 2017). Synergistically using FGF2, VEGF, and
BMP4 efficiently induced vascular progenitors from hiPSC-
derived mesodermal progenitors through regulation of the
ETS family transcription factors, ETV2, ERG, and FLI1 via
MAPK and PI3k signaling (Harding et al., 2017). Combining
VEGF with inhibitor of Notch signaling pathway in the
second stage and converted >50% hPSCs to ECs in 6 days
(Sahara et al., 2015).
In addition to small molecules and growth factors, genetic
modification has been applied to enhance EC differentiation.
Wang K. et al. (2020) transfected mesodermal progenitors
with modified mRNA encoding ETV2, a master transcription
factor in EC development. This efficiently converted mesodermal
progenitors into ECs rapidly and robustly. The implementation
of exogenous ETV2 may overcome the issues of inefficient
activation of ETV2 during EC differentiation and hiPSCs
reprogrammed from various somatic cells.
Specification of Arterial, Venous, and
Lymphatic Endothelial Cells
hPSC-derived ECs are heterogeneous (Rufaihah et al., 2013).
They displayed arterial, venous, and, to a lesser degree, lymphatic
lineage markers (Rufaihah et al., 2013). The traditional ECs
isolated were based on CD31 and/or VE-cadherin, which cannot
discern between EC subtypes. Therefore, it is necessary to develop
methods to derive or purify iPSC-EC–specific subtypes. Several
studies developed protocols to derive more homogenous hPSC-
EC subtypes (Rufaihah et al., 2013;Sivarapatna et al., 2015;Zhang
et al., 2017;Rosa et al., 2019).
VEGF concentration has been shown to affect differentiated
EC subtypes. Rosa et al. (2019) demonstrated that modulation of
VEGF concentration (10 vs. 50 ng/mL) can direct mesodermal
Frontiers in Cell and Developmental Biology | www.frontiersin.org 6May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 7
Gao and Pu Review of hPSCs-CVCs
FIGURE 3 | Schematic diagram of differentiation of hPSCs into endothelial cells. (A) Schematic diagram of chemically defined protocol. Cells were differentiated to
mesoderm by GSK3 inhibition or BMP4 treatment and treated with VEGF to induce ECs. (B,C) Schematic diagram of two-stage treated EB protocols. (D,E)
Schematic diagram of single-cell and EB protocols, respectively, and both methods applied a two-stage cytokine treatment procedure.
progenitor cell into venous-like versus arterial-like ECs in a
chemically defined and serum-free condition. Rufaihah et al.
(2013) confirmed this and further showed that hiPSC-derived
ECs are mainly arterial subtype in the presence of high
concentrations of VEGF-A (50 ng/mL) and 8-bromoadenosine-
30:50-cyclic monophosphate (0.5 mmol/L), as they expressed
higher levels of ephrin B2, whereas lower concentrations
of VEGF-A favored venous subtype and combination of
VEGF-C, and angiopoietin-1 promoted the expression of
lymphatic phenotype.
Biomimetic flow bioreactors have been employed to facilitate
the induction of arterial ECs (Sivarapatna et al., 2015). hiPSC-
ECs were purified by CD31+magnetic beads and cultured
on bioreactor membrane and ensembled into bioreactors.
Flow generated shear stress on hiPSC-ECs, which induced the
expressed arterial EC markers: ephrin B2, CXCR4, connexin40,
and Notch-1. Zhang et al. (2017) demonstrated that combination
of FGF2, VEGFA, SB431542, RESV, and L690 in the absence
of insulin greatly improved arterial EC differentiation, whereas
venous-like ECs were derived by treating cell with VEGF-A and
BMP4 only. The arterial ECs expressed arterial genes, such as
CXCR4, DLL4, Notch4, ephrin B2.
There are limited studies on lymphatic endothelial lineage
differentiation from hPSCs. Rufaihah et al. (2013) showed
that combination of VEGF-C and angiopoietin-1 promoted the
expression of lymphatic phenotype. Lee et al. (2015) compared
three different culture conditions: spontaneous differentiation
through EB formation, coculture with OP9 cells, and a feeder-
free culture with gelatin, and found that the coculture system
most effectively induced lymphatic endothelial differentiation of
hPSCs. Lymphatic ECs expressed key markers, including PROX1,
LYVE1,VEGFR3, and PODOPLANIN. These cells promoted
wound healing through lymphatic neovascularization. More
recently, it was shown that low-dose (<1 ng/mL) BMP9 promotes
early lymphatic-specified ECs (Subileau et al., 2019).
DIFFERENTIATION OF hPSCs INTO
SMCs
The sources of vascular smooth muscle in the embryonic
development process are multiple lineages (Majesky, 2007;Sinha
et al., 2014), such as neural crest (Jiang et al., 2000), secondary
heart field (Waldo et al., 2005), proepicardial organ, lateral
plate mesoderm (Mikawa and Gourdie, 1996), and the paraxial
mesoderm (Wasteson et al., 2008). Detailed description of the
embryonic origins of human vascular SMCs can be found in
reviews by Majesky (2007) and Sinha et al. (2014).
Protocol-directed hiPSC-SMC differentiation is quite different
in 3D (Xie et al., 2007;Ge et al., 2012;Kinnear et al., 2013;
Wang et al., 2014;Kinnear et al., 2020) and 2D (Huang et al.,
2006;Patsch et al., 2015;Yang et al., 2016) culture systems. Cells
derived from the outgrowth of human EBs cultured in SMC
differentiation condition, which was only composed of Dulbecco
modified eagle medium (DMEM) +5% fetal bovine serum (FBS)
and a gelatin-coated surface, produced SMCs expressing smooth
muscle MHC (SMMHC) and α-smooth muscle actin (α-SMA)
(Xie et al., 2007). Surprisingly, when outgrowing cells were
cultured in growth condition, which was composed of smooth
muscle growth medium and Matrigel-coated surface, <10% of
cells expressed SMMHC and α-SMA.
Ge et al. (2012) cultured hiPSC in EBs for 6 days in
differentiating medium, which was composed of DMEM medium
containing 10% FBS, 1% non-essential amino acids, 0.1 mM
mercaptoacids, and 1% L-glutamine. Then, EBs were cultured on
0.1% gelatin–coated surface with fresh differentiation medium
Frontiers in Cell and Developmental Biology | www.frontiersin.org 7May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 8
Gao and Pu Review of hPSCs-CVCs
for another 6 days. Furthermore, cells were dissociated and
transferred to Matrigel–coated plates in SmGM-2 media for
1 week. At last, cells were passaged and cultured on 0.1% gelatin–
coated culture dishes and cultured with 5% FBS differentiation
medium for at least 5 days to complete differentiation. This
produced highly homogenous SMC-like cells (around 96%)
(Ge et al., 2012). This protocol has been used to differentiate
elastin mutant hiPSC into SMCs to model elastin insufficiency
phenotype in SMCs (Kinnear et al., 2020) and Williams–Beuren
syndrome in vitro (Kinnear et al., 2013). Furthermore, the same
protocol has efficiently induced SMCs used for manufacturing
of macroporous and nanofibrous poly(L-lactic acid) scaffold
(Wang et al., 2014). These studies suggest that human EB
mediated SMC differentiation from hPSCs is highly efficient
in the condition of DMEM supplemented with FBS and using
gelatin as extracellular matrix.
In addition to the differentiation through induced EB,
monolayer culture differentiation system has been explored
(Figure 4). Huang et al. used 10 µM all-trans retinoid acid to
differentiate hESCs in monolayers. It was shown that >93%
of the cells expressed SMC-marker genes, such as SMMHC and
α-SMA, and proteins and were able to contract (Huang et al.,
2006). Combining GSK3 inhibition and BMP4 to commit hPSCs
to mesodermal cells followed by platelet-derived growth factor
two B subunits (PDGF-BB), SMCs can be generated >80%
efficiency within 6 days (Patsch et al., 2015).
Yang et al. (2016) used iPSCs and ESCs from different
sources to obtain contractile and synthetic SMC by monolayer
cell culture. CHIR99021 and BMP4 were used to induce
mesodermal lineage from hPSCs followed by VEGF-A and
TGF-βtreatment to induce vascular progenitors. Differentiation
medium was switched to PDGF-βand TGF-βeither on day 7
or 10 to induce contractile or synthetic SMCs. Contractile SMCs
expressed higher levels of MHC11 and calponin and had stronger
contraction activity, whereas synthetic SMCs expressed more
collagen and had stronger proliferation activity. Both protocols
converted ∼45% of hPSCs to SMC phenotypes, and the purity
could be increased to ∼95% in 4 mM lactate acid in RPMI1640
metabolic medium.
It seems that EB-mediated SMC differentiation can reach
similar differentiation efficiency as monolayer differentiation by
modulating WNT, PDGF-β, and TGF-βpathways. They may
represent SMCs with different lineage background (Majesky,
2007;Sinha et al., 2014). Thus, it is necessary to identify signature
markers in SMCs from different lineage and define the lineage
specification of SMCs differentiated from protocols. This helps
to apply hPSC-SMCs in understanding vascular development in
embryo and use disease-specific hPSC-SMCs in disease modeling
and drug screening.
