Stemcell Cardiovascular

HHS Public Access
Author manuscript
J Cardiothorac Vasc Anesth. Author manuscript; available in PMC 2020 January 01.
Author Manuscript
Published in final edited form as:

J Cardiothorac Vasc Anesth. 2019 January ; 33(1): 209–222. doi:10.1053/j.jvca.2018.04.048.
Stem Cell Therapies in Cardiovascular Disease

Maia Terashvili, PhD and
Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road,
Milwaukee, Wisconsin, 53226, USA
Zeljko J. Bosnjak, PhD, FAHA*

Departments of Medicine and Physiology, Medical College of Wisconsin, 8701 Watertown Plank
Author Manuscript
Road, Milwaukee, Wisconsin, 53226, USA
Abstract
Despite considerable advances in medicine, cardiovascular disease is still rising; with ischemic
heart disease being the leading cause of death and disability worldwide. Thus extensive efforts are
continuing to establish effective therapeutic modalities that would improve both quality of life and
survival in this patient population. Novel therapies are now being investigated not only to protect
the myocardium against ischemia-reperfusion injury but also regenerate the heart. Stem cell
therapy, such as potential use of human mesenchymal stem cells, induced pluripotent stem cells
and their exosomes will make it possible not only to address molecular mechanisms of cardiac
conditioning, but also develop new therapies for ischemic heart disease.
Despite all the studies and progress made over the last 15 years on the use of stem cell therapy for
Author Manuscript
cardiovascular disease, the efforts are still in their infancy. While the expectations have been high,
the findings indicate that most of the clinical trials are generally small and the results are
inconclusive. Because of many negative findings, there is certain pessimism that cardiac cell
therapy is likely to yield any meaningful results over the next decade or so. Similar to other new
technologies, early failures are not unusual and they may be followed by impressive success.
Nevertheless, there has been considerable attention to safety by the clinical investigators since the
adverse events of stem cell therapy have been impressively rare. In summary, while the
regenerative biology might not help the cardiovascular patient in the near term, it is destined to do
so over the next several decades.
Introduction
Author Manuscript
Cardiovascular disease is the leading global cause of death, accounting for over 17 million
deaths per year. The number of cardiovascular deaths is expected to grow to more than 23
*
Corresponding author: Zeljko J. Bosnjak, PhD, FAHA, Department of Medicine, Medical College of Wisconsin, 8701 Watertown
Plank Road, Milwaukee, Wisconsin, 53226, USA, ([email protected]).
Disclosure
The authors declare that they have no disclosures.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our
customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of
the resulting proof before it is published in its final citable form. Please note that during the production process errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Terashvili and Bosnjak Page 2
million by 2030, according to a report from the American Heart Association.1 In 2011
Author Manuscript
nearly 787,000 people died from heart disease, stroke and other cardiovascular diseases in
the United States. Two new approaches have been identified that have the potential of added
benefits to the current therapeutic strategies. The first focuses on enhancing the heart/
myocardium’s tolerance to ischemia-reperfusion injury using cardiac conditioning that will
be covered here only briefly as a historical background. The second approach is to create an
environment within the heart muscle that will result in repair of the damaged myocardium; a
topic of this review.
Considerable experimental evidence obtained in multiple models and species has

demonstrated that all forms of myocardial ischemic conditioning (pre-conditioning, per-
conditioning, post-conditioning and remote preconditioning) induce very potent
cardioprotection in animal models.2–5 In healthy, young hearts, many of these conditioning
methods can significantly increase the heart’s resistance against ischemia and reperfusion
Author Manuscript
injury. However, essentially none of these forms of myocardial ischemic conditioning have
been effective in patients. Remote ischemic pre-conditioning using transient arm ischemia–
reperfusion did not improve clinical outcomes in the ERICCA study, with 1,612 patients
undergoing elective on-pump coronary artery bypass grafting.6 Additionally, upper-limb
remote ischemic preconditioning performed in 1,385 patients did not show any significant
benefit among patients undergoing elective cardiac surgery.7 Therefore, these large
multicenter trials have not only proved that ischemic conditioning was unsuccessful in
cardiac surgeries; they also failed to confirm the presence of initial cardioprotection by
ischemic conditioning-induced reduction of cardiac troponin release,8, 9 which is a standard
diagnostic indicator of myocardial injury. The lack of clinical success most likely is due to
underlying risk factors that interfere with cardiac conditioning, along with the use of
cardioprotective agents that activate the endogenous cardioprotective mechanisms. Future
Author Manuscript
preclinical validation of drug targets and cardiac conditioning will need to focus more on
comorbid animal models (such as age, diabetes, and hypertension) and choosing the relevant
endpoints for assessing the efficacy of cardioprotective procedures to have a successful,
clinical translation.
While the existing therapies for the ischemic heart disease lower the early mortality rates,
prevent additional damage to the heart muscle, and reduce the risk of further heart attacks,
most of the patients are likely to have worse quality of life including frequent
hospitalizations. Therefore, there is an ultimate need for a treatment to improve the clinical
conditions by either replacing the damaged heart cells and/or improve cardiac performance.
Thus, the cardiac tissue regeneration with the application of stem cells, or their exosomes,
may be an effective therapeutic option.10 Stem cells, both adult and embryonic stem cells
Author Manuscript
(ESCs) have the ability to self-replicate and transform into an array of specialized cells.
Stem cells are becoming the most important tool in regenerative medicine since these cells
have the potential to differentiate into cardiomyocytes. It would, therefore, be useful to find
out if the differentiated cells can restore and improve cardiac function safely and effectively.
The purpose of this review is to present the current state of knowledge of potential use of
human stem cells, induced human pluripotent stem cells (hiPSCs), and stem cell-derived
exosomes as a cell based therapeutic strategy for the treatment of the damaged heart. These
stem cells also provide feasibility to address fundamental research questions directly
Author Manuscript
relevant to human health, including their challenges, limitations, and potential, along with
future prospects. Human induced pluripotent stem cell technology, in particular, patient-
specific hiPSC-derived cardiomyocytes (hiPSC-CMs) recently has enabled modeling of
human diseases, offering a unique opportunity to investigate potential disease-causing
genetic variants in their natural environment.
Stem Cells
Although there are many different kinds of stem cells, in this review we will include only
those that have been used for most current cardiac regeneration studies.
Embryonic stem cells are obtained from the inner cell mass of the blastocyst that forms three
to five days after an egg cell is fertilized by a sperm. They can give rise to every cell type in
Author Manuscript
the fully formed body, but not the placenta and umbilical cord.
Tissue-specific stem cells (also referred to as somatic or adult stem cells) are more
specialized than embryonic stem cells. The primary roles of adult stem cells in a living
organism are to maintain and repair the tissue in which they are found.
Mesenchymal stem cells are multipotent stromal cells which can be isolated from the bone
marrow. They are non-hematopoietic, multipotent stem cells with the capacity to
differentiate into mesodermal lineage such as bone cells, cartilage cells, muscle cells and fat
cells.
Induced pluripotent stem cells, or iPS cells, are cells taken from any tissue (usually skin or
blood) and are genetically modified to behave like an embryonic stem cell. They are
Author Manuscript
pluripotent, which means that they have the ability to form all adult cell types.
Umbilical cord blood stem cells are collected from the umbilical cord at birth and they can
produce all of the blood cells in the body.
There is great potential with the recent advances in stem cell research and hiPSC-CMs, as
these cells express the same ion channels and signaling pathways as primary human
cardiomyocytes, can be cultured for a long time and are available in sufficient quantity. In
addition, hiPSCs derived from diseased patients may be able to provide new forms of
treatment of ischemic heart disease due to their potential for repairing damaged cardiac
tissue, as shown in the Wu’s laboratory.11 Apart from their more direct role of tissue
regeneration, stem cells may also have a clinical impact by secreting multiple growth factors
and cytokines. Trophic mediators secreted by stem cells improve cardiac function by a
Author Manuscript
combination of various mechanisms such as attenuating tissue injury, inhibiting fibrotic

remodeling, promoting angiogenesis, mobilizing host tissue stem cells, and reducing
inflammation. The cardioprotective panel of stem cell secreted factors are considerable and
include, but not limited to bFGF/FGF-2, IL-1β, IL-10, PDGF, VEGF, HGF, IGF-1, SDF-1,
thymosin-β4, Wnt5a, Ang-1 and Ang-2, MIP-1, EPO and PDGF.12–18 FGF-2 reduces
ischemia-induced myocardial apoptosis, cell death and arrhythmias, and stimulates increased
expression of anti-apoptotic Bcl-2.19, 20 HGF, bFGF, Ang-1 and -2, and VEGF secreted by
BMMSCs lead to augmented vascular density and blood flow in the ischemic heart21–23,
Author Manuscript
whereas SDF-1, IGF-1, HGF facilitate circulating progenitor cell recruitment to injury sites
thereby promoting repair and regeneration.24–27 Stem cells also secrete ECM components
including collagens, TGF-β, matrix metalloproteinases (MMPs) and tissue-derived inhibitors
(TIMPs) that inhibit fibrosis.28–30
Therefore, the use of the right mediator may contribute to a better outcome in cell therapy.
Many stem cell types have been used in regenerative cardiac research, including bone
marrow-derived cells, myoblasts, endogenous cardiac stem cells, umbilical cord-derived
mesenchymal stem cells and embryonic cells. However, an exciting new milestone in the
field of regenerative and precision medicine was the development of hiPSCs. The
therapeutic potential of hiPSCs is considerable, as they are patient-specific stem cells that do
not face the immunologic barrier, in contrast to embryonic stem cells. Furthermore, there are
sources of tissue to be reprogrammed into hiPSCs that are easily accessible, such as the
Author Manuscript
donor’s skin, fat, or blood. Their use may avoid common legal and ethical problems that
arise from the use of embryonic stem cells; they can differentiate into functional
cardiomyocytes and they are now one of the most promising cell sources for cardiac
regenerative therapy.
Pluripotent Stem Cells

Pluripotent stem cells (PSCs) have been derived by explanting cells from embryos at
different stages of development under various growth conditions. PSCs can be classified into
two distinct states, naive and primed, which are believed to represent successive snapshots of
pluripotency as embryonic development proceeds.31, 32 Naïve pluripotent stem cells can be
maintained in vitro by supplying leukocyte inhibitory factor combined with inhibition of
Author Manuscript
mitogen-activated protein kinase/extracellular regulated kinase and glycogen synthase kinase

3 signaling, and are characterized by two active X chromosomes. Primed pluripotent stem
cells are dependent on fibroblast growth factor 2 signaling and transforming growth factor-β
signaling, and display inactivation of one X chromosome.31 Human embryonic stem cells
and induced PSCs (iPSCs) are considered to share some properties of naïve mouse
embryonic stem cells.33 Naïve human iPSCs can be derived by reversion of primed iPSCs
into a state that resembles naïve mouse ESCs.34
Fibroblasts are the most commonly used primary somatic cell type for the generation of
iPSCs. Fibroblasts can be reprogrammed to stable self-renewing iPSCs which resemble
ESCs by enforced expression of a cocktail of transcription factors consisting of octamer-
binding protein (Oct4), SRY-box containing gene 2 (Sox2), Kruppel-like factor 4 (Klf4), c-
myelocytomatosis oncogene (c-Myc), Lin28, and Nanog gene.35, 36 iPSCs can be generated,
Author Manuscript
expanded, and then differentiated into any cell types including endothelial cells (ECs) and
cardiomyocytes for in vitro studies or, ultimately, cell therapy.37, 38
In recent years, it has been shown that somatic cells can be directly converted to
cardiomyocytes, although the efficacy is extremely low. Transgenic expression of three
cardiac-specific transcription factors (Gata4, Mef2c, and Tbx5) resulted in the trans-
differentiation of fibroblasts into contracting cardiomyocytes referred to as induced
cardiomyocytes (iCMs). In addition, other reports have also shown that direct
Author Manuscript
reprogramming of somatic cells to iCMs is also feasible using various small molecules and
microRNAs (miRNAs), such as Hand2, Mesp1, Myocardin, ESRRG, miR-1, and
miR-133.39–42 Subsequently, alternative approaches have succeeded in generating human
iCMs with gene expression profiles and functional characteristics similar to those detected in
ESC-CMs.43
Induced Pluripotent Stem Cells

