Cellular and Engineered Organoids For Cardiovascular Models

Circulation Research
COMPENDIUM ON BASIC MODELS OF CARDIOVASCULAR DISEASE
Cellular and Engineered Organoids for

Cardiovascular Models
Dilip Thomas ,* Suji Choi ,* Christina Alamana , Kevin Kit Parker, Joseph C. Wu
ABSTRACT: An ensemble of in vitro cardiac tissue models has been developed over the past several decades to aid our
understanding of complex cardiovascular disorders using a reductionist approach. These approaches often rely on
recapitulating single or multiple clinically relevant end points in a dish indicative of the cardiac pathophysiology. The
possibility to generate disease-relevant and patient-specific human induced pluripotent stem cells has further leveraged
the utility of the cardiac models as screening tools at a large scale. To elucidate biological mechanisms in the cardiac
models, it is critical to integrate physiological cues in form of biochemical, biophysical, and electromechanical stimuli to
achieve desired tissue-like maturity for a robust phenotyping. Here, we review the latest advances in the directed stem
cell differentiation approaches to derive a wide gamut of cardiovascular cell types, to allow customization in cardiac model
systems, and to study diseased states in multiple cell types. We also highlight the recent progress in the development of
several cardiovascular models, such as cardiac organoids, microtissues, engineered heart tissues, and microphysiological
systems. We further expand our discussion on defining the context of use for the selection of currently available cardiac
tissue models. Last, we discuss the limitations and challenges with the current state-of-the-art cardiac models and highlight
future directions.
Key Words: cardiovascular disease ◼ heart ◼ organoids ◼ phenotype ◼ pluripotent stem cells ◼ tissue engineering
F
or decades, stem cell scientists have attempted to and human contribute to the key differences in the kinet-
deconvolute developmental processes using human ics and propagation of cell populations.3 Therefore, the
and early embryonic tissues that are scarce. From a use of human models is considered more reliable to
cardiovascular disease modeling and drug development uncover molecular underpinnings of human cardiac disor-
perspective, animal models have played an important role ders and the development of targeted therapies.
in the validation of several common and rare diseases.1 The discovery of pluripotent stem cells (PSCs), such
Both safety and efficacy parameters continue to be tested as human inner cell mass embryonic stem cells (ESCs)4
in animals before the commencement of first-in-human and induced pluripotent stem cells (iPSCs),5 has emerged
clinical trials. Despite the vital role of animal models in as a groundbreaking tool to study human cardiomyo-
research and pharmaceutical development, the discor- genesis and the basis of congenital and mature heart
dance between animal and human physiology and patho- diseases. Particularly, the iPSC technology has solved
physiology is evident with over 80% drug attrition rate in the issues surrounding availability of primary cells from
clinical studies.2 Thus, animal models have not been able patients, resulting in the development of more accu-
to mirror the complexity or pathological diversity present in rate disease modeling platforms. Moreover, using iPSC-
human systems, thereby limiting clinical success. During derived cardiomyocytes (iPSC-CMs) and genome editing
embryonic development, there is a notable divergence in approaches, a wide number of monogenic and complex
the pattern and timing of tissue morphogenesis between cardiac pathogenesis have been studied in vitro, provid-
humans and animals. For instance, the differences in the ing newer insights into disease mechanisms.6 Human
stem cell surface marker repertoire between the mouse iPSCs-CMs has also emerged as a cornerstone in drug
Correspondence to: Joseph C. Wu, MD, PhD, Stanford Cardiovascular Institute, 265 Campus Dr, G1120B, Stanford, CA 93404. Email joewu@stanford.edu; or Kevin
Kit Parker, PhD, Harvard University, 150 Western Ave, Boston, MA 02134. Email kkparker@g.harvard.edu
*D. Thomas and S. Choi contributed equally.
For Sources of Funding and Disclosures, see page 1797.
© 2022 American Heart Association, Inc.
Circulation Research is available at www.ahajournals.org/journal/res
1780 June 10, 2022 Circulation Research. 2022;130:1780–1802. DOI: 10.1161/CIRCRESAHA.122.320305

Thomas et al Engineered Cardiovascular Models
COMPENDIUM ON BASIC MODELS

cell types such as iPSC-derived endothelial cells (iPSC-
OF CARDIOVASCULAR DISEASE
Nonstandard Abbreviations and Acronyms ECs),9 iPSC-derived cardiac fibroblasts (iPSC-CFs),10 and
iPSC-derived smooth muscle cells (iPSC-SMCs),11 has
2D 2-dimensional significantly enriched the cellular diversity in cardiac mod-
3D 3-dimensional els, supporting physiological maturation and recapitulation
ACTA2 α-smooth muscle actin of properties like that of an adult heart.
AM atrial myocyte In this review article, we discuss the recent develop-
APLNR apelin receptor ments in the generation of state-of-the-art protocols for
BMPs bone morphogenetic proteins
engineering cellular diversity in 3D cardiac model systems,
translational utility of miniaturized cardiac models in basic
CF cardiac fibroblast
research, and high-throughput screening studies. Finally,
CM cardiomyocyte
we discuss the need for standardization to fully exploit
CNN1 calponin 1 the in vitro cardiac platforms using unprecedented devel-
CO cardiac organoid opments in precision genomics and artificial intelligence.
Cx43 connexin 43
EB embryoid body
EC endothelial cells OVERVIEW OF METHODOLOGIES TO
ECM extracellular matrix GENERATE CARDIOVASCULAR CELL
EHT engineering heart tissue TYPES IN A DISH
EMP erythromyeloid progenitor
In vivo cardiovascular cells are obtained through cardiac
ESC embryonic stem cell morphogenesis, which occurs via modulation of care-
FGFs fibroblast growth factors fully orchestrated pathways. The main pathway modula-
FLT3L fms-like tyrosine kinase 3 ligand tors include repressors of canonical Wnt/β-catenin bone
GM-CSF granulocyte-macrophage colony-stimu- morphogenetic proteins (BMPs) (bone morphogenetic
lating factor proteins), nodal/Activin-A, FGFs (fibroblast growth fac-
IFN-γ interferon γ tors), and VEGF (vascular endothelial growth factor) sig-
IL interleukin naling pathways. Among these, Wnt/β-catenin is one of
iPSC induced pluripotent stem cell the key signaling pathways required to exit pluripotency
M-CSF macrophage colony-stimulating factor and induce formation of early mesodermal intermediates
MPS microphysiological system expressing T-box transcription factors Brachyury (T) and
MTF muscular thin films eomesodermin which gives rise to multipotent progenitors,
NK natural killer cell
such as KDR (kinase insert domain receptor)+CD235a/
b+ and MesP1+ (mesodermal posterior 1) cells.12,13 The
NM nodal myocyte
manufacturing of iPSC-CMs through stage-wise speci-
PDGFRB PDGF receptor B
fication in vitro has been one of the major milestones
PECAM platelet endothelial cell adhesion in cardiovascular research for understanding disease
molecule
mechanisms and testing therapeutic strategies for clinical
PSC pluripotent stem cell translation. The most common and efficient methods to
RA retinoic acid drive cardiogenic program are based on temporal modula-
RALDH-2 retinaldehyde dehydrogenase 2 tion of Wnt, Activin-A, BMP2/4, and TGF (transforming
SCF stem cell factor growth factor)-β signaling pathways (Figure 1). Among
SHF second heart field cardiomyocyte subpopulations, the available methods pre-
SMC smooth muscle cell dominantly generate ventricular myocytes (VMs); however,
TGF transforming growth factor small proportions of atrial, pacemaker, and nonmyocyte
TNF-α tumor necrosis factor α populations introduce heterogeneity that requires further
TPO thrombopoietin purification or selection.14,15
VEGF vascular endothelial growth factor
VM ventricular myocyte
METHODOLOGIES TO GENERATE
CHAMBER-SPECIFIC CARDIOMYOCYTE
development and drug repurposing.7 Two-dimensional PROPORTIONS FROM PLURIPOTENT
(2D) iPSC-CM culture was built in part on in vitro system
using neonatal rat myocytes to explain the biophysics of STEM CELLS
conduction block.8 Parallel development in 3-dimensional Obtaining chamber-specific cardiac myocytes is important
(3D) cardiac models and generation of several supportive for many reasons such as understanding altered cardiac
Circulation Research. 2022;130:1780–1802. DOI: 10.1161/CIRCRESAHA.122.320305 June 10, 2022 1781

