1

Resumo:

A doença cardiovascular é uma das principais causas de morte no mundo ocidental. As

terapêuticas atuais para enfarte agudo do miocárdio simplesmente travam a evolução da doença,

sem uma verdadeira regeneração do tecido cardíaco ou vascular. Nos últimos anos, o uso de

células na terapêutica cardiovascular tem sido alvo de grande interesse devido ao seu potencial

para recuperação da função cardíaca através da regeneração de tecido perdido. Até à data foram

estudados vários tipos de células estaminais. Alguns apenas em laboratório, mas já foram

concluídos vários ensaios clínicos, e muitos outros ainda se encontram em andamento. Apesar

dos resultados promissores obtidos em modelos animais, os ensaios clínicos apresentam

resultados conflituosos. Muitas variáveis estão a ser estudadas para possibilitar uma melhoria da

sobrevivência e do acoplamento das células implantadas, e para permitir a sua diferenciação de

forma mais eficiente para células do tipo cardíaco. No entanto, a terapêutica celular deu um

grande passo em frente com a descoberta das células estaminais pluripotentes induzidas e com o

avanço dos tecidos cardíacos artificiais. É digno de nota que, em estudos recentes foi possível

regenerar tecido cardíaco através da reimplantação de células pluripotentes numa matriz

extracelular decelularizada. O objetivo deste trabalho é fazer uma revisão dos diferentes tipos de

células estaminais disponíveis e do seu potencial terapêutico, dos métodos que podem melhorar

os resultados da terapêutica celular, e compreender os potenciais benefícios destas recentes

descobertas e que mudanças podem elas trazer ao futuro da terapêutica cardiovascular.

Palavras-chave:

Células estaminais; Insuficiência cardíaca; Terapêutica celular; Diferenciação; Regeneração

vascular; Matriz de suporte biológico; Coração bioartificial;

2

Abstract:

Cardiovascular disease is a leading cause of death in the western world. Current therapies for

myocardial infarction merely block disease progression without actual regenerating either the

cardiac or vascular tissue. In the past few years, cell therapy has attracted great interest with the

potential of recovering cardiac function by regeneration of lost tissue. Various types of stem

cells have been studied so far. Some only in laboratory, but several clinical trials have been

completed and many more are still ongoing. Despite the promising results of animal studies,

clinical trials show conflicting results. Many variables are being taken into consideration to

improve the survival and engraftment of the implanted cells, and to allow a more efficient

differentiation into cardiac lineage cells. Cell therapy has taken a new step further, however,

with the discovery of induced pluripotent stem cells and the advances of cardiac engineered

tissues. Recent studies have, remarkably, been able to regenerate cardiac tissue by means of re-

implantation of pluripotent cells into a decellularized extracellular matrix. The focus of this

review is to discuss the different types of stem cells available and their therapeutic potential, the

methods to improve cell therapy outcome, and to understand the potential benefits of these

recent discoveries and what changes they might bring to the future cardiovascular therapy.

Keywords:

stem cells; heart failure; cell therapy; differentiation; vascular regeneration; biological scaffolds;

bioartificial heart;

3

Index

Resumo ............................................................................................................................. 1

Abstract ............................................................................................................................. 2

Keywords .......................................................................................................................... 2

Index ................................................................................................................................. 3

List of abbreviations ......................................................................................................... 5

List of Figures ................................................................................................................... 7

List of Tables .................................................................................................................... 7

Introduction ...................................................................................................................... 8

Material and methods ..................................................................................................... 10

Stem Cells………………………………………………………………………………10

Cell types…………………………………………………………………….…...11

Bone Marrow Derived Stem Cells…………………………………………11

Mesenchymal Stem Cells………………………………………………….15

Stem cell related factors………………………………………………...…17

Adipose tissue derived Stem Cells………………………………………...18

Cardiac stem/progenitor cells…………………………………………...…20

Skeletal Myoblasts…………………………………………………………21

Embryonic Stem cells……………………………………………………...24

Induced Pluripotente Stem Cells…………………………………………..25

Differentiation of pluripotent stem cells into cardiomyocytes……………………...….29

Embryoid body……………………………………………………………......…31

Coculture of pluripotent stem cells……………………………………………....31

Growth factors……………………………………………………………...…....32

Others factors and molecules……………………………………………...……..33

Spontaneous calcium oscilations………………………………………..…34

Micro-RNAs…………………………………………………...…………..35

Vascular regeneration…………………………………………………………………..37

Bioartificial Heart............................................................................................................39

Conclusion ...................................................................................................................... 44

4

Acknowledgements ........................................................................................................ 45

References ...................................................................................................................... 46

5

List of abbreviations:

α-MHC- α-myosin heavy chain

AMI- Acute myocardial infarction

ADMSC- Adipose tissue–derived mesenchymal stem cells

Akt/PkB- Protein kinase B

Alk5- Activin-like kinase 5

Apo- Apolipoprotein

ASCs/ADSC - Adipose tissue derived stem cells

BM-MNC- Bone marrow mononuclear cells

BMSC/BMC – Bone marrow stem cells

BMP4- Bone morphogenetic protein 4

BNP- Brain natriuretic peptide

CDC- Cardiosphere-derived cells

CLC - Cardiomyoblast-like cells

CM- Cardiomyocytes

CMPC-Cardiomyocyte progenitor cells

CPC- Cardiac progenitor cells

CsA- Cyclosporine A

CSC-Cardiac stem cells

CT1- Cardiotrophin-1

CVB3- Coxsackievirus B3

DNA- Deoxyribonucleic acid

EC-Endothelial cell

ECM- Extracellular matrix

EGFP- Enhanced green fluorescent protein

EDTA- Ethylenediamine tetraacetic acid

END-2- Endoderm-like cells

EPC- Endothelial progenitor cell

ERK- Extracellular signal-regulated kinase

ESC- Embryonic stem cells

FLK-1- Fetal Liver Kinase 1

G-CSF- Granulocyte colony-stimulating factor

HF –Heart failure

HGF- Hepatocyte Growth Factor

HIF- Hypoxia-inducible factor

hMSC- Human mesenchymal stem cells

6

IFN-γ - Interferon gamma

IGF-1- Insulin-like growth factor-1

IL- Interleukin

IP3- Inositol trisphosphate

IP3R- Inositol trisphosphate receptor

iPS – Induced pluripotent stem cells

KDR- Kinase insert domain receptor

LV- Left ventricle

LVEF- Left ventricle ejection fraction

LVSD - Left ventricle systolic dysfunction

MAPK- Mitogen-activated protein kinase

MDR-1- Multidrug resistance protein 1

miRNA- Micro RNA

mRNA- Messenger RNA

MSC- Mesenchymal stem cells

PGI2- Prostacyclin

PLC- Phospholipase C

PKC- Protein kinase C

RNA- Ribonucleic acid

Sca-1- Stem cell antigen 1

SERCA- Sarcoplasmic reticulum Ca2+

-ATPase

SM- Skeletal myoblasts

SMC- Smooth muscle cell

SM-MHC- Smooth muscle myosin heavy chain

SRF- Serum response factor

SSEA-1- Stage Specific Embryonic Antigen-1

STEMI- ST segment elevation myocardial infarction

TGF- transforming growth factor

TnT- Troponin-T

VEGF- Vascular endothelial growth factor

VPC- vascular progenitor cell

7

List of Figures:

Figure 1: Different types of stem cells sources for cardiac cell therapy……………….….....11

Figure 2: Different methods to obtain induced pluripotent stem cells………….…….….…..26

Figure 3: Differentiation approaches used for cardiomyocyte differentiation from pluripotent

stem cells………………………………………………………………………..………….…30

Figure 4: Application of a biological scaffold on an infarcted heart………………...…..…..40

List of Tables:

Table 1: Clinical trials using BMSCs and their respective outcomes………………………......14

Table 2: Clinical trials with various different types of cells and/or growth factors, and their

respective outcomes……………………………...………………...…………….……………...22

Table 3: Autologous cells vs Allogenic cells. Comparing characteristics and potential uses....42

8

Introduction

Heart failure is a leading cause of mortality and morbidity in the western world1. The presence

of heart failure is usually diagnosed by clinical criteria and also includes objective assessment of

left ventricular (LV) function2, like echocardiographic evaluation

3. Approximately 2.9% of

patients under 75 years and up to 7.5% in patients between 75-84 years show left ventricular

systolic dysfunction (LVSD).4 Heart Failure incidence in the U.S. approaches 10 per 1000

population after the 65 years of age. The annual rates per 1000 population of new HF events for

caucasians are 15.2 for those between 65-74 years of age, 31.7 for those between 75-84 years of

age, and 65.2 for those with or over 85 years of age5. In Australia the numbers are even greater,

with more than 10% of people aged over 65 being affected6. Around 50% of people diagnosed

with HF will succumb within five years of diagnosis5. There is an increase in prevalence of

heart failure derived from post- ischemic cardiac dysfunctions, like acute myocardial infarction

and ischemic cardiomyopathy7. Up to a billion cardiomyocytes can be lost after myocardial

infarction, and endogenous regeneration cannot restore heart’s function8. Review of the

available data suggests that, after acute myocardial infarction (AMI), approximately 30%– 45%

of patients will develop heart failure, and approximately 25%– 60% will show LVSD. Also

about 50% of patients with LVSD early after AMI will develop heart failure as well9. The 30-

day risk-standardized mortality rates in patients with heart failure early after AMI, was 15.8% in

20069, 10

. A considerable proportion of these fatal cases was due to a significant cardiomyocyte

loss, that resulted in either in a more or less exuberant heart failure. Medical interventions for

heart failure, like adjustment of the preload, afterload and contractility11

, provide some benefits

and an improvement of symptoms, but they do not recover the function of the damaged tissue.

The only therapeutic approach, used routinely, to replace the injured tissue is heart

transplantation1. However, supply of donor organs is limited, and once a heart is transplanted,

patients face a lifetime of immunosupression and there is an increased risk to develop

hypertension, diabetes and renal failure12

.

With the recent advances in stem cells research, the potential of replacing the injured

tissue has generated tremendous interest worldwide1. Stem cells may be able to provide new

forms of treatment of ischemic heart disease, due to their potential for repairing damaged

cardiac tissue13

. Several cell types have been used in research, including bone marrow- (BM)

derived cells, myoblasts, endogenous (resident) cardiac stem cells, embryonic cells and induced

pluripotent stem cells (iPS)1.

However, in several studies, both preclinical and clinical, the improvement of cardiac

function has been proven to be marginal and even temporary. The level of regeneration of

damaged cardiac tissue was very low, and below the level required to achieve clinical benefits13

.

9

One of the main drawbacks is that stem cells alone, regardless of origin, are not sufficient to

assure an efficient regeneration of the lost myocardium, and after an initial functional

improvement, they can even aggravate the situation by promoting the formation of new fibrotic

tissue at the site of a pre-existing scar1. The revascularization of the infarcted heart could play a

major beneficial role in the treatment of myocardial infarction. The differentiation of vascular

endothelial cells in clinically relevant numbers for injection into the ischemic area could

stimulate angiogenesis, and therefore preventing cell death, limiting the development of scar

tissue and improving cardiac function. This is also true for peripheral ischemia, were injection

of cells into the ischemic regions to stimulate new blood vessel growth could also be

beneficial14

. Whatever used cell type or approach, it should address the regeneration of not only

cardiac tissue, but also vascular tissue.

However, there are some issues that prevent a routine use of stem cells, both in cardiac

and vascular regeneration. Issues like limited engraftment, limited proliferation and

differentiation potential of the transplanted cells within the host tissue, identification of the

optimal cell type and administration route, the improvement of cell survival, the possible

induction of paracrine functions, and the possible activation of local spontaneous regenerative

activity1. To date, many studies have investigated the potential of cell therapy for the treatment

of heart failure in both animal models and humans, with varying success. Nonetheless,

unavoidable limitations are inherently present within the host tissue restricting cell survival and

subsequent cell therapy success. In a long term follow up study, less than 2% of the injected

stem cells survived 8 weeks after myocardial infarction15

. Apoptosis resulting from mechanisms

including loss of cell–cell contact and lack of oxygen and nutrient delivery are thought to be

responsible for the cell death observed within the myocardium16

.

