Studies Relating to the Differentiation of Human Embryonic Stem Cells

Studies relating to the differentiation of human

embryonic stem cells

A thesis submitted to the

University of Manchester

for the degree of

Doctor of Philosophy

in the Faculty of Life Sciences

2015

Georgios Anyfantis

List of Figures ...... 10

List of Tables ...... 13

Declaration ...... 15

Acknowledgements ...... 16

Abbreviations ...... 17

Chapter 1: Introduction ...... 23

1.1 Diabetes Mellitus ...... 23

1.2 Pancreatic function and Organogenesis ...... 24

1.2.1 Pancreatic Function ...... 24

1.2.2 Pancreatic Organogenesis ...... 26

1.3 Signalling Pathways associated with pancreas development ...... 26

1.3.1 Signals from Endothelial Cells ...... 26

1.3.2 Fgf Signalling ...... 28

1.3.3 TGFβ signalling ...... 28

1.3.4 Transcription Factors ...... 29

1.4 Markers of Differentiation ...... 30

1.4.1 Using Animal Models in Developmental Studies ...... 34

1.4.2 Differences between human and animal models ...... 35

1.5 The Use of hESC ...... 36

1.5.1 Embryonic Stem Cells ...... 36

1.5.1.1 Historical Perspective ...... 36

1.5.2 Derivation of human embryonic stem cells...... 37

1.5.3 Characteristics of ESC ...... 37

1.6 Self-renewal and pluripotency ...... 38

1.6.1 The LIF Pathway ...... 39

1.6.2 The FGF Pathway ...... 40

1.6.3 The TGFβ family ...... 41

1.6.4 The PI3K/Akt Signalling Pathway ...... 41

1.6.5 Transcription Factors ...... 41

1.6.6 Telomerase Activity ...... 42

1.6.7 Epigenetic Mechanisms ...... 43

1.6.8 Cell Cycle Regulation in hESC ...... 43

1.6.9 The Study of hESC - Potential ...... 44

1.6.10 Definitive Endoderm (DE) – Insulin producing cells ...... 45

1.7. hESC for the production of insulin producing cells ...... 47

1.7.1 The first step – Insulin producing cells from mESC ...... 47

1.7.2 Genetic manipulation of human embryonic stem cells ...... 49

1.7.3 Potential problems with the generation of insulin producing cells from hESC 50

1.7.4 ATP and Purinergic Receptors ...... 50

1.7.5 Identification of functional purinergic receptors ...... 53

1.7.6 The Missing Links - Role of Neural Signalling in Pancreatic Development ..... 54

1.7 Aims and Objectives ...... 55

Chapter 2: Materials and Methods ...... 57

2.1 Tissue culture ...... 57

2.1.1 Human embryonic stem cell culture ...... 57

2.1.2 MEF cell culture ...... 57

2.1.3 Mitotic inactivation of MEFs ...... 60

2.1.4 Generation and collection of hESC conditioned medium ...... 60

2.1.5 Freezing down MEFs and hESCs ...... 61

2.1.6 Thawing hESCs onto mitotically inactivated MEFs ...... 61

2.1.7 Enzymatically passaging the HUES1 hESCs with trypsin ...... 62

2.1.8 Enzymatically passaging the HUES1 and H7 hESCs with collagenase IV ...... 63

2.1.9 Mechanically passaging the HUES1 and H7 hESCs ...... 63

2.1.10 Removing the differentiated parts of a hESC colony between passages ...... 64

2.1.11 Differentiation of HUES1 and H7 hESCs to definitive endoderm (DE) ...... 65

2.1.12 Total RNA extraction from HUES1 and H7 hESC...... 65

2.1.13 DNase I digestion treatment of the extracted RNA samples ...... 66

2.1.14 Assessing RNA Integrity ...... 67

2.1.15 Conversion of Total RNA to cDNA for use in QPCR analysis ...... 67

2.1.16 PCR Amplification of cDNA...... 69

2.1.17 TAE agarose gel electrophoresis of DNA ...... 70

2.1.18 Real-time qPCR analysis of cDNA ...... 71

2.1.19 Choice of Housekeeping Genes ...... 71

2.1.20 Primer Design for PCR Amplification ...... 72

2.2 Calcium fluorescence imaging and microfluorimetry ...... 73

2.2.1 Plating HUES1 hESC on coverslips...... 76

2.2.2 Loading the colonies with Fura-2/pluronic acid and mounting of coverslips on

the experimental platform ...... 77

2.2.3 Test solutions ...... 78

2.2.4 Fitting of the dose response curve ...... 79

2.2.5 P2Y Receptors – Study of Functional Profile ...... 79

2.2.5.1 Stimulation with ATP in the absence of extracellular Ca2+ ...... 79

2.2.5.2 Stimulation with ATP in the presence of cyclopiazonic acid (CPA)...... 80

2.2.5.3 Stimulation with ATP in the absence of extracellular calcium, with the addition

of MRS2578...... 80

2.2.6 Immunostaining of coverslips ...... 80

2.2.7 Confocal Microscopy ...... 82

2.3. Alkaline Phosphatase Pluripotency Assays ...... 82

2.4. Creating a Lentiviral Vector containing the GFP under the influence of the

Pdx1 gene promoter...... 83

2.5 Lentiviral production and testing ...... 85

2.5.1 The pTiger-mPdx1 I-II-III-RFP/rIns2-eGFP vector ...... 88

Chapter 3: Facilitating attachment of HUES1 human embryonic stem cells to glass coverslips ...... 90

3.1 Introduction ...... 90

3.2 Aims ...... 92

3.3 Materials and Methods ...... 92

3.3.1 Preparation of Matrigel coated glass coverslips in 24 well plates ...... 92

3.3.2 Placing glass coverslips on 6 well plates and covering them with Fibronectin

and Laminin ...... 93

3.3.3 Transferring undifferentiated colonies to the glass coverslip coated 24 well

plates...... 94

3.3.4 Observation of colonies and karyotypic analysis ...... 94

3.3.5 Immunostaining for Oct4 ...... 95

3.4 Results ...... 95

3.5 Discussion ...... 100

3.5.1 Matrigel supports low levels of undifferentiated colony attachment ...... 100

3.5.2 Synergistic effects of fibronectin and laminin to support colony attachment

...... 101

3.6 Conclusions ...... 103

Chapter 4: Purinergic Signalling ...... 104

4.1 Introduction ...... 104

4.2 Aims & Objectives ...... 108

4.3 Results ...... 108

4.3.1 Desensitisation of purinergic receptors in hESCs ...... 108

4.3.2 Responses of undifferentiated and randomly differentiated HUES1 hESCs to

ATP stimulation ...... 109

4.3.3 Study of the P2Y purinoreceptor associated signalling in hESCs ...... 112

4.3.4 Responses of undifferentiated and randomly differentiated HUES1 hESCs to

α,β-meATP stimulation ...... 116

4.3.5 Responses of undifferentiated and randomly differentiated HUES1 hESCs to

UTP stimulation ...... 116

4.3.6 Responses of undifferentiated and randomly differentiated HUES1 hESCs to

UDP stimulation ...... 117

4.3.7 Assessment of the effects of purinergic receptor agonists on the pluripotency

of HUES1 hESCs...... 119

4.4. Discussion ...... 124

4.4.1 Purinergic Signalling in hESCs ...... 124

4.4.2 The P2Y Purinergic Profile of hESCs – HUES1 hESCs express functional P2Y6

receptors ...... 127

4.4.3 The pharmacological profile of purinergic receptors expressed in both HUES1

hESCs and their differentiated counterparts ...... 129

4.5 Critical discussion and further experiments ...... 131

4.5.1 The use of Fura-2 AM in our experiments ...... 131

4.5.2 ATP Hydrolysis and degradation in solution – A potential problem (or not)?

...... 132

4.5.3 Further experiments ...... 133

4.6 Conclusions ...... 134

Chapter 5: Development of a reporter construct for the differentiation of stem cells ...... 135

5.1 Introduction ...... 135

5.1.1: The regulation of Pdx-1 expression ...... 137

5.2 Aims ...... 139

5.3 Results ...... 140

5.3.1 Construction and testing of the pLenti-Pdx1 I-II-III-mCherry vector ...... 140

5.3.2 Testing of the pTiger-mPdx1 I-II-III-RFP/rIns2-eGFP vector ...... 146

5.4 Discussion ...... 148

5.5 Conclusions and Future Experiments ...... 151

Chapter 6: The effects of modified glucose levels on monolayer differentiation of hESCs towards definitive endoderm ...... 152

6.1 Introduction ...... 152

6.2 Aims ...... 154

6.3 Materials and Methods ...... 155

6.3.1 Differentiation of H7 hESCs ...... 155

6.3.2 Transfer of differentiating cells to coverslips ...... 158

6.3.3 Passaging differentiating cells on coverslips ...... 159

6.3.4 Immunofluorescent staining ...... 159

6.3.5 PCR Gene Expression Analysis ...... 160

6.4 RESULTS ...... 160

6.4.1 Gene and protein expression of pluripotency markers ...... 160

6.4.2 Gene expression of definitive endoderm associated markers...... 163

6.4.3 Gene expression of pancreatic endoderm and pancreatic progenitor

associated cell markers ...... 165

6.4.4 Morphological and physiological changes of the differentiating cells ...... 167

6.5 Discussion ...... 169

6.5.1 Expression of pluripotency markers during differentiation ...... 169

6.5.2 Expression of differentiation markers during differentiation...... 170

6.5.3 The effects of modified glucose concentrations on the differentiation of hESC

after the establishment of definitive endoderm...... 173

6.5.4 Impaired ability of cells obtained at the end of the definitive endoderm

differentiation protocol to attach on a range of different matrices...... 174

6.5.5 Further experiments ...... 174

Chapter 7: General Discussion ...... 176

Chapter 8: Conclusions ...... 182

REFERENCES ...... 183

WORD COUNT: 62,044

List of Figures

Fig.1.1: Development of the pancreas ...... 26

Fig.1.2: Derivation of ESC line by using immunosurgery ...... 38

Fig.1.3. Stepwise protocol to differentiate hESC to insulin producing cells ...... 46

Fig. 1.4: The Jiang protocol ...... 47

Fig. 1.5: The principle of the Lumelsky Protocol ...... 48

Fig 2.1: Observation of HUES1 hESCs under the brighfield microscope ...... 58

Fig 3.1: Differentiated and undifferentiated colonies ...... 97

Fig 3.2: Oct4 expression in hESC colonies ...... 98

Fig.3.3: Karyotypic analysis of the HUES1 hESC line ...... 99

Fig 3.4. The effects of different cell matrices on the pluripotency of attached hESC colonies ...... 100

Fig. 4.1: P2 Signalling mechanisms ...... 106

Fig 4.2: Expression of P2 genes in HUES7 hESCs ...... 107

Fig 4.3: Desensitization of purinergic receptors expressed on HUES1 hESCs upon repeated stimulation with ATP ...... 109

Fig. 4.4: The pharmacological profile of ATP related purinergic signalling in HUES1 hESCs and randomly differentiated HUES1 hESC cells ...... 111

Fig 4.5: HUES7 hESCs express functional P2Y6 receptors ...... 113

Fig 4.6: mRNA gene expression analysis of the P2Y6 expression in HUES1 hESCs and

MEFs ...... 114

Fig 4.7: Identification of P2Y6 protein expression in HUES1 hESCs, Min6 cells and

MEFs using western blot analysis ...... 114

Fig 4.8: Immunofluorescent and confocal microscopy – Localisation of P2Y6 in

HUES1 hESCs...... 115

Fig. 4.9: Studies on the pharmacological properties of UTP related purinergic signalling in HUES1 hESCs and randomly differentiated HUES1 hESC cells ...... 118

Fig. 4.10: The response of HUES1 hESCs and randomly differentiated HUES1 hESCs to UDP ...... 119

Fig 4.11: Control experiment for the alkaline phosphatase assay ...... 121

Fig 4.12: Purinergic signalling can affect the spontaneous differentiation of HUES1 hESCs...... 123

Fig 5.1: Graphical representation of the structure of the Pdx1 promoter areas I, II and III ...... 139

Fig 5.2: Restriction analysis of the cloned DNA of the constructed pLenti-Pdx1-GFP lentiviral vectors ...... 141

Fig 5.3: Digestion analysis of the pBK mPdx1 I-II-III KSII with the NotI enzyme .. 142

Fig. 5.4: Testing two different methods for the transfection of HEK293T cells with a lentiviral vector ...... 143

Fig. 5.5: Control transfection of HEK293T cells and control transduction of Min6 cells ...... 144

Fig. 5.6: Confirmation of Pdx-1 gene expression in Min6 cells ...... 144

Fig. 5.7: The lentiviral construct had been integrated into the Min6 cell's genome and is expressed into cDNA ...... 146

Fig. 5.8: Restriction analysis of DNA extracted from pTIGER-mPdx1-RFP-rIns1-eGFP clones ...... 147

Fig 6.1: Differentiation of H7 hESCs towards definitive endoderm ...... 157

Fig. 6.2: Expression of pluripotency markers Oct-4 and Nanog ...... 162

Fig. 6.3: Expression of definitive endoderm markers ...... 164

Fig. 6.4: Expression of pancreatic markers ...... 166

Fig. 6.5: The morphological characteristics of differentiating hESCs ...... 168

List of Tables

Table 1.1. Common markers of differentiation ...... 31

Table 1.2: Presentation of cell surface markers specific for ESC from human or mouse origin ...... 39

Table 1.3: Transcription factors expressed both in human and mouse embryonic stem cells, together with a brief description of their known function ...... 42

Table 2.1: Culture medium composition and karyotypic analysis HUES1 and H7 hESC lines ...... 59

Table 2.2: The composition of a typical 50 µl PCR reaction ...... 69

Table 2.3: PerfectProbe Genorm Kit Selection of Endogenous Genes ...... 73

Table 2.4: Alkaline Phosphatase Assay ...... 83

Table 5.1: A summary of the percentages of positive eGFP cells in three runs of

Flow Cytometry Analysis ...... 145

Abstract

Human embryonic stem cells (hESCs) have been a useful tool in the study of the embryo development and could be used by drug developing companies to create disease models and assist in the production of new medicines. One of the models that has been studied before, is the development of the pancreas. Scientists have obtained mixed results so far in the generation of functional pancreatic  cells from hESCs. We studied the differentiation potential of hESCs. As purinergic signalling is involved in may physiological processes, including cell proliferation and differentiation, a study of purinergic signalling in hESCs would help us deeper understand the hESC physiology. In order to study the purinergic profile of hESCs we established a culture system that allowed the transfer and attachment of pluripotent hESC colonies on glass coverslips. We then studied the functional purinergic profile of hESCs and found that they do not express functional P2X1 receptors, but they do express functional P2Y6 receptors, which might be implicated in the hESC differentiation. In parallel to these studies, we developed a reporter gene lentivirus, where the mouse Pdx-1 promoter area controlled the expression of a reporter fluorochrome, eGFP.

We managed to generate a functional lentivirus, however, further analysis is needed in order to be able to use it in developmental studies. Finally, we tested the hypothesis that glucose affects the differentiation of hESCs towards pancreatic endoderm. Our preliminary results suggested that glucose does affect the differentiation potential of hESCs.

Declaration

No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.

i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The

University of Manchester certain rights to use such Copyright, including for administrative purposes.

ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988

(as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made.

iii. The ownership of certain Copyright, patents, designs, trademarks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual

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Acknowledgements

I would like to take this chance to thank my supervisor Dr Karen Cosgrove for her help, advice and encouragement that helped me in all sorts of ways to study for my PhD and finish this PhD thesis. I also have to say a big thank you to my advisor, Prof Susan Kimber, as she was there for me whenever I needed her support and advice. I would also like to thank Dr Dennis Linton, my PhD tutor for his useful advice during the write up period of this

PhD thesis.

I also have to thank Mavvi for sharing her experience with me about the culture of the

HUES1 hESCs, as well as all members of the Cosgrove/Dunne group past and present who helped create a good environment in the laboratory and outside. I have to give special thanks to Lauren for being a good flatmate and a good company inside and outside the laboratory environment, as well as Anastasia for being a good coffee mate during those morning breaks. I also have to thank my good friends, Yannis and Vanessa, as well as all the Greek Cypriot friends in Manchester for their patience and support, as well for good company during my PhD studies. In addition, I have to thank Giannis, Chris, Maria and Evi for being good friends and for being the people who helped me "let it all out" during the difficult times of the PhD through our music filled Friday nights at the, legendary now,

Thalassa by Mykonos.

I would also like to thank Linda, Lyle and all that group I worked with between 2006 and 2008 in Newcastle, every single person from that group, for their continuous friendship, support, advice and for believing in me.

Finally, this PhD study and the thesis would have been incomplete if I did not thank my family, my parents, my brother and my sister, for all the help, support, encouragement, advice and so much more that I cannot express on a piece of paper such as this page of my

PhD thesis. A simple "thank you" cannot fit in the tiniest sense how much grateful I feel towards them for being where I am now.

Abbreviations

2+ [Ca ]i Intracellular Calcium concentration

3D 3-dimensional

ActR Activin Receptor

ADP Adenosine-5'-diphosphate

AP Alkaline Phosphatase

Arx Aristaless related homeobox

ATP Adenosine-5'-triphosphate

-meATP -methylene ATP bFGF basic Fibroblast Growth Factor

CD31 cluster of differentiation 31

PECAM-1 Platelet endothelial cell adhesion molecule

CD9 Cluster of Differentiation 9

Cdc Cell Division Cycle

CDK cyclin dependent kinase cDNA Complementary DNA

CK19 Cytokeratin 19

CPA Cyclopiazonic Acid

CXCR4 C-X-C chemokine receptor type 4

CYC KAAD-Cyclopamine

DAG Diacylglycerol

DAPI 4',6-diamidino-2-phenylindole

DE Definitive Endoderm

DM Diabetes Mellitus

DMEM Dubeco’s Modified Eagle Medium

DMEM-F12 Dubeco's Modified Eagle Medium with Ham's F12

DMSO Dimethyl Sulphoxide

DNA deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate dpc days post conception

DTT Dithiothreitol

E Embryonic Day

EB embryoid body

EC embryonic carcinoma

EDTA Ethylenediaminetetraacetic acid

EGF Epidermal growth factor

EGTA ethylene glycol tetraacetic acid

EN hormone-expressing endocrine cells

ERK extracellular signal-regulated kinases

FBS Fetal Bovine Serum

FGF Fibroblast Growth Factor

FGFR Fgf-receptor

FITC Fluorescein isothiocyanate

Flk-1 Fetal Liver Kinase 1

Flt-1 FMS-like tyrosine kinase

FoxA2 forkhead box protein A2

FoxD3 Forkhead box D3

Fura 2-AM Fura 2-acetoxymethyl ester

GABA -Amino-Butyric Acid

GAPDH glyceraldehyde-3-phosphate dehydrogenase

GATA4 GATA-binding factor 4

GATA6 GATA-binding factor 6

GCK Glucokinase

GDF11 Growth Differentiation Factor 11

GFP Green Fluorescent Protein

GH Growth Hormone

GLP1 Glucagon-like peptide-1

GLP2 Glucagon-like peptide-2

GLUT1 Glucose Transporter 1

GLUT2 Glucose Transporter 2

GLUT3 Glucose Transporter 3

GLUT4 Glucose Transporter 4

Gpc1 Glypican 1

GPRCs G-protein coupled receptors hESC Human Embryonic Stem Cells hGDF3 human Growth differentiation factor-3

Hh Hedgehog

Hk1 Hexokinase 1

Hk2 Hexokinase 2

Hk3 Hexokinase 3

Hk4 Hexokinase 4

HLXB9 Homeobox Gene HB9

HMG-Box High Mobility Group box

HNF1 Hepatocyte Nuclear Factor 1A

HNF4 Hepatocyte Nuclear Factor 4A

Hox Homeobox hTERT human Telomerase Reverse Transcriptase unit

ICAM-2 Intercellular adhesion molecule 2

ICM Inner Cell Mass

IGF Insulin Growth Factor

IgM Immunoglobulin M

IL-6 Interleukin 6

IL-6R IL-6 receptor

ILGF-1 Insulin-like growth factor 1

Ins Insulin

IP3 inositol-1,4,5-trisphosphate

IPF1 Insulin Promoter Factor 1

ISL1 Islet 1 transcription Factor

ITSFn Insulin-Transferrin-Selenium-Fibronectin

JAK Janus Kinase

KODMEM Knock-Out Dubeco’s Modified Eagle Medium

KO-SR Knock-Out Serum Replacement

Kv voltage dependent potassium channels

LB Luria Broth

LIF Leukaemia Inhibitory Factor

LIF-R Leukaemia Inhibitory Factor Receptor

MafA V-maf musculoaponeurotic fibrosarcoma oncogene homolog A

MafB V-maf musculoaponeurotic fibrosarcoma oncogene homolog B

MAP Mitogen-Activated Protein

ME Mesoderm

MEF Mouse Embryonic Fibroblasts mESC Mouse Embryonic Stem Cells

MIXL1 Mix-1 Homeobox Like Protein 1

MODY early Maturity Onset Diabetes of the Young mRNA messenger RNA mTC mouse Teratocarcinoma

NDGF Nerve-derived growth factor

NeuroD1 Neurogenic Differentiation Factor 1

Ngn3 Neurogenin 3

Nkx6.1 NK Homeobox, Family 6, Member A

Oct4 octamer-binding transcription factor 4

O/N Overnight

P2R P2 plasma membrane receptor

P2X Purinergic Receptor Type 2, Subtype X

P2Y Purinergic Receptor Type 2, Subtype Y

Pax4 Paired Box Gene 4

Pax6 Paired Box Gene 6

PBS Phosphate Buffered Saline

PBX1 Pre-B-Cell Leukaemia Transcription Factor 1

PC1/3 Proprotein convertase 1

PCR Polymerase Chain Reaction

PDGF Platelet-derived growth factor

PDK1 Pyruvate Dehydrogenase Kinase

Pdx1 Pancreas/Duodenum Homeobox Protein 1

PE Pancreatic Endoderm and Endocrine Precursor

PF Posterior Foregut Endoderm

PG Primitive Gut Tube

PI3K Phosphatidylinositol-4,5-bisphosphate 3-kinase

PKC Protein Kinase C

PP Pancreatic Polypeptide

PtdIns(4,5) Phosphatydyl Inositol-4,5-bisphosphate

PTF1a Pancreas Transcription Factor 1, subunit A

RA Retinoic Acid

RB Retinoblastoma Protein

RNA Ribonucleic Acid

RNAt Total RNA

RPMI Roswell Park Memorial Institute

RT Room Temperature

S.E.M. Standard Error of the Mean

S.O.C. Medium Super Optimal Catabolite repression Medium shh Sonic Hedgehog sIL-6R soluble IL-6 receptor

Sox17 Sex determining region Y-box 17

Sox2 Sex determining region Y-box 2

Sox4 Sex determining region Y-box 4

Sox9 Sex determining region Y-box 9

SS Somatostatin

SSEA Stage Specific Embryonic Antigen

STAT Signal Transducer and Activator of Transcription

STF1 Somatostatin Transcription Factor 1

TAE Tris Acetate EDTA

Tbn Taube nuss

TGF Tumour Growth Factor beta

UDP Uridine-5'-diphosphate

UTP Uridine-5'-triphosphate

VE-cadherin Vascular Endothelial Cadherin

VEGF Vascular Endothelial Growth Factor

Wnt-3a Winged 3A

Chapter 1: Introduction

1.1 Diabetes Mellitus

Diabetes mellitus (DM) is a metabolic syndrome that comprises of many associated metabolic related conditions. However, the main characteristic of diabetes is the high level of sugars in the blood, called hyperglycaemia. Hyperglycaemia can either be the result of defective insulin production by the pancreas causing an insulin levels deficiency or the inability of the cells to respond to insulin signalling. When the insulin levels deficiency is due to an autoimmune destruction of the pancreatic insulin secreting  cells, the diabetic condition is termed "type 1 diabetes mellitus" or DM1. The type 2 diabetes mellitus is associated with the inability of cells to respond to insulin signalling. It usually occurs after prolonged increasing insensitivity of the insulin target tissues (in particular muscle, liver and fat) to the action of insulin, a condition that is commonly known as insulin resistance (Drong et al., 2012). It is a multifactorial disease, with risk factors including the genetic profile of an individual, as well as age and lifestyle (Drong et al., 2012). The prevalence of diabetes mellitus (DM) has been increased during the past few decades. For example, in places such as the USA, the incidence of DM increased by 49% within the decade of the 90s (Triplitt,

2012). Rough estimations in the US population predict that almost 30% of people over 65 years old are affected by the condition (Triplitt, 2012). In a recent study, it was reported that a 6.8% proportion of all worldwide deaths is attributable to diabetes (Roglic et al., 2010).

Most of the health problems that are attributable to diabetes are associated with the diabetes-specific microvascular disease, which can in turn cause secondary conditions like renal failure, blindness and nerve damage (Brownlee, 2001). The impact such conditions can have in one's life, as well as the financial health costs associated with diabetes-related conditions (Drong et al., 2012) make the research into diabetic treatment essential.

Apart from the pharmaceutical interventions, research has recently turned towards other routes of diabetes management. One of them is the whole pancreas transplantation, which can completely restore insulin secretion levels, leading to normalised blood glucose

23 concentration (Boggi et al., 2012). However, even though the average pancreas transplant life has been increased to almost 17 years, the pancreas transplantation carries on itself some complications, since receiving patients need to be immunosuppressed for long times and there is always the danger of transplant rejection (Boggi et al., 2012). However, factors such as the aging of deceased donors makes the availability of such transplants limited

(Boggi et al., 2012). Pancreatic islet transplantation is a less risky operation, where sufficient numbers of pancreatic islets are extracted from the donor's pancreas and are infused through the portal vein into the patient's liver (Johnson et al., 2012). This technique has led to a proportion of up to 85% of adult patients remaining insulin independent for a year after the transplantation has taken place (Johnson et al., 2012). However, despite the recent advances in immunosuppressive therapies (Shapiro, 2011), the risks associated with the needed immunosuppression are still there. In addition, the steroid based immunosuppressive therapies can be cytotoxic to the transplanted cells (Jamiolkowski et al., 2012). In addition, the islet transplantation usually requires multiple donors in order to collect a number of islets that can be clinically significant (Rickels, 2012), this can create a lack of enough donors.

This creates a need for alternative sources of functional  cells that can produce insulin for the treatment of diabetic patients. This has created a research area that studies and attempts to treat diabetes through the application of a strategy called the three "R"s, which involves cell replacement -through islet transplantation or stem cell differentiation- cell reprogramming -usually from the exocrine part of the adult pancreas- and cell regeneration, which involves the replication and induction of endogenous adult stem cells (Dominguez-

Bendala et al., 2012). A good knowledge of the way the pancreas develops in the early embryo and operates is essential before we are able to study diabetes and investigate such potential treatments.

1.2 Pancreatic function and Organogenesis

1.2.1 Pancreatic Function

The pancreas contains exocrine and endocrine areas (Edlund, 2002). Acinar cells constitute the exocrine pancreas, which produces and secretes a variety of digestive

24 enzymes (e.g. α-amylase and chymotrypsinogen) (Edlund, 2002). The digestive enzymes and bicarbonate ions produced by the acinar cells are transported to the intestine through the highly branched ductal epithelium, which is another part of the exocrine pancreas

(Edlund, 2002). The endocrine pancreas is only a small part of the entire pancreas (Edlund,

2002). It consists of different cell types, namely α-, β-, and δ- pancreatic-polypeptide cells

(Edlund, 2002). These produce the hormones glucagon, insulin, somatostatin (SS) and pancreatic polypeptide (PP) respectively (Edlund, 2002). A new population of pancreatic cells has been identified recently, the ε- cells, which produce ghrelin (Prado et al., 2004). In the mature rodent endocrine pancreas, these cells form clusters called pancreatic islets of

Langerhans, where β-cells are in a central core and they are surrounded by α-, δ- and PP cells (Edlund, 2002). However, controversial reports failed to agree for a long time on the architecture of the human pancreatic islets, with lots of different arrangement models having been proposed (Bosco et al., 2010).

β-cells release insulin in response to increased levels of blood sugar, acting as a signal for target tissues (liver, muscle and fat) to uptake glucose by removal from the blood

(Edlund, 2002). Insulin inhibits the production of glucose in the liver (Edlund, 2002). In contrast to this, glucagon is secreted in response to low blood-sugar levels (Edlund, 2002).