DIFFERENTIATION OF hPSCs INTO
CPCs
Recently, induction of CPCs is gaining attention as they are
able to self-renew and predetermined to differentiate into cardiac
lineage cells in vitro and in vivo. This saves time and is
cost-effective as compared to derive CMs, ECs, and SMCs in vitro
and further transplantation in vivo. Various CPC markers, such
as stage-specific embryonic antigen 1 (SSEA-1) (Bellamy et al.,
2015), MESP1 (Bondue et al., 2008), and Nkx2.5 (Birket et al.,
2015), have been investigated (Figure 5).
hPSCs treated with BMP2 give rise to an early population
of cardiovascular progenitors, characterized by SSEA-1 (Brade
et al., 2013;Bellamy et al., 2015). This progenitor population was
multipotential and able to generate CMs, SMCs, and ECs in vitro.
When purified SSEA-1+progenitors implanted into non-human
primates (NHPs), they differentiated into ventricular CMs.
However, non-purified SSEA-1+progenitor cell implantation
resulted in teratomas in the scar tissue.
Cardiogenic mesodermal cells (CMCs) expressing MESP1
have been shown to differentiate into almost all cardiac cell types
both in vitro and in vivo (Bondue et al., 2008;Brade et al., 2013;
Den Hartogh et al., 2015;Lescroart et al., 2018). To monitor
early cardiac mesoderm in hPSCs, a dual MESP1 (mCherry/w)–
NKX2-5 (eGFP/w) reporter line was developed in hESCs (Den
Hartogh et al., 2015). Induction of cardiac differentiation in this
reporter line resulted in transient expression of MESP1-mCherry,
followed by expression of NKX2.5-eGFP. MESP1-mCherry cells
showed increased expression of mesodermal markers. Whole-
genome microarray profiling and fluorescence-activated cell
sorting analysis of MESP1-mCherry cells showed enrichment for
mesodermal progenitor cell surface markers, such as PDGFRα,
CD13, and ROR-2. MESP1-mCherry derivatives contained an
enriched percentage of Nkx2.5-eGFP and CMs, SMCs, and ECs.
Vahdat et al. (2019a; 2019b) established a protocol for
maintenance and large-scale expansion of early CPCs, so-called
CMCs in a defined culture system. Through chemical screening,
they developed a medium containing three factors, A83-01, bFGF,
and CHIR99021, which generated CMCs expressing cardiac
mesoderm markers and cardiac-specific transcription factors
MESP1, SSEA1, ISL1, PDGFRα, NKX2.5, and MEF2c; 1014 CMCs
were generated after 10 passages and were able to differentiate
into CMs, ECs, and SMCs in vitro. To monitor CPC derivation,
selection, and maintenance, Birket et al. (2015) engineered
hPSCs to carry a cardiac lineage reporter to enable robust
expansion of MYC expression primitive pre–NKX2.5+CPCs.
Through regulation of FGF and BMP signaling, NKX2.5+CPCs
can be differentiated into ventricular-like cells, pacemaker-like
cells, ECs, and SMCs. Yap et al. (2019) developed a chemically
defined, xeno-free, laminin-based differentiation protocol to
generate CPCs from hESCs. Laminin-221, an abundant laminin
isoform in heart extracellular matrix, induced a transcriptomic
signature with up-regulated markers for cardiac development.
CPCs appeared on day 9 or 11 of differentiation and highly
expressed ISL1,TBX5,MEF2C,C-KIT, and GATA3. Single-
cell RNA sequencing of CPCs identified three main progenitor
subpopulations, including CMs, SMCs, and small population
of epithelial cells. The CPCs generated human heart muscle
bundles in mouse heart post–ischemia/reperfusion (I/R) injury.
Uosaki et al. (2012) showed that coaggregation of endodermal
cell line End2 with hESCs significantly promoted the induction
of KDR+PDGFRα+CPCs, suggesting a direct contact with
endoderm-like cells can induce cardiac progenitors from hPSCs.
Frontiers in Cell and Developmental Biology | www.frontiersin.org 8May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 9
Gao and Pu Review of hPSCs-CVCs
FIGURE 4 | Schematic diagram of differentiation of hPSCs into smooth muscle cells. (A,B) Schematic diagram of chemically defined protocols. GSK3 inhibition or
BMP4 treatment followed by activin-A and PDGF-BB treatment induced VSMCs from hPSCs, and subsequent applications of PDGF-BB or heparin with activin-A
obtained synthetic or contractile VSMCs, respectively. (C) Schematic diagram of EB protocol for differentiation of VSMCs from hPSCs. (D,E) Schematic diagram of
chemically defined protocols that efficiently induced hPSCs to differentiate into VSMCs with different phenotypes. Both methods first used GSK3 inhibition and
BMP4 to stimulate differentiation into mesoderm cells and then treated with VEGF-A and FGFβ. Synthetic VSMCs (D) were produced by culturing the cells with
VEGF-A and FGFβand with PDGF-βand TGF-βin order. Contractile VSMCs (E) were induced by culturing the cells with PDGF-βand TGF-βdirectly.
FIGURE 5 | Schematic diagram of differentiation of hPSCs into cardiac progenitor cells. (A) MESP1+cells were obtained after treatment of GSK3 inhibition and then
were cultured in four different conditions: (a) suspension culture of spheroids, (b) adherent culture of spheroids on gelatin, (c) adherent culture of single cells on
gelatin, and (d) adherent culture of single cells on Matrigel. (B) Isoxazole (ISX-9), a cardiogenic small molecule, induced CPCs from hPSCs, which further
differentiated into three cardiac lineages in vitro.
Bylund et al. (2017) found that BMP antagonist GREMLIN
2 is linked to cardiogenesis. Inhibition of canonical BMP
signaling followed by JNK pathway activation by GREM2
induced cardiac differentiation of hiPSCs. Furthermore, GREM2
promoted proliferation of CPCs.
Other factors have been shown to be effective in deriving
CPCs. Xuan et al. (2018) demonstrated that hiPSCs treated
with isoxazole 9 (ISX-9), a potent inducer of adult neural
stem cell differentiation, for 3 days stimulated hiPSCs to
become CPCs expressing NKX2.5, GATA4, ISL1, and MEF2C
and were able to generate CMs, SMCs, and ECs in vitro
and in vivo. ISX-9 activated multiple pathways including
TGF-β–induced epithelial–mesenchymal transition signaling and
canonical and non-canonical WNT signaling at different stages of
cardiac differentiation. Cyclosporin-A, an immunosuppression
drug, has been shown to stimulate differentiation of FLK1+
mesodermal cells into FLK1+/CXCR4+/VE-cadherin−CPCs
and CMs (Fujiwara et al., 2011). The beating colonies from
hiPSCs were increased approximately 4.3 times by addition of
cyclosporin-A at mesoderm stage.
One feature associated with hPSC-derived CPCs is their great
extracellular vesicle (EV) secretory profile (El Harane et al.,
2018). EVs are rich in miRNAs, and most of the 16 highly
abundant, evolutionarily conserved miRNAs are associated with
tissue-repair pathways. In vitro, EV increased cell survival, cell
proliferation, and EC migration and stimulated tube formation.
In vivo, EV significantly improved cardiac function through
decreased LV volume and increased LV ejection fraction.
Although CPCs are emerging as a better option as compared
to CM transplantation, several issues need to be solved before
it can be fully translated into clinic: (1) Purified versus non-
purified. Non-purified CPCs have the risk to form teratoma
after implantation. It is possible that early CPC population may
contain pacemaker cells to behave as foci of automaticity and
Frontiers in Cell and Developmental Biology | www.frontiersin.org 9May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 10
Gao and Pu Review of hPSCs-CVCs
cause arrhythmias. Thus, purified cell population is preferred. (2)
Purification method. Except for SSEA1, MESP1, and NKX2.5 are
intracellular markers; if we use genetically modified CPC based
on MESP1 or Nkx2.5 expression, there is a concern of safety
issue. (3) Electrical coupling: CPCs are not CMs. Although they
will develop into CM in heart eventually after implantation, the
early developed CMs may cause electrical uncoupling leading to
ventricular arrhythmia.
CARDIOMYOPLASTY IN LARGE ANIMAL
MODELS USING hPSC-DERIVED
CARDIOVASCULAR CELLS
The continuous improvement of differentiation efficiency of
hPSCs has made large quantities of human CPC, CM, EC, and
SMC reality. They are being tested as cell transfer therapy for
cardiac repair not only in small, but also in large animal heart
models of heart diseases (Kawamura et al., 2012, 2013;Xiong
et al., 2012;Chong et al., 2014;Ye et al., 2014;Shiba et al., 2016;
Gao et al., 2018;Ishigami et al., 2018;Zhu W. et al., 2018;Ishida
et al., 2019;Romagnuolo et al., 2019) (Table 1).