Due to the aforementioned advances in iPSC-derived CMs, it is now possible to generate an
unlimited quantity of a patient’s own heart cells. This new model allows researchers to study
and understand the molecular and cellular mechanisms of inherited cardiomyopathies,
channelopathies, as well as model acquired heart diseases. Although additional studies are
needed to test their safety and efficacy, these heart cells may be also used for regenerative
Author Manuscript
medicine applications following myocardial infarction.
It was shown that hiPSCs may lose their pluripotency when transplanted into a border zone
of infarcted cardiac tissue, and engraft into native myocardium where they only partially
differentiate into cardiac myocytes. In Yan’s study, they reported that iPS cell transplantation
in the infarcted diabetic db/db and nondiabetic mice resulted in an increase in vascular
smooth muscle and endothelial cells in the infarcted heart, leading to a significantly
improved cardiac function (Figure 1).44
Another study demonstrated that iPSC derived progenitor cells differentiated into a
cardiomyocyte phenotype and developed contracting areas in mice heart tissue. Beneficial
remodeling and improved ventricular function were observed despite the lack of well-
aligned mature donor cardiomyocytes.45
Author Manuscript
In regards to safety, an important obstacle to the clinical use of hiPSCs for the regenerative
purposes is their great heterogeneity in terms of plasticity and epigenetic landscape. There is
a potential that allogeneic hiPSC transplantation into the heart may cause in situ
tumorigenesis.46 In addition, the heterogeneity of the cardiac cells produced from
pluripotent hiPSCs administration and their random implantation is likely to cause cardiac
arrhythmias. One of the main limitations of the hiPSC-derived cardiomyocytes is that they
are embryonic in nature as compared to adult cells. Many laboratories are still trying to
make these myocytes more mature and to make lineage-specific cells so as to obtain a pure
population of atrial cells, nodal cells, or ventricular cells. iPSC-derived cardiomyocytes
exhibit an immaturity of the sarcoplasmic reticulum, and a β-adrenergic response that is
significantly different from native ventricular tissue of a comparable age. Once the cells are
Author Manuscript
mature, it is also likely that investigators will be able to test the effects of various drugs
using hiPSC-CMs from a diverse population of patients with different sexes, ethnicities, and
cardiovascular diseases.
We are utilizing a model of the patient-specific hiPSCs differentiated into cardiac lineage in
order to delineate the environmental and cellular mechanisms responsible for impaired
cardioprotection in diabetes. The advantage of this approach is that the effect of
cardioprotection can be evaluated in human cells, thereby capturing the complex physiologic
Author Manuscript
interactions at the patient-specific myocyte level. Our results indicate that iPSC-derived
cardiomyocytes are not only a viable model to investigate the underlying mechanisms of
anesthetic cardioprotection,47 but they also respond similarly to human myocytes48 and
human embryonic stem-cell-derived cardiomyocytes.49 Isoflurane preconditioning protected
hiPSC-derived cardiomyocytes from oxidative stress-induced lactate dehydrogenase release
and mitochondrial permeability transition pore opening at normal glucose concentrations
(Figure 2).50
Anesthetic preconditioning also protects cardiomyocytes indirectly through its action on

other cell types in the heart, such as endothelial cells,51 or by modulation of inflammatory
response. However, hyperglycemia undermines the effectiveness of anesthetic-induced
cardioprotection by dysregulating cellular signaling.47 In addition, this study demonstrated
that the cardioprotective effects of isoflurane in elevated glucose conditions can be restored
Author Manuscript
by scavenging reactive oxygen species or inhibiting mitochondrial fission. These findings

may contribute to further understanding and guidance for restoring pharmacological
cardioprotection in hyperglycemic patients. Cardiomyocytes derived from healthy donors
and patients with a particular disease (such as diabetes) open new possibilities of studying
genotype and phenotype related pathologies in a human-relevant model. Such diseases were
nearly impossible to investigate in the past due to the lack of human cardiac cells available
for experimental investigation.
Intra-myocardial Cell-Based Therapy

Preclinical studies
Some preclinical studies provide evidence that bone marrow stem cells contribute to cardiac
Author Manuscript
function and reverse remodeling after ischemic damage52 acting both locally53 and remotely.
54 In studies to date, bone marrow stem cells have been either infused55 or injected56 in
areas that were undergoing revascularization. In preclinical studies, Bolli’s group reported
that multiple treatments are necessary to properly evaluate the full therapeutic potential of
cell therapy.57, 58 In their study on mice with a myocardial infarction received one or three
doses of cardiac mesenchymal cells through the percutaneous infusion into the left
ventricular cavity, 14 days apart. The single-dose group showed improved left ventricular
ejection fraction after the 1st infusion but not after the 2nd and 3rd-vehicle infusion. In
contrast, in the multiple-dose group, left ventricular ejection fraction improved after each
cardiac mesenchymal cell-infusion. The multiple-dose group also exhibited less collagen in
the non-infarcted region vs. the single-dose group. Engraftment and differentiation of
cardiac mesenchymal cells were negligible in both groups, indicating paracrine effects.58
Author Manuscript
There appear to be at least three dominant mechanisms that underlie the cardiac reparative
response: reduction in tissue fibrosis, neovascularization, and neomyogenesis.54, 59
Human ESC-CMs isolated from embryoid bodies have also been used for replacing
myocardial scar tissue with new, functional cardiac cells, and therefore achieving actual
myocardial regeneration. The ESC-CMs behave structurally and functionally like
cardiomyocytes, expressing characteristic morphology, cell marker and transcription factor
expression, sarcomeric organization and electrophysiological properties, including
spontaneous action potentials and beating activity.60 Mouse and human cardiac-committed
Author Manuscript
ESCs have been transplanted into small and large animal models of acute and old MI.
Although these studies have demonstrated durable in vivo engraftment, proliferation and
differentiation of ESC-CMs, as well as electromechanical integration with host
cardiomyocytes61, they have not universally shown improvement in myocardial remodeling
and function. While the reported benefits can thus be attributed to a potential synergy62
between the favorable environment created by the revascularization of the region and the
mesenchymal stem cells, the precise delineation of each contribution, however, remains
unknown. In addition, so far there is no evidence that critical number of new cells is
regenerated or injected stem cells survive following the transplant. Animal studies have
shown that only 1% of the stem cells injected into the heart tissue are detectable after 1
month. Nevertheless, in one of the recent systemic reviews and meta-analysis studies of
preclinical work, cardiac stem cells treatment resulted in significant improvement of ejection
fraction compared with placebo.63 In addition, there was a reduction in the magnitude of
Author Manuscript
effect in large compared with small animal models.
A cell-based therapy could offer additional clinical benefits for post-ischemic heart by
improving revascularization along with structural and functional properties.12, 64, 65 There
are several limitations for effective stem cell therapy, but the major problems deal with their
delivery, type of cells to be used, limited retention of the cells in the heart and the risk of
immune rejection. Direct injection of stem cells into the heart muscle results in significant
cell death and washout resulting in majority of cells being removed from the heart soon after
the injection. Many preclinical studies have reported that intravenously administered MSCs
for acute myocardial infarction attenuate the progressive deterioration in LV function and
adverse remodeling in mice with large infarcts, and in ischemic cardiomyopathy, they
improve LV function.63 Moreover, the cardiac phenotype of human embryonic stem cell-
Author Manuscript
derived cardiac myocytes and human induced pluripotent stem cell-derived cardiac
myocytes salvages the injured myocardium better than undifferentiated stem cells through
their differential paracrine effects.66
Clinical studies
In the clinical studies, the investigators have used mainly two approaches of cell
administration: intramyocardial delivery and intracoronary injection. Direct cardiac muscle
injections can be performed either surgically or using percutaneous endocardial injection
catheters, while coronary injection of stem cells can be done using an antegrade
intracoronary artery injection or a retrograde sinus injection. The antegrade intracoronary
artery injection is more attractive because it is the least invasive but some microvascular
plugging can occur as a result of stem cell injection leading to microinfactions when the
Author Manuscript
cells injected are too large for the capillary bed. Since the stem cells also need to cross the
capillary wall, this approach has been found to be less effective as compared to
intramyocardial delivery. Although the cell type, dosage, concentration, and delivery
modalities are important considerations for regenerative cell therapy clinical trials, the
available data are inconclusive and additional early phase studies will be needed before
proceeding to pivotal clinical trials.67
The stem cells derived from the bone marrow of the healthy donors have been used in
Author Manuscript
majority of clinical trials as briefly summarized in Table 1. So far, the clinical trials for
cardiac regeneration have mainly used cell-based therapies, including bone marrow-derived
cells, mesenchymal stem cells and cardiac progenitor cells. While the listed studies have met
safety end points either with autologous or allogeneic cell sources68, the effect on cardiac
function has been somewhat disappointing. One of the largest multicenter clinical trials
using bone-marrow cells given via intracoronary injection for myocardial infarct patients,
failed to reinforce the notion that these therapies are efficacious since it did not meet its
primary goal.69 A recently published Cochrane review of bone-marrow trials for heart attack
patients also found no benefits for various primary goals such as mortality, morbidity, life
quality and LVEF.70 An additional Cochrane review using bone-marrow-derived stem/
progenitor cells as a treatment for chronic ischemic heart disease and congestive heart failure
identified low-quality evidence of reduction in mortality and improvement of LVEF.71
Author Manuscript
The limited clinical success of stem-cell injections for the treatment of myocardial infarction
or heart failure has been mainly attributed to the low retention and survival of injected cells.
One of the clinical trials for treatment of heart failure resulting from ischemic heart disease
used autologous c-kit(+) cardiac stem cells and produced a significant improvement in both
global (Figure 3) and regional LV function (Figure 4), a reduction in infarct size, and an
increase in viable tissue that persisted at least 1 year after cardiac infusion (SCIPIO trial).72
Another study, also involving small number of patients, used intracoronary administration of
autologous cardiosphere-derived cells and the treatment led to a decreased scar size,
increased viable myocardium, and improved regional function of infarcted myocardium at 1
year post-injection (CADUCEUS trial) (Figure 5).73
Cells harvested from the umbilical cord

Author Manuscript
Umbilical cord blood has been demonstrated as a very useful and rich source of stem and
progenitor cells, capable of restoring blood formation and immunological functions after
transplantation. Cord blood stem cells are currently used to treat a range of blood disorders
and immune system conditions such as leukemia, anemia and autoimmune diseases. These
stem cells are used largely in the treatment of children but have also started being used in
adults following chemotherapy treatment. Another type of cell that can also be collected
from umbilical cord blood is mesenchymal stromal cells. These cells can grow into bone,
cartilage and other types of tissues and are being used in many research studies to see if
patients could benefit from these cells too. The fact that cord blood can be frozen and stored
for later use led, in 1991, to the establishment of the first cord blood bank from voluntary
donors in New York. To date, there are over 54 public cord blood banks in different parts of
the world with more than 350,000 units frozen and ready to be used.74 Indeed cord blood
Author Manuscript
transplantation is being used as an alternative to bone marrow transplantation, and more than
14,000 transplants have been documented. Cord blood stem cell treatments differ from bone
marrow stem cell treatments in three key areas: increased tolerance of the human leukocyte
antigen-mismatching, decreased risk of graft-versus-host disease, and enhanced proliferation
ability.75 Recent results of the RIMECARD study by Bartolucci et al. in human subjects
using umbilical cord-derived MSCs as potential heart failure therapy are quite encouraging.
76 The patients had stable heart failure (HF), with reduced ejection fraction of less than 40.
Although the sample size was small (15 controls and 15 HF patients treated with UC-MSCs)
Author Manuscript
to establish either safety or efficacy, the echocardiographic and cardiac MRI evaluations
demonstrated improvements in ejection fraction, starting at 3 months, and persisting through
12 months. The patients treated with placebo did not improve in either left ventricular
ejection fraction or clinical functional class. As indicated by the authors, it is tempting to
speculate that the robust paracrine secretion of various factors, including hepatocyte growth
factor, might play an important role in mediating the therapeutic effects of the UC-MSCs.
The main disadvantage of cord blood use is that the volume collected is fixed and relatively
small. Therefore, the number of stem cells available for transplantation is low compared to
the number of cells that can be collected in customizable bone marrow or peripheral blood
stem cell harvests. Nevertheless, there are many opportunities for further development of
this technology such as the cord blood selection algorithms that are currently heavily
weighted toward maximizing cell doses at the expense of the human leukocyte antigen-
Author Manuscript
matching.77
Tissue Engineering
Beside the stem cell injection therapy, currently there are non-cardiogenic and cardiogenic
stem-cell tissue patches, for the repair of myocardial infarction. Recent studies have utilized
non-cardiogenic tissue patches made of skeletal myoblasts78–81, bone marrow-derived stem
cells82, 83, or endothelial progenitor cells84 for the repair of damaged heart. Compared with
the injection of a cell suspension, the implantation of tissue sheets composed of skeletal
myoblasts has been proven more advantageous for the treatment of myocardial infarction in
rats78, 85, and dilated cardiomyopathy in hamsters.86 Moreover several cardiogenic cardiac
tissue-engineering methodologies have been developed for use with primary neonatal
cardiomyocytes. These include: injection of a mixture of bioactive hydrogels and cells
Author Manuscript
followed by cell-hydrogel polymerization in situ87 and the epicardial implantation of a