Glucagon acts to promote glycogenolysis and gluconeogenesis and together with insulin acts to keep glucose blood levels steady (Edlund, 2002). Somatostatin and pancreatic polypeptide act as inhibitors on the pancreatic endocrine and exocrine secretion (Edlund,

2002). Finally, ghrelin plays multiple physiological roles, since it is involved in the stimulation of growth hormone release, its role as a hunger signal has been suggested in humans

(Prado et al., 2004). In addition, it inhibits insulin release in rodents and humans by activating voltage dependent potassium channels (Kv) and acts directly on β cells by

2+ attenuating [Ca ]i through its interaction with the growth hormone (GH) secretagogue receptor, a receptor that is expressed in pancreatic islets, amongst other tissues (Dezaki et al., 2004; Prado et al., 2004).

1.2.2 Pancreatic Organogenesis

The intracellular mass (ICM) of the human blastocyst gives rise to the primitive endoderm and the epiblast. The primitive ectoderm arises from the epiblast and it then progresses through gastrulation, a key developmental step. During gastrulation, a major cellular reorganization occurs, giving rise to the three primary germ layers of ectoderm, mesoderm and endoderm. This is the stage when the germ cell lineage is also generated.

The endoderm gives rise to the primitive gut tube, which is then divided into the foregut, midgut and hindgut. Precursor cells at the distal end of the gut tube (at the foregut) give rise to the pancreas amongst other organs (Best et al., 2008). In vertebrates, the embryonic pancreas is created from a dorsal and ventral protrusion of this primitive gut epithelium

(Edlund, 2002), with the dorsal protrusion appearing in humans at 26 days post-conception

(d.p.c.) (Piper et al., 2004). The definitive pancreas is created by the growth, branching and fusion of these two buds (Edlund, 2002) (Fig.1.1).

1.3 Signalling Pathways associated with pancreas development

1.3.1 Signals from Endothelial Cells

The endothelial cells of blood vessels that arise from the mesoderm play an important role in pancreatic development (Lammert et al., 2001), by inducing the dorsal Pdx-1+ endoderm region to express the pancreatic transcription factor Ptf1a and maintain Pdx1 expression (Yoshitomi et al., 2004). Early endoderm expresses VEGF, which attracts the

VEGF receptor expressing blood vessels (Lammert et al., 2001). In addition, the laminin  chains 4 and 5, which are produced by the vascular endothelial cells of the islets upregulate insulin gene expression (Nikolova et al., 2006). Furthermore, the absence of vascular endothelial cells prevents the induction of glucagon expression (Yoshitomi et al.,

2004), confirming the importance of endothelial cells in pancreatic development.

Fig.1.1: Development of the pancreas. (A) Drawings showing initiation of the human dorsal and ventral pancreatic buds at approximately 30 days post-fertilization. Note that the buds appear from opposite sides of the gut tube and that the ventral bud arises from a

26 common outgrowth that will form the liver and gall bladder. (B) A slightly later stage in pancreas development showing the ventral bud moving posteriorily to join the dorsal bud. Endoderm from the gut tube forms the parenchyma of the pancreas, gall bladder, and liver; whereas surrounding mesoderm forms the connective tissue. (C,D) At week 6 of development showing fusion of the dorsal and ventral buds. The main duct is derived from the distal part of the dorsal bud and the proximal part of the ventral. In 10% of cases, the entire dorsal duct remains to form an accessory pancreatic duct. Adapted from (Sadler, 2000)

1.3.2 Fgf Signalling

Fgf-signalling is a key pathway in mouse embryo development. The Fgf family of molecules lists 22 members in mouse and human, which bind and activate the Fgf receptors

(FGFRs). The four types of FGFRs found in mice and humans (Itoh, 2007), exist in several isoforms (Arnaud-Dabernat et al., 2007) and show cytoplasmic tyrosine kinase enzymatic activity and they act by inducing ERK phosphorylation (Arnaud-Dabernat et al., 2007).

FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b and FGFR4 have all been detected in total cellular extracts of the mouse embryonic pancreas by semi-quantitative RT-PCR (Arnaud-

Dabernat et al., 2007), with the FGFR1-IIIb isoform being a pancreatic progenitor cell marker, based on in vitro studies and the wide distribution of its transcripts during the pancreatic development (Cras-Meneur et al., 2002). The FGFR2-IIIb isoform and its ligand,

Fgf-7, have been shown to be essential for the endocrine progenitor cell proliferation

(Elghazi et al., 2002), but they also control the growth, morphogenesis, and differentiation of the exocrine cells of the rat pancreas (Miralles et al., 1999). Fgf-7, together with Fgf-10 in human pancreas development promote epithelial cell proliferation (Ye et al., 2005). Finally,

FGFR3 and its ligands, Fgf-2 and Fgf-9, have been detected in the developing mouse pancreas (Arnaud-Dabernat et al., 2007). Signalling by Fgf-9/FGFR3 limits the expansion of mouse pancreatic epithelial cells (Arnaud-Dabernat et al., 2007), while Fgf-2 induces epithelial cell proliferation in the developing pancreas of rat models (Le Bras et al., 1998).

The importance of Fgf-2 in pancreatic development has also been demonstrated in chick embryos, where it represses Sonic Hedgehog (Shh), allowing the expression of Pdx1 and insulin (Hebrok et al., 1998).

1.3.3 TGFβ signalling

Signalling molecules of the TGFsuperfamily include Activin and Growth Differentiation

Factor 11 (GDF11). Activin induced signalling has been shown to regulate the axial patterning, cell differentiation, and function of foregut-derived organs, like the pancreas. This importance of Activin signalling has been demonstrated by the fact that mutations of Activin receptors, such as the ActRIIA and ActRIIB, can disrupt pancreas formation (Kim et al.,

2000). Another member of the family, GDF11, which can bind the same Activin receptors as above, is also important in pancreatic differentiation. GDF11 null mice show an increase in the numbers of neurogenin 3 positive (Ngn3+) endocrine precursors, while this is accompanied by a decrease in beta-cell numbers, demonstrating its importance in endocrine differentiation (Harmon et al., 2004). The results by Harmon et al. supported the idea that the GDF11 signalling is not directly interconnected with the Notch signalling, which also regulates Ngn3+ cell expansion in the developing pancreas (Harmon et al., 2004).

1.3.4 Transcription Factors

There are many transcription factors that have been identified to be involved in pancreatic development. These include Ptf1, Sox9 and Pdx1, which are involved in different stages of early pancreatic development, others like HNF1, Ngn3 and Pax4, which are found in islet progenitors and more that are involved in the final stages of pancreatic progenitor differentiation, like Nkx6.1, which marks the terminal differentiation of pancreaticcells

(Oliver-Krasinski et al., 2008). One of the most important early pancreatic development transcription factors is Pdx1, (pancreatic and duodenal homeobox 1). Pdx1, also known as

STF1, IDX1, IPF1, IUF1, (Babu et al., 2007) is critical for the regulation of beta cell development at the transcriptional level (Deramaudt et al., 2006). It has been identified as a

β cell-specific transactivator of the insulin and somatostatin genes (Babu et al., 2007). It seemed to be a mammalian ortholog of the endoderm-specific Xenopus laevis XlHbox8 homeobox protein (Babu et al., 2007). The gene encoding Pdx1 belongs to the family of the mammalian Parahox genes cluster on mouse chromosome 5 (Babu et al., 2007). The name

Parahox comes from the fact that these genes represent a group of genes important in development in mammals, but they are found outside the classical Hox (homeobox) cluster of genes (Babu et al., 2007). Pdx1 is needed during the pancreatic buds appearance from the foregut (Deramaudt et al., 2006). However, the expression of Pdx1 at the stage of the bud formation from the foregut does not seem to be linked to the actual bud formation, but it seems to be specifying the early pancreatic epithelium, which will later proliferate, branch and differentiate to produce the mature pancreas (Edlund, 1998). It also plays a role in the maintenance of pancreatic progenitor cells during development. Mice deficient for Pdx1 do

29 not develop a pancreas; while humans with germ line Pdx1 mutations develop early maturity onset diabetes of the young (MODY4) and late-onset forms of type 2 diabetes (Hui et al.,

2002). Besides its importance in early pancreatic development, it is essential later for the formation of the exocrine acinar cells (Deramaudt et al., 2006). It has been reported that maintenance of mature pancreas function also requires Pdx1 (Babu et al., 2007; Gannon et al., 2001). Tissue-specific experiments where Pdx1 is inactivated, have shown that the lack of Pdx1 function in  cells causes dramatic decreases in insulin expression (Gannon et al.,

2001). Pdx1 has also been shown to activate the transcription of genes involved in glucose sensing and metabolism such as GLUT2 and glucokinase (Gannon et al., 2001) The promoter related regions for Pdx1 have been shown to occupy a 6.2kb sequence upstream of the transcription initiation site (Gannon et al., 2001). Characteristic of the Pdx1 promoter are the areas I, II and III, which show between 78 and 89% sequence conservation in blocks of 200–300 nucleotides between humans, mice and chicks. These areas contain potential binding sites for other endodermal transcription factors (Gannon et al., 2001).

1.4 Markers of Differentiation

Other problems that can be created by the use of many cell lines include the potential that some cells might not be fully differentiated in a given protocol. So far, there is no definitive approach applied on mESC or hESC that can give a 100% yield of cells with the required phenotype without applying a selection method. Methods to select for a desired cell lineage include the use of antibodies against a surface marker to positively select for a given cell population (Kobari et al., 2001) or the use of antibiotic resistance imparted under the promoter of a gene of interest to remove cells not of the correct lineage –as in a cell trapping system used by Soria et al. (2000). For a clinical translation, it is essential to be able to purify populations of specific cell types from a complex mix of differentiating stem cells. The removal of undifferentiated stem cells prior to clinical use is critical to prevent the possibility of teratoma formation (He et al., 2003). Table 1.1 summarises the differentiation markers during different stages of pancreatic differentiation, as well as the ones used to monitor vascular differentiation.

Table 1.1. Common markers of differentiation. Adapted from (Asundi et al., 1998; Best et al., 2008; Lipsett et al., 2007; Lyttle et al., 2008; Rylova et al., 2008; Wang et al., 2002b)

Cell type Transcription factor Function Gut tube / DE Sox17 Formation of gut endoderm GATA4 Foregut morphogenesis; differentiation of both exocrine and endocrine lineage; transactivates glucagon gene MIXL1 Mesendoderm development and differentiation FoxA2 Homozygous knockout lethal in mice due to lack of endoderm and notochord; required for alpha-cell lineage - regulates the expression of genes important for glucose sensing in pancreatic beta- cells Gpc1 A glypican: member of a family of glycosylphosphatidylinositol anchored heparan sulfate proteoglycans expressed in specific patterns in different cells at different stages of development CXCR4 Originally a marker of stem cells derived from different adult tissues (Koblas et al., 2007) Pancreas Specification Pdx1 Essential for pancreatic development; differentiation of α-cells and β-cells; transactivator of insulin gene

Sox9 Expressed in all pancreatic progenitor cells; islet organisation; restricted to duct cells later in development Sox4 Broadly expressed in pancreatic buds with subsequent restriction to islets HLXB9 Expressed in all pancreatic progenitors with restriction to beta-cells during differentiation PBX1 Expressed in pre-pancreatic epithelium; required for both exocrine and endocrine differentiation PTF1a Expressed in all pancreatic progenitor cells; essential for exocrine development HNF1 Expressed in early endoderm, regulator of HNF6 expression HNF6 Pancreatic precursor cell specification; regulates PDX1 and NGN3 expression; islet and duct cell formation Endocrine Specification Ngn3 Differentiation of endocrine lineage ISL1 Differentiation of all islet cell types NeuroD1 Differentiation of all islet cell types; islet organisation; transactivates insulin gene IA1 Differentiation of alpha-, beta- and delta-cells Islet Cell Subtypes HNF4 Transactivation of HNF1

and insulin gene HNF1 Transactivation of PDX1 and insulin gene Pax6 Differentiation of all islet cell types; transactivates glucagon gene Pax4 Formation of alpha- and beta-cells Nkx2.2 Transactivation of NKX6.1 and insulin genes in beta- cell precursors Nkx6.1 Differentiation of beta-cells Brn4 Expressed in alpha-cells; transactivates glucagon gene Arx Co-expressed with PAX4; formation of alpha- and beta-cells MafA Transactivates insulin gene MafB Formation of alpha- and beta-cells; controls glucagon expression GATA6 Differentiation of endocrine lineage; expressed in beta- cells GLUT2 Glucose transporter GCK (Glucokinase) Glucose Sensor Insulin Glucose regulator Glucagon Glucose Regulator

Exocrine Pancreas (duct Amylase Enzyme breaking down epithelial cells) starch into simple sugars CK19 Highly expressed in the pancreatic ducts of the adult pancreas (Deramaudt et al., 2006), but is absent

or weak in acini and islets (Means et al., 2008) Vascular Differentiation CD31 (PECAM-1) undifferentiated ES cells express high levels - primarily located at cell-cell junctions – expression sharply down-regulated during early ES cell differentiation (Li et al., 2005) ICAM-2 endothelial adhesion receptor (Bautch et al., 2000) VE-cadherin Regulates endothelial permeability – part of cell- cell adherens junctions - member of cadherin family (adhesion proteins) binds to several protein partners (Dejana et al., 2008) Flk-1 a receptor for vascular endothelial growth factor (VEGF) (Yang et al., 1996)

Flt-1 soluble VEGFR-1 – regulates Flk-1 activation (Purpura et al., 2008)

1.4.1 Using Animal Models in Developmental Studies

One of the proposed forms of staging for human pancreas development defines three developmental stages (Lyttle et al., 2008). According to this staging system, human foetal pancreas development presents three developmental stages. In the first one, between 8–12 weeks, the human developing pancreas primarily contains numerous undifferentiated Pdx-1+ ductal cells, with only a few scattered single endocrine cells and rare small islet clusters by

11 weeks. The second stage lies between 14–16 weeks and is characterised by the

34 presence of numerous small vascularised islet-clusters. Finally, in the last stage, between weeks 19 and 21, adult islet structures with a rich vascular network can be observed.

In similar studies in rats, a different staging system has been proposed, which includes three stages again (Le Bras et al., 1998). During the first phase, no exocrine cells can be seen by immunohistological techniques, but there are some clusters of glucagon- and insulin-expressing cells. During the second stage, while there is not much differentiation towards endocrine or exocrine cells, epithelial cell proliferation is high, causing the rapid growth and lobulation of the epithelial component of the early pancreas and the formation of a network of small ducts. Finally, the third phase is characterized by a massive increase in the activity of the exocrine enzymes, while the number of insulin-expressing cells is rising at the same time (Le Bras et al., 1998).

The above is an example of the use of an animal model in the study of embryo development. In human embryos, gastrulation is not accessible to study, as it occurs during the third week of embryo development. This is the reason animal models are valuable tools in studying embryo development (Best et al., 2008).

1.4.2 Differences between human and animal models

It is important to remember however, that there are some differences in the pancreatic development between different species. The ventral and dorsal outgrowths of the foregut endoderm in mouse embryos appear from embryonic day (E) 9·5, with hormonal expression commencing at E9·5–10, when glucagon expression precedes that of insulin. SS and PP mRNA can be detected at the same time – even though the proteins only appear from E15·5 and E16 respectively. This information displays the rapid progression from foregut endoderm to an insulin-synthesising cell in the mouse (E9·5 to E10) (Piper et al., 2004).

It has been shown that pancreatic differentiation from foregut endoderm in human is initiated at an equivalent time (26 d.p.c) (Piper et al., 2004). However, corresponding embryonic staging between mouse and human would predict significant expression of insulin by 33 d.p.c., which is equivalent to E11. In contrast, a study of human pancreatic development showed that the expression of insulin only becomes apparent more than 2

35 weeks later, at a stage towards the end of the human embryonic period (Piper et al., 2004).

The same study also proved that insulin expression appears before glucagon and all four proteins are expressed at the same time. Furthermore, it was demonstrated that PC1/3, an enzyme that cleaves pro-insulin and is also required for the synthesis of the glucagon-like peptides GLP-1 and GLP-2 is present in mouse -cells, but absent from the human ones

(Piper et al., 2004).

Further differences between human and rodent models, include the expression of

Ngn3, which peaks at 8 to 10 weeks in human embryos and is co-expressed with insulin and glucagon in the early stages. However, such co-localisation has not been observed in rodent models of pancreatic development (Lyttle et al., 2008). These differences however, necessitate the study of pancreatic development using a human model, such as the human embryonic stem cells. 1.5 The Use of hESC

1.5.1 Embryonic Stem Cells

1.5.1.1 Historical Perspective

As early as in 1970, scientists had discovered the potential of cells from mouse teratocarcinomas (mTC), which arise from abnormally proliferating primordial germ cells in male embryos (Artzt et al., 1973) to differentiate into different types of tissues (Kahan et al.,

1970; Rosenthal et al., 1970). Some mTCs could be transplanted intraperitoneally, giving rise to "embryoid bodies" (EBs). These EBs had the potential to differentiate into different cell types when cultured, as well as give rise to primitive teratocarcinoma cells (Artzt et al.,

1973), which are also known as Embryonic Carcinoma cells (EC) (Nicolas et al., 1976). The

ECs can be used to generate cell lines that remain undifferentiated, which when injected into mice give rise to tumours containing tissues corresponding to derivatives of the 3 germ layers (Nicolas et al., 1976). The potential of using the EC cells to study early embryonic differentiation became clearer when Artzt (1973) discovered that they share common surface antigens with early cleavage mouse embryos (Artzt et al., 1973).

Later, Martin reported the generation of a cell line from preimplantation mouse embryos, by culturing them in conditioned medium from teratocarcinoma cells. This cell line

36 could lead to the formation of teratocarcinomas when injected into mice and was also pluripotent, being able to give a wide variety of tissue types when cultured (Martin, 1981).

These cells were then called mouse embryonic stem cells (mESC) (Martin, 1981), a term that was used to distinguish these embryo-derived pluripotent cells from the teratocarcinoma-derived pluripotent EC cells mentioned above (Thomson et al., 1998).

Following the development of mESC from Martin (1981), Bongso et al. (Bongso et al.,

1994) managed to generate human embryonic stem cell (hESC) lines from the ICM of human blastocysts, but these were maintained for two passages only. Later, Thomson generated and characterized a hESC line that was able to grow for long periods (Thomson et al., 1998).

1.5.2 Derivation of human embryonic stem cells

Human embryonic stem cells are derived from the inner cell mass (ICM) of the blastocyst. The separation of the ICM from the blastocyst can be done either by immunosurgery (Fig. 1.2) or by the means of mechanical dissection to remove the zona pellucida. Alternatively, the zona pellucida can be removed using a needle. In order to give rise to hESC the ICM initially had to be cultured on mitotically inactivated mouse embryonic fibroblasts (MEF) (Koestenbauer et al., 2006). However, it has been possible to derive hESC by culturing the ICM on fetal human fibroblasts and postnatal human-skin fibroblasts

(Hovatta et al., 2005) and in a feeder free environment (Klimanskaya et al., 2005). If the trophoblast is not completely removed, the blastocyst can attach to the feeder layer and flatten. This allows the continuous growth of the ICM with the remaining surrounding trophoblast as a monolayer. The expanded ICM can then be broken into smaller pieces, some of which, when subcultured, can give rise to the ESCs (Koestenbauer et al., 2006).

ICM expansion is a technique that can be used to produce both human and murine ESC

(Martin, 1981; Thomson et al., 1998).

1.5.3 Characteristics of ESC

Human and mouse ESCs are characterised by a number of common features ( Box

1.1).

Besides the characteristics defined by Thomson et al. (1998), all ESC share a high alkaline phosphatase activity (Wobus, 2001) – which was a feature first observed in human

ECs (Andrews et al., 1984). They also both express some stage specific embryonic antigens (Wobus, 2001) –SSEA– even though there are differences in the types presented on human and mouse ESC (Table 1.2). In addition, both human and mouse ESC express the germ line transcription factor Oct3/4 (also known as POU5F1) as well as the transcription factors Sox2, FoxD3, Nanog etc. (refer to Table 1.2 for an indication of common transcription factors in mouse and human ESC). A high telomerase activity is another feature shared between the two types of ESC. Finally, they both have a similar cell morphology, with high nuclear to cytoplasm ratio (Wobus, 2001).

Box 1.1: The essential characteristics of embryonic stem cells (Thomson

et al., 1998).

1. They should be derived from the pre-implantation or peri-implantation embryo

2. They should be capable of prolonged undifferentiated proliferation

3. They should have a stable developmental potential to form derivatives of all three embryonic germ layers even after prolonged culture

1.6 Self-renewal and pluripotency

A common feature between mESC and hESC, is their ability to self-renew while staying in a pluripotent state. As many of the studies on hESC are based on similar earlier studies using mESC, it is important to note that the molecular mechanisms behind pluripotency and self renewal are similar in mouse and human ESC but with some known differences including the LIF pathway (see below). Known mechanisms involve both intercellular and intracellular signalling pathways, transcription factor regulation, telomerase activity and epigenetic mechanisms. These are briefly outlined in the following sections.

Fig.1.2: Derivation of ESC line by using immunosurgery. (A) Blastocyst stage. (B) Incubation with whole serum human or mouse antibodies (according to the host organism) and complement; these attack the outer layers of the blastocyst, leaving the ICM intact, as

38 they are unable to break the cell-cell connection barrier of the outer layer of the trophoblast cells. (C) Lysis of trophoblast cells. (D) ICM is cultured on mitotically inactivated feeder cells. Adapted from (Koestenbauer et al., 2006)

Table 1.2: Presentation of cell surface markers specific for ESC from human or mouse origin. (+) is positive, (–) is negative. (Adapted from Koestenbauer et al., 2005)

Surface Antigen Human ESC Mouse ESC SSEA-1 (–) (+) SSEA-3 (+) (–) SSEA-4 (+) (–) TRA-1-60 (+) (–) TRA-1-81 (+) (–) GCTM-2 (+) (–) CD9 (+) (+) Osteopontin (+) (+)

1.6.1 The LIF Pathway

One of the first factors to be discovered in the hunt of molecules which can help maintain pluripotency of ESC was Leukaemia Inhibitory Factor (LIF). LIF is a molecule that had previously been known to induce differentiation of M1 Myeloid Leukemic cells (Williams et al., 1988). The effect of LIF on mESC is to inhibit their differentiation and support their proliferation. Its action is based on two members of the class I cytokine receptors, the low-

39 affinity LIF receptor (LIF-R) and gp-130, which form a heterodimer (Niwa et al., 1998). The gp-130 receptor can also form a homodimer, being able to sustain the undifferentiated status of mESC. This occurs when the cells are exposed to Interleukin-6 (IL-6) in the presence of soluble IL-6 receptor (sIL-6R) – gp-130 was originally identified as the signal transducing subunit of the IL-6R complex. When the sIL-6R is occupied by IL-6, it can bind to anchored gp-130 to activate cellular signalling processes, which suggests that the interaction between

IL-6R and gp-130 occurs extracellularly and that the cytoplasmic region of IL-6R is not required for signalling (Yoshida et al., 1994). Dimerisation of the gp-130 receptors leads to phosphorylation and activation of associated JAK tyrosine kinases. These in turn activate the Ras mitogen-activated protein (MAP) kinase called "extracellular signal-regulated kinase"

(ERK) which follows a downstream signalling cascade and of the STAT factors STAT1 and

STAT3. STAT3 acts to suppress differentiation of the mESC (Niwa et al., 1998). However, even though human LIF can induce STAT3 phosphorylation and nuclear translocation in hESCs, it is unable to maintain the pluripotent state of hESCs (Daheron et al., 2004).

1.6.2 The FGF Pathway

Important signalling molecules in hESC growth and pluripotency include members of the FGF family. In particular, both endogenously produced and externally added basic fibroblast growth factor (bFGF, also known as FGF-2) may be involved in keeping hESC undifferentiated (Dvorak et al., 2005). This point was strengthened by indications that hESC could stay undifferentiated in feeder free conditions when bFGF was present in the feeding medium (Xu et al., 2005). Similar mode of action to bFGF has been attributed to FGF4

(Mayshar et al., 2008). Both FGF2 and FGF4 work in a similar way, by binding to the FGF

Receptor (FGFR) (Xu et al., 2005) and activating the MAPK/Erk1-Erk2 signalling pathways

(Mayshar et al., 2008; Xu et al., 2005), as well as the PI3K/AKT pathway (Li, J. et al., 2007).

However, bFGF expression may continue even after prolonged differentiation (Mayshar et al., 2008) although this is somewhat controversial (Armstrong et al., 2006) and may depend to some degree upon the differentiation pathway taken.

1.6.3 The TGFβ family

FGF also works together with members of the TGF family, namely Activin/Nodal. It seems that the Activin/Nodal signalling pathway is important in the pluripotency status maintenance of hESC and that bFGF action also depends on this pathway (Vallier et al.,

2005).

1.6.4 The PI3K/Akt Signalling Pathway

The MAP/ERK signalling pathway that was mentioned above lies downstream to the

PI3K/Akt signalling cascade (Armstrong et al., 2006; Li et al., 2007) and most of the active components of the PI3K/AKT pathway, including the phosphorylated forms of AKT and

PDK1, are downregulated during hESC differentiation to EBs (Armstrong et al., 2006). The

MEK/ERK kinases have also been found to be targets of the fibroblast growth factor (FGF) pathway in hESCs (Li et al., 2007). However, high basal MEK/ERK activity was required for maintaining hESCs in an undifferentiated state, which does not apply to mESCs (Li et al.,

2007). Furthermore, in contrast to PI3K/AKT signalling, MEK/ERK signalling has been shown to have little or no effect on regulating hESC proliferation and survival (Li et al., 2007).

1.6.5 Transcription Factors

Transcription factors are known to be important in the maintenance of pluripotency of hESC and include the POU domain transcription factor Oct-4, the HMG-box transcription factor Sox-2 and the homeobox protein Nanog (Babaie et al., 2007). Oct-4 acts either on its own or in combination with either Sox-2 or Nanog or both, controlling the expression of a large number of genes (Babaie et al., 2007). The proposed three modes of action of Oct-4 include control of its own expression, the upregulation of genes involved in pluripotency and finally, the downregulation of pro-differentiating genes (Babaie et al., 2007). The detection of

Oct-4 expression in human peripheral blood mononuclear cells (Zangrossi et al., 2007), which would compromise the inclusion of Oct-4 in the family of pluripotency markers, may be attributed to a different Oct-4 isoform, Oct-4B, which is cytoplasmic and does not protect

41 hESC pluripotency (Lee et al., 2006). Table 1.3 presents some of the transcription factors that are involved in the pluripotency of hESCs, as well as in mESCs.

The pathways/factors mentioned above show how complex the regulation of pluripotency is in hESC. There are many pathways which are interconnected, where one of the factors in the pathway acts as its own feedback mechanism or feeds into the other pathways, linking them to each other (Neganova et al., 2008).

Table 1.3: Transcription factors expressed both in human and mouse embryonic stem cells, together with a brief description of their known function. Table adapted from Koestenbauer et al. ( 2005). Surface Antigen Function Oct4 Affects several other transcription factors, e.g. Sox2, Fgf4 and Rex-1 Sox2 Acts synergistically with Oct4 on specific genes Fgf4 Target gene for Oct4/Sox2, Promotes growth of hESC (Mayshar et al., 2008), but not of mESC (Wilder et al., 1997) Utf1 Target gene for Oct4/Sox2 – Maintains Proliferation Rate (Nishimoto et al., 2005) Rex-1 No functional significance (Masui et al., 2008) FoxD3 Repressing differentiation, promoting self-renewal and maintaining survival (Liu et al., 2008) TDGF-1 Direct Target of Oct-4, Co-receptor for NODAL, Antagonist of Activin A (Babaie et al., 2007) Nanog Represses NFkappaB preventing differentiation and acts synergistically with Stat3 to maintain pluripotency (Torres et al., 2008) Taube nuss (Tbn) In mice it has been shown to be required for ICM survival (Voss et al., 2000) hGDF3 BMP4 Inhibitor, Higher Levels in hESC, Lower Levels in mESC support “stemness” (Levine et al., 2006) Fbx15 Not needed for pluripotency (Gearhart et al., 2007)

1.6.6 Telomerase Activity

Another defining factor of the hESC pluripotency, cell cycle regulation, and in vitro differentiation capacity is the Telomerase Reverse Transcriptase unit (hTERT). hTERT is

42 downregulated upon differentiation of hESC (Yang et al., 2008). Its upregulation has a variety of effects on hESC, ranging from an enhanced proliferation and colony-forming ability, to an increase in the S phase duration of the cell cycle at the expense of a reduced

G1 phase; all these can be attributed to its ability to increase Cyclin D1 and CDC6 expression, as well as cause hyperphosphorylation of Retinoblastoma protein (RB) (Yang et al., 2008). Downregulation of hTERT on the other hand, resulted in reduced hESC proliferation, increased G1, and reduced S phase duration (Yang et al., 2008). But the most important effect of hTERT downregulation, is the loss of pluripotency and the initiation of differentiation of the hESC (Yang et al., 2008).

