OPEN ACCESS | Review

Stem cell therapy for cardiac regeneration: past, present,

and future
Jaideep Kaur Gill, Sargun Kaur Rehsia, Elika Verma , Niketa Sareen, and Sanjiv Dhingra
Institute of Cardiovascular Sciences, St. Boniface Hospital Albrechtsen Research Centre Regenerative Medicine Program,
Department of Physiology and Pathophysiology, Rady Faculty of Health Sciences, Biomedical Engineering Program, University of
Manitoba, Winnipeg MB, R2H2A6, Canada
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Corresponding authors: Niketa Sareen (email: nsareen@sbrc.ca); Sanjiv Dhingra (email: sdhingra@sbrc.ca)

Abstract
Cardiac disorders remain the leading cause of mortality worldwide. Current clinical strategies, including drug therapy, surgi-
cal interventions, and organ transplantation offer limited benefits to patients without regenerating the damaged myocardium.
Over the past decade, stem cell therapy has generated a keen interest owing to its unique self-renewal and immune privileged
characteristics. Furthermore, the ability of stem cells to differentiate into specialized cell types, has made them a popular
therapeutic tool against various diseases. This comprehensive review provides an overview of therapeutic potential of differ-
ent types of stem cells in reference to cardiovascular diseases. Furthermore, it sheds light on the advantages and limitations
associated with each cell type. An in-depth analysis of the challenges associated with stem cell research and the hurdles for
its clinical translation and their possible solutions have also been elaborated upon. It examines the controversies surround-
ing embryonic stem cells and the emergence of alternative approaches, such as the use of induced pluripotent stem cells for
cardiac therapeutic applications. Overall, this review serves as a valuable resource for researchers, clinicians, and policymak-
ers involved in the field of regenerative medicine, guiding the development of safe and effective stem cell-based therapies to
revolutionize patient care.
Key words: induced pluripotent stem cells, mesenchymal stem cells, cardiac regeneration, myocardial ischemia

Introduction
Heart failure continues to be a leading cause of mor-
tality around the world. In 2019 alone, around 17.9 mil-
lion people died of cardiovascular diseases, globally (CVDs
2021). It is anticipated that the number of patients suf-
fering from the disease will soar as the elderly popula-
tion is growing (Groenewegen et al. 2020). Besides the el-
derly, younger adults too are predisposed to cardiac disor-
ders due to risk factors such as obesity, high cholesterol,
high blood pressure, and sedentary lifestyle (Kelishadi and
Poursafa 2014). It is evident that the primary etiology of
heart failure is coronary artery disease and myocardial is-
chemia. These aetiologies commonly exist together and aug-
ment the risk of developing a heart failure (Fox et al. 2001).
Conventional therapies to treat cardiovascular diseases in-
volve catheter-based interventions, including angioplasty for
unclogging the arteries, and surgical interventions, includ-
ing coronary artery bypass surgery or organ transplant in
case of end-stage heart failure (Freystaetter and Akowuah
2021). These treatments might also include administration
of cardioprotective drugs like beta blockers, calcium chan-
nel blockers, or oral diuretics. These cardioprotective treat-
ments have been proven to enhance cardiac function in pa-
tients with coronary heart disease and help patients pre-
vent any severe cardiac effects after an initial episode of
cardiac arrest (Rossignol et al. 2019). However, these ther-
apies provide only marginal benefits in terms of long-term
recovery from disease and their continuous use might be
associated with severe side-effects masking the positive im-
pact they have on the patients. Nevertheless, these thera-
pies are insufficient to regenerate or repair the cardiac mi-
lieu, so their role in cardiac remodeling is limited (Tamargo
and López-Sendón 2011). In addition to this, for patients
with organ transplantation as the only option, surgeries are
limited by the number of organs available for transplanta-
tion. Therefore, new therapeutic options that outperform
the conventional approaches for treatment of cardiac disor-
ders are being investigated. Tissue regeneration using cell
therapy is an active area of research due to limited regen-
erative capacity of the heart following an ischemic injury.
Cell therapy provides a reliable solution for replacing the
terminally differentiated cardiomyocytes (CMs) lost during
myocardial ischemia. Stem cell therapy is a novel treatment
approach involving the use of undifferentiated stem cells to
improve a given cardiac condition (Wei et al. 2013; Padda et
al. 2015).

