i

Towards Standardized Vascular Assays for Mesenchymal

Stem Cells

Ana Teresa de Sousa Valinhas

Thesis to obtain the Master of Science Degree in

Biological Engineering

Supervisors

Doctor Ivan Wall

Professor Doctor Cláudia Alexandra Martins Lobato da Silva

Examination Committee

Chairperson: Professor Doctor Duarte Miguel de França Teixeira dos Prazeres

Professor Doctor Cláudia Alexandra Martins Lobato da Silva

Member of the committee: Doctor Pedro Miguel Zacarias Andrade

September 2015

ii

ACKNOWLEDGEMENTS

I would like to start by thanking my UCL supervisor, Dr. Ivan Wall, who made this

internship a reality. He gave me all the necessary support and valuable insights that

made it possible for me to progress in this project.

Thank you to all my Professors at IST who during these five years inspired me and

ignited my curiosity towards science.

A big thank you to all the RegenMed group for receiving me with open warms, for the

valuable discussions and for making this an unforgettable experience. A special thanks

to Carlotta and Fatumina who taught me an enormous amount of techniques and

helped me with experimental hurdles.

Thank you Gerardo and Rana for always cheering me up in the bad moments.

I want to thank Inês, Rita and Mafalda for their friendship and for being my “partners in

crime” during these five years.

My friends in Portugal deserve a big thanks for putting up with me, namely, Rita,

Francisco, Mariana, and Walter. Although far way, they were a driving force.

Lastly, I would like to pay a tribute to my family, especially my parents who went out of

their way to support me in every sense. Thank you for believing in me. All my success

is devoted to you.

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ABSTRACT

To develop and improve cellular therapies for cardiac regeneration it is important to

determine Mesenchymal Stem Cells’ (MSCs) modes of action and how can their

function be improved in the site of injury. These aspects can be studied through

appropriate in vitro assays that mimic the physiologic environment faced by MSCs

when seeded in the injured heart tissue.

In this project, a co-culture of bone marrow derived human Mesenchymal Stem Cells

(hMSCs) and Human Umbilical Vein Endothelial Cells (HUVECs) was studied through

a vascular assay. In order to study the behavior and robustness of this system several

parameters were tested such as hMSCs to HUVECs ratio (1:4, 1:2, 1:1, 2:1 and 4:1),

hMSCs age in vitro and quantity of matrix used per well. Furthermore, the effects of

pre-stimulating the hMSCs with two soluble ligands were tested.

hMSCs interacted with HUVECs in a pericyte-like way, lining the outside of the vessels

and bridging in between HUVECs. Co-cultures with 1:4 and 1:2 ratios showed

increased vessel formation with structures that had a higher resemblance with

capillaries compared to networks formed solely by HUVECs. Although, using 35 µL of

Matrigel per well leads to a reduction of 11% in the total cost, 55 µL shown less variable

results. Regarding the hMSCs age in vitro, cells had a tendency to decrease their

proliferative capacity after passage 4 and shown to be bigger in size at passage 6 and

7. Cells decreased their capacity to form vessels after reaching 16 population

doublings (passage 6). Finally, regarding the effects of hMSCs pre-stimulation: while

co-cultures where hMSCs were at passage 4 or 7 (cumulative population doublings of

12 and 19) appeared to be stimulated by ligand 2 to form more vessels, for hMSCs at

passages 5 and 6 (cumulative population doublings between 14.5 and 16.5) ligand 1

had a more positive effect.

Keywords: co-culture vascular assay, mesenchymal stem cells, angiogenesis,

cardiac ischemia.

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RESUMO

O desenvolvimento de terapias celulares para a regeneração cardiáca implica que se

determinem quais os modos de acção das células estaminais do mesênquima

(MSCs), bem como formas de melhorar a sua funcionalidade no local afectado. Estes

aspectos podem ser estudados através de testes in vitro, que replicam o ambiente

fisiológico que as MSCs enfrentam após tranplantadas no tecido afectado.

Neste projecto, a co-cultura de células humanas endoteliais do cordão umbilical

(HUVECs) e células estaminais humanas do mesênquima (hMSCs) da medula óssea

foi estudada através de um ensaio vascular. O comportamento e robustez deste

sistema foram testados através da variação de parâmetros como a razão entre hMSCs

e HUVECs (1:4, 1:2, 1:1, 2:1 e 4:1), idade das hMSCs in vitro e quantidade de matriz

utilizada por poço. O efeito da pré-estimulação de hMSCs com dois ligandos distintos

foi também averiguado.

As hMSCs interagiram com as HUVECs de uma forma semelhante aos pericitos,

revestindo o exterior dos vasos e preenchendo espaços entre as HUVECs. Co-

culturas com ratios de 1:4 e 1:2 mostraram uma maior vascularização, com estruturas

mais semelhantes a capilares do que culturas onde apenas HUVECs foram usadas.

Apesar do uso de 35 µL de Matrigel por poço levar à redução do custo total em 11%,

devem ser usados 55 µL dado que os resultados obtidos são menos variáveis. No que

diz respeito à idade celular das hMSCs in vitro, observou-se a diminuição da sua

capacidade proliferativa após a passagem 4 e mostraram ser maiores em tamanho na

passagem 6 e 7. As mesmas diminuiram a sua capacidade de formar vascularização

após atigirem um número de gerações igual a 16 (passagem 6). No que diz respeito

ao pré-estimulo das hMSCs: enquanto co-culturas onde estas estavam na passagem

4 ou 7 (número de gerações de 12 ou 19) foram mais estimuladas pelo ligando 2, co-

culturas onde as hMSCs estavam entre a passagem 5 ou 6 (número de gerações entre

14.5 e 16.5) mostraram melhores resultados quando estimuladas com o ligando 1.