1.6.7 Epigenetic Mechanisms

Pluripotency of hESC is also guided by epigenetic mechanisms. Epigenetic modification is the process where regulation of gene activation and silencing at the level of transcription takes place by regulating how genomic DNA is packaged along with histones into chromatin. Therefore, DNA sequences can either be “tightly” wrapped with histones and condensed into highly folded chromatin fibres, making them resistant to transcriptional activation, or “loosely” associated with histones creating “looped” structures, which makes them permissive to transcriptional activation (Gan et al., 2007). It appears that a variety of

“bivalent” chromatin structure marks are placed on key developmental genes in ESCs, which means that histone around untranscribed genes is modified carrying both active and repressive epigenetic marks, priming the genes for activation or repression upon differentiation. At the same time, genes responsible for pluripotency markers, like Oct-4, are marked with an “active” status (Gan et al., 2007).

1.6.8 Cell Cycle Regulation in hESC

It has been shown that hESC express the cell cycle proteins Cdk2, Cdk4, Cdk6, cyclin

D1, cyclin D3, Cdc25A and c-Myc. These molecules create complexes that control cell cycle progression. Their levels of expression are partially controlled by ubiquitin dependent

43 degradation (Neganova et al., 2008). It has been shown that quite a few of the components involved in cell cycle regulation show cell cycle-dependent expression. An important part of the hESC cell cycle, is the p53 checkpoint signalling pathway. This mechanism can act to protect hESCs by inducing differentiation, cell cycle arrest or apoptosis of those ESCs that have suffered DNA damage. In this way, it can eliminate them from the pool of undifferentiated ESC (Neganova et al., 2008). The hESC have got more mechanisms that respond rapidly to DNA damage for the same reasons. Such an example is the rapid and robust induction of the cyclin dependent kinase (CDK) inhibitor p21(WAF1/CIP1) upon gamma-irradiation, which leads to cell cycle arrest (Becker et al., 2007). Other factors, such as the cell cycle inhibitor p27 also contribute to the hESC cell cycle regulation (Egozi et al.,

2007). The cell cycle of hESCs is also regulated by pluripotency genes, such as Nanog.

Nanog overexpression enhances the G1 to S transition by direct binding to the regulatory regions of Cdk6 and Cdc25A in hESC (Neganova et al., 2008). Oct4 has also been shown to regulate genes linked to the control of cell cycle progression in hESC. Together with

Sox2, it binds to a conserved promoter region of miR-302 (Card et al., 2008). miR-302 is a cluster of eight microRNAs and they are expressed specifically in ESCs and pluripotent cells

(Card et al., 2008). miR-302 targets many cycle regulators, including cyclin D1, linking Oct-

4/Sox2 signalling to hESC cell cycle regulation (Card et al., 2008). It has also been suggested that the regulation of G1 to S transition of ESCs by master pluripotency factors such as Oct4 and Nanog, and perhaps the subsequent shortening of the G1 phase resulting from this control shield the ESCs from activities that will induce their differentiation

(Neganova et al., 2008).

1.6.9 The Study of hESC - Potential

But why should we study hESCs? One of the reasons is their ability to form mesoderm, endoderm and ectoderm tissue components when differentiated in an EB formation or when injected into immunodeficient mice. This can make them valuable tools to study early development in human, solving problems with the study of animal models, which often have different size, growth or anatomy. Furthermore, by learning how to manipulate hESCs using guided differentiation, they could potentially be used for cell based therapies,

44 as they could supply unlimited cells for transplantation. Such technologies will also be valuable to the pharmaceutical industry as the ideal platform for studying toxicology and efficacy of new chemical entities to treat a wide range of diseases. Human embryonic can also be used to study genetic defects associated with disease. Genetic defects could be studied either by direct manipulation and introduction of an artificial genetic mutation or by establishing them from blastocysts already carrying a genetic defect.

1.6.10 Definitive Endoderm (DE) – Insulin producing cells

The genetic manipulation method followed for the mESC differentiation before, was applied later in hESC (Lavon et al., 2006). By overexpressing Pdx1 or FoxA2 in hESC, significant changes were found in pancreatic development markers expression during hESC differentiation (Lavon et al., 2006). However, insulin production was minimal. FoxA2 overexpression was found to have little effect on the differentiation of hESC towards pancreatic insulin secreting cells.

The attempt to differentiate hESC towards insulin secreting cells was a target of more studies. One protocol attempted to replicate the ontogeny of the β-cell on the production of insulin secreting cells from hESC, trying to first obtain DE before moving on to full β-cell differentiation (D'Amour et al., 2005). This protocol was based on the differentiation of hESC in the presence of low serum and Activin A. The resulting cell population consisted of 80%

DE cells, a number they managed to enrich even more by selecting cells that were positive for the surface marker CXCR4. Building up on this knowledge, by adding RA and cyclopamine, a Hedgehog (Hh) inhibitor, further modification to that protocol finally led to the generation of pancreatic hormone expressing cells (Fig.1.3) (D'Amour et al., 2006) –this protocol is commonly called the “D’Amour Protocol”. However, even though the cells produced by this protocol expressed C-peptide, the cells showed minimal response to glucose. This characteristic can be associated with the foetal pancreas, which behaves in a similar way, and suggested that the resulting cells might not have been mature enough

(D'Amour et al., 2006). Later, it was shown that inclusion of PI3K inhibitors could enhance the yield of insulin producing cells (McLean et al., 2007) -similar findings were produced by

45 other studies applied in mESC differentiation (Hori et al., 2002). Use of this information led to the generation of a different protocol, which included Insulin Growth Factor (IGF) in the late differentiation stages (Fig. 1.4) (Jiang et al., 2007). IGF can inhibit the PI3K signalling pathway (Soto-Gutierrez et al., 2008). However, in a similar pattern to the cells originally obtained by the D’Amour protocol, the differentiated cells produced by the protocol from

Jiang (2007) expressed C-peptide, but showed minimal responses to glucose.

Fig.1.3. Stepwise protocol to differentiate hESC to insulin producing cells. Protocol based on knowledge of normal development (Best et al., 2008), showing the expression of key markers for each transition cell population. The differentiation protocol is divided into five stages and the growth factors, medium and range of duration for each stage are shown. CYC: KAAD-Cyclopamine, RA: all-trans Retinoic Acid, DAPT: γ-secretase inhibitor, Ex4: Exendin-4, ME: mesendoderm, PG: primitive gut tube, PF: posterior foregut endoderm, PE: pancreatic endoderm and endocrine precursor, EN: hormone-expressing endocrine cells. Adapted from D’Amour et al, 2006.

Fig. 1.4: The Jiang protocol (Jiang et al., 2007). In this 36-day protocol, hESCs were treated with sodium butyrate and Activin A to generate definitive endoderm co-expressing CXCR4 and Sox17, and CXCR4 and Foxa2. The endoderm population was then converted into cellular aggregates and further differentiated to Pdx1-expressing pancreatic endoderm in the presence of epidermal growth factor, basic fibroblast growth factor, and noggin. Soon thereafter, expression of Ptf1a and Ngn3 was detected, indicative of further pancreatic differentiation. The aggregates were finally matured in the presence of insulin-like growth factor II and nicotinamide.

1.7. hESC for the production of insulin producing cells

1.7.1 The first step – Insulin producing cells from mESC

The first successful attempt to generate insulin producing cells from ESC was described by Soria et al. (2000). Mouse ES cells were differentiated in the absence of LIF and nicotinamide was added at a later stage. This gave only a small population of insulin secreting cells. However, when transplanted into diabetic mice, the population of insulin- secreting cells was able to restore blood glucose levels. This study laid proof-of-concept that it was possible to correct diabetes using insulin-producing cells from embryonic stem cells.

Following this landmark study, many others approached the problems of obtaining β-cells from embryonic stem cells. Later studies have often used relatively complex step-wise approaches and the first of these described was a five-stage protocol based on the generation and use of nestin expressing cells as a starting material (Lumelsky et al., 2001).

Nestin is an intermediate filament protein and it is normally found in neural precursor cells.

The Lumelsky protocol was based on identification of nestin in a population of immature pancreatic cells which did not produce any hormones. When these cells were allowed to differentiate in vitro they generated insulin and glucagon expressing cells. The Lumelsky protocol is summarised in Fig. 1.5.

Fig. 1.5: The principle of the Lumelsky Protocol (adapted from Lumelsky et al. 2001)

Other studies were later based on the Lumelsky protocol. One of the derived protocols included a PI3K inhibitor in the selection/differentiation medium at stage 5 (Hori et al., 2002).

The resulting cells expressed GLUT2, Pdx-1 and glucokinase, which had not been observed with Lumelsky’s protocol before (Hori et al., 2002) and their functionality was confirmed by transplantation in diabetic mice. However, the generation of insulin secreting cells using mESC was later questioned by several pieces of work which failed to confirm these results

(Hansson et al., 2004; Rajagopal et al., 2003; Sipione et al., 2004). The developmental basis of the protocol was criticised as the protocol was designed originally to producing neuronal cells which have a different embryonic origin to pancreatic β-cells (Sipione et al.,

2004). Other criticisms included the fact that insulin immunoreactivity could have been the consequence of insulin uptake from the medium and not endogenous insulin production.

Use of the Lumelsky’s protocol in both human and mouse ESC by another group (Hansson et al., 2004) demonstrated the lack of endogenous insulin production. The resulting differentiated cells could release insulin in the medium, but C-peptide was not detectable, demonstrating that the insulin detected in the cells was not internally produced –insulin could have initially been uptaken from the medium (Kania et al., 2004). An insulin-promoter- controlled GFP vector was used in a later study to exclude this possibility from a

48 differentiation protocol (Shi et al., 2005). This new protocol was different from the Lumelsky protocol in steps like the application of Activin A and RA in the early stages of the mESC differentiation –during EB formation (Shi et al., 2005). It also differed on the presence of serum in the differentiating medium and it did not include the selection of nestin expressing cells, but followed a similar strategy for the expansion and late differentiation of the pancreatic progenitor cells (Shi et al., 2005). In this way, the achievement was the generation of insulin secreting cells that were able to respond to glucose stimulation. These cells were also able to normalize blood glucose levels and rescue the survival of streptozocin-induced diabetic mice (Shi et al., 2005).

1.7.2 Genetic manipulation of human embryonic stem cells

Lumelsky’s attempt was followed by several others trying to produce insulin secreting cells from mESC using genetic manipulation (Blyszczuk et al., 2003; Miyazaki et al., 2004).

Overexpression of Pdx1 and Pax4 in differentiating mESC was found to significantly upregulate pancreatic development related genes, which was more notable in the Pax4 overexpressing cells (Blyszczuk et al, 2003). In addition, when Pax4 overexpression was combined with the selection of nestin-positive cells, the yield of insulin expressing cells was increased by ~30% (Blyszczuk et al, 2003). In another study, mESC were transfected so that the cells would selectively express Pdx-1 when tetracycline-regulated transcriptional activator was present and tetracycline was absent (Miyazaki et al., 2004). The differentiation protocol that was followed afterwards included ITSFn (insulin-transferrin- selenium-fibronectin) serum-free medium, which was used in the Lumelsky protocol before.

However, Pdx-1 expression was induced during EB differentiation and other growth factors were included at different stages of the protocol (Miyazaki et al., 2004). It was found that the cells produced with this protocol expressed insulin 2, somatostatin, and glucokinase, but insulin 1, Glut2, or endogenous Pdx-1 gene expression were undetectable. The knowledge that insulin 1 is pancreatic cell specific in rodents, whereas insulin 2 appears both in pancreatic  cells and in the developing brain, led to the conclusion that the cells obtained through this differentiation protocol were possibly immature (Miyazaki et al., 2004). An

49 alternative approach was to overexpress Ngn3 in differentiating mESC both in an externally inducible system and under the influence of the Pdx1 promoter (Dominguez-Bendala et al.,

2005). A reporter gene controlled under the beta2/NeuroD promoter was also included in the study since beta2/NeuroD is a downstream target of Ngn3. In this way, it was shown that

Ngn3 is essential for pancreatic endocrine differentiation (Dominguez-Bendala et al., 2005).

1.7.3 Potential problems with the generation of insulin producing cells from hESC

While mature, fully differentiated cells could potentially be used for transplantation, the possibility of some cells staying undifferentiated could lead to tumour formation (Best et al,

2008). Furthermore, there has not yet been a fully successful production of insulin producing cells directly from hESC in vitro, that would be responsive to glucose, and this can be attributed to the complex developmental cues that are needed while the pancreas is actually developing, such as 3D assembly, which gives the cells the ability for cell-cell interactions

(Best et al., 2008). Finally, it is important to remember that different laboratories use different cell lines. It has been shown that different cell lines present variable characteristics in aspects such as expression of pluripotency marker molecules, transcriptional profiling and genetic stability (reviewed in (Allegrucci et al., 2007)). This might cause hESC from different lines to behave in a different way to differentiation signals and a differentiation protocol that may be working for one cell line might not act in the same way with another one. As a result, many of the developments made by individual laboratories on the cell lines they use might not be generically applicable. This may cause delays in the potential of transferring hESC- based therapies to the clinic (Allegrucci et al., 2007).

1.7.4 ATP and Purinergic Receptors

ATP was for a long time considered the "energy currency" of intracellular metabolism.

However, studies by Burnstock (Burnstock, 1972; Burnstock et al., 1970), which were initially dismissed or received a lot of scepticism by other scientists (Burnstock, 2009),

50 suggested that ATP was involved in extracellular signalling as well, in particular, in a "novel" for the time mode of neurotransmission, which was neither adrenergic or cholinergic.

Later, in 1978, Burnstock suggested that there were two types of purinergic receptors, one of them being selective to adenosine (he named it P1) and another one that was selective for ATP and ADP, which he called P2 (Burnstock, 1978) . At a later stage,

Burnstock suggested the division of P2 purinergic receptors in two subcategories, defined as

P2X and P2Y (Burnstock et al., 1985) a suggestion that was confirmed in later studies after the successful cloning of several purinergic receptors, which investigated the second messenger mechanisms behind P2 purinergic signalling and revealed that the classification between P2X ion channel receptors and P2Y G-protein coupled receptors was valid (Brake et al., 1994; Lustig et al., 1993; Valera, S. et al., 1994; Webb et al., 1993). These two categories of purinergic receptors presented different potencies to respond to signals from

ATP and its analogues, -methylene ATP (-meATP), -methylene ATP (-meATP) and 2-methylthio-ATP, while they also differ in the ability of -methylene ATP to desensitize some P2 purinoreceptors but not others, as well as the ability of ATP to produce excitatory/inhibitory responses in different tissues or in the same tissue under different conditions (Kennedy et al., 1985). There have been 7 subtypes of P2X receptors identified so far, as well as 8 subtypes of P2Y receptors (Burnstock, 2011). It was initially suggested that only some P2X purinergic receptor subtypes could combine as homo- or heteromultimers of to form ion channels (Nicke et al., 1998). However, more recent studies have suggested that P2Y receptors have the ability to form not only homo-dimers, which has been suggested to be the case for P2Y1, P2Y12 and P2Y13 (Nakata et al., 2010), but also hetero- oligomers, including a hetero-dimer formed between P2Y4 and P2Y6 (Nakata et al., 2010). In addition, hetero-trimers have been reported more recently, such as the one that consists of one P1 receptor (A2A) and two P2Y receptors (P2Y1, P2Y12) (Suzuki et al., 2013) The existence of such hetero-multimers is something that possibly needs to be taken into account when someone studies the pharmacological properties of purinergic receptors. This is a conclusion that stems from the proposed suggestion that hetero-multimerisation can have effects on the metabolism and recycling of a purinergic receptor, or even on the receptor's ligand specificity (Suzuki et al., 2013).

From a functional perspective, the P2X family receptors are ligand-gated ion channels, activated by the binding of ATP to their extracellular domain (Dubyak, 1991). It has been suggested that any functional P2X receptor consists of three subunits, which are arranged in such a way so that they create a central pore (Burnstock, 2007). Each of the three P2X receptor subunits that form the fully functional receptor consist of two hydrophobic membrane-spanning segments, joined by an extracellular loop (ectodomain) containing several conserved amino acids, with roles such as in ATP binding and in receptor polymerisation (Coddou et al., 2011). The current hypothesis for the mechanism of ligand binding suggests that one single ATP molecule binds to the extracellular domain of each of the three subunits. This leads to a conformational change of the receptor, changing its status from a closed state to an open one (Burnstock, 2007). P2X channels are non- selective to the cations they allow through towards the intracellular space, so, both Ca2+ and

Na+ can enter the cell following the receptors activation (North, 2002). The C terminus of the receptor subunits has been shown to affect the pharmacological properties of the receptors, such as their desensitisation rate or their sustained facilitation (with these two terms being antonyms, where the desensitisation is a decrease in the signaling amplitude following sustained agonist application) (Coddou et al., 2011), therefore modulating the length of the associated response.

In contrast to P2X receptors, the P2Y receptors are G-protein coupled receptors

(GPRCs), with P2Y1, P2Y2, P2Y4 and P2Y6 coupling to the Gq/11 G-protein, P2Y12 and P2Y13 associating with Gi and, finally, P2Y14 binding to Gi/o (Burnstock, 2007). The P2Y11 receptor is the only one that can bind to either the Gq/11 or the Gs G-proteins (Burnstock, 2007). The binding of nucleotides through G-protein binding leads to changes in phospholipase C activity, which in turn leads to the generation of inositol (1,4,5) triphosphate (IP3) and diacylglycerol (DAG). In purinergic signalling, IP3 will bind IP3 receptors, which are calcium channels that are found on endoplasmic reticulum (ER) membranes, causing the subsequent release of Ca2+ into the cytoplasm (Saxena et al., 2012), while DAG activates further downstream signalling effects by activating Protein Kinase C (PKC) (Dubyak, 1991).

2+ Increases in intracellular calcium concentrations ([Ca ]i) influence a range of cellular activities including cell proliferation and differentiation. It has recently been shown that ATP

52 as a signalling molecule may affect the mESC cell cycle by promoting their proliferation through effects on protein kinase C (PKC), PI3K/Akt and MAPK/ERK pathways (Heo et al.,

2006).

Even though in vivo calcium signals are usually transient, lasting between a few milliseconds up to a few minutes, the resulting gene expression changes can be long lasting

(Roche et al., 1994). One of the mechanisms explaining this property of calcium signalling is the fact that it influences the expression of the so-called “immediate-early response” genes

(Roche et al., 1994). The transcription of immediate-early response genes, many of which encode transcription factors, is activated almost as soon as the cell is exposed to an extracellular stimulus (Greenberg et al., 1992). The control of transcription factor expression allows the immediate-early response genes to mediate the expression and alter the transcription rate of secondary response genes, resulting in cell specific or ligand specific responses (Herschman, 1989; Roche et al., 1994). The ability of calcium to control the expression of many genes downstream of the immediate-early response genes has increased the interest in trying to understand how calcium homeostasis is linked to gene expression. One way to elucidate the role of calcium in signalling events inside a cell, is the

2+ study of the mechanisms that affect the [Ca ]i levels and the effects produced by the induction of these mechanisms.

Because of the involvement of Ca2+ and all the other second messengers mentioned above, purinergic signalling is involved in almost every aspect of development, pathophysiology, neurotransmission, and neuromodulation (Dalziel et al., 1994), which makes their identification important in understanding processes of the cell. Identification of purinergic receptors in cells can be either direct or indirect. The direct way involves analysis at the mRNA and protein level, using assays such as RT-PCR and western blotting. The indirect way of purinergic signalling detection is based on the downstream effect of

2+ purinergic signalling, which is an increase in [Ca ]i (and in some cases cAMP).

1.7.5 Identification of functional purinergic receptors

In this mode of purinergic signalling detection, which we will from now on call functional analysis, a range of agonists and antagonists of different receptor subtypes is

53 used to assist in receptor identification. Indeed, it has been shown that different subtypes of purinergic receptors respond to different agonists and antagonists. So, we know for example that in heterologous systems, the P2X1 and P2X3 receptors form channels that are activated by both ATP and -meATP, while when P2X2, P2X4 and P2X5 create heterologous receptors, these only respond to ATP and not -meATP (MacKenzie et al., 1999). On the other hand, P2Y receptors present a higher variety in their selectivity for different agonists, so that P2Y1, P2Y12 and P2Y13 are showing a preference for ADP, P2Y2 is equally stimulated by ATP and UTP, P2Y4 shows a preference for UTP, while P2Y6 and P2Y14 show a preference for UDP and P2Y11 is mainly activated by ATP (von Kugelgen et al., 2011). In addition to selective agonists, different purinergic receptors respond to different antagonists, such as the non-selective antagonists suramin and reactive-blue-2 or others that selectively antagonise P2X (eg. NF023) or P2Y (eg. MRS2179) receptors (Ralevic et al., 1998). The differences in the potency of purinergic receptors when it comes to their response to selective agonists or antagonists has been used to identify the functional receptors present in cells. Recent studies have also demonstrated that identification of the presence of purinergic receptors could also be shown by studying the calcium spikes following stimulation of the receptors (Saxena et al., 2012).

1.7.6 The Missing Links - Role of Neural Signalling in Pancreatic

Development

ATP has been shown to stimulate mESC proliferation and differentiation, by causing changes in intracellular Ca2+ levels (Heo et al., 2006). Furthermore, the P2X7 ATP-gated receptor has been detected in early rat pancreas development (Cheung et al., 2007). It is also known that the autonomic nervous system nerves can release ATP as a co-transmitter to noradrenaline (Mortani Barbosa et al., 2000). This could imply a potential role of neural signalling in pancreatic development during pancreatic innervation, in the same way as vasculature formation affects pancreatic development. The neuron types that innervate the pancreas are of the sympathetic, parasympathetic types of the autonomous nervous system, the sensory type and there is an astroglial population as well (Burris et al., 2007). The

54 sympathetic and parasympathetic branches play a role in the maintenance of blood glucose homeostasis by responding to changing energy demands (Burris et al., 2007). The sympathetic neurons act by inhibiting insulin secretion and up-regulating glucagon release in the pancreatic islets of Langerhans. This results to the conversion of glycogen stores to blood glucose so that immediate energy demands are met (Burris et al., 2007). The parasympathetic neurons respond to feeding and stimulate insulin secretion leading to the removal of glucose from the blood and its storage as glycogen in the liver and pancreas, repressing glucagon release at the same time (Burris et al., 2007). The three main neurotransmitters released by the neurons of the central nervous system are -Amino-Butyric

Acid (GABA), glutamate and acetylcholine (Moore, 1993), but the catecholamines adrenaline and noradrenalin are also known to be secreted by neurons, inhibiting insulin secretion

(Baltrusch et al., 2007). It has been shown that innervation and encapsulation of islets occur in tandem with islet maturation (Burris et al., 2007). Even though pancreatic neurons do not form direct synapses with the pancreatic islets, there have been suggestions that sympathetic neurons may be physiologically able to accumulate and secrete noradrenaline during the maturation of endocrine pancreas (Burris et al., 2007). The possibility of the neural signalling affecting pancreatic development is strengthened by the fact that it has been shown that innervation during pancreatic development presents a peak in weeks 14

(for the head of the pancreas) and 20 (for the pancreatic body) (Amella et al., 2008). Week

14 represents the middle stage of pancreatic development (Lyttle et al., 2008), when the endothelial precursors mature.

There has not been an extensive study of the action of purinergic signalling in hESC or the role of it in pancreatic development yet. Such a study would be important, as both the

P2X and P2Y receptors are also linked with the release of insulin from the adult pancreatic  cells (Novak, 2008).

1.7 Aims and Objectives

Even though the research into hESCs has advanced in the past few years, there are still questions on their ability to efficiently produce pancreatic  cells that can respond to glucose stimulation and secrete insulin. In the present study, we investigated the potential of

55 the hESCs to differentiate towards definitive endoderm. In parallel, we studied the functional purinergic profile of hESCs, which is defined as the functional response of hESCs in terms of

2+ changes in [Ca ]i to purinergic receptor agonists and antagonists that are selective for specific purinergic receptor subtypes, with the aim of using the results of our investigation in the probing of the mechanisms of hESC differentiation. We also explored the hypothesis that the mouse promoter region for Pdx-1 is capable of inducing the expression of a reporter gene in a lentiviral delivery system. For this part of the study, we generated a lentivirus that contained the mouse Pdx-1 promoter regions I-II-III controlling the expression of GFP.

Finally, we investigated the potential of glucose to affect the differentiation of hESCs, by differentiating hESCs towards definitive endoderm and subsequently subjecting the cells to increasing glucose concentrations.

Chapter 2: Materials and Methods

2.1 Tissue culture

All tissue culture procedures were carried out under aseptic conditions. All media and solutions that were used in tissue culture, except Phosphate Buffered Saline (PBS) and the trypsin containing solution, were filter sterilised with a 0.22 m syringe filter (Millipore,

o Wattford, UK) before use. Incubators set at 37 C, 5% CO2 were used for all tissue culture incubations.

2.1.1 Human embryonic stem cell culture

The hESC line HUES1 (HSCI iPS Core, Harvard University, USA) were cultured on tissue culture plastic surfaces pre-treated with 0.1% gelatin (type B from bovine skin, Sigma-

Aldrich, Dorset, UK) in autoclaved, distilled and deionised H2O (MilliQ, Millipore, Wattford,

UK) for 1 hr at room temperature and pre-seeded with a feeder layer of MEFs (CD-1 strain,

BSU, University of Manchester) at a suitable concentration (Section 2.1.6). Table 2.1 contains the karyotype, as well as the hESC culture medium constitution for the two different hESC lines used in the duration of the project, namely, HUES1 (Cowan et al., 2004) and H7

(Wicell, Wisconsin, USA). Fig 2.1 presents HUES1 hESC colonies, cultured on a feeder layer of mouse embryonic fibroblasts.

2.1.2 MEF cell culture

The MEFs that were used for hESC culture were previously extracted from embryonic day

13.5 mouse embryos of pregnant CD-1 mice (BSU, University of Manchester, UK) and frozen down in cryovials (Nunc, Denmark) at passage 2 (Section 2.1.5). A cryovial was thawed out for the minimum length of time in a 37oC waterbath and its contents decanted to a 50 ml centrifuge tube (Corning, Amsterdam, The Netherlands) containing 10 ml of pre- warmed MEF medium -Dubeco's Modified Eagle Medium (DMEM) (4.5 mg/ml glucose, no L-

57 glutamine, no sodium pyruvate), with the addition of 10% foetal bovine serum (FBS), 1x non essential amino acids, 2mM L-glutamine and 25 g/ml of each of penicillin and streptomycin, all supplied by Invitrogen, Paisley, UK. The cells were then centrifuged down to wash the cryoprotectant Dimethyl Sulphoxide (DMSO; Sigma-Aldrich, Dorset, UK) contained in the freezing solution away from the cells. The cells were then resuspended in a suitable volume of MEF medium. The cell suspension was plated in a suitable tissue culture flask (Corning,

Amsterdam, The Netherlands).

Fig 2.1: Observation of HUES1 hESCs under the brighfield microscope. HUES1 hESCs at passage 32 can be seen under the microscope (magnification objective = x4). It can be noted that there are different size colonies on that picture and that they lie and grow between the MEF feeder layer (see 2.1.2). Light shaded areas within the colonies represent parts of the colony where the cells have started “packing up” –getting closer to each other- which is characteristic of the way they grow. White arrow: hESC colony; Blue Arrow: MEF Feeders layer

Table 2.1: Culture medium composition and karyotypic analysis HUES1 and H7 hESC lines. A presentation of the karyotypic properties and the constitution of 100ml hESC medium for the HUES1 and the H7 hESC lines. The numbers in parentheses, show the final concentration of each constituent in the medium. The Knock-Out Dubeco’s Modified Eagle Medium (KO-DMEM), Knock-Out Serum Replacement (KO-SR), Dubeco's Modified Eagle Medium supplemented with Ham's F12 supplement (DMEM-F12), L-Glutamine, Non- Essential Amino acids were supplied by Invitrogen (Paisley, UK). The recombinant human basic Fibroblast Growth Factor (bFGF) was supplied by Peprotech (London, UK).

HUES1 H7

Karyotype 46,XX 46,XX

KO-DMEM 77.6 ml -

DMEM-F12 - 77.6 ml

KO-SR 20 ml (20%) 20 ml (20%)

L-Glutamine (200 mM) 1 ml (2 mM) 1 ml (2 mM)

Non Essential Amino Acids (100x) 1 ml (1x) 1 ml (1x)

Human recombinant bFGF (2 g/ml) 400 l (8 ng/ml) 400 l (8 ng/ml)

-mercaptoethanol (55mM) 200 l (0.11 mM) 200 l (0.11 mM)

When the mitotically active MEFs reached 90% confluency, they were passaged by washing the flask with PBS and exposing the cells to a trypsin/EDTA

(Ethylenediaminetetraacetic acid) solution containing 2.5 g/l of trypsin (1:250) and 0.38 g/l of

EDTA•4Na in Hanks' Balanced Salt Solution without CaCl2, MgCl2 and MgSO4 (both PBS and trypsin/EDTA solution supplied by Invitrogen, Paisley, UK). The flasks were incubated in a tissue culture incubator for 5 min until a single cell suspension was generated. The trypsin was inactivated with 10 ml MEF Medium and the cell suspension was centrifuged at 800 x g for 5 min. The supernatant was removed and the cell pellet was resuspended in MEF medium. The cells were then split in a ratio of 1:4 into fresh tissue culture flasks. MEFs at passages 3 and 4 were mitotically inactivated to be suitable for hESC tissue culture.

