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Berkeley
Pharma Tech
Journal of Medicine

Correspondence:� 
Nabeen.chu@gmail.com

Keywords:
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Published July 19, 2022

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Abstract
Cardiac disease continues to be among the most prevalent causes of death worldwide. 
Presently, surgeries such as angioplasties, stents, and bypasses pose many risks. To improve 
outcomes in treating ischemia, researchers have been pursuing minimally invasive, 
biocompatible treatments such as gene therapies. Gene therapies are treatments that 
enhance or suppress target genes to alleviate illness. There are various applications for gene 
therapies, including, but not limited to, the treatment of cancers, diabetes, and heart 
disease. Gene and stem cell therapies can regenerate cardiac tissue that is damaged due to 
ischemia. Furthermore, gene therapies intended to evade the immune system may decrease 
infection risks due to the new tissue being better accepted by the body as it is created from 
patients’ own cells. While DNA treatments show poor results in treating cardiac illness, 
stem cells, such as mesenchymal stem cells and induced pluripotent cells, can differentiate 
into cardiomyocytes, and mRNA can be modified to express angiogenesis growth factors 
around the affected tissue. Although further research is needed to adapt these techniques 
for safe clinical use, they show potential for inducing cardiac tissue regeneration in ischemic 
injury. This paper conducts a review of the emerging techniques and evaluates gene therapy 
as a potential treatment for ischemic injury.

Novel Methods for Inducing Cardiac 
Tissue Regeneration Following 
Ischemic Injury
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Introduction
Much research has been done on various approaches to gene therapy1 to 
treat an ischemic injury, yet few techniques have gone into clinical trials, and 
these seldom show success. This paper aims to conduct a review of a few 
major techniques and evaluate the potential of gene therapy as a treatment 
of ischemic injury.

Cardiac-related illnesses and deaths have consistently been one of the top
causes of death in the U.S. and around the world2, and has also been on the
rise since the beginning of the COVID-19 pandemic, disproportionately
a�ecting minority populations3. Thus the importance of researching more
e�ective methods of treating and preventing ischemic injury is imperative.
The mortality of ischemic heart injury di�ers by demographic, including
one’s age, country, and region, with lower-income countries and older
people su�ering higher mortality rates, with little to no improvement4.
Since the rise of gene therapy as a potential treatment method for various
illnesses in the 1990s, the �eld has been consistently researched5. The
precision of the ability to modify speci�c genes o�ers great potential.
However, due to obstacles such as high cost, ethical concerns, and di�culty
translating into clinical applications5, there is still much to uncover.

Cardiac ischemic injury refers to cell damage due to diminished �ow of
blood, containing nutrients and oxygen, through the vessel. If ischemic
injury is not caught quickly and treated in a timely manner, cardiomyocyte
death can occur, with permanent e�ects6. Ironically, surgically restoring

blood �ow to these regions may actually cause cell death and organ failure7.
Gene therapy thus presents as a favorable option to instead promote
angiogenesis through growth factors such as VEGF-A or stem cell therapies
to develop new paths of blood �ow rather than risking further
ischaemia-reperfusion injury (IRI).

We have decided to look not only into gene cascades that are often targeted
by gene therapies, but various approaches to gene modi�cation, including
stem cell research using mesenchymal stem cells (MSC), modi�ed mRNA
(modRNA), and induced pluripotent stem cells (iPSC).

Berkeley Pharma Tech Journal of Medicine | 2



Mesenchymal Stem Cells to Treat Ischemic Injury

Myocardial infarction (MI), more commonly known as heart attack, and 
related injuries are a leading cause of death globally, and despite the 
applications of extensive novel cardiac surgery and grafting to lengthen and 
ameliorate life, long-term cardiac damage may persist8 . The usage and 
application of mesenchymal stromal cells (MSCs) in heart tissue has become 
an increasingly popular candidate in the treatment of myocardial 
infarctions, as they exhibit factors that increase cardiomyocyte survivability8. 
There is an abundance of research aimed toward understanding the 
implementation of MSCs in the prognosis and improved function of heart 
tissue, due to their desirable trait of being able to di�erentiate into disparate 
cellular lineages9. It is by far one of the most promising treatments in 
regenerative medicine. However, it is di�cult to gather results from existing 
clinical trials due to suboptimal reproducibility and e�cacy outcomes,10 

and the exact mechanism of the cell treatment is still largely unknown9. 
Previous and ongoing research has shown that the usage of mesenchymal 
stromal cells has proved to have signi�cant promise in treating ischemic 
myopathies11. Many clinical trials have shown that the application of 
mesenchymal stromal cells to infarcted cardiac tissue has been shown to 
reduce �brosis12, improve regional contractility11, and reduce arrhythmias 
amongst other outcomes13. In clinical trials with mice, it has been 
demonstrated that direct application of MSCs could aid in angiogenesis and 
myogenesis of ischemic myocardium in murine post-acute myocardial 
infarction14. This is a signi�cant e�ect of MSCs, since heart failure can be 
caused by death of large numbers of cardiomyocytes, so the induction of 
angiogenesis and myogenesis can prove to be a desirable outcome14. Clinical 
studies have also shown that application of autologous bone marrow MSCs 
(BMSCs) correlated to reduced scar sizes, reduced infarct sizes, improved 
left ventricular ejection fraction (LVEF), and a signi�cant increase of viable 
tissue in patients who underwent coronary artery bypass graft surgery 
(CABG) compared to controls12. One particular study aimed to investigate 
the direct e�ect of bone marrow-derived MSCs on cardiac function 
immediately after MI by injecting the cell sample into infarcted