Figure 1. Deconstructing cardiogenesis using induced pluripotent stem cell (iPSC) technology.
An overview of cell lineage trajectory to derive cardiac cellular subtypes in vitro from pluripotent stem cells. Key factors that induce cell-
specific morphogenesis and their identity is depicted in the development pathway. Most commonly, all cardiac cell subtypes originate from the
mesendoderm progenitors (KDR [kinase insert domain receptor] and MESP1 [mesodermal posterior 1]) that arise from the primitive streak (PS).
Further biphasic Wnt modulation leads to the generation of cardiomyocytes (CMs), endothelium. Chamber-specific myocyte cell types are obtained
through bone morphogenetic protein (BMP) and retinoic acid (RA) mediated NOTCH signaling activation. Smooth muscles cells and pericytes
develop from lateral plate mesoderm (LPM) and paraxial mesoderm (PM). Cardiac fibroblasts are derived from epicardial and mesodermal
progenitors. Macrophages and natural killer (NK) cells that naturally reside in the cardiac tissue can be successfully derived from hemogenic
precursors. Improving cellular diversity in the human cardiac model systems will offer higher resemblance to cardiac tissue composition. APS
indicates anterior primitive streak; ESC, embryonic stem cell; ETV2, ets variant transcription factor 2; FGF, fibroblast growth factor; IFN, interferon;
IL, interleukin; SAN, sinoatrial node; TPO, thrombopoietin; and VEGF, vascular endothelial growth factor.32,33, 42,51,58,62,64,69,82,84
physiology, pharmacology, and cell-specific toxicology is desirable. For more precise modeling of a cell-specific
responses. Particularly, a limitless supply of patient-spe- disease, it is essential to limit heterogeneity which may
cific iPSC sources can serve as an invaluable resource lead to confounding results. For example, atrial fibrillation
to understand inherited, acute or chronic, and even racial is a phenotype associated with atrial cells, hence deriva-
differences to existing or new pharmaceutics. To generate tion of pure iPSC-AMs is desirable to model arrhythmic
enriched myocyte population, a guided iPSC differentia- phenotype in familial16 or sodium channel-linked17 atrial
tion for VM, nodal myocyte (NM), and atrial myocyte (AM) fibrillation. Similarly, iPSC-VMs can be used to understand


mechanisms that drive channelopathies and cardiomyopa- phase by glucose starvation that drives the myocytes to
thies caused by long-QT syndrome,18–20 dilated cardiomy- shift to oxidative metabolism and simultaneous elimina-
opathy,,22 hypertrophic cardiomyopathy21 among others. In tion of glucose-dependent nonmyocytes.36 In a normal
addition to inherited cardiac disorders, metabolic diseases heart, >95% of ATP is generated in the mitochondria from
such as diabetic cardiomyopathy,22,23 iron-overload car- oxidative phosphorylation and 2% to 5% is derived from
diomyopathy,24 and ischemia-perfusion injury25 have also glycolysis. Hence, supplementation with optimal oxida-
been successfully modeled using iPSC-VMs. From a cell tive substrates such as lipids in a maturation medium will
therapy perspective, obtaining pure chamber-specific cell significantly improve function and mitochondrial output.29
types may prevent abnormal automaticity or arrhythmia Overall, this 3-stage protocol is shown to generate >90%
due to differing action potentials and conduction velocities cardiac troponin T (TNNT2, cardiac troponin T) express-
from the mixture of different cardiac myocytes. Several ing myocytes reproducibly in several iPSC lines.35 iPSC-
selection strategies have been developed for purification derived VMs typically express genes, such as HAND1
such as genetically coded fluorescent reporters,26,27 modi- (heart and neural crest derivatives expressed 1), HEY2
fication of culture conditions,28 metabolic selection,29 and (hes related family bHLH transcription factor with YRPW
addition of cell-selective factors.30 However, it is important motif 2), MYL2 (myosin light chain 2), MYH7 (myosin
to note that despite the enrichment techniques, residual heavy chain 7), GJA1 (gap junction alpha-1), and KCNJ2
fractions of other cell types may remain. (potassium inwardly rectifying channel subfamily J mem-
ber 2) at higher levels compared with common myocyte
genes such as NKX2-5, phospholamban, TNNT2, and
Ventricular Cardiomyocytes CASQ-2 (calsequestrin 2). In addition, iPSC-derived VMs
Differentiation methods to derive PSC-CMs aim to mimic when stimulated at 0.5 Hz display a ventricle-like tracing
the developmental trajectory of cardiomyogenesis in vivo. with longer action potential duration at 50% repolariza-
In vivo, left VMs develop from first heart field (FHF) and tion (action potential duration 50: ≈350–400 ms) with a
right VMs develop from the second heart field (SHF). slower rise (time to peak: ≈300 ms) and decay (≈800 ms)
First heart field cardiac progenitor (TBX5+/NKX2-5+) in Ca2+ transients.37 However, most iPSC-CMs derived
and SHF cardiac progenitor (TBX5−/NKX2-5+) popula- using this approach are deemed immature in terms of
tions have been isolated from reporter lines to generate ultrastructural features, gene expression and function. To
VMs.26,27 However, due to the multipotent nature of these tackle this maturity issue, several 2D and 3D techniques
cardiac progenitors, it currently remains a challenge to have been developed that significantly improve iPSC-CM
obtain pure left or right VMs to model ventricular-specific maturity.38
diseases. Current protocols using both ESCs and iPSCs
generate a mixture of right and left VMs that arise from
the cardiac mesoderm induced by manipulating BMP and Atrial Cardiomyocytes
Activin-A signaling followed by Wnt inhibition.31,32 Due to For the generation of AMs, several in vitro17,30,39 and in
the lack of control and diffusional barrier posed by earlier vivo studies40,41 have demonstrated the role of retinoic
embryoid body (EB) methodology, a monolayer approach acid (RA) as a key regulator in the formation of atrial
was introduced that reported higher efficiency and yield.33 and pacemaker cell lineages. RA-induced differentia-
Delivery of these endogenously long-range acting mor- tion of AMs is primarily driven by the transcription factor
phogens, such as BMP and Activin-A, was limited due to COUP-TFII (chicken ovalbumin upstream promoter-tran-
the lack of tunability of growth factor concentrations and scription factor 2). Progenitors that express RALDH-2
timing to obtain reproducible results. Therefore, a small (retinaldehyde dehydrogenase 2) in the lateral plate
molecule-based approach was developed for exogenous mesoderm give rise to AMs. Building on this understand-
activation of Wnt signaling using CHIR99021 (an inhibitor ing, several protocols have been developed where the
of glycogen synthase kinase 3β) that led to endogenous first primitive streak-like induction is followed by cardiac
upregulation of BMP and nodal pathways.34,35 Adoption mesoderm specification via biphasic modulation of Wnt
of small molecule-based approach significantly improved pathway. After this stage, enrichment to obtain AMs is
differentiation efficiency and reduced the reliance on guided by stimulation of the cardiac mesoderm (between
recombinant proteins that were expensive and less stable. days 3–5) with RA to enrich the AM population up to
This approach simplified the VM derivation in 3 defined 60%.42,43 Lineage tracing studies have shown that dif-
stages: (1) induction phase, where T-Brachyury express- ferentiation efficiency can be increased to 20- to 120-
ing mesendoderm induction is mediated by Wnt activation fold via activation of JNK signaling pathway using BMP
with small molecules such as CHIR99021, followed by antagonist (GREM2, gremlin 2) which acts upstream
(2) cardiac specification phase wherein Wnt inhibition by of atrial-specific transcriptional factors COUP-TFII and
small molecules such as IWP-2 (chemical inhibitor of Wnt HEY1.44 However, it is important to note that expression
pathway) or C59 to promote upregulation of cardiac-spe- of COUP-TFII has an inverse effect on NOTCH signaling,
cific markers, such as NKX2-5 (NK2 homeobox 5) and which is important in ventricular development. Hence,
ISL-1,34 and (3) the enrichment or metabolic selection the inhibition of NOTCH pathway can stunt ventricular
development but enhance atrial specification without Cardiac Fibroblasts

exogenous RA.45,46 iPSC-derived AMs show higher gene Both CFs and SMCs in vivo originate from the epicar-
expression of HEY1, NRF1, TBX5, ATP2A2, MLC2a, dium, endocardium and neural crest progenitors. Com-
and MYH6, in addition to potassium ion channel genes, paction of the epicardium brought about by these cell
such as KCNJ3, KCNA5, and KCNK3. Electrophysiolog- types during development further promotes proliferation
ical characterization of these cells show an atrial-specific of adjacent cardiomyocytes. Epicardial cells express-
action potential tracing with a shorter plateau (action ing markers, such as WT1 and TBX18, are the major
potential duration 50: ≈160–180 ms), and an overall source of CFs. These epicardial cells further undergo
rapid Ca2+ kinetics with a faster rise (time to peak: ≈150 epithelial-to-mesenchymal transition giving rise to CFs
ms) and decay (≈600 ms).37 and SMCs.54 Following a similar trajectory, in vitro differ-
entiation of CFs from iPSCs is guided through epicardial
NMs or Pacemaker Cells transition and epithelial-to-mesenchymal transition55,56
via Wnt and FGF signaling pathways.10 Since FGF2 is
NMs of the conduction system are responsible to initi- one the potent inducers of fibrogenesis, direct stimula-
ate contractions of the heart by relaying electrical signals tion of mesodermal progenitors was shown to generate
from the atria to the ventricles. One of the main molec- CFs without the intermediate epicardial cells.57 Similar to
ular hallmarks of NMs is the absence of transcription fetal and adult ventricular CFs, iPSC-CFs derived using
factor NKX2-5. The earliest attempt to generate NMs this method show a comparable expression of fibroblast
from PSCs involved activation of mesoderm using BMP/ marker, TE-7, in 80% to 90% cells during early passage.
Activin-A followed by supplementation with neureglin However, iPSC-CFs generated using these protocols
neutralizing antibody and ErbB signaling antagonist, to express low levels of TBX20 which is more consistent
yield >50% pacemaker cells.47 Recent protocols devel- with embryonic CF phenotype.58
oped to generate NMs have taken 3 distinct approaches:
(1) overexpression of transcription factors that control Endothelial Cells
NM development, such as TBX348 and SHOX2,49 (2) The majority of the EC population in the heart emerge
enrichment of NKX2-5− population through overexpres- from the endocardium forming a significant portion of
sion of c-MYC oncogene,50 and (3) activation of BMP- both coronary and valvular endothelium.59 Recent lin-
RA signaling and blocking FGF signaling after mesoderm eage tracing studies have also demonstrated the forma-
induction.51 Typically the differentiation efficiency of NMs tion of valvular interstitial cells through VEGF and FGF
using BMP-RA signaling is around 4% to 10%, but a sig- stimulation of CD31 expressing precursor derived from
nificant improvement in enrichment up to 35% can be MESP1 endocardial progenitors.60 ECs from iPSCs or
achieved by fine-tuning of BMP-4 concentrations in the ESCs are generated from CD34+/KDR+ hematoen-
presence of TGF-β inhibitor. Unlike strong expression of dothelial or cardiogenic precursors.61 With subsequent
HCN4 in vivo, NMs derived from iPSCs show a low dif- cardiomyocyte protocol development, earlier EC proto-
fuse expression. Hence for marker-based selection and cols initially relied on EBs and cardiogenic mesoderm
enrichment, it is important to characterize NMs for the induction followed by addition of angio-inductive fac-
presence of several additional nodal ion channel genes, tors, such as DKK1, VEGF-A, and FGF2.31 However,
such as KCNJ and HCN1. Typical NMs reveal small action this method was unable to generate high yield of ECs
potential amplitudes and duration (action potential dura- due to uncontrolled endothelial-to-mesenchymal tran-
tion 50: <100 ms) with upstroke velocities of <30 V/s.51 sition. Taking advantage of the progress of differen-
tiation protocols developed in EBs, several monolayer
protocols were developed using CHIR99021, wherein
Derivation of Nonmyocytes to Engineer Cellular T-Brachyury mesodermal cells primed in endothelial
Diversity in Cardiovascular Models growth media were sufficient to drive endothelial spe-
Other than cardiomyocytes, the heart is composed of non- cific fate determination.62 Several modifications to this
myocytes, such as endothelial cells (ECs), cardiac fibroblasts protocol with the addition of endothelial-inductive fac-
(CFs), smooth muscle cells (SMCs), tissue-resident macro- tors, such as VEGF, BMP-4, and forskolin, were found
phages, and natural killer (NK) cells. The nonmyocytes are to enhance the yield of ECs in the presence of TGF-
functionally intertwined with the cardiomyocytes and are β inhibitor to limit endothelial-to-mesenchymal transi-
important in physiology and pathophysiology. Development tion.62–64 ECs have also been derived from PSCs from
of protocols focused on derivation of nonmyocyte subtypes hemogenic precursors such as CD34+ cells either by
helps to discern mechanisms that drive both maturation and delivery of exogenous hematopoietic cytokines65 or
cardiac dysfunction due to cell-cell interactions in coculture culturing CD34+ cells61 in endothelial growth media. A
studies.52,53 Given the important role of nonmyocytes in car- recent study explored derivation of ECs using BMP/
diac physiology, it is pertinent to derive them reproducibly Activin-A signaling to generate progenitors of both mid-
from iPSCs to understand disease processes that result primitive-streak cardiac mesoderm and posterior hemo-
from cellular crosstalk in a de novo manner. genic mesoderm.66 Such scalable approach allows for