Palavras-chave: ensaio vascular em co-cultura, células estaminais do mesênquima,

angiogenese, isquémica cardiaca.

v

CONTENTS

1. BACKGROUND AND AIM OF STUDIES ............................................................ 8

2. LITERATURE REVIEW ..................................................................................... 10

2.1 ISCHEMIC CARDIAC INJURIES ........................................................................... 10

2.1.1 Symptoms and diagnostics ................................................................... 10

2.1.2 Treatments ............................................................................................ 11

2.1.3 Clinical trials .......................................................................................... 12

2.2 STEM CELLS .................................................................................................. 13

2.2.1 Mesenchymal stem cells ....................................................................... 14

2.3 THE ROLE OF MSCS IN CARDIAC REGENERATION .............................................. 16

2.3.1 Mobilization of MSCs to areas of injury ................................................. 16

2.3.2 Differentiation of MSCs into cardiomyocytes, smooth muscle cells and

endothelial cells ................................................................................................. 17

2.3.3 Trophic effects ...................................................................................... 19

2.4 HUMAN UMBILICAL CORD VEIN ENDOTHELIAL CELLS (HUVECS) ....................... 20

2.4.1 Characterization of HUVECs ................................................................ 21

2.5 BLOOD VESSELS ............................................................................................ 21

2.6 ANGIOGENESIS .............................................................................................. 22

2.7 METHODS FOR ASSAYING ANGIOGENESIS ......................................................... 24

2.7.1 Types of vascular assays ...................................................................... 24

vi

LIST OF ABBREVIATIONS

BM-MSCs Bone marrow mesenchymal stem cells

BNP Brain natriuretic peptide

BSA Bovine serum albumin

CFU-Fs colony-forming unit fibroblast

DAPI 4',6-diamidino-2-phenylindole

DMSO Dimethyl sulfoxide

ECM Extracellular matrix

EDTA Ethylenediaminetetraacetic acid

EGF Endothelial growth factor

EGM-2 Endothelial growth medium 2

ESCs Embrionic stem cells

FGF Fibroblast growth factor

G-CSF Granulocyte colony-stimulating factor

GFP Green fluorescent protein

HGF Hepatocyte growth factor

hMSCs Human mesenchymal stem cells

HUVECs Human umbilical cord vein endothelial cells

iPSCs Induced pluripotent stem cells

L1 Ligand 1

L2 Ligand 2

LV Left ventricular

LVEF Left ventricular ejection fraction

vii

MI Myocardial infarction

MSCs Mesenchymal stem cells

PBS Phosphate buffer saline

PCA Principal component analysis

PD Population doublings

PDGF-B Platelet-derived growth factor B

RAM Rbp-associated molecule

SCs Stem cells

SDF-1 Stromal-cell-derived factor

TGF Transforming growth factor

TM Transmembranar

UT Untreated

VEGF Vascular endothelial growth factor

VEGF-A Vascular endothelial growth factor A

vSMC Vascular smooth muscle cells

VWF Von Willebrand Factor

8

1. BACKGROUND AND AIM OF STUDIES

Ischemic heart disease, which can ultimately cause myocardial infarction, is one of the

leading causes of death in the world (Finegold et al. 2013). Therapies available in the

market delay the effects of this disease. The only solution which copes with the irreversible

loss of myocardial tissue is heart transplantation which is dependent on donor availability

and patients have to undergo long-term immunosuppressive treatments (Abrams 2005;

Segers & Lee 2008). Thus, cellular therapies come as a solution for the high demand of

alternative treatments. (Segers & Lee 2008). In the last two decades, mesenchymal stem

cells (MSCs) have shown to be a promising alternative to treat cardiac ischemia. These

cells are easily isolated and amplified being capable of yielding large amounts of cells

needed for these treatments and show the ability to improve cardiac performance and

vascularization after myocardial infarction (Williams & Hare 2011). Animal studies and

several clinical trials have demonstrated encouraging results, although the benefits shown

are modest and often inconsistent (Terrovitis et al. 2010). This is highly influenced by the

poor engraftment and survival of MSCs at the injured site (Gnecchi et al. 2008).

The mechanisms through which MSCs act to achieve cardiac regeneration are not entirely

known. MSCs have the ability to migrate to the infarcted myocardium due to cytokines

released by ischemic and hypoxic tissues that promote expression of adhesion molecules

(Wang & Li 2007). Several authors hypothesized that MSCs can differentiate into

endothelial cells, smooth muscle cells and cardiomyocytes, which is still a contradictory

subject in the scientific community (Segers & Lee 2008; Wang & Li 2007). Other authors

defend that MSCs rescue myocardium as a consequence of spontaneous cell fusion

rather than differentiation (Terada et al. 2002). Both effects would not be sufficient to

explain the improvements seen in these studies, since that the amount of cells that are

thought to have differentiated or fused are very low (Gnecchi et al. 2012). It is agreed that

the major contribution of cardiac tissue regeneration is achieved through paracrine

signalling which leads to anti-apoptotic, anti-scarring, and angiogenic effects (Segers &

Lee 2008; Caplan & Correa 2011). Recently, it has been proposed that the beneficial

paracrine effects observed after MSC therapy might be mediated by exosomes (Lai et al.

9

2010). Other contributions for cardiac tissue regeneration might include endogenous

repair by stem cell niches present in the heart that after an enhanced injury signal are

stimulated to migrate to the injured area and become activated (Wang & Li 2007).