Berkeley Pharma Tech Journal of Medicine | 3



intramyocardial tissues15. Although the experiment showed that the donor
cells minimally remained in the myocardium after implantation, and the
exact mechanism is still unknown, there was a signi�cant reduction in
infarct sizes, improved cardiac function, expression of pro-angiogenic
factors, all occurring in a paracrine manner15.

Speci�c types of MSCs like umbilical MSCs, which promote vascular
regeneration and cardiomyocyte protection, are accessible and easily
expandable in experiments13. However, with the varying stem cell types,
there are also many di�erent constraints that have hindered more innovative
research into the application of MSCs. The application of bone-marrow
derived MSCs in particular include invasive or unethical harvesting
procedures, and decreased proliferation among varying donors13. But in a
more promising facet, umbilical-derived MSCs demonstrate more safe and
feasible qualities, including being more easily attainable, posing less ethical
concerns, and undergoing less cellular aging13.

Safety of MSC application is an important facet of the treatment to ensure
since there are several potential health concerns surrounding the treatment,
including tumor formation, organ toxicity, and ectopic tissue formation in
the vasculature16. Safety pro�les of intravenous MSC application in acute
MI patients are successfully present, showing signi�cant results such as
reduced tachycardic episodes, improved forced expiratory volumes, and no
concerns for ectopic tissue creation in the long term16. However, one study
aimed to evaluate the safety of human bone marrow-derived mesenchymal
cell application in patients su�ering from acute MI by using a multicenter
trial16. In the study, the Data Safety and Monitoring Board approved the
application of MSC dosage in each experimental cohort, and primary safety
assessments were carried out to monitor any adverse reactions to the
treatment16. The results of this study showed that there was no apparent
evidence of increased toxicity with the application of the MSCs and there
the administration was well tolerated in the cohorts at all doses16. There was
also evidence that the arrhythmia ratio was signi�cantly lower in the MSC
administered patients vs. the placebo group, and also showed improved
function in cardiac performance and pulmonary function in comparison
with the placebo group16. There was no report of long-term ectopic tissue

Berkeley Pharma Tech Journal of Medicine | 5



formation either, which showed the signi�cant therapeutic bene�ts of the
administration of the hMSCs overall16. Another study aimed to evaluate the
safety of intravenous administration of umbilical cord-derived MSCs
(UC-MSCs) as well. The experimental group involved patients with heart
failure and reduced ejection fraction, and were presently treated with
application of intravenous infusion of allogeneic umbilical-cord derived
MSCs13. The results of this study showed that the UC-MSC treated patients
expressed no adverse reactions to the infusion treatment and showed
signi�cant improvement in left ventricular ejection fraction over multiple
time checkpoints post-administration13. The results of these studies show
that the incorporation of MSCs as a treatment option is safe and feasible
with di�erent types of MSCs, and there were no noteworthy deleterious
reactions to this treatment. The results described above also showed
improvement and potential in recovery and o�ers a potential usage in future
medicine and therapy.

Many questions are still unanswered regarding the mechanism of action of
MSC treatment9. Studies have shown that injection of MSCs in a�ected
heart tissues reduced infarct size and induced angiogenesis and
myogenesis14. One main mechanism of action is injection of bone
marrow-derived-MSCs directly into infarcted heart tissue to promote
cardiac function via expansion of the cells11. This is usually caused by

MSCs’ ability to expand and di�erentiate into cardiomyocyte-like cells13.
One experiment conducted coronary artery ligations in mice and then
implemented MSCs to note any changes in grafting in the ischemic
myocardium and proliferation into cardiomyocytes14. This particular
experiment showed results illustrating improved cardiac function after
MSC implementation through the enhancement of myogenesis and
angiogenesis. Other experiments involving injection of MSCs or BMCs into
infarcted tissue have shown some results correlating with improved regional
contractibility, and no signs of ventricular arrhythmias, worsening cardiac
function, pulmonary embolisms, or cardiac tamponade11. There are still
suboptimal results correlating MSC transplantation and therapeutic
improvement of cardiac function, since the mechanisms of action are quite
complex and not fully understood9. But transplantation of MSCs in many

Berkeley Pharma Tech Journal of Medicine | 6



recent reviews and studies have proven to be safe13 and the selective study
results shown could be bene�cial.