2.1.3 Mitotic inactivation of MEFs

The mitotically active MEFs were inactivated by exposure to 10 mg/ml of mitomycin C

(Sigma-Aldrich, Dorset, UK) in KO-DMEM (Invitrogen, Paisley, UK). They were incubated with Mitomycin C in a tissue culture incubator for 2 hr. Following inactivation, the MEFs were washed x3 in PBS to remove all traces of mitomycin C. They were then collected using trypsin (section 2.2) and counted using a Neubauer Haemocytometer (Weber Scientific

International, UK). After counting, the cells were either plated down for use to collect conditioned medium (see 2.1.4) or the cells were frozen down in 10% DMSO/FBS at suitable numbers to be used as inactivated feeders at a later stage.

2.1.4 Generation and collection of hESC conditioned medium

In certain experiments, where the HUES1 hESC needed to be cultured in the absence of

MEFs, the use of hESC conditioned medium was employed. The conditioned hESC medium should contain the growth factors that the MEFs secrete to support the growth of pluripotent hESCs. For the production of hESC conditioned medium, the mitotically inactivated cells, after trypsinisation (Sections 2.1.2, 2.1.3), were plated in MEF medium in flasks pre-treated with 0.1% gelatin (type B from bovine skin, Sigma-Aldrich, Dorset, UK) for 1 hr at room temperature, at a concentration of 280,000 cells / cm2 of flask area. After 24 hr incubation in a tissue culture incubator, the MEF medium was removed and the feeder layer was washed once with PBS (Invitrogen, Paisley, UK). After the removal of the PBS, the tissue culture flasks were filled with hESC medium at a volume of 0.4 ml/cm2 of flask area. The hESC medium was collected and replaced daily for one week. The collected conditioned hESC medium from each day was aliquoted immediately upon collection in suitable volumes and frozen down at -20oC. Before being used, the frozen down conditioned hESC medium was thawed out in a waterbath set at 37oC and supplemented with an additional 40 ng/ml of human recombinant bFGF (Peprotech, London, UK).

2.1.5 Freezing down MEFs and hESCs

The mitotically active MEFs at passage 2, the mitotically inactivated MEFs at passages 3 and 4 and the HUES1 hESCs were kept in liquid nitrogen for long term storage. The pellets of cells generated after trypsinisation were resuspended in 200 l of 10% DMSO (Sigma-

Aldrich, Dorset, UK) in FBS (Invitrogen, Paisley, UK). The cells were then transfered to cryovials (Nunc, Denmark), placed in polystyrene boxes and stored at – 80oC overnight, prior to storage in liquid nitrogen.

2.1.6 Thawing hESCs onto mitotically inactivated MEFs

On the previous day to the HUES1 thawing or passaging, mitotically inactivated MEFs were seeded in MEF medium as in Section 2.2 on tissue culture plastic surfaces, at a suitable concentration - 150,000 cells per well of a 6 well plate, 25,000 cells per well of a 24 well plate (both plates from Corning, Amsterdam, The Netherlands) or 50,000 cells per 60 mm x 15 mm organ culture dish (OCD) (Beckton Dickinson, Oxford, UK). Before seeding the cells, the plastic surfaces were treated with 0.1% gelatin (type B from bovine skin, Sigma-

Aldrich, Dorset, UK) in autoclaved, distilled and deionised H2O (MilliQ, Millipore, Wattford,

UK) for 1 hr at room temperature. Prior to thawing or passaging the HUES1 hESCs, the

MEF medium was removed from the tissue culture plates/dishes and the feeder layer was washed once with PBS, before being replaced by hESC medium.

The HUES1 frozen cells contained in a cryovial were thawed using a waterbath set at

37oC for the minimum amount of time needed. The contents of the cryovial were then removed into a centrifuge tube (Corning, Amsterdam, The Netherlands) containing 10 ml of pre-warmed KO-DMEM. The cell suspension was centrifuged at 650 x g for 3 min. The supernatant was removed and the cell pellet was resuspended in a further 10 ml of pre- warmed KO-DMEM. The centrifugation step was repeated in order to remove any traces of the cryoprotectant DMSO containing solution. The supernatant was removed once more and the cell pellet was resuspended in a suitable volume of hESC medium. The cells were then plated on the mitotically inactivated feeders.

2.1.7 Enzymatically passaging the HUES1 hESCs with trypsin

The HUES1 hESCs in culture form colonies that need to be passaged at regular intervals, in order to keep the cells growing in an undifferentiated state.

Enzymatic passage of the HUES1 hESCs was employed when the growing cells presented minimal differentiation, with colonies presenting clearly defined and smooth edges and cells that presented the morphological characteristics of hESCs (visible nucleoli, high nucleus to cytoplasm ratio). It was used as a way to bulk up the numbers of growing cell colonies.

The hESC medium was removed and replaced with the minimum volume of trypsin/EDTA solution (Invitrogen, Paisley, UK) to cover the tissue culture area. The cells were then incubated in a tissue culture incubator until the feeder layer was sufficiently retracted from the colonies. At this stage, the hESC colonies presented a rough edge. The trypsin/EDTA solution in the tissue culture area was then diluted with 10x volume of hESC medium. The cells were then resuspended by being pipetted up and down through a 5 ml pipette (Corning,

Amsterdam, The Netherlands) (when they would be passaged onto wells of a 6 well plate) or through a Gilson 1ml pipette (Anachem, Luton, UK) (when they would be passaged onto wells of a 24 well plate). The different pipettes were used to generate different sizes of cell clumps -the 5ml pipette would generate clumps that would be too big for a 24 well plate, which would cover a lot of the surface of the well and would present higher differentiation rates. The hESC cell suspension was then split at a suitable ratio on new plates/dishes pre- seeded with mitotically inactivated feeders (Section 2.1.6).

On the first day after passaging, the hESC medium already present in the tissue culture plates/dishes was supplemented with fresh medium. From the second day after passaging onwards, the hESC medium was changed every day.

2.1.8 Enzymatically passaging the HUES1 and H7 hESCs with collagenase IV

An alternative way of passaging the HUES1, when they presented minimal differentiation, was by using Collagenase Type IV (Invitrogen, Paisley, UK). This was also the method of preference when passaging the H7 hESCs, as trypsin single-cell passaging in this cell line has been associated with the accumulation of karyotypic abnormalities, since the most commonly acquired ones have been shown to offer affected hESC populations a growth advantage (Chan et al., 2008).

The hESC medium was removed and replaced with 1 mg/ml collagenase IV in KO-DMEM

(Invitrogen, Paisley, UK). The cells were returned back to the tissue culture incubator until the hESC colonies started to retract from the feeder layer and their edges started to lift from the plastic culture surface. The collagenase was then diluted with hESC medium, pre- warmed at 37oC, and the undifferentiated colonies or the undifferentiated parts of them

(identified by light intensity differences over the phase contrast microscope) were selectively resuspended and broken into smaller pieces with the use of a Gilson 200l pipette

(Anachem, Luton, UK), while viewed under a dissecting microscope (Model SMZ1000,

Nikon) in a class II safety cabinet. The colony suspension was then re-plated on a MEF feeder layer (Section 2.6) at a suitable ratio and returned to the cell culture incubator.

The cells were fed with hESC medium as previously described (Section 2.1.1).

2.1.9 Mechanically passaging the HUES1 and H7 hESCs

When the tissue culture of the HUES1 or H7 hESCs contained a mixture of non- differentiated and differentiated colonies, mechanical passaging them was employed. This method is based on the use of a hand-held needle to selectively cut and lift from the tissue culture plate/dish the complete non-differentiated colonies or the undifferentiated parts of them, in the presence of freshly changed hESC medium. The technique was carried out aseptically under a dissecting microscope (Model SMZ1000, Nikon) in a class II safety

63 cabinet. The colony suspension was then re-plated on a MEF feeder layer (Section 2.1.6) at a suitable ratio and returned to the cell culture incubator.

The cells were fed with hESC medium as previously described (Section 2.1.1).

2.1.10 Removing the differentiated parts of a hESC colony between passages

During culture of the hESC colonies, some of the cells in the colonies were spontaneously differentiating. This is a normal feature of all hESC lines. As the colonies grew on the tissue culture area, the colonies would become compact, starting from the colony centre (Johkura et al., 2004). For the HUES1 cell line, the differentiation seemed to be affecting the whole colony most of the times. So, when a colony reached a critical size, it would differentiate.

For the H7 cell line, the differentiation presented a pattern, where it would start both from the centre of the colony spreading outwards, as well as from the periphery of the colony spreading inwards. The differentiated outer edges of a colony would show up as brighter areas under a dissecting microscope, while the differentiated colony centres would present as a “mushroom” formation, extending vertically from the colony.

A hESC colony that is surrounded by differentiated cells has been shown to have inhibited expansion of the undifferentiated areas (Johkura et al., 2004). Therefore, it was essential to monitor the growth of hESCs regularly and to remove their differentiated parts. In the case of HUES1, this could involve the removal of whole colonies that had undergone spontaneous differentiation.

Removal of the differentiated parts of the hESC colonies was performed using a BD

Microlance 3 Nr.14 (23G x 11/4”) hand-held needle (Beckton Dickinson, Oxford, UK). The needle was gently dragged along the periphery of each single colony, removing differentiated parts of the colony on the way, as they were more loosely attached to the tissue culture plastic. The “mushroom” formations at the centre of the colony were also removed by flicking them off with the use of the needle.

Following the cleaning of the hESC colonies, the hESC medium was replaced with fresh medium, in order to remove the differentiated parts from the culture space and prevent them from re-attaching onto the tissue culture plastic.

2.1.11 Differentiation of HUES1 and H7 hESCs to definitive endoderm (DE)

In these experiments, HUES1 cells were subjected for 24hr to 100 ng/ml activin A in RPMI

(Roswell Park Memorial Institute) basal medium (Invitrogen, Paisley, UK) with the addition of

2mM L-glutamine, in the absence of any serum. On the second and third day of differentiation, 0.2% FBS (Invitrogen, Paisley, UK) was added to the cells. At the end of this three day differentiation protocol, total RNA was extracted from the cells (see 2.1.12) to be used in QPCR studies of the expression profile of important development markers, like Pdx1,

CXCR4, FoxA2 and Sox17. Suitable controls were employed for this experiment, which included the extraction of total RNA from undifferentiated HUES1 hESC, as well as from

HUES1 hESC that had been purposely left to randomly differentiate for the same time period

(three days) in the presence of medium containing DMEM (Invitrogen, Paisley, UK) including

10% FBS.

For the H7 cell line, the protocol followed was exactly the same, apart from the first 24 hr of differentiation, where the RPMI medium was also supplemented with 25 ng/ml Wnt-3a.

The Wnt-3a was not used earlier as it is quite expensive, however, there were reports that suggested it improved the survival of the differentiating cells (Hussain et al., 2004), so, it was decided that it would be included in our protocol.

All differentiations were performed on 6 well tissue culture plates (Corning, Amsterdam,

The Netherlands).

2.1.12 Total RNA extraction from HUES1 and H7 hESC

In order to study the gene expression profile of the hESCs before and during the differentiation towards a definitive endoderm phenotype, RNA was extracted from the cells,

65 using the RNEasy kit (QIAGEN, Crawley, UK). All steps were carried out at room temperature. The centrifugation steps, when these were used, involved running the samples in a microcentrifuge at 12,000 x g for 15 s, except where otherwise stated.

The cells were washed once with PBS (Invitrogen, Paisley, UK). 600 l of RLT lysis buffer from the kit, with the addition of 10 l mercaptoethanol per ml of buffer, was added directly to the cell layer in each well of the 6 well plate. The cells were lysed by pipetting up and down, using a 1ml Gilson pipette (Anachem, Luton, UK). The cell lysate was transferred to a 1.5ml eppendorf (Starlab, Milton Keynes, UK). The lysate was then homogenised, by being passed repeatedly through a 21G needle (Beckton Dickinson, Oxford, UK). This was followed by addition of 1x volume (600 l) of 70% ethanol. The sample was mixed by pipetting up and down through a 1ml Gilson pipette and 700 l of the mix was transferred to an RNeasy column. The column was then centrifuged in a microcentrifuge. The flow through was discarded, the column was re-filled with the rest of the mix and the centrifugation step was repeated. Following that step, 700 l of the kit buffer RW1 was added to each RNeasy column, which was then centrifuged and the flow through was discarded. The next step involved washing of the column twice with 500 l of the kit buffer

RPE, with centrifugation of the column after each wash and disposal of the flow through.

After the second wash and centrifugation step, the RNeasy column was placed in a fresh

1.5ml collection tube, supplied with the kit, and centrifuged for a further 2 min, to remove all traces of the RPE buffer. The column was finally transferred to a fresh 2 ml tube, supplied with the kit. 50 l of RNAse/DNAse free water (supplied with the kit) was applied to the centre of the column membrane. It was centrifuged for 1 min. The flow through contained the extracted RNA.

2.1.13 DNase I digestion treatment of the extracted RNA samples

Before the extracted RNA could be used to monitor gene expression in the cells, it should undergo DNase I digestion, to eliminate the presence of any contaminating DNA, which would interfere with the gene expression analysis.

The Ambion DNA-free DNase treatment kit (Applied Biosystems, Warrington, UK) was used for this step. 5 l of DNA-free 10x buffer were added to the extracted RNA sample, together with 1 l of the supplied DNase I solution. The samples were then incubated for 30 min at 37oC in a thermal cycler (Techne, TC-512). Following that step, the samples were supplemented with 5 l of DNase inactivation buffer (supplied with the kit) and incubated for

2 min at room temperature, with gentle flicking of the tube containing the sample to mix the contents at regular intervals. The samples were then centrifuged at 10,000 x g and the supernatant was carefully removed into a fresh 1.5ml eppendorf tube (Starlab, Milton

Keynes, UK)

2.1.14 Assessing RNA Integrity

The integrity of the extracted RNA was analysed using the Agilent 2100 bioanalyser using the RNA 6000 Nano LabChip® kit (both from Agilent Technologies, South Queensway, UK), in order to identify any possible degradation of the RNA during the extraction procedure.

The Agilent analyser ran the RNA samples and a ladder on the LabChip® and separated them according to size. The bioanalyser then used an algorithm to determine the integrity of the RNA samples. Each sample was given an RNA integrity number, which, as per the manufacturer's instructions, defined whether the RNA in the sample was intact or degraded.

2.1.15 Conversion of Total RNA to cDNA for use in QPCR analysis

Before PCR amplification could be used to study the gene expression in the extracted

RNA, the mRNA contained in the extracted total RNA was converted to cDNA.

cDNA conversion was performed either with the Superscript II reverse transcriptase enzyme (Invitrogen, Paisley, UK), for the HUES1 hESCs, or with the RT-QScript kit

(PrimerDesign, Southampton, UK), for the H7 hESCs. This was due to better price negotiated for the PrimerDesign kit during our study. For all parts of each reaction that required heat, a thermal cycler was used (Techne, TC-512)

For the Superscript II protocol, a 20 μl reaction volume was used for 1 μg of total RNA.

The following components were mixed for each reaction: 1 μl Oligo (dT)12-18 (500 μg/ml)

(Invitrogen, Paisley, UK) and 0.1 μl of a 100 mM dNTP mix (Bioline, London, UK) were added to 1 μg of total RNA and the reaction volume was brought up to 12 μl with sterile, distilled water. The mixture was heated to 65°C for 5 min, in order to unfold the total RNA, and quickly chilled on ice. The contents of the tube were briefly centrifuged in a microcentrifuge and the following components were added to each reaction: 4 μl of 5x First-

Strand Buffer, 2 μl 0.1 M dithiothreitol (DTT) and 1 μl RNaseOUT recombinant ribonuclease inhibitor (40 units/μl) (all from Invitrogen, Paisley, UK). The contents of the tube were mixed gently by flicking the tube. The reaction was then incubated at 42°C for 2 min, to allow the binding of the oligo (dT)12-18 primers to the RNA template. Finally, 1 μl (200 units) of

Superscript II reverse transcriptase were added to each reaction, the contents were mixed by pipetting gently up and down and the tubes were incubated for 50 min at 42°C, to allow the extension of the cDNA strand. The reaction was inactivated by heating at 70°C for 15 min. The cDNA could now be used as a template for amplification in PCR.

The primerdesign Precision Reverse Transcription Kit (Primerdesign, Southampton,

UK) was used for the studies in H7 cells due to its lower price compared to other options. For the conversion of mRNA into cDNA using this kit, initially, 1 μl of Oligo-dt primer was applied to 1μg of total RNA and the volume was subsequently adjusted to 9 μl with nuclease-free water (Promega, UK). Samples were then incubated at 65 oC for 5 min, a step that should prevent the formation of any RNA template secondary structures. The samples were then immediately cooled by placing on ice. With the samples remaining on ice, a master mix was prepared consisting of 2 μl qScript (10x) buffer, 1 μl of 10 mM dNTP mix, 2 μl of 0.1 M DTT,

4 μl RNAse- and DNAsefree water and 1 μl of qScript enzyme per reaction. 10 μl of the master mix was then added to each sample. The samples were then briefly vortexed and centrifuged at 8,000 x g using a bench top mini spin plus microcentrifuge (Eppendorf,

Histon,UK). This was followed by incubation of the samples at 55 oC for 20 min, when the reverse transcription actually occurred. Finally, the reaction was stopped by heat-inactivation at 75 oC for 15 minutes.

Each reverse transcription reaction, whether conducted with the Superscript or the qScript reverse transcriptase, included a control which had been processed in the same way as all other samples, minus the addition of the reverse transcriptase enzyme, the volume of which was substituted with H2O. Absence of a signal (gel band or real-time QPCR product) when running this sample would confirm that no DNA contamination was carried over from the RNA isolation procedure.

All cDNA samples were stored in a -20 oC freezer until needed for further analysis.

2.1.16 PCR Amplification of cDNA

PCR amplification was used to study the gene expression of the cDNA produced from the procedure described in 2.1.13-2.1.15.

Each PCR reaction was set to a final volume of 50 l. All steps of the PCR amplification were carried out in a thermal cycler (Techne, TC-512).

Table 2.2: The composition of a typical 50 µl PCR reaction.

10x PCR Buffer (Invitrogen, Paisley, UK) 5 L (1X)

50 mM MgCl2 (Invitrogen, Paisley, UK) 1.5 l (1.5 mM)

100 mM dNTPs (Bioline, UK) 0.1 l (0.2 mM)

Forward primer (10 M) 1 l (0.2 M)

Reverse primer (10 M) 1 l (0.2 M)

Taq Polymerase (5 Units/l; Invitrogen, 0.4 l (2 Units)

Paisley, UK)

cDNA (see 2.1.15) 2 l

Autoclaved, distilled water 38.1 l

The components of each PCR reaction are shown in table 2.2. A master mix was always prepared on ice, containing everything apart from the cDNA. The master mix was aliquoted

69 into a suitable number of 0.2 ml thin-wall PCR tubes (Starlab, Milton Keynes, UK) and the cDNA was added to each single PCR tube. Water was added to one PCR tube, instead of cDNA, to act as a negative control, to eliminate the possibility of DNA contamination in any of the buffers and components used in the reaction. In addition, one PCR tube contained

100 g human genomic DNA (Promega, Southampton, UK). This sample acted as the positive control, to confirm that the reaction components were working.

2.1.17 TAE agarose gel electrophoresis of DNA

A 1.5% agarose gel (Melford Laboratories Ltd, Ipswich. UK) in TAE buffer (Bio-Rad

Laboratories, Hemel Hempstead, UK), which contained 2.86 x 10-4 mgI/ml of ethidium bromide (Promega, Southampton, UK), was prepared. The DNA was mixed with an appropriate volume of blue/orange 6x loading dye (Promega, UK), to facilitate the gel loading and provide a way of tracking the migration progress of the PCR products during the agarose electrophoresis. A DNA ladder was loaded together with the DNA samples. The

DNA ladders used were either the bench top premixed 100 base pair (bp) DNA ladder

(Promega, UK) or the Hyperladder I (1Kb; Bioline, UK), depending on the size of the expected product. In general, PCR reaction products were run with the 100 bp ladder, while the Hyperladder I was used for digested products from the restriction analysis of vectors.

After the DNA samples had been mixed with the loading dye, they were loaded on the agarose gel in a suitable gel electrophoresis apparatus (Biorad, UK) and run at 100 Volt until suitable separation of the DNA marker bands was achieved. The gel was then removed from the electrophoresis apparatus and viewed using a UVIDoc gel documentation system

(UVItec, UK). Images of the full gel, as well as of the areas of interest, were then taken using the UVIDoc system, and downloaded on a personal computer by means of a network connection to the system.

2.1.18 Real-time qPCR analysis of cDNA

In order to analyse gene expression, the cDNA samples were amplified using realtime qPCR. The qPCR reactions were set with the Precision-LR kit (PrimerDesign, UK), using their SYBR-Green master mix, which contained a ROX reference dye (Invitrogen, UK), a dye that is designed to normalize the fluorescent reporter signal in real-time quantitative PCR. In more detail, the ROX reference dye is used by the qPCR machine to normalise any fluctuations in fluorescence between reactions. A master mix was created, which contained

4 ml nuclease-free water, 10 l SYBR-Green Precision-LR master mix and 1 l of primer. 15 ml of this master mix were added into each well of a 96-well optical plate (Applied

Biosystems, UK). This was followed by the addition of 5 ml of cDNA to give a final reaction volume of 20 μl. As an internal control, it was ensured that all cDNA samples were at the same concentration before adding to the reaction. This was an assumption made by the fact that all mRNA to cDNA reverse transcriptions were run in batches for all samples needed for a real-time qPCR analysis, using the same amount of mRNA for all cDNA conversions. The optical plate containing the qPCR reactions was sealed with nuclease free optical film

(Applied Biosystems, UK) and centrifuged at 1000 xg for 5 minutes at 4 oC. The plate was then loaded in an ABI 7500 thermal cycler (Applied Biosystems, UK) and run for 50 cycles, using the conditions recommended by PrimerDesign (UK) in their SYBR-Green master mix protocol. This consisted of the following cycling parameters: 95 oC for 10 min, 95 oC for 30 sec, 50 oC for 30 sec, 72 oC for 15 sec. A melting curve analysis was set to run at the end of each qPCR reaction in the Applied Biosystems thermal cycler. This step was utilized in order to confirm the specificity of the primers used in the qPCR reactions. The results were collected and analysed using the 7500 system sequence detection software v1.2.3 (Applied

Biosystems, UK).

2.1.19 Choice of Housekeeping Genes

Much of the cell work that was carried out during this study included differentiation of hESCs. This could imply that the expression of any potential housekeeping genes could

71 vary amongst different cell populations before and after differentiation. Therefore, the housekeeping genes to use for the real-time PCR amplification had to be chosen carefully, in order to conform to the basic principle of housekeeping genes selection, which is the least possible expression level variation within the test conditions. For that reason, the

PerfectProbe Genorm kit (PrimerDesign, UK) was employed. The Genorm kit offered a total of 12 housekeeping genes (Table 2.3) to be tested at the same time in triplicates for all the culture conditions tested, before and after differentiation. The results were then loaded on the QBase+ software, supplied together with the kit. An algorithm was run by the software that defined the housekeeping genes whose expression was less variable within the different cell populations. This led to the conclusion that a combination of two housekeeping genes

(beta actin and ATP synthase) was the most reliable choice of housekeeping genes.

2.1.20 Primer Design for PCR Amplification

The primers for the PCR amplifications were designed to have a 19-25 base length, with 40-60% GC content. The primers were either designed using the PrimerPremier software (Premier Biosoft International, Palo Alto, USA) or were chosen from a database of pre-tested primers (Primer Bank, University of Harvard, USA, http://pga.mgh.harvard.edu/primerbank), when these were available. When possible, it was intended to design or pick human genome specific PCR primers. In order to check the primer specificity to the human genome, the primers were run in the Primer BLAST analysis of the

National Centre for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/tools/primerblast/index.cgi?LINK_LOC=BlastHome) against the genomic sequence of Homo sapiens and Mus musculus. This action was performed in order to minimise the presence of false positive results, induced by the contamination of the hESC

RNA by RNA from the feeder layer of the hESC culture. The primers were then synthesised by MWG Eurofins (London, UK). The primers were sent out in lyophilised form and, upon receiving, were reconstituted with nuclease-free water (Promega, UK) to a concentration of

100 mM. This was the stock concentration and was stored in a -20 oC freezer. In order to avoid possible contamination, the tubes containing the stock solutions of primers were only opened when needed to make a 1:10 dilution of a primer, the working solution.

Table 2.3: PerfectProbe Genorm Kit Selection of Endogenous Genes. Presenting the housekeeping genes that were compared for the least expression variability between the undifferentiated hESCs and their counterparts that had been subjected to directed differentiation. Human geNorm kit gene list Homo sapiens actin, beta (ACTB), mRNA. Homo sapiens glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mRNA. Homo sapiens ubiquitin C (UBC), mRNA. Homo sapiens beta-2-microglobulin (B2M), mRNA. Homo sapiens phospholipase A2 (YWHAZ), mRNA. Homo sapiens Ribosomal protein L13A (RPL13A), mRNA. Homo sapiens 18S rRNA gene Homo sapiens cytochrome c-1 (CYC1), mRNA. Homo sapiens eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), mRNA. Homo sapiens succinate dehydrogenase complex, (SDHA), mRNA. Homo sapiens topoisomerase (DNA) I (TOP1), mRNA. Homo sapiens ATP synthase, (ATP5B), mRNA

2.2 Calcium fluorescence imaging and microfluorimetry

The fluorescent dye fura-2 acetoxymethyl ester (Fura-2 AM) (Invitrogen, Paisley, UK) was

2+ used to estimate changes in the concentration of intracellular calcium ions ([Ca ]i). Its use is based on chemical properties, which include the ability to cross the cell membrane as a permeable ester. Once inside the cell, it undergoes cleavage to yield an active form that is impermeable to the cell membrane and can bind free Ca2+.

2+ The property of fura-2 as a fluorochrome dye that makes it useful for the study of [Ca ]i is its property to be excited at different light wavelengths when it is bound to Ca2+ and when it is in an unbound state. The non-bound version of Fura-2 absorbs light maximally at 362 nm, emitting light at 518 nm, while the Ca2+-bound complex of the dye absorbs light maximally at

335 nm, emitting light at 510 nm. This makes it possible by knowing the ratio R of the dye's fluorescence intensities F1 and F2 at two excitation wavelengths 1 and 2 to calculate

[Ca2+], independent of total dye concentration, path length or the absolute sensitivity of the instrument (Grynkiewicz et al., 1985). The two wavelengths used correspond to the one at which the dye absorbs maximally when it is bound to Ca2+ (e.g. 340 nm) and the one at which it absorbs when it is in its unbound state (e.g. 380 nm). While the unbound to calcium fura-2 has a maximum absorbance at 362 nm, this wavelength is not used in the ratio

73 calculations, as closely to that wavelength, at 360 nm, fura-2 shows what is called an isosbestic point, where the fluorescent intensity of fura-2 is insensitive to free calcium concentration (Kong, 1995).

The Fura-2, in each of its Ca2+ free and bound states, has got a proportionality coefficient, called Sf and Sb for each of the absorption wavelengths 1and 2These coefficients connect the emission intensity F with the Ca2+ concentration –and since the fluorescence contribution from any given molecular species is proportional to the concentration of that species, they are called proportionality coefficients. In a calibration solution containing

2+ known low concentrations of free (Cf) and Ca -saturated bound dye (Cb), the total fluorescence intensities F1 and F2 at wavelengths 1 and 2 will be given by the following formula (from (Grynkiewicz et al., 1985):

F1 = (Sf1 * Cf) + (Sb1 * Cb) (Equation 1)

F2 = (Sf2 * Cf) + (Sb2 * Cb) (Equation 2)

2+ However, the Cf and Cb are associated to [Ca ] by the following formula, considering a

1:1 of dye to Ca2+ molecules:

2+ Cb = Cf * [Ca ] / Kd (Equation 3)

where Kd is the effective dissociation constant. If the fluorescence ratio F1:F2 is R, then

R = (Sf1 * Cf + Sbl * Cb) / (Sf2 * Cf + Sb2 * Cb) 

2+ 2+  R = (Sf1 + Sb1 * [Ca ] / Kd) / (Sf2 + Sb2 * [Ca ] / Kd) (Equation 4)

 [Ca2+] = (Equation 5)

2+ Note that Sf1/Sf2 is simply the limiting value that R can have at zero [Ca ] and so may be considered Rmin. At the same time, Sb1/Sb2 is the analogous limiting Rmax that the ratio has at saturating [Ca2+]. The above equation may therefore be written as:

[Ca2+] = (Equation 6)

So, the ratio of emission at 510 nm from the two excitation wavelengths 1 and 2 can be used to estimate [Ca2+] levels (Grynkiewicz et al., 1985).