further investigation into EC phenotypes that are car- process together with vascular SMCs from the epicardial
diac tissue-specific versus the arteriovenous EC deriva- progenitors expressing PDGFR-β, whereas PDGFR-α
tives to study the cell-specific roles in a disease. expressing epicardial cells diverge and give rise to CFs.
PDGFR-β-derived cardiac pericytes are characterized
Smooth Muscle Cells
by expression of NG2 and ACTA2.79 Recent reports also
Vascular SMCs play an important role in maintaining
indicate that cardiac pericytes can arise from endocar-
vascular tone and blood pressure. During development,
dial endothelial intermediates,80 which develop as low
SMCs primarily emerge from lateral mesoderm, paraxial
NG2-expressing PDGFR-β cells, eventually leading to
mesoderm, and neuroectoderm. Several methodologies
high NG2 expressing pericytes after integration into the
have been introduced based on EB differentiation67 or
capillaries.
differentiation of selective CDH5 (cadherin 5)− cell popu-
lations68 in smooth muscle medium containing VEGF and Tissue-Resident Macrophages and NK Cells
bFGF.11 SMCs show specific markers such as ACTA2 Cells of the monocytic lineage originate from the hemo-
(α-smooth muscle actin) and CNN1 (calponin 1). Based genic progenitors from the mesoderm. Most tissue-res-
on these markers, >90% purity was obtained through ident innate immunity cells such as macrophages arise
negative sorting or metabolic selection, although the lin- from circulating blood monocytes. Monocyte differentia-
eage origin of the SMCs was unclear. The discovery of tion cascade begins with CD34+/KDR+ hematopoietic
Wnt mediated mesodermal induction made it feasible to progenitors to erythromyeloid progenitors (EMPs), which
derive SMCs of the mesodermal origin that exclusively further give rise to cells of the erythroid and myeloid lin-
expressed PDGFRB (PDGF [platelet-derived growth eage. Further differentiation of EMPs to monocytes is
factor] receptor B).64 Similarly, it was shown that meso- dependent on exogenous stimulation with hematopoi-
dermal mesenchymal progenitors expressing APLNR etic cytokines. For monocyte derivation, CD45+/CD73+
(apelin receptor) and the PDGFA receptor when exog- EMPs is induced with exogenous cytokines such as IL
enously treated with TGF-β and sphingophospholipid (interleukin)-3, IL-6, TPO (thrombopoietin), SCF (stem
led to SMCs expressing ACTA2 and CNN1.69 To mimic cell factor), and GM-CSF (granulocyte-macrophage col-
generation of in vivo-like lineage-specific SMCs, Cheung ony-stimulating factor). Continued stimulation with IL-3,
et al. introduced a stepwise protocol that gave rise to IL-6, and M-CSF (macrophage colony-stimulating factor)
mesodermal and ectodermal progenitors via exogenous further gives rise to CD45+/CD14+ monocytes. These
delivery of FGF2, BMP-4, and PI3K inhibitor, followed by monocytes can be further polarized using lipopolysaccha-
TGF-β inhibition to derive neural crest lineage through ride and IFN (interferon)-γ to derive M1 macrophages,
ectodermal precursors. These precursors led to the for- or via IL-4 stimulation to derive M2 macrophages.81,82
mation of SMCs when supplemented with (PDGF)-BB Other than the monocytic-derived macrophages from
and TGF-β for 6 days.70 Due to the immature pheno- the blood, recent lineage tracing studies have shown the
type of the SMCs, several attempts have been made presence of 2 unique subsets of cardiac tissue macro-
to obtain a mature contractile SMC phenotype through phages expressing CCR2+/Ly6c+ and CCR2−/Ly6c+.83
overexpression of myosin heavy chain MYH11.69,71 To One of the key questions that requires further investi-
date, iPSC-SMCs have been used to study pathologi- gation is whether we can generate macrophages that
cal mechanisms in several vascular diseases, such as resemble cardiac tissue-resident macrophages. In a
hypertension,72 atherosclerosis,73 and supravalvular aortic similar fate determination route as the macrophages, NK
stenosis among others.74,75 cells also emerge from EMPs. To direct in vitro differen-
tiation of NK cells, EMPs are supplemented with FLT3L
Pericytes
(fms-like tyrosine kinase 3 ligand), IL-3, IL-15, and IL-7
Pericytes together with SMCs stabilize newly formed
over 4 weeks to generate CD45+/CD56+ NK cells.84
vasculature through physical and molecular interactions
The resulting NK cells have been shown to be function-
with the endothelium. In the heart, the absence or deple-
ally active, producing cytokines, such as IFN-γ and TNF
tion of pericytes can result in vascular leakage and hem-
(tumor necrosis factor)-α. Studies using these cells in a
orrhaging in the microvasculature. The loss of pericytes
coculture or with cardiac organoid (CO) models will help
due to disease or toxicity can change the shape of the
reveal inflammatory processes that lead to monocyte
blood vessels and compromise permeability.76 Pericytes
recruitment in inflammatory cardiac diseases.
emerge mainly from the epicardium,77 lateral mesoderm,
and paraxial mesoderm.78 Mesodermal pericytes are
obtained through endothelial intermediates expressing Identifying Biological Variability for Better
CDH5 and PECAM (platelet endothelial cell adhesion Differentiation Outcomes
molecule) which in the presence of FGF2 and PDGF-
BB give rise to NG2 (neural/glial antigen 2)-expressing Despite the advances made in deriving cardiovascular
immature pericytes.69 Cardiac tissue-specific pericytes cell types in a dish, there are several challenges that
develop through an epithelial-to-mesenchymal transition stem from the inherent variability among patient-derived