In order to develop and improve cellular therapies it is important to determine which MSCs

modes of action contribute to tissue heart regeneration, to what extent and how MSCs

function can be improved in the site of injury (Bain et al. 2014). It is imperative to design

appropriate in vitro assays that mimic the physiologic environment faced by these cells

when seeded in the injured heart. Thus, these assays should use substrates that are

present in the ECM after ischemic injury such as fibronectin and collagen (Sutton &

Sharpe 2000), low oxygen pressure (1-7% oxygen) (Carreau et al. 2011), multiple

endogenous cell types and growth factors that will interact with MSCs in a living organism.

In vitro assays will play a major role in the selection of populations suitable for the intended

clinical application in a cheaper and less time-consuming way compared to in vivo assays

(Sorrell et al. 2009).

One of the most important processes involved in the regeneration of wound sites is

angiogenesis, since that vascularization delivers a supply of several cell populations and

bioactive factors that initiate and support tissue regeneration (Sorrell et al. 2009).

Improvement of MSCs function in the injured myocardium after transplantation can be

achieved through pretreating these cells with cytokines, growth factors, pro-survival

factors and physical stimuli (Terrovitis et al. 2010).

Given that MSCs have shown the capacity to improve vascularization by interaction with

endothelial cells (Caiado et al. 2008), the co-culture of this cells was studied through a

vascular assay. In order to achieve standardization of this assay, several parameters were

studied such as the effect of using hMSCs between the fourth and seventh passage; the

variation of cell ratio between 4:1, 2:1, 1:1, 1:2 and 1:4 and, the usage of 35 µL and 55 µL

of Matrigel per well. Furthermore, the effects of pre-stimulating the hMSCs with two

different soluble ligands was tested.

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2. LITERATURE REVIEW

2.1 Ischemic cardiac injuries

Ischemic heart disease is characterized by reduced blood supply to the heart muscle. It is

the primary cause of death throughout the world, including most low-income and middle-

income countries (Segers & Lee 2008).

2.1.1 Symptoms and diagnostics

Myocardial ischemia is caused by coronary heart disease, blood clot and coronary spasm

which can cause abnormal heart rhythms and myocardial infarction (Nesto & Kowalchuk

1987).

Coronary heart disease, the most common cause of myocardial ischemia, consists of the

partial or complete occlusion of the heart’s arteries due to accumulation of plaques made

of cholesterol on artery walls (Abrams 2005). When a rupture of the plaques that develop

in the walls of arteries occurs, a blood clot is formed which may lead to severe myocardial

ischemia, resulting in myocardial infarction (Abrams 2005). A coronary artery spasm is a

brief, temporary tightening of the muscles in the artery wall that can decrease or stop

blood flow to a part of the myocardium (Maseri 1987).

Signs and symptoms of myocardial ischemia include angina pectoris which consists on

chest pressure or pain, typically felt of the left side of the body, neck or jaw pain, shoulder

or arm pain, fast heartbeat and shortness of breath (Abrams 2005). Many patients suffer

from silent ischemia, meaning they do not experience signs or symptoms (Maseri 1987).

Diagnostics strategies include stress testing and coronary angiography (Abrams 2005).

Stress testing, which can be induced by exercise or drug administration, is a noninvasive

test where the response of the patient’s heart to external stress is tested (Abrams 2005).

These tests are useful providers of prognostic information but might be inconclusive.

Usually it is necessary to perform coronary angiography to obtain conclusive results. This

test consists on the injection of a dye into the patient’s bloodstream that enables

11

visualization of the arteries by x-ray, making it possible to identify possible artery

blockages (Abrams 2005).

2.1.2 Treatments

The treatment of ischemia makes use of therapeutic drugs such as antianginal (anti-

ischemic), vasculoprotective therapy and revascularization (Abrams 2005).

Antianginal agents include nitrates, beta-adrenergic blockers and calcium-channel

blockers. All three work by decreasing the myocardial oxygen consumption through

different mechanisms. Combination therapies, consisting of two or more of these

therapies, can be also prescribed to patients. (Abrams 2005).

Vasculoprotective therapy is based on the reduction of causes of ischemia. This is

achieved by doing regular exercise and by pharmacology therapy, which makes use of

agents such as aspirin, statins, beta-blockers, clopidogrel and ACE inhibitors (Abrams

2005).

Revascularization is achieved through invasive treatments which include percutaneous

coronary intervention (balloon angioplasty and stenting) or coronary-artery bypass

surgery (Abrams 2005).

Depending on the extent of ischemia, the tissue might be salvaged by appropriate and

timely intervention through the therapies previously referred (Abrams 2005). When

ischemia occurs, the heart undergoes a set of adaptive responses that change the

hemodynamics of the tissue as less myocardium attempts to maintain the same cardiac

output as before. This remodeling consists on neurohormonal responses, cytokine

activation, cardiomyocyte loss due to necrosis or apoptosis, cardiomyocyte hypertrophy,

disruption of the ECM and collagen accumulation followed by scar formation, which can

ultimately cause heart failure (Wang & Li 2007; Li et al. 2014). After myocardial infarction,

there is a loss of cardiomyocytes and vessel regression. The only therapy that addresses

12

the loss of cardiomyocytes is heart transplantation which has limitations such as donor

supply and the need for long-term immunosuppressive therapy (Segers & Lee 2008).