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Fig. 1. Types of stem cells for cardiac regeneration and source of stem cells. Different types of stem cells are employed for
cardiac therapy, including mesenchymal stem cells (MSCs) and pluripotent stem cells. MSCs can be derived from marrows of
iliac crest and femur (BM-MSCs); umbilical cord (UC-MSCs); adipose tissue (AD-MSCs); or from amniotic fluid. Pluripotent stem
cells can be isolated from blastocyst stage embryo (ESCs) or by de-differentiation of somatic cells from skin or blood, in the
presence of growth factors (iPSCs). Created using BioRender (BioRender.com).
Bone marrow
(for BM-
MSCs)
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Blastocyst
(for ESCs)
Somatic cells
+
Reprogramming factors
SKIN
(Oct3/4, Sox2, Klf4, c-Myc)
(for iPSCs)
Stem cells are the undifferentiated cells in our body that
possess the ability of self-renewal and differentiation into
multiple cell types of our body (Zakrzewski et al. 2019).
Based on their differentiation abilities, stem cells can be
either totipotent, pluripotent, or multipotent in nature.
Totipotent cells possess the ability to generate all cell types
in the human body, including the embryonic and extra
embryonic tissues. Pluripotent cells are generally derived
from inner cell mass of the embryo. They have the capac-
ity to divide into cell types from all three lineages (endo-
dermal, mesodermal, or ectodermal), except the extraem-
bryonic tissues. These cells are referred to as embryonic
stem cells (ESCs). Additionally, pluripotent stem cells can
also be induced from adult somatic cells like skin tissue or
blood cells by de-differentiating them in the presence of re-
quired growth factors known as Yamanaka factors (Rikhtegar
et al. 2019). These induced pluripotent stem cells (iPSCs)
have gained a lot of attention lately, for their flexibility
in being re-programmed from any somatic cell and dif-
ferentiating them into cells of interest (Yamanaka 2020).
Multipotent mesenchymal stem cells (MSCs) have a lim-
ited differentiation potential and can generate only cer-
tain cell types. These stem cells can be isolated from
adult tissues, including bone marrow (BM), blood, adi-
pose tissue (AD), or neonatal sources like umbilical cord
(UC), placenta, or amniotic fluid (Guo et al. 2020) (Fig.
1). Based on the donor, the transplantation of stem cells
can be either autologous (cells from the patient are in-
jected back in the same person for treatment) or allo-
geneic (where the cells from a healthy donor are in-
jected into the diseased, non-self host) (Zakrzewski et al.
2019).
162
Umbilical cord
(for UC-SCs)
Adipocytes
Stem cell (for AD-MSCs)
culture Heart
Amniotic fluid
(for MSCs)
BLOOD
Different types of stem cells for heart
repair
Pluripotent stem cells
Embryonic stem cells
ESCs are pluripotent stem cells obtained from the inner
cell mass of day 4 to 7 blastocyst stage embryo (Zakrzewski
et al. 2019). ESCs are promising candidates for the treat-
ment of cardiac diseases due to their ability to proliferate
indefinitely and differentiate into cells of all three germ
layers (Menasché 2020; Zhu et al. 2009). Following their first
isolation in 1994, human ESCs (hESCs) have been widely
used for the generation of different cell types for multiple
therapeutic applications (Hentze et al. 2009; Pré et al. 2013).
Directed differentiation of hESCs into CMs can be achieved
under appropriate culture conditions and CM-specific factors
(Dixon et al. 2011). However, a small percentage of hESCs
(5%–15%) spontaneously differentiate into CMs under the
required conditions (Wong and Bernstein 2010). Current re-
search is focused on controlling the differentiation of hESCs
into the cardiac lineage to increase the yield of hESC-derived
CMs for cell therapy. The techniques for enrichment, purifi-
cation, and selection have been developed to direct cardiac
differentiation toward comparatively pure homogeneity.
The strategies such as long-term culture, three-dimensional
(3D) tissue engineering, mechanical loading, electrical stim-
ulation, modification of substrate stiffness, and therapy
with neurohormonal substances have been tested in hESC-
derived CMs to improve their differentiation potential and
maturation status (Yang et al. 2014).
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ESCs have shown the potential to promote cardiac re-
modeling in pre-clinical trials by differentiating into car-
diac progenitors, which engraft into the host myocardium
and reduce scar formation. A study reported that extracel-
lular vesicles (EVs) secreted by human pluripotent stem cells
(hPSCs)-derived cardiovascular progenitor cells (hPSCs–CPCs)
exhibited cardioprotective effects under hypoxic conditions,
which in turn increased the expression levels of metasta-
sis associated lung adenocarcinoma transcript 1 (MALAT-1).
The MALAT-1 was found to promote tube formation in hu-
man umbilical vein endothelial cells by targeting miR-497
(Wu et al. 2020). Further, Chong et al. (2014) demonstrated
the ability of hESC-derived CMs to remuscularize infarcted
monkey hearts through intramyocardial (IM) administration.
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They found that the grafted cells formed electromechanical
connections with host myocytes, indicating significant poten-
tial for regeneration of infarcted heart muscle in humans.
Although sustained engraftment of ESCs is rare, ESC-derived
progenitors have been observed to positively influence the re-
modeling of infarcted heart tissue through paracrine mecha-
nisms (Chong et al. 2014). In the first clinical case report on
a 68-year old patient suffering from severe HF, hPSCs–CPCs
displayed promising results with no complications pertain-
ing to arrhythmias, tumor formation, or immunosuppres-
sion along with an increased left ventricular ejection frac-
tion (LVEF) (36%) following transplant (Menasché et al. 2015).
Menasche et al. evaluated the safety of hESCs in patients with
severe ischemic LV failure and explored the feasibility of pro-
ducing clinical-grade CPCs. In this ESCORT trial, around 8.2
million hESC-derived cardiovascular progenitors embedded
in a fibrin patch were delivered epicardially during a coro-
nary artery bypass procedure to six patients with a median
LVEF of 26%. The patient safety was evaluated after a year of
treatment based on the imaging for cardiac or off-target tu-
mors and serial interrogations of cardioverter–defibrillators
placed in every patient to screen for arrhythmias. The study
also checked for the presence of donor-specific antibodies in
recipients to assess alloimmune response. It was observed
that few patients acquired clinically silent alloimmunization.
Additionally, most of the patients were symptomatically re-
lieved, with cell-treated segments displaying an increased sys-
tolic motion. This trial helped to establish a protocol to ob-
tain highly purified (97.55%) cardiovascular progenitor popu-
lation from hESCs (Menasché et al. 2018). Although there are
well-established in vitro cell differentiation techniques and
encouraging results from in vivo preclinical research, several
obstacles must be overcome before wide acceptance of ESCs
as clinical candidates for patients with myocardial infarction
(MI). For instance, due to their embryonic origin, isolation
of hESCs is associated with ethical issues in many countries
(Volarevic et al. 2018). Furthermore, some studies also express
the concern of teratoma formation following ESCs injection
(Hentze et al. 2009; Yamanaka 2020).
Induced pluripotent stem cells
Since 2006, iPSCs have emerged as a revolutionary devel-
opment in the field of regenerative medicine. These cells are
generated by reprogramming adult somatic cells using four
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Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc) (Rikhtegar
et al. 2019). Exhibiting unique characteristics of pluripotency
and self renewal, iPSCs are becoming an ideal choice for the
field of regenerative medicine. Additionally, there are sev-
eral modifications in cell culture methods and protocols that
have been optimized to promote the chamber-specific differ-
entiation of iPSCs to generate ventricular, nodal, and atrial
CMs, smooth muscle cells, and cardiac fibroblasts (Batalov
and Feinberg 2015; Batalov et al. 2021; Wang et al. 2021).
Unlike their embryonic counterparts, iPSCs are being highly
considered for disease modeling and drug screening owing
to their accessibility and ethical acceptability (Cong et al.
2019; Rikhtegar et al. 2019). A study demonstrated that iPSC-
derived CM transplantation improved functional recovery in
macaque monkey hearts via reduction in the scar size. Fur-
thermore, after 1 month of transplantation, the CM-treated
hearts exhibited significant recovery in global LVEF of around
10.6% ± 0.9% (Liu et al. 2018). In a study by Ye et al., the
efficacy of iPSCs in acute MI was investigated in a porcine
model. Three types of cells derived from iPSCs were intramy-
ocardially administered, namely CMs, endothelial cells, and
smooth muscle cells. Additionally, a 3D fibrin patch was ap-