iPSC lines.85 Hence, it is important to consider genetic cells undergo forced aggregation in a defined cavity
background and the effect of a known variant within a or mold. The common denominator between these 2
specific genetic background for both iPSC derivation classes of models is that they do not require exogenous
and differentiation. Some genetic changes are amplified extracellular matrix (ECM) as a primer to initiate cellular
in culture in both ESC and iPSC lines. For example, in self-assembly, but they have major differences in their
nearly 18% to 20% of the lines screened by the Interna- patterning or spatially organized features. However, the
tional Stem Cell Initiative, a gain of prosurvival oncogenic main difference between the resulting constructs after
mutations such as 20q11.21 is seen.86 Karyotypically spontaneous cardiogenesis and cellular assemblage lies
increased expression of such oncogenes often down- in the patterning or spatially organized features.99
regulate differentiation associated genes,87 although COs are generally used as broad term for precardiac
the influence of epigenetic factors is mostly removed structures and feature-specific organoids, which, in part,
due to the reprogramming process.88 Some residual constitute the cell or cardiac tissue-like assembly in a min-
DNA methylations could influence the differentiation iature form. Terminologies, such as gastruloids, cardiods,
efficiency based on source of the donor cells.89 Large- heart-forming organoids, and heart organoid, are confus-
scale iPSC expression quantitative trait loci studies fur- ing due to their striking differences in the features exhib-
ther point to the differences in genetic variants across ited by different CO models. In addition, it is challenging
different individuals are higher than interindividual dif- to identify the model based on the in vivo morphogenetic
ferences.85,90 Such interdonor variability introduced by stage of cardiac development. Due to the current limita-
the variants that affect the differentiation genes91 and tions in the organoid models which do not resemble mor-
by the protocols themselves, affect the activation of phologically identifiable heart or heart chambers, there
gene regulatory networks that define cell fate.92,93 On a is a need for unifying classification based on the level of
protein level, similar studies with >200 iPSC lines from patterning and cavity formation. COs that do not exhibit
151 donors that compare protein quantitative trait loci any morphogenetic or chamber-like features can be col-
suggest phenotypic outcomes are also influenced by lectively categorized as amorphic COs, whereas single
protein expression levels. In cases of rare or unknown or multicavity-forming organoids can be categorized as
variants, such differences may confound our under- cavity-forming COs. Cardiac microtissues or assembloids
standing when investigating risk factors for a specific are user-defined multicellular assemblies that are formed
disease causing phenotype.94 One of the approaches to with predifferentiated cardiomyocytes with or without
tackle the variability is to apply rigorous characterization nonmyocytes, such as CFs, ECs, and SMCs. These mod-
protocols to determine genomic integrity between pas- els are limited in terms of cellular diversity but allow for
sages within the same line,95 across different clones,96 dissecting cell-cell interactions in a controlled microenvi-
and across lines from different donors. Outliers based on ronment. Most CO and assembloid models do not adopt
variation in genes pertinent for somatic differentiation a rod-shaped morphology reminiscent of an adult cardio-
can be screened out by defining an outlier enrichment myocyte, which is a hallmark of maturation. Using tissue
scale based on whole genome, transcriptomic, and pro- engineering techniques, the differentiated cell types can
teomic profiles.97 Such a system can also be established be embedded in a suitable ECM to achieve cardiac tis-
for genetic and nongenetic disorders that are classified sue-like anisotropy, and exert strain in a uniaxial direction
based on disease-specific and rare variants.98 due to passive tension offered by deformable substrates.
In general, these ECM-guided constructs are broadly
classified under engineered heart tissues (EHTs). How-
SEMI-HIGH THROUGHPUT STEM CELL- ever, the term EHT does not provide a reasonable com-
DERIVED 3D CARDIAC STRUCTURES mensurability to morphology or the tissue composition of
Redefining Terminologies: Distinguishing CO a human heart. Regardless of the form factor (strip, ring,
or patch), ECM-based 3D tissue preparations composed
Models, Assembloids, and Anisotropic Cell- of ventricular cardiomyocytes with or without supporting
Based Models nonmyocytes is a tissue-like representation of the myo-
COs or self-assembled microtissues or assembloids are cardium.100 Therefore, engineered heart muscle would
broadly defined as 3D tissue surrogates composed of be an apt terminology for better comprehension for both
different cell types that reasonably mimic cellular het- users and a nonspecialized audience.
erogeneity and finite functions of the target organ. Gen-
eration of COs involves germ-layer specification through
activation or nodal/activin/BMP signaling using natu- COs: Mimetics for Cardiogenesis
rally occurring morphogens or small molecule chemical In the past several years, there has been tremendous
analogs. Developmental COs are believed to undergo efforts focusing on generating developmental COs to
developmental trajectories as seen in vivo, whereas study cardiac biology and regeneration.101 In vivo, the
microtissues assembled from terminally differentiated chronology of heart formation begins with the appearance


of first heart field giving rise to the cardiac crescent, fur- organoids introduced by Drakhlis et al114 presented a
ther merging into a linear heart tube that gives rise to left layered self-assembly of the COs consisting of endo-
ventricle and parts of atria. The emergence of SHF drives dermal core, endocardial cell layer, a myocardial layer,
arterial and venous polarity in the tube-like stage, further and interspersed liver anlagen and septum-like cells. In
guiding the folding and chamber formation that gives rise this protocol, the bulk of the cardiomyocytes arose from
to right ventricle, outflow tract and parts of atria.102,103 The SHF giving rise to higher ventricular and a smaller frac-
myocardium formed from the mesoderm is sandwiched tion of atrial-like myocytes.
between the endocardium and epicardium, demarcating One of the main reasons for the disproportionality of
a layered assembly. germ-layer assembly and maturation could be because
Most in vitro CO models are derivatives of previous in vivo Wnt pathway modulation occurs in a region-spe-
protocols that first demonstrated the role of transcrip- cific and cyclically controlled manner, which is challeng-
tional regulators driving mesoderm and cardiac dif- ing to achieve in vitro while using small molecules as
ferentiation in vivo.31,32 Wnt modulation using naturally they randomly diffuse in a static culture setting. There
occurring in vivo morphogens such as BMP and Activin- are other examples wherein cavity-forming COs have
A or chemically synthesized small molecules such as been developed using BMP and Activin-A molecules to
CHIR/IWP-2 is used to obtain cardiomyocytes from induce microcavities within the COs (Figure 2A). These
ESCs or iPSCs. Before the adoption of 2D culture pro- cardiac morphogens dictate the cell fate and trajec-
tocols for higher yield of cardiomyocytes35,104,105, earlier tory in a dose-dependent manner as shown in one
methods obtained cardiomyocytes from spheroid EBs example wherein a higher BMP-4-to-Activin-A ratio
formed from aggregates of ESCs or iPSCs.31,32,106–110 doubled the atrial cell fraction compared with that of
Building on the EB differentiation approach, recently ventricular cells within the COs, leading to the enrich-
developed CO models have been successful in dem- ment of nonmyocytes from epicardial and endocardial
onstrating hallmarks of early cardiomyogenesis through progenitors.115 Similarly, in an improved cavity-forming
the formation of cardiac mesoderm, endoderm, coemer- CO model called cardiods, a multitude of cardiogenic
gence of gut-like structures, chamber-like cavities, and factors including both naturally occurring and chemi-
EC networks induced by vascular growth factors. In one cally derived small molecules were shown to drive
of the first examples, EBs were subjected to Activin- cavity formation. The resulting concentric assembly
A and BMP that gave rise to distinct first heart field of epicardium and myocardium enveloped by a single
and SHF regions but lacked polarity and structural fea- central cavity was lined by a layer of ECs.116 The cav-
tures of a cardiac crescent or tube-like structures, for ity formation was a result of higher Wnt activation that
which it was aptly termed as a pre-CO. Using a small adversely affected development of a myocardium. The
molecule-based approach, the emergence of multiple addition of epicardial cells and subsequent self-assem-
lineages during gastrulation was demonstrated with bly led to repatterning which mimic the exterior epicar-
mouse ESCs that gave rise to cardiac crescent progeni- dial cell layer. However, it is important to note that this
tors, neural, mesodermal, endodermal derivatives, and model lacks endodermal cells and temporal chronol-
hematopoietic progenitors.111 ogy of in vivo development from crescent-like stage,
Applying the same principles of gastrulation that tube formation, and looping that precede chamber for-
provides a primer for multiple germ-layers and isotropic mation. The unique characteristics of each model are
assembly, Rossi et al112 using mouse iPSC-derived EBs dissimilar to the early heart due to lack of fine-tuned
were able to enrich cardiac structures within gastruloids control over spatially controlled signaling aspects of
by modulating Wnt signaling and addition of cardio- morphogenesis (Figure 2B). Despite the concerns of
genic factors such as ascorbic acid, bFGF, and VEGF. reproducibility, a thorough interrogation of these pro-
The gastruloids supported the formation of primitive cesses may allow the tracing of cellular trajectories
gut-like structures that codevelop with cardiomyocytes useful to studying congenital diseases or aid in the
during embryogenesis. However, despite the appear- drug development process. Therefore, it is imperative
ance of cardiac progenitors and tube-like formation, it that a translational fit for purpose be defined based on
does not follow in vivo stages that lead to diversification the similarity between the CO model of choice and the
of atrio-ventricular population followed by looping to disease being modeled compared with the relevant in
form 4 chambers. In a slightly different approach, Silva vivo heart development stages.
et al113 used human iPSCs to form gastruloid structures
but deviated from spontaneous assembly by early dis-
sociation during the organoid formation, followed by Cardiac Assembloids and Microtissues
cellular reconstitution into spheroids. It remains unclear Cardiac assembloids or microtissues are beating clus-
how disruption of self-organization may provide in vivo- ters of cells formed by controlled aggregation and
like patterning in the resulting gastruloid. Heart-forming self-organization of stem cell-derived cardiovascular

Figure 2. Embryoid body-like human

cardiac models.
Summary of recent noteworthy human
cardiac organoid (CO) models that mimic
some features of heart development in
vivo. A, The COs can be classified based
on the presence or absence of cavity
that resemble chamber-like features
during early heart development. B, A brief
summary of the morphogenetic factors
or small molecules used to direct the
formation of distinct precardiac structures
in vitro. Act-A indicates Activin-A; BMP,
bone morphogenetic protein; CHIR, an
inhibitor of glycogen synthase kinase 3β;
ECM, extracellular matrix; FGF, fibroblast
growth factor; RA, retinoic acid; and VEGF,
vascular endothelial growth factor.
cell types or from established human cell lines. In com- function by improving cardiomyocyte Ca2+ kinetics
parison to COs, the user-defined control achieved in and electrophysiology. Owing to such benefits of non-
these types of models depends on its composition, size myocytes and their role in maturation of CMs, recently
and shape, simplicity in fabrication, and high reproduc- developed protocols118,119 now combine all 3 major
ibility. Several different cell types, such as CMs, ECs, cell types to dissect cell-cell responses in disease and
and CFs, can be coaggregated in an ECM-free form drug responses. Applications of well-defined microtis-
to derive beating spheroids of uniform size and struc- sues have also been demonstrated for the evaluation
tures. These resulting structures are specifically termed of cardiotoxicity and injury-related fibrotic response or
as cardiac microtissues or assembloids. Giacomelli et ischemia to the myocardium. For example, Richard et
al53,117 developed a microtissue model through codif- al120 demonstrated that cardiomyopathy due to injury
ferentiation of CMs and ECs wherein the presence of or cardiotoxicity can be recapitulated on a microtissue
cardiac stromal cells enhanced gene expression and platform using primary human CFs and umbilical vein