The fundamental dogma that the heart is a post-mitotic organ has recently been

challenged with the identification of resident cardiac stem cells. These Sca-1+ or c-kit+ cells

although comprising only about 2% of total heart cells, they contribute to the turnover and

growth of vascular smooth muscle and endothelial cells, as well as cardiomyocytes17, 18

.

Recently there have been a few studies that show an alternative way to treat heart failure,

within the range of heart transplantation. The twist is, the use of a bio-artificial heart generated

from stem cells.

These recent “discoveries” have paved the way to the progressive involvement of

regenerative medicine in the treatment of cardiac diseases as significant as acute myocardial

infarction and heart failure, regardless of etiology.

The purpose of this paper is to review the biology and characteristics of various types of

stem cells, and their therapeutic potential, exclusively in the field of post-myocardial infarction

and related heart failure, as a paradigm of a cardiovascular disease real model.

10

Methods:

The present review was written based in a bibliographic research using the PubMed’s data base.

The search was conducted based on MeSH terms, using the following combinations:

[stem cells heart failure] search produced 786 results;

[stem cells AND bone marrow AND heart AND trial ] search produced 183 results;

[stem cells AND bone marrow AND heart failure] search produced 286 results;

[(stem cell AND adipose) AND (trial OR study) AND (heart OR cardiac)] search produced 85

results;

[(stem cell AND skeletal myoblasts) AND (heart OR cardiac) AND (study OR trial)] search

produced 160 results;

[resident cardiac progenitor] search produced 105 results;

[embryonic stem cells AND heart failure AND cell therapy] search produced 101 results;

[(induced pluripotent stem cells) AND (heart OR cardiac)] search produced 262 results;

[stem cells AND differentiation factors AND cardiomyocytes] search produced 406 results;

[taylor AND bioartificial heart] search produced 2 results;

[taylor[Author] AND heart AND matrix] search produced 13 results;

[heart AND matrix AND decellularized OR decellularization] search produced 222 results;

In every search made, only items with links to full text, and a publication date no further back

than 2006 were selected.

Other articles referred in this review that were not produced in the original search, were located

in PubMed’s data base trough other articles’ own references, or were obligingly referred by the

guidance counselor. In total, 190 articles are referred in this review.

Stem cells:

First discovered in 1963, stem cells possess self-renewal capacity, long term viability and multi-

lineage potential.

They are an effective source of cells for new tissue generation. To date, multiple types of stem

cells have been identified as having clinical potential in cell therapy and tissue engineering.

However, potential only goes so far. To be used with success in a clinical context, a stem cell

must possess multiple characteristics. In the case of cardiac tissue engineering, cells should

permit easy harvest using routine surgical procedures, they should be easy to expand and

generate nontumorogenic, patient-specific cardiac cells (multipotency) for autologous

application. If from an allogenic source, they should also be immune privileged. In either case,

minimal or no ethical dilemmas associated with their use, is also an advantage19

.

11

Cell-Types:

There are several potential sources of cardiac progenitor cells. Pluripotent stem cells from

different sources can be expanded in vitro and differentiated into cardiac progenitor cells and

mature cardiac cell types, thus enabling cell replacement therapy or tissue engineering (fig 1).

Herein, it will be presented the biology, advantages and disadvantages and potential therapeutic

uses of different stem cells, such as bone marrow derived stem cells, mesenchymal stem cells,

adipose tissue derived stem cells, cardiac stem/progenitor cells, skeletal myoblasts, embryonic

stem cells and induced pluripotent stem cells in cardiac pathology, namely after myocardium

infarction and in ischemic cardiomyopathy.

Figure 1: Different types of stem cells sources for cardiac cell therapy.

Adapted from Wollert and Drexler, Nat. Rev. Cardiol. 2010, 7; 204–215

Bone Marrow Derived Stem Cells (BMCs)

BM stem cells are endowed with great plasticity. They are able to differentiate in vitro into cells

of different lineages, including cardiac cells1. There are at least four main characteristics that

make BMCs so attractive: (a) they are easy to harvest, (b) high income cell numbers do not limit

clinical applications, (c) BMCs contain an heterogeneous composition of cells including

fractions of stem and progenitor cells, and (d) their preparation does not require prolonged ex

vivo manipulation7.

12

However, in vivo studies seem to have controversial results. Some groups showed extensive

differentiation of donor BM-derived cells to both vascular and cardiac cells in the host20

. Others

show a contribution mainly to the formation of new vasculature, and not to the generation of

new cardiomyocytes21

.

Clinical trials have been initiated using BM infusion, but the results of these trials are not

consistent. The TOPCARE-AMI trial was a randomized study on the effects of intracoronary

infusion of circulating progenitor or bone marrow-derived progenitor cells in patients with

reperfused AMI. Five-year follow-up data demonstrates that intracoronary cell treatment is

indeed safe and is associated with a sustained LV functional improvement, 5 years after acute

myocardial infarction. Although this trial did lack a placebo-treated control group, it showed an

excellent safety profile, with a 97% event-free survival without death, myocardial infarction or

re-hospitalization for heart failure. Plus, it did not show significant difference between

circulating progenitor cells and BMC treatment results22

. The placebo-controlled, double-blind,

randomized multicenter REPAIR-AMI study demonstrated that intracoronary administration of

BMC improved left ventricular contractile function in patients with impaired function after ST

segment elevation myocardial infarction, after both 4 months and 1-year follow-up, and

lessened post-infarction left ventricular remodeling23,24

. Similar studies assessed that

intracoronary injection of BMSCs early after reperfusion of an acute myocardial infarction

significantly improves the recovery of regional myocardial function25

. Another double-blind,

placebo-controlled, multicenter trial demonstrated a significant reduction of the occurrence of

major adverse cardiovascular events, after the intracoronary administration of BMC. According

to the follow-up data this event reduction is maintained for 2 years after the AMI26

. This is in

agreement with other study results27

. Further evidence from more clinical trials suggests that

injection of BMCs in patients with end-stage ischemic HF is safe28

, and there are no procedural

or acute complications, and no long-term safety concerns, such as ventricular arrhythmias29

or

the development of tumor or ectopic tissue. Cell therapy improves symptoms and quality of life

and may have beneficial effects on myocardial perfusion30, 31

. Also improves exercise tolerance,

survival and myocardial performance32

. The STAR-heart study showed that in chronic heart

failure there are cell-induced improvements in hemodynamics at rest, exercise capacity and

oxygen uptake at rest and exercise, left ventricular contractility indexes and left ventricular

geometry. Over a 5-year follow-up intracoronary BMC therapy was not associated with adverse

events, but it has been associated with improved LV performance, quality of life, and survival33

.

In other words, BMC transfer in CHF may result in smaller infarct size, better LV-EF, and

better wall movement7, 34

. This improvement in results may be due to four factors: improved

intrinsic myocardial function, a decrease in loading (volume or pressure), an increase in

elasticity or stimulated neovascularization that improves microcirculation after AMI25

.

13

On the other hand, other clinical trials have not shown such promising results. A randomized,

placebo-controlled, double-blind trial failed to demonstrate that cell therapy was superior to

placebo due to similar improvement of LVEF in both groups, at 6 months. Although cell

therapy had a beneficial effect on LV remodeling, and provided a reduction in major adverse

CV events35

. Another study did not demonstrate any improvement in LV systolic function after

two sequential treatments with intracoronary infusion of bone marrow cells in patients with

chronic ischemic heart failure28

. The ASTAMI study failed to demonstrate significant effects of

mBMC therapy on LV global systolic function, remodeling, regional systolic function, or

diastolic function during 3 years follow-up36

. G-CSF therapy aiming to mobilize bone marrow

stem cells, has failed to improve left ventricular recovery,37

although the relation between acute

myocardial infarction and the activation of bone marrow progenitor cells by Wnt signaling has

been documented38

. A randomized study demonstrated that the treatment with intracoronary

injection of BMSCs in patients early after anterior MI had no influence on exercise capacity or

on heart rate and systolic blood pressure response to exercise. It also has no significant

influence on other indices related to prognosis and ventricular arrhythmia occurrence, and no

influence in pulmonary function39

. Another study in swine models shows that an intracoronary

injection of both MNC and unselected BM one week after MI does not improve LV function

after 4 weeks. BM-derived MNC or unselected BM treatment were useless in reversing the

fibrotic remodelling induced by the MI40

. In the REGENT trial, treatment with BMCs did not

lead to a significant improvement of LVEF or volumes, in patients with AMI. However, in the

patients that presented the most severely impaired LVEF, cell therapy produced some

advantages41

.

Possible reasons for the result discrepancy found in many of these studies, may include

differences in study protocol and design, including time from reperfusion to cell injection, type,

number, and isolation technique of cells, cell delivery routes or follow-up design7, 42

.

Test results evidencing better myocardial perfusion in a study in the BMSC group, confirm the

hypothesis that BMSCs may react mainly through the enhancement of neovascularization21

.

And since improvement in cardiac function has been observed even without significant

engraftment and differentiation of transplanted cells, it is believed that paracrine mechanisms

play an important part in the therapeutic benefits of BM–derived cells1, 43

.

There is still much that is unknown about the potential therapeutic role of BMSCs in heart

disease. There are several ongoing studies that are expected to shed some light on this subject.

For example the SWISS-AMI study aims to confirm the therapeutic benefit of intracoronary

administration of autologous BM-MNC after AMI, and also to define the optimal time slot for

cell therapy as well as clinical criteria for patients that can qualify for such therapy44

. The TAC-

HFT trial, a randomized, double-blind, placebo controlled study aims to demonstrate the safety

14

and efficacy of BMCs and MSCs administered transendocardially in patients with chronic

ischemic cardiomyiopathy45

. And the COMPARE-AMI trial will evaluate the impact of BMSCs

in the change in coronary atherosclerotic burden progression, proximal and distal to a coronary

stent in infarcted arteries, and also whatever major adverse cardiac events may occur46

.

Table1: Clinical trials using BMSCs and their respective outcomes.

Trial Number of

patients

Cell Type Number of

cells (median)

Delivery Route Results

REPAIR-AMI 187 BMNCs 198x106 Intracoronary

administration

Improvement of

left ventricular function.

STAR-heart study

191 BMCs 6,6+3,3x107 intracoronary Improvement of ventricular

performance,

quality of life and

survival.

TOPCARE-

AMI

59 BMCs/CPCs 5.5 + 3.9x106 Intracoronary

infusion

Long term safety

of cell therapy, and favorable

effects on LV

function.

FOCUS-HF 30 ABMMNC 3x107 Transendocardial

delivery

Therapy is safe,

improves

symptoms and quality of life.

Cardiac Study 38 BMCs 418x106 Intracoronary infusion

Decrease in the occurrence of

heart failure.

Grajek et al. 45 BMCs 1x106 Intracoronary

transplantation

No increase in

EF. Slight

improvement of

myocardial perfusion.

ASTAMI 100 mBMCs 68x106 Intracoronary injection

No differences between groups

indicating

beneficial effect

REGENT 200 Non-selecetd

BMCs

1,90x106 Intracoronary

infusion

No significant

improvement of

LVEF or volume.

DanCell-CHF 32 BMCs 1st infusion:

647+382x106 2nd infusion:

889+361x106

Intracoronary

injection

No change in LV

function

Traverse et al. 40 BM-MNCs 100x106 Intracoronary

infusion

No improvement

in LVEF at 6

months, but

favorable effect on LV

remodeling

Adapted from Wollert and Drexler, Cell therapy for the treatment of coronary heart disease: a

critical appraisal, Nat. Rev. Cardiol. 2010, 7, 204–215 Several studies seem to indicate beneficial effects of BMCs therapy in patients with either AMI

or ischemic heart disease. The clinical use of autologous BMC therapy implies no ethical

problems and there are no major stem cell-related side effects. There was an overall good safety

in BMCs use.

15

Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are stromal cells present in various tissues, such as BM and

adipose tissue. MSCs can participate in cardiac tissue repair not only by direct

transdifferentiation but also by reducing cell damage and activating endogenous mechanisms of

tissue regeneration. Because of this paracrine activity, they exert anti-inflammatory, anti-

apoptotic and angiogenic effects47, 48

. The mechanisms underlying these effects are controversial

and most likely multi-factorial, but it is clear that this therapeutic approach is not devoid of

merit and therefore will be further evaluated49

. Also MSCs can be easily isolated, and greatly

expanded ex vivo without loss of phenotype or differentiation capacity. Importantly, MSCs are

also immunoprivileged, so it is possible to used them in an allogeneic host50

.