Two large animal models, pig and NHP, have been used as
preclinical models to investigate feasibility, efficacy, and safety
of hiPSC-derived cardiac cells. Among them, more studies used
pigs as the pig’s heart is very similar to human’s in terms of
morphology, size, electrophysiology, and metabolic physiology
(Lelovas et al., 2014). Comparatively, relatively fewer studies
with NHPs have been reported. Although NHPs are more
closely resembled to human anatomy, physiology, function,
and metabolism (Cox et al., 2017) and are more valuable
from an experimental perspective, they are not cost-effective
and are associated with ethical issues (Zhu K. et al., 2018;
Cong et al., 2019).
Transplantation of hPSC-CMs Only
Cardiac cells differentiated from hiPSCs have been either
directly intramyocardially injected or applied epicardially using
cells sheets or patches. In many studies, transplantation
of hPSC-CMs into cardiovascular disease model animals
could improve heart function and reduce the ventricular
remodeling. Kawamura et al. (2012) generated hiPSC-CM sheets
using 6-cm thermoresponsive dishes. The cell sheets were
approximately 30- to 50 µm thick. Eight hiPSC-CM sheets were
implanted through median sternotomy with chronic MI.
The transplanted hiPSC-CM sheets attenuated LV remodeling
and increased neovascularization without teratoma formation.
To enhance survival of hiPSC-CMs posttransplantation, the
same group implanted hiPSC-CM sheets with an omentum
to enhance blood supply to cell sheets (Kawamura et al.,
2013). Histology showed the transplanted tissues contained
abundant cTnT+cells surrounded by vascular-rich structures.
In addition, it has been found that the transplantation
of hESC-CMs can promote remuscularization to a certain
extent in both pig and NHP models (Chong et al., 2014;
Romagnuolo et al., 2019).
Transplantation of Multilineage Cardiac
Cells
In addition of CMs, VCs, including ECs and SMCs, differentiated
from hESCs (hESC-VCs) or hiPSCs (hiPSC-VCs), have been
investigated. Implantation of VCs promoted survival of ischemic
cells, angiogenesis, and antiapoptotic effect through paracrine
factors released. Implantation of hESC-VCs or hiPSC-VCs
seeded in fibrin/thrombin patch alleviated LV contractile
dysfunction and wall stress and improved myocardial energetics
(Xiong et al., 2012) and attenuated the reduction of ATP
utilization at infarct border zone (Xiong et al., 2013) in porcine
heart model of I/R.
On this basis, researchers began to combine these different
cells and transplant them into animal models together. Ye et al.
(2014) implanted trilineage cardiac cells, including CMs, ECs,
and SMCs, derived from hiPSCs in combination with a fibrin
patch loaded with insulin growth factor into porcine heart post–
acute I/R. The transplantation of trilineage cardiac cells makes the
efficacy of cell therapy more comprehensive and effective: hiPSC-
CMs regenerated CM, whereas hiPSC-VC improved donor and
host CM viability and stimulated neovascularization. Molecular
factors having antiapoptotic (angiogenin, angiopoietin, IL-6,
matrix metalloproteinase-1, PDGF-BB, TIMP Metallopeptidase
Inhibitor 1, urokinase receptor, and VEGF), promoting cell
homing (IL-8, monocyte chemoattractant protein-1, Monocyte
chemoattractant protein-3, matrix metalloproteinase-9), and
inducing cell division (angiogenin, angiopoietin, PDGF-BB,
VEGF) properties were identified in paracrine factors released by
hiPSC-CMs and hiPSC-VCs.
Gao et al. (2018) manufactured human cardiac muscle patch
(hCMP) using 4 million hiPSC-CMs, 2 million each of hiPSC-
ECs and hiPSC-SMCs. They implanted two hCMPs into pig heart
model of acute MI. The hCMP transplantation was associated
with significant improvements in LV function; reduced cardiac
apoptosis, infarct size, and myocardial wall stress; and reversed
some MI-associated changes in sarcomeric regulatory protein
phosphorylation. Ishigami et al. (2018) generated cardiac tissue
sheets using simultaneously induced hiPSC-CMs and hiPSC-VCs
in temperature-responsive culture dishes. They transplanted four
cardiac tissue sheets on the epicardium of infarcted myocardium
in a porcine model of chronic MI. Transplantation resulted in
significant increases of circumference strain and capillary density
and reduction of fibrotic tissue in infarct and border regions
after transplantation.
Limitations and Improvements in
Transplantation Treatment of Animal
Models
Histological analysis revealed that only a few implanted hiPSC-
CMs survived at week 8 after implantation. Thus, the improved
cardiac function was achieved mainly through the paracrine
factors instead of regeneration of CMs (Kawamura et al.,
2012). Therefore, the recovery of heart function involves
many aspects, and it is necessary to use multilineage cell
transplantation to improve blood vessel supply, inflammation
regulation, and metabolism.
Frontiers in Cell and Developmental Biology | www.frontiersin.org 10 May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 11
Gao and Pu Review of hPSCs-CVCs
TABLE 1 | Cellular cardiomyoplasty in large animal models using hPSC-derived cardiovascular cells.
Animal model Cell type Cell number Teratoma Arrhythmia
Pig model of acute
ischemia/reperfusion
hiPSC-CMs
hiPSC-ECs
hiPSC-SMCs
2.0 ×106
2.0 ×106
2.0 ×106
N.A. Not detected Ye et al., 2014
Pig model of acute
ischemia/reperfusion
hiPSC-CMs
hiPSC-ECs
hiPSC-SMCs
4.0 ×106
2.0 ×106
2.0 ×106
N.A. Not detected Gao et al., 2018
Pig model of acute
ischemia/reperfusion
hESC-ECs
hESC-SMCs
2.0 ×106
2.0 ×106
N.A. During surgery Xiong et al., 2012
Pig model of
chronic ischemia
hiPSC-CMs 2.5 ×107Not detected N.A. Kawamura et al., 2012
Pig model of chronic
myocardial infarction
hiPSC-CMs
hiPSC-ECs
hiPSC-VMCs
1.0 ×107Not detected Not detected Ishigami et al., 2018
Pig model of chronic
myocardial infarction
hiPSC-CMs 1.0 ×108N.A. N.A. Ishida et al., 2019
Monkey model of
chronic ischemia
hESC-CMs 1.0 ×109Not detected Within 24 h after delivery Chong et al., 2014
Monkey model of
chronic ischemia
hiPSC-CMs 4.0 ×108N.A. Within 4 weeks after
delivery
Shiba et al., 2016
N.A. means that there was no relevant data or information in the article.
To improve cell engraftment and reduce immunogenicity
of allogeneic iPSC-CMs, Kawamura et al. (2016) injected
allogeneic monkey iPSC-CMs into MHC-matched or non-
matched NHPs. The transplantation of allogeneic iPSC-CMs in
MHC-matched NHP had increased cell engraftment with less
immune-cell infiltration. Shiba et al. (2016) injected 4 ×108
major histocompatibility complex (MHC) matched allogeneic
iPSC-CMs into NHPs post–chronic MI. Transplantation of the
iPSC-CMs improved heart contractile function at 4 and 12 weeks
posttransplantation. Although electrical coupling was established
between donor and host CMs as assessed by use of the fluorescent
calcium indicator G-CaMP7.09, the incidence of ventricular
tachycardia was transiently, but significantly, increased when
compared to control animal group. Furthermore, no macroscopic
or microscopic tumor formation was detected. Wang et al. (2019)
determined the efficacy of hESC-derived CPCs in NHPs. They
found that implantation of hESC-CPCs into acutely infarcted
myocardium significantly ameliorated the functional worsening
and scar formation, concomitantly with reduced inflammatory
reactions and CM apoptosis, as well as increased vascularization.
Moreover, hESC-CPCs modulated cardiac macrophages toward a
reparative phenotype in the infarcted hearts.
CHALLENGES OF hPSC TECHNOLOGY
IN THE TREATMENT OF
CARDIOVASCULAR DISEASES
Cell Quality
Although various protocols have been developed to induce hPSCs
into CPCs, CMs, ECs, and SMCs, which have been extensively
evaluated in small and large animal models of heart diseases,
there is a lack of commonly accepted standards to evaluate
and control the quality of hPSC-derived cardiac cells. This
especially applies to hiPSC-derived cardiovascular cells, as the
reprogramming may change the genetic stability, and epigenetic
memory may compromise therapeutic outcome.
Immunogenicity of hPSCs and Their
Derivatives
The second issue is related to the immunogenicity of hPSCs.
It has been shown that hESCs have low expression of MHCI
and complete absence of MHCII antigens and costimulatory
molecules, such as CD80 and CD86 (Li et al., 2004;Wu et al.,
2008). The expression levels of the above molecules in hiPSCs
are almost same as those in hESCs (Suarez-Alvarez et al.,
2010). Thus, hPSCs may possess immune privilege property.