Given that heart tissue does not regenerate spontaneously, stem cells constitute a

promising alternative treatment for ischemic heart disease due to their ability to promote

regeneration (Li et al. 2014). Mesenchymal stem cells, a subpopulation of stem cells, have

been studied as a cell-based therapeutic strategy of cardiac repair. This is due to their

unique properties such as ease of isolation and amplification, multilineage potential and,

low immunogenicity which makes them tolerable as an allogeneic transplant (Williams &

Hare 2011). MSCs secrete a range of bioactive molecules, including a broad array of

paracrine factors, that give them the capacity to participate in the injury response (Caplan

2007). There is evidence that these cells promote angiogenesis, have anti-sacarring

properties, decrease cardiomyocyte apoptosis and increase nerve sprouting (Caplan &

Dennis 2006).

2.1.3 Clinical trials

The majority of clinical trials where stem cells were used as therapeutic agents for the

treatment of ischemic heart disease, have involved cells of the bone marrow given that

they are readily available and easy to expand (Michler 2013). Some examples are given

below.

In 2004, a study was done on effects of intracoronary infusion of autologous BM-MSCs

(8-10x109, n=34) or saline (n=35) in patients with acute myocardial infarction (MI) (Chen

et al. 2004). After 3 months there was an improvement in perfusion as well as

improvements in left ventricular (LV) chamber size in patients treated with infused BM-

MSCs (Chen et al. 2004).

In 2005, another study showed the results of transplanting autologous MSCs and

endothelial progenitors (n=11) into MI patients with doses of 1-2x106 cells (Katritsis et al.

2005). LV function was improved, myocardial scarring was reduced and there were no

adverse effects reported in this study (Katritsis et al. 2005).

13

In 2006, a study demonstrated that plasma levels of brain natriuretic peptide (BNP) fell at

three and six months, while function capacity improved with no effect on LV function or

survival (Wang et al. 2006). This test was performed in a randomized trial of 24 patients

with non ischemic dilated cardiomyopathy receiving standard drug therapy in which the

treatment group (n=12) was treated with intracoronary injections of MSCs and the control

group (n=12) received saline (Wang et al. 2006).

In 2009, a randomized, double-blind clinical trial of allogeneic MSCs (n=39) and placebo

(n=21) administered intravenously after acute MI using a dose of 0.5, 1.6, 5 (x106) cells/Kg

was performed (Hare et al. 2009). Treated patients had fewer arrhythmic events and

improved pulmonary function (Hare et al. 2009).

In 2010, 16 patients with anterior MI were subjected to intracoronary transplantation of

autologous MSCs using doses between 1.22±1.77x107 (grp-I) and 1.32±1.76x107 (grp-II)

(Yang et al. 2010). Patients showed no adverse effects and improved LVEF at six months

(Yang et al. 2010).

Other studies have shown contradictory results, such as the one where sudden death of

pigs undergoing intramyocardial injection of MSCs was observed, compared with the

placebo group (Amado et al. 2005).

Several clinical trials have shown to attenuate LV dilation, although none of these have

yet proven true myocardial regeneration with the development of new cardiomyocytes or

new blood vessels originating from the administered cells (Michler 2013).

2.2 Stem cells

Stem cells have both the ability to self-renew, that is, to divide and create additional stem

cells, and also to differentiate along a specified molecular pathway originating progeny

with more restricted potential (Fuchs & Segre 2000). The potency or differentiation

potential of a stem cell is an important parameter that defines a hierarchy. The three first

types of potency that appear in this hierarchy are totipotency, pluripotency and

14

multipotency. Totipotent stem cells, which include the zygote and early embryonic

blastomeres up to at least the 4-cell stage embryo, give origin to the cells of a whole

organism, being able to differentiate into the first two lineages, the pluripotent inner mass

of the blastocyst and the trophectoderm (Mitalipov & Wolf 2009). Pluripotent stem cells,

such as embryonic stem cells (ESCs) obtained from the inner mass of the blastocyst and

induced pluripotent stem cells (iPSCs) obtained through molecular reprogramming of

somatic cells, have the capacity to differentiate into cell types of all the three germ layers,

namely, ectoderm, endoderm and mesoderm in vitro and in vivo (Thomson et al. 1998;

Takahashi & Yamanaka 2006). Lower down the hierarchy appear multipotent stem cells.

Consisting of some fetal, adult stem cells and progenitor like-cells, these are responsible

for maintaining tissue homeostasis and to renew certain tissues throughout life.

In adults, multipotent stem cells can be divided into MSCs and parenchymal SCs. The first

group generates new somatic connective tissue while the second generates new

nonconnective tissues. While MSCs are extensively distributed, parenchymal SCs

aggregate in organs niches in a quiescent state, at a low rate of basal division, protected

and supported by MSCs (Alvarez et al. 2012).

2.2.1 Mesenchymal stem cells

Friedenstein and colleagues were the first to demonstrate that the bone marrow (BM)

contains a population of hematopoietic stem cells and a rare population of stromal cells

(Friedenstein et al. 1968; Friedenstein et al. 1970). The last were distinguished from the

majority of hematopoietic cells by their rapid adherence to tissue culture vessels and by

the fibroblast-like appearance (spindled shape) of their progeny (Friedenstein et al. 1970).

These authors also showed that BM suspensions results in the establishment of discrete

colonies initiated by single cells defined by them as CFU-Fs (the colony-forming unit

fibroblast) (Friedenstein et al. 1968). They were also the first to demonstrate the ability of

MSCs to differentiate into mesoderm-derived tissue and to identify the importance of these

cells in controlling the hematopoietic niche (Fridenshteĭn 1990).