Clinical data are scarce, but previous endomyocardial injection of allogeneic MSCs after acute

MI did not raise safety concerns, and was not associated with long-term clinical events or

pathologic effects. It did reduce MI size, but did not alter the LVEF, nor was its effect dose-

dependent51

.

This was the scene with an endomyocardial approach, but a quantitative, randomized study

compared the three most common MSCs delivery approaches: intravenous (IV), intracoronary

(IC) and endocardial (EC) delivery. The results showed that IC and EC injection of allogeneic

MSCs post-MI achieve a greater increase in cell engraftment when compared with IV infusion,

and that IC was more efficient than EC injection. However, IC delivery was also associated with

a higher incidence of decreased coronary blood flow, while EC delivery was safer and better

tolerated than IC and IV deliveries52

.

The area of greatest engraftment and regeneration usually resides in the border zone between

infarcted tissue and viable myocardium53

.

A double-blind, placebo-controlled, dose-ranging study using allogeneic hMSCs, demonstrated

safety of hMSCs infusion after AMI, as well as long-term absence of ectopic tissue formation.

Cell-treated patients had improved outcomes regarding cardiac arrhythmias, pulmonary

function, left ventricular function, and symptomatic global assessment54

.

Although an allogenic approach is possible, autologous cells may offer advantages, by avoiding

late immunologic events49

.

A randomized, blinded, placebo-controlled study of bone-marrow (BM)-derived MSCs in adult

mini-swine, showed that autologous MSCs can be safely and effectively prepared post-MI and

delivered surgically in a porcine model of ischemic HF49

. The use of MSCs in therapy can have

an extraordinary magnitude of benefits including reduced infarct size, 51

formation of new

contractile and perfused tissue, and increase in LVEF49

. It’s likely that MSCs engraft,

differentiate into myocytes, promote neovascularization, and maintain reservoirs of immature

cells. Even late injection of cells into the post-infarct tissue can have a desirable outcome53

.

16

Evidences of a dose–response effect to the MSC therapy can be found in the same randomized

trial. In summary, it shows that a high-dose group (200x106 MSCs) develops reverse

remodeling at structural and functional levels (the low-dose only prevents infarct expansion),

and improves contractile function in the infarct zone followed by a substantial increase in

LVEF, unlike in lower-dose (20x106 MSCs) or placebo groups

49. Autologous MSCs

implantation can also promote expression of IGF-1, HGF, and VEGF growth factors, which in

turn enhance cardiomyogenesis and angiogenesis in the failing myocardium, at the same time

improving cardiac remodeling and function. IGF-1, a growth hormone mediator, plays an

important role in myocardial growth, and the expression of IGF-1 mRNA and protein

dramatically increases with MSCs addition. The HGF secreted by the MSCs or by triggered

cells in their vicinities, might have a beneficial effect on the amelioration of cardiac remodeling

and heart function. VEGF is a highly specific mitogen for vascular endothelial cells. It’s no

surprise that, capillary density significantly increases with MSC treatment. The augmented

vascular beds might better supply the cardiac tissue with blood, therefore improving

cardiogenesis, cardiac remodeling and function55

.

MSCs also seem to be useful in the treatment of myocarditis. They improve experimental acute

coxsackievirus B3 (CVB3)-induced myocarditis in mice, via their anti-apoptotic and

immunomodulatory properties, since they cannot be infected by CVB3 and reduce direct CVB3-

induced cardiomyocyte injury in an NO-dependent way and require priming by IFN-γ47

.

Atorvastatin treatment may protect the cardiac tissue undergoing acute infarction and

reperfusion, by creating a better environment for the survival and differentiation of implanted

MSCs. The beneficial effects occur as a synergism between Atorvastatin and MSCs. Treatment

with either alone could not achieve these effects. Atorvastatin administration enhances the

survival and differentiation of implanted MSCs, decreases the infarcted area, promotes

angiogenesis, and reverses negative ventricular remodeling in swine hearts56

. MSCs exposed to

hypoxic conditions before transplantation also show a higher survival rate. This is due in part to

upregulation of survival proteins such as Akt57

. MSCs appear to activate antiinflammatory

pathways. The beneficial effects of MSCs are partly dependent on the secretion of IL-10, an

anti-inflammatory cytokine58

.

Despite the positive outcome of these studies, others did not show such improvement. The use

of these cells is also not without complications or side-effects: MSCs sometimes exhibit

chromosomal disorders, cause tumors in rodent models, and can cause ectopic bone and fat

tissue formation. Some MSCs also form encapsulated structures containing calcifications after

intramyocardial delivery49

.

17

Stem cell related factors

Apart from their more direct role of tissue regeneration, stem cells, namely BMSCs and

MSCs, may also have a clinical impact by secreting multiple growth factors and cytokines.

Stem cell transplantation and cytokine secretion may enhance and prolong the mobilization of

more stem cells59

. Trophic mediators secreted by stem cells improve cardiac function by a

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

remodelling, promoting angiogenesis, mobilizing host tissue stem cells, and reducing

inflammation60

. This mechanism works both ways, since the use of the right mediator may

contribute to a better outcome in cell therapy. Most studies relating to this subject were

conducted using BMCs or MSCs. The possibility of using growth factors capable of mobilizing

stem cells to peripheral blood and induce their homing to the infarcted heart has therefore great

therapeutic appeal since it would avoid invasive procedures61

.

G-CSF is a potent cytokine that influences the proliferation, survival, maturation, and the

functional activation of granulocytes, and it is involved in mobilization of granulocytes, stem,

and progenitor cells from the bone marrow37

.

Studies using G-CSF alone showed no improvement in cardiac function, neither with classical

nor with long term treatment61

. However, cardiac function and cardiomyocyte number were

significantly increased in animals with G-CSF plus dipeptidylpeptidase-IV inhibition treatment.

Also, adverse thinning of the infarcted LV free wall was lessened as a result of the treatment62

.

A multicentre, randomized, single-blind, placebo-controlled trial concluded that the G-CSF

therapy attenuates LV remodelling 6 months after mechanical reperfusion of a large anterior

STEMI. Also in patients with an EF ≤45% after reperfusion and a time to reperfusion of >2 and

<12 h, early administration of high-dose G-CSF may attenuate unfavorable remodelling at 6

months63

.

Another trial, on the other hand, demonstrated that although G-CSF administration could be

associated with improvement in a range of subjective outcomes, adverse events were common,

with several episodes of myocardial ischemia and angina, while objective measures of cardiac

perfusion remained unchanged64

.

Overall, short-term cell mobilization with G-CSF seems safe in patients with AMI. In patients

with myocardial infarction, most studies showed a significant recovery of left ventricular

function. The current results allow the possibility that G-CSF might be an effective part of a

treatment strategy combining several cytokines and/or local stem cell delivery65

.

VEGF also is a key therapeutic trophic factor in MSC-mediated myocardial regeneration. VEGF

hMSC derived cardiomyocytes improved cardiac function, highlighting the role of VEGF in

mediating the therapeutic function of MSCs60

. But its potential is not limited to the MSCs.

18

Overexpression of VEGF promotes the migration of the resident cardiac stem cells into the

infarcted region of the hearts after MI, via stimulation of Flk-1 and activation of PI3K/Akt57

.

Paracrine pigment epithelium-derived factor (PEDF) plays a critical role in the regulatory

effects of MSCs against MI injury. PEDF is a strong anti-angiogenic factor. Compared to young

MSCs, old MSCs secrete more PEDF in the infarct region, and increase the fibroblast

population. It is therefore conceivable that increased PDEF levels exert more significant

biological effects resulting in an infarct region containing fewer ECs and VSMCs, inhibiting

angiogenesis. The impaired therapeutic efficacy of aged MSCs is mostly due to the age-

dependent, increased PEDF secretion66

.

Adipose tissue derived Stem Cells (ASCs/ADSC)

Adipose tissue contains a population of stem cells that express mesenchymal cell specific

markers and most of them lack the expression of hematopoietic and endothelial cell markers.

Also, freshly isolated cells express the OCT4 that is a molecule typical of embryonic stem cell

phenotype. These cells were identified positive for CD29 and CD44 markers. Their capacity for

differentiation toward adipogenic, cardiogenic and neurogenic lineages suggest that ADSC is a

good multipotential cell candidate for the future cell replacement therapy67

.

Unlike embryonic stem cells (ES cells), autologous ASCs do not raise ethical, immunorejection,

or oncological concerns. Also, adipose tissue is abundant in most individuals and can be easily

harvested with little discomfort and damage to the donor68

. Adipose tissue has a significantly

higher stem cell density than bone marrow69

, which means that even a small amount of adipose

tissue can be used for potentially successful autologous cell transplantation68

. Unlike BMSCs,

which require processing and culturing after aspiration, therefore making them difficult to use in

an acute clinical setting, ASCs are promising cell candidates for myocardial regeneration,

especially in acute situations68,70

. It has been demonstrated that, like cultured human ASCs,

freshly isolated hASCs have the ability to engraft and improve cardiac function when

transplanted directly into the hearts of acute MI-subjected mice. Reduction of perfusion defects

and greater LV wall thickness is also present. This means that the use of fhASCs obviates the

need for prior cell expansion in vitro, allowing for immediate autologous cell transplantation70

.

In mice and porcine studies, enhanced LV fraction, greater LV wall thickness and cardiac

function improvement were observed after ASC transplantation68, 71

. ASCs, like BMSCs have

significant clinical benefits in treating cardiovascular diseases. Both hASCs and hBMSCs are

resistant to hypoxic conditions, and can both successfully engraft after coronary delivery,

increasing the EF percentage, resulting in a significant improvement in ventricular function.

Their influence in myocardial cytokine levels shows no detectable inflammatory reaction in

either cell type. But, by attenuating the contractile dysfunction and pathologic remodeling,

ASCs can contribute better than BMSCs to the recovery of ventricular performance after

19

myocardial infarction. They also show better cell proliferation and cardiomyocyte

differentiation potential72

.

ADSCs have the ability to differentiate into cardiomyocytes and vascular cells. Studies

demonstrate that adipose derived cells improve heart function by increasing angiogenesis

besides decreasing the degree of fibrosis in the infarcted tissue73

. Therefore they show great

potential to differentiate into endothelial cells, incorporate into vessels, and promote post-

ischaemic neovascularization69, 71

.

ASCs can also exert paracrine effects in myocardial regeneration, by secreting significant

amounts of angiogenic and antiapoptotic factors70, 71

.

However, there have been some studies in which the therapeutic potential of ADSCs alone has

not been demonstrated. In fact, there are cases in which ASC rapidly die off after injection into

the infarcted heart, and are not capable of significantly preventing left ventricular remodeling or

subsequent loss of cardiac function74

. Others took adipose tissue–derived mesenchymal stem

cells (ADMSCs), and used dimethylsulfoxide to differentiate them into cardiomyoblast-like

cells (CLCs). Only the transplantation of the hADMSC-derived CLC could regenerate

myocardial tissues, rescue cardiac dysfunction after MI, and improve long-term survival rate.

With the non-committed hADMSCs this was not verified75

.

Combinations of stem cell therapy with pharmacological treatments usually provide added

benefits, when compared to stem cell therapy alone. The combination of ADSCs and PGI2

strongly increases ADSC delivery and entrance, without affecting ventricular and metabolic

functions. PGI2 exerts effects on vasodilatation, platelet aggregation inhibition, cell–cell

interactions, cell permeability and blood cell adhesion to injured endothelium, as well as

cardioprotection in ischaemia/ reperfusion damage76

. And while some studies have reported that

the treatment of hADSCs with 5-azacytidine results in the differentiation of the cells into the

cardiomyocyte phenotype72

, others showed that the use of 5-azacytidine alone could not induce

early stages of cardiomyogenic differentiation in human ADSCs77

.