However, increased MHC expression and immunogenicity have
been documented after differentiation (Swijnenburg et al., 2005;
Suarez-Alvarez et al., 2010). Although immunosuppressive drug
regimens can be used to suppress recipients’ immune response
to transplanted allogenic hPSC-derived cells, optimal dose and
combination of different drugs to achieve minimal drug toxicity
are still far from optimization. Universal hESC or hiPSC cell
lines, which have human leukocyte antigen (HLA) class I (HLA-
I) and II (HLA-II) knock-out (Han et al., 2019;Xu et al., 2019;
Wang X. et al., 2020), may be a solution. HLA-I and HLA-II
knockout hiPSCs can generate immunocompatible and ready-to-
use cardiovascular cells.
Defects of hiPSCs Derivatives
The third issue is specifically related to hiPSCs. Although hiPSCs
can differentiate into “personalized” patient-specific cells and
Frontiers in Cell and Developmental Biology | www.frontiersin.org 11 May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 12
Gao and Pu Review of hPSCs-CVCs
tissues to circumvent both immunogenicity barriers, they may
have limited therapeutic potential if they are reprogrammed
from patients with diseases caused by genetic mutations. Again,
derivatives of universal hESCs or hiPSCs will be a good option for
allogeneic transplantation.
Optimal Cell Types and Numbers for
Cardiac Repair or Regeneration
The fourth issue is associated with cell type and cell dosing.
Currently, most studies determined the efficacy of one cell
type, either hPSC-CMs or CPCs, whereas only a few have
compared different stage-specific cardiac cells. Thus, it is
hard to provide unequivocal evidence for the superiority
of one type over the other. In large animal heart models,
transplanted hPSC-CM numbers ranged between 4 ×108and
1×109(Chong et al., 2014;Shiba et al., 2016;Liu et al.,
2018;Romagnuolo et al., 2019). Although from a clinical
perspective, a higher number of hPSC-CMs may be more
beneficial to cardiac function, a mixed cardiac cell population
may be a cost-effective way as compared with pure CM
transplantation. Genetic modification hPSC derivatives with
genes to enhance their reparability may be a cost-effective option
(Tao et al., 2020).
AUTHOR CONTRIBUTIONS
YG and JP conceived the design of the work. YG wrote the
manuscript with support from JP. Both authors contributed to
the article and approved the submitted version.
ACKNOWLEDGMENTS
We apologize to our colleagues whose work could not be cited
due to limitations to the length of this manuscript.
REFERENCES
Adams, W. J., Zhang, Y., Cloutier, J., Kuchimanchi, P., Newton, G., Sehrawat, S.,
et al. (2013). Functional vascular endothelium derived from human induced
pluripotent stem cells. Stem Cell Rep. 1, 105–113.
Anderson, D., Self, T., Mellor, I. R., Goh, G., Hill, S. J., and Denning, C. (2007).
Transgenic enrichment of cardiomyocytes from human embryonic stem cells.
Mol. Ther. 15, 2027–2036. doi: 10.1038/sj.mt.6300303
Assmus, B., Rolf, A., Erbs, S., Elsasser, A., Haberbosch, W., Hambrecht, R., et al.
(2010). Clinical outcome 2 years after intracoronary administration of bone
marrow-derived progenitor cells in acute myocardial infarction. Circ. Heart
Fail. 3, 89–96. doi: 10.1161/circheartfailure.108.843243
Ban, K., Bae, S., and Yoon, Y. S. (2017). Current strategies and challenges for
purification of cardiomyocytes derived from human pluripotent stem cells.
Theranostics 7, 2067–2077. doi: 10.7150/thno.19427
Ban, K., Wile, B., Kim, S., Park, H. J., Byun, J., Cho, K. W., et al. (2013). Purification
of cardiomyocytes from differentiating pluripotent stem cells using molecular
beacons that target cardiomyocyte-specific mRNA. Circulation 128, 1897–1909.
doi: 10.1161/circulationaha.113.004228
Bauwens, C. L., Peerani, R., Niebruegge, S., Woodhouse, K. A., Kumacheva, E.,
Husain, M., et al. (2008). Control of human embryonic stem cell colony and
aggregate size heterogeneity influences differentiation trajectories. Stem Cells
26, 2300–2310. doi: 10.1634/stemcells.2008-0183
Bellamy, V., Vanneaux, V., Bel, A., Nemetalla, H., Emmanuelle Boitard, S., Farouz,
Y., et al. (2015). Long-term functional benefits of human embryonic stem cell-
derived cardiac progenitors embedded into a fibrin scaffold. J. Heart Lung
Transplant. 34, 1198–1207. doi: 10.1016/j.healun.2014.10.008
Birket, M. J., Ribeiro, M. C., Verkerk, A. O., Ward, D., Leitoguinho, A. R.,
den Hartogh, S. C., et al. (2015). Expansion and patterning of cardiovascular
progenitors derived from human pluripotent stem cells. Nat. Biotechnol. 33,
970–979. doi: 10.1038/nbt.3271
Bondue, A., Lapouge, G., Paulissen, C., Semeraro, C., Iacovino, M., Kyba, M.,
et al. (2008). Mesp1 acts as a master regulator of multipotent cardiovascular
progenitor specification. Cell Stem Cell 3, 69–84. doi: 10.1016/j.stem.2008.06.
009
Brade, T., Pane, L. S., Moretti, A., Chien, K. R., and Laugwitz, K. L. (2013).
Embryonic heart progenitors and cardiogenesis. Cold Spring Harb. Perspect.
Med. 3:a013847. doi: 10.1101/cshperspect.a013847
Bruneau, B. G. (2013). Signaling and transcriptional networks in heart
development and regeneration. Cold Spring Harb. Perspect. Biol. 5:a008292.
doi: 10.1101/cshperspect.a008292
Buikema, J. W., Lee, S., Goodyer, W. R., Maas, R. G., Chirikian, O., Li, G., et al.
(2020). Wnt activation and reduced cell-cell contact synergistically induce
massive expansion of functional human iPSC-derived cardiomyocytes. Cell
Stem Cell 27, 50–63.e5.
Burridge, P. W., Anderson, D., Priddle, H., Barbadillo Munoz, M. D., Chamberlain,
S., Allegrucci, C., et al. (2007). Improved human embryonic stem cell embryoid
body homogeneity and cardiomyocyte differentiation from a novel V-96 plate
aggregation system highlights interline variability. Stem Cells 25, 929–938. doi:
10.1634/stemcells.2006-0598
Burridge, P. W., Matsa, E., Shukla, P., Lin, Z. C., Churko, J. M., Ebert, A. D., et al.
(2014). Chemically defined generation of human cardiomyocytes. Nat. Methods
11, 855–860. doi: 10.1038/nmeth.2999
Bylund, J. B., Trinh, L. T., Awgulewitsch, C. P., Paik, D. T., Jetter, C., Jha,
R., et al. (2017). Coordinated proliferation and differentiation of human-
induced pluripotent stem cell-derived cardiac progenitor cells depend on bone
morphogenetic protein signaling regulation by GREMLIN 2. Stem Cells Dev. 26,
678–693. doi: 10.1089/scd.2016.0226
Chen, H. I., Sharma, B., Akerberg, B. N., Numi, H. J., Kivela, R., Saharinen, P.,
et al. (2014). The sinus venosus contributes to coronary vasculature through
VEGFC-stimulated angiogenesis. Development 141, 4500–4512. doi: 10.1242/
dev.113639
Chong, J. J., Yang, X., Don, C. W., Minami, E., Liu, Y. W., Weyers, J. J., et al. (2014).
Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human
primate hearts. Nature 510, 273–277.
Cohen, E. D., Tian, Y., and Morrisey, E. E. (2008). Wnt signaling: an essential
regulator of cardiovascular differentiation, morphogenesis and progenitor self-
renewal. Development 135, 789–798. doi: 10.1242/dev.016865
Cong, X., Zhang, S. M., Ellis, M. W., and Luo, J. (2019). Large animal models for the
clinical application of human induced pluripotent stem cells. Stem Cells Dev. 28,
1288–1298. doi: 10.1089/scd.2019.0136
Cox, L. A., Olivier, M., Spradling-Reeves, K., Karere, G. M., Comuzzie, A. G.,
and VandeBerg, J. L. (2017). Nonhuman primates and translational research-
cardiovascular disease. ILAR J. 58, 235–250. doi: 10.1093/ilar/ilx025
Den Hartogh, S. C., Schreurs, C., Monshouwer-Kloots, J. J., Davis, R. P., Elliott,
D. A., Mummery, C. L., et al. (2015). Dual reporter MESP1 mCherry/w-NKX2-5
eGFP/w hESCs enable studying early human cardiac differentiation. Stem Cells
33, 56–67. doi: 10.1002/stem.1842
Den Hartogh, S. C., Wolstencroft, K., Mummery, C. L., and Passier, R. (2016). A
comprehensive gene expression analysis at sequential stages of in vitro cardiac
differentiation from isolated MESP1-expressing-mesoderm progenitors. Sci.
Rep. 6:19386.