tissue-engineered cardiac patch.88, 89
Generally, implantation of the engineered myoblast sheets over an infarction site yielded
improved neovascularization, attenuated left ventricular dilatation, decreased fibrosis,
improved fractional shortening, and prolonged animal survival compared to the delivery of
the same number of myoblasts by cell injection.85 In addition, the bone marrow-derived,
spatially arranged SMC-endothelial progenitor bi-level cell sheet interactions between
SMCs and endothelial progenitor cells augment mature neovascularization, limit adverse
remodeling, and improve ventricular function after myocardial infarction.90 In diabetic
patients treatment of diabetes mellitus-induced cardiomyopathy with tissue-engineered
smooth muscle cell-endothelial progenitor cell bi-level cell sheets prevented cardiac
Author Manuscript
dysfunction and microvascular disease associated with diabetes mellitus-induced

cardiomyopathy.91 As indicated before, the main disadvantage of injecting the cell-
suspensions directly into the heart muscle compare to engineered heart tissue technique is
that most of the injected cells are washed out of the heart or do not survive the injection.
This is inefficient and can also be dangerous if some cells have not yet fully developed into
myocardial cells and are therefore still pluripotent. These cells could survive in other parts
of the body and form tumors. The advantage of the tissue patches is that significantly fewer
of the stem cell-derived heart cells are required and fewer cells undergo apoptosis. Some of
Author Manuscript
the major drawbacks currently encountered with regeneration using tissue patches, include
the problems with electrical continuity and patch vascularization. Using a similar tissue-
engineering strategy, Shimko et al. formed cardiac constructs using pure differentiated
cardiomyocytes derived by genetic selection from D3 mouse ESCs with a neomycin-
resistance gene driven by the α-MHC promoter.92 They found that 10% cyclic stretch at rate
of 1–3 Hz for 3 days increased the expression of cardiac markers such as α-cardiac actin, α-
MHC and Mef-2c, but the resulting cardiac tissues were noncontractile. Immunostaining
showed that pure cardiomyocytes were present, but they had disorganized sarcomeres and a
relatively rounded appearance.92
Recently, in a study published by Nummi et al., they reported that during on-pump coronary
artery bypass graft surgery, part of the right atrial appendage can be excised upon insertion
of the right atrial cannula of the heart-lung machine and the removed tissue can be easily cut
Author Manuscript
into micrografts for transplantation.93 Appendage tissue is harvested during cannulation of

the right atrium, and therefore, no additional procedure is needed. Isolation of the cells and
preparing the matrix for transplantation is done simultaneously with the coronary artery
bypass graft operation in the operating room, so the perfusion time and the aorta clamp time
are not increased. After the bypass anastomoses, the atrial appendage sheet is placed on the
myocardium with three to four sutures allowing the myocardium to contract without
interference. They believe that atrial appendage-derived cells therapy administered during
CABG surgery will have an impact on patient treatment in the future.93
While some of the outcomes of these trials have been modest at best, it is now evident that
the success of future cardiac cell therapies will be highly dependent on the ability to
overcome the problem of low retention and survival of implanted cells.94 Potential
Author Manuscript
approaches to address this issue include: coinjecting cells with bioactive, in situ
polymerizable hydrogels87, preconditioning cells with hypoxia or prosurvival factors95,
genetic engineering of cells to enhance their angiogenic and/or antiapoptotic action96 and
the epicardial implantation of a preassembled tissue-engineered patches.27, 85, 97 In
particular, tissue patch implantation, although surgically more complex than cell or cell/
hydrogel injections, may support long-term survival of transplanted cells and exert a more
efficient structural and functional cardiac tissue reconstruction at the infarct site.98
Cellular Reprogramming
The adult human heart lacks sufficient ability to replenish the damaged cardiac muscles
since the rate of cardiomyocyte renewal activity is less than 1% per year. The mechanical
and electrical engraftment of injected cardiomyocytes is largely not feasible at the scale that
Author Manuscript
would be necessary for cardiac improvement. On the other hand, the human heart contains
large population of fibroblasts that could be used for direct reprograming. As such, direct
fibroblasts reprogramming in vivo has emerged as a possible approach for cardiac
regeneration. With considerably better understanding of the various molecular mechanisms,
direct fibroblast reprogramming has improved considerably but still lacks sufficient efficacy
using human cells (Figure 6).
Stem Cell-Derived Exosomes and Small Vesicles

Author Manuscript
As indicated earlier, the survival of transplanted stem cells is dismal and the beneficial
effects of stem cell therapies is not due to their differentiation into new cardiomyocytes but
instead because they are the temporary source of the exosomal growth factors. Therefore,
despite the stem cells’ early demise, there are some limited cardiac benefits from this
treatment, including decreased cardiomyocyte apoptosis, reduced fibrosis, enhanced
neovascularization and improved left ventricular ejection fraction. It is for that reason why
the exosome therapy recapitulates the benefits of stem cell therapy,99 and many studies have
shown that the activation of cardioprotective pathways obtained by stem cell therapy can be
reproduced by the injection of exosomes produced by the stem cells.100 An additional
benefit of using exosomes for cardioprotection and regeneration is the lack of tumor-forming
potential of exosomes. However, the underlying mechanisms of stem cells or hiPSC-derived
exosome therapy are still unclear. Numerous scientific investigations have identified recent
Author Manuscript
applications of exosomes in the development of molecular diagnostics, drug delivery

systems and therapeutic agents.
Exosomes are small membrane vesicles (30–100 nm) of endocytic origin that are secreted by
most cells after being formed in the cellular multivesicular bodies. The fusion of
multivesicular bodies into the plasma membrane leads to the release of their intraluminal
vesicles as exosomes. Once released in the extracellular environment, their cargo of
functional molecules can be taken up by recipient cells via several mechanisms including
fusion with the plasma membrane, phagocytosis and endocytosis. The formation and release
can be upregulated through different steps based on environmental stimuli such as stress or
hypoxia. There are two main mechanisms responsible for exosome release. First, there is a
constitutive mechanism that is mediated by specific proteins involved in membrane
Author Manuscript
trafficking, such as RAB heterotrimeric G-proteins and protein kinase D. Second, there
exists an inducible mechanism that can be activated by several stimuli including increased
intracellular Ca2+ and DNA damage. Studies have used different approaches to also increase
the angiogenic potential of exosomes released by the stem cells.101 Exosome release with a
basal angiogenic potential can be substantially increased in vitro using stress conditions that
mimic organ injury, such as hypoxia, irradiation, or drug treatments. Changes in exosomal
composition facilitate angiogenesis and tissue repair most likely via enhanced level of
growth factors and cytokines.
Exosomes contain various molecular constituents of their cell of origin, including proteins
and RNA. The cargo of mRNA and miRNA in exosomes was first discovered at the
University of Gothenburg in Sweden.102 In that study, the differences in cellular and
Author Manuscript
exosomal mRNA and miRNA content were described, as well as the functionality of the
exosomal mRNA cargo. Exosomes facilitate cell-cell communication to the recipient cell
membrane and deliver effectors including transcription factors, oncogenes, small and large
non-coding regulatory RNAs (such as microRNAs) and mRNAs into recipient cells and can
be used for cardiac protection and repair. Exosomes have also been shown to carry double-
stranded DNA. Exosomes can be derived from many different types of stem cells including
umbilical cord, cardiosphere-derived cells, cardiac stem cells, embryonic, induced
pluripotent, mesenchymal and endothelial progenitor cells. They can carry and deliver
mRNAs, miRNAs and proteins to the injured heart muscle and play a significant role in
Author Manuscript
resident cardiac stem cell activity, cardiomyocyte proliferation, beneficial cardiac

remodeling, apoptosis reduction, angiogenesis, anti-inflammatory response and a decrease in
infarct size. The advantages for effective exosome therapy include the cell free component,
log-term stability and low or no immune response. Some of the limitations include the
necessity of repeated injections, target cell selection and the random packing of the exosome
cargo.
Some preliminary reports have demonstrated that exosomes released from cardiac progenitor
cells can improve cardiac function in the damaged heart.103, 104 It has been proposed that
exosomes released from transplanted cardiomyocytes are involved in metabolic events in
target cells by facilitating an array of metabolism-related processes, including modulation of
gene expression. Moreover, exosomes secreted from the hiPSC-derived cardiomyocytes
exert protective effects by transferring endogenous molecules to salvage injured neighboring
Author Manuscript
cells by regulating angiogenesis, apoptosis, fibrosis, and inflammation. It has been shown
that ischemic preconditioned hearts promote exosome release and help spread
cardioprotective signals within the myocardium.105, 106 Also, the administration of
mesenchymal stromal cell-secreted exosomes demonstrated improved cardiac function in the
acute myocardial infarction mouse model. Mesenchymal stem cell-derived exosomes
increased adenosine triphosphate levels, reduced oxidative stress, and activated the PI3K/Akt
pathway to enhance cardiomyocyte viability after ischemia-reperfusion (I/R) injury.107
Recently, it was shown that ischemic preconditioning of mesenchymal stromal cells
increased levels of miR-21, miR-22, miR-199a-3p, miR-210, and miR-24 in exosomes
released by the cells, and the administration of mesenchymal stromal cell-ischemic
preconditioning exosomes resulted in a reduction of cardiac fibrosis and apoptosis compared
with the hearts treated with control exosomes. Stem cell-derived exosomes possess the
Author Manuscript
ability to modulate cardiomyocyte survival and confer protection against angiotensin II-
induced hypertrophy by activating PI3K/Akt/eNOS pathways via RNA enriched within the
exosomes. Additionally, it has been shown that exosome treatment increased levels of
adenosine triphosphate and NADH, decreased oxidative stress, increased phosphorylated-
Akt and phosphorylated- glycogen synthase kinase 3, and reduced phosphorylated-c-JNK in
hearts after I/R. Subsequently, both local and systemic inflammations were significantly
reduced 24 hours after reperfusion.107 Intact exosomes restore bioenergetics, reduce
oxidative stress, and activate pro-survival signaling, thereby enhancing cardiac function and
geometry after myocardial I/R injury.107 Clearly, stem cell-derived exosomes may be a
potential adjuvant to reperfusion therapy for myocardial infarction and heart failure.
Conclusions
Author Manuscript
The existing therapies for the ischemic heart disease have many limitations and efforts are
underway for new treatments using the stem cell therapy to improve the clinical conditions
by either replacing the damaged heart cells and/or improve cardiac performance. The cardiac
tissue regeneration with stem cells, their exosomes or small vesicles and tissue engineering
may be effective therapeutic options. Although the expectations have been high, the results
from majority of clinical trials are negative. Due to very low engraftment and survival of
stem cells injected into a cardiac muscle, there is convincing evidence that the release of
paracrine factors from the stem cells contributes to myocardial cardioprotection and
Author Manuscript
regeneration. It is likely that the future research will be focused on the biology of these
endogenous signaling pathways, and will lead the way for different applications of exosomes
and small vesicles in regenerative medicine. In the future there might be more successful
approaches that would utilize stem cell technology with various bioengineering constructs
having not only cardiomyocytes but other cardiac cells.
Regarding the regulatory agencies, one would need a significant efficacy/safety data and
meaningful end points when compared with standard-of-care drugs that are used today for
heart attacks and heart failures. So where are we today since most of the clinical trials did
not achieve their primary efficacy end points? Because the cell-therapy studies for heart
disease did not achieve this so far, more preclinical work might be necessary using current
and other approaches in order to demonstrate compelling rationale for new clinical trials.
Although the regenerative biology might not be very helpful to the cardiovascular patient in
Author Manuscript
the near term, it is most likely that we will witness very impressive and exciting results over
the next several decades.
Acknowledgments
This work was supported in part by grants P01GM066730 and T32 GM089586 from the National Institutes of
Health, Bethesda, MD.
Abbreviations
Ca2+ calcium ion
Ecs endothelial cells