Another important variable to consider is the route of ADSCs administration. A study compared

intracoronary and transendocardial administration of autologous ADSCs in a porcine model of

myocardial infarction. The results demonstrated that both pathways are feasible, with a similar

number of engrafted and differentiated ADSCs at 3 weeks follow-up, although intracoronary

administration of ADSCs demonstrated a greater increase in neovascularization78

. On the same

subject, it has been reported that the transplantation of adipose-derived stem cells with aid of a

fibrin glue, enhances cell retention and survival. In a previous experiment, it improved the

infarcted heart function, reduced the infarct size, increased wall thickness and promoted

angiogenesis in a rat model of MI79

. Another study showed that the epicardial delivery of

trilayered ADSC sheets is associated with a greater number of retained cells, a better

20

preservation of LV geometry, and ultimately, higher rates of post-infarction survival, when

compared to other more traditional delivery methods80

.

Cardiac stem/progenitor cells

The heart was considered to be a post-mitotic organ, but in fact, it has a resident population of

stem cells which are multipotent and can regenerate myocardium in the event of injury81

. This

means that cardiac myocyte regeneration can take place in adult hearts82

, and although CSCs are

involved in myocyte turnover and replacing occasional cell dropouts, they are incapable of

regenerating large injuries such as an infarct83

. Like any other stem cell, cardiac stem/progenitor

cells show the properties of self- renewal and differentiation potential and participate in the

myocardial repair process84

by mobilization from cardiac stem cell niches to the site of injury in

response to the cues from the cytokine rich microenvironment of the infracted heart20

. Resident

hCPCs are most abundant in the neonatal period and rapidly decrease over time85

. The niches

where cardiac stem cells are stored are preferentially located in the atria and apex but are also

detectable in the ventricle. They can be isolated from routine biopsy specimens and can be

expanded to obtain clinically relevant numbers of cells in a short period83

. hCPCs derived from

spherical clusters of cells known as cardiospheres, can be reproducibly isolated and expanded

from young human myocardial samples regardless of age or congenital cardiac diagnosis85

.

These cells can be propagated in vitro and differentiated into spontaneously beating

cardiomyocytes, after 5-azacytidine stimulation, avoiding the need for co-culture with neonatal

cardiomyocytes86

. They express stem cell markers such as c-kit, MDR-1, Islet-1, SSEA-1, and

Sca-1, depending on the cell category. Studies using Sca-1+ cardiac progenitor cells, dictated

these cells as one of the best sources for cell transplantation87

. They also harbor telomerase

activity, which is only present in replicating cells83

. The most used marker for studies in CPCs is

the c-kit, but Wnt-1 is also a possibility. In fact, data from a recent study indicates that the adult

heart can respond to injury with a modest increase in Wt11 progenitors. Thymosin β4 enhances

this response, via a significant reactivation of Wt1 expression ultimately resulting in

cardiomyocyte restitution. The derived cardiomyocytes were shown to structurally and

functionally integrate with resident muscle88

. CPCs also express Nkx2.5, which is a target for

Notch1. Activation of Notch1 favors the commitment of CPCs to myocytes and regulates the

compartment of transit amplifying myocytes in vitro and in vivo. However, it maintains the

newly formed cells in a highly proliferative state89

.

CSCs are clustered together in the niche and are coupled with the surrounding cells through the

expression of gap and adherens junctions; adherens junctions appear to be involved in the

preservation of the undifferentiated state of CSCs18

.

CSCs can also differentiate towards the endothelial and smooth muscle lineage86

.

21

The formation of cardiospheres is an alternative technique to single-cell deposition and clonal

expansion. A fraction of cells present in the core of the cardiospheres express the stem cell

antigen c-kit and is surrounded by an outer layer made of cells positive for CD105.

Cardiosphere-derived cells can spontaneously mature towards the myocyte lineage, by

undergoing co-culture with neonatal ventricular myocytes. Connexin 43 is expressed between

highly dividing cells within the cardiospheres and in the expanded differentiating cardiosphere-

derived cells18

.

CSCs offer many advantages for regenerative therapy, since they can be autologous and thus

unlikely to trigger infectious or immunological complications, they are more cardiogenic than

other adult stem cells, and can trigger angiogenic responses after myocardial transplantation83

.

The CADUCEUS study for example, showed an increase in viable myocardial tissue as a result

of cell therapy. However, the changes achieved in scar size were not accompanied by clear

changes in ejection fraction90

. According to the SCIPIO trial, intracoronary infusion of

autologous CSCs is not associated with apparent adverse effects, and it results in a substantial

improvement in LV systolic function 1 year after infusion. Also it is associated with increased

functional capacity, improved quality of life, and reduced LV scar size91

.

Skeletal Myoblasts

Skeletal myoblasts, or satellite cells, are found in the basal membrane of muscle fibers. They

can be easily isolated and expanded in culture, and show a worthy resistance to hypoxia-induced

apoptosis1. SMs have multiple advantages including safety, easy availability from an autologous

source without any ethical or religious concerns, ease of in vitro expansion to large numbers and

myogenic differentiation without the fear of tumorigenicity20

.

Results of clinical trials have demonstrated the safety and feasibility of percutaneous delivery of

myoblasts. Treated patients showed improvement in ventricular viability, and evidence of

reverse ventricular remodeling. Although a couple of arrhythmic events have been reported92

.

In the MARVEL trial for example, in HF patients with chronic postinfarction cardiomyopathy,

administration of myoblasts was feasible and lead to important clinical benefits. Improvements

in functional capacity were observed as well. It was concluded that ventricular tachycardia may

be provoked by myoblast injection, but appears to be a transient and treatable problem93

.

Although there is evidence of the positive and beneficial effects of SM implantation, there are

some studies in which this is not so simple.

The first randomized controlled trial of autologous myoblast transplantation in patients with

severe ischemic heart disease failed to detect a clinical significant improvement in regional or

global LV function. In this named MAGIC study, the loss of injected cells was largely caused

by apoptosis due to cell detachment from the extracellular matrix and by ischemia. This factor

22

was certainly critical in that the myoblast-injected areas were not vascularized because most of

the infarct-related arteries were unsuited for direct revascularization94

.

In another recent study, ventricular injection of skeletal myoblasts was associated with a

negative inotropic effect. In addition, independently of the global beneficial effect on heart

function after treatment, the frequency of irregular contraction was increased. However, even

though the function of isolated cardiomyocytes appeared to be compromised in infarcted

animals treated by intramoycardial myoblast injection, the global ventricular function of these

hearts was indeed stable. These results imply that, after intramyocardial injection of myoblasts,

altered contractile properties may occur at a cellular level, without deleterious effect on the

global heart function95

.

It seems that myoblasts do not exhibit any electromechanical coupling with the host

cardiomyocytes, because they fail to form gap junctions, which increases the chances of

arrhythmias20

.

Table 2: Clinical trials with various different types of cells and/or growth factors, and their

respective outcomes.

Trial Number of

patients

Cell/factor

type

Number of

cells/factor

dose

Delivery route Results

Prochymal 53 Bone

marrow-

derived

hMSCs

0.5/1.6/ 5.0

x106

Intravenous

infusion

hMSCs are

safe in patients

after AMI

Arguero et al. 39 GSF

mobilized

stem cells

3-5 x 106

Intramyocardial

injection

Cell therapy is

a safe and

useful patients

with ischemic

dilated CM.

STEMMI 78 G-CSF

mobilized

CD34+

+

MSC

25x109

4,9x1011

Intracoronary

injection

G-CSF

induced a

dissociated

pattern in

circulating

CD34+ cells

STEM-AMI 60 G-CSF 5 mg/kg percutaneous

coronary

intervention

G-CSF

attenuates

ventricular

remodelling in

anterior

STEMI

SITAGRAMI 36 Mobilized

stem cells

G-CSF 10

μg/ kg/d 5

days

+

Sitagliptin

100 mg 28

days

Percutaneous

coronary

administration

Sitagliptin and

G-CSF are

safe and

feasible

after AMI in

short term

23

GAIN I 20 G-CSF +

CD133+

cells

10 μg/kg

G-CSF

+

0.75-

1.0×106

cells

intracoronary

infusion

Significant

rate of

ischemia and

other adverse

effects

MAGIC 120 SM 400-800

x106

Intramyocardial

injection

Therapy failed

to improve

heart function.

Early

postoperative

arrhythmic

events

CAuSMIC 23 SM <600 x106

Endomyocardial

injection

Myoblast

transplantation

in HF is safe,

feasibile.

Improves

ventricular

viability and

remodeling

MARVEL 23 SM 400-800x106

Intramyocardial

injection

Myoblast cells

therapy is

feasible and

has clinical

benefits. VT

may occur.

CADUCEUS 31 CDCs 12,5-25 x106

Intracoronary

infusion

Infusion of

CDCs after MI

is safe. No

change in end-

diastolic

volume, end-

systolic

volume,

LVEF.

SCIPIO 14 CSCs 1x106

Intracoronary

infusion

Autologous

CSCs improve

LV systolic

function,

reduce infarct

size in patients

with HF after

MI

Adapted from Wollert, Drexler, Cell therapy for the treatment of coronary heart disease: a

critical appraisal H. Nat. Rev. Cardiol. 2010:7; 204–215

Stem cells of different origins produce similar results in cardiac cell therapy. Although clinical

trials show an overall safety and good outcomes, adverse events may occur.

24

Embryonic Stem cells

Embryonic stem cells (ESC) are pluripotent stem cell lines that have the potential to give rise to

virtually every cell type in the body. This makes them very useful for tissue regeneration,

although it makes them hard to control96

. These cells are not present in the adult organism, but

in the inner cell mass (ICM) of the developing embryo97

, approximately 5 days after

fertilization98

.

ES cells are easily identifiable due to a higher nuclear/cytoplasmic ratio and distinctive activity

of alkaline phosphatase and expression of a cell surface marker stage specific embryonic

antigen-1 (SSEA-1).20

If ES cells can differentiate into the derivatives of all three embryonic germ layers, this means

that, in theory, ESCs can survive in injured hearts, form stable intracardiac grafts and

differentiate into cardiomyocytes. However, only a small portion of ESCs actually does this. It

is believed that the infarcted cardiac microenvironment cannot selectively promote ESCs

cardiac differentiation. One of the possible explanations is that cytokines and secreted growth

factors in infarcted myocardium may not provide a constructive microenvironment for ESCs to

acquire a cardiac phenotype99

. Directed differentiation can be done by three main approaches,

embrioid body formation, stromal cell co-culture, BMP4 and Activin A supplement, all of

which will be addressed further in this review100

. Transplantation of hESC-derived

cardiomyocytes (CM) after extensive myocardial infarction in rats results in the formation of

stable cardiomyocyte grafts, attenuation of the remodelling process, and functional benefit101

although, the obtained cells mostly have features of fetal rather than adult cardiomyocytes1.

There is evidence that transplantation of allogeneic embryonic stem cells achieves functional

and structural repair in stress-precipitated, nonischemic genetic cardiomyopathy102

. The increase

in systolic function does suggest that the transplanted population induce local contractile force.

One concern, however, is the potential for arrhythmia, particularly given that both nodal and

ventricular cells can be generated from immature cardiomyocytes96

. Besides their ability to

repopulate the infarcted myocardium via neoangiomyogenesis, ESC cells release paracrine

factors which contribute to preserve the function of host cardiac tissue20

. Strategies are also

being developed to prime ES cells prior to transplantation and manipulate their paracrine

behavior20

. Also inhibition of p38MAPK during differentiation of hESC results in accelerated

differentiation of hCM. This enrichment is dependent on inhibiting p38MAPK at the time of

ectoderm–mesoendoderm discrimination. Besides, p38MAPK inhibition does not abolish

pluripotency, and the resulting hCM are genetically stable, and are viable upon in vivo

transplantation into mouse cardiac tissue103

.