plied, which released insulin-like growth factor-1 (IFG-1) to
increase survival. Significant improvements were observed in
many parameters of cardiac function, such as LV function and
arteriole density. Additionally, myocardial metabolism and
ATP turnover rate were significantly improved, while apop-
tosis and infarct size were considerably reduced without any
ventricular arrhythmias (Ye et al. 2014). Another approach
to using iPSCs in heart disease is through iPSC-derived EVs
(iPSC-EVs). These vesicles contain molecular cargo such as
iPSC-released miRNAs and additional factors that aid in cel-
lular differentiation and angiogenesis (Rikhtegar et al. 2019).
Adamiak et al. compared the efficacy of iPSCs and iPSC-EVs
in a mice model of acute MI. The mice were divided into
three groups, namely IM injection of vehicle, iPSCs, or iPSC-
EVs obtained from murine iPSC supernatants. While both iP-
SCs and iPSC-EVs showed similar improvements in functional
myocardium, iPSC-EVs showed a more significant decrease in
apoptosis. Additionally, teratoma formation was observed in
the iPSC group, but not in the iPSC-EV group (Adamiak et al.
2018). One of the most fruitful applications of iPSCs that is be-
ing widely exploited is the development of disease models for
different pathological conditions. Several researchers have
reported isolation and application of patient-specific iPSCs as
disease models, since these cells offer patient-specific geno-
type that can be studied for its genetic abnormalities without
epigenetic effects on cells (Musunuru et al. 2018). These mod-
els have been reported for valvular defects, rhythm disorders,
metabolic disorders, and patient-specific iPSCs for dilated car-
diomyopathy (Lin et al. 2015; Blinova et al. 2019; Sequiera et
al. 2022).
Several clinical trials are currently investigating the ap-
plication of ESCs and iPSCs for cardiac therapy (Okano et
al. 2013; Deinsberger et al. 2020). An ongoing phase I trial
(NCT03763136) is evaluating the safety and efficacy of al-
logeneic iPSC-derived CM transplantation in patients with
severe heart failure. With around 20 patients, this study
is currently recruiting and is estimated to complete this
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year (Help Therapeutics 2022). Another phase I clinical trial
(NCT04696328) is investigating the safety and efficacy of al-
logeneic iPSC-derived CM sheets for treating ischemic car-
diomyopathy. This trial will focus on improvement of LVEF
alongside other safety-related evaluations (Toda 2021).
These clinical trials are evaluating the safety and efficacy
of stem cell therapy for heart disease, and their results may
provide important insights into the potential of these ther-
apies for clinical use in cardiac patients (Shiba et al. 2016).
However, to ensure a successful clinical translation, it is im-
portant to address the challenges associated with the use of
iPSCs, such as the risk of teratoma formation and the high
cost of production (Ye et al. 2014). Recently, in a study, in-
ducible caspase-9 (iC9) was introduced into iPSCs, and the
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exposure of these iPSCs to chemical inducer of dimerization
(CID) lead to initiation of a caspase cascade, which helped in
elimination of tumor-forming cells, thereby creating an effi-
cient iC9/CID safeguard system (Ando et al. 2015). The trans-
plantation of iPSCs in the heart is also reported to cause
arrhythmogenic response; in large animal models, there is
a greater risk of developing temporary ventricular arrhyth-
mia compared to small animal models (Kadota et al. 2020).
Okawa et al. compared the transcriptomic profile of fetal
heart and found that iPSC-derived CMs (iPSC-CMs) had similar
immature phenotype and electrophysiology, which may con-
tribute to arrhythmia (van den Berg et al. 2015). To overcome
this challenge and achieve a mature phenotype, Parikh et al.
(2017) used a combination of triiodo-l-thyronine and dexam-
ethasone to increase the formation of T-tubules in iPSC-CMs,
which resulted in subsequent calcium release and excitation–
contraction coupling that were similar to adult myocardium.
MSCs for cardiac therapy
MSCs are multipotent cells with the ability to differenti-
ate into cells of specific lineages, including osteogenic, chon-
drogenic, and adipogenic (Poomani et al. 2022). MSCs can
be isolated from several adult or neonatal tissues, including
BM, AD, endometrium, dental pulp, and UC along with sev-
eral other sources (Guo et al. 2020). Their ease of isolation
and maintenance, as well as least teratoma formation ten-
dency makes MSCs favorable candidates for clinical studies.
Currently, MSCs from BM and UC are most widely used in
clinics for cardiac therapy, followed by AD derivatives (Kabat
et al. 2020). MSCs mediate their action by secreting various
paracrine factors involved in tissue regeneration pathways,
including vascular endothelial growth factor (VEGF), hepa-
tocyte growth factor (HGF), and transforming growth factor
(TGF)-beta, which aid in angiogenesis and wound repair. They
also release immunosuppressive molecules such as IL-10,
prostaglandin E2 (PGE2), macrophage colony-stimulating fac-
tor, and tumor necrosis factor (TNF), which assist in prevent-
ing adverse immune response in the ischemic myocardium,
thus repairing the tissue (Dhingra et al. 2013; Yan et al. 2019;
Jiang and Xu 2020; Sareen et al. 2020).
Bone marrow-derived MSCs
Bone marrow-derived MSCs (BM-MSCs) are commonly iso-
lated from blood obtained from the marrow in iliac crest
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of pelvis or from marrow in the femur (Deans and Moseley
2000). These cells are easy to isolate and offer easy expan-
sion in vitro without loss of function. In addition to this, they
can be obtained from young healthy donors and transplanted
into allogeneic, diseased, and aged hosts (Da Silva and Hare
2013). Numerous experimental studies have reported sup-
porting evidence for the use of BM-MSCs in improving the
function of damaged myocardium by secretion of soluble
factors and promoting angiogenesis, reducing immune re-
sponse, and preventing fibrosis (Ammar et al. 2015; Stephen
et al. 2016; Shafei et al. 2017). Boomsma et al. demonstrated
that after injection of murine BM-MSCs into the infarcted
myocardium, systolic function was preserved post injection.
Also, the MSCs were able to migrate to the site of damage
and restore contractility of the heart by secreting paracrine
factors (Boomsma et al. 2007). In a rabbit model of dilated
ischemic cardiomyopathy, Mu et al. demonstrated that trans-
plantation of autologous BM-MSCs ameliorated cardiac func-
tion through paracrine interaction with endogenous CMs.
The BM-MSCs also resulted in upregulation of VEGF recep-
tors, thus suggesting their pro-angiogenic potential (Mu et
al. 2011). In swine model of MI-induced injury, IM injection
of BM-MSCs improved cardiac function and ventricular wall
thickness while reducing the scar size (Schuleri et al. 2009).
Cai et al. reported similar findings in acute MI models where
injection of BM-MSCs in swine hearts resulted in improved
cardiac function by upregulation of mTOR and improved glu-
cose metabolism in the heart by increasing the expression
of GLUT1 and 4 transporters (Cai et al. 2016). In CHART-1
study, the efficacy of autologous cardiopoietic MSCs was as-
sessed in 351 participants, the MSC-treated groups showed
progressive reduction in LV end-diastolic and end-systolic vol-
umes within 1 year of stem cell injection (Teerlink et al. 2017).
Despite the promising results obtained in several studies,
some of the reports have demonstrated increased expression
of several apoptotic and senescence genes with age, which
might affect the functions and proliferation of autologous
stem cells isolated from older patients, limiting the use of
autologous MSCs in clinical trials (Choudhery et al. 2012).
However, aging itself does not affect the positive outcomes
of MSCs therapy, which was shown in a study by Golpanian
et al. (2015) where the patients receiving MSCs were segre-
gated into different age groups and the results of improved
cardiac function and scar size did not vary based on the re-
cipient’s age. In addition to improving cardiac function in
vivo, BM-MSCs have also been reported to stimulate the dif-
ferentiation as well as proliferation of ckit+ cells in the my-
ocardium. In pig MI models, treatment with BM-MSCs re-
sulted in increase in ckit+ and GATA4+ cardiac stem cells
compared to control animals. In this study, MSCs stimulated
generation of Nkx and troponin positive adult cardioblasts
in vitro (Hatzistergos et al. 2010). Furthermore, Huang et al.
(2013) showed that transplantation of sheet fragments of au-
tologous BM-MSCs post MI into the infarcted myocardium
of a porcine model significantly improved ventricular dila-
tion and maintained cardiac function after surgically induced
MI. Hare et al. (2009) tested the efficacy of allogeneic MSCs
in acute MI patients in Prochymal trial and found improved
LVEF and reverse remodeling in BM-MSC-treated patients