15

Mesenchymal stem cells, a term first used by Caplan, can be defined as a heterogeneous

subset of multipotent stromal cells that differentiate into a variety of end-stage cell types

(Caplan 1991). It is well documented that these cells can differentiate into bone

(Haynesworth et al. 1992), cartilage (YOO et al. 1998), muscle (Wakitani et al. 1995),

marrow stroma (Majumdar et al. 1998), tendon and ligament (Young et al. 1998), fat

(Dennis et al. 1999), and a variety of other tissues (Studeny et al. 2004).

MSCs have been isolated from amniotic fluid, periosteum, peripheral blood, umbilical

cord, synovial tissue and lung tissue. In adult mice, MSCs have been isolated from nearly

every tissue type, suggesting that these cells reside in all postnatal organs (Williams &

Hare 2011). The different populations of MSCs are different in terms of gene and cytokine

expression (Sakaguchi et al. 2005; Hwang et al. 2009). However, a set of core gene

expressions are preserved (Tsai et al. 2007) as well as other properties, allowing their

identification as MSCs (Dominici et al. 2006).

2.2.1.1 Characterization of MSCs

A cell is considered an MSC if it satisfies the following criteria: it presents a fibroblast like

morphology, it is able to differentiate into osteoblasts, adipocytes and chondroblasts when

stimulated with the right cues in vitro, and it expresses a set of surface markers while lacks

the expression of others. (Sharma et al. 2014).

There is not a unique cell surface marker that distinguishes MSCs from HSCs, thus the

minimal criteria to identify them is by the expression of nonspecific markers including

CD105 (SH2 or endoglin), CD73 (SH3 or SH4), CD90 (Thy-1), and the lack of expression

of hematopoietic markers such as CD14 or CD11b, CD34, CD45, CD79α or CD19 and

HLA-DR. It is important to refer that a portion of MSCs derived from adipose tissue

expressed CD34. In addition MSCs have been reported to express CD13, CD29, CD166,

CD44, platelet-derived growth factor α, CD49b, CD49e, CD54, CD71, CD106, β2

microtubulin, nestin, p75, HLA ABC, SSEA-4 and TRK (A,B,C) (Bernardo et al. 2009;

Williams & Hare 2011; Rankin 2012; Dominici et al. 2006).

16

2.3 The role of MSCs in cardiac regeneration

MSCs have an important role in the wound-healing process specifically in difficult non-

healing wounds resulting from trauma, diabetes, vascular insufficiency and numerous

conditions. These cells participate in the inflammatory, proliferative, and remodeling

phases of wound healing (Maxson et al. 2012).

Traditionally, MSCs were thought to be interesting for their multipotent differentiation

capacity when stimulated with the right cues in vitro. Several authors hypothesize that

MSCs can differentiate into endothelial cells, smooth muscle cells and cardiomyocytes,

which is still a contradictory subject in the scientific community (Segers & Lee 2008; Wang

& Li 2007). Other authors defend that MSCs rescue myocardium as a consequence of

spontaneous cell fusion rather than differentiation (Terada et al. 2002). However, these

cells have a much broader application as therapeutic agents due to the secretion of

bioactive molecules and interactions with other types of cells that lead to immumodulatory

effects and trophic effects such as anti-apoptotic, anti-scarring, angiogenic and mitotic

effects (Caplan & Correa 2011). The effects of MSC-secreted molecules can be direct

(which lead to intracellular signalling), indirect or even both (Caplan & Dennis 2006).

Recently, it has been proposed that the beneficial paracrine effects observed after MSC

therapy might be mediated by exosomes (Lai et al. 2010). Other contributions for cardiac

tissue regeneration might include stimulation of endogenous repair by stem cell niches

present in the heart by enhancing an injury signal and stimulating the migration and

activation of this cell niche (Wang & Li 2007).

2.3.1 Mobilization of MSCs to areas of injury

MSCs have the ability to migrate to the infarcted myocardium probably due to cytokines

released by ischemic and hypoxic tissues that promote expression of adhesion molecules

(Wang & Li 2007). They might originate from the BM or from the vicinity. Details about the

migration and the mode of action of MSCs are still not fully understood due to the lack of

reliable tracing markers (Shi et al. 2012).

17

It has been shown that stromal-cell-derived factor (SDF-1) induces stem cell homing to

injured myocardium immediately post-infarction (Askari et al. 2003). In another study,

MSCs were directly implanted into the bone marrow of mice that were later injected with

granulocyte colony-stimulating factor (G-CSF), which promoted migration of the marrow

MSCs into the heart after myocardial infarction (Kawada et al. 2004). This suggests that

G-CSF is implicated as a general mobilization factor and SDF-1 as a chemotactic factor

(Caplan & Dennis 2006)

A therapy based on endogenous mobilization is particularly interesting for patients with

acute myocardial infarction or heart failure who might not be able to go through the

invasive procedures necessary for the harvest and transplantation of MSCs (Wang & Li

2007).

2.3.2 Differentiation of MSCs into cardiomyocytes, smooth muscle

cells and endothelial cells

Studies in animal models of myocardial infarction have shown the ability of transplanted

MSCs to differentiate into cardiomyocytes and vascular cells (endothelial cells and

vascular smooth muscle cells) (Gnecchi et al. 2012). On the one hand, some authors

failed to detect permanent engraftment of transplanted BM-MSCs (Müller-Ehmsen et al.

2006) and on the other hand it has not been possible to reproducibly induce a functional

cardiac phenotype in BM-MSCs in vitro using physiological growth factors or non-toxic

chemical compounds (Gnecchi et al. 2012). These findings question the plasticity of BM-

MSCs.