Despite their cardiogenic potential, there are problems that hinder clinical application. When

injected into cardiac tissue, hESC-CM can engraft, survive and form grafts with striated

25

cardiomyocytes, at least in an acute myocardial infarction model. However, although hESC-CM

transplantation can slow the progression of heart failure in an AMI, the same hESC-CM

injection protocol is insufficient to restore heart function or improve cardiac remodeling in case

of chronic myocardial infarction104

. But it’s the teratogenicity of ES cells that raises the major

safety concerns20

. The inadvertent introduction of any undifferentiated ESC into host tissue will

result in teratomas100

. A pre-differentiation of hESCs before injection could present a potential

solution to this problem. Although this apparent tumor-free application of pre-differentiated

hESC has been reported in animal models, 105

this does not exclude its potential risk if used in a

human clinical setting98

. The development of teratoma, the substantial ethical and regulatory

concerns in their availability, not to mention the immunological considerations, are the major

limitations of ES cells in their progress to routine clinical application20

. And also most studies

do not show characteristics of fully matured hCM in the injected cells103

. Finally, transplantation

of hESCs into patients is also limited by potential HLA incompatibility. Life-long

immunosuppressive therapy, which can lead to infections and organ-based toxic side effects,

might be required to prevent graft rejection106

.

Induced pluripotent stem cells (iPS)

An exciting new milestone in the field of regenerative medicine was the development of the

induced Pluripotent Stem Cells (iPS)107

. In 2006, it was first reported that differentiated mouse

fibroblasts could be reprogrammed into stem cells, that like ESC, could be propagated

indefinitely11,108

, had the capacity to form all three germ layers (endoderm, mesoderm and

ectoderm)7,108

, and subsequently, were capable of differentiating into adult cells107

. These cells

were termed iPS. Nuclear reprogramming with ectopic stemness factors has opened the

opportunity to generate autologous patient-derived iPS from adult somatic cells108

. The

therapeutic potential of iPSCs is considerable, because they are patient-specific stem cells that

do not face the immunologic barrier, like ESCs. Furthermore, there are plentiful and easily

accessible sources of tissue, such as the donor’s skin, fat, or hair. Thus, immediate advantages

over other progenitors include their immune-privileged status as autologous tissue and their

potential abundance107

.

The generation and use of iPSC is non-controversial, and rise above the ethical concerns and

political barriers faced by ESCs107

.

Induced pluripotent stem cells were first obtained by infecting adult fibroblasts with viral

vectors expressing four transcription factors108

. These factors included Oct 3/4, Sox2, Klf4, and

c-Myc107-109

. Later studies using retrovirus-mediated transduction of four other transcription

factors (Oct3/4, SOX2, Nanog, Lin28) were also capable of establishing human iPS11, 110

. This is

26

because the c-Myc factor (a proto-oncogene) was found to induce tumors in mice and hence was

excluded from the reprogramming pot. However, this seemingly subtle modification rendered

the process more time consuming and less efficient , since c-Myc plays a critical role in the rate

of proliferation of the somatic cells111

.

The fact that gene manipulation is being used increases safety concerns and promotes regulatory

barriers. For example, the use of retroviruses leads to integration of viral DNA into the

chromosome, which raises the risk of silencing indispensable genes or inducing oncogenes107,

112,113. The integration of viral vectors in the genome may promote malignancy

1. Lentiviruses

can be used to produce iPS cells with reduced numbers of viral integrations, and although they

don’t completely eliminate the risks of insertional mutagenesis and viral reactivation, they

significantly reduce them114

.

These concerns are overcome in part with the use of adenoviruses or plasmid constructs, but

even these episomal vectors carry a risk of DNA integration. Accordingly, any iPSCs created

with DNA-based strategies need to be screened carefully to exclude any DNA integration107,

115,116. But there are other ways to create iPS. (Fig2)

Figure 2: Different methods to obtain induced pluripotent stem cells. Original figure.

They have also been derived with transposons1. Transposons are discrete elements of DNA that

have the distinctive ability to move from one chromosomal location to another. DNA

27

transposons that move directly as DNA, are particularly attractive as gene delivery tools117

.

Chemical or physical methods are utilized to introduce new genes or DNA segments into cells.

The introduced DNA usually does not integrate with the chromosomal DNA and hence does not

affect host cell replication115

. One of these transposon methods is the piggyBac transposition

system. It includes a mobile genetic element that can be used to integrate transgenes into host

cell genomes. The piggyBac system is able to deliver large genetic elements. Unlike viral

vectors, this system does not require special storage or quality control conditions. It does not

need to be prepared in high titers and does not have a limited lifetime118

. These strategies offer

certain advantages in that the elements can be silenced or excised, which decreases the

possibility of reactivation107

. Another non-viral method to achieve iPS cells generation goes by

direct delivery of recombinant reprogramming proteins. Unlike the use of genome-integrating

viruses this method does not cause mutagenesis or genetic dysfunction119

. However, this

protein-based approach requires a significant amount of protein for the reprogramming process.

Producing them in the large quantities needed for this approach using traditional heterologous in

vivo production methods, is difficult111

. However these methods, which rely on repeated

administration of transient vectors, have shown so far very low iPS derivation efficiencies,

probably because of weak or inconstant expression of reprogramming factors1.

Nonviral methodologies that may overcome these concerns include the administration of

synthetic messenger RNAs. This is one of the most recent advances in iPS technology.

By repeated administration of mRNAs carrying specific modifications, together with a soluble

interferon inhibitor to overcome innate antiviral response, human somatic cells can be

reprogrammed to iPS with efficiency and kinetics superior to other established protocols.

Moreover, since this technology is RNA based, it completely eliminates the risk of genomic

integration and insertional mutagenesis inherent to all DNA-based methodologies, including

those that are ostensibly nonintegrating. With this approach one can avoid the random variation

of expression, typical of integrating vectors, as well as the uncontrollable effects of viral

silencing. RNA reprogramming may produce higher-quality iPSCs, possibly owing to the fact

that they are transgene free, thus potentially leading to further enhancements of the efficiency

and kinetics of reprogramming1, 120

.

Various growth factors and chemical compounds have recently been found to improve the

induction efficiency of iPS cells.

Inhibition of TGF-β signaling helps in the reprogramming of mice fibroblasts by enabling

faster, more efficient induction of iPSCs, whereas activation of TGF-β signaling blocks the

reprogramming. In other words, the use of a TGF-β receptor I kinase or an activin-like kinase 5

(Alk5) inhibitor could enhance the efficiency of iPSC derivation121

.

28

Vitamin C (Vc), a common nutrient vital to human health, enhances the reprogramming of

somatic cells to pluripotent stem cells. By adding Vc to the culture medium, iPSCs from mouse

and human cells can be better obtained. It is possible that Vc allows this by facilitating histone

demethylation122

. It has been determined that p53 small interfering RNA (siRNA) and UTF1 are

able to increase the generation efficiency of fully reprogrammed iPSCs from fibroblasts.

Silencing p53 expression could promote the immortalization of fibroblasts. p53 siRNA may

function as an antagonist of cellular apoptosis and consequently contribute to the

reprogramming process. UTF1, on the other hand, may favor the switch from differentiated to

pluripotent through the establishment of an epigenetic profile or specific chromatin state likely

to appropriate cell fate stimuli123

.

The loss of p53 may play several roles in facilitating the reprogramming process to form iPSCs.

This is because p53 constitutes a main barrier to the reprogramming of differentiated cell

types124

. Studies have shown that p53 suppresses iPS cell generation. It is due to its suppressive

effects on cell proliferation, survival, or plating efficiency. In addition, they may have direct

effects on reprogramming. So, transient suppression of p53 by siRNA or other methods may be

useful in generating iPS cells for future medical applications124-126

. Also it has been studied that

hypoxic conditions can improve the efficiency of iPS cell generation from mouse and human

somatic cells. In fact, cell cultivation under 5% O2 favors more efficient iPSC generation127

.

iPS have a great therapeutic potential. Since they can be established from human adult tissues,

they can avoid legal and ethical problems, they can differentiate into functional

cardiomyocytes128

and are now one of the most promising cell sources for cardiac regenerative

therapy.

Studies suggest that iPS cells, when inserted into infarcted heart, lose their pluripotency and

engraft into native myocardium and differentiate into cardiac myocytes. Improved cardiac

function is also observed129

.

Another study demonstrated that iPS derived progenitor cells differentiated into a

cardiomyocyte phenotype and developed contracting areas in mice heart tissue. Despite the lack

of well-aligned mature donor cardiomyocytes, beneficial remodelling and improved ventricular

function were observed. Positive effects can be explained by the formation of larger grafts and

enhanced neovascularization130

. Regarding this, another study reported that iPS cell

transplantation in mice resulted in an increase in VSM and ECs in the infarcted heart, and a

significantly improved cardiac function. Also it showed that iPS can differentiate into vascular

smooth muscle cells and endothelial cells in vitro, and enhance neovascularization131

.

An important safety concern holding back the clinical application of iPS, is their great

heterogeneity in terms of plasticity and epigenetic landscape1. A study demonstrates that,

allogeneic iPSC transplantation into the heart can cause in situ tumorigenesis in

29

immunocompetent recipients, and that cells leaking from the beating heart, will likely serve as a

source of tumor spread132

.

Besides, the heterogeneity of the cardiac cells produced from pluripotent iPSCs is likely to

cause arrhythmias111

.

Another limitation observed with the in vitro differentiation of iPSCs, is that the myocytes, even

after 2 months of standard two-dimensional tissue culture conditions, remain embryonic in

phenotype based on their size, organization and electrical properties111

. These cardiomyocytes

exhibit immaturity of the sarcoplasmic reticulum, and a β-adrenergic response that is

significantly different from native ventricular tissue of a comparable age133

. So, in addition to

safety concerns, there are manufacturing hurdles to overcome for therapeutic application.

Differentiation of pluripotent stem cells into cardiomyocytes:

Despite having obtained some positive results with BMSCs, MSCs and even with CSCs therapy

after AMI (like increased LVEF and improvement of quality of life over a short term), mid- and

long-term results are still unsatisfactory36

. Especially when it comes to patient survival rate and

the long-term safety profiles regarding this kind of therapy. Transplantation of ADSCs or iPS

seems very promising, but lacks a sturdy validation from clinical trials. Many outcomes have

fallen below expectations due to several obstacles that have yet to be overcome. One of the

major issues in cell therapy is that most of the stem cells do not differentiate completely into

fully mature, electrically-coupled cardiomyocytes. In fact, they resemble more fetal

cardiomyocytes, with impaired contractile properties and immature intercellular

communications, lacking an adequate vascularization and present in insufficient numbers. And

that is when they go down the cardiac lineage in first place. This means that most of the

transplanted cells do not engraft properly and eventually die out. To prevent this, differentiation

processes must be improved. The following text explores the differentiation steps required to go

from pluripotent stem cells to functioning adult cardiomyocytes. The current methods used to

achieve these mature cells and the various factors that influence the process and their isolation

are also discussed.

30

Figure 3: Differentiation approaches used for cardiomyocyte differentiation from pluripotent

stem cells. Original figure.

The efficiency of cardiomyocyte differentiation is poor and the differentiated cells are usually a

heterogeneous mixture of various types of cells11

. The main problem associated with the use of

pluripotent stem cells is the necessity to obtain purified populations of differentiated or

committed cells, in order to avoid the risk of teratomas1. In order to find a way to obtain purified

populations of differentiated cells, it is necessary to understand the necessary steps of stem cell

differentiation into heart cells. Four main steps are required to generate cardiomyocytes from

pluripotent stem cells: (a) formation of mesoderm, (b) the patterning of mesoderm toward

anterior mesoderm or cardiogenic mesoderm, (c) formation of cardiac mesoderm, and (d)

maturation of early cardiomyocytes134

.

Regarding the first step, studies show that activation of both the Wnt and nodal (TGF-β)

signaling pathways are required together to efficiently induce mesoderm in stem cell

differentiation cultures135

.

For cardiogenic mesoderm, studies report that Mesp1 induces this transition in differentiating

ESCs and promotes development of mesoderm precursors of the cardiovascular lineage.

Transient expression of Mesp1 is sufficient to allow development of a restricted set of

31

cardiovascular mesoderm, including endothelial cells, smooth muscle cells, and

cardiomyocytes, as well as repressing the expression of key genes regulating other early

mesoderm derivatives136

.

Regarding cardiac mesoderm, there have been reports of Gata5 and Smarcd3b promoting the

development of CP cells in vivo137.

The last step in the differentiation stages to cardiomyocytes is the differentiation of committed

cardiac progenitors to beating cardiomyocytes, a process that can be controlled by factors such

as Wnt11138, 139

. Transduction of Wnt11 into MSCs increases their differentiation into CMs by

upregulating GATA-4140. Maturing cardiomyocytes can be identified by the expression of

cardiac structural proteins such as α-actin, α-myosin heavy chain (α-MHC), BNP, islet-1140

or

the cardiac isoform of Troponin-T (cTnT).