MSCs have been used as a therapeutic application for many degenerative
diseases, mainly due to their various di�erentiation potential , paracrine
factors17 and secretory products like angiogenic factors, mitogenic factors,

antiapoptotic factors, and growth factors18. In particular MSC’s have been

frequently involved in experimental models of cardiovascular diseases13 and
express attributes that make them desirable for use in cardiac function
modulation after myocardial infarction9 including secretions that could

prevent cardiac in�ammation and aid in cardiac injury repair19. Although
the underlying detailed mechanism of MSC transplantation is still mostly
unknown and incomplete19, and clinical trial results are still suboptimal9,

transplantation treatments show promising e�ects15 regarding improvement

in myocardial function in experimental models13. Additionally, the
trajectory of evidence shows that transplanted cells have a bene�cial e�ect
on overall cardiac function after myocardial infarction20 and ischemic heart

disease18 due to varying di�erent results. Transplanted MSCs from red bone
marrow in adult rat specimens showed di�erentiation into cardiomyocytes
by the presence of cardiac proteins like desmin, and also showed to form
connections with native cardiomyocytes in the rat sample18. This
di�erentiation into both vascular endothelial cells and smooth muscle cells
promoted cardiac function via myogenic and angiogenic e�ects14. Engrafted
cells harvested from a healthy human sample also showed similar results,
with the stem cells strongly morphologically resembling host
cardiomyocytes and also expressing desmin, myosin heavy chain, actinin,
and cardiac troponin amongst others, each of which are native cardiac tissue
proteins21.

Echocardiographic assessment of transplanted bone marrow MSCs into
post-MI tissues also showed that the cells relieved the restrictive e�ects of
left ventricular function and geometry caused by MI20. Additionally,
intravenous application of umbilical cord MSCs showed improved left
ventricular ejection fraction and left ventricular end diastolic volume in
control groups for patients with systolic heart failure or reduced ejection

Berkeley Pharma Tech Journal of Medicine | 7



fraction13. Novel experiments have also shown that direct injection of MSC
exosomes also reduced infarct sizes, improved cardiac function, and o�ered
protection of cardiomyocytes from hypoxia8.

Fig 1. MSC Differentiation Potential. Based on the diagram of
differentiation potential for MSC terminal phenotypes, we can see how

bone-marrow MSCs or umbilical cord derived MSCs have the potential to
differentiate to different cell types including cardiomyocytes, vascular endothelial

cells, and skeletal muscle cells amongst other cells. This differentiation into
cardiomyocytes explains why the expansion of the cells in cardiac tissue settings is very

desirable. The differentiation into cardiomyocytes has been measured by proteins
like desmin and troponin, and is also noted to form connections with native

cardiomyocytes within the tissue.

Although much of the application of MSCs is still subject to more extensive
clinical testing and development15, there is a huge promise of the already
performed experiments discussed above to show safety of the clinical
application and the therapeutic bene�ts of administration. The studies
above have depicted the many potential and con�rmed bene�ts of
administration of either bone-marrow derived MSCs or umbilical
cord-derived MSCs, including reduced infarct sizes, overall improved

Berkeley Pharma Tech Journal of Medicine | 8



cardiac function and LVEF, cardiomyocyte production and protection, 
angiogenesis, and myogenesis. Safety of administration of BMSCs and 
UC-MSCs were both measured with signi�cant results in cohort groups, 
showing that overall, administration is safe and feasible with generally little 
to no adverse e�ects13. Although these studies presented signi�cant 
outcomes, it has been suggested that more extensive and larger clinical trials 
are needed to fully understand the clinical bene�ts of administration of 
certain MSCs13. Due to the many measured bene�cial implications of 
MSCs, including overall cardiac function, we can conclude that MSC 
transplantation can be a new therapeutic strategy for treating patients 
su�ering from myocardial infarction14.

Modifed RNA as a Method of Gene Therapy
DNA modi�cation comes with many risks, including immunological 
response, and lack of speci�city regarding its locus of action. Consequently, 
RNA therapy is becoming a further researched �eld, including clinical 
applications for modRNA and miRNA23.

Modi�ed mRNA (modRNA) is becoming a prevalent research topic in
many di�erent �elds, such as diabetes, ischemic injury, and the mechanism
of a COVID-19 vaccine24. In this method of gene therapy, mRNA is
injected into the heart to enhance target protein translation, which in this
case is cardiomyocytes and angiogenesis to repair the heart after massive cell
death25. However, mRNA is short lived and cannot provide lasting e�ects
for cardiomyocyte regeneration. They often trigger an immune response,
and are also broken down by RNAases. Thus the degree of protein
expression is dependent on mRNA stability26 and they must be engineered
to be more e�cient in their expression in the cell, or be longer lasting. So far,
this is achieved through four di�erent methods: altering nucleotides,
mRNA capping, untranslated region manipulation, and the length of the
poly A chain27.