ECs, together with iPSC-CMs. The microtissues thus misalignment of the sarcomere or exhibit a short sar-
formed showed the presence of a putative EC net- comere length, leading to reduced contractility. These
works resembling a vascular network with lumen-like results suggest that the mechanical and geometric
structures. environment of ECM affect the heart function and dis-
Cardiac microtissues or assembloids has a unique ease development.133–135
niche in disease modeling and cardiotoxicity high-
throughput screening due to its facile and cost-effective
fabrication methodology.121 More importantly, the con- Structural, Electrophysiological, and Mechanical
trol of over cellular composition allows for ease of dis- Connection of Cell-Cell to Cardiac Tissue
secting cellular crosstalk between cardiomyocytes and Cardiac tissue is formed as the cells are connected
nonmyocytes in the context of in vitro maturation and end-to-end (Figure 3C). Here, ECM geometry also
disease progression in a dish. Further development of plays a vital role in regulating cell-cell coupling.136 In
the platforms will be crucial to understand the long-term the in vitro cell-pair model, cell-ECM adhesion con-
benefits or consequence of in vitro maintenance of such strains the cell-to-cell junction morphology and regu-
heterotypic microtissues.122 lates tissue assembly (Figure 3D).137 As the assembly
of FAs leads to ECM shape-dependent cell-migration
and myofibrillogenesis at an early stage of cell culture,
ECM FOR STRUCTURE AND FUNCTION OF the FAs near the cell-cell interface are disassembled
THE HEART to interconnect their myofilament. The ECM geometry
In addition to cell-cell interaction in a confined geom- also organizes the laminar architecture of cardiac tis-
etry, cell-ECM interactions are essential to obtain cel- sue.138 Geometric line patterns of ECM proteins such
lular polarity and cell-cell junctions in axial direction for as fibronectin allow for inter/intracellular organization
mechanical and electrical coupling. In 2D cultures, the of cardiac tissue (Figure 3E). This affects the align-
cell-cell junctions are isotropic whereas in native heart ment of cytoskeleton architecture.
tissue the CMs are highly polarized within fibrous ECM- Cell-to-cell junction location also affects the struc-
forming intercalated discs with adjacent cells axially, ture and function of the heart. The intercellular junc-
while forming costamere complexes with surrounding tions provide mechanical binding, force transmission, and
ligands in the lateral direction.123,124 Hence, engineering ionic communication among cells.139 Adherens junctions
the right extracellular environment is not only significant and desmosomes form direct links to the cytoskeletal
for functional coupling but also for force transduction structure, thus transmitting contractile force. Gap junc-
through load bearing contractions. tions play a critical role in intercellular communication
by transmitting electrical impulses. Cx43 (Connexin 43)
is the most abundant gap junction protein in the myo-
ECM in Regulation of Myofibrillogenesis cardium. In a normal adult heart, Cx43 expression and
Cell-ECM binding plays an important role in myofibrillo- localization mainly occurs at the longitudinal cell ends,
genesis during cell development. When the cells adhere called intercalated discs. A direct correlation between
to the ECM, focal adhesions (FAs) which are multipro- Cx43 immunosignal and electrical intercellular conduc-
tein complex linking the ECM and cytoskeletal actin tance suggests that Cx43 plays an important role in cell
are assembled,125,126 activating downstream signaling communication throughout the heart.140 In in vitro stud-
to regulate cytoskeletal and myofibril assembly along ies have found an intimate correlation between mechani-
the ECM structures (Figure 3A).127,128 When cardiomyo- cal adherens junctions and electrical coupling junctions,
cytes are cultured on the single-cell sized rectangular and Cx43 expression occurs after the formation of the
micropatterned ECM, localized FAs are observed at the adhesion junction protein, N-cadherin.141 Therefore, the
corner as the adhered cells are spread to ECM pat- remodeling of cell-to-cell adhesion is occurred due to a
tern. From the corner, FAs initiate actin polymerization stiff ECM environment, may influence gap junction redis-
and organize myofilaments parallel to the boundary of tribution, which can disrupt abnormal electrophysiologi-
ECM pattern,129 resulting in anisotropic organization cal leading to arrhythmogenesis that is associated with
of cytoskeletal architectures.127 Rectangular patterned many cardiomyopathies.137
myocytes with a 7:1 aspect ratio show anisotropic The end-to-end connections of cardiomyocytes
cytoskeleton structure, exhibiting maximum contractil- are arranged on cardiac tissue within a collagen ECM
ity and fast Ca2+ handling compared with shapes with fibrous network in the heart (Figure 3E).142 In in vitro
other aspect ratios (Figure 3B).130,131 This corresponds cardiac tissue models, the inter/intracellular organi-
to cardiomyocytes found in the healthy adult heart.132 zation is also affected by geometric patterns of ECM
However, ECM patterns with aspect ratios lower proteins, such as fibronectin or gelatin.143 The aligned
or higher than 7:1 and stiff ECM substrates cause cytoskeleton structure and junction proteins results

Figure 3. Hierarchical structure of cardiomyocyte and extracellular matrix (ECM) structure in the heart.
A, Cardiomyocytes shows aligned myofilament structure which is connected to ECM through costamere. Costameres contain focal adhesions
(FAs) complex connecting with cytoskeletal actin filaments. B, A cardiomyocyte cultured on 1:7 aspect ratio of rectangular microcontact printed
ECM pattern, showing intracellular organization using a DIC image and immunostaining images for vinculin, F-actin (Actin filament), and sarcomeric
Alpha-actinin in (i–iv), respectively. Scale bar, 10 µm. Reprinted from Bray et al130 with permission. Copyright ©2008, Wiley-Liss, Inc. C, Junction
formation is occurred at the intercalated disc where the cells are connected end-to-end. D, Immunostained images of cardiomyocytes cell-pair
images showing FAs and junction formation and intracellular organization according to culture days. Scale bar, 10 µm. Reprinted from McCain et
al137 with permission. Copyright ©2012, NAS. E, Fibrous ECM structures supporting cardiomyocytes with their intra/extracellular organization. F, In
vitro cardiac tissue organization by ECM geometry patterns. Scale bar, 20 µm. Reprinted from Lee et al143 with permission. Copyright ©2022, AAAS.
G, Hierarchical cardiac muscle tissues are organized into the heart, inducing cyclic blood pumping with coordinated tissue contraction.
in faster action potential propagation in the longitudi- An important step in constructing the 3D organ-level
nal tissue direction than the lateral direction.138,144 A in vitro heart is assembling cardiomyocytes into a 3D
monolayer of anisotropic neonatal rat VMs exhibited a chamber structure that pumps fluid in and out cyclically.
longitudinal to transverse conduction velocity ratio of Collagen, the most abundant protein in cardiac ECM, is
1.89±0.38, similar to the ratio found in the in vivo rat mixed with cardiomyocytes and cultured for 10 days to
heart.145 This, in turn, activates the organized cardiac fabricate a balloon-shaped chamber.150,151 The chamber
tissue that leads to synchronized contraction. Healthy also exhibited responses to drugs that induced a fast-
cardiomyocytes with well-organized tissue structure beating frequency or weaker pumping function. Three
show strong contractile stress that can be analyzed dimensional printing is another promising manufacturing
by muscular thin film (MTF) cantilever146 or EHT plat- method for building organ models due to its simple design
forms.147,148 Therefore, the intra/extracellular structure control and reproducibility. However, ECM-based hydro-
of cardiac tissue is inextricably related to electrical and gel inks used in 3D printing are usually soft or fluidic,
mechanical functions which are important factors in and do not retain 3D shape without additional support.
determining cardiac function. To overcome this challenge, researchers have developed
sacrificial baths that support 3D structure temporarily
during printing.152,153 Collagen gel inks and cardiomyo-
In Vitro Organ Model of the Heart cyte inks are printed in the shape of a ventricle chamber.
The in vitro heart has been studied on multiple scales, These printed ventricle models show synchronized con-
from cellular assays to engineered tissue by recapit- traction, resulting in anisotropic Ca2+ wave propagation,
ulating the structure and function of the heart (Fig- reaching 16% wall thickening at peak systole,153 and a
ure 3F).149 The human heart consists of hierarchical 0.7% ejection fraction.154
cardiac muscle tissues organized into chambers which Considering the main functions of ECM other than
pump blood throughout the body with coordinated tis- cell adhesion, tissue scaffolds provide structural integ-
sue contraction (Figure 3G). Three dimensional organ- rity in cell assembly, as well as mechanical support to
level in vitro models of the heart provide a direct in vivo the tissue. Early 3D in vitro organ-level tissue models
comparison of clinically relevant functional parameters used decellularized rat heart matrix as a tissue scaf-
such as pressure-volume change, stroke volume, and fold to preserve the underlying ECM.155 Recellularizing
Ca2+ propagation signal. the heart by intramural injection of cardiac-derived cells