Currently, cardiomyocytes can be differentiated from pluripotent stem cells either by

spontaneous embryoid body (EB) differentiation in suspension, coculture with mouse

endoderm-like cells (END-2 cells), or guiding the cardiac differentiation with defined growth

factors either in suspension or in monolayer culture138

.

Embryoid body

The differentiation properties of iPS and ESC are almost completely identical141

. hESCs or iPS

can mature into spontaneously contracting cardiomyocyte-like cells if cultured in suspension.

They form three-dimensional aggregates called embryoid bodies. Within the embryoid body,

derivatives of the three germ layers (ectoderm, endoderm, and mesoderm) develop

spontaneously142

. Within these mixed population of cells contracting areas with functional

properties of cardiomyocytes can be detected138

. Though the EB method is convenient for

inducing differentiation, it possesses several weak-points, such as difficulty to dissect the

differentiation mechanisms, difficulty to directly observe differentiating cells by microscopy,

and difficulty to conduct single cell analysis of differentiation143

. Cardiomyocyte induction in

the embryoid body-based differentiation system is also quite variable, partly due to the

heterogeneity among the aggregates138

. However, the embryoid body formation in suspension

cultures remains widely applied method to induce cardiomyocyte differentiation largely due to

its simple and inexpensive nature138

.

Coculture of pluripotent stem cells

Another method is based on coculture of pluripotent stem cells with a visceral endoderm-like

cell line (END-2)138

. When seeded on visceral endoderm-like (END-2) cells, hESC and iPSC

form rhythmically contracting areas within a few days following initiation of co-culture144

.

The cardiac differentiation efficiency of this method can be enhanced in the absence of serum

and with ascorbic acid138

. Also selective p38MAPK inhibitors were found to significantly

32

contribute to the generation of hESC-derived cardiomyocytes in a dose-dependent manner when

used with an END-2 conditioned medium145

.

However, the cardiac differentiation efficiency from standard END-2 co-culture experiments is

usually fairly low138

.

In other studies, co-culture of MSCs and CMs also enhanced the cardiomyogenic differentiation

of MSCs146

.

Ggrowth factors

The signaling pathways regulating the cardiogenesis can be recapitulated in cell culture by the

addition of specific growth factors138

.

A part of the cardioinductive activity of anterior endoderm is mediated by growth factors

belonging to the TGF-β superfamily, and at least two TGF-β family members improve the

efficiency of the directed differentiation of ES/iPS cells into cardiomyocytes: Activin A and

BMP411, 147

.

Another important factor is Wnt. Transduction of Wnt11 into MSCs increases their expression

of cardiac markers and promotes their differentiation into cardiac phenotypes. Loss-of-function

experiments suggest that these effects are associated with upregulation of GATA-4140

. It has

also been determined that Wnt/β-catenin signaling modulates activin A/BMP4-mediated cardiac

differentiation of hES cells. During early stages of differentiation, prior to gastrulation, addition

of exogenous Wnt3a enhances cardiac differentiation, while inhibition of Wnt factors with Dkk

decreases cardiogenesis. This indicates that the ability of activin A/BMP4 to induce cardiac

differentiation depends on endogenous Wnt signaling. On the other hand, addition of Dkk1 at

later stages of differentiation, after gastrulation, actually increases cardiogenesis, showing that

Wnt/b-catenin signaling has a biphasic effect in hES cells139, 148

.

Also using Wnt activators (Wnt8) and Wnt repressors (Dkk1), demonstrates that if Wnt

activation occurs prior to gastrulation, the amount of lateral mesoderm tissue is increased, which

subsequently increases the number of cardiac progenitors (determined by Nkx2.5 expression).

If, however, Wnt overexpression is induced after gastrulation, cardiac progenitor numbers are

reduced96

.

BMP2 acts in a combinatorial manner with Wnt3a to drive pluripotent stem cells toward an

early mesodermal and cardiogenic fate in vitro149

.

None of the currently available protocols results in homogenous populations of cardiomyocytes.

One of the challenges over the last years has been to develop robust isolation techniques that

allow scalable purification of cardiomyocytes and specific cardiac subtypes. Although it is not

the objective of this review to discuss or compare the different types of isolation techniques, a

brief reference will be made to the most recent ones.

33

There have been some approaches developed, like mechanical isolation or Percoll

centrifugation. But the highest levels of cardiac purity have been obtained using genetic

selection techniques. Undifferentiated pluripotent stem cells are genetically modified to carry

either a reporter gene, usually a green fluorescent protein (EGFP) or mammalian selection gene

under the transcriptional control of a cardiac-specific promoter138

.

These techniques are not without some disadvantages, there for it is necessary to continue

research and develop additional more effective strategies.

Other factors and molecules:

Cyclosporin-A (CsA) can induce cardiogenic differentiation in mouse ESCs, iPSCs and human

iPSCs. But the molecular mechanisms conducting this effect are still unknown.

CsA-expanded cardiomyocytes from human iPSCs exhibit many features of functional

cardiomyocytes. Nevertheless, they are still immature and display some structural features of

fetal cardiomyocytes, such as relatively low global electron density, sparse myofibrils, and

abundant ribosome granules150

.

Exogenous expression of apoA-I promotes cardiac differentiation and enables maturation of

calcium handling properties of pluripotent stem cell-derived cardiomyocytes. ApoA-I over-

expression promotes cardiac differentiation of pluripotent stem cells, it increases cardiac

specific gene expression during ESC differentiation, and enhances the expression of calcium

handling proteins (RyR2, NCX-1 and SERCA-2a) with a corresponding maturation of calcium

handling properties in ESC-derived cardiomyocytes. ApoA-I-induced cardiac differentiation is

dependent on a BMP4-SMAD1/5 signaling cascade. The cardiogenic effects of apoAI are also

observed in human iPSCs151

.

Activation of the HIF-1 pathway promotes cardiac differentiation and maturation of cultured

ECSs at atmospheric oxygen levels. Although physical hypoxia can cause a dramatic increase in

HIF-1α protein levels, such drastic treatment is not beneficial to cardiac differentiation. The

expression of exogenous HIF-1α alone is sufficient to promote the cardiac differentiation of the

ESCs. This increase in cardiac differentiation is due to upregulation of the early cardiac

transcription factors (GATA4, GATA6 and Nkx2.5) and the cardiogenic factors (VEGF and

CT1) by HIF-1α. In addition to cardiac differentiation, increased HIF-1α expression also drives

the maturation of the ESC-derived cardiomyocytes, improving Ca2+

handling and sarcoplasmic

reticulum function152

.

Gata5 and Smarcd3b can promote the development of CPC-like cells in vivo. Gata5 alone has

pro-myocardial activity, which is stimulated by endogenous Smarcd3b137

.

Activation of PKC-delta induces the expression of multiple cardiomyogenic genes in ADSCs.

Although transient activation of this PKC isoform alone seems to be insufficient to generate

34

well-differentiated cardiomyocyte-like cells, the overexpression of cDNA for PKC-delta results

in marked increases in cardiac mRNA expression153

.

TGF-β1 promotes ADSC cardiomyogenic differentiation in vitro. ADSCs treated with only

TGF-ß for 1 or 2 week showed enhanced expression of cardiac MHC, troponin I, and a-

sarcomeric actin without any multiple lineage differentiation146

.

Icariin also facilitates the differentiation of ADSCs into CMs. It seems that Icariin markedly

enhances mRNA levels of GATA-4 and NKX-2.5 (markers of cardiac differentiation) during

the early stage of differentiation. Also it seems that the extracellular signal-regulated kinase

(ERK) pathway is activated and involved in ICA promoted cardiac differentiation. This means

that CM differentiation can be at least partially inhibited by an ERK inhibitor154

.

Thyroid hormones have a positive effect on cardiac differentiation of ESCs. T3-treated ESCs

show an increase in differentiation into cardiomyocytes, and increases gene expression of

cardiogenesis (Nkx2.5) and myofibrillogenesis (MLC-2v, α- and β-MHC). T3 enhances

cardiogenesis of ESCs through the classical genomic pathway but not the Akt- and MAPK-

signaling cascades. T3 supplementation leads to a more mature cardiac phenotype and also

enhances the expression of calcium-handling proteins (RyR2 and SERCA-2a) with

corresponding maturation of calcium-handling properties in ESC-derived cardiomyocytes155

.

Cultured mesoangioblast-like cells and single cell-derived colonies also express pluripotency

genes. The transduction with Sox2 enhanced the differentiation capacity of circulating

mesangyoblasts to the cardiovascular lineages and significantly improved the therapeutic

potential compared to control cMABs in the myocardial infarction model156

.

Spontaneous calcium oscillations

Changes of calcium levels in CPCs occur and trigger a cascade of events by which CPCs divide

and acquire the myocyte phenotype.

Normally, hCPCs display spontaneous elevations in intracellular Ca2+

attributable to IP3R-

mediated Ca2+

release from the ER. The most significant regulator of this mechanism is Ca2+

itself. The probability of IP3R open the channel is stimulated at low Ca2+

concentrations,

whereas high concentrations exert an inhibitor effect.

Reuptake of Ca2+

into the ER is done by SERCA, which replenishes the Ca2+

stores, allowing

repetitive oscillations with preserved amplitude and duration157

. Although the Na+/Ca

2+

exchanger, plasma membrane Ca2+

pump, and store operated channels are functional and

contribute to Ca2+

homeostasis in hCPCs, they are not implicated in the initiation or incidence of

Ca2+

oscillations in these undifferentiated cells. Only later in the differentiation stage does the

exchanger molecule regulate the calcium transients158

. This means that neither cell-to-cell

communication or the interstitial matrix are implicated in the rapid and transient elevations of

35

Ca2+

, and that Ca2+

cycling in myocytes appears to have no influence regarding this ions

oscillations in CPCs.

However, agonists of Gq protein–coupled receptors, histamine and ATP stimulate PLC and IP3

formation, leading to an increase in the number of activated hCPCs and frequency of Ca2+

oscillations in vitro. These Ca2+

oscillations promote hCPC proliferation, therefore showing that

cytosolic calcium plays a primary role in hCPC growth157

. ATP-mediated CPC growth may be

critical for cardiac repair, and it is possible that ATP and histamine may complement each other

in the modulation of hCPC function.

Induction of Ca2+

oscillatory events in hCPCs before their intramyocardial delivery in vivo

enhances engraftment of these cells within the infarcted heart, their expansion in the harsh

environment of the necrotic tissue, and the generation of a differentiated myocyte progeny157

.

Micro-RNAs

Myocyte dropout by wear and tear, aging, or cardiotoxicity is readily recovered by activation of

CSCs. This phenomenon occurs with aging, but is no longer effective after severe myocardial

injury. In fact, CSCs delivered to the infarcted heart generate a large number of small fetal-

neonatal cardiomyocytes that fail to acquire the differentiated phenotype. But the interaction of

CSCs with postmitotic myocytes results in cells with adult characteristics. This occurrence can

be explained by the influence of micro-RNAs159

. MicroRNAs control gene expression160

. They

are small non-coding RNAs that play important gene-regulatory roles, like controlling

translation of messenger RNA (mRNA) by induction of mRNA degradation or blockade of

translation161

.

They have the ability to transverse gap junctions and so may migrate from cardiomyocytes to

CSCs dictating their destiny159

.

In the nucleus, the primary transcript (pri-miRNA) is primed through the activity of an enzyme

called Drosha. This results in a pre-miRNA, usually 60-100 nucleotides long. The pre-miRNA

is transported from the nucleus to the cytoplasm by exportin-5. In the cytoplasm, pre-miRNA is

further trimmed by an enzyme Dicer into a 22- nucleotide miRNA. The two strands of the

newly formed miRNA are then separated. One strand is loaded into the RISC complex (RNA-

induced silencing complex) 162

, there by regulating their expression by transitional repression or

mRNA degradation163

.

miRNAs can be expressed in a variety of cell types160

. In this review the focus remains in those

miRNA associated with cardiomyocyte differentiation. One example in particular is miR-499.