Author Manuscript
eNOS endothelial nitric oxide synthase
eNOS/PKG endothelial nitric oxide synthase/protein kinase G
ERK1/2 Extracellular signal-regulated protein kinases 1 and 2
hiPSCs human stem cells, induced pluripotent stem cells
HiPSC-CMs human stem cells, induced pluripotent stem cells-derived

cardiomyocytes
IPSC induced pluripotent stem cells
NADH Nicotinamide adenine dinucleotide

Author Manuscript
JAK/STAT Janus kinase and Signal Transducer and Activator of

Transcription pathways
p42/p44 MAPK/ERK specific isoforms of mitogen-activated protein kinase and

extracellular regulated kinase
PI3K/Akt/mTOR Phosphatidylinositol 3-kinase, serine/threonine kinase also

Author Manuscript
known as protein kinase B, and mammalian target of

rapamycin pathway
PKC protein kinase C
PKG cGMP protein kinase G pathway
References
1. Benjamin EJ, Blaha MJ, Chiuve SE, Cushman M, Das SR, Deo R, de Ferranti SD, Floyd J, Fornage
M, Gillespie C, Isasi CR, Jimenez MC, Jordan LC, Judd SE, Lackland D, Lichtman JH, Lisabeth L,
Liu S, Longenecker CT, Mackey RH, Matsushita K, Mozaffarian D, Mussolino ME, Nasir K,
Neumar RW, Palaniappan L, Pandey DK, Thiagarajan RR, Reeves MJ, Ritchey M, Rodriguez CJ,
Roth GA, Rosamond WD, Sasson C, Towfighi A, Tsao CW, Turner MB, Virani SS, Voeks JH,
Willey JZ, Wilkins JT, Wu JH, Alger HM, Wong SS, Muntner P. American Heart Association
Author Manuscript
Statistics C, Stroke Statistics S. Heart Disease and Stroke Statistics-2017 Update: A Report From
the American Heart Association. Circulation. 2017; 135:e146–e603. [PubMed: 28122885]
2. Heusch G, Botker HE, Przyklenk K, Redington A, Yellon D. Remote Ischemic Conditioning.
Journal of the American College of Cardiology. 2015; 65:177–195. [PubMed: 25593060]
3. Heusch G. Cardioprotection: chances and challenges of its translation to the clinic. Lancet. 2013;
381:166–175. [PubMed: 23095318]
4. Schwartz Longacre L, Kloner RA, Arai AE, Baines CP, Bolli R, Braunwald E, Downey J, Gibbons
RJ, Gottlieb RA, Heusch G, Jennings RB, Lefer DJ, Mentzer RM, Murphy E, Ovize M, Ping PP,
Przyklenk K, Sack MN, Vander Heide RS, Vinten-Johansen J, Yellon DM. New Horizons in
Cardioprotection Recommendations From the 2010 National Heart, Lung, and Blood Institute
Workshop. Circulation. 2011; 124:1172–1179. [PubMed: 21900096]
5. Heusch G. Molecular Basis of Cardioprotection Signal Transduction in Ischemic Pre-, Post-, and
Remote Conditioning. Circulation Research. 2015; 116:674–699. [PubMed: 25677517]
6. Hausenloy DJ, Candilio L, Evans R, Ariti C, Jenkins DP, Kolvekar S, Knight R, Kunst G, Laing C,
Author Manuscript
Nicholas J, Pepper J, Robertson S, Xenou M, Clayton T, Yellon DM. Investigators ET. Remote
Ischemic Preconditioning and Outcomes of Cardiac Surgery. New England Journal of Medicine.
2015; 373:1408–1417. [PubMed: 26436207]
7. Meybohm P, Kohlhaas M, Stoppe C, Gruenewald M, Renner J, Bein B, Albrecht M, Cremer J,
Coburn M, Schaelte G, Boening A, Niemann B, Sander M, Roesner J, Kletzin F, Mutlak H,
Westphal S, Laufenberg-Feldmann R, Ferner M, Brandes IF, Bauer M, Stehr SN, Kortgen A,
Wittmann M, Baumgarten G, Meyer-Treschan T, Kienbaum P, Heringlake M, Schoen J, Treskatsch
S, Smul T, Wolwender E, Schilling T, Fuernau G, Bogatsch H, Brosteanu O, Hasenclever D,
Zacharowski K. RIPHeart (Remote Ischemic Preconditioning for Heart Surgery) Study
Collaborators. RIPHeart (Remote Ischemic Preconditioning for Heart Surgery) Study: Myocardial
Dysfunction, Postoperative Neurocognitive Dysfunction, and 1 Year Follow-Up. J Am Heart Assoc.
2018 Mar 26.7(7)
8. Hausenloy DJ, Mwamure PK, Venugopal V, Harris J, Barnard M, Grundy E, Ashley E, Vichare S,
Di Salvo C, Kolvekar S, Hayward M, Keogh B, MacAllister RJ, Yellon DM. Effect of remote
ischaemic preconditioning on myocardial injury in patients undergoing coronary artery bypass graft
Author Manuscript
surgery: a randomised controlled trial. Lancet. 2007; 370:575–9. [PubMed: 17707752]

9. Thielmann M, Kottenberg E, Kleinbongard P. Cardioprotective and prognostic effects of remote
ischaemic preconditioning in patients undergoing coronary artery bypass surgery: a single-centre
randomised, double-blind, controlled trial. Lancet. 2013; 382:597–604. [PubMed: 23953384]
10. Fernandez-Aviles F, Sanz-Ruiz R, Climent AM, Badimon L, Bolli R, Charron D, Fuster V,
Janssens S, Kastrup J, Kim HS, Luscher TF, Martin JF, Menasche P, Pinto FJ, Simari RD, Stone
GW, Terzic A, Willerson JT, Wu JC, Group TW. Global Overview of the Transnational Alliance
for Regenerative Therapies in Cardiovascular Syndromes (TACTICS) Recommendations: A
Comprehensive Series of Challenges and Priorities of Cardiovascular Regenerative Medicine. Circ
Res. 2018; 122:199–201. [PubMed: 29348246]
11. Wu JX, Li J, Zhang NN, Zhang CH. Stem cell-based therapies in ischemic heart diseases: a focus
on aspects of microcirculation and inflammation. Basic Research in Cardiology. 2011; 106:317–
Author Manuscript
324. [PubMed: 21424917]

12. Chen L, Tredget EE, Wu PY, Wu Y. Paracrine factors of mesenchymal stem cells recruit
macrophages and endothelial lineage cells and enhance wound healing. PLoS One. 2008; 3:e1886.
[PubMed: 18382669]
13. Korf-Klingebiel M, Kempf T, Sauer T, Brinkmann E, Fischer P, Meyer GP, Ganser A, Drexler H,
Wollert KC. Bone marrow cells are a rich source of growth factors and cytokines: implications for
cell therapy trials after myocardial infarction. Eur Heart J. 2008; 29:2851–8. [PubMed: 18953051]
14. Kubal C, Sheth K, Nadal-Ginard B, Galinanes M. Bone marrow cells have a potent anti-ischemic
effect against myocardial cell death in humans. Journal of Thoracic and Cardiovascular Surgery.
2006; 132:1112–1118. [PubMed: 17059931]
15. Xu M, Uemura R, Dai Y, Wang Y, Pasha Z, Ashraf M. In vitro and in vivo effects of bone marrow
stem cells on cardiac structure and function. J Mol Cell Cardiol. 2007; 42:441–8. [PubMed:
17187821]
16. Uemura R, Xu M, Ahmad N, Ashraf M. Bone marrow stem cells prevent left ventricular
Author Manuscript
remodeling of ischemic heart through paracrine signaling. Circulation Research. 2006; 98:1414–
1421. [PubMed: 16690882]
17. Takahashi M, Li TS, Suzuki R, Kobayashi T, Ito H, Ikeda Y, Matsuzaki M, Hamano K. Cytokines
produced by bone marrow cells can contribute to functional improvement of the infarcted heart by
protecting cardiomyocytes from ischemic injury. American Journal of Physiology-Heart and
Circulatory Physiology. 2006; 291:H886–H893. [PubMed: 16603697]
18. Pallante BA, Duignan I, Okin D, Chin A, Bressan MC, Mikawa T, Edelberg JM. Bone marrow
Oct3/4+ cells differentiate into cardiac myocytes via age-dependent paracrine mechanisms. Circ
Res. 2007; 100:e1–11. [PubMed: 17122441]
19. Xie Z, Koyama T, Suzuki J, Fujii Y, Togashi H, Sawa H, Nagashima K. Coronary reperfusion
following ischemia: different expression of bcl-2 and bax proteins, and cardiomyocyte apoptosis.
Jpn Heart J. 2001; 42:759–70. [PubMed: 11933925]
20. Nishida S, Nagamine H, Tanaka Y, Watanabe G. Protective effect of basic fibroblast growth factor
against myocyte death and arrhythmias in acute myocardial infarction in rats. Circulation Journal.
2003; 67:334–339. [PubMed: 12655165]
Author Manuscript
21. Schinkothe T, Bloch W, Schmidt A. In vitro secreting profile of human mesenchymal stem cells.
Stem Cells Dev. 2008; 17:199–206. [PubMed: 18208373]
22. Schenke-Layland K, Strem BM, Jordan MC, Deemedio MT, Hedrick MH, Roos KP, Fraser JK,
Maclellan WR. Adipose tissue-derived cells improve cardiac function following myocardial
infarction. J Surg Res. 2009; 153:217–23. [PubMed: 18694573]
23. Estrada R, Li N, Sarojini H, An J, Lee MJ, Wang E. Secretome from mesenchymal stem cells
induces angiogenesis via Cyr61. J Cell Physiol. 2009; 219:563–71. [PubMed: 19170074]
24. Askari AT, Unzek S, Popovic ZB, Goldman CK, Forudi F, Kiedrowski M, Rovner A, Ellis SG,
Thomas JD, DiCorleto PE, Topol EJ, Penn MS. Effect of stromal-cell-derived factor 1 on stem-cell
homing and tissue regeneration in ischaemic cardiomyopathy. Lancet. 2003; 362:697–703.
[PubMed: 12957092]
25. Neuss S, Becher E, Woltje M, Tietze L, Jahnen-Dechent W. Functional expression of HGF and
HGF receptor/c-met in adult human mesenchymal stem cells suggests a role in cell mobilization,
tissue repair, and wound healing. Stem Cells. 2004; 22:405–14. [PubMed: 15153617]
Author Manuscript
26. Mourkioti F, Rosenthal N. IGF-1, inflammation and stem cells: interactions during muscle
regeneration. Trends Immunol. 2005; 26:535–42. [PubMed: 16109502]
27. Cabrera-Fuentes HA, Aragones J, Bernhagen J, Boening A, Boisvert WA, Botker HE, Bulluck H,
Cook S, Di Lisa F, Engel FB, Engelmann B, Ferrazzi F, Ferdinandy P, Fong A, Fleming I, Gnaiger
E, Hernandez-Resendiz S, Kalkhoran SB, Kim MH, Lecour S, Liehn EA, Marber MS, Mayr M,
Miura T, Ong SB, Peter K, Sedding D, Singh MK, Suleiman MS, Schnittler HJ, Schulz R, Shim
W, Tello D, Vogel CW, Walker M, Li QOY, Yellon DM, Hausenloy DJ, Preissner KT. From basic
mechanisms to clinical applications in heart protection, new players in cardiovascular diseases and
cardiac theranostics: meeting report from the third international symposium on “New frontiers in
cardiovascular research”. Basic Research in Cardiology. 2016; 111(6):69. [PubMed: 27743118]
Author Manuscript
28. Xu X, Xu Z, Xu Y, Cui G. Effects of mesenchymal stem cell transplantation on extracellular matrix