Understanding the alterations of mRNA requires an understanding of
mRNA synthesis. Often, a DNA plasmid is transcribed in vitro and the
resulting mRNA is used for therapy. In this process, substitutions can be
made in the nucleotide pairing process. Kierzek et al. showed in 2014 the

Berkeley Pharma Tech Journal of Medicine | 9



speci�c e�ects of substituting pseudouridine for uridine with the di�erent
nucleic acids, in which Ψ-A pairing demonstrated greatest enhanced
stability due to hydrogen bonding, at about 0.5kcal/mol. Due to the
increased stability and because pseudouridines are found naturally in the
body, these modRNA are able to avoid detection by the immune system,
speci�cally by TLR3, TLR7, and TLR828. Furthermore, it was found that
VEGF mRNA whose uridines were completely replaced by pseudouridine,
did not trigger the immune system and remained locally in the target tissue
of cynomolgus monkeys or rats while promoting angiogenesis. In this
experiment, the mRNA was injected intradermally or intravenously29.

mRNA caps are methylated guanosine groups attached to the 5’ end by a
triphosphate group present in all eukaryotic mRNA30. These give identity

to the mRNA strand as well as signal for degradation when removed31.
Thus the synthetic mRNA must also be capped adequately to increase
translation of protein.

The untranslated region of RNA, called the UTR, appears on both the 5’
and 3’ ends of the mRNA strand, and plays a role in mRNA translation
e�ciency and localization32. Thus UTR of modRNA must also be
manipulated adequately to optimize translation rate and half-life. For
example, the addition of AU-rich elements (ARE) weakened protein
expression, and the more stable UTR was able to increase transcription
stability33. Synthesizing UTR speci�c to the needs of the modRNA is
imperative to its e�ective protein synthesis, and also allows for more precise
control of the therapy.

mRNA is modi�ed by extending the polyA tail to increase its translation.
This modRNA is administered through intramyocardial injection to a�ect a
large area of cardiomyocyte and non-cardiomyocyte cells23. modRNA is able
to stay longer in the cell than RNA by changing its structure with
pseudouridine and evading RNAase34, but is still shorter-lived than DNA,

thus reducing the risk of mutation and multiple transcriptions over time25.

Rather, it is described as a “pulse”35, producing a rapid episode of protein
synthesis that can be better controlled than DNA therapy methods.

Berkeley Pharma Tech Journal of Medicine | 10



Currently, due to the temporary nature of modRNA, treatments may need
to be administered multiple times to demonstrate e�ectiveness, which not
only causes trauma on the cardiac tissue but also will become expensive25.

modRNA is already required in large doses36, thus production or
administrative costs must be reduced to make this treatment clinically
feasible. Furthermore, injecting these genes into the intracardiac tissue is
invasive and can be risky for patients who have already su�ered injury to the
heart. Further research must be done on less invasive gene and drug delivery
methods that are still able to target speci�c cells and promote angiogenesis25.
One such method is cell-penetrating peptides that have been demonstrated
to carry DNA, RNA, or proteins into the cell via endocytosis37.

Figure 2. The Structure of a Modified mRNA. The poly A tail increases
translation and allows it to remain longer within a cell.

Despite these intricacies of modifying mRNA, it has been demonstrated
that modRNAs can deliver relatively quick local responses both in vitro and
in vivo. Mouse hearts were able to retain the e�ects of a cardiac injection of
modRNA for 24 hours after surgery38. Furthermore, modRNA coding for
VEGF-A, a growth factor that promotes revascularization was
demonstrated to successfully induce angiogenesis following myocardial
infarction39. The modRNA injected into the site of injury promoted heart
progenitor cells to di�erentiate into vascular cells rather than muscle cells.
In this experiment, the mice injected with VEGF-A DNA and VEGF-A
RNA both formed vessels, but the mice who received the RNA injection
presented with vasculature that was less permeable and more similar in
shape to control hearts. The vessels of VEGF-A DNA injected mice showed
edema, and had higher mortality rates. These di�erences, likely due to the

Berkeley Pharma Tech Journal of Medicine | 11



di�erences in acting speed of RNA and DNA, display the potential of
modRNA in the �eld of cardiology. Moreover, because VEGF-A, a
commonly used and researched growth factor, can cause increased vessel
permeability at long exposure times, the shorter “pulses” of RNA may be
more e�ective in treating ischemic heart injury.

VEGF-A’s results are much more promising with mRNA than DNA largely
due to its rapid and local characteristics29. This e�ect can be further
enhanced by moving away from lipid carriers, sometimes called
nanoparticles, that are often used to encapsulate proteins or genetic
material. Removing this layer prevents the mRNA from entering
circulation and traveling away from the target tissue, and the naked delivery
of the modRNA increased protein was found to increase translation 53-226
fold38. However, it must be noted that while for ischemic, local injury,
removing the nanoparticles improves translation, for drugs that are aimed
for general circulation, such as mRNA vaccines, the lipid nanoparticles are
an essential part of drug delivery40.

Carlsson et al. (2018)29 demonstrated the e�ects of VEGF mRNA after
further replacing the pseudouridine with 1-methylpseudouridine and
injecting naked mRNA in a citrate-saline bu�er in swine. The treatment
resulted in e�cient and long-lasting protein expression, local e�ects, and
improvement in cardiac function at 1 week and 2 months after injection29.
Improvement here was de�ned as increase in muscle contractility and
decrease in �brosis, and were dose-dependent. These �ndings further
support and build upon Zangi et al.’s �ndings in 201339 but with greater
speci�city in the injections’ e�ects, as measured through bioluminescence,
and with higher e�ciency. With cardiac angiogenesis in both species of mice
and swine, modRNA appears to be a strong candidate for carrying out
cardiac regeneration after an ischemic injury. However, these studies will
have to be carried out over longer periods to ensure whether the modRNA
must be reinjected, and for any side e�ects.