compared to untreated controls. To address the effect of MSC
dose in modulating cardiac function, Florea et al. conducted
a TRIDENT trial where 30 ischemia patients received differ-
ent doses (20 million or 100 million) of MSCs. Patients receiv-
ing higher dose of MSCs showed improved EF compared to
the patients treated with lower numbers of MSCs; however,
both groups showed reduction in the scar size (Florea et al.
2017).
UC-derived MSCs
UC-derived MSCs (UC-MSCs) are formed during fifth week
of embryogenesis. The UC is a rich source of MSCs that can
be isolated from its different parts, including blood, UC
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endothelium, and wharton’s jelly (Mushahary et al. 2018).
The UC-MSCs express both adult stem cell markers and ESC
markers like Tra 1-60, Tra 1-81, and stage-specific embryonic
antigen 1 and 4 along with alkaline phosphatase (Carlin et
al. 2006). These MSCs exhibit higher multipotency compared
to adipose or BM derivatives (Fong et al. 2011). Similar to BM-
MSCs, UC-MSCs are known to suppress T cell proliferation,
but their rejection time is slower compared to BM-MSCs;
therefore, they are favored for allogeneic transplantation
(Weiss et al. 2008; Deuse et al. 2011). Additionally, UC-MSCs
exhibit longer telomeres and higher telomerase activity,
higher proliferation rates, and ease of isolation through least
invasiveness, compared to other multipotent stem cell types,
making them a promising source of MSCs for cardiac stem
cell therapy (Yannarelli et al. 2013b; Fazzina et al. 2016). In a
study using mouse MI models, animals receiving human UC-
MSCs (hUC-MSCs) sheets showed significant improvement
in EF and improved infarct size, compared to untreated
controls. The authors also reported low oncogenicity as well
as reduced TNF-α secretion (Gao et al. 2022). Another group
of researchers showed antiapoptotic and proangiogenic role
of UC-MSCs in murine MI models. They also found enhanced
activation of resident cardiac progenitors cells in the UC-
MSC-treated animals (Nascimento et al. 2014). Additionally,
UC-MSCs-conditioned media have also been reported to
reduce inflammation in porcine myocardium by prevent-
ing the activation of NF-κB pathway. Treated animals also
exhibited improved cardiac function and reduced fibrosis
as seen by TGF-β and IL-6 levels (Liu et al. 2016). A similar
study in rats with dilated cardiomyopathy showed reduced
expression of TNFα, TGF-β1, and ERK1/2 in UC-MSC-treated
animals, highlighting its antifibrotic role (Zhang et al. 2018).
In a study by Yannarelli et al. (2013a), it was shown that cul-
ture of embryonic CMs with UC-MSCs resulted in significant
upregulation of cardiac-specific genetic markers compared to
CMs that were co-cultured with BM-MSCs. Some studies have
also shown the differentiation of UC-MSCs into myotubes,
smooth muscle cells, and beating CMs by culturing them un-
der defined conditions (Hollweck et al. 2011; Tompkins et al.
2018; Chen et al. 2021). Qui et al. show that cardiac allograft
transplantation that was accompanied by UC-MSC injection
had lesser major histocompatibility complex class-II (MHC-II)
expression and survived longer compared to the animals
without UC-MSCs (Liu et al. 2016). Of the few clinical trials
conducted yet in cardiac patients using UC-MSCs, RIMECARD
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trial, assessed the efficacy and safety of UC-MSCs in patients
with chronic HF. The patients who received UC-MSCs showed
significant improvement in LVEF with no adverse events
till 12 months follow-up period. Additionally, there were no
alloantibodies generated against the transplanted cells till
90 days of injection (Bartolucci et al. 2017). Currently, a ran-
domized clinical trial is underway, involving 20 MI patients
undergoing coronary artery bypass grafting (CABG), to study
the effect of hUC-MSCs in improving the heart function of
these patients (Shanghai East Hospital 2021).
AD-derived MSCs
AD was first identified as a source of MSCs for cell therapy
by Zuk et al. (2001). The AD-derived MSCs (AD-MSCs) are
isolated from stroma of the AD using collagenase-mediated
degradation, yielding progenitor cells that can differentiate
into osteogenic, adipogenic, or chondrogenic cell types (Zuk
et al. 2002). Due to easier access to autologous AD compared
to BM and UC, as well as the abundance of fat tissue for their
isolation, AD-MSCs are sought after candidates for multiple
pathologies (Zuk 2013; Frese et al. 2016). In addition to this,
these cells are also advantageous in terms of shorter dou-
bling time and generation of significantly higher stem cell
progenitors compared to BM-MSCs (Zhu et al. 2012). AD-MSCs
can differentiate into CMs and display a later onset of senes-
cence, signifying their potential for use in treating chronic
cardiac conditions (Tambrchi et al. 2022). Some studies report
the differentiation of AD-MSCs into cells of different vascular
lineages, aiding in cardiovascular remodeling (Ni et al. 2019).
In addition to this, generation of CMs has also been reported
by several researchers through spontaneous or chemical-
induced differentiation in vitro, indicating the potential of
AD-MSCs for cardiac regeneration (Planat-Bénard et al. 2004;
Choi et al. 2010; Jiang et al. 2018). In one such study, Chang et
al. demonstrated that rats receiving AD-MSCs after MI showed
improved heart function. After 12 weeks of transplantation,
the authors also observed expression of CM markers, includ-
ing GATA1 and myosin heavy chain in transplanted AD-MSCs
(Chang et al. 2011). Ammar et al. showed that AD-MSCs were
able to alleviate the symptoms of cardiotoxicity in doxoru-
bicin (Dox)-treated diabetic rats. AD-MSC-treated diabetic
rats showed reduced collagen deposition and smooth muscle
actin (SMA) expression as well as reduced infiltration of im-
mune cells in Dox-treated myocardium (Ammar et al. 2015).
In porcine models of acute MI, intracoronary (IC) injection of
autologous AD-MSCs was associated with increased cardio-
protection and better cardiac perfusion with no significant
changes in EF or cardiac volumes (Bobi et al. 2017). Besides
the use of AD-MSCs alone, several studies showed positive
effects following the transplantation of AD sheets as such
in the ischemic myocardium (Hamdi et al. 2011). Kim et al.
(2017) showed that the use of AD-MSC sheets in acute MI mod-
els resulted in better cell engraftment and higher growth
factor and cytokine secretion to prevent adverse cardiac
remodeling compared to hearts that received AD-MSCs in-
jections. Given the positive outcomes of pre-clinical studies,
several clinical trials have been performed using AD-MSCs to
assess changes in heart function following transplantation.
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In a randomized, double-blinded APOLLO trial, the safety
of AD-MSCs was tested for the first time in ST-elevated MI
(STEMI) patients. IC infusion of AD-MSCs was deemed safe
without coronary flow impediment. The authors reported
improved cardiac function and 50% reduction in scar forma-
tion (Cytori Therapeutics 2013b). PRECISE clinical trial was
performed in 27 patients with ischemic cardiomyopathy.
Patients receiving AD-MSCs showed better maximal oxygen
consumption at 18 months follow-up period, compared to
untreated controls. The patients also showed improved LV
mass and wall motion score index while preserving exercise
capacity in AD-MSC-treated patients (Perin et al. 2014). The
Athena trial program conducted double-blinded study in 31
ischemic cardiomyopathy patients. Although there was no
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difference in LV function in AD-MSCs and placebo patients,
the AD-MSC-treated patients showed improved cardiac per-
fusion and reduced 12 month hospitalization rate (Henry et
al. 2017). In a recent phase II clinical trial involving patients
with heart failure with reduced EF (HFrEF), the patients who
received AD-MSCs showed improved quality of life; however,
the ventricular function and dimensions did not change
compared to placebo group, after a 3-year follow-up period
(Qayyum et al. 2023). Given the mixed results from different
trials, there is a need to perform more extensive studies to
understand the role of AD-MSCs in improving heart function.
Delivery of stem cells to the heart
The route and technique of stem cell delivery to the heart
are crucial factors in contributing towards the clinical out-
come of cell therapy (Labusca et al. 2018). Stem cells can
be delivered to the injured myocardium via IM, intravenous
(IV), and IC routes, or using 3D scaffolds (Rheault-Henry et al.
2021). IM injection involves direct delivery of stem cells in the
ischemic myocardial tissue. It is considered to be most precise
approach to deliver cells and can be carried out with the help
of catheters in patients with coronary heart disease (Sheng et
al. 2013). Though IM injection offers targeted delivery of stem
cells, this mode is invasive and can result in severe negative
effects, including patient deaths. For similar reasons, it is not
feasible to inject multiple doses of cells in the same patient
(Sheng et al. 2013). On the other hand, IV approach involves
infusion of stem cells in the blood stream using IV drip. It is
generally preferred in patients after an acute MI, and due to
its least invasive nature, it is a viable route to deliver multiple
cell infusions. However, despite being amongst the common
methods of cell delivery, not many of the cells transplanted
via IV mode survive and might end up being trapped in the
blood vessels or untargeted organs (Yukawa et al. 2012). Addi-
tionally, this technique relies on homing of stem cells in the
injured myocardium using paracrine signaling (Sheng et al.
2013). IC infusion allows the cells to be delivered in the my-
ocardium via one of the coronary arteries. It is the most preva-
lent mode of cell delivery since it can be accompanied by the
surgical intervention in patients with MI. This method, how-
ever, might not be feasible in patients with less perfused ves-
sels. In addition to this, it might be important to consider the
size of cells and the arteries before delivery to prevent cells
from being obstructed in smaller capillaries (Bilewska et al.
166 Can. J. Physiol. Pharmacol. 102: 161–179 (2024) | dx.doi.org/10.1139/cjpp-2023-0202
2022). In a study involving porcine MI model, the efficacy of
different delivery approaches was tested. Amongst endocar-
dial, IM and IC routes, IC injection showed the largest average
number of engrafted cells compared to other ways of MSC
administration (Moscoso et al. 2009). One such study states
that only 3.5% of the injected MSCs were confined in the
heart after 6 weeks of injection. However, it was also found
that highest number of these cells were retained when MSCs
were injected a day after MI (Kurtz 2008). Some of the stud-
ies have also reported the use of biomaterial-based patches to
deliver stem cells. There was improvement in the retention
and engraftment of stem cells when the cells were delivered
through patches (Park et al. 2019; Tabei et al. 2019). However,
given the variations in the cell type and characteristics, one
mode of delivery may be preferred over the other based on
the target organ and patient condition to achieve the best
possible outcomes (Campbell and Suzuki 2012; Li et al. 2021).
Challenges involved in clinical translation of
stem cells
Stem cell technology has certainly shown progress towards
clinical use in treatment of heart diseases (Table 1). However,
there are several challenges and shortcomings that need to be
abridged before stem cell therapy becomes a norm in clinical
settings (Fig. 2). Following are some of the hurdles for the
translation of stem cell therapy.
Heterogeneity of stem cells
To begin with, it is very challenging to characterize MSCs,
since these cells lack the expression of unique surface mark-
ers and express markers that are present on other cell types
as well. Therefore, even though there are standards defined
by the International Society for Cellular Therapy, these are
quite ambiguous and can be applied to other cell types
(Nesselmann et al. 2008). For instance, MSCs show marked
expression of CD29 (integrin ß1), which is also expressed
by epithelial and myoepithelial cells (Togarrati et al. 2018).
Similarly, CD90 (Thy-1) is co-expressed by hematopoeitc stem
cells and keratinocytic stem cells (Kisselbach et al. 2009). In
addition to this, CD105 (endoglin), is expressed by endothe-
lial and certain hematopoietic stem cells as well (Nesselmann
et al. 2008; Rossi et al. 2019). Besides MSCs, pluripotent stem
cells have also been reported for heterogeneity at different
levels. Since ESCs and iPSCs are known for their indefinite
multiplication and pluripotent nature, it is challenging to
control the differentiated cell population generated by these
cells. Each ESC and iPSC cell line exhibits differences in dif-
ferentiation potential favoring variable cell types (Yamanaka
2020). Amongst other factors, major contributors towards
interculture variability include the time they have been in
culture, as well as genetic factors (Hayashi et al. 2019). Lately,
analysis of single cell cultures has revealed the presence of
different cell types in a single population of pluripotent stem
cells, adding to the complexity of stem cell hetrogeneity
(Yang et al. 2021). In a study by Yang et al., exome sequencing
revealed the presence of almost 400 variants of iPSCs in
mouse fibroblast-derived 24 iPSC clones (C. Li et al. 2015).
 Canadian Science Publishing