after myocardial infarction in rats. Coron Artery Dis. 2005; 16:245–55. [PubMed: 15915077]
29. Ohnishi S, Yasuda T, Kitamura S, Nagaya N. Effect of hypoxia on gene expression of bone
marrow-derived mesenchymal stem cells and mononuclear cells. Stem Cells. 2007; 25:1166–77.
[PubMed: 17289933]
30. Ohnishi S, Ohgushi H, Kitamura S, Nagaya N. Mesenchymal stem cells for the treatment of heart
failure. International Journal of Hematology. 2007; 86:17–21. [PubMed: 17675261]
31. Nichols J, Smith A. Naive and Primed Pluripotent States. Cell Stem Cell. 2009; 4:487–492.
[PubMed: 19497275]
32. Kumari, D. States of Pluripotency: Naïve and Primed Pluripotent Stem Cells. INTECH; 2016. ()
33. Stadtfeld M, Hochedlinger K. Induced pluripotency: history, mechanisms, and applications. Genes
& Development. 2010; 24:2239–2263. [PubMed: 20952534]
34. Guo G, Yang J, Nichols J, Hall JS, Eyres I, Mansfield W, Smith A. Klf4 reverts developmentally
programmed restriction of ground state pluripotency. Development. 2009; 136:1063–1069.
Author Manuscript
[PubMed: 19224983]
35. Kelaini S, Cochrane A, Margariti A. Direct reprogramming of adult cells: avoiding the pluripotent
state. Stem Cells Cloning. 2014; 7:19–29. [PubMed: 24627642]
36. Ebert AD, Diecke S, Chen IY, Wu JC. Reprogramming and transdifferentiation for cardiovascular
development and regenerative medicine: where do we stand? Embo Molecular Medicine. 2015;
7:1090–1103. [PubMed: 26183451]
37. Rufaihah AJ, Huang NF, Kim J, Herold J, Volz KS, Park TS, Lee JC, Zambidis ET, Reijo-Pera R,
Cooke JP. Human induced pluripotent stem cell-derived endothelial cells exhibit functional
heterogeneity. American Journal of Translational Research. 2013; 5(1):21–35. [PubMed:
23390563]
38. Zhang JH, Wilson GF, Soerens AG, Koonce CH, Yu JY, Palecek SP, Thomson JA, Kamp TJ.
Functional Cardiomyocytes Derived From Human Induced Pluripotent Stem Cells. Circulation
Research. 2009; 104:E30–E41. [PubMed: 19213953]
39. Jayawardena TM, Egemnazarov B, Finch EA, Zhang L, Payne JA, Pandya K, Zhang Z, Rosenberg
Author Manuscript
P, Mirotsou M, Dzau VJ. MicroRNA-mediated in vitro and in vivo direct reprogramming of

cardiac fibroblasts to cardiomyocytes. Circ Res. 2012; 110:1465–73. [PubMed: 22539765]
40. Nam YJ, Song K, Luo X, Daniel E, Lambeth K, West K, Hill JA, DiMaio JM, Baker LA, Bassel-
Duby R, Olson EN. Reprogramming of human fibroblasts toward a cardiac fate. Proc Natl Acad
Sci U S A. 2013; 110:5588–93. [PubMed: 23487791]
41. Protze S, Khattak S, Poulet C, Lindemann D, Tanaka EM, Ravens U. A new approach to
transcription factor screening for reprogramming of fibroblasts to cardiomyocyte-like cells. J Mol
Cell Cardiol. 2012; 53:323–32. [PubMed: 22575762]
42. Wada R, Muraoka N, Inagawa K, Yamakawa H, Miyamoto K, Sadahiro T, Umei T, Kaneda R,
Suzuki T, Kamiya K, Tohyama S, Yuasa S, Kokaji K, Aeba R, Yozu R, Yamagishi H, Kitamura T,
Fukuda K, Ieda M. Induction of human cardiomyocyte-like cells from fibroblasts by defined
factors. Proc Natl Acad Sci U S A. 2013; 110:12667–72. [PubMed: 23861494]
43. Fu JD, Stone NR, Liu L, Spencer CI, Qian L, Hayashi Y, Delgado-Olguin P, Ding S, Bruneau BG,
Srivastava D. Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state. Stem
Cell Reports. 2013; 1:235–47. [PubMed: 24319660]
Author Manuscript
44. Yan B, Abdelli LS, Singla DK. Transplanted induced pluripotent stem cells improve cardiac
function and induce neovascularization in the infarcted hearts of db/db mice. Mol Pharm. 2011;
8:1602–10. [PubMed: 21851072]
45. Mauritz C, Martens A, Rojas SV, Schnick T, Rathert C, Schecker N, Menke S, Glage S, Zweigerdt
R, Haverich A, Martin U, Kutschka I. Induced pluripotent stem cell (iPSC)-derived Flk-1
progenitor cells engraft, differentiate, and improve heart function in a mouse model of acute
myocardial infarction. European Heart Journal. 2011; 32:2634–2641. [PubMed: 21596799]
46. Gutierrez-Aranda I, Ramos-Mejia V, Bueno C, Munoz-Lopez M, Real PJ, Macia A, Sanchez L,
Ligero G, Garcia-Parez JL, Menendez P. Human induced pluripotent stem cells develop teratoma
more efficiently and faster than human embryonic stem cells regardless the site of injection. Stem
Cells. 2010; 28:1568–70. [PubMed: 20641038]
Author Manuscript
47. Canfield SG, Sepac A, Sedlic F, Muravyeva MY, Bai XW, Bosnjak ZJ. Marked Hyperglycemia
Attenuates Anesthetic Preconditioning in Human-induced Pluripotent Stem Cell-derived
Cardiomyocytes. Anesthesiology. 2012; 117:735–744. [PubMed: 22820846]
48. Mio Y, Bienengraeber MW, Marinovic J, Gutterman DD, Rakic M, Bosniak ZJ, Stadnicka A. Age-
related attenuation of isoflurane preconditioning in human atrial cardiomyocytes - Roles for
mitochondrial respiration and sarcolemmal adenosine triphosphate-sensitive potassium channel
activity. Anesthesiology. 2008; 108:612–620. [PubMed: 18362592]
49. Sepac A, Sedlic F, Si-Tayeb K, Lough J, Duncan SA, Bienengraeber M, Park F, Kim J, Bosnjak ZJ.
Isoflurane Preconditioning Elicits Competent Endogenous Mechanisms of Protection from
Oxidative Stress in Cardiomyocytes Derived from Human Embryonic Stem Cells. Anesthesiology.
2010; 113:906–916. [PubMed: 20823757]
50. Canfield SG, Zaja I, Godshaw B, Twaroski D, Bai X, Bosnjak ZJ. High Glucose Attenuates
Anesthetic Cardioprotection in Stem-Cell-Derived Cardiomyocytes: The Role of Reactive Oxygen
Species and Mitochondrial Fission. Anesth Analg. 2016; 122:1269–79. [PubMed: 26991754]
Author Manuscript
51. Novalija E, Varadarajan SG, Camara AKS, An JZ, Chen Q, Riess ML, Hogg N, Stowe DF.
Anesthetic preconditioning: triggering role of reactive oxygen and nitrogen species in isolated
hearts. American Journal of Physiology-Heart and Circulatory Physiology. 2002; 283:H44–H52.
[PubMed: 12063273]
52. Schuleri KH, Feigenbaum GS, Centola M, Weiss ES, Zimmet JM, Turney J, Kellner J, Zviman
MM, Hatzistergos KE, Detrick B, Conte JV, McNiece I, Steenbergen C, Lardo AC, Hare JM.
Autologous mesenchymal stem cells produce reverse remodelling in chronic ischaemic
cardiomyopathy. European Heart Journal. 2009; 30:2722–2732. [PubMed: 19586959]
53. Zhao YS, Li TL, Wei XF, Bianchi G, Hu JP, Sanchez PG, Xu K, Zhang P, Pittenger MF, Wu ZJJ,
Griffith BP. Mesenchymal Stem Cell Transplantation Improves Regional Cardiac Remodeling
Following Ovine Infarction. Stem Cells Translational Medicine. 2012; 1(9):685–95. [PubMed:
23197875]
54. Suzuki G, Iyer V, Lee TC, Canty JM. Autologous Mesenchymal Stem Cells Mobilize cKit(+) and
CD133(+) Bone Marrow Progenitor Cells and Improve Regional Function in Hibernating
Myocardium. Circulation Research. 2011; 109:1044–1054. [PubMed: 21885831]
Author Manuscript
55. Hu SS, Liu S, Zheng Z, Yuan X, Li LH, Lu MJ, Shen R, Duan FJ, Zhang XL, Li J, Liu XW, Song
YH, Wang W, Zhao SH, He ZX, Zhang H, Yang KM, Feng W, Wang X. Isolated Coronary Artery
Bypass Graft Combined With Bone Marrow Mononuclear Cells Delivered Through a Graft Vessel
for Patients With Previous Myocardial Infarction and Chronic Heart Failure A Single-Center,
Randomized, Double-Blind, Placebo-Controlled Clinical Trial. Journal of the American College of
Cardiology. 2011; 57:2409–2415. [PubMed: 21658561]
56. Stamm C, Kleine HD, Choi YH, Dunkelmann S, Lauffs JA, Lorenzen B, David A, Liebold A,
Nienaber C, Zurakowski D, Freund M, Steinhoff G. Intramyocardial delivery of CD133(+) bone
marrow cells and coronary artery bypass grafting for chronic ischemic heart disease: Safety and
efficacy studies. Journal of Thoracic and Cardiovascular Surgery. 2007; 133:717–25. [PubMed:
17320570]
57. Tokita Y, Tang XL, Li QH, Wysoczynski M, Hong KU, Nakamura S, Wu WJ, Xie W, Li D, Hunt
G, Ou QH, Stowers H, Bolli R. Repeated Administrations of Cardiac Progenitor Cells Are
Markedly More Effective Than a Single Administration: A New Paradigm in Cell Therapy.
Circulation Research. 2016; 119:635–651. [PubMed: 27364016]
Author Manuscript
58. Guo YR, Wysoczynski M, Nong YB, Tomlin A, Zhu XP, Gumpert AM, Nasr M, Muthusamy S, Li
H, Book M, Khan A, Hong KU, Li QH, Bolli R. Repeated doses of cardiac mesenchymal cells are
therapeutically superior to a single dose in mice with old myocardial infarction. Basic Research in
Cardiology. 2017; 112(2):18. [PubMed: 28210871]
59. Karantalis V, DiFede DL, Gerstenblith G, Pham S, Symes J, Zambrano JP, Fishman J, Pattany P,
McNiece I, Conte J, Schulman S, Wu K, Shah A, Breton E, Davis-Sproul J, Schwarz R,
Feigenbaum G, Mushtaq M, Suncion VY, Lardo AC, Borrello I, Mendizabal A, Karas TZ, Byrnes
J, Lowery M, Heldman AW, Hare JM. Autologous Mesenchymal Stem Cells Produce Concordant
Improvements in Regional Function, Tissue Perfusion, and Fibrotic Burden When Administered to
Patients Undergoing Coronary Artery Bypass Grafting. Circulation Research. 2014; 114:1302–
1310. [PubMed: 24565698]
Author Manuscript
60. Mummery CL, Zhang JH, Ng ES, Elliott DA, Elefanty AG, Kamp TJ. Differentiation of Human
Embryonic Stem Cells and Induced Pluripotent Stem Cells to Cardiomyocytes A Methods
Overview. Circulation Research. 2012; 111:344–358. [PubMed: 22821908]
61. Shiba Y, Fernandes S, Zhu WZ, Filice D, Muskheli V, Kim J, Palpant NJ, Gantz J, Moyes KW,
Reinecke H, Van Biber B, Dardas T, Mignone JL, Izawa A, Hanna R, Viswanathan M, Gold JD,
Kotlikoff MI, Sarvazyan N, Kay MW, Murry CE, Laflamme MA. Human ES-cell-derived
cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature. 2012;
489:322–5. [PubMed: 22864415]
62. Gokhale AG, Chelluri LK, Kumaresan K, Subramanyam G, Sudhakar K, Vemuri S, Debnath T,
Ratnakar KS. Evaluation of the autologous bone marrow mononuclear therapy and functional
restoration in the scarred myocardium by imaging analysis. J Cardiovasc Dis Res. 2011; 2:133–6.
[PubMed: 21814420]
63. Zwetsloot PP, Vegh AM, Jansen of Lorkeers SJ, van Hout GP, Currie GL, Sena ES, Gremmels H,
Buikema JW, Goumans MJ, Macleod MR, Doevendans PA, Chamuleau SA, Sluijter JP. Cardiac
Author Manuscript
Stem Cell Treatment in Myocardial Infarction: A Systematic Review and Meta-Analysis of

