Modulating the Regenerative Features of Human

Mesenchymal Stem/Stromal Cells with SDF-1α:

A Gene Therapy Approach Using Minicircles

Tiago Miguel Ricardo Ligeiro

Thesis to obtain the Master of Science Degree in

Biotechnology

Supervisor: Professor Doctor Duarte Miguel de França Teixeira dos Prazeres

Co- Supervisor: Professor Doctor Cláudia Alexandra Martins Lobato da Silva

Examination Committee

Chairperson: Professor Doctor Arsénio do Carmo Sales Mendes Fialho

Supervisor: Professor Doctor Duarte Miguel de França Teixeira dos Prazeres

Member of the Committee: Doctor Evguenia Pavlovna Bekman

November 2018

I

Abstract

SDF-1α, a chemoattractant involved in angiogenesis and hematopoiesis, has emerged

as an alternative or complement to VEGF delivery for the treatment of ischemic diseases. The

aim of this thesis was to construct and manufacture minicircle (MC) encoding human SDF-1α to

transfect mesenchymal stem/stromal cells (MSCs) and augment their regenerative capacity. A

parental plasmid (PP) was constructed by cloning the SDF-1α gene into a pre-existing backbone.

Following transformation, colonies harboring the construction were confirmed by restriction

analysis and sequencing. In vivo PP amplification and recombination were performed in E. coli,

yielding supercoiled MC (scMC) and sc miniplasmid (scMP). Primary purification removed most

impurities, while RNase and Nickase-assisted chromatography efficiently isolated scMC from

other contaminants. Subsequently, MC (500 ng) harboring either SDF-1α or VEGF were used to

lipofect BM-MSCs from two donors. 24 h post-transfection cell recoveries ranged from 50-80%,

which worsened at 72 h (30-40%). qPCR revealed 4000 and 750-fold-up increases in SDF-1α

and VEGF mRNA expression, respectively. Transfection at 95% confluency with fewer (250 ng)

MC led to lower mRNA expression, but higher recoveries at 72 h (~55%). ELISA revealed

increased VEGF concentration, 3500pg/mL (up from 2500pg/mL), at 72 h, while HUVEC

migration increased by 30%, under modified protocol. Consequently, improved protein content

and regenerative potential are achieved through a balance between expression and cell densities,

modulated by DNA quantity. Furthermore, co-culture of hematopoietic progenitors with modified

MSCs revealed increased expansion with minimal impact on regenerative features. Overall, these

findings indicate that MSCs overexpressing SDF-1α have an improved regenerative potential.

Keywords: Angiogenesis, Hematopoiesis, Ischemic Diseases, Gene and Cell Therapy,

Mesenchymal Stem/Stromal Cells;

II

Resumo

SDF-1, uma quimiocina envolvida na angiogénese e hematopoiese, tem surgido como

alternativa ou complemento ao VEGF no tratamento de doenças isquémicas. O objetivo deste

trabalho foi construir e produzir minicirculos (MC) codificando SDF-1 para transfectar células

estaminais/estromais mesenquimais (CEM), para aumentar a sua capacidade regenerativa. O

plasmídeo parental (PP) foi desenvolvido clonando o gene SDF-1 num PP pré-existente. Após

transformação, análise de restrição e sequenciação foram utilizados para confirmar presença

construção desejada em colónias selecionadas. A amplificação e recombinação do PP in vivo

decorreram em E. coli, originando MC superenrolados (seMC) e miniplasmídeos superenrolados

(seMP). A purificação primária removeu a maioria das impurezas, enquanto cromatografia

assistida por Nickase e RNase isolou de forma eficiente os seMC. Subsequentemente, MC (500

ng) codificando SDF-1 ou VEGF foram lipofectados em CEM da medula óssea. 24 h após

transfeção, recuperação celular variou entre 50-80%, piorando às 72 h (30-40%). qPCR revelou

aumentos de 4000x e 750x na expressão de mRNA de SDF-1 e VEGF, respetivamente.

Transfeção a densidades mais elevadas (95% de confluência) com menos DNA (250 ng)

causaram diminuição na expressão de mRNA, mas melhor recuperação às 72 h (~55%). ELISA

revelou aumento na concentração VEGF, 3500 pg/mL (de 2500 pg/mL), às 72 h, enquanto a

migração de HUVECs aumentou por 30%, com o novo protocolo. Consequentemente, aumento

do conteúdo proteico e potencial regenerativo são alcançados através de um equilíbrio entre

expressão e densidade celular, modulados pela quantidade de DNA. Co-cultura de progenitores

hematopoiéticos com CEM modificadas revelou maior expansão com impacto mínimo nas

características regenerativas. Estes resultados indicam que CEM sobre-expressando SDF1 têm

potencial regenerativo aumentado.

Palavras-Chave: Angiogénese, Hematopoiese, Doenças isquémicas, Terapia Génica e Células,

Células Estaminais/Estromais Mesenquimais

III

Acknowledgements

I would like to acknowledge, appreciate and thank to all individuals who contributed to

this work and made my master’s thesis easier, better, possible.

First of all, I would like to acknowledge professor Joaquim Sampaio Cabral for allowing

me to develop my master’s thesis at iBB, namely at the BERG and SCERG facilities of Instituto

Superior Técnico.

To all of my supervisors, professor Miguel, professor Cláudia and professor Gabriel, for

introducing me to the field of gene and cell therapy and to the topic of this work. Also, for their

complete availability to receive me in their offices, answer my constant e-mails and texts, and for

all of their guidance and support. Not only their input and contribution to this work was immense,

but their guidance also helped towards my own personal development. For all this, I am incredibly

grateful.

To all of my lab colleagues at BERG, especially to Cláudia Alves and Ana Rita, for taking

the time to help me, teach me and pass on all of their lab knowledge and expertise. It was crucial

throughout all my work and substantially improved the scientist within me.

To all the colleagues at SCERG, where the positive environment and complete availability

from every lab member contributed tremendously to thesis. I would like to especially thank Joana

Serra, Marília Silva, Diogo Pinto, Ana Carina, Sara Bucar and Sara Morini. Without their help,

teaching and guidance, my integration and work at the stem cell laboratory would not have been

so easy and joyful.

To my fellow master students, for all the support, discussions and conversations that

made the rough times easier to handle, but also the master’s degree much more interesting and

joyful. In particular, I would like to thank to Cristiana Ulpiano, with whom I shared this incredible

experience, and who was a true companion all throughout this work. Words do not suffice to

describe the roller coaster of emotions and experiences that we shared and endured, which, in

the end, we managed to overcome.

Finally, to my friends and family. Although not directly related with the work that I

developed, they were, and continue to be, the bedrock in which my life is built, and without whom,

this master’s thesis would not be possible.

To all of you, I am truly grateful! Thank You.

IV

Table of Contents

Abstract .......................................................................................................................................... I

Resumo .......................................................................................................................................... II

Acknowledgements ...................................................................................................................... III

Table of Contents ......................................................................................................................... IV

List of Abbreviations .................................................................................................................... VII

List of Figures ............................................................................................................................. VIII

List of Tables ................................................................................................................................. IX

List of Equations ........................................................................................................................... IX

1. Introduction............................................................................................................................... 1

1.1 Ischemic Diseases ................................................................................................................ 1

1.2 Mesenchymal Stem/Stromal Cells ...................................................................................... 2

1.2.1 The Physiological of Role of MSCs ................................................................................ 3

1.2.2 Regenerative Features ................................................................................................. 4

1.2.3 Immune system and MSCs ........................................................................................... 5

1.2.4 Tissue Sources and Isolation of MSCs .......................................................................... 5

1.2.5 Identity and Characterization of MSCs ......................................................................... 6

1.2.6 Translation to Clinical Environment ............................................................................. 7

1.3. The Angiogenic Process ...................................................................................................... 8

1.3.1 Triggering Angiogenesis: EC Activation and Sprouting ................................................ 9

1.3.2 Vessel Sprouting: Tip and Stalk Cell Specialization ...................................................... 9

1.3.3 Vessel Elongation ....................................................................................................... 10

1.3.4 Lumen Formation: Tip Cell Anastomose and Stalk Cell Coalescence ......................... 10

1.3.5 Vessel Maturation: Mural Cell Differentiation and Recruitment ............................... 10

1.4. The Role of MSCs in Revascularization ............................................................................. 11

1.4.1 Pro-Angiogenic Properties of MSC’s Secretome: Key Signaling Factors .................... 11

1.4.2 MSCs and Hypoxia ...................................................................................................... 12

1.5. Stromal-Derived Factor-1 ................................................................................................. 13

1.5.1. The Role of SDF-1 in Angiogenesis: Physiology and Therapeutics ............................ 14

1.5.2. SDF-1 and Hematopoiesis ......................................................................................... 15