While clinical trials of VEGF modRNA for cardiac regeneration are scarce,
its safety and e�ectiveness were observed in treating patients with type II
diabetes mellitus. The study was conducted in a randomized, double-blind,

Berkeley Pharma Tech Journal of Medicine | 12



placebo-controlled nature, splitting participants into three groups to receive
either placebo or VEGF-A mRNA at various doses41. The results re�ect that

of Carlsson et al.29 that demonstrated dose-dependent results. VEGF-A
mRNA was able to successfully promote basal skin blood �ow at injection
sites of the forearm, and the results were dose-dependent. At 7-14 days after
injection, vasodilation and neovascularization were induced, demonstrating
the potential for VEGF-modi�ed mRNA applications in humans41.
Observed adverse events were of onsite reactions, which occurred in all
participants, but with only mild severity. The e�ects of cardiac modRNA
injection in humans as of yet unknown, though the gene therapy method is
considered safe to be researched further in the context of cardiac ischemic
injury. However, this clinical trial only had male participants, thus women
must also be tested to ensure comparable results, and whether there are sex
di�erences in dosing.

After the safety and biocompatibility of VEGF modi�ed mRNA were
con�rmed, it was applied in the cardiac �eld for patients undergoing
Coronary artery bypass grafting (CABG) by AstraZeneca in a study called
EPICCURE. This is a very common surgical procedure that aims to
revascularize the heart by attaching grafts bypassing the clogged artery.
However, there are still many risks, especially for those who have a history of
renal disease or stroke. Adverse events include infection, atrial �brillation,
myocardial dysfunction, which are at higher rates for those who are
undergoing dialysis or have other underlying cardiac conditions such as
peripheral artery disease or pericarditis42. AZD861, a VEGF mRNA drug in
citrate-bu�ered saline, will be administered immediately after CABG before
reperfusion at various doses to determine whether angiogenesis will be
promoted, and if it will improve outcomes of the surgery36. While no paper
published in a scienti�c journal has reported the results, AstraZeneca
published data on the company’s website and presented it at the American
Heart Association’s Scienti�c Sessions in 2021. The treated groups
demonstrated the biocompatibility and safety of AZD8601, and in
agreement with the phase 1 clinical trial41, there were no infections or severe
adverse events. Furthermore, because the injections in this trial were
epicardial injections, they better demonstrate that VEGF mRNA is safe for

Berkeley Pharma Tech Journal of Medicine | 13



human use. But more research must be done to increase e�ciency of the 
treatment for signi�cant bene�ts in cardiac angiogenesis43.

Induced Cardiomyocytes for Cardiac Regeneration
Induced cardiomyocytes (iCMs) are cardiomyocyte-like cells that are 
derived from the reprogramming of other cells, the most common being 
induced pluripotent stem cells (iPSCs)44. Culturing iPSCs with speci�c 
media allows them to be di�erentiated into the target cell, in this case, 
iCMs. iPSCs provide a solution to the long-standing di�culty of the lack 
of regenerative abilities of cardiomyocytes; their ability to proliferate 
allows treatment to surpass the stagnant number of normal 
cardiomyocytes in the body, a revolutionary possibility for regenerative 
medicine44. iPSCs also provide a safer alternative to potentially toxic 
drug treatments and can serve as excellent disease models44, 45.

Though iPSCs remain the most common cell that are used in iCM 
derivation, cardiomyocyte-like cells have also been derived from the 
reprogramming of �broblasts. Derived from direct lineage reprogramming 
via upregulation of genetic reprogramming factors, several studies have 
reported the generation of functional iCMs from cardiac �broblasts in both 
in vitro and in vivo mouse models46, 47. Qian et al. (2012)46 examined the 
conversion of murine cardiac �broblasts to cardiomyocyte-like cells via the 
expression of three cardiac-speci�c transcription factors (Gata4, Mef2c, and 
Tbx5) in vitro and observed a large percentage of cells that were partially 
reprogrammed. They applied their �ndings to an in vivo model and 
observed a similar rate of conversion with an even higher level of success in 
fully reprogramming the �broblasts into cardiomyocyte-like cells46. Wada et 
al. (2013)47 also previously reported conversion using the aforementioned 
transcription factors to be successful for mouse �broblasts in vitro47. 
Following their in vitro study, they observed the generation of new 
cardiomyocytes from endogenous cardiac �broblasts with the aid of the 
same transcription factors, and various improved aspects of overall cardiac 
function.

Following these discoveries, the possibility of directly reprogramming 
human cardiac �broblasts (HCFs) was also studied. While success in the

Berkeley Pharma Tech Journal of Medicine | 14



conversion of HCFs to iCMs was observed, the process was observed to be
more complex than the mouse model, requiring additional reprogramming
factors47, 48. The three transcription factors used in the in vitro and in vivo
rodent models were not su�cient; the addition of Mesp1 and Myocd
allowed for the generation of human iCMs in vitro47. However, the iCMs

derived from this process “did not beat spontaneously”47. To di�erentiate
into functional, beating iCMs, “human iCMs [required] coculture with
murine cardiomyocytes,” unlike their mouse counterparts47. Finally, the
level of authenticity of the replicated iCMs in comparison to natural
cardiomyocytes was unable to be conclusively de�ned47.