Dubois, N. C., Craft, A. M., Sharma, P., Elliott, D. A., Stanley, E. G., Elefanty, A. G.,
et al. (2011). SIRPA is a specific cell-surface marker for isolating cardiomyocytes
derived from human pluripotent stem cells. Nat. Biotechnol. 29, 1011–1018.
doi: 10.1038/nbt.2005
Frontiers in Cell and Developmental Biology | www.frontiersin.org 12 May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 13
Gao and Pu Review of hPSCs-CVCs
El Harane, N., Kervadec, A., Bellamy, V., Pidial, L., Neametalla, H. J., Perier,
M. C., et al. (2018). Acellular therapeutic approach for heart failure: in vitro
production of extracellular vesicles from human cardiovascular progenitors.
Eur. Heart J. 39, 1835–1847. doi: 10.1093/eurheartj/ehy012
Elliott, D. A., Braam, S. R., Koutsis, K., Ng, E. S., Jenny, R., Lagerqvist, E. L., et al.
(2011). NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors
and cardiomyocytes. Nat. Methods 8, 1037–1040. doi: 10.1038/nmeth.1740
Evans, S. M., Yelon, D., Conlon, F. L., and Kirby, M. L. (2010). Myocardial lineage
development. Circ. Res. 107, 1428–1444. doi: 10.1161/circresaha.110.227405
Filipczyk, A. A., Passier, R., Rochat, A., and Mummery, C. L. (2007). Regulation
of cardiomyocyte differentiation of embryonic stem cells by extracellular
signalling. Cell. Mol. Life Sci. 64, 704–718. doi: 10.1007/s00018-007-6523- 2
Fonoudi, H., Ansari, H., Abbasalizadeh, S., Larijani, M. R., Kiani, S.,
Hashemizadeh, S., et al. (2015). A universal and robust integrated platform for
the scalable production of human cardiomyocytes from pluripotent stem cells.
Stem Cells Transl. Med. 4, 1482–1494. doi: 10.5966/sctm.2014-0275
Freund, C., Ward-van Oostwaard, D., Monshouwer-Kloots, J., van den Brink, S.,
van Rooijen, M., Xu, X., et al. (2008). Insulin redirects differentiation from
cardiogenic mesoderm and endoderm to neuroectoderm in differentiating
human embryonic stem cells. Stem Cells 26, 724–733. doi: 10.1634/stemcells.
2007-0617
Fujiwara, M., Yan, P., Otsuji, T. G., Narazaki, G., Uosaki, H., Fukushima, H.,
et al. (2011). Induction and enhancement of cardiac cell differentiation from
mouse and human induced pluripotent stem cells with cyclosporin-A. PLoS One
6:e16734. doi: 10.1371/journal.pone.0016734
Gao, L., Gregorich, Z. R., Zhu, W., Mattapally, S., Oduk, Y., Lou, X., et al. (2018).
Large cardiac muscle patches engineered from human induced-pluripotent
stem cell-derived cardiac cells improve recovery from myocardial infarction in
swine. Circulation 137, 1712–1730. doi: 10.1161/circulationaha.117.030785
Ge, X., Ren, Y., Bartulos, O., Lee, M. Y., Yue, Z., Kim, K. Y., et al. (2012). Modeling
supravalvular aortic stenosis syndrome with human induced pluripotent stem
cells. Circulation 126, 1695–1704. doi: 10.1161/circulationaha.112.116996
Gil, C. H., Ki, B. S., Seo, J., Choi, J. J., Kim, H., Kim, I. G., et al. (2016). Directing
human embryonic stem cells towards functional endothelial cells easily and
without purification. Tissue Eng. Regen.Med. 13, 274–283. doi: 10.1007/s13770-
016-9076- 3
Goldie, L. C., Nix, M. K., and Hirschi, K. K. (2008). Embryonic vasculogenesis
and hematopoietic specification. Organogenesis 4, 257–263. doi: 10.4161/org.
4.4.7416
Han, X., Wang, M., Duan, S., Franco, P. J., Kenty, J. H., Hedrick, P., et al. (2019).
Generation of hypoimmunogenic human pluripotent stem cells. Proc. Natl.
Acad. Sci. U.S.A. 116, 10441–10446.
Harding, A., Cortez-Toledo, E., Magner, N. L., Beegle, J. R., Coleal-Bergum, D. P.,
Hao, D., et al. (2017). Highly efficient differentiation of endothelial cells from
pluripotent stem cells requires the MAPK and the PI3K pathways. Stem Cells
35, 909–919. doi: 10.1002/stem.2577
Harris, I. S., and Black, B. L. (2010). Development of the endocardium. Pediatr.
Cardiol. 31, 391–399. doi: 10.1007/s00246-010-9642-8
Hayashi, Y., and Furue, M. K. (2016). Biological effects of culture substrates on
human pluripotent stem cells. Stem Cells Int. 2016:5380560.
He, J. Q., Ma, Y., Lee, Y., Thomson, J. A., and Kamp, T. J. (2003). Human
embryonic stem cells develop into multiple types of cardiac myocytes: action
potential characterization. Circ. Res. 93, 32–39. doi: 10.1161/01.res.0000080317.
92718.99
Huang, H., Zhao, X., Chen, L., Xu, C., Yao, X., Lu, Y., et al. (2006). Differentiation
of human embryonic stem cells into smooth muscle cells in adherent monolayer
culture. Biochem. Biophys. Res. Commun. 351, 321–327. doi: 10.1016/j.bbrc.
2006.09.171
Hudson, J., Titmarsh, D., Hidalgo, A., Wolvetang, E., and Cooper-White, J. (2012).
Primitive cardiac cells from human embryonic stem cells. Stem Cells Dev. 21,
1513–1523. doi: 10.1089/scd.2011.0254
Ikuno, T., Masumoto, H., Yamamizu, K., Yoshioka, M., Minakata, K., Ikeda, T.,
et al. (2017). Efficient and robust differentiation of endothelial cells from human
induced pluripotent stem cells via lineage control with VEGF and cyclic AMP.
PLoS One 12:e0173271. doi: 10.1371/journal.pone.0173271
Ishida, M., Miyagawa, S., Saito, A., Fukushima, S., Harada, A., Ito, E., et al.
(2019). Transplantation of human-induced pluripotent stem cell-derived
cardiomyocytes is superior to somatic stem cell therapy for restoring cardiac
function and oxygen consumption in a porcine model of myocardial infarction.
Transplantation 103, 291–298. doi: 10.1097/tp.0000000000002384
Ishigami, M., Masumoto, H., Ikuno, T., Aoki, T., Kawatou, M., Minakata, K., et al.
(2018). Human iPS cell-derived cardiac tissue sheets for functional restoration
of infarcted porcine hearts. PLoS One 13:e0201650. doi: 10.1371/journal.pone.
0201650
Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M.,
et al. (2000). Differentiation of human embryonic stem cells into embryoid
bodies compromising the three embryonic germ layers. Mol. Med. 6, 88–95.
doi: 10.1007/bf03401776
Jiang, X., Rowitch, D. H., Soriano, P., McMahon, A. P., and Sucov, H. M.
(2000). Fate of the mammalian cardiac neural crest. Development 127,
1607–1616.
Jung, J. J., Husse, B., Rimmbach, C., Krebs, S., Stieber, J., Steinhoff, G.,
et al. (2014). Programming and isolation of highly pure physiologically and
pharmacologically functional sinus-nodal bodies from pluripotent stem cells.
Stem Cell Rep. 2, 592–605. doi: 10.1016/j.stemcr.2014.03.006
Kattman, S. J., Witty, A. D., Gagliardi, M., Dubois, N. C., Niapour, M., Hotta, A.,
et al. (2011). Stage-specific optimization of activin/nodal and BMP signaling
promotes cardiac differentiation of mouse and human pluripotent stem cell
lines. Cell Stem Cell 8, 228–240. doi: 10.1016/j.stem.2010.12.008
Kawamura, M., Miyagawa, S., Fukushima, S., Saito, A., Miki, K., Ito, E., et al.
(2013). Enhanced survival of transplanted human induced pluripotent stem
cell-derived cardiomyocytes by the combination of cell sheets with the pedicled
omental flap technique in a porcine heart. Circulation 128(11Suppl. 1), S87–
S94.
Kawamura, M., Miyagawa, S., Miki, K., Saito, A., Fukushima, S., Higuchi,
T., et al. (2012). Feasibility, safety, and therapeutic efficacy of human
induced pluripotent stem cell-derived cardiomyocyte sheets in a
porcine ischemic cardiomyopathy model. Circulation 126(11Suppl. 1),
S29–S37.
Kawamura, T., Miyagawa, S., Fukushima, S., Maeda, A., Kashiyama, N., Kawamura,
A., et al. (2016). Cardiomyocytes derived from MHC-homozygous induced
pluripotent stem cells exhibit reduced allogeneic immunogenicity in mhc-
matched non-human primates. Stem Cell Rep. 6, 312–320. doi: 10.1016/j.
stemcr.2016.01.012
Kehat, I., Kenyagin-Karsenti, D., Snir, M., Segev, H., Amit, M., Gepstein, A.,
et al. (2001). Human embryonic stem cells can differentiate into myocytes
with structural and functional properties of cardiomyocytes. J. Clin. Invest. 108,
407–414. doi: 10.1172/jci200112131
Kempf, H., Kropp, C., Olmer, R., Martin, U., and Zweigerdt, R. (2015). Cardiac
differentiation of human pluripotent stem cells in scalable suspension culture.