1.6. Gene Therapy: Increasing MSCs’ Regenerative Features ................................................ 16

1.6.1 Genetic Modifications: Where to Aim? ...................................................................... 16

1.6.2 Gene and Genome Editing ......................................................................................... 17

1.6.3 Vectors: Viral and non-viral ........................................................................................ 18

1.7. Clinical Trials: Gene Therapy and MSCs ........................................................................... 20

V

2. Aim of Studies ......................................................................................................................... 22

3. Materials and Methods ........................................................................................................... 23

3.1. General and Analytical Techniques .................................................................................. 23

3.2. Parental Plasmid Construction ......................................................................................... 23

3.3. Minicircle Production and Purification............................................................................. 24

3.3.1. HIC-HPLC Quantification of pDNA Production .......................................................... 24

3.3.2. Minicircle Production ................................................................................................ 25

3.3.3. Primary Purification ................................................................................................... 25

3.3.4. Nickase (Nb.BbvCI) Restriction .................................................................................. 26

3.3.5. Multimodal Chromatography .................................................................................... 26

3.3.6. Supercoiled Minicircle De-salting and Concentration. .............................................. 26

3.4. Mesenchymal Stem Cell Culture ...................................................................................... 26

3.4.1. MSCs Cell Thawing .................................................................................................... 27

3.4.2. Cell Passaging ............................................................................................................ 27

3.5. Human Mesenchymal Stem Cell Transfection. ................................................................ 27

3.5.1. MSC Lipofection ........................................................................................................ 27

3.5.2. MSC Microporation ................................................................................................... 28

3.5.3. Medium Collection and Cell Harvest ......................................................................... 28

3.5.4. RNA Extraction and cDNA Synthesis ......................................................................... 28

3.5.5. mRNA Quantification with Quantitative Polymerase Chain Reaction ...................... 29

3.5.6. ELISA: Quantifying VEGF Protein Concentration in MSC Culture Supernatants ....... 29

3.5.7. Immunophenotyping MSCs by Flow Cytometry ....................................................... 29

3.5.8. Angiogenic Functional Assays ................................................................................... 29

3.6. Co-culture of MSCs and HSCs ........................................................................................... 30

3.6.1. Hematopoietic Stem/Progenitor Cell Thawing and Recovery .................................. 30

3.6.2. Magnetic Activated Cell Sorting: Purifying CD34+ Cells............................................. 30

3.6.3. Co-culturing MSCs and HSCs ..................................................................................... 31

4. Results and Discussion ............................................................................................................ 32

4.1 Construction of the Parental Plasmid pMini-CSDF1N2 ..................................................... 32

4.2 Minicircle Production ........................................................................................................ 33

4.2.1 HPLC analysis: Identifying the best pDNA producing clone ....................................... 33

4.2.2 MC Production in E. coli BW2P by in vivo Recombination of PP ................................ 34

4.3 Minicircle Purification ....................................................................................................... 36

4.3.1 Primary Purification: Retrieving MC molecules ......................................................... 36

4.3.2 Miniplasmid Relaxation with Nickase ........................................................................ 37

4.3.3 Supercoiled Minicircle Isolation with Multimodal Chromatography ......................... 37

VI

4.4 Transfection of Human Mesenchymal Stem/Stromal Cell with SDF-1α encoding

Minicircles ............................................................................................................................... 40

4.4.1 HPL vs FBS: Comparing Transfection Under Differential Supplementation ............... 40

4.4.2 Modulating VEGF Overexpression with Varying Quantities of Minicircle ................. 42

4.4.3 Transfection of BM-MSC with MC-SDF1 and MC-VEGF: Exploiting the Synergy

Between SDF-1α and VEGF ................................................................................................. 43

4.4.4 Functional Assays: Evaluating the Angiogenic Potential of Modified MSCs .............. 47

4.4.5 Optimizing the Lipofection Protocol .......................................................................... 50

4.4.6 ELISA Quantification of Gene Expression at the Protein Level .................................. 53

4.4.7 Microporation of MSC: Testing an Alternative Gene Delivery Method ..................... 56

4.5. Co-culturing MSCs and HSCs: The effects of overexpressing SDF-1α .............................. 57

5. Conclusions & Future Work..................................................................................................... 62

6. References ............................................................................................................................... 65

7. Supplementary Data ................................................................................................................ 77

VII

List of Abbreviations

AT – Adipose Tissue

bFGF – basic Fibroblast Growth Factor

BGH – Bovine Growth Hormone

polyadenylation site

BM – Bone Marrow

bp – base-pair

CAM – Chick Chorioallantoic Membrane

CM – Conditioned medium

CMV – Cytomegalovirus Immediate Early

Promoter

CRISPR – Clustered Regulatory Interspaced

Short Palindromic Repeats

CVDs – Cardiovascular Diseases

CXCL – C – X- C motif ligand

CXCR – CXC chemokine receptor

DLL4 – Delta-Like 4

DMEM – Dulbecco’s Modified Eagle’s Medium

DMEM+10% FBS – DMEM supplemented with

10% FBS

DMEM+5%HPL – DMEM supplemented with

5% HPL

DNA – Deoxyribonucleic acid

EBM – Endothelial basal medium

ECM – Extracellular Matrix

ECs – Endothelial Cells

EDTA – Ethylenediaminetetracetic Acid

EGM-2 – Endothelial growth medium 2

ELISA – Enzyme-linked Immunosorbent Assay

EMA – European Medicine Agency

EPCs – Endothelial Progenitor Cells

FBS – Fetal Bovine Serum

FDA – Food and Drug Administration

GAPDH – Glyceraldehyde 3-phosphate

Dehydrogenase

GMP – Good Manufacturing Practices

GvHD – Graft versus host disease

HDL – High-Density Lipoprotein

HGF – Hepatocyte Growth Factor

HIC-HPLC – Hydrophobic-Interaction - High-

Performance Liquid Chromatography

HIF-1 – Hypoxia-Inducible Factor - 1

hMSCs – Human MSCs

HPCs - Hematopoietic Progenitor Cells

HPL – Human Platelet Lysate

HRE – Hypoxia-Response Element

HSCs – Hematopoietic Stem Cells

HUVEC – Human Umbilical Vein Endothelial

Cells

IDs – Ischemic Diseases

IFN-γ – Interferon gamma

LB – Luria-Bertani

LDL – Low-Density Lipoprotein

MACS – Magnetic Activated Cell Sorting

MC – Minicircle

MHC – Major Histocompatibility complex

MMC – Multimodal Chromatography

MP – Miniplasmid

MRS – Multimer Resolution Site

MSCs – Mesenchymal Stem/Stromal Cells

oc – open circular

PBS – Phosphate Buffer Saline

PCR – Polymerase chain reaction

pDNA – Plasmid DNA

PEG – Polyethylene glycol

PGE-2 – Prostaglandin E2

PP – Parental Plasmid

qPCR – Quantitative (or Real time) PCR

RNA – Ribonucleic acid

RT-PCR – Reverse Transcriptase – PCR

sc – supercoiled

SDF-1 - Stromal-Derived Factor 1

SMCs – Smooth Muscle Cells

Tris - Tris(hidroximetil)aminometano

UCB – Umbilical Cord Blood

UCM – Umbilical Cord Matrix

VEGF – Vascular Endothelial Growth Factor

VEGFR – VEGF Receptor

VIII

List of Figures

Figure 1 – Representation of the Regenerative Features of MSC Secretome. ............................ 4

Figure 2 - General Representation of Angiogenesis. .................................................................... 8

Figure 3 – Construction of a New Parental Plasmid: Cloning SDF-1α in pMinili-CVGN2 by

Replacing VEGF-GFP. ........................................................................................................ 32

Figure 4 – Growth Kinetics of E. coli BW2P (pMini-CSDF1N2) During a MC Production Run. .. 34

Figure 5 – Visualization of Recombination Efficiency and Screening of the Migration Pattern of

the Various Isoforms............................................................................................................ 35

Figure 6– Primary Purification and Miniplasmid Relaxation with Nickase. ................................. 36

Figure 7 – Primary Purification and Miniplasmid Relaxation with Nickase. ................................ 38

Figure 8 - RNase and Nickase-Assisted Multimodal Chromatography ....................................... 39

Figure 9 – Polyacrylamide Gel Electrophoresis of Fraction 5 from RNase-Assisted Multimodal

Chromatography. ................................................................................................................. 39

Figure 10 - Agarose Gel Electrophoresis of scMC Samples After Concentration and De-Salting.