Somatic �broblasts were also found to give rise to iCMs through
upregulation of cardiac gene expression, which can occur both in vitro and
in vivo49. Further studies involving human cardiomyocytes in vitro and in
vivo are needed to understand the speci�c mechanisms required to produce
an e�ective treatment based on this technology.

While iCMs are a very promising �eld of cardiac gene therapy, they are, of
course, not without their limitations. Horikoshi et al. (2019)44 observed that
iCMs derived from iPSCs appear to lack the mature characteristics of
normal adult cardiomyocytes. Based on the natural process of
cardiomyocyte maturation, they cultured iPSC-derived iCMs with fatty
acids, the basis for the fatty acid β-oxidation process that supplies mature
cardiomyocytes with energy44. Their study revealed that a fatty acid culture
can promote the maturation process of iPSC-derived iCMs in order to allow
them to function as fully-matured adult iCMs44.

Another limitation observed in the �eld is the applicability of iCM-based
treatment to chronic heart failure, a condition for which regenerative
therapy is in high demand48. While iPSCs have provided excellent platforms
for disease modeling thus far, in vivo cardiac reprogramming studies have
been focused on models in the acute stage of myocardial infarction (MI), so
its potential to alleviate chronic heart failure has yet to be determined48.
However, given the marked improvement of overall cardiac function
observed in numerous studies, there seems to be a de�nite potential for
chronic heart failure reversal through treatment with iCMs.

Berkeley Pharma Tech Journal of Medicine | 15



iCMs themselves have not been observed to proliferate independently, a
characteristic which would greatly improve the e�cacy of regenerative
therapy48. Instead of relying on the proliferation capabilities of the source
cells, such as iPSCs, development of iCMs that are able to endogenously
proliferate would remove the necessity for repeated administration of iCMs.
Certain critical factors have been identi�ed in addition to the possibility of
using a third iCM derivation source in the form of induced
cardiac-progenitor cells (iCPCs) that would be converted into iCMs;
however, further study is needed to solidify the technology and translate it
to clinical application48.

Figure 3. Common Derivation Methods of iCMs. Diagram outlining two 
major sources, iPSCs and fibroblasts, used for the derivation of iCMs. Details the 

processes by which both sources can be used to create iCMs in vitro that can be inserted 
for treatment. Also touches on how both of the sources can be directly inserted and 

used to develop functional iCMs in vivo as part of treatment regimens.

Berkeley Pharma Tech Journal of Medicine | 16



Gene Cascades to Target for Cardiac Regeneration
The manipulation of certain cardiac genes has shown signi�cant progress in 
improving ischemic heart disease. Changes in the expression of certain genes 
in�uence cardiac cell proliferation and di�erentiation, demonstrating gene 
therapies’ potential e�cacy in treating cardiac disease. This section examines 
multiple therapies that concentrate on changing the expression of target 
genes. Two main techniques were investigated, one focusing on the 
transplantation of BMSCs with speci�c gene deletion and the other 
concentrating on the protein-protein interactions that in�uence a particular 
gene involved in cardiac cell signaling.

Recent studies have shown that hepcidin, an iron-regulating protein 
primarily in the liver, is also present in low amounts in the heart, to regulate 
iron homeostasis and ferritin content in cardiomyocytes53. Injuries in the 
heart due to ischemic disease result in the destruction of cardiomyocytes, 
which releases a high concentration of iron into the extracellular space54. 
The increased iron content outside cardiomyocytes is associated with 
myocardial �brosis and with the formation of reactive oxygen species 
(ROS)53. These conditions cause an intracellular iron de�ciency that leads 
to the overexpression of hepcidin proteins55. In e�orts to understand how 
hepcidin a�ects ischemic heart disease and to study the possibilities of 
related therapies, the gene coding for hepcidin, Hamp, was manipulated in 
deletion experiments56.

Despite the lack of knowledge on how cardiac hepcidin functions and 
a�ects heart injuries, researchers �rst carried out direct gene deletions of 
Hamp in mice to prevent the production of hepcidin and study its e�ect56. 
Nonetheless, this gene therapy demonstrated unpromising results, causing 
scientists to quickly transition into the transplantation of either mice with 
bone-marrow-derived cells that are Hamp de�cient or with myeloid cells 
with Hamp deletions. The investigation was carried out on mice that were 
deliberately in�icted with myocardial infarction. Those with Hamp 
expression in macrophages were shown to produce high levels of hepcidin as 
a result of increased interleukin (IL-6) production, which is responsible for 
Hamp’s transcription57.

Berkeley Pharma Tech Journal of Medicine | 17



This new study on the e�ect of hepcidin on heart injuries revealed that this
protein is related to the secretion of IL-4 and IL-13 which are each
important for inducing cardiac repair56. Hamp de�cient mice with
myocardial infarction displayed a signi�cant reduction in infarct size and
tissue �brosis as well as increased cardiomyocyte renewal. This result was
related to the lack of hepcidin that led to improved receptor functions, such
as C–C motif receptor 2 (CCR2)+, in macrophages. This improvement
enhanced the secretion of IL-4 and IL-13 from the macrophage increasing
cardiac healing as well as cardiomyocyte regeneration after a heart injury56.