There is evidence that MSCs express cardiomyocyte markers in vitro when cultured in the

presence of DNA demethylating agent 5-azacytidine (Hattan et al. 2005; Makino et al.

1999). Other authors reported the differentiation of murine MSCs into cardiac tissue in

mouse and chick embryos (Pochampally et al. 2004; Fluri et al. 2012). Kawada et al

18

presented evidence that MSCs might differentiate into cardiomyocytes when in the

presence of cardiac injury although with a low efficiency. Acute myocardial infarction was

induced in lethally irradiated syngeneic mice that after transplantation of BM-MSCs

(GFP+) were treated with G-CSF. After eight weeks, GFP+/actin+ cells were found in the

heart suggesting that the BM-MSCs differentiated into cardiomyocytes (Kawada et al.

2004).

It is not clear whether stem cells transdifferentiate through a fusion-dependent or –

independent manner (Gnecchi et al. 2012). Although with low efficiency, it was

demonstrated that mouse BM-MSCs injected into infarcted hearts can fuse with resident

cardiomyocytes (Noiseux et al. 2006).

MSCs could differentiate into endothelial and smooth muscle cells under specific growth

conditions (Jiang et al. 2002; Galmiche et al. 1993). Furthermore, MSCs cultured in vitro

and then transplanted into infarcted rat hearts expressed smooth muscle as well as

endothelial cell markers (Davani 2003). Two other studies showed that MSCs

transplanted into infarcted rat hearts or into chronically ischemic dog hearts, resulted, for

the first, in endothelial differentiation with enhanced microvascular density as well as

improved heart function (Psaltis et al. 2008), and for the second, greater vascular density

with evidence of engrafted MSCs with EC and VSMC markers (Silva et al. 2005).

Some authors defend the hypothesis that instead of differentiating into EC and VSMC,

MSCs turn into pericytes that stabilize and stimulate the maturation of new blood vessels

(Gnecchi et al. 2012).

Besides all the controversy around this subject, it is agreed that the number of cells that

appeared to have a cardiomyocyte, endothelial or smooth muscle cell like phenotype in

animal studies were not sufficient to explain the beneficial effects on heart regeneration

and functionality generated after MSC transplantation (Gnecchi et al. 2012). This suggests

that the main contribution of MSCs to heart regeneration might be due to trophic effects.

19

2.3.3 Trophic effects

Trophic effects are those chemotactic, mitotic, and differentiation-modulating effects

which emanate from cells as bioactive factors that act primarily on neighboring cells and

whose effects never result in differentiation of the producer cell (Caplan & Dennis 2006).

It was demonstrated that MSCs implanted into ischemic myocardium stimulated and

increased production of vascular endothelial growth factor (VEGF), increased both

vascular density and blood flow, and decreased apoptosis, all of which were probably

influenced by the secretion of bioactive molecules (Tang et al. 2004). Furthermore, other

authors demonstrated that cell culture medium conditioned by hypoxic MSCs can reduce

apoptosis and necrosis of isolated rat cardiomyocytes exposed to low oxygen tension.

MSCs overexpressing Akt-1 had better cytoprotective effect in vitro (Gnecchi et al. 2005).

The concentrated culture medium from Akt-MSCs was injected into a rat experimental

model of permanent coronary occlusion and compared with the controls. The infarct size

and cardiomyocyte apoptotic effect was significantly reduced in rats treated with

concentrated conditioned medium from Akt-MSCs (Gnecchi et al. 2006).

MSCs might affect the extracellular matrix through paracrine signalling, having a positive

effect on post-infarction remodeling and strengthening of the infarcted scar (Gnecchi et

al. 2012). It was demonstrated that injection of MSCs directly into ischemic rat hearts

decreases fibrosis, apoptosis and left ventricular dilatation and increases myocardial

thickness (Berry et al. 2006). MSCs might also improve cardiac function by stimulating the

growth of different types of cells in the infarcted area and by the decreasing the production

of extracellular matrix proteins such as collagen type I and II and tissue inhibitor of

metalloproteinase-1 (Xu et al. 2005).

MSCs might also have a role on cardiac metabolism and contractibility. MSC

transplantation attenuates bioenergetic abnormalities in the border zone of pig infarcted

hearts (Feygin et al. 2007). Furthermore, Gnecchi et al. demonstrated that Akt-MSC

conditioned medium prevents metabolic remodeling in infarcted rat hearts. Akt-MSC

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treated hearts had normal pH and better contractibility compared with MSC treated hearts

and the controls (Gnecchi et al. 2009).

Some authors suggest MSCs might also contribute for endogenous cardiac repair by

stimulating resident cardiac stem cells or cardiomyocyte replication through paracrine

signalling (Gnecchi et al. 2012; Wang & Li 2007). Newly formed cardiomyocytes after

injection of MSCs into infarcted pig hearts was observed (Amado et al. 2005). Recently,

GFP-MSC were injected into the heart of a pig model I/R injury. The results suggested

that MSCs could stimulate cardiomyocyte endogenous turnover by stimulating c-kit+

cardiac stem cells and by enhancing cardiomyocyte cell cycling (Hatzistergos et al. 2010).

It was proposed that MSC therapy can be mediated by exossomes. The authors

demonstrated that exosomes obtained from culture medium of MSCs derived from hESCs

reduced infarcted size while the culture medium lacking exosomes did not (Lai et al. 2010).