............................................................................................................................................. 40

Figure 11 - Cell Population Analysis After Lipofection of M79A15 BM-MSCs Under DMEM

Supplemented with Either 10% FBS or 5%HPL .................................................................. 41

Figure 12 -VEGF and SDF-1α Relative mRNA Expression of M79A15 BM-MSCs After Lipofection

Under FBS or HPL Supplementation. ................................................................................. 42

Figure 13 - Cell Population Analysis After Lipofection of M79A15 BM-MSCs with Varying

Quantities of MC-VEGF. ...................................................................................................... 42

Figure 14 - Relative Expression of VEGF in M79A15 BM-MSCs After Lipofection with Varying

Amounts of MC-VEGF ......................................................................................................... 43

Figure 15 - Cell Dynamics Analysis After Lipofection of M79A15 BM-MSCs ............................. 44

Figure 16 – Relative mRNA Expression of SDF-1 and VEGF After Lipofection of M79A15 BM-

MSCs with MC-SDF1 and MC-VEGF .................................................................................. 45

Figure 17 - Cell Population Analysis After Lipofection of M48A08 BM-MSCs with MC-VEGF and

MC-SDF1. ............................................................................................................................ 46

Figure 18 – Relative mRNA Expression of SDF-1 and VEGF After Lipofection of M48A08 BM-

MSCs with MC-SDF1 and MC-VEGF .................................................................................. 46

Figure 19 -HUVEC Migration Assay with CM After 24 h and 48 h of Conditioning for both BM-

MSC Donors ........................................................................................................................ 47

Figure 20 – HUVEC Tube Formation Assay with CM After 24 h and 48 h of Conditioning for Both

BM-MSC Donors ................................................................................................................. 48

Figure 21 - Cell Population Dynamics of M79A15 BM-MSCs After Lipofection at 95% Confluency

with 250 ng of DNA.............................................................................................................. 51

Figure 22 – Relative Gene Expression of M79A15 BM-MSCs After Lipofection with MC-SDF1 and

MC-VEGF at 95% Confluency with 250 ng of MC. ............................................................. 51

Figure 23 - HUVEC Migration Assay with CM After 48 h of Conditioning with M79A15 BM-MSC

Lipofected at 95% Confluency with 250 ng of MC. ............................................................. 52

Figure 24 - VEGF Quantification with ELISA: Specific Expression ............................................. 54

Figure 25 - VEGF Concentration in Culture Media and Conditioned Media in Different Donors and

Lipofection Protocols ........................................................................................................... 55

Figure 26 – Evaluating Cell Numbers and RelativeGene Expression of M79A15 BM-MSCs

Following Microporation with MC-SDF1. ............................................................................. 57

Figure 27 – Flow Cytometry Analysis of MACS Enrichment of CD34+ Cells from Umbilical Cord

Blood. .................................................................................................................................. 58

Figure 28–Numbers of CD34+ Hematopoietic Cells After Four Days of Co-Culture with MSCs

Overexpressing SDF-1α ...................................................................................................... 59

Figure 29 - Flow Cytometry Analysis of Hematopoietic Cells at the Fourth Day of Co-Culture with

MSCs Overexpressing SDF-1α ........................................................................................... 60

IX

List of Tables

Table 1 – Summary of Selected Clinical Trials Involving Gene Therapy and MSCs from the

American NIH Clinical Trial Database (ClinicalTrials.gov) .................................................. 20

Table 2 – HIC-HPLC Quantification of pDNA Production by E. coli BW2P (pMini-CSDF1N2)

Clones. ................................................................................................................................ 34

List of Equations

Equation 1 ................................................................................................................................... 24

Equation 2 ................................................................................................................................... 28

Equation 3 ................................................................................................................................... 28

1

1. Introduction

1.1 Ischemic Diseases

Ischemic diseases (IDs) are a subset of cardiovascular diseases (CVDs), that arise from

vascularization problems. These are characterized by the development of ischemia, a state where

diminished blood supply causes a decrease in oxygen and nutrient availability, leading to stress,

damage and ultimately cell death1. IDs can affect various body regions such as limbs, heart and

brain, in the cases of peripheral artery disease, myocardial infarction and stroke, respectively1.

Moreover, different manifestations of ischemia within the same organ are possible. An example

of this phenomenon is ischemic heart disease, in the cases of myocardial infarction and angina

pectoris. The former presents itself as an acute event, in which symptoms appear suddenly and

severely, while the latter displays slow progression with mild symptomatology that worsens with

time.

The principal cause of IDs is the build-up of atheromatous plaque in the arterial lumen

due to atherosclerosis. While the exact mechanisms triggering atherogenesis are still unclear,

years of data and studies have established that lipids, cholesterol and low-density lipoprotein

(LDL) play an essential role in this process2,3. Additionally, IDs are also caused by other events

and conditions that restrict or block blood supply such as embolism, thrombus, trauma,

aneurysms, among others. Furthermore, underlying conditions including diabetes, rheumatic

heart, hypercholesterolemia and high blood pressure have also been correlated with an increased

risk of developing ischemia4,5.

CVDs are the leading cause of morbidity and mortality worldwide with 17.7 million deaths

reported in 2015, corresponding to 31% of all deaths1. Of these, IDs such as stroke and

myocardial infarction account for the majority of deaths, specifically, 14.1 out of 17.7 million1. This

group of diseases is also responsible for the majority of deaths from non-communicable disease,

while also being the principal cause of premature death (people under 75 years of age)6. Several

studies have concluded that the global burden of CVDs has been increasing in the last decades

- in 1990 CVDs represented 25.9% of global mortality6, 5% less than current values. Interestingly,

the number of deaths in high-income countries has remained stable in the last years, whereas in

middle and low-income countries it augmented immensely7. Conversely, age-standardized

mortality rates for CVDs have been decreasing for the past 30 years worldwide, especially in high

income countries7,8. This tendency has been explained by increased prevention efforts, with the

promotion of healthy lifestyles and check-ups, and by medical advances which enable better care

and management9. In addition to their burden on human lives, CVDs also represent a tremendous

strain on global economy, with overall costs of €211 billion and $555 billion for the EU and US,

respectively, in 20178,10. As with mortality, the majority of costs are due to ischemic heart disease

and stroke, which represent nearly three quarters of these expenses8.

Present day treatment for IDs varies from case to case, depending on underlying causes,

affected body region and extent of the disease. First line treatment usually involves lifestyle

management with diet and exercise, which aim to reduce cholesterol intake and maintain blood

pressure4. However, the predominant approach is medication targeting cholesterol synthesis,

2

platelet aggregation and blood pressure, with statins, aspirin, nitroglycerin and others11. In acute

or severe cases surgical intervention is usually required, which can either be artery bypass,

endarterectomy or angioplasty5. In the former, a blood vessel is engrafted with each end at the

extremities of the obstruction, thus creating an alternative route bypassing the blockage.

Endarterectomy is a procedure where there is removal of the atheroma, clearing up the arterial

lumen. Angioplasty is a technique where a balloon catheter is inserted in the affected area,

followed by inflation, which widens the artery and improves blood flow. Usually, the balloon

catheter is accompanied by a stent, a rigid tubular device that is positioned during inflation and

stays inside the artery to prevent re-occlusion.

Despite their efficacy in restricting damage and maintaining function, available treatment

options fail to improve or restore previous tissue function. Frequently, these manage to prevent

deaths, but are incapable of reversing morbidity acting as palliative measures12. Consequently,

to tackle the challenges and limitations of the available therapies and diminish the global burden

of IDs, new approaches are required. Recent years have shown that various types of stem cells

can either elicit or participate in repair and revascularization of ischemic tissues. Of these,

mesenchymal stem/stromal cells (MSCs) have been reported to possess intrinsic pro-angiogenic

activity, i.e. the capacity to promote blood vessel formation, thus being a suitable candidate as

cellular therapy for IDs13. These properties have enabled progression of MSCs into pre-clinical

and clinical trials, whilst attracting bioengineering approaches to increase their natural activity.

1.2 Mesenchymal Stem/Stromal Cells

MSCs were first identified and described as non-hematopoietic bone-marrow stromal

cells in the late 1960’s by Friedenstein et al.14. Their discovery followed observation of the

capacity of bone marrow (BM) stromal cells to originate bone, fat and cartilage after heterotopic

transplantation, thus hinting the existence of multilineage precursors14. Further studies showed

these were a subpopulation of plastic adherent and spindle-shaped cells, which proliferated and

formed colonies in vitro, being termed colony forming units – fibroblasts (CFU-Fs)15.

Subsequently, reports demonstrated their ability to maintain multipotency during in vitro

proliferation, suggesting clonality16. Consequently, their capacity to differentiate into various

mesodermal lineages in vitro16, and their clonality were considered evidence for multipotency and

self-renewal, both hallmarks of stemness, leading to their acceptance as stem cells.17.