Nat. Protoc. 10, 1345–1361. doi: 10.1038/nprot.2015.089
Kinnear, C., Agrawal, R., Loo, C., Pahnke, A., Rodrigues, D. C., Thompson, T.,
et al. (2020). Everolimus rescues the phenotype of elastin insufficiency in
patient induced pluripotent stem cell-derived vascular smooth muscle cells.
Arterioscler. Thromb. Vasc. Biol. 40, 1325–1339. doi: 10.1161/atvbaha.119.
313936
Kinnear, C., Chang, W. Y., Khattak, S., Hinek, A., Thompson, T., de Carvalho
Rodrigues, D., et al. (2013). Modeling and rescue of the vascular phenotype
of Williams-Beuren syndrome in patient induced pluripotent stem cells. Stem
Cells Transl. Med. 2, 2–15. doi: 10.5966/sctm.2012-0054
Kita-Matsuo, H., Barcova, M., Prigozhina, N., Salomonis, N., Wei, K., Jacot, J. G.,
et al. (2009). Lentiviral vectors and protocols for creation of stable hESC lines
for fluorescent tracking and drug resistance selection of cardiomyocytes. PLoS.
One 4:e5046. doi: 10.1371/journal.pone.0005046
Klaus, A., Muller, M., Schulz, H., Saga, Y., Martin, J. F., and Birchmeier, W. (2012).
Wnt/beta-catenin and Bmp signals control distinct sets of transcription factors
in cardiac progenitor cells. Proc. Natl. Acad. Sci. U.S.A. 109, 10921–10926.
doi: 10.1073/pnas.1121236109
Koyano-Nakagawa, N., Kweon, J., Iacovino, M., Shi, X., Rasmussen, T. L., Borges,
L., et al. (2012). Etv2 is expressed in the yolk sac hematopoietic and endothelial
progenitors and regulates Lmo2 gene expression. Stem Cells 30, 1611–1623.
doi: 10.1002/stem.1131
Laflamme, M. A., Chen, K. Y., Naumova, A. V., Muskheli, V., Fugate, J. A., Dupras,
S. K., et al. (2007). Cardiomyocytes derived from human embryonic stem cells
in pro-survival factors enhance function of infarcted rat hearts. Nat. Biotechnol.
25, 1015–1024. doi: 10.1038/nbt1327
Frontiers in Cell and Developmental Biology | www.frontiersin.org 13 May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 14
Gao and Pu Review of hPSCs-CVCs
Lee, S. J., Park, C., Lee, J. Y., Kim, S., Kwon, P. J., Kim, W., et al. (2015). Generation
of pure lymphatic endothelial cells from human pluripotent stem cells and their
therapeutic effects on wound repair. Sci. Rep. 5:11019.
Lelovas, P. P., Kostomitsopoulos, N. G., and Xanthos, T. T. (2014). A comparative
anatomic and physiologic overview of the porcine heart. J. Am. Assoc. Lab.
Anim. Sci. 53, 432–438.
Lerman, A., and Zeiher, A. M. (2005). Endothelial function: cardiac events.
Circulation 111, 363–368. doi: 10.1161/01.cir.0000153339.27064.14
Lescroart, F., Wang, X., Lin, X., Swedlund, B., Gargouri, S., Sanchez-Danes, A.,
et al. (2018). Defining the earliest step of cardiovascular lineage segregation by
single-cell RNA-seq. Science 359, 1177–1181. doi: 10.1126/science.aao4174
Levenberg, S., Golub, J. S., Amit, M., Itskovitz-Eldor, J., and Langer, R. (2002).
Endothelial cells derived from human embryonic stem cells. Proc. Natl. Acad.
Sci. U.S.A. 99, 4391–4396.
Li, L., Baroja, M. L., Majumdar, A., Chadwick, K., Rouleau, A., Gallacher, L., et al.
(2004). Human embryonic stem cells possess immune-privileged properties.
Stem Cells 22, 448–456. doi: 10.1634/stemcells.22-4-448
Li, Z., Hu, S., Ghosh, Z., Han, Z., and Wu, J. C. (2011). Functional characterization
and expression profiling of human induced pluripotent stem cell- and
embryonic stem cell-derived endothelial cells. Stem Cells Dev. 20, 1701–1710.
doi: 10.1089/scd.2010.0426
Lian, X., Hsiao, C., Wilson, G., Zhu, K., Hazeltine, L. B., Azarin, S. M., et al. (2012).
Robust cardiomyocyte differentiation from human pluripotent stem cells via
temporal modulation of canonical Wnt signaling. Proc. Natl. Acad. Sci. U.S.A.
109, E1848–E1857.
Lim, J., and Thiery, J. P. (2012). Epithelial-mesenchymal transitions: insights from
development. Development 139, 3471–3486. doi: 10.1242/dev.071209
Lin, B., Kim, J., Li, Y., Pan, H., Carvajal-Vergara, X., Salama, G., et al. (2012). High-
purity enrichment of functional cardiovascular cells from human iPS cells.
Cardiovasc. Res. 95, 327–335. doi: 10.1093/cvr/cvs185
Lin, H., McBride, K. L., Garg, V., and Zhao, M.-T. (2021). Decoding genetics
of congenital heart disease using patient-derived induced pluripotent stem
cells (iPSCs). Front. Cell Dev. Biol. 9:630069. doi: 10.3389/fcell.2021.63
0069
Lin, Y., Linask, K. L., Mallon, B., Johnson, K., Klein, M., Beers, J., et al. (2017).
Heparin promotes cardiac differentiation of human pluripotent stem cells in
chemically defined albumin-free medium, enabling consistent manufacture of
cardiomyocytes. Stem Cells Transl. Med. 6, 527–538. doi: 10.5966/sctm.2015-
0428
Liu, F., Li, D., Yu, Y. Y., Kang, I., Cha, M. J., Kim, J. Y., et al. (2015). Induction of
hematopoietic and endothelial cell program orchestrated by ETS transcription
factor ER71/ETV2. EMBO Rep. 16, 654–669. doi: 10.15252/embr.201439939
Liu, X., Qi, J., Xu, X., Zeisberg, M., Guan, K., and Zeisberg, E. M. (2016).
Differentiation of functional endothelial cells from human induced pluripotent
stem cells: a novel, highly efficient and cost effective method. Differentiation 92,
225–236. doi: 10.1016/j.diff.2016.05.004
Liu, Y. W., Chen, B., Yang, X., Fugate, J. A., Kalucki, F. A., Futakuchi-Tsuchida,
A., et al. (2018). Human embryonic stem cell-derived cardiomyocytes restore
function in infarcted hearts of non-human primates. Nat. Biotechnol. 36, 597–
605.
Ma, J., Guo, L., Fiene, S. J., Anson, B. D., Thomson, J. A., Kamp, T. J., et al. (2011).
High purity human-induced pluripotent stem cell-derived cardiomyocytes:
electrophysiological properties of action potentials and ionic currents. Am. J.
Physiol. Heart Circ. Physiol. 301, H2006–H2017.
Majesky, M. W. (2007). Developmental basis of vascular smooth muscle diversity.
Arterioscler. Thromb. Vasc. Biol. 27, 1248–1258. doi: 10.1161/atvbaha.107.
141069
Mikawa, T., and Gourdie, R. G. (1996). Pericardial mesoderm generates a
population of coronary smooth muscle cells migrating into the heart along with
ingrowth of the epicardial organ. Dev. Biol. 174, 221–232. doi: 10.1006/dbio.
1996.0068
Mohr, J. C., de Pablo,J. J., and Palecek, S. P. (2006). 3-D microwell culture of human
embryonic stem cells. Biomaterials 27, 6032–6042. doi: 10.1016/j.biomaterials.
2006.07.012
Mohr, J. C., Zhang, J., Azarin, S. M., Soerens, A. G., de Pablo, J. J., Thomson,
J. A., et al. (2010). The microwell control of embryoid body size in order to
regulate cardiac differentiation of human embryonic stem cells. Biomaterials
31, 1885–1893. doi: 10.1016/j.biomaterials.2009.11.033
Ng, E. S., Davis, R. P., Azzola, L., Stanley, E. G., and Elefanty, A. G. (2005). Forced
aggregation of defined numbers of human embryonic stem cells into embryoid
bodies fosters robust, reproducible hematopoietic differentiation. Blood 106,
1601–1603. doi: 10.1182/blood-2005-03-0987
Nourse, M. B., Halpin, D. E., Scatena, M., Mortisen, D. J., Tulloch, N. L., Hauch,
K. D., et al. (2010). VEGF induces differentiation of functional endothelium
from human embryonic stem cells: implications for tissue engineering.
Arterioscler. Thromb. Vasc. Biol. 30, 80–89.