2.4 Human Umbilical Cord Vein Endothelial cells (HUVECs)

The endothelium consists of a single cell monolayer composed of endothelial cells that

lines the inside of all blood vessels. The endothelium controls gas exchanges at the

pulmonary level, regulates the flow of circulating blood cells of various bioactive molecules

such as growth factors, coagulation proteins, lipoproteins and hormones, especially

through the presence of membrane bound-receptors. Furthermore, it participates in the

regulation of homeostasis and, in cooperation with the ECM and smooth muscle cells, it

controls the level of vascular constriction and contributes to the metabolism of amines. It

also participates in the inflammatory and immune response through surface antigens,

adhesive molecules and through the synthesis of cytokines (Jaffe 1987; Baudin et al.

2007). Thus, it is important to have models that can recapitulate the properties of

endothelial cells in vitro.

HUVECs are considered a model for ECs by many researchers around the world.

However, these cells are not able to represent all the metabolic properties and the

responses in physiopathology and toxicity of all types of ECs in an organism.

21

Nevertheless, HUVECs are the most simple and available human EC type, adequate for

the preparation of large quantities of cells (Baudin et al. 2007).

2.4.1 Characterization of HUVECs

HUVECs are thin, flattened, with pinocyte vesicles and many cytoplasmatic vesicles. The

cytoplasm also contains clusters of free ribosomes, smooth and rough endoplasmatic

reticulum, clumps of coarse filaments, fine filaments, prominent microtubules and Weibel-

Palade bodies (Jaffe et al. 1973).

Like other types of endothelial cells, HUVECs have the ability to spontaneously form

tubule-like structures when seeded on top of Matrigel (Chung-Welch et al. 1989). HUVECs

can be characterized using EC markers, such as ACE (peptidyl-dipeptidase EC.3.4.15.1),

CD143 and Von Willebrand Factor/Factor VIII RA, even if the latter is weakly expressed

in these particular ECs (Baudin et al. 2007). Other markers can be used such as CD31,

CD34, CD45 or ICAM-I, CD62E (E-selectin), CD106 (VCAM-1), which can be more or less

specific for ECs and are expressed often in HUVECs after a particular stimulation

(Nakache et al. 1986). Furthermore, the lectin from Ulex europaeus (anti-H blood group

specificity) binds to most ECs (Jaffe et al. 1973).

2.5 Blood Vessels

Blood vessels are composed of two cell types: endothelial cells which reside in the lumen

and perivascular cells that line the outer part of the vessels (Bergers & Song 2005).

Perivascular cells are a group of cells that include vascular smooth muscle cells (VSMCs)

and pericytes. Arteries and veins are surrounded by VSMCs, intermediate-size vessels

by cells that have properties similar to VSMCs and pericytes and finally, the smallest

capillaries are lined by a single lawyer of pericytes (Gerhardt & Betsholtz 2003). While

VSMCs provide structural support and blood flow control in large vessels without

interacting with ECs, pericytes share a basement membrane with the late. Thus, pericytes

are in direct contact with ECs through pores in the basement membrane, making cell-to-

22

cell contact possible as well as paracrine signalling (Gerhardt & Betsholtz 2003; Bergers

& Song 2005).

A group of markers that unequivocally identifies pericytes has not been identified, since

these cells present distinct characteristics and functions according to their locations in

different organs (Bergers & Song 2005). A pioneer study by Crisan et al. defined,

confirmed and validated the link between MSCs and perivascular cells. In this study,

pericytes recovered from different organs shown the expression of MSC markers, they

were myogenic in culture and in vivo, and exhibited at the clonal level, osteogenic,

chondrogenic and adipogenic potential (Crisan et al. 2008). These findings suggest that

pericytes might give origin to MSCs and other types of stem cells or that MSCs are in fact

pericytes (Caplan 2008).

2.6 Angiogenesis

Angiogenesis is the process that leads to the formation of new vessels through the

sprouting of new capillaries from existing ones (Olsson et al. 2006), making it an important

factor in the regeneration of ischemic myocardium. This process not only allows the

inflammatory cells to migrate to the wound site, but also provides the supply of oxygen

and nutrients necessary to support the tissue’s function (Tonnesen et al. 2000; Aguirre et

al. 2010).

In mature organisms, ECs remain in a quiescent, nonproliferative state. These cells

become active when provoked by pathological conditions such as wounding and

inflammation, until normal function is achieved and quiescence can be re-established

(Ware & Simons 1997; Phng & Gerhardt 2009). The sprouting process is led by

endothelial tip cells that are stimulated by a vascular endothelial growth factor-A (VEGF-

A) gradient to produce long, dynamic falopodia containing tyrosine kinase receptor

VEGFR2/KDR/Flk-1 and other receptors which are used to probe the environment for

directional cues (Gerhardt et al. 2003). Another population of ECs, called the stalk cells,

follow the tip cells. These produce fewer falopodia and are stimulated by VEGF-A to

proliferate (Gerhardt et al. 2003). These cells cease proliferation behind the migration

23

zone and end up forming the vascular lumen by establishing adherens and tight junctions

to maintain the integrity of the new sprout and to create luminal/abluminal polarity (Phng

& Gerhardt 2009). Paracrine VEGF-A production is then down regulated by flow-

dependent tissue oxygenation and a quiescent state is established in the new vessels

(Phng & Gerhardt 2009).

In relation to pericyte recruitment there are two different views described in the literature.