More recently, questions have been raised regarding the applicability of the term

mesenchymal stem cell to these BM stromal cells18,19. The prototypical MSC is a self-renewing

cell, which sits at the top of the mesenchymal hierarchy, giving rise to skeletal, muscle and tendon

lineages in vivo. However, definitive evidence supporting the aforementioned biological activity in

vitro and in vivo is still lacking. In part, this results from the inability to probe isolated cells directly,

with most studies being performed after in vitro expansion of clonal populations. Furthermore, the

existence of a single common precursor has been considered unlikely, since during embryonic

development muscle and skeletal lineages derive from distinct progenitors20. Additionally, muscle

differentiation in vivo has not been convincingly demonstrated. As consequence, alternative

nomenclatures such as mesenchymal stromal cells or skeletal stem cells have been proposed.

3

To avoid inaccuracy and confusion, the International Society for Cellular Therapy (ISCT),

suggested the use of mesenchymal stromal cell to describe in vitro expanded cells, limiting the

use of mesenchymal stem cell to designate the in vivo counterpart18.

Regardless of these nomenclature issues, mesenchymal stem/stromal cells have

gathered tremendous attention over the recent years. First, their heterogenous nature, intrinsic

properties and behavior have been under study to enable better comprehension and

characterization21. Furthermore, although identified in various tissues, MSCs are frequently

present in low numbers which has puzzled the scientific community regarding their role in the

body. Moreover, interest in these cells has expanded beyond basic research to applied research.

Initially, this was due to their mesodermal multipotency, which granted tremendous potential for

tissue engineering applications. However, in recent years, the secretive activity of MSCs

harnessed attention, since it has been demonstrated to possess various regenerative features.

Accordingly, their trophic behavior motivated approaches to directly use MSCs as therapeutic

agents in cellular therapies.

1.2.1 The Physiological of Role of MSCs

Although originally discovered as a bone-marrow cells, MSCs have already been found

in various tissues throughout the body22, but always in low frequencies23, which hinted for

widespread function in the body. In the BM, they seem to play a role in hematopoiesis by aiding

and interacting with hematopoietic stem cells (HSCs), also helping to maintain HSC homeostasis

and forming the niche24. Firstly, through their multipotency MSCs are believed to undergo

osteoblast, adipocyte and fibroblast differentiation, originating the non-hematopoietic components

of the niche, the confined compartment where stem cells, in this case HSCs, lie. The osteoblasts

in the BM are known to regulate HSCs either by cell-to-cell contact and the secretion of soluble

factors25. In turn, the MSCs-derived fibroblasts, known as reticular cells, localize in perivascular

and secrete C-X-C motif ligand 12 (CXCL12) in abundancy, a chemokine that regulates HSC

maintenance, localization of HSC in the BM, and also their mobilization from the BM into the blood

stream. Moreover, further studies have revealed a role of nestin+ MSCs, which also reside close

to vascular site, in supporting HSCs proliferation and homeostasis through the secretion of an

array of factors, including SCF and others26,27.

There is evidence that MSCs reside near blood vessels as it occurs within BM, hinting a

possible role28. Pericytes, cells living in perivascular tissue which are responsible for supporting

blood vessels, have been described to display MSC-like features29. In agreement, authors

suggested that pericytes were either specialized MSCs or cells immediately derived from MSCs30.

However, a recent report by Guimarães-Camboa et al provided evidence that pericytes isolated

from various tissues fail to exhibit MSCs characteristics and activity, challenging previous

observations31. Despite controversy regarding pericyte nature, MSCs location close to vascular

site seems indisputable. There, MSCs are believed to secrete a variety of factors contributing to

blood vessel maintenance and maturation, but also, to blood vessel formation by setting up to the

appropriate environment and stimuli32, which will be discussed in further parts of this manuscript.

4

1.2.2 Regenerative Features

In addition to these classical physiological roles, several studies show that MSCs also

participate and aid in injury containment and regeneration. In the first instance, MSC appear to

migrate towards and attach to injury sites. This has been attributed to the expression of receptors

to specific chemokines and other surface markers. An example of this is endothelium adherence

in lesions, where endothelial cells become activated and express E-selectin and vascular cell

adhesion molecule, both of which match the CD49d and CD44 receptors in MSCs, resulting in

docking33.

Figure 1 – Representation of the Regenerative Features of MSC Secretome. MSCs have anti-apoptotic and anti-fibrotic capacity. Also, secreted factors can also promote angiogenesis and cell migration to injury sites, while also promoting growth and differentiation of specific cells, by setting up the appropriate environment. A list of the processes and of the factors secreted is presented. Figure from Silva-Meirelles et al.34

In addition to multilineage differentiation potential, an attractive feature of MSCs is the

capacity to secrete a wide variety of molecules, named the secretome, which modulate the

microenvironment, and influence surrounding cells activity and also has remarkable regenerative

potential34. Studies have shown that MSC infusion into injury sites revealed decreased apoptosis

of surrounding cells, thus an anti-apoptotic effect. These events correlated with elevated

expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and

insulin-like growth factor 1 (IGF-1), factors that promote endothelial cell survival35. Additionally,

an anti-scarring activity of MSCs has also been described. The release of basic fibroblast growth

factor (bFGF) at injury sites activates MSCs, through the c-Jun N-terminal kinase (JNK) pathway,

leading to their proliferation, HGF expression and reduced fibrogenesis36.

5

Another interesting property is the capacity to recruit other types of cells to inflammatory,

angiogenic and injury locations. MSCs secrete a variety of chemokines, including CXCL12,

CXCL8, CXCL2, CXCL10, CCL2, CCL3 and other factors such as the HGF and VEGF, which

have chemotactic effect and contribute to cellular docking37. Nevertheless, in vitro the proportion

and the molecules secreted vary between experiments, a probable consequence of culture

conditions, but also suggesting a differential recruitment in the presence of different cells and

environment. This capacity is in line with MSC regenerative features, as recruiting ECs and SMCs

and others can facilitate revascularization, elimination of necrotic tissue and repair.

Initial approaches to translate MSCs into clinical environment aimed at harnessing their

multipotency for tissue engineering and regenerative medicine38. However, insights into their

paracrine activity, ability to set-up the microenvironment and modulate several regenerative

processes have broadened the spectrum of applications to cellular therapies39.

1.2.3 Immune system and MSCs

MSCs possess another remarkable characteristic that distinguish them from other cells,

a unique relation with the immune system. In vitro, MSCs were reported to have

immunomodulatory activity, as they inhibit T-cell and B-cell proliferation, and NK cell activation,

while also influencing the cytokine secretion profiles of macrophage and dendritic cells. Despite

other molecules being at play, prostaglandin E2 (PGE-2) and indolamine-2,3-dioxygenase,

appear to be central to this phenomenon. Furthermore, immunomodulation is also observed in

vivo, as MSC infusions were proven effective in treating graft versus host disease (GvHD)

following HSC transplantation 40, although the molecules involved are still unclear due to the

complexity of these interactions. Nevertheless, MSCs effect on the immune system is not

exclusively inhibitory. Changes in interferon γ (IFN-γ) concentration have differential effect on

major histocompatibility (MHC) class II expression in MSCs41. Also, the MSC-like pericytes display

pro-inflammatory activity in early stages of wound healing and anti-inflammatory in later phases37.

As for their recognition, MSCs exhibit low immunogenicity. In part, this can be explained

by their absence of class II MHC, low expression of class I MHC, and lack of various co-

stimulatory surface markers, such as CD40, CD8042. This absence allows infused MSCs to avoid

immune surveillance, despite MHC mismatch. Furthermore, their immunomodulatory behavior

allows them to evade further encounters, escaping immune aggressiveness. In agreement, a shift

paradigm of MSCs interaction with the immune system has shifted. Previously, MSCs were

regarded as immune privileged, while currently it is accepted that MSCs are immune evasive43.

Nevertheless, it is clear that MSCs stand in a unique position for cell therapy application, since

their low immunogenicity facilitates allogeneic use, not only boosting MSC effect after infusion,

but also enabling the possibility of progressing to an “off-the-shelf” application.

1.2.4 Tissue Sources and Isolation of MSCs

Although initially described as BM cells, MSCs have already been isolated from various

other tissues such as, adipose, brain, skin, urine and others. Moreover, neonatal tissues including

amniotic fluid, umbilical cord matrix (or Wharton Jelly) and placenta were also described as MSCs

6

sources44. However, some reports indicate high variability amongst cells isolated from different

tissue sources and donors45. From a clinical perspective, it is crucial to understand the features

of cells harvested from each source, whereas from an engineering standpoint it is important to

have precise description of the isolation procedure and yields in order to assess the feasibility of

widespread application and production. In spite of the variety of sources, there are three that due

to their characteristics have stepped up as the preferred: bone-marrow, adipose tissue and the

umbilical cord matrix44.