Olsson, A. K., Dimberg, A., Kreuger, J., and Claesson-Welsh, L. (2006). VEGF
receptor signalling - in control of vascular function. Nat. Rev. Mol. Cell Biol.
7, 359–371. doi: 10.1038/nrm1911
Patsch, C., Challet-Meylan, L., Thoma, E. C., Urich, E., Heckel, T., O’Sullivan, J. F.,
et al. (2015). Generation of vascular endothelial and smooth muscle cells from
human pluripotent stem cells. Nat. Cell Biol. 17, 994–1003.
Perea-Gomez, A., Vella, F. D., Shawlot, W., Oulad-Abdelghani, M., Chazaud, C.,
Meno, C., et al. (2002). Nodal antagonists in the anterior visceral endoderm
prevent the formation of multiple primitive streaks. Dev. Cell 3, 745–756. doi:
10.1016/s1534-5807(02)00321- 0
Protze, S. I., Lee, J. H., and Keller, G. M. (2019). Human pluripotent stem
cell-derived cardiovascular cells: from developmental biology to therapeutic
applications. Cell Stem Cell 25, 311–327. doi: 10.1016/j.stem.2019.07.010
Romagnuolo, R., Masoudpour, H., Porta-Sanchez, A., Qiang, B., Barry, J., Laskary,
A., et al. (2019). human embryonic stem cell-derived cardiomyocytes regenerate
the infarcted pig heart but induce ventricular tachyarrhythmias. Stem Cell Rep.
12, 967–981. doi: 10.1016/j.stemcr.2019.04.005
Rosa, S., Praca, C., Pitrez, P. R., Gouveia, P. J., Aranguren, X. L., Ricotti, L., et al.
(2019). Functional characterization of iPSC-derived arterial- and venous-like
endothelial cells. Sci. Rep. 9:3826.
Rufaihah, A. J., Huang, N. F., Kim, J., Herold, J., Volz, K. S., Park, T. S., et al.
(2013). Human induced pluripotent stem cell-derived endothelial cells exhibit
functional heterogeneity. Am. J. Transl. Res. 5, 21–35.
Sahara, M., Hansson, E. M., Wernet, O., Lui, K. O., Spater, D., and Chien, K. R.
(2014). Manipulation of a VEGF-Notch signaling circuit drives formation of
functional vascular endothelial progenitors from human pluripotent stem cells.
Cell Res. 24, 820–841. doi: 10.1038/cr.2014.59
Sahara, M., Hansson, E. M., Wernet, O., Lui, K. O., Spater, D., and Chien, K. R.
(2015). Manipulation of a VEGF-Notch signaling circuit drives formation of
functional vascular endothelial progenitors from human pluripotent stem cells.
Cell Res. 25:148. doi: 10.1038/cr.2015.2
Shiba, Y., Gomibuchi, T., Seto, T., Wada, Y., Ichimura, H., Tanaka, Y., et al. (2016).
Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates
primate hearts. Nature 538, 388–391. doi: 10.1038/nature19815
Sinha, S., Iyer, D., and Granata, A. (2014). Embryonic origins of human vascular
smooth muscle cells: implications for in vitro modeling and clinical application.
Cell. Mol. Life Sci. 71, 2271–2288. doi: 10.1007/s00018- 013-1554-3
Sivarapatna, A., Ghaedi, M., Le, A. V., Mendez, J. J., Qyang, Y., and Niklason,
L. E. (2015). Arterial specification of endothelial cells derived from human
induced pluripotent stem cells in a biomimetic flow bioreactor. Biomaterials
53, 621–633. doi: 10.1016/j.biomaterials.2015.02.121
Spater, D., Hansson, E. M., Zangi, L., and Chien, K. R. (2014). How to
make a cardiomyocyte. Development 141, 4418–4431. doi: 10.1242/dev.09
1538
Su, L., Kong, X., Lim, S., Loo, S., Tan, S., Poh, K., et al. (2018). The prostaglandin
H2 analog U-46619 improves the differentiation efficiency of human induced
pluripotent stem cells into endothelial cells by activating both p38MAPK and
ERK1/2 signaling pathways. Stem Cell Res. Ther. 9:313.
Suarez-Alvarez, B., Rodriguez, R. M., Calvanese, V., Blanco-Gelaz, M. A., Suhr,
S. T., Ortega, F., et al. (2010). Epigenetic mechanisms regulate MHC and antigen
processing molecules in human embryonic and induced pluripotent stem cells.
PLoS One 5:e10192. doi: 10.1371/journal.pone.0010192
Subileau, M., Merdzhanova, G., Ciais, D., Collin-Faure, V., Feige, J. J., Bailly, S.,
et al. (2019). Bone morphogenetic protein 9 regulates early lymphatic-specified
endothelial cell expansion during mouse embryonic stem cell differentiation.
Stem Cell Rep. 12, 98–111. doi: 10.1016/j.stemcr.2018.11.024
Swijnenburg, R. J., Tanaka, M., Vogel, H., Baker, J., Kofidis, T., Gunawan, F., et al.
(2005). Embryonic stem cell immunogenicity increases upon differentiation
after transplantation into ischemic myocardium. Circulation 112(Suppl. 9),
166–172.
Frontiers in Cell and Developmental Biology | www.frontiersin.org 14 May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 15
Gao and Pu Review of hPSCs-CVCs
Tan, S., Tao, Z., Loo, S., Su, L., Chen, X., and Ye, L. (2019). Non-viral vector
based gene transfection with human induced pluripotent stem cells derived
cardiomyocytes. Sci. Rep. 9:14404.
Tan, Y., Han, P., Gu, Q., Chen, G., Wang, L., Ma, R., et al. (2018). Generation
of clinical-grade functional cardiomyocytes from human embryonic stem cells
in chemically defined conditions. J. Tissue Eng. Regen. Med. 12, 153–163. doi:
10.1002/term.2381
Tao, Z., Loo, S., Su, L., Tan, S., Tee, G., Gan, S. U., et al. (2020). Angiopoietin-1
enhanced myocyte mitosis, engraftment, and the reparability of hiPSC-CMs for
treatment of myocardial infarction. Cardiovasc. Res. 116:cvaa215.
Ting, S., Chen, A., Reuveny, S., and Oh, S. (2014). An intermittent rocking platform
for integrated expansion and differentiation of human pluripotent stem cells to
cardiomyocytes in suspended microcarrier cultures. Stem Cell Res. 13, 202–213.
doi: 10.1016/j.scr.2014.06.002
Tohyama, S., Hattori, F., Sano, M., Hishiki, T., Nagahata, Y., Matsuura, T., et al.
(2013). Distinct metabolic flow enables large-scale purification of mouse and
human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell 12, 127–
137. doi: 10.1016/j.stem.2012.09.013
Uosaki, H., Andersen, P., Shenje, L. T., Fernandez, L., Christiansen, S. L., and
Kwon, C. (2012). Direct contact with endoderm-like cells efficiently induces
cardiac progenitors from mouse and human pluripotent stem cells. PLoS One
7:e46413. doi: 10.1371/journal.pone.0046413
Uosaki, H., Fukushima, H., Takeuchi,A., Matsuoka, S., Nakatsuji, N., Yamanaka, S.,
et al. (2011). Efficient and scalable purification of cardiomyocytes from human
embryonic and induced pluripotent stem cells by VCAM1 surface expression.
PLoS One 6:e23657. doi: 10.1371/journal.pone.0023657
Vahdat, S., Pahlavan, S., Aghdami, N., Bakhshandeh, B., and Baharvand, H.
(2019a). Establishment of A protocol for in vitro culture of cardiogenic
mesodermal cells derived from human embryonic stem cells. Cell J. 20, 496–
504.
Vahdat, S., Pahlavan, S., Mahmoudi, E., Barekat, M., Ansari, H., Bakhshandeh,
B., et al. (2019b). Expansion of human pluripotent stem cell-derived early
cardiovascular progenitor cells by a cocktail of signaling factors. Sci. Rep.
9:16006.
Virani, S. S., Alonso, A., Benjamin, E. J., Bittencourt, M. S., Callaway, C. W.,
Carson, A. P., et al. (2020). Heart disease and stroke statistics-2020 update: a
report from the American Heart Association. Circulation 141, e139–e596.
Waldo, K. L., Hutson, M. R., Ward, C. C., Zdanowicz, M., Stadt, H. A., Kumiski,
D., et al. (2005). Secondary heart field contributes myocardium and smooth
muscle to the arterial pole of the developing heart. Dev. Biol. 281, 78–90.
doi: 10.1016/j.ydbio.2005.02.012
Wang, J., Liu, M., Wu, Q., Li, Q., Gao, L., Jiang, Y., et al. (2019). Human embryonic
stem cell-derived cardiovascular progenitors repair infarcted hearts through
modulation of macrophages via activation of signal transducer and activator
of transcription 6. Antioxid. Redox Signal. 31, 369–386. doi: 10.1089/ars.2018.
7688
Wang, K., Lin, R. Z., Hong, X., Ng, A. H., Lee, C. N., Neumeyer, J., et al. (2020).