Table 1. Summary of clinical trials completed in the past decade using stem cells from different sources, including bone
marrow, adipose tissue, and umbilical cord.
Route of National clinical trial
Study, phase (Year) Phase Cell type administration Major findings number (reference)
Safety and efficacy of 2 Autologous CD34+ stem Intracoronary –Improved LVEF NCT00629018
autologous stem cells Increased 6 min walk (Vrtovec 2015)
cells for dilated distance
cardiomyopathy –Reduced levels of
(2006–2013) NT——proBNP
APOLLO Trial 1 Adipose tissue-derived Intracoronary –Improved cardiac NCT00442806 (Cytori
(2007–2012) stem and regenerative function and perfusion Therapeutics
cells (ADRCs) defect 2013b)
–Decreased scar size
–Evidence on
pro-angiogenic,
anti-apoptotic, and
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immunomodulatory
effects of ADRCs
PRECISE Trial 1 ADRCs Direct injection in –Maximal oxygen NCT00426868 (Cytori
(2007–2012) the left ventricle consumption and Therapeutics
metabolic equivalents 2013a)
preserved
–Improved left ventricular
mass and wall motion
score index
TAC–HFT (2008–2014) 1/2 Autologous MSCs and Injections with an –No adverse events after a NCT00768066 (Hare
bone marrow infusion catheter 1-month follow-up 2015a)
mononuclear cells Minnesota Living with
(BMCs) Heart Failure score
improved at 1 year
follow-up for both
treatment groups
PROCHYMAL 2 Allogeneic MSCs Intravenous –Reduction in ventricular NCT00877903 (Hare
(2009–2016) tachycardia et al. 2009)
POSEIDON–pilot 1 Autologous and Transendocardial –Reduction in infarct size NCT01087996 (Hare
study (2010–2013) allogeneic BM-MSCs –Improved ventricular 2015b)
remodeling
MESAMI (2010–2014) 1/2 Autologous MSCs Transendocardial –Improved EF and LVESV NCT01076920
(University
Hospital, Toulouse
2014)
POSEIDON–DCM 1/2 Autologous and Transendocardial –Improved endocardial NCT01392625 (Hare
(2011–2017) allogeneic BM-MSCs function and increased 2018)
EF
–TNFα suppression
RIMECARD Trial 1/2 Umbilical cord-derived Intravenous –Improved LVEF NCT01739777
(2012–2015) MSCs (UB-MSCs) –Improved functional (Bartolucci et al.
capacity 2017)
–Improved quality of life
TRIDENT (2013–2017) 2 Allogeneic MSCs Transendocardial –Increases EF NCT02013674 (Florea
–Reduced scar size et al. 2017)
To assess the safety of 2 Allogeneic BM-MSCs Intravenous –Improved functional NCT02467387
ischemia tolerant capacity (CardioCell LLC
MSCs (2014–2017) –Improved 2020)
immunomodulatory
markers
SCIENCE Trial 2 Allogeneic AD-MSCs Intramyocardial –Slight improvement in NCT02673164
(2016–2021) New York Heart (JKastrup 2021)
Association (NYHA) class
To assess the safety of 1/2 Hydrogel-packed Intramyocardial –No adverse effects 1 year NCT02635464 (Dai
UC-MSCs with UC-MSCs after therapy 2020)
injectable collagen Enhanced cardiac function
scaffold
(2015–2019)