Preclinical Studies. Circ Res. 2016; 118:1223–32. [PubMed: 26888636]
64. Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stem cell signaling and
therapy. Circ Res. 2008; 103:1204–19. [PubMed: 19028920]
65. Burchfield JS, Dimmeler S. Role of paracrine factors in stem and progenitor cell mediated cardiac
repair and tissue fibrosis. Fibrogenesis Tissue Repair. 2008; 1:4. [PubMed: 19014650]
66. Tachibana A, Santoso MR, Mahmoudi M, Shukla P, Wang L, Bennett M, Goldstone AB, Wang M,
Fukushi M, Ebert AD, Woo YJ, Rulifson E, Yang PC. Paracrine Effects of the Pluripotent Stem
Cell-Derived Cardiac Myocytes Salvage the Injured Myocardium. Circ Res. 2017; 121:e22–e36.
[PubMed: 28743804]
67. Golpanian S, Schulman IH, Ebert RF, Heldman AW, DiFede DL, Yang PC, Wu JC, Bolli R, Perin
EC, Moye L, Simari RD, Wolf A, Hare JM. Cardiovascular Cell Therapy Research N. Concise
Review: Review and Perspective of Cell Dosage and Routes of Administration From Preclinical
and Clinical Studies of Stem Cell Therapy for Heart Disease. Stem Cells Transl Med. 2016;
5:186–91. [PubMed: 26683870]
Author Manuscript
68. Bouchard A. Stem Cells in the Treatment of Heart Failure. MyHeartnet. 2017; 1:4.
69. Quyyumi AA, Vasquez A, Kereiakes DJ, Klapholz M, Schaer GL, Abdel-Latif A, Frohwein S,
Henry TD, Schatz RA, Dib N, Toma C, Davidson CJ, Barsness GW, Shavelle DM, Cohen M,
Poole J, Moss T, Hyde P, Kanakaraj AM, Druker V, Chung A, Junge C, Preti RA, Smith RL,
Mazzo DJ, Pecora A, Losordo DW. PreSERVE-AMI: A Randomized, Double-Blind, Placebo-
Controlled Clinical Trial of Intracoronary Administration of Autologous CD34+ Cells in Patients
With Left Ventricular Dysfunction Post STEMI. Circ Res. 2017; 120:324–331. [PubMed:
27821724]
70. Fisher SA, Zhang H, Doree C, Mathur A, Martin-Rendon E. Stem cell treatment for acute
myocardial infarction. Cochrane Database Syst Rev. 2015:CD006536. [PubMed: 26419913]
71. Fisher SA, Doree C, Mathur A, Taggart DP, Martin-Rendon E. Stem cell therapy for chronic
ischaemic heart disease and congestive heart failure. Cochrane Database Syst Rev. 2016;
12:CD007888. [PubMed: 28012165]
72. Chugh AR, Beache GM, Loughran JH, Mewton N, Elmore JB, Kajstura J, Pappas P, Tatooles A,
Author Manuscript
Stoddard MF, Lima JA, Slaughter MS, Anversa P, Bolli R. Administration of cardiac stem cells in
patients with ischemic cardiomyopathy: the SCIPIO trial: surgical aspects and interim analysis of
myocardial function and viability by magnetic resonance. Circulation. 2012; 126:S54–64.
[PubMed: 22965994]
73. Malliaras K, Makkar RR, Smith RR, Cheng K, Wu E, Bonow RO, Marban L, Mendizabal A,
Cingolani E, Johnston PV, Gerstenblith G, Schuleri KH, Lardo AC, Marban E. Intracoronary
cardiosphere-derived cells after myocardial infarction: evidence of therapeutic regeneration in the
final 1-year results of the CADUCEUS trial (CArdiosphere-Derived aUtologous stem CElls to
reverse ventricUlar dySfunction). J Am Coll Cardiol. 2014; 63:110–22. [PubMed: 24036024]
74. Mummery, CL, Stolpe, Avd; Roelen, BAJ, Clevers, H. Stem cells: scientific facts and fiction. 2.
London: Elsevier/AP, Academic Press Elsevier; 2014.
Author Manuscript
75. Thomas ED. A history of haemopoietic cell transplantation. British Journal of Haematology. 1999;
105:330–339. [PubMed: 10233401]
76. Bartolucci J, Verdugo FJ, Gonzalez PL, Larrea RE, Abarzua E, Goset C, Rojo P, Palma I, Lamich
R, Pedreros PA, Valdivia G, Lopez VM, Nazzal C, Alcayaga-Miranda F, Cuenca J, Brobeck MJ,
Patel AN, Figueroa FE, Khoury M. Safety and Efficacy of the Intravenous Infusion of Umbilical
Cord Mesenchymal Stem Cells in Patients With Heart Failure: A Phase 1/2 Randomized
Controlled Trial (RIMECARD Trial [Randomized Clinical Trial of Intravenous Infusion Umbilical
Cord Mesenchymal Stem Cells on Cardiopathy]). Circ Res. 2017; 121:1192–1204. [PubMed:
28974553]
77. Van Besien K, Liu H, Jain N, Stock W, Artz A. Umbilical cord blood transplantation supported by
third-party donor cells: rationale, results, and applications. Biol Blood Marrow Transplant. 2013;
19:682–91. [PubMed: 23142329]
78. Memon IA, Sawa Y, Fukushima N, Matsumiya G, Miyagawa S, Taketani S, Sakakida SK, Kondoh
H, Aleshin AN, Shimizu T, Okano T, Matsuda H. Repair of impaired myocardium by means of
Author Manuscript
implantation of engineered autologous myoblast sheets. Journal of Thoracic and Cardiovascular

Surgery. 2005; 130:1333–1341. [PubMed: 16256786]
79. Hata H, Matsumiya G, Miyagawa S, Kondoh H, Kawaguchi N, Matsuura N, Shimizu T, Okano T,
Matsuda H, Sawa Y. Grafted skeletal myoblast sheets attenuate myocardial remodeling in pacing-
induced canine heart failure model. J Thorac Cardiovasc Surg. 2006; 132:918–24. [PubMed:
17000305]
80. Siepe M, Giraud MN, Pavlovic M, Receputo C, Beyersdorf F, Menasche P, Carrel T, Tevaearai HT.
Myoblast-seeded biodegradable scaffolds to prevent post-myocardial infarction evolution toward
heart failure. J Thorac Cardiovasc Surg. 2006; 132:124–31. [PubMed: 16798312]
81. Giraud MN, Ayuni E, Cook S, Siepe M, Carrel TP, Tevaearai HT. Hydrogel-based engineered
skeletal muscle grafts normalize heart function early after myocardial infarction. Artif Organs.
2008; 32:692–700. [PubMed: 18684206]
82. Chen CH, Wei HJ, Lin WW, Chiu I, Hwang SM, Wang CC, Lee WY, Chang Y, Sung HW. Porous
tissue grafts sandwiched with multilayered mesenchymal stromal cell sheets induce tissue
regeneration for cardiac repair. Cardiovasc Res. 2008; 80:88–95. [PubMed: 18539631]
Author Manuscript
83. Potapova IA, Doronin SV, Kelly DJ, Rosen AB, Schuldt AJ, Lu Z, Kochupura PV, Robinson RB,
Rosen MR, Brink PR, Gaudette GR, Cohen IS. Enhanced recovery of mechanical function in the
canine heart by seeding an extracellular matrix patch with mesenchymal stem cells committed to a
cardiac lineage. Am J Physiol Heart Circ Physiol. 2008; 295:H2257–63. [PubMed: 18835924]
84. Derval N, Barandon L, Dufourcq P, Leroux L, Lamaziere JM, Daret D, Couffinhal T, Duplaa C.
Epicardial deposition of endothelial progenitor and mesenchymal stem cells in a coated muscle
patch after myocardial infarction in a murine model. Eur J Cardiothorac Surg. 2008; 34:248–54.
[PubMed: 18457957]
85. Bursac N. Cardiac tissue engineering using stem cells. IEEE Eng Med Biol Mag. 2009; 28:80–6.
82, 84–6, 88–9. [PubMed: 19353830]
86. Kondoh H, Sawa Y, Miyagawa S, Sakakida-Kitagawa S, Memon MA, Kawaguchi N, Matsuura N,
Shimizu T, Okano T, Matsuda H. Longer preservation of cardiac performance by sheet-shaped
myoblast implantation in dilated cardiomyopathic hamsters. Cardiovascular Research. 2006;
69:466–475. [PubMed: 16423569]
Author Manuscript
87. Davis ME, Hsieh PC, Takahashi T, Song Q, Zhang S, Kamm RD, Grodzinsky AJ, Anversa P, Lee
RT. Local myocardial insulin-like growth factor 1 (IGF-1) delivery with biotinylated peptide
nanofibers improves cell therapy for myocardial infarction. Proc Natl Acad Sci U S A. 2006;
103:8155–60. [PubMed: 16698918]
88. Zimmermann WH, Didie M, Doker S, Melnychenko I, Naito H, Rogge C, Tiburcy M, Eschenhagen
T. Heart muscle engineering: An update on cardiac muscle replacement therapy. Cardiovascular
Research. 2006; 71:419–429. [PubMed: 16697358]
89. Nguyen PK, Neofytou E, Rhee JW, Wu JC. Potential Strategies to Address the Major Clinical
Barriers Facing Stem Cell Regenerative Therapy for Cardiovascular Disease: A Review. JAMA
Cardiol. 2016; 1:953–962. [PubMed: 27579998]
90. Luger D, Lipinski MJ, Westman PC, Glover DK, Dimastromatteo J, Frias JC, Albelda MT, Sikora
S, Kharazi A, Vertelov G, Waksman R, Epstein SE. Intravenously Delivered Mesenchymal Stem
Author Manuscript
Cells: Systemic Anti-Inflammatory Effects Improve Left Ventricular Dysfunction in Acute