Firstly, for being the source where initial studies were conducted and also the best

characterized, MSCs from the BM are considered the “gold standard”, hence the BM still being

considered the primary option46. Nevertheless, MSCs are a rare cell type within the BM,

representing less than 1% of all nucleated cells, which limits the obtention of large numbers.

Harvesting MSCs from the BM involves a simple aspiration protocol. Subsequently, cells are

subject to density centrifugation and plating, taking advantage of MSCs adherence to plastic to

remove hematopoietic cells47. Nevertheless, this is a highly invasive collection approach, which

poses the risk of infection for the donor, yielding isolated cells less prone to ex-vivo expansion,

when compared to neonatal-derived MSCs, namely umbilical cord matrix (UCM)48.

Adipose tissue (AT) has raised interest as an alternative source to BM cells, due to the

availability of material and cellular yield. Frequently, AT used for MSC isolation originates from

leftover material of liposuction, lipectomy and lipoplasty. Isolation protocols from AT involves

enzymatic treatment, followed by centrifugation to recover the pre-adipocyte stromal vascular

fraction49. Similarly to BM-MSCs, AT-MSCs also show low expansibility when compared to

neonatal-derived MSCs, which has been explained by their adult-tissue origin. Conversely,

purification from AT has been shown to have high isolation yields49. Consequently, limitations

from adult sources ranges between availability and “older” cell state, as they show reduced

proliferation and possibly accumulated genomic alterations44.

MSCs isolated from the UCM show higher pre-disposition to expansion, have reduced

contamination risk with blood-borne viruses and lack accumulated genomic mutations, when

compared to adult tissues. Additionally, they benefit from wide availability, as well as easier

collection44. Isolation of MSCs from UCM can be accomplished either with enzymatic treatment,

followed by solid-liquid separation and plating, or by culturing tissue explants49,50.

1.2.5 Identity and Characterization of MSCs

Whether in the clinical setup or at the laboratory scale, it is crucial to proceed with

characterization of isolated MSCs populations. This provides a confirmation that the isolated cells

are MSCs and in fact possess MSCs properties. Cellular characterization differs between cell

type, but usually aims to screen the presence of specific surface markers, gene expression

patterns, particular mechanical-physical properties and biological potential (e.g. stem cells are

usually tested for the presence of specific surface proteins and also for differentiation potential).

As previously mentioned, MSCs are very heterogenous, not only from donor to donor, but

also amongst different tissue sources51. This variability has hampered the establishment of

precise criteria for a population to qualify as MSCs. Moreover, the absence for characterization

7

has hindered comparison and reproducibility between reports. Therefore, the ISCT has issued a

set of guidelines with recommended standards for BM-derived MSCs19. Accordingly, BM-MSCs

must be plastic adherent and present fibroblast-like morphology. They must also be capable of

differentiation into osteocytes, adipocytes and chondrocytes in vitro. Furthermore, more than 95%

of the population is required to express CD73 (ecto 5’ nucleotidase), CD90 (thy-1), and CD105

(endoglin) in a flow cytometry measurement. Conversely, the population must lack (more than

98% of the population in flow cytometry measurement) endothelial and hematopoietic lineages

markers CD34, CD45, CD19, CD14, CD11b, CD79α, and the immune receptor HLA-DR19. The

absence in these receptors assures a reduced confounding effect from other cells, such as

macrophages, monocytes, HSC, endothelial cells and B-cells. In turn, Gimble et al52 reviewed the

list of criteria for the characterization of AT-derived MSCs. Despite the differences between AT-

MSCs and BM-MSCs, the immunophenotypic characterization should identify expression of

surface markers described by Dominici et al19 for BM-MSCs, namely positive for CD73, CD90 and

CD105 and negative for CD14, CD31, CD34 and HLA-DR. Moreover, AT-derived MSCs should

also be capable of differentiation into chondrogenic, adipogenic and osteogenic lineages52.

1.2.6 Translation to Clinical Environment

In light of such attractive properties, MSCs have gathered widespread interest, which led

to multiple efforts to translate MSCs into the clinical set-up as a therapy for various illnesses.

However, with the exception to GvHD, successful establishment of MSCs as a cellular therapy is

yet to be achieved53,54. A major cause for this is the high dosages required, typically ranging

between 106-107 cells per kilogram of body weight55. The efficacy of MSC infusions seems to be

limited as cells fail to migrate and engraft into desired body locations, with the majority of cells

being retained in the lungs56,57 and liver 57, and consequently failing to act as intended.

Furthermore, MSCs have been demonstrated to be a very heterogenous cell population, with

variability not only from donor to donor, but also amongst different tissue sources within the same

donor51. Consequently, MSC usage is highly subject to batch-to-batch variations, hindering not

only their therapeutic potential, but also consistency among experimental results. Therefore, to

address these paramount issues in MSC translation, several approaches are being considered

to augment MSCs therapeutic capacity, such as expansion, genetic engineering, and others.

Since MSCs exist in very low numbers within the body, donation is an inviable approach

for large-scale application, hence alternative routes are necessary. Accordingly, there has been

increasing demand in protocols and platforms for ex-vivo expansion of MSCs to satisfy required

cell numbers for clinic55. Recently, bioreactors such as stirred-tank bioreactors, vertical wheel

bioreactor, hollow-fiber reactors and others, have emerged as alternatives to standard planar

systems (flasks), as they offer large scalability, better homogenization and monitoring, while also

enabling higher cell densities58. Furthermore, to tackle the need for an adhesion substrate, macro

and micro carrier particles have been developed, which provide surface for cellular adhesion

permits MSC adhesion, expansion and augmented cell densities59.

However, many of the culture methods for expansion involve steps and components

which are not compliant with good manufacturing practices (GMP), a set of rules and production

8

criteria that manufacturers must follow to ensure safety and quality of drugs and biologicals used

in clinical settings60. In this context, recommendations to avoid the use of: serum of animal origin

(e.g fetal bovine serum, FBS), other animal (xenogeneic) origin supplements and enzymes, and

undefined media components60,61. The latter problem has been tackled at the research level with

the use of defined and xenogeneic-free culture systems avoiding the use of serum and animal

products, resorting to small molecules and other supplements, such as human platelet lysate

(HPL), as alternatives62.

Another strategy that has been employed to improve the therapeutic features of MSC,

namely in the context of angiogenesis, is genetic engineering63. This aims at manipulating the

genetic content of target cells in order to modify their gene expression and subsequently their

phenotype, physiology and biological activity/potency63,64. Frequently, extra copies of specific

genes are introduced in cells to increase their capacity to produce a certain factor, boosting its

presence and effect. An example of this is the overexpression of VEGF to increase the capacity

of MSCs in triggering angiogenesis65. Notably, alteration of the expression profile of specific

genes not only reduces cellular variability and lot-to-lot variation by standardizing its expression,

but also increase the overall potency of MSCs. Nonetheless, genetic engineering will be

addressed in greater detail in forthcoming chapters.

1.3. The Angiogenic Process

There are two main processes of blood vessel formation and growth: angiogenesis and

vasculogenesis. The latter refers to de novo assembly of vessels due to the differentiation of

precursors, whereas angiogenesis is the process of vessel sprouting from pre-existent ones66.

These processes occur actively during development, but in adulthood they come to halt, with

sprouting happening rarely as vessel cells, mostly endothelial cells (ECs), are in a quiescent state.

Nevertheless, it has been found that ECs can become active and initiate sprouting under

appropriate stimuli67. Here, angiogenesis will be focused not only for simplicity, but also because

it is the type of blood vessel formation targeted with this work. A brief representation of

angiogenesis is depicted in Figure 2, which was taken from Brudno et al. 201368.

Figure 2 - General Representation of Angiogenesis. (A) Signaling factor VEGF and Ang2 activate endothelial cells (B) and pericyte detachment; (C) Sprout formation commences leading to formation of endothelial tip and stalk cells with secretion of Platelet Derived Growth Factor; (D) After stalk cell coalesce and tip cell anastomose there is mural cell recruitment leading to (E) vessel maturation. Image from Brudno et al. 201368

9

1.3.1 Triggering Angiogenesis: EC Activation and Sprouting

In the normal state, quiescent ECs locate adjacent to each other, forming a cylindrical

layer and a tube. Mural cells, both smooth muscle cells (SMCs) and pericytes, surround the

tubular ECs, providing physical and physiological support. In mature vessels, these cells are

interconnected through a basement membrane composed of extracellular matrix (ECM)

molecules forming a sheath around tubes. Moreover, this envelope around ECs traps them,

ensuring their positional permanence and structural stability69.