and perfusion of ECs allowed the building of an artificial periods by months, allowing for the assessment of drug
heart after 8 days of culture. Alternatively, synthetic bio- efficacy and toxicity with chronic drug administration or
compatible fiber scaffolds also replicate the role of ECM gene therapy treatments (as opposed to acute studies).
by facilitating cell alignment and providing mechanical However, the maturity of human iPSC-CMs represents
support to cardiac tissue.156 Aligned microscale fibers in significant obstacle when attempting to replicate com-
a ventricle-shaped scaffolds can be used to build orga- plex cardiomyopathies in vitro. For that reason, rodent
nized tissue structure in a 3D in vitro model of the heart cell models, coupled to well understood animal models
with electromechanically coupled cardiomyocytes, result- (eg, spontaneously hypertensive rat), suggest that in so
ing in pressure-volume change by the tissue-engineered far as diseases that affect adults are concerned, animal
ventricle contraction. models will remain an important platform for drug discov-
However, in vitro ventricle models pump fluid with 50 ery for the near future.
to 200 times lower efficiency than in vivo ventricles.156 For Microphysiological systems (MPSs), commonly
better performance, additional anatomic features need to referred to as organs-on-a chip, are great potential
be recapitulated in in vitro heart models. Tissue matura- tools that can minimize the uncertainty of the existing
tion significantly affects pumping strength.147 Although preclinical animal model testing.165,166 Cardiac MPS can
long culture periods often prove challenging, mature be defined to replicate the cellular microenvironment of
cells tend to show stronger contractility. This requires the diseased heart with well-defined geometric, topo-
advancement in better long-term cell culture techniques logical, mechanical, and biochemical cues drawn from
highly relevant to building accurate heart models. postmortem histological studies of explanted hearts.
In vitro tissue thickness is another challenge. Ven- Compared with conventional biochemical assays, car-
tricular myocardium consists of laminar tissue that is diac MPSs allows high throughput and real-time moni-
approximately 4 cells thick.149,157 However, cells situated toring of electrophysiological or contractile functional
deeper than 100 µm in in vitro tissue do not survive due changes upon electrical or biochemical intervention.167
to the lack of nutrient and oxygen diffusion. Integrating More recently, methods to conduct real-time immuno-
vascular structure within 2D cardiac muscle tissue is assays on organ chips further enhance the ability to
one approach to solving the thickness limitations.158,159 understand the mechanisms of efficacy or toxicity.168
Another approach is to build 3D scaffolds that allow for Eventually, these tools will be used to screen patients
cell infiltration or in situ cell differentiation to recapitu- for efficacy and toxicity before their enrollment in a clin-
late the layered laminar tissue structure of the heart.154 ical trial, lowering the risk associated with the trial and
In addition, studies have found that cardiac output per- reducing the time and cost of bringing medicinal and
formance is highly influenced by tissue direction, with genetic therapies to market.169,170
helical direction exhibiting better performance than
circumferential direction.160,161 Developing advanced
techniques for precisely controlling tissue direction will Electrophysiological Assessment of Cardiac
improve cardiac output in future 3D organ models, and Tissue on a Chip
will allow us to develop more powerful preclinical test- Cardiomyocytes generate action potential and pass the
ing platforms. electrical impulses in an organized tissue direction that
leads to coordinated actions. Optical mapping is widely
used as a noninvasive assessment tool to visualize action
INTEGRATIVE CARDIAC
potential and its wave propagation to study the cardiac
MICROPHYSIOLOGICAL SYSTEMS electrophysiology (Figure 4A). Voltage sensitive dyes
Pharmaceutical research for drug development involves (eg, Di-4-ANEPPS) or Ca2+ sensitive dyes (eg, X-Rhod,
high-risk, long-term process, and substantial costs. Dur- Fluo-4) are used to monitor transmembrane potential
ing the drug development’s preclinical stages, animal changes or Ca2+ transient propagation, respectively.171,172
models are historically, and remain, the primary platform Measuring Ca2+ transient provides quantitative informa-
for drug efficacy and toxicity. However, fundamental tion of Ca2+ handling properties of the cardiac tissue,
differences between animal models and humans often such as conduction velocity and signal intensity.173 This
cause safety issues or low efficacy. For example, the allows us to investigate clinical phenotype of inherited
resting heartbeat in mice (≈600 bpm) is higher than heart diseases in vitro, such as recapitulating hallmark
human (≈60 bpm). Additionally, Ca2+ handling proper- features of catecholaminergic polymorphic ventricular
ties, myosin expression, and ion channel expression vary tachycardia by showing ectopic Ca2+ propagation from
significantly between animal models and humans.162,163 patient-derived cardiac tissues with rapid electrical pac-
Two-dimensional and 3D iPSC platforms offer advan- ing or adrenergic stimulation.172
tages over animal models in several respects.164 Human Multielectrode arrays are another tool to record elec-
iPSC-CMs can recapitulate inherited cardiomyopathies trophysiological function for high-throughput and long-
by using patient-derived iPSCs and also extend culture term readout platforms (Figure 4B).174–176 High-resolution

Figure 4. Cardiac microphysiological system for electrical and mechanical functional assessment.
A, Optical mapping system from in vitro cardiac tissue showing anisotropic electrical potential propagation in aligned tissue direction and
arrhythmias in disease model. B, Microelectrodes arrays measuring the local electrical potential changes, providing high spatiotemporal bioactivity
of the cardiac tissues. C, Muscular thin film (MTF) system to measure contractility by measuring bending force of the laminar tissues that are
formed on the cantilevers. D, Engineering heart tissues (EHT) system allowing for monitoring contractility of the cardiac tissue that causes cyclic
movement of the posts. E, Biowires deformation caused by contractile cardiac tissue as the tissue is sustained by wires.
electrode arrays can measure bioelectric activity with 2D laminar cardiac tissue layers on the elastomeric
high spatiotemporal resolution at multiple length scale thin films are one of the new cardiac MPS platforms
from cellular to tissue levels. As the electrodes are in con- for measuring the contractility of in vitro cardiac tissue
tact with outside cells, these devices monitor extracellu- (Figure 4C). The systolic shortening and diastolic resto-
lar electrophysiological signals, providing cardiac activity ration of cardiac myocytes organized into a syncytium
information in relative values, such as beat frequency and potentiate a cyclic bending motion of the laminated film
wavefront propagation. However, measuring the action allowing measurement of contractile stress.179 These
potentials in the intracellular space is also imperative to tools have been used to study complex diseases such
understanding quantitative electrophysiological functions as Barth syndrome, a genetic disease of mitochondrial
such as ion current modulation and membrane potential function that is characterized by muscle weakness and
changes. Membrane poration techniques such as elec- poor myofibrillogenesis.180 Engineered Barth syndrome
troporation or laser optoacoustic poration overcome iPSC-CM tissues from patients in the form of a MTF plat-
those limitations of the multielectrode arrays system form recapitulated the weak contractile function of the
by enabling the electrode arrays to assess intracellular disease, showing slow and weak bending deformation.
space.177,178 Integrating the advanced nano-fabrication Histological studies of the engineered tissues revealed
technologies and in vitro cardiac human tissue models that sarcomeres were poorly formed and aligned, and
in cardiac MPS will enable the real-time monitoring of that myofibrils were not laterally coupled, hence sug-
the intracellular action potential of cardiac tissues that gesting that the mitochondrial dysfunction has the col-
are exposed to test drugs178 or disease-inducing environ- lateral effect of rendering the myocytes unable to build
ments like hypoxia or ischemia.177 and maintain the contractile apparatus. Further experi-
ments went on to demonstrate the appropriate rescue of
the diseased tissue by therapeutic means. Studies like
Contractile Assessment of Cardiac Tissue on a these can be further enhanced by using MTF platforms
Chip integrated with strain sensors to enable real-time moni-
The cardiac ventricles are a 2D laminar tissues wrapped toring of contractile responses to changes in drug dose
around the ventricular cavities to form the 3D form. for high-throughput drug response studies.181,182 These
To approximate this architecture, MTF composed of technical capabilities are important because they will