The first one is based on studies from retinal angiogenesis where vessels started to

maturate after pericyte recruitment, suggesting that endothelial cells secrete growth

factors, particularly platelet-derived growth factor B (PDGF-B), that recruited pericytes,

which have a PDGF-β receptor (Bergers & Song 2005; Gerhardt & Betsholtz 2003). The

other view was based on studies performed in the corpus luteum where pericytes were

the first cell type to invade the ruptured follicle. In this case, pericytes were a source of

VEFG-A stimulated by endothelial cells, suggesting a paracrine loop between these two

types of cells (Gerhardt & Betsholtz 2003).

There are several growth factors and cytokines known to be involved in the induction and

promotion of angiogenesis that stimulate endothelial growth and migration besides the

VEGF family. Others include the fibroblast growth factor (FGF) family which bind heparin-

like components of the extracellular matrix on the surface of ECs and SMCs, facilitating

attachment (Ware & Simons 1997). These factors also stimulate the production of

proteases by ECs that digest the extra cellular matrix before the sprouting process (Ware

& Simons 1997). Other growth factors include the transforming growth factor (TGF) α and

the hepatocyte growth factor (HGF) (Ware & Simons 1997).

Specific signalling pathways are known to control angiogenesis such as angiopoietin/Tie2,

Notch, Wnt, TGFβ/Alk1, S1P/Edg1, Slit/robo, Semaphorin/Plexin, Netrin/Unc5b and cell

matrix/integrin signalling (Phng & Gerhardt 2009). The understanding of these

mechanisms will provide important clues for modulation of strategies for therapeutic

neovascularization.

24

2.7 Methods for assaying angiogenesis

The development of large-scale viable cell therapy processes implies the creation of

adequate assays that can validate the process and function as quality control

measurements that ensure product safety and identity (Wall & Brindley 2013).

Angiogenesis has been assayed both in vivo and in vitro. In the next section a brief

description of the assays available nowadays is made.

2.7.1 Types of vascular assays

Angiogenesis assays in vivo include the chick chriollantoic membrane assay (CAM),

corneal micropocket assay, the rodent mesentery angiogenesis assay and the zebrafish

assay (Staton et al. 2004).

The CAM assay uses the membrane which results from the fusion of the somatic

mesoderm of the chorion with the splanchnic mesoderm of the allantois (a highly

vascularized membrane that serves as the initial respiratory system of the avian embryo)

to assess the effects of different substances on the development of vessels (Norrby 2006).

The quantification in a specific area is achieved through microscopy techniques,

determinations of content and synthesis of DNA, protein and basement membranes

(Norrby 2006).

In the corneal micropocket assay, a pocket is made in the stroma within the cornea and a

slow release polymer is placed in it (Norrby 2006). Given that the cornea is a tissue that

lacks vascularization, true neovascularization is observed in this assay (Norrby 2006).

This procedure is not easy to perform due to the accessibility and reduced size of the

cornea. Results are assessed through transmission and scanning electron microscopy

(Norrby 2006).

The mesentery is a very thin membrane, easily exteriorized from the gut of small rodents,

which can be used to analyze cellular and vascular components in detail (Norrby 2006).

In the mesentery assay, the effect of several anti or pro-angiogenic factors are studied

25

through the formation of microvessels with the aid of microscopic techniques (Norrby

2006).

Zebrafish are small freshwater fish with a generation time of about three months that are

largely used as a model for drug discovery and developmental biology (Staton et al. 2004).

The embryonic development of zebrafish is external and its optical transparency during

the first few days enables continuous microscopy inspections (Norrby 2006). While small

and lipophilic molecules are added to the water that holds the fish, peptides or proteins

have to be directly injected into the yolk sacs (Staton et al. 2004).

In vivo vascular assays are time consuming, costly, prone to high variability and are often

difficult to quantify (Staton et al. 2004). Furthermore, these are not well translatable to a

commercially viable process that has to follow strict GMP guidelines and there are moral

issues associated to these practices. Thus, in vitro assays come as a solution to overcome

these issues.

The ideal in vitro assay, which is the focus of this study, has to be robust, simple to

perform, easily quantifiable and physiologically relevant (Staton et al. 2004). These

assays are either focused on proliferation, migration, tubule formation or differentiation of

ECs.

Proliferation assays are based on the usage of markers of cell division to assess

endothelial cell proliferation in culture (Staton et al. 2004). There are two types of

proliferation assays: the ones that determine net cell number and others that evaluate cell-

cycle kinetics (Staton et al. 2004).

Migration assays make use of the ability of endothelial cells to move according to a

gradient of an angiogenesis-inducing factor. Usually, endothelial cells are plated on top of

a filter and migrate across it in response to the angiogenic factor placed in the lower

chamber (Staton et al. 2004). This assay is highly sensitive to differences in concentration

gradients (Falk & Leonard 1982).

26

Differentiation assays make use of matrices such as fibrin, collagen or Matrigel, where

ECs are seeded on and stimulated to form capillary-like structures (Lawley & Kubota

1989). While fibrin and collagen are mammalian ECM components, Matrigel is a mixture

of extracellular and basement membrane proteins derived from the mouse Engelbreth-

Holm-Swarm sarcoma on which endothelial cells attach and rapidly form tubules, within 4

to 12 hours (Kleinman & Martin 2005). A second form of Matrigel was developed called

growth factor-reduced Matrigel where the levels of stimulatory cytokines and growth

factors have been reduced (Kleinman & Martin 2005).

Angiogenesis is a complex process, making it necessary to study further aspects, such

as the interaction of these cells with other cell types and ECM components. So far there

is not an in vitro assay capable of model angiogenesis, which is why it is necessary to use

a combination of different assays to assess this process (Staton et al. 2004).

27

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