The commitment of hCSCs to the myocyte lineage and the generation of functionally competent

adult cardiomyocytes are influenced by miR-499, which is barely detectable in primitive cells

but is highly expressed in postmitotic human cardiomyocytes159

. miR-499 is expressed in

36

differentiated hCMPCs and, together with its gene MYH7β, is strongly enriched in cardiac

tissue163

. In short, the miR-499 enhances the hypertrophic response of hCSC-derived

cardiomyocytes, promoting a more effective functional and structural recovery of the damaged

heart. Sox6 and Rod1 are factors that govern hCSC growth, and appear to be regulated by

junctional coupling and the translocation of miR-499 from postmitotic myocytes159

. Sox6 is a

transcription factor implicated in early myocyte commitment in embryos163

, and Rod1 is an

RNA binding protein that negatively modulates cell differentiation. Moreover, knockdown of

Sox6 induces cardiogenic differentiation of hCMPCs, confirming the role of Sox6 in muscle

differentiation. Induction of miR-499 represses the expression of Sox6 in hCMPCs, leading to a

reduction in cell proliferation and enhanced myocyte differentiation. Thus, when cells are

committed to the cardiac lineage and start to express MYH7Bβ, miR-499 is coexpressed,

thereby repressing Sox6 to further induce differentiation and modulate fiber expression in

developing cardiomyocytes163

. In conclusion, the miR-499 expression and the Sox6 and Rod 1

inhibition enhance the differentiation of the heterogeneous population of fetal cardiac human

cells159

.

There is also miR-1, which is a highly conserved miRNA with a cardiac and skeletal muscle–

specific expression pattern, able to bind to the promoters of several essential cardiac

transcription factors, such as Mef2, SRF, Nkx2.5, and GATA4. miR-1 contributes to the balance

between cardiomyocyte proliferation and differentiation, thereby repressing cardiac progenitor

cell proliferation. A prerequisite for differentiation is inhibition of proliferation, and

introduction of miR-1 into human cardiac-derived CMPCs results in a reduction in proliferation

rate163

.

MiR-133 overexpression in either hESC or mESC inhibits expression of cardiac genes and

stimulates myoblast proliferation by repression of SRF and cyclin D2. It is thought that miR-

133 acts prior to miR-1 to enhance differentiation toward a mesoderm and myocyte lineage, but

then miR-1 is required to continue differentiation toward the cardiomyocyte cell lineage164

.

Interestingly, deletion of miR-208b and miR-499 lacks an overt cardiac phenotype, suggesting

that exists a significant degree of redundancy among myogenic miRs159

.

miR-21, together with miR-1, miR-133a and miR-133b, is a good candidate to elucidate the

maturation phase of cardiac-specific differentiation. miR-145 on the other hand, is not165

.

Neovascularization and vascular regeneration capacity are some of the most important factors in

host tissue that determine the administered stem cells fate. They influence proliferation,

differentiation and the establishment of effective connections with the host neighboring cells.

This makes the role of endothelial progenitor cells, a crucial one.

37

Vascular regeneration

Vascular regeneration, by definition, includes the restoration of normal vascular function and

structure, the reversal of vascular biological aging, and the growth of new blood vessels.

Therapeutic applications of vascular regeneration for coronary or peripheral arterial diseases are

directed to three major goals: (a) relieving symptoms of ischemia, (b) preventing target-organ

damage due to hypoxia, reperfusion, or capillary leak, and (c) avoiding cardiovascular

complications due to acute thrombosis, embolism, plaque rupture, or dissection107

.

The type of cells most used in clinical development of vascular regeneration is adult stem cells.

In the heart, vascular progenitor cells (VPCs) are located in the proepicardium from where they

migrate into the myocardium and differentiate into endothelial cells (ECs) and smooth muscle

cells (SMCs) organized in coronary vessels, but they can also be found in vascular niches,

composed of clusters of cells expressing c-kit and KDR (kinase insert domain receptor), in

epicardial coronary arteries, arterioles, and capillaries. These c-kit-KDR-positive cells are

located in the intima, media and adventitia, and connexin 43 and N-cadherin can be detected at

the interface with ECs and SMCs166

.

Even so, there are no surface markers that clearly distinguish early endothelial progenitors. In

addition, methods for harvesting, purifying, and culturing these progenitor cells are still in

development. This means that the EPCs used are, in fact, a mixed population of progenitor cells

of different lineages. Within this population of cells, there are true endothelial progenitors that

can incorporate into the vascular network, and hematopoietic progenitors that may contribute by

secreting angiogenic cytokines and matrix metalloproteinases107

.

But regarding smooth muscle, both smooth muscle myosin heavy chain (SM-MHC) and

smoothelin are currently recognized as the best markers to define the phenotype of mature

contractile vascular smooth muscle cells167

.

Although adult stem cells bring many advantages to vascular regeneration, they are not without

problems. They are an autologous source of stem cells, but this means that there is a delay in

treatment due to the time needed to collect the cells, isolate them and then propagate progenitors

ex vivo, to obtain adequate numbers before injection. Adverse effects of their delivery could

include microvascular embolism, pathological neovascularization. Besides, in patients who

most need EPC therapy, these cells are rare, have limited replicative capacity, and are often

dysfunctional. In older individuals, for example107

.

Also, hESC-derived endothelium are able to differentiate into functional vascular beds by

treating EBs with VEGF96

. In fact, hESC-derived endothelial cells can survive transplantation

and incorporate into existing vasculature96

. Also ES-CM stimulates resident c-kit+ cardiac

progenitor cells (CPCs) and circulating FLK-1 cells from the storage site to the injury and their

differentiation into ECs168

.

38

iPSCs have been shown to differentiate into each of the major cardiovascular components,

including smooth muscle cells, ECs169

, vascular mural cells, and cardiomyocytes. iPSC derived

ECs (iPSC-ECs) can be allogenic or autologous. Allogeneic iPSCs may be of some utility since

they do not induce formation of teratoma, and maybe immune mechanisms might be modulated

to reduce the concerns of teratoma formation during therapy with autologous iPSC derived

cells. With autologous iPSC-derived cells, there is also the concern that genetic or acquired

abnormalities that predisposed a patient to a particular disease will be recapitulated in their

iPSCs. In such a case, the patient-derived iPSCs may be dysfunctional. A dysfunctional iPSC-

EC graft could potentially contribute to vascular inflammation by manifesting endothelial

adhesion molecules, chemokines, and prothrombotic factors. iPSC-EC also may promote tumor

angiogenesis, pathological retinopathy, or neovascularization and progression of atheromatous

plaque107

. Nonetheless, studies have demonstrated that it is possible to induce and isolate human

vascular cells from iPS cells. It seems to indicate that the properties of differentiation are nearly

identical to those of hES cells169

. Increases in VSM and ECs in the infarcted heart following iPS

cell transplantation have been reported in animals. Furthermore, there is an increase in coronary

artery vessels, suggesting neovascularization131

.

Studies in BMSCs suggest that combined patching and cell transplantation is suitable for

angiogenesis and arteriogenesis, but it does not produce better results than simple cell injection

into the myocardium170

.

A clinical trial demonstrates that the cell therapy using intramuscular implantation of BMSCs

leads to the extension of amputation-free interval and improvement in the ischemic pain, ulcer

size, and pain-free walking distance. The safety and efficacy are not inferior to the conventional

revascularization therapies171

.

There are several growth factors that play an essential role in vascular cell differentiation. The

NOXs-ROS axis system plays an important role in EC and VSMC differentiation from

stem/progenitor cells, as well as in EC proliferation, migration and apoptosis167

. Studies

determined the presence of anti-apoptotic and pro-angiogenic factors in ES-CM. Data suggests

that ES-CM contains increased amount of anti-apoptotic (TAC and IGF-1), and pro-angiogenic

factors (IGF-1, HGF, and VEGF). So, these released factors provide beneficial effects by

enhancing neovascularization and inhibiting apoptosis168

.

A further understanding of the function and contribution of these cells is necessary to harvest

their full potential. Indeed, several of these vascular stem cells have only just been described,

and our overall knowledge is yet limited.

39

Bioartificial Heart

Cardiovascular cell therapy is performed with a population of stem or progenitor cells that can

differentiate into multiple lineages. The use of exogenous stem cells has had some encouraging

results, but these were not demonstrated after longer follow-up times172

. Also, injection therapy

techniques of these cells into the cardiac tissue have shown some limitations. Ideally, such a

population of cells would survive in injured myocardium, give rise to new mature

cardiomyocytes and vasculature and integrate with surrounding host tissue173

. This scenario is

feasible, although it is rare. Mostly due to poor cell survival, the lack of cell integration and

poor differentiation of stem cells174

. Potential causes for this poor cell survival are ischemia and

the occurrence of apoptosis at the injection site, due to poor vascularization172

. And even when

successfully injected into injured or infarcted heart, stem cells can develop into fibroblasts,

cartilage, and fat, or in the case of embryonic stem or iPS cells, even into benign tumors or

teratomas173

. Integration and coupling of the newly formed heart cells are also critical to achieve

a successful result, since it is required that the contractile tissue will beat synchronously with

the remaining myocardium172

.

But recent advances in stem cell (SC) biology have increased the likelihood that personalized

cardiac tissue can be generated in the laboratory. First is the isolation of SCs from adult heart

that can originate beating cardiocyte- like tissues, and vascular progenitors173

. Second is the

ability to generate adult-derived pluripotent SCs that can differentiate towards the cardiac

lineage. And third, the engineering of cardiac tissues175

. Cardiac tissue engineering has strongly

evolved over the past decade19

. The idea of cardiac bioengineering has primarily involved

seeding an artificial scaffold (e.g. hydroxyapatite, collagen, and fibrin) with cells and cultivating

it in the laboratory to be used as a cardiac patch. In principle, such a patch could be applied to

the scar, lessening it’s expansion and slowing progression to HF173

. These scaffolds function as

a carrier platform, for in vivo cell delivery172

.

Numerous approaches have been taken to engineer simpler cardiac tissues such as acellular or

simple cell-based patches173

. Several methods have been studied, including biodegradable

scaffolds, supporting matrices, and cell sheets. Ideally, cardiac constructs should contain

contractile properties that respond to adrenergic stimulation, improve the function of the

damaged host tissue, survive transplantation and integrate through extensive vascular networks,

be of suitable scale and engineered from clinically applicable cell types19

. In order to be

clinically useful, an engineered cardiac tissue must mimic the functional and morphological

properties of the native myocardium and remain viable after implantation (Figure 4). One of the

major concerns is oxygen diffusion, 175

since the creation of cardiac patches even beyond a few

40

hundred microns in depth (>200 µm) has been limited by an inability to create the geometry

necessary to support the high oxygen and energy demands of cardiomyocytes12, 175

.

The fact is that cardiac muscle is very hypoxia-sensitive, such that cardiomyocytes must be

intricately coupled with patent vasculature to survive and contract appropriately173

. Researchers

have tried combinations of channeled synthetic extracellular matrix (ECM) constructs with

oxygen carriers, or stacking single layer cardiac cell sheets, to engineer thick and compact

scaffolds173, 175

. Also, cardiac muscle is a contractile tissue with specific structural and energetic

specifications. Cardiomyocytes demand specific spatial orientation as well as functional

coupling of cells and matrix both electrically and mechanically. These cells are very sensitive to

their microenvironment, which is the extracellular matrix, composed mainly of collagen type I

and basement membrane proteins172, 173

. The advantage of synthetic scaffold materials lies in

their controlled, well-characterized composition, degradation and physical properties176

. There

have been reports of grafted myocardial cell sheets which functionally integrated and where

able to contract simultaneously with the host heart. Meaning there was a bidirectional electrical

communication between the host heart and grafted cell sheet177

. It has also been demonstrated

that other engineered heart tissues, by integrating and electrically coupling to host myocardium,

can exert beneficial effects on systolic and diastolic ventricular function178

.

However, engineering cardiac tissues with a thickness on the order of human heart remains a

challenge and is unlikely to occur in the absence of concomitantly engineered vascular beds. To

maximize tissue thickness, it is possible to use a vascularized tissue-engineering chamber,

which allows the generation of a spontaneously beating 3-dimensional mass of cardiac tissue179

.