The microscope rig which was used to measure the fluorescent emission at two different excitation wavelengths was calibrated by the use of three solutions. One of them contained

10 mM Ca2+ and 8 M fura-2 AM –this was called the “High [Ca2+]” solution. The second one was Ca2+-free and also contained 8 M fura-2 AM –this was referred to as the “Low [Ca2+]” solution. Finally, the third solution contained neither Ca2+ nor Fura-2 AM –this was called the

“Background” solution.

The equipment used to measure the fura-2 ratio consists of a chamber, that was connected with rubber tubing to a series of syringes which were linked together with a series of taps that allowed each syringe to be open or closed in the circuit independent of the others. The chamber was set up with 2 coverslips. On this occasion, where the calibration took place, both coverslips were empty. In an experimental situation, one coverslip would contain the cells to be studied and the other would act as a lid, creating a gap between them, where the control and agonist containing solutions would go through. After focusing on the lower coverslip, the microscope was set slightly out of focus by moving the focus point up – so that it would focus between the two coverslips of the chamber. Background solution was introduced to the chamber and the 340 nm and 380 nm counts were recorded using the

MetaFluor Fluorescence Ratio Imaging Software (Molecular Devices, LLC, USA) that was supplied with the calcium rig. Afterwards, the “High [Ca2+]” solution was applied to the chamber and the 380 nm counts were checked to make sure that they were twice the 380 nm “Background” solution counts. This would reassure us that there was a measurable difference in the ratio between the background and a calcium containing solution. Then, the

“Low [Ca2+]” solution was applied to the chamber and it was checked that the counts were not saturating the ratio panels on the MetaFluor software, which would reassure us that there is a measurable range of calcium concentrations to use the ratio on. If either the “High

[Ca2+]” solution 380 nm counts were less than twice the same counts of the Background

75 solution or the “Low [Ca2+]” solution counts were saturating, this would indicate that the Fura-

2 AM concentration would have to be re-adjusted by preparing new “High [Ca2+]” and “Low

[Ca2+]” solutions.

Several readings from several high, low and background introductions to the chamber were taken, ensuring that the readings were made whilst flow through the chamber was steady and no bubbles were present during each application.

From the spreadsheet produced by the MetaFluor software, the mean values for 340 and

380 nm for Background, High and Low [Ca2+] solutions were calculated. So, in Equation 6, the following values could be used in the Metafluor software in order to be able to estimate

2+ the [Ca ]i :

2+ Rmin = 340 nm counts / 380 nm counts in Low [Ca ] solution

2+ Rmax = 340 nm counts / 380 nm counts in High [Ca ] solution

2+ 2+ (Sf2 / Sb2) = 380 nm counts in Low [Ca ] solution / 380 nm counts in High [Ca ] solution.

2.2.1 Plating HUES1 hESC on coverslips

Intracellular Ca-measurements were undertaken by mounting HESC on glass coverslips

24 hours prior to experimentation. This procedure may induce differentiation of the cells since it removes them from feeders. For this reason several experimental procedures were undertaken to identify the most effective way of undertaking experiments on undifferentiated cells. These included:-

(1) Plating HUES1 hESC colonies on coverslips pre-covered with matrigel basal matrix

(Beckton Dickinson, Oxford, UK).

(2) Plating HUES1 hESC colonies on coverslips pre-covered with 50 g/ml fibronectin

(Chemicon, UK) in PBS (Sigma-Aldrich, Dorset, UK)

(3) Plating HUES1 hESC colonies on coverslips pre-covered with 20 g/ml laminin from

Human Placenta in PBS (both from Sigma-Aldrich, Dorset, UK)

For each of these conditions, 300 l of the matrix solution was applied onto coverslips that were pre-plated on 24 well plates. The plates were then incubated at either room temperature or at 37 oC for 2hr. This was followed by washing of the plates with PBS, removing the PBS and applying 600 l of conditioned medium. Based upon cell adherence, maintenance and morphology, the option of using a mixture of 50 g/ml fibronectin and 20

g/ml laminin in a ratio of 3:2 provided the most appropriate option for these studies.

Verification that HESC had retained their undifferentiated status was carried out by immunohistochemistry for Oct4 (see 2.2.4).

In more detail, coverslips (15mm, Scientific Laboratory Supplies, Nottingham, UK) were placed into wells of a 24-well plate (Corning, Amsterdam, The Netherlands). The coverslips were then covered with a solution containing 50 g/ml Fibronectin (Chemicon, UK) and 20

g/ml laminin from Human Placenta (Sigma-Aldrich, Dorset, UK) at a ratio of 3:2 v/v –the solutions were created in ice cold PBS (Sigma-Aldrich, Dorset, UK). The fibronectin/laminin coated coverslips were incubated in a tissue culture incubator for 2 hr. The fibronectin/laminin solution was then removed and the coverslips were washed once in PBS

(Invitrogen, Paisley, UK). Each coverslip was then coated with 500 l of freshly thawed conditioned hESC medium (Section 2.1.4).

Complete HUES1 hESC undifferentiated colonies were then mechanically passaged onto the coverslips as described in Section 2.1.10. After all the colonies were transferred onto the coverslips, a needle was further used to gather the colonies towards the centre of the coverslip. The 24-well plate was then incubated overnight in a tissue culture incubator.

2.2.2 Loading the colonies with Fura-2/pluronic acid and mounting of coverslips on the experimental platform

The HUES1 hESC colonies were observed under a brightfield microscope the next day and photographs of the colonies containing non-differentiated cells were taken using a brightfield microscope (Olympus CKX41 microscope, CQ Imaging Micropublisher 5.0 camera, Qcupture Pro software, Qimaging, UK).

Approximately 5 M Fura-2 AM (Invitrogen, Paisley, UK) in DMSO with 6% added pluronic acid/DMSO in the form of the nonionic detergent pluronic F-127 (Invitrogen, Paisley, UK) was then applied to each of the wells containing undifferentiated colonies. The use of pluronic F-127 is recommended to prevent the formation of fura-2 micelles (Yates et al.,

1992).

The cells were then incubated for 2 hr in a tissue culture incubator. This was followed by removal of a single coverslip each time from a well of the 24 well plate. The coverslip with the cells was placed on the fluorescence imaging rig perifusion chamber platform (RC-20H,

Harvard Apparatus, UK) and the cells were bathed in a Ca2+ containing control basal solution. The basal solution consisted of the following: 137mM NaCl, 5.36mM KCl, 0.81mM

MgSO4, 0.34mM Na2HPO4, 0.44mM KH2PO4, 4.17mM NaHCO3, 10mM HEPES, 1.26mM

CaCl2 and 5mM D-glucose.

The platform was placed on the Ca2+ imaging microscope (Axiovert S100, Carl Zeiss linked to a Quantix Photometrics CCD camera, UK). Areas that contained cells with hESC morphology –from brightfield microscopy photographs- were chosen in the MetaFluor software (MDS Analytical Technologies, UK). These areas were monitored during each experiment and the ratios of fura-2 intensity were recorded by the software, based on the in vitro calibration values. Analysis was performed on the data to assess the estimated rise in

2+ [Ca ]i throughout each experiment.

2.2.3 Test solutions

The undifferentiated regions of the HUES1 hESC colonies were tested for their ability to respond to different concentrations of the nucleotides ATP, UTP, UDP and b-meATP (all supplied from Sigma-Aldrich, Dorset, UK), each nucleotide being an agonist of a single or more purinergic receptor subtypes. Each nucleotide was dissolved in water to give 100 mM stock solutions, which were frozen and thawed as required, trying to keep the freeze-thaw cycles at a minimum. Working concentration solutions were prepared using serial dilutions of the stock solutions and the appropriate basal calcium containing or calcium-free buffer,

78 depending on the experimental procedure. The concentrations used for the dose response profile of all the purinergic receptor agonists mentioned above, were in the range between 1

M and 100 M. Further to that, the ability of the hESC to recover after repeated applications of a high concentration of ATP (100 M) was investigated.

2.2.4 Fitting of the dose response curve

2+ The response results in terms of changes in [Ca ]i from the application of different nucleotides to the cells were recorded and used to fit a dose response curve. In particular,

2+ all the estimated [Ca ]i raw values were loaded onto Prism6 (Graphpad). The analytic function of the software was used to plot the data of agonist concentration vs % percentage

2+ [Ca ]i on a plot with a logarithmic x-axis. The data were fitted to the one-site dose response model equation, called "the 4 Parameter Logistic" nonlinear regression model.

The equation for that model is the following: y = (A+((B-A)/(1+(10((C-x)*D))))), where A is the

lowest value of the response, Rmin and B is the highest value of the response, Rmax, C is the

EC50 and D is the Hill Slope. From the resulting plot, the values of the pEC50 and the maximum response Rmax were determined.

2.2.5 P2Y Receptors – Study of Functional Profile

2.2.5.1 Stimulation with ATP in the absence of extracellular Ca2+

In order to study the functionality of any P2Y receptors in HUES1 hESCs, the cells were initially perfused with Ca2+ containing medium (see 2.2.2). This was followed by perfusion in medium containing no calcium. This medium was the same as in 2.2.2, except for the substitution of the calcium salt by 1 mM ethylene glycol tetraacetic acid (EGTA). EGTA acts as a chelating agent, making metals like Ca2+ unavailable in the extracellular medium. The cells were then stimulated with the addition of 100 M ATP. Any recorded responses would indicate that internal calcium stores were operating, suggesting the involvement of P2Y receptors in the signalling process.

2.2.5.2 Stimulation with ATP in the presence of cyclopiazonic acid (CPA)

The coverslip with the HUES1 hESCs was perfused with EGTA-containing medium. This was followed by the addition of 30 M CPA into the EGTA-containing perfusion medium.

CPA inhibits the sarcoplasmic reticulum Ca2+ pump (Laursen et al., 2009), meaning that after repeated stimulations of the P2Y receptors, the intracellular stores would be almost depleted of calcium. The cells were stimulated with 100 M ATP, until there was no response shown

2+ in the [Ca ]i. Then, the perfusion medium was changed to calcium containing medium (see

2.2.2). After the coverslip had been incubated for 4 min in that medium, enough time to replenish the intracellular calcium stores, the cells were stimulated again with the addition of

100 M ATP.

2.2.5.3 Stimulation with ATP in the absence of extracellular calcium, with the addition of MRS2578.

In order to study the role of the P2Y6 purinergic receptor in the responses to ATP in hESCs, MRS2578 (Tocris Biosciences, Bristol, UK) was used at a concentration of 10 M.

MRS2578 is a selective inhibitor of the P2Y6 purinergic receptor. The MRS2578 was received in a lyophilised form and was diluted to a stock concentration of 100 M in DMSO and aliquots were stored in the -20oC freezer. It was freshly diluted with Ca2+-free basal medium to the appropriate working concentration, immediately before the initiation of each experiment. The coverslips with the HUES1 hESC colonies were incubated for 5 min in

EGTA containing perfusion medium, with the addition of 10 M MRS2578. Following this step, the cells were stimulated by the addition of 100 M ATP, in EGTA containing perfusion medium, in the presence of MRS2578.

2.2.6 Immunostaining of coverslips

After Ca2+ imaging, the coverslips were recovered for immunostaining with a mouse anti- human Oct3/4 Antibody (Beckton Dickinson, Oxford, UK, UK). This allowed the confirmation of the areas of the monitored colonies that contained undifferentiated hESCs.

Each coverslip was transferred into a well of a 24 well plate after the end of the relevant

Ca2+ imaging experiment. The cells were fixed with 4% paraformaldehyde in PBS (both from

Sigma-Aldrich, Dorset, UK) for 1 hr at room temperature. Following fixation, the cells were washed x3 in PBS (Sigma-Aldrich, Dorset, UK). The cells of the colonies were then permeabilised and blocked with PBS (Sigma-Aldrich, Dorset, UK) solution, containing 10% normal goat serum (Jackson Immuno Research Laboratories Inc., West Grove, USA) and

0.1% TritonX-100 (Sigma-Aldrich, Dorset, UK) for 1 hr at room temperature. The cells were then washed x3 in PBS (Sigma-Aldrich, Dorset, UK). The 1o antibody (Oct3/4) was then applied to the coverslip after it was diluted to a concentration of 2.5 g/ml in PBS containing

3% normal goat serum and 0.01% Tween-20 (Sigma-Aldrich, Dorset, UK). The coverslips were incubated with the primary antibody for 1 hr at room temperature. This was followed by

3x washes with 0.05% Tween-20 in PBS (Sigma-Aldrich, Dorset, UK). The secondary antibody (Alexa Fluor® 488 goat anti-mouse IgM, Molecular Probes, Invitrogen, Paisley, UK) was diluted to a concentration of 5 g/ml in a 1x PBS solution, containing 3% normal goat serum and 0.01% Tween-20 and applied to the coverslips. The coverslips were incubated for

40 min in the dark at room temperature. At the end of the incubation, the coverslips were washed x4 with 0.05% Tween-20 in PBS (Sigma-Aldrich, Dorset, UK). The coverslips were then mounted on microscope slides using Prolong® Gold Anti-fade Reagent with DAPI

(Molecular Probes, Invitrogen, Paisley, UK). They were incubated at room temperature overnight in the dark, prior to viewing under a widefield fluorescent microscope (Olympus

BS51, UK, linked to a Coolsnap EZ camera from Photometrics, Germany). The images were recorded using the Metavue software package (MDS Analytical Technologies Ltd., Canada) and analysed using the ImageJ image analysis software (http://rsbweb.nih.gov).

Sometimes more than one primary antibodies were combined, like in the case of double staining for P2Y6 and Oct4. In these cases, the antibodies were combined in the same solution. In the case of the P2Y6 receptor, the antibody used was purchased from Alomone

Labs (Jerusalem, Israel) (rabbit, polyclonal, anti-human anti-P2Y6) and it was used at a working dilution of 1:100. In this case, the secondary antibody used was the Alexa 594 goat anti-rabbit antibody from Invitrogen (Paisley, UK) and it was used at a working dilution of

1:200.

2.2.7 Confocal Microscopy

In order to pinpoint the location of the P2Y6 purinergic receptor in the hESCs, confocal microscopy was employed. The procedure used to stain the coverslips containing colonies of HUES1 hESCs was performed as described in section 2.2.4. Images of cells that were stained for Oct4 (Alexa488), P2Y6 (Alexa594) and DAPI (4',6-diamidino-2-phenylindole, a fluorochrome that emits fluorescence when bound to A/T-rich regions in the DNA) were collected using a Nikon C1 confocal on an upright 90i microscope with a 60x/ 1.40 Plan Apo objective [and 3x optical confocal zoom]. The confocal settings were set in the following way: pinhole 30μm, scan speed 400Hz unidirectional, format 1024 x 1024. The images for

DAPI, Alexa488 and Alexa594 were excited with the 405nm, 488nm and 543nm laser lines respectively (Alexa488 has similar excitation-emmision spectrum as FITC, while Alexa594 has a similar excitation-emmission spectrum as Texas Red). When acquiring 3D optical stacks the confocal software (Nikon) was used to determine the optimal number of Z sections between the top and the bottom layer of the cells. Only the maximum intensity projections of these 3D stacks are shown in the results section. 2.3. Alkaline Phosphatase Pluripotency Assays

HUES7 hESCs were enzymatically passaged onto 24 well plates in the presence of MEF feeders using trypsin/EDTA (Section 2.6.2). Two days after plating down the hESCs, 100 M of each of ATP, UTP, UDP and 300 M of Suramin -a well established general purinergic singalling antagonist (Mallard et al., 1992)- (all from Sigma, UK) in hESC medium were each applied to 4 wells of a 24 well plate. An additional 4 wells of a 24 well plate were used as non-differentiated control, supplemented with hESC medium. In addition, a well of each 24 well plate was used as a positive control for differentiation, by incubating with DMEM

(4.5mg/ml glucose, no L-glutamine, no pyruvate, Invitrogen, UK) containing 10% FBS

(Invitrogen, UK). The agonist containing solutions were replaced daily. The cells were incubated in a tissue culture incubator for 24, 36 and 72 hr. At each of these time-points, the cells were fixed with 4% paraformaldehyde in PBS (both from Sigma, UK). This was followed by analysis of alkaline phosphatase expression, using the standard protocol that came with the kit (Millipore, UK). High alkaline phosphatase activity is an inherent feature of hESCs

(Richards et al., 2002). Measuring this activity allowed us to define non-differentiated and randomly differentiated hESC colonies.

Phase contrast images were taken at 4x magnification using a phase contrast microscope

(Olympus CKX41 microscope, CQ Imaging Micropublisher 5.0 camera, QCapture Pro software, Qimaging, UK). The data were presented as percentages of alkaline phosphatase positive vs alkaline phosphatase negative colony areas, which were identified following specific criteria, summarised on table 2.4. They were subsequently analysed using the

SigmaStat software (Aspire Software International, USA).

Table 2.4: Alkaline Phosphatase Assay. The criteria used to choose which colonies to use for inclusion in the alkaline phosphatase assay analysis.

• Chose 20 colonies from each well of the 24 well plate, for each time-point and for each agonist/control condition • Colony size to be included in results should exceed the area of 6,000 mm2 • Partial colonies at the edges of the image were not counted in • Positive control cell colour was set • Negative Control cell colour was set

2.4. Creating a Lentiviral Vector containing the GFP under the influence of the Pdx1 gene promoter.

The MultiSite Gateway Pro cloning kit (Invitrogen, UK) was used to clone the areas I-III of the Pdx1 promoter and the Cherry Red expression gene to a Lentiviral vector. The pLNTGateway ® destination vector was used in order to create the expression vector. It had been developed by Dr Howe in the laboratory group of Prof Thrasher (University College

London, UK).

The sequences for the areas I-II-III of the mouse Pdx-1 promoter and the sequence for the eGFP fluorochrome were cloned into a pLenti backbone lentiviral vector (Van Velkinburgh et al., 2005), using the Multisite Gateway® technology (Invitrogen, Paisley, UK). The Pdx1 promoter areas and the GFP gene were each cloned into a PDONR vector by Dr Tristan

McKay. The resulting vectors were named PDONR 1-5r Pdx1 and PDONR 5-2 GFP and they will be referred to as the entry clones from now on. The recombination reaction to

83 generate the lentiviral expression vector consisted of 10 fmole of each of the entry clones and 20 fmole of the accepting LNT-WTR vector, made up to 8 l using 1x Tris EDTA (TE) buffer, pH 8.0. The LR Clonase™ II Plus enzyme mix (Invitrogen, UK), part of the cloning kit, was thawed on ice. After briefly vortexing it, 2μl of the enzyme mix was added to the recombination reaction. The reaction was incubated at room temperature (RT) for 16 hr, as in the manufacturer's manual. This incubation was followed by the addition of 1ml of a 1μg/μl proteinase K solution -part of the kit- and incubation of the reaction for 10 min on a heated block at 37 oC.

The reaction contents were then used to transform One Shot® Mach1™ T1R chemically competent E. coli cells (Invitrogen, UK). A transformation reaction consisted of 2μl of the recombination reaction added to a vial of One Shot Mach1 T1R cells. A positive control was also included, in which the competent cells were transformed with 1ng of a control pUC19 vector. The cells with the recombination reaction components were incubated on ice for 30 min. The cells were then heat-shocked for 30 s at 42°C without shaking. The transformation reaction tube was immediately transferred to ice for 2 min.

Super optimal catabolite repression (S.O.C.) medium (part of the kit, Invitrogen, UK) was added to the reaction (250μl). The tube was then incubated at 37°C for 1 hour while being vigorously shaken. The contents of the transformation reaction tube were plated in S.O.C. medium on pre-warmed ampicillin-LB (Luria Broth) agar plates (50μg/ml ampicillin, Sigma,

UK).

The plates were incubated overnight (O/N) at 37°C. A suitable number of colonies was picked from the plates of the recombination test reaction. They were inoculated into 5ml of

LB with 50μg/ml ampicillin and grown O/N at 37°C with vigorous shaking. A DNA extraction was performed for each of the inoculated clones using the QIAGEN Miniprep Plasmid Kit

(QIAGEN, UK), while a small volume of the grown culture was kept aside for a future bigger scale DNA preparation of any positive clones.

After having generated enzymatic digestion maps of each of the expected lentiviral vectors, we decided to check that the cloning reactions had worked by using restriction enzymes. Any clones with the right recombination events should give the right sizes of bands on an agarose gel. The extracted DNA from each clone was digested with NotI

84 enzyme (Roche, UK). Each digestion reaction contained 10x buffer H (Roche, UK), 5 units of

NotI enzyme and 100mg of extracted DNA, made up to a total volume of 50μl by the addition of Tris/EDTA buffer, pH 8.0. The reactions were incubated O/N at 37°C and they were run on a 1.2% agarose gel (Melford Laboratories Ltd., UK), in Tris-Acetate-EDTA (TAE) buffer

(BioRad Laboratories, USA), which contained 2.86 x 10-4 μg/ml ethidium bromide (Promega,

UK). 2.5 Lentiviral production and testing

In order to generate lentiviral particles using our lentiviral construct, it needed to be transfected into a packaging cell line, which would provide the machinery for the lentiviral vector and two other packaging and envelope vectors to form lentiviral particles. The lentiviral construct was made in a way that would prevent its uncontrolled spread. In order to achieve this, a lentiviral vector was used, where the sequences that control the packaging of the lentiviral construct had been removed prior to the cloning reaction. This would ensure that the lentivirus would be unable to infect cells in the absence of sequences that encode its packaging and envelope associated proteins. The packaging cell line that was used was the

HEK293T cells (kindly donated by Lisa Blackenbury, a PhD student in the group of Prof

Streuli, University of Manchester). These cells have been modified to stably express the large T-antigen of the simian vacuolating virus 40 (SV40). This modification allows the high replication rate of any vectors containing the SV40 origin of replication.

Viral particle production was performed by transfecting the HEK293T cells with what is called a 2nd generation lentiviral packaging system. Successful viral particle production with this system requires the transfection of the packaging cells with a total of three vectors. In our case, one was the lentiviral construct we had designed, the second one was a plasmid encoding the gag and pol viral proteins, the pCMV-DR8.73, and the third plasmid, pMD2.G, encoded the VSV-G envelope glycoprotein of the vesicular stomatitis virus (both supplied from Plasmid Factory, Bielefeld, Germany). Two methods were tested for the transfection of the HEK293T cells: calcium phosphate precipitation and Lipofectamine2000® (Invitrogen,

UK). Lipofectamine2000® was used according to the manufacturer's instructions. For the transfection using calcium phosphate precipitation, approximately 5 x 104 HEK293T cells were plated into each well of 6 well cell culture dishes (BD Biosciences, Oxford, UK) and

o were allowed to attach by overnight incubation at 37 C, 5% CO2. This was followed by preparation of the plasmids for transfection. Our lentiviral construct was mixed with the two packaging plasmids, so that the final concentration ratio of the plasmids in the mix was 6:3:2

(construct: pCMV-DR8.73:pMD2.G). They were then resuspended in a pH 7.1 solution containing DMEM supplemented with HEPES buffer (25 mM) (both from Invitrogen, UK). A stock solution of 2 M calcium chloride was added to the resuspended plasmid mix, to give a final CaCl2 concentration of 20 mM. The solution was then incubated at room temperature for 20 min. During that time, a second solution with DMEM (Invitrogen, UK) containing 10 %

FBS (Invitrogen, UK) and 25 mM HEPES (pH 7.9) was prepared, its pH was adjusted to pH7.9 and it was then filter sterilised using a .22μm acrodisc filter (Millipore, UK) attached to a sterile syringe. The medium from the cultured HEK293T cells was then removed and replaced with the pH 7.9 DMEM solution, which had been pre-warmed at 37oC in a waterbath. 1 ml of this solution was placed in each well of the 6 well plate. This was supplemented with 250 μl of the lentivirus suspension and the dish was gently shaken to assist the spreading of the viral particles. As a positive control, the HEK293T cells were also transfected with a lentiviral construct encoding eGFP under the control of a Spleen Focus-

Forming virus (SFFV) promoter (kindly donated by Dr T. McKay, University of Manchester,

UK). This additional construct –consisting of the same backbone as our generated reporter lentiviral construct- was used as a reference point, in order to assess the level of cell transfection.

The transfected HEK293T cells were then incubated overnight in 5% C02 at 37 °C

(Galaxy R, Wolf Laboratories, York, UK). This was followed by removal of the medium and replacement with medium for the Min6 cells -Min6 cells inherently express Pdx-1

(Deramaudt et al., 2006), so, they were used to test the successful operation of the lentiviral vector- which consisted of DMEM, 20% FBS and 2 mM L-glutamate.

The positive control virus transfected cells were checked approximately one day later for signs of eGFP expression. Images were obtained using an Olympus CKX41 inverted microscope (Olympus, Watford. UK) at x4 and x20 magnification. Images were captured on a Retiga-SRV camera (QImaging. Marlow, UK) through QCapture Pro software (QImaging,

Marlow, UK).

Following the detection of eGFP in the HEK293T cells, it was assumed that the plasmids that encoded the virus proteins were functioning properly and that the lentivirus of our construct was being successfully produced. The medium of the transfected HEK293T cells was substituted with fresh medium, suitable for Min6 cell culture, 24 hr post transfection.

Then, at 48 hr post transfection initiation time, the virus containing medium was used to transduce the Pdx-1 expressing cell line Min6. In more detail, the virus containing supernatant was collected from the HEK293T cells, centrifuged at 5,000 x g for 5 min and was then filtered through a .45 μm acrodisc filter attached to a sterile syringe. The filtered medium was applied to Min6 cells that had been plated during the previous day and were about 30% confluent at the time point of the initiation of transduction, which started with the application of the viral particles to the cells. The fluorescence of the Min6 cells that had been transduced with the control eGFP reporter lentivirus was assessed under a fluorescent microscope.

At 72 hr past the transduction initiation time, the cells were analysed using flow cytometry.

In more detail, the Min6 cells, including some non-transduced cells, were trypsinised, centrifuged and washed in pre-warmed PBS (Sigma-Aldrich, UK) twice. The eGFP expression of 10,000 cells from each condition was assessed using flow cytometric analysis

(Flow Cytometry Facility, University of Manchester, UK). Flow cytometric analysis is a high throughput method can isolate cell populations and identify single cells according to various characteristics, such as cell size, granularity or intensity of fluorescent signal. The cell samples that were run through the flow cytometric analysis included a negative control, with non-transduced cells, which was used as a background reference for the assessment of the native Min6 autofluorescence at different excitation/emission wavelengths. In order to prevent the inclusion of any dead cells or debris in the results, the cell populations were gated during the flow cytometric analysis, so that only live single cells were monitored.

Gating is the process of creating different sub-populations of cells on a resulting histogram of the data from a flow cytometer and it can apply in real-time during data acquisition. Regions of interest were set up during the flow cytometric analysis run, so that the total number of cells was presented. In this setup, the total number of fluorescent cells was presented as a percentage of the total number of sorted alive single cells. Following the flow cytometric

87 analysis of the one potentially positive result, where the cells transduced with our lentiviral construct presented eGFP expression, genomic DNA was extracted from transduced cells that had not been run through the analysis. In addition, total RNA was extracted from transduced unsorted cells and was later converted into cDNA. In order to assess the integration and operational status of the lentiviral construct in the transduced Min6 genome, a PCR reaction was set up, using a primer for eGFP, with the genomic DNA and cDNA from the unsorted cells used as template.

2.5.1 The pTiger-mPdx1 I-II-III-RFP/rIns2-eGFP vector

During the course of construction and testing of our own reporter lentiviral vector, a similar approach was taken by other researchers elsewhere and published (Szabat et al.,

2009). This vector was obtained and tested in our hands (kind gift from Dr Jim Johnson,

University of British Columbia, Vancouver, BC, Canada).

This lentivirus was based on the pTIGER backbone and comprised of the mouse Pdx-

1 promoter regions I-II-III driving the expression of red fluorescent protein (RFP), in addition to the promoter region of the rat Insulin 1 gene, controlling the expression of eGFP. An additional vector was obtained from the same group. This was a control vector. Therefore, it would be used to test the transfection and transduction efficiency of the test vector, similar to the pLenti-SFFV-eGFP that had been used in our earlier experiments. It comprised of the pTIGER backbone, with a CMV promoter driving the expression of eGFP.