allow unmanned study of acute responses to medicinal ECM hydrogel to support cell culture environments.
therapeutics in vitro. Bioreactors can also be used to incorporate microfluidic
Another approach for measuring contractility is using channels not only to support cell culture in 3D scaffolds
EHTs that are formed and suspended around 2 elas- but also providing a platform to analyze fluid movement
tomeric poststructures (Figure 4D).183 In some studies, generated by in vitro 3D chamber-shaped ventricle
fibroblasts have been cocultured on the EHT platforms models.156 While the tissue-engineered ventricle pumps
to promote cardiac tissue compaction, cell assembly, and fluid in and out of the chamber, the bioreactor can apply
spontaneous contraction.147,184 The tension between the external pulsatile pressure through the fluidic channel,
posts applies an auxotonic load to the papillary muscle- driving the intraventricular fluid flow. Integration of the
like tissues, which facilitates the longitudinal alignment valve structure restricts unidirectional flow, enabling
of the embedded cardiomyocytes. In addition, applying functional evaluation of engineered ventricular models
electrical stimulation with gradually increasing intensity through measurements of physiological pressure and
can accelerate the cardiac tissue maturation, resulting volume dynamics.
in physiologically relevant sarcomere length (2.2 µm), In addition, while drug testing in static cardiac MPS
mitochondria density (30%), and improved Ca2+ han- relies on diffusion-driven drug molecules, microfluidic
dling properties.147 The advanced maturation technique cardiac MPS offers a spatiotemporal gradient of drugs
enables testing drug effects that are consistent with clin- and biochemicals that can be recapitulated through fluid
ical outcomes. This property allows the EHT platform to flow across the cardiac tissue.188 Microfluidic MTF plat-
serve as a tool for studying patient-specific pathophysiol- forms can evaluate cardiac function under rapid and
ogy and disease mechanisms by recapitulating electrical continuous perfusion of drug solution, facilitating drug
and mechanical pathologies reported in cardiac disease efficacy testing with real-time continuous monitoring and
such as arrhythmogenic cardiomyopathy. Cell junctional high throughput. A microfluidic heart-on-a chip is also
protein mutation in the arrhythmogenic cardiomyopathy amenable to control medium oxygenation, creating cell
leads to disruption of sarcomere stability and organiza- culture conditions to induce acute hyperoxia to the in
tion, resulting in the impaired contractility.185,186 vitro cardiac tissue.177 This microfluidic MPS integrated
Biowires are another class of new cardiac tissue with multielectrode arrays provides assessment of real-
culture platforms. As the mixture of cell and ECM gel time disease progress and are useful for high-through-
is casted, the wire structure promotes self-assembly of put pharmacological studies by monitoring electrical and
cardiomyocytes, forming aligned tissue along the wire mechanical function of cardiac tissue.
roughly approximating ventricular papillary muscle.171
Applying electrical stimulation to the tissue during cul-
Multiorgan Assemblies to Model
ture promotes tissue maturity, resulting in increased
structural myofibril organization and Ca2+ handling prop- Pharmacodynamic Drug Interactions
erty. A more recent version of Biowire (Biowire II), which Medicinal therapeutics are transported to the target
allows cardiac tissue to be generated between 2 parallel organ(s) via blood circulation which takes them on a cir-
wires, enables contractility measurements (Figure 4E).187 cuitous route through organs that both absorb, react to,
Deformation of the wires due to tissue contraction was and metabolize the drugs. Multiorgans-on-a-chip, which
optically measured and translated into contractile force. interconnect 2 or more organ models in a single MPS,
Biowire II also provides a heteropolar tissue model that can recapitulate those dynamic processes of absorption,
combines tissues of different cell type formed on each distribution, metabolism, and excretion to provide accu-
wire, facilitating comparison of the drug effects on a spe- rate and efficient preclinical predictions of drug response
cific target tissue versus the control tissue. For example, in the human body.
heteropolar cardiac tissues with distinct atrial and ven- The endothelium of the vasculature plays an impor-
tricular chamber models have demonstrated chamber- tant role in regulating the delivery of drugs or molecules
specific drug responses because the administered drug to the heart muscle. Combining endothelial barrier and
only affects the target tissue model.187 With this tech- cardiac MTF tissue in a platform can enable the evalua-
nique, antifibrotic compounds can be tested on hetero- tion of the effect of endothelial barrier on drug transport.
polar cardiac tissues composites of fibrous and healthy When Ca2+ channel blocker isradipine is administered
heart tissues by the contractile functional and electro- through the endothelial barrier, the disruption of cardiac
physiological readout. contractile function was significantly delayed compared
with direct drug exposure to the cardiac tissues.182 In
addition, modulating permeability of the in vitro endothe-
Microfluidic Integrated Cardiac Microphysiology lial barrier can help regulate the temporal onset of car-
System diac drug responses.
Organs on chips with integrated microfluidics allow for Microfluidic systems provide platforms to simu-
the delivery of precise and predictable nutrients and late drug transportation from organ-to-organ in vitro,

enabling pharmacological studies in more physiologi- being correlated with profibrotic remodeling197 and IL-6
cally relevant conditions by recapitulating organ cross- being linked to reduced Ca2+ transients.193 Due to the
talk. For example, effects of an anti-cancer drug were complexity of these interactions, and their inherent mul-
different in a microfluidic organ-on-a-chip model that ticellular nature, limited works with in vitro models have
connected bone Ewing Sarcoma tumor and heart mus- been reported for mechanistically probing how these
cle tissue. The model’s results demonstrated lower car- processes evolve. In addition, further progress in obtain-
diotoxicity and tumor response in an integrated setting. ing more accurate quantitative information from the mul-
Whereas direct drug exposure to each isolated tissue tiorgans-on-a-chip and modeling methods to translate
significantly reduced both tumor viability and cardiac the information into in vivo results will help us achieve
function.189 Depending on the drugs, metabolites can the goal of developing human surrogate systems that
be also toxic to other organs or become an active form can replace animal models in the future.
to improve drug efficacy, which is one of the main rea-
sons for discrepancies between single organ in vitro
studies and clinical trial outcomes. Integrated liver and BREAKING THE HYPE CYCLE: CHOOSING
heart models in a single MPS can help evaluate the
THE RIGHT MODEL IN THE RIGHT
on-target drug efficacy on integrated liver tissue and
decrease off-target cardiac toxicity as the parent drug CONTEXT
is metabolized in the liver tissue model.190,191 As the depth of technical knowledge and subject matter
To further simulate the complexity of the human body expertise grows with the precise and accurate modeling
that involves immune, nervous, and vascular systems, of cardiovascular diseases using 2D and 3D models, the
multiorgans-on-a-chips have been developed by inte- challenge of fit to purpose design of the model remains.
grating with key functional organs models, such as liver, Lack of established quality control standards for iPSC-
kidney, heart, and brain.192,193 As researchers seek to CMs198,199 and limited regulatory guidance on the use of
make accurate quantitative predictions of human phar- models represents both an opportunity and a hurdle for
macological responses that are translated from accurate the field. Whereas toxicological studies may benefit from
in vitro test results, it is important to integrate physiologi- standardized models, drug discovery models may require
cally appropriate cardiovascular systems for organ-organ that MPSs are developed in such a manner as to yield a
connections. To build a semivascularized multiorgan-on- unique readout that maps to a clinical diagnostic.167 The
a-chip platform, researchers installed endothelial barriers primary context of use of the iPSC-based cardiac mod-
into a series of organ models and connected the organ els may be based on where the model fits among the 4
chips via vascular perfused fluid that was delivered by categories: (1) screening and drug response, (2) mature
an automated robot system.194 The endothelial barriers mono or polygenic disease mechanisms, (3) develop-
mimic physiological systemic transportation of small mol- mental disorders, and (4) environmental disorders and
ecules between organs, providing better predictions of cardiac diseases with late onset (Figure 5). Lack of con-
the relevant pharmacokinetic response. Arteriovenous sensus or diligence on the appropriateness of choosing
reservoir modules from multiorgan-on-a-chip are also a model is based on several such factors.200 One of the
used to mix drugs that pass through each organ com- main factors is the missing microenvironmental compo-
partments, simulating systemic circulation of the body.195 sition and cues (eg, cellular heterogeneity, flow, ECM,
Both microstructural recapitulation and functional mechanical stretch) in the model that is clinically known
analysis of cardiac MPSs are essential to study disease to contribute toward a disease phenotype. For example,
phenotypes and drug treatment responses. Integrating to accurately model interstitial tissue or aortic diseases
cardiac functional readout system into large number of that lead to fibrosis, in addition to the relevant cell types
the in vitro cardiac tissue have a potential to overcome such as patient-specific fibroblasts, SMCs, the model
the limitation of low throughput in vivo animal studies must also include a tailored, structural ECM component.
that minimize the cost and times in the drug discov- Second, clinical phenotypic manifestation should in some
ery process. But still more efforts are required to build form be measurable using assays that are precise, repro-
multiorgan-on-a-chips that incorporate cardiac tissue ducible, and devoid of bias.201,202
models close to the human body environment, such as
cardiac immune response. The inflammatory response Predictive Disease Modeling for Functional and
of the peripheral and central nervous systems plays a
key role in the development and persistence of cardiac
Preclinical Toxicology Responses Using High-
pathologies, with autoimmune diseases, such as rheu- Throughput Platforms and Multiomic Profiling
matoid arthritis, being linked with increases in cardiac Human iPSC-CMs have proven to be instrumental in the
inflammation, such as endocarditis, myocarditis, and evaluation of drug-induced toxicity for the past several
pericarditis.196 Furthermore, the ILs have been shown years. High-throughput studies using iPSC-CMs have
to have maladaptive effects on cardiac tissue, with IL-4 been performed for screening novel drug candidates,


Figure 5. The evolving paradigm of in vitro cardiovascular disease modeling.
A plethora of in vitro cardiac models have been developed over several decades. Induced pluripotent stem cell (iPSC) technology has fueled the
development of human cardiac models to accelerate predictability of cardiovascular disorders and drug responses to complement in vivo studies.
The flowchart offers a novice guide on general principles for choosing a more relevant model in the context of the disease in question, and
validity of the model based on an understanding of cellular contribution toward the disease, obtaining clinically translatable phenotype, and most
importantly the reliability and reproducibility for disease modeling. ECM indicates extracellular matrix.
off-target drug effects, and drug repurposing. Several surrogates for testing drug sensitivities but disorders
examples of chemotherapeutics used in the treatment that are caused by disruption of more complex cardiac
of various cancers have been profiled using iPSC-CM structures such as gap junctions can be well studied
models that show bystander cardiotoxicity.171,203 in 3D cardiac constructs. For example, 2D iPSC-CMs
Several pathological mechanisms with dominant generated from LQT patients showed spontaneous
phenotype can be assayed on 2D such as cytoskele- and frequent cellular arrhythmias, whereas in 3D EHTs
tal disarray caused due to truncation of proteins or ion arrhythmias were only observed when challenged with a
channels that result in arrhythmic disorders. Although QT prolongation agent.205 EHTs have also been used to
the success of modeling of the molecular events that study cardiotoxicity under loading conditions. Sunitinib,
lead to the disorder depends on the degree of matura- a TKI (tyrosine kinase inhibitor) at a clinically relevant
tion achieved in vitro using well-defined methodologies. concentration was shown to induce caspase-induced
For example, the β-adrenergic signaling is important toxicity in EHTs under afterload conditions.203 For high-
in cAMP signaling to model chronotropic responses. throughput toxicity profiling, cardiac microtissues that
In diseases such as Takotsubo syndrome where over are roughly a third in size to that of an EHT have been
stimulation of β-adrenergic leads to stress inotropy and used for high-content imaging in multi-well formats to
lipid accumulation can be faithfully demonstrated at monitor dynamic changes in structure and metabolism.
a cellular level given the comparable level of receptor Drug or small molecule diffusion kinetics in 3D culture
expression to that of an adult myocyte, and sensitiza- systems better represent in vivo diffusion barriers.206
tion as seen in patients with Takotsubo syndrome under Cardiac microtissues comprised of iPSC-CMs, primary
adrenergic stress.204 As alluded to previously, 2D iPSC- ECs and fibroblasts treated with sunitinib showed cyto-
based cardiovascular derivatives can serve as excellent toxicity above 10 µmol/L, whereas lower concentrations