Myocardial Infarction and Ischemic Cardiomyopathy. Circ Res. 2017; 120:1598–1613. [PubMed:
28232595]
91. Kawamura M, Paulsen MJ, Goldstone AB, Shudo Y, Wang H, Steele AN, Stapleton LM, Edwards
BB, Eskandari A, Truong VN, Jaatinen KJ, Ingason AB, Miyagawa S, Sawa Y, Woo YJ. Tissue-
engineered smooth muscle cell and endothelial progenitor cell bi-level cell sheets prevent
progression of cardiac dysfunction, microvascular dysfunction, and interstitial fibrosis in a rodent
model of type 1 diabetes-induced cardiomyopathy. Cardiovasc Diabetol. 2017; 16:142. [PubMed:
29096622]
92. Shimko VF, Claycomb WC. Effect of mechanical loading on three-dimensional cultures of
embryonic stem cell-derived cardiomyocytes. Tissue Eng Part A. 2008; 14:49–58. [PubMed:
18333804]
93. Nummi A, Nieminen T, Patila T, Lampinen M, Lehtinen ML, Kivisto S, Holmstrom M, Wilkman
E, Teittinen K, Laine M, Sinisalo J, Kupari M, Kankuri E, Juvonen T, Vento A, Suojaranta R,
Harjula A. consortium A. Epicardial delivery of autologous atrial appendage micrografts during
Author Manuscript
coronary artery bypass surgery-safety and feasibility study. Pilot Feasibility Stud. 2017; 3:74.
[PubMed: 29276625]
94. Menasche P, Alfieri O, Janssens S, McKenna W, Reichenspurner H, Trinquart L, Vilquin JT,
Marolleau JP, Seymour B, Larghero J, Lake S, Chatellier G, Solomon S, Desnos M, Hagege AA.
The Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial: first randomized
placebo-controlled study of myoblast transplantation. Circulation. 2008; 117:1189–200. [PubMed:
18285565]
95. Laflamme MA, Chen KY, Naumova AV, Muskheli V, Fugate JA, Dupras SK, Reinecke H, Xu C,
Hassanipour M, Police S, O’Sullivan C, Collins L, Chen Y, Minami E, Gill EA, Ueno S, Yuan C,
Gold J, Murry CE. Cardiomyocytes derived from human embryonic stem cells in pro-survival
factors enhance function of infarcted rat hearts. Nat Biotechnol. 2007; 25:1015–24. [PubMed:
17721512]
96. Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE. Cardiomyocyte grafting for cardiac
repair: graft cell death and anti-death strategies. J Mol Cell Cardiol. 2001; 33:907–21. [PubMed:
Author Manuscript
11343414]
97. Eschenhagen T, Zimmermann WH. Engineering myocardial tissue. Circ Res. 2005; 97:1220–31.
[PubMed: 16339494]
98. Sekine H, Shimizu T, Dobashi I, Matsuura K, Hagiwara N, Takahashi M, Kobayashi E, Yamato M,
Okano T. Cardiac cell sheet transplantation improves damaged heart function via superior cell
survival in comparison with dissociated cell injection. Tissue Eng Part A. 2011; 17:2973–80.
[PubMed: 21875331]
99. Kishore R, Khan M. Cardiac cell-derived exosomes: changing face of regenerative biology. Eur
Heart J. 2017; 38:212–215. [PubMed: 28158461]
100. Davidson SM, Takov K, Yellon DM. Exosomes and Cardiovascular Protection. Cardiovasc Drugs
Ther. 2017; 31:77–86. [PubMed: 27796607]
101. Alcayaga-Miranda F, Varas-Godoy M, Khoury M. Harnessing the Angiogenic Potential of Stem
Cell-Derived Exosomes for Vascular Regeneration. Stem Cells Int. 2016; 2016:3409169.
[PubMed: 27127516]
102. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of
Author Manuscript
mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nature Cell
Biology. 2007; 9:654–9. [PubMed: 17486113]
103. Barile L, Lionetti V, Cervio E, Matteucci M, Gherghiceanu M, Popescu LM, Torre T, Siclari F,
Moccetti T, Vassalli G. Extracellular vesicles from human cardiac progenitor cells inhibit
cardiomyocyte apoptosis and improve cardiac function after myocardial infarction.
Cardiovascular Research. 2014; 103:530–541. [PubMed: 25016614]
104. Gray WD, French KM, Ghosh-Choudhary S, Maxwell JT, Brown ME, Platt MO, Searles CD,
Davis ME. Identification of Therapeutic Covariant MicroRNA Clusters in Hypoxia-Treated
Cardiac Progenitor Cell Exosomes Using Systems Biology. Circulation Research. 2015;
116:255–63. [PubMed: 25344555]
Author Manuscript
105. Giricz Z, Varga ZV, Baranyai T, Sipos P, Paloczi K, Kittel A, Buzas EI, Ferdinandy P.
Cardioprotection by remote ischemic preconditioning of the rat heart is mediated by extracellular
vesicles. Journal of Molecular and Cellular Cardiology. 2014; 68:75–78. [PubMed: 24440457]
106. Li J, Rohailla S, Gelber N, Rutka J, Sabah N, Gladstone RA, Wei C, Hu PZ, Kharbanda RK,
Redington AN. MicroRNA-144 is a circulating effector of remote ischemic preconditioning.
Basic Research in Cardiology. 2014; 109(5):423. [PubMed: 25060662]
107. Arslan F, Lai RC, Smeets MB, Akeroyd L, Choo A, Aguor ENE, Timmers L, van Rijen HV,
Doevendans PA, Pasterkamp G, Lim SK, de Kleijn DP. Mesenchymal stem cell-derived
exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to
enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/
reperfusion injury. Stem Cell Research. 2013; 10:301–312. [PubMed: 23399448]
108. Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C, Desmet W, Kalantzi M, Herbots L,
Sinnaeve P, Dens J, Maertens J, Rademakers F, Dymarkowski S, Gheysens O, Van Cleemput J,
Bormans G, Nuyts J, Belmans A, Mortelmans L, Boogaerts M, Van de Werf F. Autologous bone
Author Manuscript
marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction:

double-blind, randomised controlled trial. Lancet. 2006; 367:113–21. [PubMed: 16413875]
109. Meyer GP, Wollert KC, Lotz J, Pirr J, Rager U, Lippolt P, Hahn A, Fichtner S, Schaefer A,
Arseniev L, Ganser A, Drexler H. Intracoronary bone marrow cell transfer after myocardial
infarction: 5-year follow-up from the randomized-controlled BOOST trial. Eur Heart J. 2009;
30:2978–84. [PubMed: 19773226]
110. Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M, Egeland T, Endresen K, Ilebekk A,
Mangschau A, Fjeld JG, Smith HJ, Taraldsrud E, Grogaard HK, Bjornerheim R, Brekke M,
Muller C, Hopp E, Ragnarsson A, Brinchmann JE, Forfang K. Intracoronary injection of
mononuclear bone marrow cells in acute myocardial infarction. N Engl J Med. 2006; 355:1199–
209. [PubMed: 16990383]
111. Tendera M, Wojakowski W, Ruzyllo W, Chojnowska L, Kepka C, Tracz W, Musialek P,
Piwowarska W, Nessler J, Buszman P, Grajek S, Breborowicz P, Majka M, Ratajczak MZ.
Investigators R. Intracoronary infusion of bone marrow-derived selected CD34+CXCR4+ cells
and non-selected mononuclear cells in patients with acute STEMI and reduced left ventricular
Author Manuscript
ejection fraction: results of randomized, multicentre Myocardial Regeneration by Intracoronary

Infusion of Selected Population of Stem Cells in Acute Myocardial Infarction (REGENT) Trial.
Eur Heart J. 2009; 30:1313–21. [PubMed: 19208649]
112. Assmus B, Rolf A, Erbs S, Elsasser A, Haberbosch W, Hambrecht R, Tillmanns H, Yu J, Corti R,
Mathey DG, Hamm CW, Suselbeck T, Tonn T, Dimmeler S, Dill T, Zeiher AM, Schachinger V.
Investigators R-A. Clinical outcome 2 years after intracoronary administration of bone marrow-
derived progenitor cells in acute myocardial infarction. Circ Heart Fail. 2010; 3:89–96. [PubMed:
19996415]
113. Perin EC, Willerson JT, Pepine CJ, Henry TD, Ellis SG, Zhao DX, Silva GV, Lai D, Thomas JD,
Kronenberg MW, Martin AD, Anderson RD, Traverse JH, Penn MS, Anwaruddin S, Hatzopoulos
AK, Gee AP, Taylor DA, Cogle CR, Smith D, Westbrook L, Chen J, Handberg E, Olson RE,
Geither C, Bowman S, Francescon J, Baraniuk S, Piller LB, Simpson LM, Loghin C, Aguilar D,
Richman S, Zierold C, Bettencourt J, Sayre SL, Vojvodic RW, Skarlatos SI, Gordon DJ, Ebert
RF, Kwak M, Moye LA, Simari RD. Cardiovascular Cell Therapy Research N. Effect of
transendocardial delivery of autologous bone marrow mononuclear cells on functional capacity,
Author Manuscript
left ventricular function, and perfusion in chronic heart failure: the FOCUS-CCTRN trial. JAMA.
2012; 307:1717–26. [PubMed: 22447880]
114. Hare JM, Fishman JE, Gerstenblith G, DiFede Velazquez DL, Zambrano JP, Suncion VY, Tracy
M, Ghersin E, Johnston PV, Brinker JA, Breton E, Davis-Sproul J, Schulman IH, Byrnes J,
Mendizabal AM, Lowery MH, Rouy D, Altman P, Wong Po Foo C, Ruiz P, Amador A, Da Silva
J, McNiece IK, Heldman AW, George R, Lardo A. Comparison of allogeneic vs autologous bone
marrow-derived mesenchymal stem cells delivered by transendocardial injection in patients with
ischemic cardiomyopathy: the POSEIDON randomized trial. JAMA. 2012; 308:2369–79.
[PubMed: 23117550]
115. Leistner DM, Fischer-Rasokat U, Honold J, Seeger FH, Schachinger V, Lehmann R, Martin H,
Burck I, Urbich C, Dimmeler S, Zeiher AM, Assmus B. Transplantation of progenitor cells and
Author Manuscript
regeneration enhancement in acute myocardial infarction (TOPCARE-AMI): final 5-year results

suggest long-term safety and efficacy. Clin Res Cardiol. 2011; 100:925–34. [PubMed: 21633921]
116. Heldman AW, DiFede DL, Fishman JE, Zambrano JP, Trachtenberg BH, Karantalis V, Mushtaq
M, Williams AR, Suncion VY, McNiece IK, Ghersin E, Soto V, Lopera G, Miki R, Willens H,
Hendel R, Mitrani R, Pattany P, Feigenbaum G, Oskouei B, Byrnes J, Lowery MH, Sierra J, Pujol
MV, Delgado C, Gonzalez PJ, Rodriguez JE, Bagno LL, Rouy D, Altman P, Foo CW, da Silva J,
Anderson E, Schwarz R, Mendizabal A, Hare JM. Transendocardial mesenchymal stem cells and
mononuclear bone marrow cells for ischemic cardiomyopathy: the TAC-HFT randomized trial.
JAMA. 2014; 311:62–73. [PubMed: 24247587]
117. Mathiasen AB, Qayyum AA, Jorgensen E, Helqvist S, Fischer-Nielsen A, Kofoed KF, Haack-
Sorensen M, Ekblond A, Kastrup J. Bone marrow-derived mesenchymal stromal cell treatment in
patients with severe ischaemic heart failure: a randomized placebo-controlled trial (MSC-HF
trial). Eur Heart J. 2015; 36:1744–53. [PubMed: 25926562]
118. Guijarro D, Lebrin M, Lairez O, Bourin P, Piriou N, Pozzo J, Lande G, Berry M, Le Tourneau T,
Cussac D, Sensebe L, Gross F, Lamirault G, Huynh A, Manrique A, Ruidavet JB, Elbaz M,
Author Manuscript
Trochu JN, Parini A, Kramer S, Galinier M, Lemarchand P, Roncalli J. Intramyocardial

transplantation of mesenchymal stromal cells for chronic myocardial ischemia and impaired left
ventricular function: Results of the MESAMI 1 pilot trial. Int J Cardiol. 2016; 209:258–65.
[PubMed: 26901787]
119. Surder D, Manka R, Lo Cicero V, Moccetti T, Rufibach K, Soncin S, Turchetto L, Radrizzani M,
Astori G, Schwitter J, Erne P, Zuber M, Auf der Maur C, Jamshidi P, Gaemperli O, Windecker S,
Moschovitis A, Wahl A, Buhler I, Wyss C, Kozerke S, Landmesser U, Luscher TF, Corti R.
Intracoronary injection of bone marrow-derived mononuclear cells early or late after acute
myocardial infarction: effects on global left ventricular function. Circulation. 2013; 127:1968–79.
[PubMed: 23596006]
120. Choudry F, Hamshere S, Saunders N, Veerapen J, Bavnbek K, Knight C, Pellerin D, Locca D,
Westwood M, Rakhit R, Crake T, Kastrup J, Parmar M, Agrawal S, Jones D, Martin J, Mathur A.
A randomized double-blind control study of early intra-coronary autologous bone marrow cell
infusion in acute myocardial infarction: the REGENERATE-AMI clinical trialdagger. Eur Heart
J. 2016; 37:256–63. [PubMed: 26405233]
Author Manuscript
121. Nicolau JC, Furtado RHM, Silva SA, Rochitte CE, Rassi A Jr, Moraes J Jr, Quintella E,
Costantini CR, Korman APM, Mattos MA, Castello HJ Jr, Caixeta A, Dohmann HFR, de
Carvalho ACC. MiHeart AMII. Stem-cell therapy in ST-segment elevation myocardial infarction
with reduced ejection fraction: A multicenter, double-blind randomized trial. Clin Cardiol. 2018;
41(3):392–99. [PubMed: 29569254]
122. Traverse JH, Henry TD, Pepine CJ, Willerson JT, Chugh A, Yang PC, Zhao DXM, Ellis SG,
Forder JR, Perin EC, Penn MS, Hatzopoulos AK, Chambers JC, Baran KW, Raveendran G, Gee
AP, Taylor DA, Moye L, Ebert RF, Simari RD. TIME Trial: Effect of Timing of Stem Cell
Delivery Following ST-Elevation Myocardial Infarction on the Recovery of Global and Regional
Left Ventricular Function: Final 2-Year Analysis. Circ Res. 2018; 122:479–488. [PubMed:
29208679]
Author Manuscript
Author Manuscript
Figure 1.
iPS cell transplantation in the infarcted diabetic db/db and nondiabetic mice resulted in an
Author Manuscript
increase in vascular smooth muscle and endothelial cells in the infarcted heart, leading to a
significantly improved cardiac function. Photomicrographs show anti-CD-31 in red (A,
panels a, e, i), anti-red fluorescence protein (RFP) in green (A, panels b, f, j) and total nuclei
stained with diamidino-phenylindole (DAPI) in blue (A, panels c, g, k). Merged images are
shown in A, panels d, h, i. Scale bar=100 μm. Panel B shows quantitative analysis of total
endothelial cells generation from transplanted iPS cells in both C57BL/6 and db/db mouse
hearts two weeks post-MI,*P < 0.001 vs MI.44
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Figure 2.
Isoflurane delayed mitochondrial permeability transition pore (mPTP) opening and protected
induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from oxidative stress in 5
Author Manuscript
mM and 11 mM glucose. mPTP opening was induced by photoexcitation-generated