Figure 4. The Effect of the Hamp Gene on Hepcidin Production.
Hamp-deficient (-/-)  mice reduced hepcidin production and induced the secretion of
IL4 and IL13 responsible for cardiomyocyte renewal, while mice with Hamp gene

(+/+) produced high levels of hepcidin reducing the release of IL4 and IL13.

While this research demonstrates a new �nding regarding the relationship
between hepcidin and IL-4/IL-13, the mechanisms of how hepcidin
in�uences the function of certain receptors and interleukins are still not
widely understood. There is limited research on the potential of using this
gene therapy due to the researchers’ e�ort to �rst better understand how
cardiac hepcidin works in the heart and related injuries54. Further

Berkeley Pharma Tech Journal of Medicine | 18



investigation on hepcidin is required to understand how the production of
cardiac hepcidin a�ects certain tissues in heart cells before diving into
particular therapies53. The study of this protein has great potential of being
utilized for improved cardiac function in ischemic heart diseases,
particularly the Hamp-de�cient BMSCs that induced cardiac repair56.

Although not yet implemented into paradigms of gene therapy, the ongoing
in-depth study of protein-protein interactions has led to the possibility of
controlling the expression of speci�c genes, essential for signaling pathways
involving cell proliferation. The detailed understanding of Wnt
protein/β-catenin pathways, responsible for cell proliferation and growth,
has led to signi�cant improvements in addressing diseases in organs such as
the lungs58. In attempts to regulate this pathway for cardiac cell
regeneration, recent studies have revealed protein interactions with Wnt
units that could inhibit injury-induced cardiomyocyte proliferation59.

In a study on heart regeneration and injury repair, scientists discovered
novel small molecules called cardiomogens (CDMG 1 and 2) that have the
ability to inhibit Wnt expression and cause β-catenin reduction
downstream59. The investigation was carried out using embryonic zebra�sh
with surgically-induced heart injuries. CDMGs inhibited Wnt by
speci�cally targeting β-catenin and Tcf/Lef-mediated transcription that is
needed to initiate the expression of particular genes speci�ed by Wnt60. The
reduction in β-catenin accelerates the proliferation of damaged heart cells,
leading to improved heart function. CDMG1, particularly, was e�ective in
healing heart injuries by increased formation of cardiomyocytes and
reduction in �brotic scar tissue59. Nonetheless, the mechanism of CDMG
on particular Wnt pathways is still not well-understood. There are a wide
variety of Wnt proteins and related signaling pathways that a�ect distinct
cellular lineages and their functions. Therefore, further studies regarding
what speci�c Wnt proteins CDMG structures inhibit and what speci�c
genes are a�ected by these molecules could potentially result in new gene
therapies, and ultimately improvements in medicine.

In addition to the newly discovered molecules, secreted frizzled-related
proteins (Sfrp) inhibit canonical Wnt signaling pathways responsible for cell

Berkeley Pharma Tech Journal of Medicine | 19



proliferation, while activating non-canonical Wnt pathways that lead to
di�erentiation61. This research was accomplished by culturing cardiac
progenitor cells (CPC) from mice. The binding of Sfrp2 to Wnt6 reduced
CPC proliferation while fostering di�erentiation. Inhibition of Wnt by Sfrp
causes an activation of non-canonical Wnt/Planar Cell Polarity (PCP)
pathway through c-Jun N-terminal kinase (JNK)58. This activation
encourages the expression of cardiac transcription factors and CPC
di�erentiation suggesting that the regulation of Wnt proteins is a possible
avenue through which clinicians can address ischemic heart injuries61. A
signi�cant limitation of this �nding is the lack of comprehension of the
particular pathways that the inhibition of Wnt6 a�ects, and what factors or
genes are involved.

Figure 5. The Role of the Wnt Pathway in β-catenin Production. Sfrp2
binds to Wnt6 proteins to activate non-canonical Wnt pathway that results in CPC

differentiation and leads to a reduction of β-catenin responsible for proliferation.

The �nding of pathways related to cardiac cell induction and regeneration
has demonstrated a possibility of manipulating these processes to address

Berkeley Pharma Tech Journal of Medicine | 20



heart injury. In the case of hepcidin proteins, Hamp de�ciency has shown to
be associated with iron-homeostasis and the secretion IL-4/IL-13 that help
with cardiomyocyte renewal. CDMG molecules and Sfrp proteins were
proven to be related to the inhibition of Wnt pathways that reduce cell
proliferation but result in di�erentiation. However, for both cases, the
pathways resulting in improved heart function are still not well-understood
and just provide a glance of future research directions. There is, thus,
signi�cant room for elaboration; further studies may implicate more speci�c
cell or gene therapies for treating ischemic heart diseases.