Robust differentiation of human pluripotent stem cells into endothelial cells
via temporal modulation of ETV2 with modified mRNA. Sci. Adv. 6:eaba7606.
doi: 10.1126/sciadv.aba7606
Wang, X., Lu, M., Tian, X., Ren, Y., Li, Y., Xiang, M., et al. (2020). Diminished
expression of major histocompatibility complex facilitates the use of human
induced pluripotent stem cells in monkey. Stem Cell Res. Ther. 11:334.
Wang, Y., Hu, J., Jiao, J., Liu, Z., Zhou, Z., Zhao, C., et al. (2014). Engineering
vascular tissue with functional smooth muscle cells derived from human iPS
cells and nanofibrous scaffolds. Biomaterials 35, 8960–8969. doi: 10.1016/j.
biomaterials.2014.07.011
Wasteson, P., Johansson, B. R., Jukkola, T., Breuer, S., Akyurek, L. M., Partanen,
J., et al. (2008). Developmental origin of smooth muscle cells in the descending
aorta in mice. Development 135, 1823–1832. doi: 10.1242/dev.020958
Willems, E., Spiering, S., Davidovics, H., Lanier, M., Xia, Z., Dawson, M., et al.
(2011). Small-molecule inhibitors of the Wnt pathway potently promote
cardiomyocytes from human embryonic stem cell-derived mesoderm. Circ. Res.
109, 360–364. doi: 10.1161/circresaha.111.249540
Wu, D. C., Boyd, A. S., and Wood, K. J. (2008). Embryonic stem cells and their
differentiated derivatives have a fragile immune privilege but still represent
novel targets of immune attack. Stem Cells 26, 1939–1950. doi: 10.1634/
stemcells.2008-0078
Xie, C. Q., Zhang, J., Villacorta, L., Cui, T., Huang, H., and Chen, Y. E. (2007).
A highly efficient method to differentiate smooth muscle cells from human
embryonic stem cells. Arterioscler. Thromb. Vasc. Biol. 27, e311–e312.
Xiong, Q., Ye, L., Zhang, P., Lepley, M., Swingen, C., Zhang, L., et al. (2012).
Bioenergetic and functional consequences of cellular therapy: activation of
endogenous cardiovascular progenitor cells. Circ. Res. 111, 455–468. doi: 10.
1161/circresaha.112.269894
Xiong, Q., Ye, L., Zhang, P., Lepley, M., Tian, J., Li, J., et al. (2013). Functional
consequences of human induced pluripotent stem cell therapy: myocardial
ATP turnover rate in the in vivo swine heart with postinfarction remodeling.
Circulation 127, 997–1008. doi: 10.1161/circulationaha.112.000641
Xu, C., Police, S., Rao, N., and Carpenter, M. K. (2002). Characterization and
enrichment of cardiomyocytes derived from human embryonic stem cells. Circ.
Res. 91, 501–508. doi: 10.1161/01.res.0000035254.80718.91
Xu, H., Wang, B., Ono, M., Kagita, A., Fujii, K., Sasakawa, N., et al. (2019). Targeted
disruption of HLA genes via CRISPR-Cas9 generates iPSCs with enhanced
immune compatibility. Cell Stem Cell 24, 566–578.e7.
Xu, X. Q., Zweigerdt, R., Soo, S. Y., Ngoh, Z. X., Tham, S. C., Wang, S. T., et al.
(2008). Highly enriched cardiomyocytes from human embryonic stem cells.
Cytotherapy 10, 376–389. doi: 10.1080/14653240802105307
Xuan, W., Wang, Y., Tang, Y., Ali, A., Hu, H., Maienschein-Cline, M., et al.
(2018). Cardiac progenitors induced from human induced pluripotent stem
cells with cardiogenic small molecule effectively regenerate infarcted hearts
and attenuate fibrosis. Shock 50, 627–639. doi: 10.1097/shk.0000000000
001133
Yamamizu, K., Kawasaki, K., Katayama, S., Watabe, T., and Yamashita, J. K. (2009).
Enhancement of vascular progenitor potential by protein kinase A through
dual induction of Flk-1 and Neuropilin-1. Blood 114, 3707–3716. doi: 10.1182/
blood-2008- 12-195750
Yang, L., Geng, Z., Nickel, T., Johnson, C., Gao, L., Dutton, J., et al. (2016).
Differentiation of human induced-pluripotent stem cells into smooth-muscle
cells: two novel protocols. PLoS One 11:e0147155. doi: 10.1371/journal.pone.
0147155
Yap, L., Wang, J. W., Moreno-Moral, A., Chong, L. Y., Sun, Y., Harmston,
N., et al. (2019). , In vivo generation of post-infarct human cardiac
muscle by laminin-promoted cardiovascular progenitors. Cell Rep. 26,
3231–3245.e9.
Ye, L., Chang, Y. H., Xiong, Q., Zhang, P., Zhang, L., Somasundaram, P., et al.
(2014). Cardiac repair in a porcine model of acute myocardial infarction with
human induced pluripotent stem cell-derived cardiovascular cells. Cell Stem
Cell 15, 750–761. doi: 10.1016/j.stem.2014.11.009
Ye, L., Haider, H., Tan, R., Toh, W., Law, P. K., Tan, W., et al. (2007).
Transplantation of nanoparticle transfected skeletal myoblasts overexpressing
vascular endothelial growth factor-165 for cardiac repair. Circulation 116(11
Suppl.), I113–I120.
Ye, L., Zhang, P., Duval, S., Su, L., Xiong, Q., and Zhang, J. (2013a). Thymosin beta4
increases the potency of transplanted mesenchymal stem cells for myocardial
repair. Circulation 128(11Suppl. 1), S32–S41.
Ye, L., Zhang, S., Greder, L., Dutton, J., Keirstead, S. A., Lepley, M., et al. (2013b).
Effective cardiac myocyte differentiation of human induced pluripotent
stem cells requires VEGF. PLoS One 8:e53764. doi: 10.1371/journal.pone.00
53764 doi: 10.1371/journal.pone.0053764
Zhang, F., Qiu, H., Dong, X., Wang, C., Na, J., Zhou, J., et al. (2020). Transferrin
improved the generation of cardiomyocyte from human pluripotent stem cells
for myocardial infarction repair. J. Mol. Histol. 52, 87–99. doi: 10.1007/s10735-
020-09926- 0
Zhang, J., Chu, L. F., Hou, Z., Schwartz, M. P., Hacker, T., Vickerman, V., et al.
(2017). Functional characterization of human pluripotent stem cell-derived
arterial endothelial cells. Proc. Natl. Acad. Sci. U.S.A. 114, E6072–E6078.
Zhang, J., Klos, M., Wilson, G. F., Herman, A. M., Lian, X., Raval, K. K., et al. (2012).
Extracellular matrix promotes highly efficient cardiac differentiation of human
pluripotent stem cells: the matrix sandwich method. Circ. Res. 111, 1125–1136.
doi: 10.1161/circresaha.112.273144
Zhang, J., Wilson, G. F., Soerens, A. G., Koonce, C. H., Yu, J., Palecek, S. P., et al.
(2009). Functional cardiomyocytes derived from human induced pluripotent
stem cells. Circ. Res. 104, e30–e41.
Zhang, M., Schulte, J. S., Heinick, A., Piccini, I., Rao, J., Quaranta, R., et al.
(2015). Universal cardiac induction of human pluripotent stem cells in two and
Frontiers in Cell and Developmental Biology | www.frontiersin.org 15 May 2021 | Volume 9 | Article 658088

fcell-09-658088 May 6, 2021 Time: 17:42 # 16
Gao and Pu Review of hPSCs-CVCs
three-dimensional formats: implications for in vitro maturation. Stem Cells 33,
1456–1469. doi: 10.1002/stem.1964
Zhang, S., Dutton, J. R., Su, L., Zhang, J., and Ye, L. (2014). The influence of a
spatiotemporal 3D environment on endothelial cell differentiation of human
induced pluripotent stem cells. Biomaterials 35, 3786–3793. doi: 10.1016/j.
biomaterials.2014.01.037
Zhao, M. T., Shao, N. Y., and Garg, V. (2020). Subtype-specific cardiomyocytes for
precision medicine: where are we now? Stem Cells 38, 822–833.
Zhu, K., Wu, Q., Ni, C., Zhang, P., Zhong, Z., Wu, Y., et al. (2018). Lack
of remuscularization following transplantation of human embryonic
stem cell-derived cardiovascular progenitor cells in infarcted nonhuman
primates. Circ. Res. 122, 958–969. doi: 10.1161/circresaha.117.3
11578
Zhu, W., Zhao, M., Mattapally, S., Chen, S., and Zhang, J. (2018). CCND2
overexpression enhances the regenerative potency of human induced
pluripotent stem cell-derived cardiomyocytes: remuscularization of
injured ventricle. Circ. Res. 122, 88–96. doi: 10.1161/circresaha.117.
311504
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2021 Gao and Pu. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The
use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with
these terms.
Frontiers in Cell and Developmental Biology | www.frontiersin.org 16 May 2021 | Volume 9 | Article 658088