Under stress or hypoxia, there is the stabilization and activation of specific transcriptional

regulators, namely the hypoxia-inducible factors (HIF), leading to the secretion of several factors,

such as VEGF, HGF, bFGF and others70. These, in turn, will trigger vessel sprouting to enable

tissue reperfusion and restore oxygen supply70. In the presence of VEGF, ECs shift towards an

active state, beginning proliferation, motility and secretive behavior67. In order to sprout, ECs must

escape the basement membrane, for which they secrete metalloproteases. These will mildly

loosen the coating by breaking the ECM and detaching mural cells, allowing ECs to grow and

migrate71. Following degradation, pro-angiogenic and anti-angiogenic factors sequestered in the

basement membrane will be released into the environment, which play a role in regulating

appropriate sprouting direction72. Nevertheless, the degradation process must be balanced, since

exaggerated ECM dismantling impairs sprout growth due to the lack of structural support, thus

requiring tight regulation73. Moreover, various chemoattractant molecules, such as the SDF-1, are

secreted in ischemic sites, which lead to EPCs recruitment and further boost vessel formation74.

1.3.2 Vessel Sprouting: Tip and Stalk Cell Specialization

At each sprouting location, ECs may specify in either tip cells or stalk cells, each

contributing differently to the angiogenic process75. Tip cells locate at the edge of the sprout and

guide sprouting during vessel formation. These cells form filopodia and express surface receptors

that are used to probe the environment for cues76. Opposite to this, stalk cells locate at the basis

of the protrude, fueling and supporting sprout elongation by VEGF-derived proliferation and

growth. These are less prone to form filopodia, but are tailored to support tubes, as they form

junctions and basement membrane with neighbor cells to ensure structural integrity75.

Despite their differences, EC specialization between tip and stalk behavior is a transient

phenotype and not a definitive cellular fate. Moreover, it is a highly dynamic process, where

neighbor ECs compete for tip cell phenotype and frequently alternate between both77.

Accordingly, EC specialization occurs through lateral inhibition mediated by notch signaling,

which works in orchestrated fashion with VEGF to form a feedback loop75. Various ligands and

receptors play a role in this process, such as the DLL4, JAG1, and the VEGFR family (1, 2 and

3)75. Elevated presence of the notch ligand DLL4, a transmembrane protein, has been found in

tip cells, but low in stalk cells. Conversely, JAG1 expression and low notch activity has been

described for tip cells, whereas stalk cells exhibit increased notch activity and JAG1 expression78.

Lateral inhibition at sprouting sites commences following VEGF exposure, where it binds

and activates VEGFR2, leading to DLL4 upregulation in ECs. In turn, elevated DLL4 presentation

10

will activate notch pathway in surrounding ECs, which will lead to a decrease in VEGFR2 and

VEGFR3 expression, while upregulating VEGFR179. Consequently, a cell pattern arises from the

contrasting VEGF receptor expression which will cause a differential response to VEGF

stimulation80. Accordingly, cells expressing VEGFR1 will display stalk cell phenotype and

proliferate, whereas cells expressing VEGFR2 will act as tip cells protruding filopodia77. Therefore,

cells exposed to the highest VEGF concentration or with higher DLL4 content have an advantage

and usually end up becoming tip cells by inhibiting neighboring ECs, which will become stalk cells.

Furthermore, the differential VEGF receptor expression caused by lateral inhibition are also

responsible for enhancing the forming pattern, as they create a feedback loop78.

1.3.3 Vessel Elongation

During vessel formation, tip cells spearhead the sprout by guiding its growth. Various

aspects of vessel growth, such as direction, filopodia and others, must be well regulated to ensure

not only appropriate oxygen and blood supply, but also vessel integrity. Accordingly, tip cells are

known express various receptors which are used to prospect the surroundings for attracting and

repulsing stimuli. Interestingly, similarities between axonal growth and blood vessel elongation

have been found, both in terms of the process and in the intervenient types of receptors81. One

of the most relevant attracting cues is VEGF, which binds the VEGFR2 and NRP82.

Robo4 is a ligand from the roundabout family which is expressed in ECs. It has been

reported to be a repulsive signaling molecule, implicated in vessel integrity and prevention of

hypervascularization and leakiness83. Robo4 antagonizes the VEGF-VEGFR2 mediated

activation of the SRC kinase, preventing permeability. Moreover, evidence suggests that Robo4

also binds the UNC5B, a receptor also associated with repulsion. This receptor is mostly a netrin

receptor, which are molecules secreted by ECs that can have an attractive or repulsive function,

e.g., netrin1 has been shown to contribute to ECs migration and proliferation, but also a repulsive

activity, depending on the receptor it binds84,85.

1.3.4 Lumen Formation: Tip Cell Anastomose and Stalk Cell Coalescence

Proper vessel formation requires the establishment of a lumen, which occur through

different mechanisms. There is evidence that lumen formation is a consequence of vacuolar

coalescence between adjacent ECs, resulting in cell-cell connection, with simultaneous ECs

reshaping to favor lumen structure86. Moreover, tip cells establish interaction with other tip cells

to expand and connect the existing sprouts and network. At certain point, interacting tip cells

anastomose, fusing, followed by consolidation with VE-cadherins. This connection process is

further supported by the deposition of basement membrane87.

1.3.5 Vessel Maturation: Mural Cell Differentiation and Recruitment

At this point, there is the onset of blood flow, which stimulates shear-stress responses in

ECs, important for vessel maturation. Afterwards, as oxygen and nutrients are delivered to target

tissue, VEGF expression decreases, resulting in ECs return to a quiescent state88. The last step

is blood vessel maturation, which occurs at different levels. In terms of structure, vessel networks

will remodel into a ordered network adapted to vascular and tissue patterning88. Another level of

11

maturation is cellular and tissue composition, that occurs through recruitment of mural cells,

mostly pericytes and smooth muscle cells, induced by TGF-β89. Pericytes will set-up a supportive

microenvironment for EC differentiation and remodeling, whereas smooth muscle cells will

provide structural and physical support and regulate blood flow90.

1.4. The Role of MSCs in Revascularization

MSCs are known to support new blood vessel formation through angiogenesis. Reports

demonstrated that the presence of MSCs augmented tube formation and migration of ECs, while

MSC infusion in models of ischemia led to increased tissue perfusion and function74.

Nevertheless, to better harness the therapeutic potential of MSCs in revascularization it is crucial

to probe the pro-angiogenic potential of these cells91. In vitro, functional assays are used to

interrogate the potential of MSCs in triggering pro-angiogenic behavior of model ECs. Various

protocols are available in which the different aspects of sprouting angiogenesis such as tube

formation92, migration93,94, proliferation95 or even wound healing96 can be tested. In these, MSCs’

secretions (i.e. the conditioned medium from MSC culture supernatants) or MSCs themselves are

contacted with ECs under defined culture conditions for a set period of time to allow ECs to

acquire pro-angiogenic behavior. ECs from different sources, such as aorta and other arteries,

and veins, can be adopted as model cells for these assays, but the human umbilical vein

endothelial cells (HUVECs) are the most used due to their availability, relatively easy isolation

and in culture maintance97,98.

In vitro functional assays enable precise control of the environment, thus reducing

confounding effects inherent to parallel processes within whole organisms91. Conversely, in vitro

platforms often fail to recapitulate the complexity of vessel sprouting, in terms of cellular

interactions, stimuli and maturation. Additionally, in vitro tests generally take place on synthetic

environments that barely resemble physiological conditions, all of which lead to disparities

between in vitro and in vivo findings91. Accordingly, in vivo assays become indispensable tools.

These involve the use of intact organisms to assess the angiogenic potential in highly complex

systems and provide an environment closer to physiological conditions. One of most used

techniques is the chick chorioallantoic membrane (CAM) assay, in which the therapeutic agent is

delivered to the CAM, and throughout the following days the CAM may be inspected for changes

in vasculature99,100. Another widespread assay is the cornea angiogenesis assay, where

therapeutic agent is implanted in the corneal stroma of mouse, rat or rabbit. After the defined

period of time, the cornea is explanted and vasculature directly analyzed and quantified101.

1.4.1 Pro-Angiogenic Properties of MSC’s Secretome: Key Signaling Factors

In addition to their mechanical and physical support roles in vessel maturation, MSC’s

paracrine signaling seems to be the major pro-angiogenic effect. Their secretome provides a

spectrum of contributions including: tube formation, EC activation, cellular recruitment, ECM

remodeling, EC proliferation and among others. The array of factors secreted by MSCs include

VEGF, SDF-1, Angiogenin, angiopoietin 1 and 2, HGH, IGF-1, TGF-β, IL-6, IL-8, placental growth

factor, FGF-2, and others, as reviewed by Bronckaers et al13. Notably, it seems that MSCs are

12

involved not only in stimulating and triggering blood vessel formation, but are also present and

preponderant throughout the process, as evident by the variety of factors and subsequent effects.