oxidative stress. Isoflurane delayed mPTP opening in iPSC-CMs in the presence of 5 mM
and 11 mM glucose concentrations (A). Isoflurane did not delay mPTP opening in the
presence of 25 mM glucose concentrations (A). *P < 0.001 versus control, n=18 cells/group.
H2O2-induced oxidative stress increased lactate dehydrogenase (LDH) release from iPSC-
CMs in 5 mM, 11 mM and 25 mM glucose concentrations (B). In iPSC-CMs, isoflurane
reduced stress-induced LDH release in 5 mM and 11 mM glucose, but not in 25 mM glucose
(B). *P < 0.05 versus stress, n=3 experiments/group. Ctrl = Control; Iso = Isoflurane
treatment; and Stress = H2O2 + 2-Deoxyglucose.50
Author Manuscript
Author Manuscript
Figure 3.
Administration of Cardiac Stem Cells (CSC) in Patients with Ischemic Cardiomyopathy.
Panel A: The mean baseline LVEF in the eight treated patients who were included in the
Author Manuscript
cardiac magnetic resonance analysis was 27.5% at baseline (4 months after CABG surgery
and before CSC infusion), and increased markedly to 35.1% (P=0.004, n=8) at 4 months and
41.2% (P=0.013, n=5) at 12 months after CSC infusion. Panel B: Change in LVEF at 4
months and 12 months after CSC infusion (absolute EF units). Data are means ± SEMs. 72
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Figure 4.
Panel A: Regional EF at baseline and 4 and 12 months after CSC infusion in the infarct-
related regions. Panel B: Change in regional EF in the infarct-related regions at 4 and 12
months after CSC infusion (absolute EF units). Panel C: Regional EF in the dyskinetic
segments of the infarct-related regions at baseline and 4 and 12 months after CSC infusion.
Panel D: Change in regional EF in the dyskinetic segments of the infarct-related regions at 4
and 12 months after CSC infusion (absolute EF units). Panel E: Regional EF in the least
functional segment of the infarct-related regions at baseline and 4 and 12 months after CSC
infusion. Panel F: Change in regional EF in the least functional segment of the infarct-
related regions at 4 and 12 months after CSC infusion (absolute EF units). Data are means ±
SEMs. 72
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Figure 5. Intracoronary Cardiosphere-Derived Cells After Myocardial Infarction

(A) Representative matched, delayed contrast-enhanced magnetic resonance images and
their corresponding cine short-axis images (at end-diastole [ED] and end-systole [ES]) at
baseline and 1 year. In the pseudocolored, delayed contrast-enhanced images, infarct scar
Author Manuscript
tissue, as determined by the full width half maximum method, appears pink. Each cardiac
slice was divided into 6 segments and the infarcted segments were visually identified from
delayed contrast enhanced images. Scar size (percentage of infarcted tissue per segment) and
systolic thickening were calculated for each individual infarcted segment at baseline and 1
year. Endocardial (red) and epicardial (green) contours of the left ventricle are shown. In the
CDC-treated patient (top row), scar decreased, viable mass increased and regional systolic
function improved over the period of 1 year in the treated infarcted segments (highlighted by
arrows). In contrast, no major changes in scar mass, viable myocardial mass or regional
systolic function were observed in the control patient (bottom row). (B) Scatterplots of the
changes in the percentage of infarcted tissue and the changes in systolic thickening for every
infarcted segment of treated and control patients. ED = end-diastole. 73
Author Manuscript
Author Manuscript
Author Manuscript
Figure 6. Cell therapy and tissue engineering approaches for cardiovascular disease therapy
There are various novel treatment options that have been tested for the heart failure due to
ischemic heart disease or genetic disorders. Previous clinical trials have employed various
adult stem cell and progenitor cell populations to test their efficacy for therapeutic
applications. Additional approaches are being explored, including implantation of in vitro
constructed cell sheets of engineered heart muscles (EHMs) as well as direct in vivo
reprogramming of cardiac fibroblasts in the scar region to cardiomyocytes. The regenerative
capacity of adult stem and progenitor cell populations is also being evaluated along with
administration of exosomes and small vesicles secreted by the stem cells.36
Author Manuscript
Author Manuscript
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Table 1
Selected list of landmark clinical trials using mostly bone marrow-derived mesenchymal stem cells conducted to treat acute myocardial infarction and
heart failure.
Study n Type of patient Cell type; Delivery Follow up (months) Outcome LVEF % Ref.
Janssens et al. 67 AMI BMD; Intracoronary 4 N.S. 106
107
Terashvili and Bosnjak
BOOST 60 AMI BMD; Intracoronary 61 N.S.
ASTAMI 100 AMI BMD; Intracoronary 36 N.S. 108
REGENT 200 AMI BMD; Intracoronary 6 N.S. 109
REPAIR-AMI 204 AMI BMD; Intracoronary 24 5% 110
MAGIC 97 HF SMB 6 N.S. 92
FOCUS-CCTRN 92 HF BMD; Transendocardial 6 N.S. 111
POSEIDON 30 ICM BMD; Transendocardial 12 N.S. 112
SCIPIO 33 IHF CPCs; Intracoronary 12 8% 70
CADUCEUS 25 AMI CDCs; Intracoronary 12 N.S. 71
TOPCARE-AMI 55 AMI BMD; Intracoronary 60 11% 113
TAC-HFT 65 ICM BMD; Transendocardial 12 N.S. 114
MSC-HF 55 IHF BMD; Transendocardial 6 5% 115
MESAMI 10 ICM BMD; Transendocardial 12 6% 116
SWISS-AMI 200 AMI BMD; Intracoronary 4 N.S. 117
REGENERATE-AMI 100 AMI BMD; Intracoronary 12 N.S. 118
PreSERVE-AMI 161 AMI BMD; Intracoronary 6 N.S. 67
MiHeart-AMI 121 AMI BMD; Intracoronary 6 N.S. 119
RIMECARD 30 HF UC-MSC; Intravenous 12 5% 74
TIME 120 AMI BMD; Intracoronary 24 N.S. 120
n: Number of patients in the study; LVEF: Changes in Left ventricular Ejection Fraction; IHF: Ischemic Heart Failure; CPCs: Cardiac Progenitor Cells; AMI: Acute Myocardial Infarction; CDCs:
Cardiosphere-derived Cells; BMD: Bone Marrow Derived; HF: Heart Failure; SMB: Skeletal Myoblasts; ICM: Ischemic Cardiomyopathy; UC-MSCs: Umbilical cord-derived mesenchymal stem cells. N.S.:
Not Significant
Page 29

Stemcell Cardiovascular

Uploaded by

Copyright:

Available Formats

Stemcell Cardiovascular

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Stemcell Cardiovascular

Uploaded by

Copyright:

Available Formats

HHS Public Access

Published in final edited form as:

Stem Cell Therapies in Cardiovascular Disease

Zeljko J. Bosnjak, PhD, FAHA*

Road, Milwaukee, Wisconsin, 53226, USA

Considerable experimental evidence obtained in multiple models and species has

combination of various mechanisms such as attenuating tissue injury, inhibiting fibrotic

Pluripotent Stem Cells

mitogen-activated protein kinase/extracellular regulated kinase and glycogen synthase kinase

Induced Pluripotent Stem Cells

medicine applications following myocardial infarction.

Anesthetic preconditioning also protects cardiomyocytes indirectly through its action on

by scavenging reactive oxygen species or inhibiting mitochondrial fission. These findings

Intra-myocardial Cell-Based Therapy

effect in large compared with small animal models.

Cells harvested from the umbilical cord

followed by cell-hydrogel polymerization in situ87 and the epicardial implantation of a

dysfunction and microvascular disease associated with diabetes mellitus-induced

into micrografts for transplantation.93 Appendage tissue is harvested during cannulation of

Stem Cell-Derived Exosomes and Small Vesicles

applications of exosomes in the development of molecular diagnostics, drug delivery

resident cardiac stem cell activity, cardiomyocyte proliferation, beneficial cardiac

Ecs endothelial cells

eNOS endothelial nitric oxide synthase

eNOS/PKG endothelial nitric oxide synthase/protein kinase G

ERK1/2 Extracellular signal-regulated protein kinases 1 and 2

hiPSCs human stem cells, induced pluripotent stem cells

HiPSC-CMs human stem cells, induced pluripotent stem cells-derived

IPSC induced pluripotent stem cells

NADH Nicotinamide adenine dinucleotide

JAK/STAT Janus kinase and Signal Transducer and Activator of

p42/p44 MAPK/ERK specific isoforms of mitogen-activated protein kinase and

PI3K/Akt/mTOR Phosphatidylinositol 3-kinase, serine/threonine kinase also

known as protein kinase B, and mammalian target of

PKC protein kinase C

PKG cGMP protein kinase G pathway

surgery: a randomised controlled trial. Lancet. 2007; 370:575–9. [PubMed: 17707752]

324. [PubMed: 21424917]

28. Xu X, Xu Z, Xu Y, Cui G. Effects of mesenchymal stem cell transplantation on extracellular matrix

P, Mirotsou M, Dzau VJ. MicroRNA-mediated in vitro and in vivo direct reprogramming of

Stem Cell Treatment in Myocardial Infarction: A Systematic Review and Meta-Analysis of

implantation of engineered autologous myoblast sheets. Journal of Thoracic and Cardiovascular

Cells: Systemic Anti-Inflammatory Effects Improve Left Ventricular Dysfunction in Acute

marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction:

ejection fraction: results of randomized, multicentre Myocardial Regeneration by Intracoronary

regeneration enhancement in acute myocardial infarction (TOPCARE-AMI): final 5-year results

Trochu JN, Parini A, Kramer S, Galinier M, Lemarchand P, Roncalli J. Intramyocardial

mM and 11 mM glucose. mPTP opening was induced by photoexcitation-generated

Figure 5. Intracoronary Cardiosphere-Derived Cells After Myocardial Infarction

BOOST 60 AMI BMD; Intracoronary 61 N.S.

ASTAMI 100 AMI BMD; Intracoronary 36 N.S. 108

REGENT 200 AMI BMD; Intracoronary 6 N.S. 109

REPAIR-AMI 204 AMI BMD; Intracoronary 24 5% 110

MAGIC 97 HF SMB 6 N.S. 92

FOCUS-CCTRN 92 HF BMD; Transendocardial 6 N.S. 111

POSEIDON 30 ICM BMD; Transendocardial 12 N.S. 112

SCIPIO 33 IHF CPCs; Intracoronary 12 8% 70

CADUCEUS 25 AMI CDCs; Intracoronary 12 N.S. 71

TOPCARE-AMI 55 AMI BMD; Intracoronary 60 11% 113

TAC-HFT 65 ICM BMD; Transendocardial 12 N.S. 114