The vectors arrived in the form of DNA blotted on filter paper. They were extracted from the filter paper by cutting the paper into small pieces and allowing it to soak O/N in

DNAse/RNAse-free water (Promega, UK) at 4 oC. The filter paper was then removed by centrifugation on a tabletop microcentrifuge at 17,900 x g for 1 min and the supernatant containing the DNA was removed into a clean microcentrifuge tube. The recovered DNA was used to transform SURE-2® competent cells (Stratagene, UK) according to the manufacturer's manual. The cells were grown overnight in liquid suspension selective LB medium containing ampicillin, at 37oC. 100 μl of that overnight growth were plated on selective Agar plates containing 100μg/ml ampicillin, which were then incubated at 37oC overnight. A total of nine colonies were picked from the agar plates and DNA was extracted

88 from them using the QIAPREP Spin Miniprep Kit (QIAGEN, UK) as in the manufacturer's instructions. The extracted DNA was digested using the BamHI and NheI enzymes as described before for the NotI digests (see chapter 2.4), in order to confirm the successful transformation and amplification of the vectors. The digested products were run on an agarose gel. The resulting bands did not correspond to the restriction profile provided to us by Dr Johnson’s group.

It was then decided to repeat the transformation of cells using the extracted DNA from the blotted filter papers. This time, a different strain of E.coli was used. The MAX

Efficiency® Stbl-2™ strain (Invitrogen, UK) were transfected according to the manufacturer's protocol. The MAX Efficiency® Stbl-2™ is a bacterial strain adapted to reduce recombination events occurring during the amplification of vectors containing repeat sequences, such as lentiviral vectors. Growing these cells at 30oC instead of 37oC may have decreased the chances of recombination events in the vector. This could be due to the low copy number of plasmids reducing the chance of recombination events, similar to what has been achieved before by using a low copy origin of replication containing plasmid (Miyatsuka et al., 2007).

Chapter 3: Facilitating attachment of HUES1 human embryonic stem cells to glass coverslips

3.1 Introduction

Since the introduction of human embryonic stem cells (hESCs) (Thomson et al.,

1998), scientists have been trying to find alternative ways to culture them in a feeder-free environment that can support their self renewal, maintaining them in their undifferentiated, pluripotent state. The main reason for that is the fact that only a xeno-free (free from animal substances) environment would be useful in a clinical setup, as there is a danger of the animal pathogens cross-transferring to the hESCs from the animal feeders (Richards et al.,

2002). But even before considering the clinical grade applications of hESCs, their culture on feeders is a challenging approach, as the feeder environment is mostly undefined, it can present batch-to-batch variation and the production of feeders is a labour intensive task

(Hannoun et al., 2010).

The feeder free environments that have proved that they can support the self renewal and proliferation of hESCs involve a combination of extracellular matrixes (ECM) and growth factors (Baxter et al., 2009; Stelling et al., 2013; Wang et al., 2005). Cells that require ECM create cell surface-ECM contact points called focal adhesions. These points of contact bind and connect cellular cytoskeletal components – through adhesion molecules - to their extracellular environment. Their role in the life of a cell is very important, as they support cell motility and transduce extracellular signals (Adams, 2001). In general, it is thought that the main function of the ECM is the support of the growth and maintenance of a variety of cells

(Hughes et al., 2010). In fact, for hESCs in particular, it has been suggested that the focal point-ECM interaction can affect their differentiation potential (Gong et al., 2008; Ma et al.,

2008).

A primary aim of this PhD study was the analysis of purinergic signalling in hESCs, by investigating the purinergic receptors that are functional in hESCs and assessing their role in hESC pluripotency. The functional analysis of the purinergic receptors present on hESCs,

90 involved the study of their calcium dynamics after stimulation with various purinergic receptor agonists. In order to study calcium dynamics, a setup called the "calcium rig" was used, as described in Chapter 2. A prerequisite for the use of the calcium rig was that the cells to be studied had to be attached on a glass coverslip. Human embryonic stem cells have been cultured before on glass coverslips and used in calcium imaging experiments in the presence of feeders (Wright, Elli Alexandra et al., 2009). However, the presence of feeders would have made the interpretation of results difficult. In addition, the feeders would potentially compete with the hESCs for the different purinergic signalling agonists/antagonists presented to the coverslips, reducing the availability of these molecules for the hESCs. Therefore, a major obstacle for performing this technique on hESCs was the fact that the hESCs needed to be attached onto glass coverslips, in the absence of feeders.

A protocol that would achieve this should have certain characteristics. In order to prevent the cells from differentiating or adapting to a different environment, the time between the removal of the colonies from feeders and the initiation of the calcium imaging experiment should be minimal. This necessitated the development of a protocol that would allow overnight attachment of the colonies to a glass coverslip.

There have been many studies about matrices that allow the attachment and proliferation of hESCs on plastic surfaces, using either human fibroblasts (HF) or mouse embryonic fibroblast feeders (MEFs) or different coating matrices, such as laminin, fibronectin and Matrigel in the presence of medium conditioned on MEFs or HF (Bigdeli et al., 2008). The first report of successful culture of hESCs on a feeder free environment was in 2001 (Xu et al., 2001). Extensive studies of the factors involved in pluripotency have allowed the development of cell culture media that allow the growth of hESCs in a feeder free environment (Baxter et al., 2009). However, their constitution, which involves many growth factors, makes them rather impractical in terms of daily having to prepare complicated media formulations, while the number of growth factors used makes these culture media not cost-effective for our purposes as well. Also, these feeder-free environments were designed for the cells to grow for long periods of time, allowing the cells to “adapt” to their environment over time. In addition, the attachment of hESCs on glass surfaces has not been achieved yet. The extracellular matrix proteins have been shown to

91 have variable efficacy as attachment molecules, according to the substrate on which they are bound (Lim et al., 2005). Some of the most important factors that play a role in this efficacy have been explored before and summarised by Cooke (Cooke et al., 2008). These include the distance of the ECM proteins from the substrate, the environment surrounding the active domain of the ECM proteins and the secondary structure it forms upon attachment to the substrate. These factors affect the accessibility of the active domains of the ECM proteins by the cells, the activity of the ECM proteins, as well as the topography of the covered substrate (Cooke et al., 2008).

3.2 Aims

The aim of this part of our study was the development of optimal conditions to culture

HUES1 hESC colonies overnight on glass coverslips without the presence of MEF feeders, so that they could be used on downstream experiments studying purinergic signalling. In more detail, this part of our study focused on the comparison of the ability of fibronectin and laminin -alone or in combination, as well as Matrigel, in supporting the overnight attachment of undifferentiated HUES1 hESC colonies onto glass coverslips in the absence of feeders.

3.3 Materials and Methods

3.3.1 Preparation of Matrigel coated glass coverslips in 24 well plates

On the day before the plating of the hESC colonies on Matrigel basal matrix (Beckton

Dickinson, Oxford, UK) covered glass coverslips, a new Matrigel bottle was allowed to thaw on ice overnight at 4 oC. At the same time, all the materials that would be used to aliquot it and place it on the wells of a 24 well plate were stored in a -20 oC freezer overnight – including the 24 well plate (Corning, Amsterdam, Netherlands), which contained sterile glass coverslips (15mm, Scientific Laboratory Supplies, Nottingham, UK), the 1.5 ml microcentrifuge tubes used for aliquoting the Matrigel, 200 μl and 1000 μl plastic pipettor tips

(Starlabs, UK). This was because the thawed Matrigel has the capacity to gel rapidly at

22oC, so, while we had to take these steps to make sure that it stayed cold for as long as possible before setting on the coverslips we were going to coat.

On the next day, all the materials described above were placed on ice, while the

Matrigel was aliquoted into ice cold microcentrifuge tubes, split into 2 mg per tube. The ice cold pipettor tips were changed to new ones each time they became clogged with the ice cold Matrigel, as this indicated that they were becoming too warm and that the Matrigel was solidifying inside them. Each aliquot was placed on ice immediately. One Matrigel aliquot was kept on ice –this one would be used to cover the 24 well plates, while the rest of them were stored at -20 oC until further use.

The Matrigel aliquot was then placed in an ice cold 15 ml tube (Starlabs, UK) and was diluted with 6 ml cold KO-DMEM (Invitrogen, UK). Enough volume of this solution to just about cover the glass coverslips (150 μl) was then applied to each glass coverslip in the 24 wells, while the plate was on an ice bucket full with ice.

The plate was allowed to sit at room temperature (RT) for 1 hr, before it was ready to be used. Following that, the surface of the Matrigel covering the coverslips was checked, in order to make sure that it had covered the coverslip with a smooth, even layer, not having any chunks of Matrigel. Only the wells that met this condition were further used in an experiment. The wells that met these specifications were then washed once with KO-DMEM prewarmed at 37 oC and filled with 600 μl of prewarmed MEF-CM (see Section 2.1.4 of PhD

Materials and Methods for MEF-CM production).

3.3.2 Placing glass coverslips on 6 well plates and covering them with Fibronectin and Laminin

Coverslips (15mm, Scientific Laboratory Supplies, Nottingham, UK) were placed into wells of a 24-well plate (Corning, Amsterdam, Netherlands). Fibronectin (Chemicon, UK) and laminin from human placenta (Sigma-Aldrich, Dorset, UK) were diluted with ice cold PBS to the appropriate concentration before use (50 μg/ml and 20 μg/ml, respectively). The coverslips were then covered with 300 μl of the stock solution containing 50 μg/ml fibronectin

(Chemicon, UK) or 20 μg/ml laminin from human placenta (Sigma-Aldrich, Dorset, UK) or a combination of the two in a ratio of 1:1 v/v. After assessment of the results from this

93 experiment (see 3.3.4), it was repeated with a set of coverslips covered with the best condition out of all the conditions used. It is important to mention that the fibronectin had an expiry date of 6 months at 4 oC, something that was checked each time before it was used.

The fibronectin/laminin coated coverslips were incubated in a tissue culture incubator at 37 oC for 2 hr. The fibronectin/laminin solution was then removed and the coverslips were washed once in PBS (Invitrogen, Paisley, UK). Each coverslip was then coated with 500 μl of freshly thawed MEF-CM, supplemented with 20 ng/ml of bFGF (R&D, UK), as in the protocol described by Tomishima (Tomishima, 2012).

3.3.3 Transferring undifferentiated colonies to the glass coverslip coated 24 well plates.

Whole undifferentiated colonies from cultures of HUES1 hESCs (5 of them per 24 well plate well) were transferred by mechanical passaging (see Section 2.1.10 of PhD Materials &

Methods) to the coated coverslip.

After all the wells had received the right number of colonies, the colonies were moved towards the centre of the well, as this is the usable area for the calcium imaging experiments.

3.3.4 Observation of colonies and karyotypic analysis

One day after plating the HUES1 hESC colonies on the glass coverslips, they were observed under an optical microscope at different magnifications (x4, x10, x20). They were assessed morphologically for the characteristics of undifferentiated hESC colonies –well defined colonies with smooth edges, containing small cells with a high nucleus to cytoplasm ratio and multiple visible nucleoli. A total of 80 attached colonies were categorised as

“undifferentiated” or “differentiated” for each condition. The experiment was repeated twice.

In parallel to the observation under the brightfield microscope, HUES1 hESCs that had attached on the glass coverslips were sent away for karyotypic analysis (TDL Genetics,

London, UK). In more detail, Karyomax Colcemid (Invitrogen, UK) was added to the tissue cells to make up a concentration of 1 g/ml. The cells were then incubated in the tissue

o culture incubator at 37 C, 5% CO2 for 1 hr. This was followed by removal of the medium, washing in PBS (Sigma-Aldrich, UK) and trypsinisation of the cells. The trypsinised cells were suspended in hESC medium and centrifuged at 400 x g for 4 min. The supernatant was aspirated, leaving only about 200 l of liquid in the centrifuge tube. Then, the bottom of the centrifuge tube was tapped gently, in order to break up any cell clumps. 5 ml of ice cold

0.56% KCl solution in PBS was added to the cells and the cells were gently mixed. The mix was allowed to stand at room temperature (RT) for 6 min and it was then centrifuged at 400 x g for 4 min. The next step involved the aspiration of the supernatant and gentle resuspension of the pellet in the remaining drops of liquid. 5 ml of fixative solution containing methanol:acetic acid at a ratio of 3:1 was added to the cell mixture drop wise, while gently shaking in order to mix. The cells were then centrifuged at 400 x g for 4 min, the liquid supernatant was aspirated and the resuspension into the fixative solution was repeated. The resulting mix of cells in fixative solution was mailed to a company specialising in cytogenetic studies, TDL Genetics, UK. They analysed 20 cells using g-banding (giemsa staining), whereupon the stained metaphase chromosomes present dark bands at A/T rich areas, which can help in the identification of chromosomal abnormalities such as inversions, deletions and translocations (Speicher et al., 2005).

3.3.5 Immunostaining for Oct4

The colonies from the condition that presented the highest percentage of colonies with an undifferentiated morphology were further assessed by immunofluorescent staining for

Oct4 (BD Biosciences, UK), a marker of pluripotency for hESC (for complete methodology, refer to Section 2.2.5 of PhD Thesis Materials & Methods) (Fig. 3.2). This step was used in order to confirm that the results obtained from the observations of the colonies under a brightfield microscope were accurate.

3.4 Results

The cells that had attached onto the glass coverslips were assessed morphologically for the characteristics of undifferentiated hESC colonies –well defined colonies with smooth

95 edges, containing small cells with a high nucleus to cytoplasm ratio and multiple visible nucleoli. The result was presented as the mean percentage of “undifferentiated” attached colonies. Fig. 3.1 presents typical colonies consisting of undifferentiated (A) or differentiated cells (B).

In addition, these results were further confirmed by fluorescent microscopy, whereby the cells that had attached onto the glass coverslips were stained for the transcription factor

Oct4 (fig. 3.2).

In parallel experiments, the cells that had attached onto the glass coverslips were processed and sent away for karyotypic analysis. The results from TDL Genetics Ltd demonstrated that the HUES7 hESCs were karyotypically normal, carrying a 46,XX karyotype (Fig. 3.3).

The Matrigel coated coverslips only presented a median value of 9 colonies out of 80 colonies with undifferentiated characteristics (two experiments, n1=7, n2=11 => 11.25% of the total of 80 colonies that were included in the experiment). At the same time, the fibronectin covered coverslips presented a median value of 25/80 colonies with undifferentiated characteristics (n1=25, n2=25 => 31.25%), while the laminin covered coverslips, presented a median value of 21/80 colonies (n1=20, n2=22 => 26.25%). The coverslips that were covered with fibronectin/laminin at a ratio of 1:1 v/v presented a median value of 28.5 colonies with undifferentiated characteristics out of 80 (n1=27, n2=30 =>

35.63%).

The experiment was repeated, this time with the condition that gave the highest percentage of undifferentiated colonies from the previous experiments (fibronectin:laminin

@1:1 v/v), with coverslips that were covered with a ratio of fibronectin:laminin of 3:2 v/v. The result of this second experiment for the 1:1 v/v ratio gave a mean value of 29.5 colonies with undifferentiated characteristics out of 80 (n1=29, n2=30). The 3:2 v/v ratio gave a mean value of 35/80 colonies with undifferentiated characteristics (n1=34, n2=36). So, the respective percentages of undifferentiated colonies for these two conditions were 36.88% and 43.75%. Fig. 3.3 summarises these results.

Fig 3.1: Differentiated and undifferentiated colonies. A typical example of a colony that was counted as a mainly undifferentiated one (A), compared to a colony that was having characteristics of differentiation, and therefore, was not considered to be undifferentiated (B). The black arrow on image B shows the area with features of differentiation, with elongated epithelial-like cells, as opposed to the small cells with a high nucleus to cytoplasm ratio and visible nucleoli, which is the characteristic morphology of hESCs. Scale bars at 100 m.

Fig 3.2: Oct4 expression in hESC colonies. Human embryonic stem cell colonies plated on glass coverslips covered with Fibronectin and Laminin at a ratio of 3:2 were used for downstream experiments. After each experiment, they were immunostained for Oct4 (green) and DAPI (blue). Figure 3.2A shows a typical example of an immunostained undifferentiated colony, while panel B shows a colony that has mostly differentiated, having lost its pluripotency status. The scale bar is at 100 m.

Fig.3.3: Karyotypic analysis of the HUES1 hESC line. The results of the karyotypic analysis with giemsa staining showed that the karyotyped hESCs were 46,XX, as expected from the HUES1 cell line and concluded that the HUES1 hESCs presented a normal karyotype, with no visible karyotypic abnormalities being present.

Fig 3.4. The effects of different cell matrices on the pluripotency of attached hESC colonies. Bar chart presenting the percentage of undifferentiated colonies (n=80, 2 experiments for each condition) when the hESC colonies were plated on glass coverslips covered with different matrices, namely Matrigel, fibronectin, laminin or a combination of fibronectin and laminin at a 1:1 ratio and a combination of fibronectin and laminin at a 3:2 ratio. (Fn = Fibronectin, Ln = Laminin).

3.5 Discussion

3.5.1 Matrigel supports low levels of undifferentiated colony attachment

Matrigel is a reconstituted basement membrane (BM) matrix gel. It is a gelatinous mixture of proteins derived from a mouse cell line from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, which produces large amounts of BM constituents. It is used as a cell culture substrate that mimics BM functions (Futaki et al., 2003). It contains laminin, entactin, type IV collagen and heparan sulfate proteoglycan, amongst other components

(Emonard et al., 1987) and several growth factors, including bFGF, TGF, EGF, etc (Benton et al., 2011).

Matrigel has been used quite extensively to study a variety of cells, including cancer cell metastasis (Benton et al., 2011), endothelial cell differentiation (Grant et al., 1995),

100 mechanisms of angiogenesis (Albini et al., 2010) and even for the culture of hESCs (Draper et al., 2004).

The Matrigel that was used in our experiments was the growth factor reduced (GFR) version of it. It is a modified version of Matrigel, which does not contain vast amounts of growth factors, such as bFGF, EGF, insulin-like growth factor 1 (ILGF-1), TGF, PDGF and nerve-derived growth factor (NDGF) that are contained in the full unmodified version of it

(Hughes et al., 2010). This particular modified version of Matrigel was chosen because many of the growth factors contained in the unmodified Matrigel can act both towards supporting hESC pluripotency, but also in making them differentiate (Wobus et al., 2005).

However, Matrigel has a major drawback: since it is not well defined, it can present batch-to- batch variation (Hughes et al., 2010; Serban et al., 2008). This could have been the reason why there were so few colonies that seemed to be undifferentiated in our experiments.

There is also a study that was published after these experiments had been concluded, which supports that glass coated with Matrigel can induce hESC differentiation much more readily than a polystyrene plastic surface covered with Matrigel (Kohen et al., 2009). Using different methods to study the Matrigel surface and characteristics, Kohen (2009) reported that

Matrigel sets in different ways on polystyrene and glass. It was suggested in that report that the higher density of the protein network that Matrigel creates on a glass surface, can possibly promote uncontrolled differentiation, without maintaining the self-renewal phenotype of hESCs (Kohen et al., 2009). So, our observation directly supports this report.

3.5.2 Synergistic effects of fibronectin and laminin to support colony attachment

Our experiments suggest a synergistic and additive effect between fibronectin and laminin, with fibronectin presenting a more enhanced role relative to laminin in keeping the

HUES1 hESCs in an undifferentiated state, while at the same time helping them attach on the glass coverslips. This is a result that could be explained by the fact that when it comes to cell survival and cell migration, fibronectin and laminin act through distinctively different pathways (Gu et al., 2002; Gu et al., 2001). In more detail, the alpha(5) chain-containing

101 laminin isoform called laminin-11 (also known as laminin-511), has been shown to preferentially activate the Rac GTPase, in contrast to fibronectin, which signals through the activation of the Rho GTPase (Gu et al., 2001). Laminin-511 has been shown to be important for the retention of hESC pluripotency in culture, since it can be used as a substitute for the absence of MEF feeders (Evseenko et al., 2009), is produced by human feeders that support hESC pluripotency (Hongisto et al., 2012) and can support long term self renewal of hESCs in culture (Rodin et al., 2010). Laminin-511 has been detected in the commercial laminin from human placenta (Sigma-Aldrich, UK) that was used throughout our experiments, even though it has been shown to exhibit batch to batch variation (Wondimu et al., 2006).

At this point, it would have been useful to make a note of the importance of migration in the survival of hESCs in general. It has been demonstrated previously that the hESCs differentiate and show poor survival when they are dissociated into single cells in culture

(Gauthaman et al., 2010). In fact, the hESCs proliferate in the form of colonies, wherein the individual cells are tightly adhered to each other (Li, D. et al., 2010). It seems that the colony formation is a function promoted by the chemotactic movement of hESCs towards each other, which is accentuated when the cells are found closer together (Li, L. et al., 2010). As both laminin and fibronectin have been shown to promote cell migration in a chemotactic way for some cell lines (Shibayama et al., 1995), this could be an additional reason behind the fact that fibronectin and laminin are better than Matrigel in maintaining hESCs in an undifferentiated status.

The fact that fibronectin alone seemed to be better than laminin at keeping HUES1 hESC colonies undifferentiated, while at the same time they presented an additive effect when mixed, since the 1:1 laminin:fibronectin solution provided us with higher numbers of attached and non-differentiated colonies, prompted us to try the 3:2 v/v ratio of fibronectin:laminin. A possible explanation of the fact that the increased fibronectin concentration in the matrix coating mixture increased even more the number of undifferentiated HUES1 hESC colonies, may come from a study published back in 1991.

This study suggested that the presence of exogenously supplied fibronectin (not secreted by

102 the cells) promotes and enhances the assembly of basement membrane matrix containing laminin (Austria et al., 1991). Fibronectin might act similarly in our experimental set up.

3.6 Conclusions

We have hereby developed a system that can allow the propagation and attachment of HUES1 hESC colonies on glass coverslips. The final constitution of our artificial matrix suggests that fibronectin could have a more important role than laminin in keeping hESCs in a pluripotent state. However, it also suggests that the laminin and fibronectin can act synergistically and additively, complementing older studies on the subject. This study, by no means presents a full characterization of the hESCs used in our experiments. Such a study would require more extensive testing –like inclusion of more pluripotency markers, such as

NANOG, Sox2, SSEA-3 and Tra1-81. However, the purpose of this work was not to fully characterise the hESCs, but to produce a cost effective system that would allow us to plate

HUES1 hESCs on glass coverslips in the absence of feeder cells, so that these can be used in downstream experiments.

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Chapter 4: Purinergic Signalling

4.1 Introduction

2+ Increases in intracellular calcium concentrations ([Ca ]i) influence a range of cellular activities including cell proliferation and differentiation. Even though in vivo calcium signals are usually transient, lasting between a few milliseconds up to a few minutes, the resulting gene expression changes can be long lasting (Roche et al., 1994). One of the mechanisms explaining this property of calcium signalling is the fact that it influences the expression of the so-called “immediate-early response” genes (Roche et al., 1994). The transcription of immediate-early response genes, many of which encode transcription factors, is activated almost as soon as the cell is exposed to an extracellular stimulus (Greenberg et al., 1992).

The control of transcription factor expression allows the immediate-early response genes to mediate the expression and alter the transcription rate of secondary response genes, resulting in cell specific or ligand specific responses (Herschman, 1989; Roche et al., 1994).

The ability of calcium to control the expression of many genes downstream of the immediate- early response genes has increased the interest in trying to understand how calcium homeostasis is linked to gene expression. One way to elucidate the role of calcium in

2+ signalling events inside a cell, is the study of the mechanisms that affect the [Ca ]i levels and the effects produced by the induction of these mechanisms.

2+ [Ca ]i can be modulated via the activation of purinergic receptors. Purinergic receptors or purinoreceptors are a membrane-bound family of receptors that respond to signals from nucleotides, such as ATP (Burnstock, 1990; Dalziel et al., 1994). ATP, apart from being associated with energy storage in cells, has been demonstrated to be a signalling molecule, which is released by nerve cells. It has also been established that non-neural cells release ATP in response to a variety of signals (Thompson et al., 2012). There are two classes of purinoreceptors, P1 and P2. The P1 purinoreceptors, also called adenosine receptors, are responsive to adenosine and AMP with higher potency than ADP and ATP

(Burnstock, 1990; Dalziel et al., 1994). In contrast, the P2 receptors, also called ATP receptors are more responsive to ATP and ADP. Furthermore, the P1 purinoreceptors are

104 prone to the action of antagonists like theophylline and caffeine (methylxanthines), while the

P2 purinoreceptors are not affected by this class of molecules (Dalziel et al., 1994).

Purinergic ATP receptors are expressed in the early stages of embryonic development of many different organisms. Their expression profile seems to be altered during development and this may indicate specific functions for purinergic signalling in the progression of differentiation and final phenotype specification (Dalziel et al., 1994). Heo and Han have previously demonstrated that ATP signalling via purinergic receptors in mouse

2+ embryonic stem cells is associated with a rise in [Ca ]i concentration (Heo et al., 2006).

They also suggested that purinergic signalling is linked to mouse embryonic stem cell proliferation and differentiation (Heo et al., 2006). With this information in mind, even though there have been no reports about the ability of the hESCs themselves to secrete ATP, it should not surprise us if hESCs secreted ATP, which would regulate their proliferation/survival/differentiation in a paracrine manner. Earlier studies have demonstrated the ability of non-secretory mammalian cells to secrete ATP under quiescent conditions, upon mechanical stimulation, or when exposed to various agonists, such as acetylcholine (Burnstock, 1990). An alternative source of ATP during embryo development would be the developing nervous system. Nerve cells can secrete ATP either as the primary neurotransmitter and pre-synaptic modulator, or as a co-transmitter (Su, 1983). This presents a clear possibility that during the innervation of different parts of the developing embryo, nerve cells may be stimulating early stem cell populations to differentiate. These possibilities prompted us to study the functional profile of the hESCs. Previous work

(Cosgrove, K.E., unpublished) has demonstrated that the HUES7 hESC line expressed purinergic receptors at the mRNA level (Fig. 4.2).

No one has so far studied the functional profile of purinergic signalling receptors and their potential role in the differentiation of human embryonic stem cells. Therefore, the functions of purinergic receptors in these cells remain to be determined. In this study, the functional profile of purinergic receptors in hESCs was investigated. We hereby define the functional profile of purinergic receptors as the process whereby the range of functional receptors is distinguished by the study of the cell responses to different purinergic signalling

105 agonists/inhibitors. At the same time, the association of purinergic signalling to the differentiation of human embryonic stem cells was explored.

For our studies, we used the HUES1 hESC line. The results of our study suggested that HUES1 hESCs did not express functional P2X1 purinoreceptors, however, they expressed functional P2Y6 receptors, which were responsible for coupling responses to ATP

2+ to changes in intracellular [Ca ]i. They also suggested that a possible link between pluripotency and purinergic signalling may exist.

Fig. 4.1: P2 Signalling mechanisms. A schematic representation of the signalling pathways involved in P2 purinergic signalling. The P2X receptors are ligand-gated ion channels, allowing entry of Ca2+ from the extracellular space, whereas P2Y receptors are G-Protein coupled receptors, mobilising calcium from the internal calcium stores through the action of phospholipase C. G/G/GQ: G-Protein subunits; PLC: Phospholipase C; PIP2: Phosphatidyl inositol (4,5) bisphosphate; DAG: Diacyl glycerol; IP3: Inositol triphosphate; ER: Endoplasmic reticulum.

Adapted from the Applied Biosystems “Gene Assist Pathway Atlas”, (2010) (Biosystems, 2010).

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Fig 4.2: Expression of P2 genes in HUES7 hESCs. Earlier results in the Cosgrove group (Wright, Elli Alexandra et al., 2009) have shown expression of purinergic receptors in HUES7 hESCs. Total RNA was extracted from HUES7 cells cultured on inactivated MEF feeder cells (n=3). RNA was also extracted from inactivated MEF cells alone (n=2). HUES7 hESCs expressed P2X1,2,4,6,7 and P2Y1,2,6,11. NT: no template control; gDNA: human genomic DNA; L: 100 bp DNA marker; ±: with/without reverse transcriptase

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4.2 Aims & Objectives

The aim of the study reported in this Chapter was to functionally characterise the purinergic profile of hESCs and compare the purinergic response profile of HUES1 hESCs against their randomly differentiated counterparts. In order to achieve this, undifferentiated

2+ and randomly differentiated hESCs were analysed using calcium microfluorimetry and [Ca ]i values were recorded upon exposure of the cells to a range of purinergic agonists.

Exposure of hESCs to purinergic receptor agonists and inhibitors was performed in the presence and absence of extracellular Ca2+. Use of receptor subtype specific agonists and inhibitors led to the identification of functional receptors. At the same time, the employment of extracellular Ca2+-free environment was used to identify the involvement of P2Y purinergic signalling in the observed cell responses. Finally, an alkaline phosphatase assay was used in order to investigate a potential role of purinergic signalling in hESC differentiation.