Nevertheless, of various factors secreted by MSCs influence blood vessel sprouting, only a few

have been further studied for translation into the clinic and progressed to clinical trials.

As far as angiogenesis is concerned, VEGF plays a central role, being the classical factor

under study. VEGF belongs to a family of five factors: A, B, C, D and placenta growth factor, with

VEGF-A being the most thoroughly studied. Additionally, several mRNA isoforms of VEGF-A

exist, the most predominant and relevant being VEGF-A165. Regarding function, it acts as a ligand

for specific tyrosine kinases, Flt-1 and KDR, commonly referred to as VEGFR-1 and VEGFR-2,

respectively102. VEGF has a pervasive role in angiogenesis, since it stimulates the shift from

dormant state to an active state in ECs, thus triggering blood vessel formation. Moreover, it

stimulates EC proliferation and tube formation. Also, its presence has been shown mildly recruit

surrounding cells to sprouting region, which has been explained by its activation of SDF-167. Being

such a pervasive pro-angiogenic factor, several attempts have been made to target VEGF in

vascularization therapies, aiming to increase its stability and expression103,104.

One key factor that has been intensively studied and that has reached clinical trials is the

hepatocyte growth factor (HGF) 105. Although less potent than VEGF, HGF has shown promising

results as therapeutic agent. It acts through tyrosine kinase receptor, c-Met, and boost cell

proliferation and motility. However, the most relevant feature is the promotion of cell survival by

preventing apoptosis, thus boosting the formation and maturation of blood vessels during

sprouting. Also, HGF stimulates the secretion of other pro-angiogenic cytokines106.

The fibroblast growth factor (FGF) family have also shown potent pro-angiogenic

responses. The most extensively studied and used member of this family for therapeutic

angiogenesis is FGF-2, also known as basic FGF (bFGF), which together with VEGF are the most

clinically researched. Resembling VEGF, members of FGF family also bind to tyrosine kinase

receptors, the FGFRs, which also activate related pathways MAPK, PI3K and PLC-γ. FGF is

known to be involved in various processes including would healing, angiogenesis and others.

Focusing in angiogenesis, FGF-2 signaling typically to stimulate migration and proliferation of

cells associated with vessel sprouting, such as ECs and SMCs. Beyond its traditional, evidence

supports an additional role in regulating vessel integrity, where FGF-2 blockade leads to

compromised endothelial junctions.107

1.4.2 MSCs and Hypoxia

Hypoxia is the physiological condition of low oxygen availability (~1-5%O2, compared to

21% in the atmosphere). Hypoxia represents a harmful condition for most fully differentiated cells,

which impairs growth and metabolism, leading to cell stress and eventually death108,109. In the

case of MSCs, however, the state of hypoxia is well tolerated. This is not entirely surprising since

stem cell niches (e.g. the BM) have low oxygen tension110,111. Moreover, hypoxia appears to have

a stimulatory effect in a biphasic fashion, distinguished by short-term and long-term exposure (as

reviewed by Bravkova et al 2014)112.

13

Although not entirely clear, it is widely accepted that the stimulating effect of hypoxia in

MSCs is linked to the HIF family of transcription factors, especially the HIF-1113. Under low oxygen

conditions, the subunits of HIF-1 are stabilized, enabling correct dimerization and activation70.

HIF-1 is a master regulator, controlling the expression of over 1000 genes, being responsible for

activating a variety of genes through the hypoxia-responsive element (HRE)114, leading to

alterations in expression profiles culminating in orchestrated cellular responses70.

Regarding revascularization, studies have shown that MSCs’ pro-angiogenic activity is

also enhanced by hypoxia. This is reflected in terms of elevated expression and secretion of

various factors: VEGF, HGF, bFGF, and others115. Furthermore, MSCs cultured under hypoxic

conditions display higher migration potential, which has been explained and shown by the

upregulation of various receptors including CXCR4, cMet, VEGFR, receptors for SDF-1α, HGF

and VEGF, respectively 116,117.

Despite their tolerance to low oxygen concentrations, the sudden exposure to ischemia

or extremely low oxygen concentration (0-0.5% O2) has been shown to be harmful to MSC,

causing senescence and programmed cell death118. This poses as problem for therapies targeting

ischemic conditions, where MSCs are usually cultured under higher oxygen tensions (9-21% O2)

and subsequently injected into ischemic sites (

14

isoforms α and β are the most pervasive128,129. The different splice forms share the first three

exons, varying only in the fourth which determines functional diversity. The first eight amino acids

are responsible for interacting with the receptor130, while the c-terminal portion stabilizes this

binding through interactions with the ECM and also regulates susceptibility to degradation130.

Furthermore, balanced SDF-1 presence is achieved through proteolytic cleavage at the N-

terminal and C-terminal131. Proteolysis at N-terminus is mediated by dipeptidyl peptidase IV

(CD26), is variant-independent since all share the initial part and impairs biological activity by

disrupting binding capacity132. In turn, C-terminal cleavage is accomplished by metalloproteases,

it is isoform dependent due to the variations presented in this portion, it does not inhibit activity,

but rather affect stability of interaction, decreasing potency and half-life133.

Notably, despite their structural differences, the various isoforms seem to have similar

mechanism of action, i.e. binding to CXCR4 to trigger downstream signaling, which is due to

common N-terminus130. On the contrary, the major difference among the SDF-1 is occurs in terms

of potency and duration of the signal, derived from resistance to degradation and cleavage, or by

stabilized binding to the cognate receptor133,134. SDF-1α is the shortest isoform and the most

active in certain locations, but since it has shorter C-terminus is more prone to degration,

especially in the blood where it has short half-life and hence poor long-distance effect.

Conversely, SDF-1β by possessing 4 extra amino acid residues in C-terminus has resistance to

degradation, having higher potency. Another layer is achieved with ECM interaction, as in the

case of SDF-1γ134. Although it is expressed in low quantities and hence having low immediate

potency, the longer and basic composition of its C-terminus provide stronger affinity with ECM

glycosaminoglycans, which inhibit degradation and promote stronger receptor-ligand binding,

achieving longer action134.

1.5.1. The Role of SDF-1 in Angiogenesis: Physiology and Therapeutics

The presence of SDF-1 in the microenvironment of vessel sprouting is known to actively

attract neighbor ECs, EPCs, SMCs and pericytes121. The recruitment of ECs and EPCs to

neovascularization sites enables docking and incorporation during vessel formation, which will

eventually complement resident ECs and fuel elongation. The attracting cue provided by this

chemokine also contributes to proper network formation, as orchestrated signaling will lead to

appropriate direction of the sprout. Furthermore, the recruitment of SMCs and pericytes will, in

latter stages, support newly formed vessels by providing physical aid and contribute to the

maturation process135. Notably, SDF-1 was found to be highly expressed in ischemic sites136,

since hypoxia and apoptosis provide stimulating cues to chemokine expression. SDF-1 is one of

the genes under HIF-1 regulation, possessing an HRE in its regulatory regions, which is

responsible for its activation in hypoxia121.

Despite central role in angiogenesis, the application of VEGF and other factors, such as

FGF-2 and HGF, in the clinic have yielded inconclusive results. VEGF derived vessels show

hyperpermeability, while also failing to mature properly137. Therefore, and given its putative

involvement in angiogenesis, SDF-1 gathered attention over time as a possible target and

therapeutic agent in revascularization. Of notice, the presence of SDF-1 promotes cell survival138,

15

which coupled to its recruitment activity, is thought to be capable of promoting docking and

permanence of supporting cells, enhancing their engraftment, boosting blood vessel formation

and maturation. In addition, SDF-1 also contributes to vessel formation due to its positive

interaction with VEGF, where its presence increases VEGF expression139. Furthermore, various

studies have been performed using SDF-1 delivery at ischemic sites for revascularization

purposes, either by direct protein injection or cell-derived expression. These have supporting

evidence for the capacity to drive neovascularization in affect body parts, hence SDF-1 holds

great promise as pro-angiogenic therapeutic target. Additionally, SDF-1 is a potential

complementary factor to VEGF-bases therapies. Since VEGF is only capable of retaining

pericytes and smooth muscle cells for a transient period, co-supplying SDF-1, along with VEGF,

could provide useful cues to support VEGF-mediated vessel formation67.

One problem hampering translation of SDF-1 relates to its susceptibility to degradation

and inactivation in the blood, following CD26 and metalloproteinase cleavage. This problem is

being addressed by designing modified versions of this protein, where key amino acids for binding

are replaced to confer resistance to degradation and consequently increase durability and

potency140.