on 2D culture format show severe toxicity due to higher technology. One of the key aspects of consideration is
drug bioavailability and uptake.207 There is substantial the materials used in the model and their compatibility
evidence that complex cardiac model systems are ame- with the intended target cells or engineered tissues. For
nable to high-throughput platforms to measure hundreds example, polydimethylsiloxane is widely used to fabri-
of parameters through an iterative approach. With the cate MPS devices because of its versatility in molding
progress in next generation sequencing technologies and prototyping. However, polydimethylsiloxane can be
and standardized approaches to perform genome-wide unsuitable for drug-based studies due to its property of
analysis, significant advances can be made in disease drug adsorption which may reduce the bioavailability in
modeling and drug discovery using in vitro cardiac mod- the device for the cells and tissues. Hence, other mate-
els for prospective clinical outcomes. rials such as silicon or thermoplastics could be widely
adopted to eliminate the concerns of unwanted sub-
strate interactions within the multicellular/tissue chips.
CHALLENGES AND OUTLOOK Standardization of these parameters will also reduce the
Advancements in iPSC technology have to a greater cost of obtaining bespoke materials and shift the focus
extent eliminated the dependence on primary cells or on quality control and end-user reliability measures for
tissue sources with the promise of large and reproduc- mass production.
ible quantities of various cardiovascular cell types for One of the major hurdles using multicellular cardiac
research. With several emerging precision medicine ini- models is to find the right balance of nutrient medium
tiatives, there is a need to obtain high-quality patient- to meet cell-specific demands. Currently, specialized
derived iPSCs at a manufacturing scale. However, the medias are developed for individual cell types. Combi-
challenge of scale also comes with the need for rig- nation of multiple cells in organoids, engineered tissues,
orous standardization methods in obtaining iPSCs for and MPS systems require a universal or customized
differentiation into cardiovascular cell types. Despite media formulations. In advance of the availability of a tai-
these standardized methodologies, several reports have lored or universal medium, an approach that can be pur-
detected significant loads of single nucleotide variants sued in the interim is to cater each cell population with
due to age of donors,208 prolonged culture,209 clonal or its specialized medium on an MPS platform in multiple
somatic cell variation,210 and genetic changes due to single-pass circuits, integrated with an external loop for
pluripotency induction methods.211 Given the variability exchange of media metabolites.213 In the meanwhile, to
that may be introduced in iPSC-derived cardiovascular develop a design criterion for optimal universal medium a
cell types, in addition to standard karyotyping assays, thorough characterization of the cell secretomes must be
whole genome exome sequencing and chromosomal made independently for each cell type using proteomic
microarrays should be employed to detect abnormali- analyses for the assessment of metabolite concentra-
ties in iPSCs.97 As previously discussed, since the tion and waste products. This will help trace cell-spe-
genetic variability is often larger than the expression of cific responses in coculture setup and reveal potential
a disease phenotype, it is important to evaluate multiple negative influence in cell behavior due to altered culture
controls and patient lines to assess the nonoverlap- environment.
ping differences with high sensitivity and specificity.95
For comparisons, both test and control lines should
be as closely matched as possible for age, sex, and CONCLUSIONS
ethnicity.212 The heart is a complex organ and has greater influence
Three dimensional cardiac models are touted as more on other organ systems due to its key role in provid-
effective predictors for disease modeling and therapeu- ing nourishment. Hence, studying tissue and organ-
tic testing due to the complexity and physiological rel- level dynamics using in vitro cardiac models offers an
evance. The common denominator in these models is unprecedent opportunity in heart regeneration, dis-
that they all are static systems which are limited in reca- ease screening, and drug development. Prima facie
pitulation of flow mediated stresses, nutrient gradients, the goal of the emerging engineered cardiac models
vascular interaction, or systems-level biomolecular inter- is to build greater physiological relevance, and confi-
actions offered by MPS systems. To develop the platforms dence in functional end points for the development of
that aid in generation of these models, standardization prospective clinical strategies. Along the same lines,
is essential for commercially availability as an off-the- long-term monitoring of the models will not only model
shelf product for widespread use. Most MPS devices acute effects but also assist in longitudinal studies
originate in the labs that develop them over many years, through the course of patient care in the clinic. One of
hence commercializability of such platforms depend on the major breakthroughs in the translation of cardiovas-
efforts made in standardization of the fabrication pro- cular disease has been our ability to model functions
cess, reproducibility of results, and transferability of the and responses to biological perturbations, stressors,


or compounds. For wider adoption of these advanced
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ARTICLE INFORMATION 10.1161/CIRCRESAHA.120.318049
12. David R, Brenner C, Stieber J, Schwarz F, Brunner S, Vollmer M, Mentele
Affiliations E, Müller-Höcker J, Kitajima S, Lickert H, et al. MesP1 drives vertebrate
Stanford Cardiovascular Institute, Stanford University School of Medicine, Stan- cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-
ford, CA (D.T., C.A., J.C.W.) Division of Cardiovascular Medicine, Department of signalling. Nat Cell Biol. 2008;10:338–345. doi: 10.1038/ncb1696
Medicine, Stanford University School of Medicine, Stanford, CA (D.T., C.A., J.C.W.) 13. David R, Jarsch VB, Schwarz F, Nathan P, Gegg M, Lickert H, Franz WM.
Disease Biophysics Group, John A. Paulson School of Engineering and Applied Induction of MesP1 by Brachyury(T) generates the common multipo-
Sciences, Harvard University, Boston, MA (S.C., K.K.P.) Harvard Stem Cell Insti- tent cardiovascular stem cell. Cardiovasc Res. 2011;92:115–122. doi:
tute, Harvard University, Cambridge, MA, Wyss Institute for Biologically Inspired 10.1093/cvr/cvr158
Engineering, Boston, MA (K.K.P.) Greenstone Biosciences, Palo Alto, CA (J.C.W.). 14. Josowitz R, Lu J, Falce C, D’Souza SL, Wu M, Cohen N, Dubois NC,
Zhao Y, Sobie EA, Fishman GI, Gelb BD. Identification and purification
Acknowledgments of human induced pluripotent stem cell-derived atrial-like cardiomyo-
We thank Michael Rosnach for Figures 3 and 4. Figures 1, 2, and 5 were created cytes based on sarcolipin expression. PLoS One. 2014;9:e101316. doi:
with BioRender.com. We also thank Blake Wu for critical reading of the article 10.1371/journal.pone.0101316
and feedback. 15. Chen Z, Xian W, Bellin M, Dorn T, Tian Q, Goedel A, Dreizehnter L, Schneider
CM, Ward-van Oostwaard D, Ng JK, et al. Subtype-specific promoter-driven
Sources of Funding action potential imaging for precise disease modelling and drug testing
We are grateful for funding support from the Tobacco-Related Disease Re- in hiPSC-derived cardiomyocytes. Eur Heart J. 2017;38:292–301. doi:
search Program (TRDRP) of the University of California (grant T29FT0380 for 10.1093/eurheartj/ehw189
D. Thomas), John A. Paulson School of Engineering and Applied Sciences at 16. Benzoni P, Campostrini G, Landi S, Bertini V, Marchina E, Iascone M, Ahlberg
Harvard University (for K.K. Parker), National Institutes of Health (NIH) and Na- G, Olesen MS, Crescini E, Mora C, et al. Human iPSC modelling of a familial
tional Center for Advances in Translational Sciences (NCATS; UH3HL141798 form of atrial fibrillation reveals a gain of function of If and ICaL in patient-
and UG3TR003279 for K.K. Parker and UH3 TR002588 for J.C. Wu) and R01 derived cardiomyocytes. Cardiovasc Res. 2020;116:1147–1160. doi:
HL150693, R01 HL163680, R01 HL113006, R01 HL141371, and R01 10.1093/cvr/cvz217
HL126527 (for J.C. Wu). 17. Hong L, Zhang M, Ly OT, Chen H, Sridhar A, Lambers E, Chalazan B, Youn
SW, Maienschein-Cline M, Feferman L, et al. Human induced pluripotent
Disclosures stem cell-derived atrial cardiomyocytes carrying an SCN5A mutation iden-
J.C. Wu is a cofounder of Greenstone Biosciences. The other authors report no tify nitric oxide signaling as a mediator of atrial fibrillation. Stem Cell Reports.
conflicts. 2021;16:1542–1554. doi: 10.1016/j.stemcr.2021.04.019
18. Itzhaki I, Maizels L, Huber I, Zwi-Dantsis L, Caspi O, Winterstern A, Feldman
O, Gepstein A, Arbel G, Hammerman H, et al. Modelling the long QT syn-
drome with induced pluripotent stem cells. Nature. 2011;471:225–229. doi:
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Cellular and Engineered Organoids For Cardiovascular Models

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Circulation Research

COMPENDIUM ON BASIC MODELS OF CARDIOVASCULAR DISEASE

Cellular and Engineered Organoids for

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development but enhance atrial specification without Cardiac Fibroblasts

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Figure 2. Embryoid body-like human

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