4.3 Results

4.3.1 Desensitisation of purinergic receptors in hESCs

As the purinergic receptors are known to desensitise fairly fast on continuous agonist presentation (Burnstock, 1990; Wright, A. J. et al., 2009), we were interested to see whether that effect would be observed in hESCs. Repeated applications of 100 M ATP suggested that the purinergic receptors present on hESCs desensitize (Fig. 4.3). In fact, the cells

2+ responded to each subsequent ATP stimulation with a smaller rise in [Ca ]i. The first application of ATP caused a rise of 47±10 nM, the second application caused a rise of 27±8 nM and the 3rd stimulation with ATP caused a rise of 18±3 nM (n=5/6 experiments), all vs. the basal level of calcium of 100 nM. This means that the reduction in response between the first and the second application of ATP reached 56.2%, while the purinergic response at the

3rd application of ATP fell down to 33% of the second application’s response. The reduction

2+ in the amplitude of response in terms of [Ca ]i suggested that the purinergic receptors that are expressed in HUES1 hESCs can desensitize. The values presented are the mean

2+ estimated percent rises in [Ca ]i ±SEM (n=5 experiments). The statistical significance of the results was confirmed by One Way ANOVA (p=0.009), which confirmed that the differences

108 in the mean values among the treatment groups were greater than would be expected by chance (Fig. 4.3).

Fig 4.3: Desensitization of purinergic receptors expressed on HUES1 hESCs upon repeated stimulation with ATP. 100 M ATP were applied repeatedly to HUES1 hESCs in the presence of extracellular Ca2+. Each application lasted 30 sec and was followed by a 4 min break. Statistical comparison of the responses (one way ANOVA) between the three different applications revealed that the results were statistically significant (**: p≤0.01, n= 5 repeats)

4.3.2 Responses of undifferentiated and randomly differentiated

HUES1 hESCs to ATP stimulation

2+ The estimated value of the average basal resting [Ca ]i was 100±8 nM (n=15 experiments, p<0.05) for the HUES1 hESCs, while it was slightly higher at about 150±12 nM for the randomly differentiated hESCs. The minimum concentration of ATP required by the

2+ undifferentiated hESC to respond by demonstrating a rise in [Ca ]i was 10 µM. The

2+ estimated rise in [Ca ]i for this study was defined as the absolute difference between the starting and highest observed concentration. In the case of 10 µM ATP, this was 62±5 nM

109

2+ [Ca ]i (n= 40 cells from 5 experiments), while maximum response was observed at 50 µM

2+ ATP (168±5 nM [Ca ]i, n=40 cells). The response to 100 M ATP was found to give an

2+ 2+ estimated rise in [Ca ]i of approximately 150 nM (150±7 nM [Ca ]i, n=40 cells) (Fig. 4.4A).

Statistical analysis using one way ANOVA (both normality and equal variance tests passed) revealed that the differences in the mean values among the treatment groups are greater than would be expected by chance, which implied that there was a statistically significant difference between them (p≤0.001). In addition, post hoc tests were performed (Holm-Sidak method), which confirmed that there was statistical significance in all pair-wise comparisons

(overall significance level=0.05). In more detail, the differences between the responses of hESCs at different concentrations of ATP were statistically significant. The data were also fitted in a dose response curve (Fig. 4.4A), using the GraphPad Prism, from which the values of pEC50 and maximum response Rmax were extrapolated. The pEC50 for ATP in hESCs was equal to 4.98 (p<0.0001), while the maximum response was determined to be 160 nM

2+ [Ca ]i. At the same time, the Hill slope of the dose response curve was determined to be equal to 10.03 (p<0.0005, r2=0.98).

2+ The rise in [Ca ]i as a result of stimulation of randomly differentiated hESCs was lower than in undifferentiated hESCs (p<0.05), while at the same time, the randomly differentiated cells presented a different stimulation profile. Our data demonstrated that the maximum response to ATP in differentiated hESCs was produced upon stimulation with 100

2+ M ATP (93±8 nM [Ca ]i, n=40 cells), while their response to 10 M ATP was at around 20

2+ nM (20±10 nM [Ca ]i, n=40 cells). The randomly differentiated cells responded to 50 M of

2+ 2+ ATP by showing an estimated rise in [Ca ]i of around 75 nM (75±8 nM [Ca ]i, n=40 cells).

One way ANOVA statistical analysis revealed that the response values for the three different concentrations of ATP, having passed the normality and equal variance test, were statistically significant different between them, with the differences in mean values among the treatment groups being greater than would be expected by chance (p≤0.001). The

ANOVA comparison was followed by the Holm-Sidak pairwise comparison test, which confirmed the statistically significant difference between the responses of the three groups

(overall significance level=0.05). The results were then fitted in a dose-response curve (Fig.

4.4B). The pEC50 for ATP in randomly differentiated hESCs was determined as 4.56

110

2+ (p<0.0001), while the maximum response Rmax was determined to be 110 nM [Ca ]i. The hill slope of the dose response curve was determined to be equal to 1.33 (p<0.0001, r2=0.97).

Comparison of the responses of randomly differentiated hESCs to different concentrations of ATP by one way ANOVA revealed that the results were statistically significant (p≤0.001), while all pairwise comparisons (Holm-Sidak test) between the different responses confirmed that the differences were significant statistically (overall significance level=0.05).

Fig. 4.4: The pharmacological profile of ATP related purinergic signalling in HUES1 hESCs and randomly differentiated HUES1 hESC cells. HUES1 hESCs and randomly 2+ differentiated HUES1 hESCs responded to ATP by showing change in [Ca ]i. The raw data were plotted on a dose response curve for the HUES1 hESCs (A) and the randomly differentiated HUES1 hESCs (B).

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4.3.3 Study of the P2Y purinoreceptor associated signalling in hESCs

Application of 100 µM ATP in the absence of extracellular Ca2+ caused an absolute

2+ level change (the absolute difference between the highest and lowest value) in [Ca ]i of

336±20 nM, n=40 cells; p<0.001 vs. basal levels of 100 nM in HUES1 hESCs, while the response of the differentiated hESCs was significantly lower (215±10 nM, n=40, paired t-test, p<0.001 vs. basal levels of 150 nM). Upon exposure of hESCs to CPA in the presence of extracellular calcium, we discovered that the purinergic response to ATP was lost. Removal of CPA exposure restored the response of the hESCs to ATP, which was observed as an

2+ increase in [Ca ]i (Fig. 4.5C), which further supported the suggestion that the increase in

2+ [Ca ]i associated with ATP in HUES1 hESCs is mainly attributed to the internal calcium stores and in consequence to P2Y signalling.

Out of the P2Y receptors that could have been involved in purinergic signalling in hESC -based on our earlier gene expression studies- we chose to focus on the P2Y6 purinoreceptor, as there was an available selective antagonist for it at that time that could assist our studies. The analysis of mRNA gene expression demonstrated that the HUES1 cells expressed the P2Y6 purinergic receptor (Fig. 4.6). This result was further confirmed by using Western Blot analysis with an anti-P2Y6 specific antibody (Alomone Laboratories,

Israel) which showed expression of the P2Y6 receptor at the protein level on protein extracts from HUES1 hESCs (Fig. 4.7). This result was also confirmed by immunofluorescent studies, which suggested that this receptor is expressed in HUES1 hESCs. In order to investigate the localisation of the P2Y6 receptor, the immunostained hESCs were studied under an upright confocal microscope. Fig 4.8 presents the immunofluorescent images with

Oct-4, DAPI and P2Y6 staining, as well as the maximum intensity projections of the 3D stack that was acquired from these immunostained coverslips under the confocal microscope.

However, the expression of a protein does not always confirm that it is functional in a cell or tissue. In order to confirm that the P2Y6 purinergic receptor was functional in the

2+ HUES1 hESCs, a selective P2Y6 antagonist, MRS2578, was used in Ca microfluorimetry experiments, where the cells were stimulated with 100 µM ATP in a calcium-free

112 environment, in the presence of MRS2578. These studies revealed that only one third of the total number of hESC cells were capable of responding (33%), which may suggest that not all, but the majority of the hESCs express functional P2Y6 receptors. In addition, they showed that undifferentiated hESCs had a significantly lower response to ATP (from approx

336±20 nM in the absence of the MRS2578 antagonist to 35±4 nM when it was present, n=50 cells), while randomly differentiated hESCs also showed a reduced response (from

215±10 nM in the absence of the MRS2578 antagonist to 109±5 nM when the antagonist was present, n=160 cells from 6 preparations, ANOVA Dunn’s Method, p<0.05). In addition, the entire population of the randomly differentiated hESCs studied herein presented a response upon ATP stimulation while under the influence of MRS2578. For a summary of these results, see Fig. 4.5 (A,B).

Fig 4.5: HUES7 hESCs express functional P2Y6 receptors. Application of 100 µM ATP in 2+ 2+ the absence of extracellular Ca caused a rise in [Ca ]i in all cells in the colonies studied (n= 2 colonies, 80 cells for each of undifferentiated and differentiated cells from 3 experiments) (A). However, stimulation of hESCs with 100 µM ATP, in a calcium-free environment and in the presence of MRS2578 – revealed that only a fraction of HUES1 hESCs (33%) were capable of responding, with all randomly differentiated cells studied showing a response (B). Panel (C) shows a characteristic trace of a hESC (n=40 cells), where the hESCs fail to respond to ATP with calcium supplied in the extracellurar environment and in the presence of CPA . The response is rescued upon the removal of CPA. (CPA: Cyclopiazonic acid). Results are expressed as mean values ±SEM; *: p<0.05.

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Fig 4.6: mRNA gene expression analysis of the P2Y6 expression in HUES1 hESCs and MEFs. mRNA was extracted from hESCs and MEFs and used to convert into cDNA. RT-

PCR analysis revealed that the HUES1 cells at 3 different passages expressed P2Y6. M = 1 kb DNA Marker (Bioline, UK). The numbers above the lanes correspond to 3 consecutive passages of HUES1 hESCs, starting from passage P32. g = human genomic DNA. NC = no template control. + is for mRNA samples that were converted to cDNA in the presence of Reverse Transcriptase, while - is for samples that were treated in the exact same way, minus the Reverse Transcriptase.

Fig 4.7: Identification of P2Y6 protein expression in HUES1 hESCs, Min6 cells and MEFs using western blot analysis. The figure shows the developed blot, after being probed with a P2Y6 antibody. The band was found to run at a molecular weight of 42 kDa. From left to right, showing protein expression in MEF cells (M), Min6 cells (M6), positive control (antigen that was supplied together with the antibody) (+), HUES1 hESCs (H).

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Fig 4.8: Immunofluorescent and confocal microscopy – Localisation of P2Y6 in HUES1 hESCs. HUES1 colonies stained for DAPI (blue channel), Oct-4 (green channel) and P2Y6 (red channel) as they appear under a snapshot widefield (A, B) and confocal (C, D) microscope.

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4.3.4 Responses of undifferentiated and randomly differentiated

HUES1 hESCs to α,β-meATP stimulation

2+ The HUES1 hESC [Ca ]i response to different concentrations of meATP was tested. No response was produced by the application of a range of concentrations of

meATP between 0.1-100 M (n=80 cells for each concentration from 5 experiments) in neither the undifferentiated nor the randomly differentiated hESCs. Towards the end of each experiment, 100 ATP were applied on the colony, to confirm the cell viability, which confirmed that the cells were alive, but unable to respond to this purinergic receptor agonist.

4.3.5 Responses of undifferentiated and randomly differentiated

HUES1 hESCs to UTP stimulation

2+ The HUES1 hESCs were also responsive to UTP, with an estimated rise in [Ca ]i

2+ appearing at 10 µM (105±12 nM [Ca ]i, n=40 cells from 4 experiments), while the maximum

2+ response was shown at 50 µM of UTP (130±10 nM [Ca ]i, n=40 cells from 4 experiments)

(p<0.05). The response of the HUES1 hESCs to 100 µM of UTP was almost similar to the

2+ response presented upon stimulation with 50 µM of UTP (120±10 nM [Ca ]i, n=40 cells from

4 experiments). The results were fitted on a dose response curve (Fig. 4.9A), which revealed that the pEC50 for UTP in HUES1 hESCs was equal to 5.13, while the maximum response

2+ Rmax was found to be 124 nM of [Ca ]i. In parallel, from the same dose response curve, we determined that the Hill slope for UTP in HUES1 hESCs was equal to 7.1 (p<0.05, r2=0.96).

In contrast, the randomly differentiated cells showed reduced responses to UTP stimulation, compared to the undifferentiated hESCs. The maximum response in terms of a

2+ 2+ rise in [Ca ]i was shown upon stimulation with 50 M UTP (38±2 nM [Ca ]i, n=40 cells from

2+ 4 experiments), while the response to 10 M UTP was at around 22 nM (22±2 nM [Ca ]i, n=40 cells from 4 experiments). Finally, the response of the randomly differentiated cells to

2+ 100 M UTP was approximately 30 nM (30±2 nM [Ca ]i, n=40 cells from 4 experiments)

(Fig. 4.9A). Fitting the results on a dose response curve (Fig. 4.9B) helped us determine the pEC50 for UTP in randomly differentiated cells, which was equal to 5, with a maximum

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2+ response Rmax calculated to be at 35 nM [Ca ]i. The Hill slope for UTP in randomly differentiated cells was also extracted from the fitted dose response curve and it was found to be equal to 8.64 (p<0.05, r2=0.95).

One way ANOVA was used to compare the mean values across all the different treatment groups in both hESCs and randomly differentiated hESCs (Diff), which revealed that all the mean values were statistically different between them (p≤0.001). Then, a post hoc test (Holm-Sidak pairwise multiple comparison) revealed that there was statistical significance in the differences between all groups (overall significance=0.05), apart from the following pairs: 10M UTP hESCs vs. 50M UTP Diff, 50M UTP hESCs vs. 100 M UTP hESCs and 50M ATP Diff vs. 100M ATP Diff, where the differences between the groups were not statistically significant.

4.3.6 Responses of undifferentiated and randomly differentiated

HUES1 hESCs to UDP stimulation

2+ The HUES1 hESCs were much more sensitive to UDP in terms of changes in [Ca ]i, showing a response after the application of only 1 M UDP, which resulted in a rise of 95 nM

2+ (95±2 nM [Ca ]i, n=40 cells from 5 experiments) (Fig. 4.10). The response to 10 M of UDP

2+ was at around 25 nM (25±2 nM [Ca ]i, n=40 cells), while the cells showed a response to 50

2+ 2+ M of UDP by demonstrating a rise in [Ca ]i of approximately 40 nM (40±3 nM [Ca ]i, n=40 cells).

The randomly differentiated cells also responded to UDP, showing changes in the

2+ 2+ levels of [Ca ]i of approximately 155±5 nM, 83±4 nM and 60±5 nM [Ca ]i upon stimulation with 1 M, 10M and 50 M UDP respectively (n=40 cells).

However, because of the apparent desensitisation of both types of cells, we were unable to fit the results on a dose response curve.

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Fig. 4.9: Studies on the pharmacological properties of UTP related purinergic signalling in HUES1 hESCs and randomly differentiated HUES1 hESC cells. HUES1 hESCs and randomly differentiated HUES1 hESCs responded to UTP by showing change in 2+ 2+ 2+ [Ca ]i. The estimated changes in [Ca ]i ([Ca ]i) were plotted in the form of a dose response curve for each of the applied concentrations of UTP for the hESCs (A) and their randomly differentiated counterparts (B).

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Fig. 4.10: The response of HUES1 hESCs and randomly differentiated HUES1 hESCs to UDP. Showing the estimated responses of hESCs and their randomly differentiated counterparts upon exposure to increasing concentrations of UDP in terms of a change in 2+ [Ca ]i (nM). The results are presented on a bar chart in the form of the mean values ±SEM. *: p≤0.05

4.3.7 Assessment of the effects of purinergic receptor agonists on the pluripotency of HUES1 hESCs.

Undifferentiated hESC are typically characterised by high expression levels of markers, such as SSEA-4, TRA-1-60, Oct-4 and alkaline phosphatase (AP). These markers are down-regulated upon spontaneous or induced differentiation. Quantification assay kits based upon the detection of AP levels are available and have been used to assess the differentiation potential of ATP and other agonists of purinoreceptors on HUES1 hESC. The assay is based upon the fact that, under alkaline conditions, ATP can catalyse the hydrolysis of p-nitrophenyl phosphate (p-NPP) into phosphate and p-nitrophenol. The amount of p- nitrophenol produced is proportional to the amount of AP present within the reaction. Since p-nitrophenol is a yellow coloured reagent, changes in expression levels of AP can be visually assessed and quantified through measurements of the optical density.

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ImagePro Express software (QImaging, UK) was used to assess the number of alkaline phosphatase positive colonies and their relative areas. Criteria for assessing the differentiation status were set by comparing data sets from hESCs that remained undifferentiated with those from cells that had been intentionally differentiated (this was the differentiation control), using a medium containing 10% FBS in DMEM-Glutamax (both from

Invitrogen, UK). Figure 4.9 illustrates the colour contrasts between colonies under both conditions (A,B), where the dark red colonies are considered pluripotent, while the light pink colonies do not have such a high AP activity and are therefore considered differentiated. Fig

4.11 also presents the results from a control experiment (C), where the alkaline phosphatase assay was tested using hESC that had been cultured in hESC medium (Ctrl) or differentiation control medium (Diff). The average pluripotency was assessed by calculating the total area of dark red (pluripotent) vs pink (differentiated) stained cells over a total of 160 colonies for each condition. The results demonstrated that the percentage of pluripotency of the HUES1 hESCs remained almost constant up to 48 hr at ~80% (n=1, 160 colonies were included in the calculations). The differentiation control medium caused a 75% decrease in pluripotency within 24 hr, between the 24 hr and the 48 hr experimental time points (n=1,

160 colonies were included in the calculations).

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Mean Mean % Pluripotency

Levels

Fig 4.11: Control experiment for the alkaline phosphatase assay. Images representing the colours that were chosen to represent a positive (A) result, where the colonies were stained dark red and a negative (B) result where the differentiating colonies have lost their high AP activity and were stained pink. Panel C shows the mean ± SEM percentage pluripotency, defined as the total area of red vs pink colonies, showing HUES1 hESCs for the control (Ctrl) HUES1 hESCs, cultured in hESC medium and the differentiation control (Diff) colonies at 24 and 48 hr. The differentiation control was incubated in medium consisting of DMEM-Glutamax with added 10% FBS. Statistical analysis: * : p≤0.05

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Further analysis was carried out by applying 100 M ATP, UTP or UDP to the HUES1 hESCs in culture. The application was repeated every 24 hr and the colonies were then stained with the alkaline phosphatase assay at 24 hr and 48 hr after the initiation of the experiment, in order to determine the amount of differentiation of the HUES1 colonies. The results were compared to control populations of HUES1 hESCs that had not received the treatment with ATP, UTP or UDP.

The average area occupied by the pluripotent cells of a number of colonies (n=160) that had been incubated with 100 M ATP was measured, together with the total area of all colonies counted. In other words, the cells were on each colony were divided into dark red and pink and the result was the percentage of the area occupied by dark red stained cells against the total area of the measured colonies,

The results suggested that ATP exposure over 48 hr may lead to early differentiation of 15% more HUES7 hESCs than the control (Fig. 4.12A). A similar effect was observed following the exposure of hESCs to 100 M UTP (Fig. 4.12B). Exposure of the cells to 100

M UDP did not show any significant effects on the differentiation status of hESCs, compared to the control (results not shown). In contrast, the application of suramin caused over 80% of the hESCs to differentiate within the first 24 hr (Fig. 4.12C)

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Fig 4.12: Purinergic signalling can affect the spontaneous differentiation of HUES1 hESCs. ATP exposure (100 M) over 48 hr led to the early differentiation of 15% more HUES7 hESCs than control, n=160 colonies, a result that was statistically significant (A). A similar effect was observed following the exposure of hESCs to 100 M UTP (B). Exposure to 10 M suramin was enough to drive the differentiation of 82% of hESCs within the first 24 hr period. Results are expressed as mean values ±SEM; *: p<0.05

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4.4. Discussion

4.4.1 Purinergic Signalling in hESCs

2+ ATP is known to induce increases in [Ca ]i through two distinct mechanisms, one involving the opening of the membrane channels of the P2X receptor family. An alternative

2+ pathway involves triggering the release of Ca from IP3-sensitive stores. This necessitates the study of the mechanism of action of ATP in the HUES1 hESCs, by determining the influence of extracellular Ca2+.

Our data demonstrated that the functional purinergic profile of the cells we studied is distinct between the undifferentiated HUES1 hESCs and their randomly differentiated counterparts. This result came in agreement with other published work, which demonstrates that the purinergic profile of stem cells changes during differentiation towards different cell types (Abbracchio, 1996; Glaser et al., 2012), which in our case, we have made it specific for hESCs and we have also suggested involvement of P2Y6 .

The fact that the hESCs responded to stimulation by ATP suggested the possible functional presence of any of the following purinoreceptors: P2X1,2,3,4,5,7 and P2Y1,2,4,6

(Burnstock, 2007). In addition, our results suggested that the HUES1 hESCs do not have functional P2X1 receptors, since they are the receptors that are mainly sensitive to - meATP (Burnstock, 2007). Interestingly, previous studies on another hESC cell line,

HUES7, have demonstrated the expression of P2X1 at the gene level (Wright, Elli Alexandra et al., 2009). We did not confirm this result by analysing the gene expression of HUES1 hESCs. However, if gene expression analysis studies confirmed the expression of P2X1 in

HUES1 cells, this would indicate that the P2X1 receptor is expressed in HUES1 cells, but is non-functional. In other words, it may be possible that the expression of P2X1 is associated with cell differentiation and is therefore kept inhibited by regulating the post-translational modification of the protein. However, there studies on a range of cells have appointed conflicting functions on P2X1 when it comes to the promotion or inhibition of cell differentiation. In P19 murine carcinoma cells, P2X1 expression only appeared after the induction of neuronal differentiation (Resende et al., 2007). However, in different studies, it has been shown that P2X1 receptor signalling protects rat osteoblasts from differentiating

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(Orriss et al., 2012). It is possible that such differences in observed patterns after stimulation with an agonist selective for P2X1 may be associated with its ability to form heteromultimers with other P2X receptor subtypes, a property of P2X1 that has been previously reviewed by

Burnstock (2007).

The differences in signalling effects after stimulation of the same receptor in different cells might also be related to the way the cells decide over which pathway to follow after the same stimulus has been received, in this case a rise in intracellular calcium ions levels. The cell responses upon activation of a purinergic receptor vary according to the variations in intracellular calcium concentration, such as the length, frequency and amplitude of calcium oscillations, or the nature of the calcium levels modification, i.e. a transient local vs. a global cell calcium level rise (Tonelli et al., 2012).

The response of cells to ATP in the absence of extracellular calcium could also indicate the involvement of another purinergic receptor in purinergic signalling, the P2Y2 purinoreceptor, which can also respond to ATP. The response of the hESCs to ATP in the absence of extracellular calcium, suggested the presence of functional P2Y receptors in the cells, since these are the receptors that are capable of utilising the intracellular calcium to produce calcium spikes upon stimulation with nucleotides. This can be explained by the P2Y purinergic receptors signalling pathway, which implies that stimulation of these receptors by the right agonist, leads to the release of Ca2+ from the intracellular calcium stores (fig. 4.1).

The suggestion that P2Y receptors play an important role in purinergic signalling in hESCs was further investigated by employing CPA in our experiments. CPA inhibits the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), the role of which is to keep the internal cell calcium stores replenished by mediating the uptake of Ca2+ ions into the endoplasmic

2+ reticulum lumen (Laursen et al., 2009). The [Ca ] inside the calcium storing organelles is controlled by a balance between the action of the SERCA channels and the passive efflux

(leakage) of Ca2+ (Hofer et al., 1996). The passive efflux of Ca2+ means that continued exposure of the cells to CPA will lead to depletion of the internal calcium stores.

The presence of functional P2Y2 receptors in HUES1 hESCs could be associated with the observations by Heo & Han, who suggested that ATP raises the proliferation rate of mESC through the action of the purinergic receptors P2X3, P2X4, P2Y1 and P2Y2 (Heo et al.,

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2006). The ability of ATP to control proliferation rate has already been demonstrated in astrocytes. Studies on purinergic signalling in astrocytes have shown that ATP acts synergistically with bFGF to promote DNA synthesis (Neary et al., 1994). It has been proposed that the bFGF and the purinergic signaling pathways merge at the level of the

MAPK cascade and that both ATP and bFGF stimulate the formation of AP-1 complexes, which are functional heterodimeric transcription complexes consisting of Fos and Jun families of transcription factors (Abbracchio, 1996). Since bFGF is an important growth factor needed by the hESCs to remain pluripotent, it is possible that ATP employs a similar mechanism in HUES7 hESCs to control cell proliferation, making the expression of P2Y2 receptors important for hESC proliferation.

On the other hand, our preliminary results suggested that ATP, together with UTP, seemed to have a negative effect on the HUES7 hESC pluripotency level, making the cells differentiate early. While there are no reports linking ATP or UTP with pluripotency or differentiation of embryonic stem cells, the ability of ATP to control the differentiation of different cells has been thoroughly investigated (Abbracchio, 1996). It has been suggested that in many instances, the phenotypic changes induced by nucleotides are serving the function of promoting the maturation of cells towards a more differentiated phenotype

(Abbracchio, 1996). The proposed mechanisms of action include a direct effect of nucleotides on target cells via receptor-dependent and receptor-independent mechanisms, stimulation of the production of secondary trophic substances active on target cells, enhancement of the effects of such trophic substances on target cells, or a combination of all the above (Abbracchio, 1996). The existence of such a diversity of pathways that can lead to differentiation following nucleotide signalling, makes it difficult to define whether the

2+ differentiation effect is a result of the changes occurring in [Ca ]i or a result of any of the other pathways that seem to be linked to purinergic signalling, like the downstream DAG and

IP3 signalling that is associated with the P2Y purinergic receptors. Investigation of the differentiation observed in the alkaline phosphatase experiments to check if it is linked to calcium signalling, would have to involve the inclusion of a higher number of inhibitors and agonists of specific purinergic receptor subtypes (Burnstock, 2007), something that we did not manage to do because of time restraints, the lack of readily available purinergic receptor

126 specific antagonists (Bornstein, 2008) and the fact that one of the commonly used purinergic signalling inhibitors described in the literature, suramin, is directly affecting the differentiation status of hESCs by removing bFGF from high-affinity plasma membrane receptors, as well as from heparin-like, low-affinity binding sites (Presta et al., 1991; Stein, 1993).

Our data also suggested that the receptors that responded to ATP desensitised. The desensitisation of P2X receptors has been documented before and it is one of the ways of modifying the responsiveness to sustained/repeated applications of ATP. It consists of a two-phase cycle (Roberts et al., 2006). In the first part of the cycle, the receptor desensitises, most likely by having a ligand-bound closed conformation, or by an agonist- induced complete change in the extracellular ligand-binding domain (Roberts et al., 2006).

The receptor can then return to its “active” conformation and is available to be stimulated again. This happens when the agonist unbinds and the receptor returns to a recovered state

(Roberts et al., 2006). G-protein-coupled receptors, like the P2Ys are also known to desensitize rapidly on agonist application (Weick et al., 2005).

4.4.2 The P2Y Purinergic Profile of hESCs – HUES1 hESCs express functional P2Y6 receptors

Removal of Ca2+ from the extracellular solution did not have any effect on the ability of

2+ 2+ ATP to increase [Ca ]i levels. This pointed to an involvement of internal Ca stores in this process, which indicates the presence of functional P2Y receptors in the HUES1 hESCs.

2+ Furthermore, according to our investigation, CPA inhibited the rise in [Ca ]i in hESCs in response to ATP in the presence of extracellular Ca2+. CPA is an inhibitor of the microsomal Ca2+-ATPases (Falcone et al., 1995; Metz et al., 1994), which are ATP-driven

Ca2+ pumps that replenish the internal Ca2+ stores with calcium (Sze et al., 2000). In the absence of any stimulus, the intracellular Ca2+ stores “leak”, steadily losing calcium ions, which are released to the cytoplasm; therefore, any inhibition of Ca2+ uptake by the microsomal ATPases induces the internal calcium stores depletion (Potten et al., 2009).

2+ This relates to our results, as the inhibition of a rise in [Ca ]i in hESCs upon ATP stimulation in the presence of extracellular calcium indicated that the cells rely mainly on the internal

2+ stores for their purinergic signalling associated [Ca ]i changes.