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 Canadian Science Publishing

Table 1. (concluded).
Route of National clinical trial
Study, phase (Year) Phase Cell type administration Major findings number (reference)
Chronic myocardial 2 Allogeneic cardiology Intramyocardial –No significant changes NCT03092284
ischemia stem cell centre observed between (JKastrup 2022)
(2015–2022) adipose tissue-derived groups with respect to
mesenchymal stromal myocardial
cell product function/structure; no
(CSCC_ASC) changes in 6 min walk
test

Fig. 2. Challenges in clinical translation of stem cell therapy for heart repair. (i) Heterogeneity of stem cells: the differentiation
potential of stem cells varies between different stem cell types as well as within same stem cell type, which contributes to
the variability in the effects seen following treatment. (ii) Dose optimization: it is important to optimise the dose of stem
cells for transplantation. Given different cell numbers can contribute to different end results based on cell type and organ
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of transplantation, cell numbers being transplanted should be optimized before clinical adaptation of stem cell therapy. (iii)
Migration of stem cells to untargeted organs can result in cell loss soon after transplantation or even obstruction of smaller
capillaries. (iv) Loss of stem cell immunoprivilege post transplantation: it results in the recognition of stem cell by host immune
response resulting in eventual loss from the site of transplantation. Immune suppression of stem cells varies with cell type
and source, which should be considered before transplantation for better survival in donor heart. Part of this figure was created
using the software bioicons (https://bioicons.com/).

Heterogeneity/
Uncontrolled differentiation

Cardiac
Patient

Stem cells
cultured in vitro

Dose optimization
tion
n
Loss of stem cell immune privilege

Migration

Using single cell RNA sequencing, another study revealed the
variation in human PSC-derived limbal stem cells, in a time-
dependent manner (Sun et al. 2021). Furthermore, lack of
availability of standard protocols contributes to generation
of variable degrees of pluripotent stem cells in different labs
(Pattison et al. 2018). All these factors contribute significantly
towards results with low reproducibility and more variability
following transplantation of ESCs and iPSCs.
Survival of transplanted stem cells in vivo
Besides the struggles associated with characterization of
stem cells, there is still a long way to go to ensure longer sur-
vival and engraftment of MSCs in the ischemic myocardium.
Some of the major contributors towards cell death in the is-
chemic myocardium include reduced blood supply, increased
oxidative stress, and enhanced inflammation in the my-
ocardium (Abdelwahid et al. 2016). Therefore, to overcome
these challenges, enhancing stem cell survival in injured
heart muscle by improving cell engraftment, inhibiting cell
death pathways, and increasing the expression of cell sur-
vival proteins might be important for their clinical transla-
tion (Abdelwahid et al. 2016). Consequently, genetic modifi-
cation of MSCs to increase the secretion of pro-survival genes
has been practiced to improve stem cell survival in the heart.
Noiseux et al. demonstrated that MSCs overexpressing Akt
show marked reduction in apoptosis and increased secretion
of VEGF and insulin growth factor (IGF), thereby showing im-