Conclusion
Mesenchymal stromal cells have been a popular and desirable candidate in 
treating function post-myocardial infarction, as they have shown promising 
e�ects in clinical trials. The administration of MSCs have shown signi�cant 
therapeutic bene�ts contributing to patients su�ering from myocardial 
infarction and other pulmonary and cardiac myopathies. Although more 
extensive and larger-scale clinical trials need to be implemented to exactly 
understand the mechanism and exact bene�ts of MSC administration, the 
existing studies have shown results indicating high e�cacy. Studies have 
shown that through both UC-MSCs, patients treated with infusion of 
MSCs have shown outcomes which include increased myogenesis and 
angiogenesis, improved left ventricular ejection reaction, reduced infarct 
sizes in heart tissue, cardiomyocyte proliferation, and generally improved 
cardiac function. The di�erence in MSC types and their correlating bene�ts 
and consequences are also discussed, as well as the safety and feasibility of 
their administration onto experimental cohorts. These studies also showed 
little to no adverse e�ects to any of the treatments, and little signi�cant 
worsening of conditions has been recorded. The exact mechanism of their 
bene�cial e�ect is still unknown and needs to be more extensively studied, 
but these studies have shown promising therapeutic bene�ts.

Modi�ed mRNA also demonstrates great potential to be used towards
cardiac regeneration, especially expressing VEGF, as it has expressed no
adverse reactions when used clinically, and successfully promoted
angiogenesis. Developments have been made in the puri�cation and evading
the immune system through RNA caps, pseudo nucleotides, and the poly A

Berkeley Pharma Tech Journal of Medicine | 21



tail. However, more research must be done speci�cally on the ischemic
injury and cardiac regeneration model for it to be used speci�cally in
response to a myocardial infarction. Before this, an understanding of the
e�ect of VEGF on humans, and increasing e�ciency of the treatment is
imperative, as there is very little data on optimal dosage concentration or
repetitions that carry out the desired e�ects. Lastly, for modi�ed mRNA to
be a viable clinical option, the shelf life and sustainable storage of the
puri�ed mRNA in bu�er must be researched to ensure minimal
degradation over time. Thus, modifying mRNA shows great potential as a
gene therapy mechanism, though more research must be done to adapt it to
be used for human cardiac regeneration.

iCMs derived from various sources have been employed in numerous
treatment approaches to cardiac illness, and continue to provide many
research opportunities in the �eld of regenerative medicine. As observed by
the evolution of iCM research over time, studies have made their way from
animal models to the use of human cell sources for experimentation,
suggesting that application of iCM-based treatment in clinical trials is not
far o�. iCMs provide a treatment option that has the potential to draw on a
abundantly-sourced treatment platform, avoid the negative side-e�ects of
toxic pharmacological treatments, and signi�cantly expand the scope of
regenerative medicine. Continued research into iCMs seems to be a very
promising path to treating ischemic injury induced by cardiac dysfunction.

Particular genes and protein interactions related to the heart have resulted in
a greater insight into cardiac regeneration to address heart injuries. Cardiac
hepcidin protein reduction through Hamp gene de�ciency was shown to be
associated with iron-homeostasis and the secretion of IL4/IL13 from
macrophages inducing cardiomyocyte regeneration. The inhibition of
pathways related to Wnt proteins using CDMG and Sfrp molecules also
revealed that cardiac progenitor cell di�erentiation is a possible metric with
which to gauge improvement in heart injuries. However, these studies are
recent research interests that have only been implemented in animal models.
The particular genes, reactions, and factors involved in these pathways are
still not well-understood. While there is a long way to go for a greater
comprehension of how they work in the human heart, the identi�cation of

Berkeley Pharma Tech Journal of Medicine | 22



these pathways and possible molecules provides an insight into future
research directions that could potentially result in novel gene therapies.

A

B C

D E

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Figure 6. Overview of Amount of Research Articles Pertaining to Each
Therapeutic Approach. To obtain a broad overview of how cardiac ischemic injury
has been approached in research, a PubMed search of the key terms “gene therapy +

cardiac regeneration + ischemic injury” was conducted on May 6, 2022. The first one
hundred relevant articles were cataloged and analyzed for the specific research topic

that was covered. Panel (A) shows the number of research articles that appeared by the
topic overall, and panels (B)-(F) show the number of articles per topic by year of

publication. “Genetic Research without Specified Method” refers to work done with
specific genes that did not mention the MSCs, modRNA, or iCMs. “Other Therapies”

refers to therapies used to treat cardiac ischemic injury that did not involve genetics,
such as cell therapy or drug therapy. “Multiple Therapies” refers to articles that

address more than one form of therapy.

Research in the �eld of gene therapy to alleviate cardiac ischemic injury has
been studied for decades and will continue to be studied in coming years. As
the current trends in the research were reviewed, all therapies covered in this
article still seem to be relevant to the �eld. Overall, the most popularly
researched therapy seems to be the direct interaction with speci�c genes,
above any other methods mentioned. However, as mentioned, the others do
not fall far behind. As the �eld continues to expand, it is hopeful that
research will make the transition from models to clinical trials and continue
to add to each of these therapies for treatment.

Berkeley Pharma Tech Journal of Medicine | 24

F  G



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