So, to summarize, generating such a 3D tissue requires: firstly, a geometrically and spatially

appropriate scaffold; secondly, vascularization for tissue perfusion; thirdly, the availability of

cells that can give rise to parenchymal and vascular components; fourthly, an ability to tune the

microenvironment to alter cell physiology and function; and fifthly, a capacity for driving

tissue/organ maturation in vitro.

Figure 4: Application of a biological scaffold on an infarcted heart.

Adapted from Stubbs et al. Toward Clinical Application of Stem Cells for

Cardiac Regeneration, Heart, Lung and Circulation 2011;20:173–179

41

But the very cardiac extracellular matrix contains a complex vascular network capable of

meeting the demands of the working heart173

. In general, it can be expected that ECM would

consist primarily of collagen, glycosaminoglycans, fibronectin,( which induces cell growth,

migration, cytoskeletal organization and promotes endothelial differentiation)174

laminin,180

(which stimulates cardiac differentiation through cell adhesion motifs)174

, and possess a diverse

variety of growth factors181

. Tissue engineering concepts that involve native ECM components

appear very promising due to their strong biocompatibility, preserved bioactivity and their

porosity which is very important for cell seeding, nutriment diffusion, and angiogenesis172, 182

. It

has been reported that human ESCs cultured in a special hydrogel of 75% cardiac ECM and

25% collagen can produce remarkable results regarding organization of cardiac proteins and

contractile behavior. Furthermore, the differentiation and maturation of cardiac progenitors can

be enhanced without the use of supplemental growth factors174

.

So, by developing a perfusion method to remove all cellular constituents from cadaveric tissue

while retaining acellular vascular networks throughout the remaining ECM, which can be re-

lined with functional endothelial cells, could create a feasible alternative to cell sheets and

patches173

. Decellularization of allogeneic heart, provides an acellular naturally occurring three-

dimensional biologic scaffold material that subsequently can be seeded with selected progenitor

cell populations181

. In most regenerative-medicine applications, the decision of an autologous

versus allogeneic cell source is based on criteria such as: (a) the number of required cells and

the timeframe in which they are needed, (b) the ease of cell harvest and expansion, and (c) the

ability to differentiate the needed cell types in vitro. (Table 3)

42

Table 3: Autologous cells vs Allogenic cells. Comparing characteristics and potential uses.

Cell Autologous Allogeneic

Therapeutic potential Non-emergent situation Acute injury

Origin Self-derived Different individuals

Harvest method Not easily be harvested Harvested in larger quantities,

possibly from younger and

healthier individuals; Can be

maintained or expanded in

advance

Time of preparation Usually weeks to prepare.

Less time to produce, less

expensive.

Advantages Less likely to be rejected or

invoke an adverse immune

response, decrease the need

for harsh immunosuppressive

antirejection drug regimens,

avoid the increased risk of

infection and cancer.

They can be grown in large

numbers, and can be readily

modified in vitro

Disadvantages The harvested cell numbers

are often insufficient to be

useful in a nascent tissue.

Increased risk of adventitial

agent transmission, potential

adverse immune reactions,

and potential for widespread

patient involvement should a

product be contaminated.

A decellularized heart would have an advantage over other scaffold types due to its preserved

spatial array of matrix components180

.

By using a detergent-based perfusion decellularization, it is possible to obtain a 3D scaffold

comprising native cardiac ECM in the original four-chambered geometry and fine architecture,

retaining both gross structural and biochemical properties of the native heart173

. Then, because

the major vascular conduits should remain in place, the next step is to recellularize the

decellularized myocardial matrix. In time, the recellularized LV wall should be capable of

contractile force with reasonable synchronicity183

. In a nutshell, the acellular native ECM can

provide perfusable vascular bed and a blueprint for ventricular geometry and heart valves,

which can transform tissue contraction into actual pump function176

.

In a recent report, rat hearts were decellulerized by coronary perfusion with detergents,

preserving the underlying extracellular matrix, and producing an acellular, perfusable vascular

architecture, competent acellular valves and intact chamber geometry. These constructs were

then reseeded with cardiac or endothelial cells. Antegrade coronary sodium dodecyl sulfate

(SDS)181

perfusion over 12 h gave better results than did polyethylene glycol, Triton-X100 or

enzyme-based protocols for full removal of cellular constituents12

, although combinations of

these various approaches for decellularization can be used to maximize the efficiency of the

process180,181

. Histological evaluation revealed no remaining nuclei or contractile elements and

43

DNA content decreased to less than 4% of that in cadaveric heart. Glycosaminoglycan content

was unchanged12

. Collagens I and III, laminin and fibronectin remained within the thinned,

decellularized heart matrix. The fiber composition and orientation of the myocardial ECM were

preserved, whereas cardiac cells were removed12

. Recellularization of this matrix was performed

by injection of neonatal cardiac derived cells into the matrix in a bioreactor. The construct

matured over time, and by day 8, it showed reasonable contractions of the recellularized LV

segments and drug responsiveness12, 183

.

However, the mechanisms by which naturally occurring scaffold materials promote functional-

tissue reconstruction are not yet fully understood181

. A report showed it is possible for either

hESC or hMSCs, when cultured in a decellularized heart, to differentiate into cells expressing

cardiac markers or cells expressing endothelial markers. This differentiation didn’t appear to be

random, since endothelial differentiation of either cell type appeared limited to the original

vascular tubes180

.

The decellularized rat hearts were placed in a bioreactor and seeded with freshly isolated

neonatal cardiac cells through intramural injection. By adjusting sterile organ-culture conditions

it was possible to provide a simulated systolic and diastolic medium flow through pulsatile

antegrade left heart perfusion and a circuit of coronary flow through a left atrial cannula.

Pulsatile left ventricular distension was gradually increased by adjusting the preload and

afterload. Electrical stimulation was provided through epicardial leads. This perfused organ

culture was maintained for 8–28 days. Towards the end, spontaneous depolarizations were

recorded and some relative pump function could be seen12

. This decellularization techonology

can also be applied to blood vessels. Human tissue engineered vessels may provide an

alternative option for small diameter vascular grafts184

.

There have been recent reports on decellularization methods of porcine hearts to produce 3-

dimensional scaffolds. There are many decellularization agents that can be used to obtain a heart

construct185

, and although the SDS is usually preferred185-187

, it is likely that a combination of

several agents may produce the most efficient results. In fact, it was reported that a technique

utilized retrograde aortic perfusion with successive hypertonic, hypotonic, enzymatic, acid, and

detergent solutions to maximize the distribution of chemicals throughout the tissue, maximize

the disruption of cells, and minimize the damage to the ECM. The protocol took less than 10 h

to complete, and effectively removed DNA from the porcine heart tissue186

. However, it has also

been shown that using only SDS as a detergent and avoiding other physical, enzymatic and

chemical treatments such as freezing, trypsin and EDTA, shows better preservation of the

extracellular structure, and avoids severe disruption of the normal ECM187, 188

. Any

decellularization protocol requires a certain degree of ECM disruption to allow for adequate

exposure to reagents. And while chemical agents provide favorable antigenic profiles, they can

44

also irreversibly damage ECM components that are essential for cellular growth, differentiation

and repair176

.

If the decellularization process is too intensive, it can increase stiffness189

or even loss of

mechanical functionality188

. On the other hand, if the process is ineffective, it can lead to a less

than complete removal of the cell remnants and increased DNA fragment size. This is

associated with a more proinflammatory macrophage response, which contributes to the

degradation of the ECM scaffold190

.

More work is necessary to achieve a definite conclusion on this matter.

Conclusion:

Cardiac cell therapy has been gaining ground during the past several years. So far many types of

cells have been investigated regarding their regenerative and therapeutic potential. Despite the

huge variability in cell types, cell processing techniques, dose, time and route of delivery, data

from clinical trials suggests that the use of stem cells for cardiac therapy is relatively safe,

frequently resulting in improvement of cardiac function or some other clinical benefit. Although

there have been many conflicting results, cell therapy holds a promise in cardiac and vascular

regeneration. Although, caution is required since there is still the risk of adverse effects, like

arrhythmic events, adverse immune reactions, and the formation of teratomas which is possible

particularly in the case of ESCs and iPS.

Paracrine factors may be important for limiting tissue injury, inhibiting cardiac remodelling,

promoting angiogenesis and reducing inflammation, but only the cells that undergo an efficient

cardiogenic differentiation into mature cells are capable of truly regenerating scarred tissue.

Procedures to drive stem cell differentiation towards cardiac lineage are being evaluated.

Regardless of cell type, allogenic cells seem more advantageous than autologous cells, despite

their immune status, since they can be grown in larger numbers, harvested from younger and

healthier individuals, can be readily modified in vitro and are less expensive.

A sustained and effective therapeutic benefit is only possible if there is a compatible survival

and engraftment rate. The use of biocompatible scaffolds could provide the protective

environment needed for cells to survive and integrate with the host tissue.

Recent cardiac tissue engineering studies proved it is possible to create a 3 dimension

perfusable whole organ scaffold, from decellularized cadaveric matrix, that can later be

populated with stem/progenitor cells. This could offer, in the future, a wide range of solutions to

heart failure and donor heart shortage for transplant.

45

Acknowledgments:

I would like to thank Prof. Dr. Manuel Joaquim Lopes Vaz da Silva for all his guidance, help

and support throughout the elaboration of this review.

46

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de: mg seamaster mg.seamaster@gmail.com

para: wollert.kai@mh-hannover.de

data: 12 de Março de 2012 20:59

assunto: Permission request for a Master thesis

To Prof Kai C. Wollert, MD

Greetings Prof. Wollert,

My name is Diogo Manuel Rego Q. Magalhães, and i am a medical student at Faculdade de

Medicina na Universidade do Porto, in Portugal. I am presently writing a systematic review,

entitled “Stem cells and cardiovascular disease”, for my Master's thesis, which is due in March

16th. I recently came across your article: Kai C. Wollert and Helmut Drexler, Cell therapy for

the treatment of coronary heart disease: a critical appraisal, Nat Rev Cardiol. 2010

Apr;7(4):204-15. Epub 2010 Feb 23, which I found to be highly informative and useful. I was

particularly drawn by Figure 2: Sources of cardiac progenitor cells, in page 212. I found it rather

interesting and I was wondering if I could be allowed to use said figure in my upcoming thesis. I

kindly request your permission to use it; if not in its entirety than perhaps I could construct a

similar figure, using only a part of the full image.

I also took particular attention at Table 1: Randomized trials in patients with acute myocardial

infarction or ischemic heart failure, page 206, and was wondering if it would be allowed for me

to construct a similar table, but using different trials with various different stem cell types.

I deeply appreciate if you could send your reply to med05182@med.up.pt and/or

mg.seamaster@gmail.com, as soon as possible, whether it is to confirm or deny this request.

I am very grateful for your time, and apologize for any inconvenient that this email might have

caused.

Best regards

Diogo Magalhães

de: mg seamaster mg.seamaster@gmail.com

para: andrew.newcomb@svhm.org.au

data: 12 de Março de 2012 21:14

assunto: Permission request

To Andrew E. Newcomb, MD

Greetings Andrew E. Newcomb, MD

My name is Diogo Manuel Rego Q. Magalhães, and i am a medical student at Faculdade de

Medicina na Universidade do Porto, in Portugal. I am presently writing a systematic review,

entitled “Stem cells and cardiovascular disease”, for my Master's thesis, which is due in March

16th. I recently came across your article: Stubbs SL, Crook JM, Morrison WA, Newcomb AE,

Toward clinical application of stem cells for cardiac regeneration, Heart Lung Circ. 2011

Mar;20(3):173-9. Epub 2010 Jul 22, which I found to be highly informative and useful. I was

particularly drawn by Figure 1. Schematic diagram identifying the steps involved in cardiac

tissue engineering, in page 174. I found it rather interesting and I was wondering if I could be

allowed to use said figure in my upcoming thesis. I kindly request your permission to use it; if

not in its entirety than perhaps I could construct a similar figure, using only a part of the full

image.

I deeply appreciate if you could send your reply to med05182@med.up.pt and/or

mg.seamaster@gmail.com, as soon as possible, whether it is to confirm or deny this request.

I am very grateful for your time, and apologize for any inconvenient that this email might have

caused.

Best regards

Diogo Magalhães