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proved heart function post MI (Kang et al. 2015; Shan et al.
2018). Similarly, another study showed the effect of increased
stretching on MSCs in improving their angiogenic potential
and decreasing apoptosis by targeting VEGF signaling (Zhu
et al. 2015). Several other studies have shown positive effects
induced by hypoxic pre-conditioning of stem cells prior to
transplantation (Yan et al. 2012; Hao et al. 2019). Induction of
chemical or physically established hypoxia had similar effects
on improving stem cell survival in vivo. It is also shown that
MSCs pre-conditioned with caspase inhibitors in the presence
of hyperoxia improved cell survival in vivo (Saini et al. 2013).
Hu et al. compared the improvement in cell survival and rate
of apoptosis between normally cultured MSCs and hypoxia
preconditioned MSCs in primate model of MI. After 3 months,
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a significant improvement in scar size and LV function was
noted due to the secretion of pro-angiogenic factors such as
HGF, erythropoietin, and angiopoietin-I (Hu et al. 2016). Many
researchers propose that due to hypoxic microenvironment
in the body, 2%–5% O2 levels is helpful for enhanced thera-
peutic potential of stem cells as well as preventing sponta-
neous differentiation of ESCs (Ezashi et al. 2005; Rodrigues
et al. 2010; Antebi et al. 2018). Furthermore, poor survival
of iPSCs due to their inability to adapt to the environment
has been reported following transplantation, due to insuffi-
cient vascularization and the incidence of necrosis (Cho et
al. 2021). To address this challenge, the co-administration of
a fibrin patch containing IGF-encapsulated microspheres has
been proposed to provide this growth factor and increase sur-
vival and cardiac function. Ye et al. (2014) found that the en-
graftment of iPSCs was improved when combined with a fib-
rin patch, resulting in around 20-fold higher engraftment.
Migration and homing of stem cells in vivo
Another major challenge associated with stem cell therapy
is the migration of cells to the site of injury as well as their
lower retention at the transplantation site (Guo et al. 2020).
According to some reports, expression of surface markers,
cell size as well as culture conditions of stem cells, plays an
important role in their engraftment and migration to the tar-
get site. In one such study, stem cells over expressing CXCR4
were able to migrate and engraft better in the ischemic my-
ocardium promoting angiogenesis (Zhang et al. 2008). Sim-
ilarly, Becker et al. showed that migration of MSCs was as-
sociated with the expression of matrix metalloprotienase-
2 (MMP-2). They also reported that higher confluence of
MSCs in cultures resulted in increased synthesis of MMP-
2 inhibitor, therefore lesser cell migration (De Becker and
Riet 2016). Furthermore, to achieve better migration of trans-
planted cells to the target organ, stem cells can be pre-treated
with certain chemokines and factors, including TNFα and in-
tegrin β1 (Segers et al. 2006; Ip et al. 2007; Wu and Zhao
2012). Inhibition of adhesion molecules including VCAM, on
the surface of MSCs has also been reported to positively affect
their translocation to the target organ (Chen et al. 2019). In
addition to this, higher culture temperatures negatively af-
fect stem cell migration through NF-κβ pathway (Sen and Ta
2020). Stem cell size is another important factor, which needs
to be considered before the injection of stem cells in heart.
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Canadian Science Publishing
The size of coronary capillaries is limited to about 7–10 μm.
Therefore, to prevent microinfarctions due to microvascular
plugging, it is important to consider the size of bigger stem
cells before injection (Gallet et al. 2015; Ishikawa 2015). Gao
et al. conducted a randomized controlled trial in 18–80-year
old patients with STEMI. According to this study, IC injec-
tion of MSCs can easily result in adherence of these cells to
the capillaries and affect the blood flow in the heart (Gao
et al. 2013). Furthermore, another study reported the trap-
ping of MSCs in lungs soon after IV injection, due to larger
size of MSCs (Eggenhofer et al. 2014; Masterson et al. 2021).
In another study comparing multipotent stem cells, neural
progenitor cells, BM mononuclear cells, and MSCs, the au-
thors found that smaller sized cells were able to bypass the
pulmonary microvascular barrier compared to larger MSCs
(Fischer et al. 2009). Similar findings were presented by an-
other study where the patients showed pulmonary embolism
after IV injection of AD-MSCs (Jung et al. 2013).
Immunogenicity of stem cells after
transplantation
MSCs are considered to be immunoprivileged; therefore,
these cells should be able to evade host immune system and
avoid immune rejection. However, several studies have re-
ported that MSCs become immunogenic after transplanta-
tion in the ischemic heart (Dhingra et al. 2013; Abu-El-Rub
et al. 2020; Sareen et al. 2020). Therefore, another major
challenge in clinical translation of MSC-based therapies is
to maintain the immunoprivilege of stem cells after trans-
plantation. Huang et al. investigated whether MSCs maintain
their immunoprivilege and functional capacity after trans-
plantation in a rat MI model. Assessment of cardiac environ-
ment at 6 months showed that allogeneic MSCs switch their
immune-tolerant state to immune-reactive state (Huang et al.
2010). Alteration in immune state has been reported due to
multiple reasons, including change in the expression of cell
surface immune antigen, MHC-II molecules on the surface
of MSCs, or reduced secretion of immunosuppressive soluble
factors such as PGE2 (Abu-El-Rub et al. 2020; Liu et al. 2020;
Sareen et al. 2020). Immune activity of stem cells can also be
attributed to age of donor as well as the source of stem cells
(Hass et al. 2011; Gao et al. 2016). Wu et al. (2014) showed that
both BM-MSCs and ADSCs from older animals show lesser
suppression of CD4+ , CD8+ T cells compared to the cell iso-
lated from younger animals, highlighting the important role
of donor age in regulating stem cell immunoprivilege. Simi-
larly, Ribeiro et al. (2013) compared the immunosuppressive
abilities of MSCs sourced from adult and neonatal tissues and
found that these cells differ in their ability to suppress the
CD4+ and CD8+ T cell activation, emphasizing further on the
importance of source of stem cells for transplantation.
Uncontrolled differentiation of stem cells in vivo
resulting in undesired phenotype
Some other studies have reported concerns over differenti-
ation of stem cells into cells of undesired phenotype. Follow-
169
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ing transplantation, when cells are exposed to host microen-
vironment, they might respond to the available factors and
undergo spontaneous differentiation (Fowler et al. 2020). In
addition to this, the CMs generated following differentiation
of iPSCs or ESCs might be of immature phenotype and there-
fore exhibit differences in calcium handling and sarcomere
organization (van den Berg et al. 2015; Rheault-Henry et al.
2021; Campostrini et al. 2022). This might result in increased
arrhythmias following transplantation of these cells in the
myocardium. Furthermore, in some of the older studies, un-
fractionated BM cells have been shown to differentiate into
osteoblasts in some cases resulting in ossification of the heart
tissue. Therefore, it is advisable to use sufficient quantities of
MSCs in the perfusate while ensuring their purity simultane-
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ously (Breitbach et al. 2007). To overcome these hurdles, it
might be better to use differentiated CMs derived from stem
cells for cardiac repair and regeneration (Hasan et al. 2016).
Transplantation of cells treated with factors for directed dif-
ferentiation might be another approach to consider. Boland
et al. suggested that adding Wnt-3 in the culture media of
MSCs represses the osteogenic capacity of MSCs (Narcisi et
al. 2015). On the other hand, it might be beneficial to rule
out the use of cells altogether and consider cell-derived prod-
ucts instead. Cell-free systems are gaining popularity since
they combine best of both the worlds. They are able to escape
the immune response since they lack the expression of anti-
gen presenting cell surface proteins. Moreover, they exhibit
similar positive effects as seen by whole cell transplantation
(Keshtkar et al. 2018; Jafarinia et al. 2020).
Challenges with large-scale production of stem
cells for clinical application
To replace the CMs lost during an ischemic event, treat-
ment using exogenous stem cells has shown a great promise.
However, to achieve expected results, these cells need to
be expanded to higher numbers before transplantation in
patients. These large stem cell numbers are obtained us-
ing bioreactors. Nevertheless, large-scale production of stem
cells faces major challenges. There are a lot of aspects that
need to be considered when executing these steps, beginning
from donor selection to the final purified product ready to
be administered in vivo. First, there is a lack of standard-
ized/optimized protocol for sourcing, isolating, and main-
taining sufficient number of high-quality stem cells (Lappin
and Cheng 2021). Heathman et al. (2016) highlighted the sig-
nificance of understanding the impact of donor features on
expanding autologous and allogeneic cell-based treatment
bioprocesses in developing manufacturing processes from
numerous BM-hMSC donors. Second, stem cells possess lim-
ited proliferative capability affecting the scope of their scala-
bility and expansion. To achieve large-scale production, stem
cells need to be expanded in vitro while maintaining their
stemness and avoiding genetic abnormalities or other alter-
ations that could affect their safety and efficacy (Brown et al.
2013; Neri 2019). Third, it is important to undergo quality
control and characterization to ensure the safety and efficacy
of stem cells for clinical use. This involves comprehensive
characterization to confirm the identity, purity, potency, and
170 Can. J. Physiol. Pharmacol. 102: 161–179 (2024) | dx.doi.org/10.1139/cjpp-2023-0202
absence of contaminants or genetic abnormalities (Guadix et
al. 2019; Henn et al. 2023). This will also be a key step in
unlocking the value of demonstrating process comparabil-
ity, allowing for any necessary process changes and multisite
manufacturing models (Martin et al. 2019). Fourth, scaling
up the production of stem cells will significantly increase the
cost. Optimizing production methods, including automation
and bioreactor systems, can help improve efficiency and re-
duce labor and material costs (Lee et al. 2022). Lastly, ethi-
cal and regulatory considerations must be addressed while
scaling up the stem cells production. Meeting regulatory
requirements and obtaining necessary approvals can be a
complex and time-consuming process, adding to the chal-
lenges of large-scale production (Bauer 2004; Lo and Parham
2009).
Addressing these challenges requires multidisciplinary
collaboration between scientists, engineers, clinicians, and
regulatory experts. Continued research and technological
advancements will play a crucial role in overcoming these
hurdles and enabling the large-scale production of stem cells
for clinical applications.
Future directions: stem cell therapy for cardiac
regeneration
Stem cell therapy has shown promise for cardiac regenera-
tion and repair. However, for clinical translation of stem cell-
based approaches, the above-described challenges need to be
addressed. The heterogeneity of stem cell isolation and cul-
ture procedures can be addressed by standardizing the me-
dia components being used for each stem cell type. Optimiz-
ing the media components for clinical use is a daunting task
at present. However, reports suggest that standardizing cul-
ture conditions is beneficial in preventing interculture vari-
ability. It has been reported that iPSCs cultured under stan-
dard conditions had lesser variation in their genome (Molina-
Ruiz et al. 2022). According to many researchers fetal bovine
serum (FBS) is the most widely used component with unde-
fined composition in the culture medium, and it might pose
some health risks associated with the transfer of xenogeneic
components into human body (Dessels et al. 2016). However,
it has also been associated with reduced expression of p53-
related genes along with increased reprogramming efficiency
of stem cells (Kwon et al. 2016). Therefore, it is inevitable for
cell culture media and requires better standardization. Des-
sels et al. highlighted various alternates for stem cell culture
replacing the serum component either through growth fac-
tors or through serum albumin, replacing fetal calf serum
completely. For human cell cultures, different options such
as plasma, platelet-rich plasma, and plasma lysate can be con-
sidered (Dessels et al. 2016). In one such study, Kocaoemer et
al. examined the effect of human AB-serum and thrombin-
activated platelet-rich plasma as substitutes for FBS. The au-
thors reported that differentiation of ADSCs into osteogenic
and adipogenic lineages was significantly lower in case of
cells cultured in bovine serum compared to the serum ob-
tained from human donors (Kocaoemer et al. 2007). Similarly,
Atashi et al. (2015) showed higher cell proliferation following