1.5.2. SDF-1 and Hematopoiesis

Hematopoiesis is the process giving rise to blood cells, such as erythrocytes, lymphocytes

and other white cells141. HSCs, which are usually identified by positive expression of the CD34

surface marker142, are responsible for this continuous process as they undergo various stages of

differentiation and commitment. Hematopoiesis starts during embryonic development in the yolk

sac, transitioning to the liver during fetal development and finally settling in the BM in late

development. The chemokine, SDF-1 and its receptor CXCR4, have been shown to be

responsible for homing of HSCs from the fetal liver to BM, as knockouts in either gene lead to

impaired BM-derived lympho- and myelopoiesis126. Moreover, the regulatory role of SDF-1

exerted in HSCs seems to be persist during adulthood, with SDF-1 regulating location and

permanence. HSCs inhabit specific sites within the BM, termed the niche, which is regulated by

CXCL12 gradients secreted by stromal-derived cells143. In agreement, knockouts in this signaling

axis have been correlated with increased circulation of HSC and hematopoietic progenitor cells

(HPCs)144.

In addition to is chemotactic effect, SDF-1/CXCR4 also affects other HSCs homeostasis.

Studies involving CXCL12 depletion have reported increase not only in HSC migration, but also

in proliferation144,145. Accordingly, authors shown the exhaustion of long-term engrafted HSCs

within the BM in absence of CXCR4/CXCL12 or in deletion mutants, coupled with increase in

hematopoietic progenitor population in spleen and blood145.

As far as therapeutic prospects of HSCs are concerned, the SDF-1/CXCR4 axis assumes

a paramount importance. Being one of the major regulators of HSC trafficking and localization,

SDF-1 has been a targeted for various applications involving HSCs. Firstly, disrupting SDF-1

signaling within the BM has been shown to induce HSC mobilization into the peripheral blood.

Such strategies are pertinent during HSC harvest for bone marrow transplantation. Although, it is

16

not the standard approach, plerixafol, a CXCR4 inhibitor, has been used in patients unresponsive

to G-CSF treatment or where its use is not indicated146. Furthermore, SDF-1/CXCR4 is also

present during HSCs engraftment during transplantation. Experiments blocking this cascade of

signaling have reported blocked HSCs engraftment and failure, while enriching harvested CD34+

HSC/HPC for CD184+ expression appears to positively improve transplantation147,148.

1.6. Gene Therapy: Increasing MSCs’ Regenerative Features

MSCs translation into the clinical environment has been hampered by poor migration and

survival, but also by potency variations across different donors53. Gene therapy has stepped as a

route to improve their regenerative potential, by altering the expression of specific genes. Given

the complexity of gene expression and the wide range of tools and technology available, there

are several possible strategies available not only in terms of vectors, but also in terms of targets

and delivery routes149.

Genetic modifications of cells for clinical applications may be performed in vivo or ex vivo.

In the former, the genetic material, a vector containing it, or a conjugation are directly delivered

into the recipients’ body, either locally or infused intravenously. Then, the construction is expected

to reach target cells and successfully cross the membrane and arrive at the nucleus or cytoplasm.

Conversely, ex vivo gene therapy involves specific cell harvest and isolation, with an expansion

step being usually associated with this process, followed by introduction of the desired genetic

modification. The last step involves re-implanting/delivering cells into the patient, which can also

be systemically or locally149,150.

1.6.1 Genetic Modifications: Where to Aim?

Gene expression is the process whereby the cellular machinery accesses a specific DNA

sequence and through a series of reactions and processes synthesizes an RNA, a peptide or a

protein. It is an extremely complex process that involves several mechanisms and tight

regulation151. Alteration of gene expression can be achieved by either targeting the processes

such as DNA availability, transcription and translation, or conversely, promote modifications

affecting products, namely, RNAs and proteins152.

In terms of process, it is possible to aim at regulatory regions, such as the promoter,

enhancers or ribosome binding regions. These sequences are responsible for driving the levels

of transcription and translations, respectively. Hence, by modifying, changing or removing such

sequences it is possible to increase, decrease, or even cancel the expression of a gene153. In

turn, targeting RNAs and proteins frequently aims to modify the stability, structure or other

properties to either activate/inactivate or increase/decrease durability. mRNA translation is

affected by several factors, such as half-life, stability and conformation, which can be manipulated

by directly changing the RNA sequence, or by supplying extra factors or even other RNAs to

stabilize/degrade the target154. One possible strategy is the use of RNA interference, a pathway

where short double stranded RNAs with sequence homology to the target gene can be used to

silence its transcripts155. Likewise, proteins are also subject to conformational changes and

stability, thus being possible to address these issues with alteration of specific nucleotides (and

the respective amino acids), or by targeting specific peptidases and inhibitors140.

17

In addition to site-specific alterations of either the regulatory sequences or of the coding

region, there is an alternative: delivery extra copies of genes or RNAs149. These approaches are

the most frequently used in gene therapy as they are usually simpler to promote and because

targeted gene editing tools are still unrefined for human use. Supplying extra copies of the gene

sequences usually aims increasing the expression of the delivered gene, but it can also be used

to supply the correct copy of a given gene156. Moreover, copies of novel (to the organism) genes

can also be delivered to cells, such as in the case DNA vaccination, where the gene of a

pathogen’s protein is introduced in the subject to drive the expression of a particular antigen,

triggering an immune response and stimulating immunization156.

1.6.2 Gene and Genome Editing

As for genome targeted alterations, these aim to change specific sequences directly

within the chromosomal DNA, thus generating a permanent modification. Although several are

available, the recent clustered regulatory interspaced short palindromic repeats (CRISPR)

associated with Cas9 (CRISPR-Cas9) technology has boosted this approach and became the

most used157. CRISPR-Cas9 allows the user to select a specific region of genome and by

delivering a short RNA oligomer including part of target sequence and the Cas9 protein, it is

possible to disrupt the genome at a precise locus. In greater detail, the Cas9 is an endonuclease

capable of causing double strand breaks in DNA, and also to associate in complexes with short

ribonucleic oligomers. However, unlike most endonucleases, the Cas9 complex does not bind to

strict and specific sequences, but rather having its activity guided by the short RNA sequence. In

the absence of a homologous sequence, following Cas9 cleavage cells will activate the non-

homologous DNA repair mechanisms, which usually lead to altered sequences and knockouts. In

the case of precise sequence modifications, this procedure should be accompanied by the

delivery of another a homologous sequence containing the desired alteration. The presence of

the alternative sequence will elicit homology directed reparation of target locus in the genome,

resulting in cells with edited genome157.

Zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN)

offer an alternative to the CRISPR-Cas9 system158. These systems act in similar fashion to

CRISPR-Cas9, in the sense that they also induce double strand breaks to elicit homologous or

non-homologous DNA repair to modify/disrupt specific sequences. However, TALEN and ZFN

use protein fusions of a DNA cleaving domain with a DNA-binding domain to achieve specific

sequence cleavage. These two strategies differ on their DNA binding domain, where ZFN use a

combination of zinc-finger protein motifs, while TALEN use transcription activator-like effectors

(TALEs). Zinc-fingers usually are domains of DNA-binding proteins that bind specific nucleotide

triplets159. Several zinc-fingers exist, which show differential specificity to different nucleotide

combinations. Therefore, by combining various zinc-finger motifs in tandem, it is possible to build

a construction with affinity to specific DNA sequences. TALEs on the other hand are a class of

proteins with conserved 33-34 amino acid sequences, except for 12th and 13th, which varies

amongst TALEs160. Like Zinc-fingers, each TALE presents binding affinity to specific nucleotide

sequences, thus combining various TALE in tandem, also leads to a protein fusion with high

18

sequence specificity160. As a DNA-cleavage domain, both systems take advantage of the non-

specific DNA cleaving domain of FokI nuclease, which is attached to either the tandem zinc-

fingers or tandem TALEs. The activity of this FokI domain is not restricted to sequence specific

targets, but unlike Cas9 it only acts as a dimer161. Accordingly, TALEN and ZFN strategies involve

designing either two ZFN or TALEN constructs, one targeting each strand of the same target DNA

region. When the two constructs in each strand come into proximity the FokI domains are capable

of dimerizing161, acquiring activity and causing a proximal double-strand break, which will activate

DNA repair causing the desired alteration162.

Although TALEN and ZFN were more established protocols, the recent description of

Cas9-CRISPR approach caused a paradigm shift. This system enables an easier targeting design

as only a ~20mer oligoribonucleotide is required for efficient Cas9 guidance157. Moreover, the fact

that short RNA sequences are easier to synthesize enabled substantial cost reduction, when

compared to TALEN and ZFN. The possibility to clone the desired gRNA sequence in a plasmid

ve