culture of stem cells in the presence of autologous platelet-
rich plasma. A meta-analysis by Palombella et al. compared
the variations in different parameters between FBS-based me-
dia versus human platelet lysate (HPL)-based culture media.
They measured doubling time, expression of stem cell mark-
ers, paracrine activity of MSCs, immunosuppressive abilities,
and cell morphology. The authors found that the doubling
time of MSCs was higher in HPL-supplemented media. How-
ever, no differences in immunosuppressive abilities were re-
ported in both the conditions (Palombella et al. 2022). On the
other hand, Copland et al. (2013) suggested that the presence
of fibrinogen in HPL could negatively impact the immuno-
suppressive ability of MSCs due to its ability to trigger the
pro-inflammatory phenotype of MSCs by activation of TLR4
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receptor. One of the potential solutions could be to reduce
the fibrinogen content in HPL. However, this issue still needs
to be addressed in future studies to ensure safety of the stem
cell procedures since most of the manufacturing facilities
have begun to use HPL to culture stem cells (Kinzebach and
Bieback 2013).
Cell-free systems for cardiac regeneration
Biomaterials for cardiac regeneration
Cell retention and survival after transplantation are the
major barriers to achieving the full therapeutic efficacy of
stem cells. Therefore, investigating newer avenues using
biomaterials with or without stem cells for regeneration of
the myocardium is becoming an active area of research. The
biomaterials offer a conducive 3D microenvironment to in-
jected stem cells while masking the surface antigens that are
responsible for triggering an immune response. In addition
to this, the biomaterials also offer efficient environment for
growth and support of endogenous or injected cells aiding
in cardiac regeneration (Majid et al. 2020). Some of the most
commonly used biomaterials for cardiac regeneration in-
clude organic compounds like polyethylene glycol; inorganic
compounds, including silica nanoparticles, gold nanopar-
ticles, carbon nanoparticles as well as biological materials,
including viral like particles, protein nano systems as well as
EVs (Rafieerad et al. 2019, 2020; Han et al. 2022). Proteins such
as laminins, fibronectins, collagen, and polysaccharides have
also been employed for use as scaffolds mimicking natural ex-
tra cellular matrix. These semi-scaffolds can serve as binding
sites for growth factors such as fibroblast growth factor (FGF),
VEGF, and HGF (Rosellini et al. 2018). In a recently concluded
clinical trial to demonstrate the safety and efficacy of colla-
gen hydrogels for stem cell delivery to the heart, the patients
receiving CABG were additionally subjected to either a “cells
with collagen” treatment or “cells” by itself. The patients
who received hydrogel-packed MSCs demonstrated improved
LVEF with no adverse effects 1 year after the therapy (He et
al. 2020). Jin et al. (2009) administered MSCs embedded in
poly(lactide-co-epsilon-caprolactone) into a rat model of MI
and noted that it considerably improved LVEF and reduced
infarct size. Rafieerad et al. examined the impact of using
0D titanium carbide MXene quantum dots (MQDs) on im-
mune response regulation with the aim of treating allograft
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Canadian Science Publishing
vasculopathy. The MQDs were found to exhibit immunomod-
ulatory effects by specifically decreasing the activation of
human CD4+ IFN-gamma+ T-lymphocytes and enhancing the
proliferation of immunosuppressive CD4+ CD25+ FoxP3+ reg-
ulatory T-cells (Rafieerad et al. 2021). Use of quantum dots to
trace the route of injected MSCs in vivo has been reported in
some recent studies. These materials can provide a substitute
for fluorescent molecules and organic dyes owing to their
photostability (Muller-Borer et al. 2007).
Exosomes as cell-free systems for cardiac
regeneration
Lately, EVs have emerged as promising cell-free systems
for cardiac regeneration due to their advantages including
lack of immune response caused by cell surface markers
and no limitation to injection doses due to smaller sizes (Ha
et al. 2016). Additionally, they can be targeted to specific
tissues or organs by modification of their cargos (Minghua
et al. 2018). Based on their size, EVs can be divided into
microvesicles (100–1000 nm), apoptotic bodies (50 nm–5 μm)
or exosomes (30–150 nm). Of these, exosomes are one of the
most studied EVs (Sanz-Ros et al. 2023). Stem cell-derived
exosomes have been reported to yield positive results similar
to cell therapy through paracrine mechanisms (Gallet et al.
2017). Exosomes contain nucleic acids, proteins, lipids, and
several other factors, which have been reported to play a
positive role in cardiac disease models, including atrial fib-
rillation, MI, and cardiac hypertrophy (Fu et al. 2020; Meng
et al. 2020). Moreover, by acting as carriers of important
miRNAs, exosomes can regulate inflammation and apoptosis
resulting in reduced expression of fibrotic and inflammatory
markers and improving heart function (Sareen et al. 2023).
In a study by Shao et al. (2017), it was demonstrated that the
miRNA sequence profile for MSC-derived exosomes (MSCs-Ex)
was similar to MSCs, indicating exosomes to be the probable
reason for positive effects of MSCs. Zhang et al. (2019) demon-
strated that exosomes secreted by hypoxia-preconditioned
BM-MSC overexpressing miRNA-24 suppressed CM death in
rats. A study conducted by Cheng et al. illustrated the effect of
EVs overexpressing HIF1α on CMs in rats. Through paracrine
signaling, these exosomes reduced scarring and increased
the survival of the CMs under hypoxic conditions (Cheng et
al. 2020). Particularly for pluripotent stem cells, exosomes
can prevent the risk of teratoma formation as demonstrated
by Adamiak et al. The authors compared the effect of iPSCs
and iPSC-EVs in MI models. Though both the groups showed
similar positive effects on heart tissue, iPSC group showed
cardiac tumors that were absent in iPSC-EV-treated animals
(Adamiak et al. 2018). Similar to MSCs, according to a study
in pluripotent cells, exosomes from iPSC-derived CMs (iPSC-
CMs Ex) were reported to have miRNA profile similar to
iPSC-CMs. The iPSC-CM Ex group showed reduced apoptosis
and cardiac fibrosis along with improved cardiac function
post treatment (Santoso et al. 2020). This study serves as
an important source of potential target transcripts of the
exosomes and thereby determine suitable cargoes that may
enhance their therapeutic benefits. It is also noteworthy that
171
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iPSC-CMs-EVs have also been shown to improve wall thick-
ness, reduce cardiac hypertrophy, and promote angiogenesis
in porcine models without any safety concerns related to fi-
brous tissues formation and unwanted remodeling (Gao et al.
2020). Currently, a clinical trial (NCT03384433) is underway
that aims to examine the effect of miR-124 containing allo-
geneic MSCs-Ex in patients with acute ischemic stroke. The
study will assess the safety of these exosomes over a 1-year
period using a modified ranking scale (Dehghani 2021).
Another promising approach includes the application of
nanoparticles imitating EVs to target MI. A study conducted
by Yao et al. demonstrates the possibility of using a nano
complex that imitates an exosome, specifically mesoporous
silica nanoparticles that are covered with MSC membranes.
Can. J. Physiol. Pharmacol. Downloaded from cdnsciencepub.com by 119.18.3.4 on 04/16/24
The membrane coating is a camouflaging strategy for the
immune system, as the nanoparticles can efficiently reach
the target area and decrease interactions with other envi-
ronments along the way. This nanocomplex also included
miRNA, specifically miR-21, which is an essential part of the
exosome. It has been shown to promote regeneration in in-
farcted cardiac muscle and decrease CM apoptosis (Yao et al.
2020). The potential use of nanoparticles in cardiac repair is
critical because this technique is less invasive and can im-
prove delivery.
Genetic manipulation of stem cells
Innumerable gene modification therapies have been inves-
tigated so far with the aim of bringing stem cell therapy
to patient-care settings. These genetic modifications can en-
hance cell survival, improve cardiac-specific differentiation
of stem cells, improve secretion of paracrine factors, or re-
duce immune response generated by transplanted stem cells
(Korshunova et al. 2020). AD-MSCs treated with lentiviral par-
ticles for enhanced secretion of IFG-1 showed improved heart
function compared to non-transduced AD-MSCs. IGF1+ AD-
MSCs also showed enhanced secretion of VEGF and HGF
(Bagno et al. 2016). Moreover, Li et al. showed that when
MSCs are genetically manipulated for increased expression
of IGF-1 and treated with 5-azacytidine (5-AZA) in a porcine
model, they triggered to differentiate into CM-like cells to
a higher degree compared to cells treated with 5-AZA alone
(J. Li et al. 2015). Another strategy used to increase the mi-
gration and survival of MSCs is genetically engineering them
to overexpress stromal-derived factor-1, which is one of the
most common chemokines playing a role in cell migration
(Nowakowski et al. 2016). Additionally, genetic modification
of MSCs to overexpress immunosuppressive factors like cy-
clooxygenase 2 has been reported to enhance their survival
rate in ischemic rat hearts (Sareen et al. 2020). The MHC-II
and human leukocyte antigen (HLA) are major contributors
towards immune response generation following stem cell
transplantation. Therefore, some studies focus on preventing
the upregulation of MHC-II to prevent immune response gen-
eration in MSCs (Abu-El-Rub et al. 2019, 2020). Another group
of researchers engineered PSCs to express HLA-E genes at B2M
locus, without surface expression of HLA-A, B, or C, making
them resistant to natural killer cell lysis and were unrecog-
nizable by CD8+ T cells (Gornalusse et al. 2017). Liang et al.
172 Can. J. Physiol. Pharmacol. 102: 161–179 (2024) | dx.doi.org/10.1139/cjpp-2023-0202
investigated whether the upregulation of ERBB4 in MSCs has
a role in increasing cardio protection in a mouse model of MI.
The ERBB4 overexpression reduced apoptosis, accelerated CM
division, and decreased the infarct size. Moreover, it also pro-
moted cell migration and rendered the MSCs resistant to hy-
poxic and low glucose conditions of the cardiac milieu (Liang
et al. 2015). Hence, it is reasonable to profess that MSCs mod-
ified to overexpress ERBB4 have significant cardioprotective
benefits.
Conclusion
Stem cell therapy has the potential to offer a long-term
promise to improve the condition of patients with heart dis-
ease. The stem cells exhibit paracrine effects to suppress the
immune response in the ischemic heart favoring reverse re-
modeling. Findings from multiple studies have supported
their role in improving heart function, reducing cardiac fi-
brosis, preventing CM death, and promoting vascular regen-
eration of the myocardium post ischemia. However, the field
of stem cells lacks standardization in terms of cell culture
techniques and cell characterization, which might be the con-
tributing factor towards the variable results seen in clinics.
These can be addressed by defining standard operating pro-
tocols for all the laboratories to follow. Stem cells also face is-
sues like host immune rejection and migration to undesired
tissues post transplantation. However, use of synthetic bio-
materials for their direct injection in the myocardium and
EVs as cargos for their paracrine factors is a viable alternate
to cell therapy. Though these strategies are feasible, more in-
depth studies are required to assess their efficacy and safety
for translational application in cardiac patients.
Acknowledgements
This work was supported by funding from the Canadian In-
stitutes of Health Research to SD. NS was supported by “Go
for it” Project of Fondazione CRUI (Italy).
Article information
History dates
Received: 2 June 2023
Accepted: 2 August 2023
Version of record online: 16 January 2024
Notes
This paper is a part of a collection entitled "Physiology of
Adaptation and Medicine." It comprises papers from the ISAM
Conference held in Orlando, Florida, October 25-28, 2022.
https://cdnsciencepub.com/topic/cjpp-physiology-adaptation-
medicine.
Copyright
© 2024 The Author(s). This work is licensed under a Creative
Commons Attribution 4.0 International License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduc-
tion in any medium, provided the original author(s) and
source are credited.

Data availability
No data were generated.
Author information
Author ORCIDs
Elika Verma https://orcid.org/0000-0002-6642-7280
Sanjiv Dhingra https://orcid.org/0000-0002-2664-7633
Author contributions
Conceptualization: JKG, SKR, NS, SD
Data curation: NS
Funding acquisition: SD
Methodology: JKG, SKR, EV, NS, SD
Can. J. Physiol. Pharmacol. Downloaded from cdnsciencepub.com by 119.18.3.4 on 04/16/24
Project administration: SD
Resources: SD
Supervision: SD
Writing – original draft: JKG, SKR, EV, NS, SD
Writing – review & editing: JKG, EV, NS, SD
Competing interests
The authors declare no conflict of interest to this work.
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