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2+ In addition, the fact that the hESCs responded to UTP with a rise in their [Ca ]i levels strengthens the suggestion that hESCs express functional P2Y receptors. As UTP is a selective P2Y2 and P2Y6 agonist (Boeynaems et al., 1996; Burnstock, 1997), the response seen in our experiments indicated that P2Y2 and P2Y6 may both contribute to purinergic signalling in undifferentiated hESCs. The presence of functional P2Y6 receptors was also confirmed by the response of hESCs to UDP, a purinergic receptor agonist that is only specific to P2Y6 (von Kugelgen, 2006). The P2Y subtype identity was further elucidated by studying responses to ATP in the presence of MRS2578, a P2Y6 selective inhibitor

(McDonald et al., 2009), which confirmed that functional P2Y6 receptors were indeed present. This was further confirmed by immunostaining studies, which showed that P2Y6 is expressed in HUES1 hESCs. The fact that P2Y6 protein expression was seen on the same confocal plane as the nuclear Oct-4 staining, could indicate expression of P2Y6 receptors in the cytoplasm of hESCs, even though that would need further experiments to confirm, such as combined immunostaining with antibodies that bind to endosomes, which have the capacity to store proteins during the protein recycling that modulates their signalling. An antibody that could be used for that purpose could be an endosome marker like Rab5 (Yang et al., 2009). If the P2Y6 receptor was also found in intracellular endosomes, such a finding would not be unexpected to find P2Y6 receptors internalised in the cell in endosomes, as this is one of the ways a cell controls the functionality of GPCRs (Yang et al., 2009), the family of receptors including P2Y6. However, the active form of the receptor should be found on the cell membrane surface. The immunofluorescent and confocal microscopy results could be further improved by double staining for a cell surface marker, in addition to the P2Y6. An example of such a marker is the surface specific embryonal antigen 4 (SSEA-4), which is also a marker of pluripotency.

Our study suggested that the hESCs responded differently to a variety of purinergic signalling agonists than their differentiated counterparts. A part of our study that confirmed this suggestion was the fact that spontaneously differentiated hESCs did not respond in the same way to purinergic signalling agonists in the presence of MRS2578 in the medium. This was contradicted by the fact that the differentiated cells seemed to express P2Y6 as well, as was confirmed by immunofluorescence. These data suggest a reduced relative importance

128 for P2Y6 in differentiated cells in the responses to ATP –this may be due to the presence of other functional purinergic receptors. This is not surprising, as purinergic signalling has been shown to change during the development and terminal differentiation of a variety of tissues, like myoblasts and osteoblasts (Martinello et al., 2011; Orriss et al., 2006). This change in purinergic receptor expression during differentiation has also been observed in the P19 murine embryonal carcinoma cells (Resende et al., 2008). The P19 embryonal carcinoma cells are pluripotent cells with the ability to differentiate towards many tissue types in a similar manner to what happens in early embryos, thus emulating the molecular and morphological events that occur during early embryonic development (McBurney et al.,

1982).

4.4.3 The pharmacological profile of purinergic receptors expressed in both HUES1 hESCs and their differentiated counterparts

The binding of ligands to cell receptors in general, can sometimes follow a trend, where the binding of one molecule of the ligand makes it easier for more molecules to bind to the same receptor. This is called cooperative binding and it is a feature of receptors with more than one binding sites (Gesztelyi et al., 2012). There are also cases where the binding of one ligand molecule to a receptor makes further molecules harder to occupy another site of the same receptor, in other words, the receptor's affinity for other ligand molecules decreases -this is called non-cooperative binding. A useful way of defining this property of the receptor is by using the Hill coefficient or Hill slope, whereby a Hill slope value of less than 1 indicates non-cooperative binding, while when the Hill slope value is higher than 1, then we can say that the receptor presents cooperative binding for its ligand (Gesztelyi et al.,

2012). Interestingly, when ATP was applied to the HUES1 hESCs, the Hill slope we extracted out of the dose response curve fitted model was higher than 1 for both the hESC and their differentiated counterparts. Our results also suggested that when it came to P2Y6 functionality for example, the hESCs and the randomly differentiated hESCs had a different purinergic profile. Knowing from earlier reports that the purinergic signalling profile of cells changes with differentiation (Burnstock et al., 2011), as well as the fact that ATP is an agonist of many purinergic receptor subtypes (Burnstock, 2006), it was not surprising that

129 the Hill slope of these two different cell types differed. The cooperative binding in hESCs could imply that the binding of the ligand to the receptor creates conformational changes that reveal further ligand sites, making them "available" to more ligand molecules. There are such examples known in purinergic signalling, such as the case with the trimeric P2X receptors, such as P2X7. The P2X7 receptor is known to have three ATP binding sites and binding of one molecule of ATP on one site causes conformational changes that affect the selectivity of the receptor towards diphosphate forms of nucleotides (Browne et al., 2013), while all the P2X receptors have been shown to appear in two different open states, each allowing different molecules to go through the gated channel (Rokic et al., 2013). While the allosteric ligand interactions related to these two different "open" states have not been studied yet, when such studies take place, they could reveal that in one of these states, the nucleotides have different access to their binding sites. Both positive and negative cooperative binding has been observed in purinergic receptors, however, this has been artificially induced by the use of positive and negative allosteric modulators, which have the potential to be used as drugs (Jacobson et al., 2011).

The pEC50 values we calculated for each dose of ATP or UTP to our hESCs and their differentiated counterparts gave us an indication of the potency of the applied agonists -the pEC50 determines the concentration of agonist that needs to be applied to the cells in order to elicit half of the maximum biological response. This means that a smaller pEC50 correlates to a smaller dose of an agonist required to give the same response, which makes an agonist more potent. The pEC50 values were found to be similar in HUES1 hESCs and their differentiated counterparts for ATP and UTP applied to the cells as agonists, however, the Rmax values differed significantly between the undifferentiated and differentiated cells.

This apparent discrepancy could be explained by the actual molecular profiling of each cell type in terms of purinergic receptors. Our hypothesis is that the purinergic profile of differentiated hESCs is different than that of the undifferentiated cells. Since ATP for example can stimulate more than one subtypes of purinergic receptors (Burnstock, 2006), a combination of purinergic receptors could be stimulated by the application of this agonist and, depending on the expression levels of each receptor subtype, the end response may appear different.

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4.5 Critical discussion and further experiments

4.5.1 The use of Fura-2 AM in our experiments

Throughout this study, when it came to measurements of Ca2+ ions inside the cell, we used the Fura-2 AM. This is one of the many chemically fluorescent indicators for Ca2+, with

a high affinity for calcium ions – the dissociation constant Kd (Ca2+) of Fura-2 is 140 nM (Gee et al., 2000). The advantage of using the acetoxymethyl ester version of Fura-2 is the fact that the acetoxymethyl ester version is less prone to leakage out of the cell. Fura-2 has been known to leak out of the loaded cells and it has been previously reported that this might be the result of a facilitated transport mechanism (Malgaroli et al., 1987). Fura-2 AM is an esterified version of Fura-2AM that is cell-permeant (Oakes et al., 1988 1325). It becomes cell-impermeant upon entering the cell and becoming trapped within the cell, when the esterified part of the molecule is cleaved by native cell esterases.

A disadvantage that is commonly associated with other fluorescent calcium indicators is the photobleaching, which can reduce the signal obtained from calcium bound versions of the indicators, as well as introduce forms of the indicators that are still fluorescent, but insensitive to Ca2+ (Becker et al., 1987). For a ratiometric indicator, such as Fura-2, this problem is diminished, since, ideally, both excitation wavelengths will cause equal amount of photobleaching, which will be disregarded later, as the ratio should stay stable. There are

2+ some disadvantages on the use of Fura-2 AM for the measurement of [Ca ]i. One is the fact that Fura-2 has been shown to compartmentalise inside the cell, causing concentration gradients in parts of the cell (Malgaroli et al., 1987; Takeuchi et al., 1989), which can make it unavailable to respond at areas of the cell where local diffusion of calcium ions occurs. The second disadvantage had to do with the calibration of the system used for using the Fura-2

AM, which was performed in the absence of cells. For optimal results, an in-situ calibration would need to be performed after each experiment, as it has been known that the affinity of

Fura-2 for Ca2+ is affected by pH, viscosity and other intracellular environment factors

(Takahashi et al., 1999). However, this would be both time consuming and not needed in

2+ our case. The reason was the fact that we were not interested in determining the [Ca ]i very accurately or to visually confirm the source of Ca2+ entering the cytoplasm. The purpose of

131 this study was to study purinergic signalling in hESCs and assess the contribution of some of the different purinergic receptor subtypes on the observed Ca2+ changes inside the cell. So,

2+ it’s worth pointing out again that all values we had been obtaining when it came to [Ca ]i were estimated values.

4.5.2 ATP Hydrolysis and degradation in solution – A potential problem (or not)?

A potential problem with the exposure of hESC cultures to purines to study their long- term action on pluripotency and proliferation is the presence of ectonucleotidases on the cells. This family of enzymes are presented on the surface of the cells and they are responsible for modulating the purinergic signalling cascade (Yegutkin, 2008). The ectonucleotidases have the ability to rapidly hydrolyse ATP to ADP, AMP and adenosine.

They are important to the cell in many ways, one of them is the fact that their topological distribution allows the segregation of micromolar concentrations of an “ATP halo” in the pericellular space (Yegutkin et al., 2008), which can be used for autocrine signalling or for the control of localised signalling responses. In fact, alkaline phosphatase itself, whose activity is monitored by the alkaline phosphatase assay, is an ectonucleotidase (Yegutkin et al., 2008). However, it is unlikely that the 100 M ATP/UTP/UDP that was used in the experiment would have managed to stay intact and non-hydrolysed in the culture medium for

24 hr, which was the time interval between changing the feeding medium on the culture.

This would seemingly present a potential problem in the study of the action of specific purinergic receptors, as for example UTP could be hydrolysed into UDP during our experiment and they both have different preferences for P2Y purinoreceptors (Burnstock,

2006). In addition to that, ATP and AMP activate completely different purinoreceptors, with

AMP activating the P1 receptors (Dalziel et al., 1994). However, since the results from the alkaline phosphatase assay were different for UTP and UDP, this indicates that ectonucleotidases might not be such a major problem. One way to exclude ectonucleotidase activity from an experiment would be the use of non-hydrolysable nucleotide analogues, such as ATPS, UTPS or UDPS (Burnstock, 2007). Generalised inhibitors of

132 ectonucleotidases would not have been suitable for our purpose, as alkaline phosphatase activity for example is an inherent feature and characteristic of hESCs. However, even in the presence of ectonucleotidases, 24 hr exposure of hESCs to purinergic agonists might not be needed in order to observe any effect, since it has been shown before that even spontaneous transients in calcium levels, such as what might be happening after we apply purinergic agonists to the cells in vitro, may be enough to drive gene transcription

(Dolmetsch et al., 1998). A set of more extensive experiments monitoring the purinergic agonist levels in the medium in parallel with proliferation, such as the use of a suitable luminescent assay and a plate reader could provide a deeper insight on the effect of agonist hydrolysis on the purinergic signalling pathways studied in this chapter,

4.5.3 Further experiments

A more thorough investigation of the function of purinergic signalling in hESC physiology still needs to be completed. In this study, we have examined the hypothesis that stimulation of purinergic receptors by ATP will generate Ca-signalling events in undifferentiated hESC and that receptor stimulation may alter the self-renewal capacity of hESC by causing differentiation. We have also shown that the P2Y6 purinoreceptor may play a role in purinergic signalling in HUES1 hESCs. Further experiments are needed in order to thoroughly study the effects of P2Y6 signalling in the differentiation of hESCs. Such experiments could include the generation of an inducible vector system controlling the expression of siRNA for P2Y6 or a conditional P2Y6 knockout model. A similar effect could be achieved by the use of a blocking antibody for P2Y6 that has an epitope outside the cell membrane. Since all active receptors are found on the cell surface, no cell permeabilisation would be needed with such antibody and one could work with live cells. Any of these techniques would allow the control of expression of P2Y6 and it would offer us the capability to study the effects of P2Y6 related signalling in the course of differentiation of the hESCs towards pancreatic progenitors. Further studies should also attempt to establish whether the purinergic profile of hESCs during their differentiation towards pancreatic progenitors resembles the purinergic receptor profile of the developing embryo. This knowledge, might give us further control on directing the differentiation of the hESCs towards a pancreatic 

133 cell phenotype. In addition, it would be interesting to study whether signalling through the purinergic receptors affects the proliferation rate or survival of the hESCs, in a similar fashion to their action in mouse embryonic stem cells (Heo et al., 2006; Thompson et al., 2012).

Even though the alkaline phosphatase assay offered some preliminary results on the differentiation status of hESCs when exposed to purinergic receptor agonists, there are methods with higher sensitivity that could be used for the study of the differentiation and proliferation. These methods include the sorting of the hESC population using a surface marker for pluripotency, such as SSEA-4 and the use of proliferation specific assays, such as a BrdU associated assay. Furthermore, since our confocal microscopy data suggested some co-localisation between Oct-4 and P2Y6, it would be interesting what percentage of the

P2Y6 is internalised at any time. The speed of receptor recycling could offer further indications on the importance of purinergic signalling in hESC physiology.

4.6 Conclusions

The results indicate that HUES1 hESCs do possess functional P2Rs, which respond

2+ to ATP, UDP and UTP with changes in [Ca ]i following receptor stimulation. The study of the functionality of more purinergic subtypes was not possible in the hESCs, due to the lack of channel selective inhibitors and/or the interaction of some of them (e.g. suramin) with pluripotency associated molecular pathways (Kim et al., 2009). The Ca2+ responses observed, in combination with gene expression studies from others in my research group, indicate that the following P2Rs may be functional in HUES1 hESCs: P2Y2 and P2Y6, with

P2Y6 suggested having a significant –but not exclusive – role in the response to ATP in undifferentiated hESCs.

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Chapter 5: Development of a reporter construct for the differentiation of stem cells

5.1 Introduction

There are protocols available for the generation of pancreatic cells from hESCs.

These mainly focus on reproducing in vitro the major steps followed in embryonic development for the generation of the pancreas. These steps have been summarised by

Oliver-Krazinski (Oliver-Krasinski et al., 2008) and include the generation of foregut endoderm, followed by pancreatic endoderm, the production of pancreatic progenitors and then endocrine progenitors, leading to the generation of mature β cells that can secrete insulin in response to glucose stimulation. As the foregut endoderm, which is part of the primitive gut, is derived from definitive endoderm, one of the initial targets of groups focusing on the differentiation of hESCs towards pancreatic progenitors, has been the development of definitive endoderm.

It has been shown before that hESCs in tissue culture may express endoderm associated genes such as -fetoprotein (Itskovitz-Eldor et al., 2000), an effect that was possibly associated with the naturally occurring spontaneous differentiation of hESCs.

However, in order to obtain a sufficient population of definitive endoderm, the hESCs had to be subjected to directed differentiation. Several protocols have been able to efficiently generate definitive endoderm from hESCs, either by monolayer culture or by the formation of embryoid bodies (D'Amour et al., 2005; D'Amour et al., 2006; Jiang et al., 2007;

Johannesson et al., 2009; Kroon et al., 2008; Phillips et al., 2007; Shim et al., 2007; Zhang et al., 2009). However, their success in producing large numbers of Pdx-1 positive cells, indicative of pancreatic endoderm, was limited, with low yields obtained compared to the 80-

90% of hESC-derived definitive endoderm starting populations. These low yields presented a limitation to progress that needed to be addressed. Establishing a protocol to increase the yield of Pdx-1 positive cells would potentially include a comparison between multiple conditions. This in turn, would require a large number of hESCs to work with. The tissue culture of hESCs is laborious and technically demanding (Oh et al., 2005), as well as expensive. Therefore, testing multiple conditions for the generation of a reliable protocol for

135 the development of Pdx-1 positive cells would be a challenge for the average laboratory. We decided to bridge this gap by developing a tool that could be used for screening multiple protocols and potential novel substances to increase the efficiency of Pdx-1 positive cells production from definitive endoderm.

There are several ways in order to achieve the selection of a particular phenotype from a mixture of cells. An example is the employment of selective culture conditions (Smith,

2001). According to this method, manipulation of culture conditions such as FBS content, or the presence of growth factors during differentiation, can affect both the fate and the purity of the resulting cell populations following either monolayer or embryoid body differentiation protocols (Barberi et al., 2007; Wiles et al., 1991). Alternatively, a pure cell population can result from lineage depletion, where usually a cell surface antigen and a suitable antibody against it can be used to purify immunologically a specific cell type (Smith, 2001). This can be achieved either by methods such as fluorescence-activated cell sorting (FACS) analysis or through the use of magnetic beads attached to the antibodies used during the selection.

In the case of the strategies pursued towards the generation of definitive endoderm and pancreatic progenitors, there are recent studies utilising such techniques (Kahan et al., 2011;

Segev et al., 2012; Wang et al., 2012). A final method for the selection of pure cell populations is the introduction of transgenes in the differentiating cells (Smith, 2001). These transgenes can confer to the target cells either antibiotic resistance or fluorescent selection capability, controlled under the transcriptional control of a promoter that is active only in the desired cell population (Smith, 2001). Thus, whenever the promoter of interest is active in the target cells, it drives the expression of either an antibiotic resistance gene or a fluorescent marker, such as GFP.

We decided to develop such a cell selection system, where a fluorochrome, eGFP, would act under the transcriptional control of the Pdx-1 promoter. The principle behind our selection system would be the implementation of the Pdx-1 promoter-controlled reporter gene into hESCs, by using a lentivirus containing the transgene. This would permit rapid monitoring of differentiating cultures via a fluorescent plate-based reader system, or simple live fluorescent microscopy, to detect expression of Pdx-1. It could also be utilised for selection of differentiating cell populations that were committed to Pdx-1 positive pancreatic

136 endoderm, using fluorescence-activated cell sorting (FACS) analysis. Such cells could then be further differentiated towards pancreatic progenitor cells, with the end target of producing mature pancreatic cells that are capable of releasing adequate amounts of insulin in response to glucose stimulation.

In addition to being useful for finding the most efficient ways to differentiate hESCs to

Pdx-1 positive pancreatic endoderm, such a tool would also be useful for commercial drug screening for type 2 diabetes, where the up-regulation of Pdx-1 in  cells or their progenitors is seen as a desired feature of new drugs (Blyth, 2012; Kaneto et al., 2007). The reason behind this desirability is the fact that Pdx-1 plays a central role in  cells, controlling the transcription of the insulin gene (Ohlsson et al., 1993), as well as the pancreatic GLUT2 expression (Waeber et al., 1996). Furthermore, it plays a central role in the differentiation of endocrine progenitors towards mature  cells, by regulating the expression of genes such as

Ngn-3 and FoxA2 (Oliver-Krasinski et al., 2009). Finally, the importance of Pdx-1 in insulin expression and cell differentiation has also been demonstrated by its effect on non-cells.

Indeed, studies have shown that exogenously introduced Pdx-1 expression can lead to transdifferentiation of non-pancreatic cells to insulin secreting cells (Kaneto et al., 2008).

A deep understanding of the mechanisms involved in regulating the Pdx-1 expression in vivo is essential in order to generate a lentiviral transgene delivery system to facilitate the monitoring of Pdx-1 positive cells.

5.1.1: The regulation of Pdx-1 expression

The fact that both human and mouse models with inactive PDX-1 present with pancreatic agenesis, where the pancreas is completely absent, has led to the belief that both human and mouse Pdx-1 expression are controlled by similar mechanisms (Melloul et al.,

2002). Indeed, it has been shown that the human and mouse Pdx-1 5’-flanking areas share three short and highly homologous regions. These homologous areas were located between

−2.81 and −1.67 kb of the human Pdx-1 gene and between −2.7 and −1.8 kb of the mouse

Pdx-1 gene (Melloul et al., 2002). These three regions are since known as PH1, PH2, and

PH3 –standing for PDX-1-homologous regions 1–3 or alternatively known as areas I, II and

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III (Fig 5.1) (Gerrish et al., 2000; Marshak et al., 2000). Gerrish et al. (2000) also demonstrated that areas I and II bind HNF3, a transcription factor alternatively known as

FoxA2. They also showed that FoxA2 is an important transcription factor in the regulation of

Pdx-1 expression. Their methodology presented an example of how embryonic stem cells can be useful when studying early embryo development. Homozygous mutant mice for

FoxA2 died early in embryogenesis, before the pancreatic development was complete, which made the in vivo study of the role of FoxA2 in the regulation of Pdx-1 expression impossible.

The study of the role of FoxA2 in the regulation of Pdx-1 expression was performed in vitro, by differentiating mouse embryonic stem cells that had been manipulated in order to produce a knockout model of FoxA2 (Gerrish et al., 2000). Finally, they demonstrated that areas I and II can mediate cell-selective gene activation. Area I of the Pdx-1 promoter region has been shown to bind HNF1(Gerrish et al., 2001), but also PDX-1 itself (Gerrish et al., 2001;

Marshak et al., 2000)–suggesting the presence of a feedback mechanism, where PDX-1 seems to regulate its own expression. Different studies on area II have shown that it is also regulated by binding to Pax6 (Samaras et al., 2002), a transcription factor essential for the differentiation of glucagon secreting cells during pancreatic development (St-Onge et al.,

1997). Interestingly, area II can act independently of area I in facilitating Pdx1 expression in pancreatic cells, but both of them actually act synergistically, with area I potentiating the area II activity in both developing and adult pancreatic islet cells (Van Velkinburgh et al.,

2005). The role of area III in Pdx-1 expression regulation was not recognised for a long time, even though it had been known that deletion of the three conserved areas I-II-III could lead to serious defects in the development of the endocrine pancreas – although they were not as serious as the effects of a null Pdx-1 mutant (Fujitani et al., 2006). It was later discovered that area III, which can drive transient gene expression of Pdx-1 in pancreatic cells, contains a binding site for PTF1, a transcription factor that is essential for pancreas development (Wiebe et al., 2007). Interestingly, heterozygous mice with a Pdx1 deletion have been shown to present a better clinical picture than heterozygous mice with a deletion of the areas I, II and III of the Pdx1 promoter, which demonstrates the importance of the

Pdx1 promoter area (Fujitani et al., 2006).

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Therefore, all these studies indicate that a reporter lentiviral vector that could be used for the monitoring of Pdx-1 expression during pancreatic differentiation should include areas

I, II and III of the promoter area that controls Pdx-1 expression. 

Fig 5.1: Graphical representation of the structure of the Pdx1 promoter areas I, II and III. A drawing of areas I, II and III of the Pdx1 promoter, showing the transcription factors that have been associated with binding each area to modulate Pdx1 expression. Adapted from various references/reports (Eto et al., 2007; Melloul et al., 2002; Miyatsuka et al., 2007; Vanhoose et al., 2008; Wiebe et al., 2007).

-4.5 +1

5.2 Aims

The aim of this study was to generate a system that would allow the monitoring of

Pdx-1 expression. In order to achieve this, a reporter vector was designed, containing the mouse related sequences for the areas I, II and III that regulate Pdx-1 expression upstream of the Pdx-1 transcription initiation site. The vector was designed in such a way, so that an eGFP fluorochrome would be put under the transcriptional regulation of these sequences.

Our hypothesis was that the binding of transcription factors to areas I, II and/or III during differentiation, or during mature beta-cell function, would then cause the expression of eGFP which would be visible in live cells via fluorescent microscopy and via flow cytometry.

An alternative lentiviral vector was also tested which was developed elsewhere

(Szabat et al., 2009). This vector was obtained and tested in our hands (kind gift from Dr

Jim Johnson, University of British Columbia, Vancouver, BC, Canada). One of the main differences of this lentivirus was the fact that, apart from the mouse Pdx-1 promoter areas I-

II-III controlling red fluorescent protein (RFP) expression, the rat Insulin-1 promoter areas

139 controlling eGFP expression were also included. We tested this lentiviral vector in Min6 cells, which inherently express both Pdx-1 and insulin (Deramaudt et al., 2006).

5.3 Results

5.3.1 Construction and testing of the pLenti-Pdx1 I-II-III-mCherry vector

Reporter vectors can be useful tools in the monitoring of differentiation of hESCs towards various cell lines. Attempts were made to generate a lentiviral vector that could potentially be used for the monitoring of hESC differentiation towards pancreatic  cells.

Eleven bacterial clones were picked from a selective, antibiotic-containing agar plate, where the cloning reaction products had been plated overnight. Plasmid DNA was extracted from them, followed by DNA digestion with NotI enzyme. It was expected that the NotI enzyme would cut the DNA at two restriction site positions to result in two clear bands of linear DNA of different specific sizes. The digested products were run side by side with undigested plasmids on an agarose gel (Fig. 5.2). The gel revealed two potential positive cloning reactions (clones 2 and 4), where the Pdx-1 promoter sites and the eGFP sequence might have been cloned correctly into the lentiviral vector. However, the existence of an extra band meant that the results should be further investigated. Initially, confirmation of the result was attempted through digestion of the pBK mPdx1 I-II-III KSII with the NotI enzyme.

The pBK KSII vector should have only a single NotI restriction site, and this should lie outside of the Pdx-1 promoter region. Therefore, digestion of this plasmid -containing the cloned sequence for the mouse Pdx-1 promoter- with NotI should confirm that the mouse

Pdx-1 promoter areas I-II-III did not have any additional NotI restriction sites. The digest analysis showed a single linear DNA band, suggesting that there were no additional restriction sites inside the cloned Pdx-1 promoter sequence (Fig. 5.3). Therefore, since the eGFP sequence was not known to contain any NotI restriction sites, it was assumed that the additional NotI site must have resided within the lentiviral backbone.

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Fig 5.2: Restriction analysis of the cloned DNA of the constructed pLenti-Pdx1-GFP lentiviral vectors. The lanes correspond to the undigested (U) and digested (D) versions of each of 11 clones that were picked from the transfected bacterial cells (A). The expected band sizes were 7,449 bp and 3,232 bp. The undigested vector size was 10,681 bp. The clone numbers 2 and 4 were closer to the expected result; however, an additional band meant that the total size of the vector was higher than expected. The red arrows show the expected band sizes for the digested products. Panel B presents the expected vector map, with its basic features, showing the position of the two NotI restriction sites. M= Hyperladder I (Bioline, UK); WPRE= Woodchuck hepatitis virus post-transcription regulatory element; cPPT= central polypurine tract; LTRs= Long Terminal Repeats; 3'-del(U3)LTR= 3' Long Terminal Repeat missing the U3 enhancer element.

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Fig 5.3: Digestion analysis of the pBK mPdx1 I-II-III KSII with the NotI enzyme. A single band on the gel, confirmed that there were no additional digest sites inside the Pdx-1 promoter area sequence. The picture presents the single band, together with the Hyperladder I DNA marker (Bioline, UK). Expected product size: 3,661 bp. Band sizes on the left from bottom to top: 2,000 bp, 3,000 bp, 4,000 bp.

DNA sequencing analysis and alignment of these potential positive clones with the expected sequences revealed that the areas I, II and III of the constructed lentiviral vector were intact and inserted in the correct orientation and that both the positive clones were similar in sequence. The eGFP sequencing analysis of our construct suggested that it included 13 single mismatched bases, out of the total of 604 bases of the published eGFP sequence (US National Library of Medicine National Institutes of Health Database).

However, it was assumed that the sequence of our vector might have represented a different version of the eGFP fluorochrome. The sequence obtained was checked for any possible

NotI restriction sites, but there were none present. It was therefore assumed that a possible recombination event might have occurred in a position of the vector outside the regions of interest.

It was then decided to go on with packaging the lentiviral constructs inside the human embryonic kidney cell line HEK293T, in order to produce lentiviral particles and check whether the lentiviral construct would work.

Alternative methods of transfection were tested, including the use of Lipofectamine

2000® (Invitrogen, UK) and calcium phosphate precipitation. The resulted transfected cells after using the two methods of transfection can be seen in Fig. 5.4. Calcium phosphate DNA precipitation has been shown to be superior in terms of viral titration to another lipid based transfection substance, FuGENE6 (Mitta et al., 2005), which prompted us to perform all

142 subsequent HEK293T transfection experiments using the calcium phosphate precipitation method.

Fig. 5.4: Testing two different methods for the transfection of HEK293T cells with a lentiviral vector. The transfection of the HEK293T cells with the control lentiviral vector ® pLNT-SFFV-eGFP was tested using Ca2PO4 precipitation (A) and Lipofectamine 2000 (B). The maximum cell transfection, as indicated by the fluorescence level of the transfected cells under a fluorescent microscope, was observed at 48 hr post-transfection time. Scale bar at 25 m.

HEK293T cells were transfected with the eGFP control lentiviral construct and the transfection rate was assessed by observation under a fluorescent microscope every 24hr.

No visible eGFP expression was seen at 24hr post transfection. The HEK293T cells showed the first signs of fluorescence at 48 hr and they seemed to be at their maximum level at 72 hr post transduction initiation (Fig. 5.5). However, when viral particles from this transfection were used to transduce the pLenti-mPdx1 I-III-eGFP into Min6 cells, a cell line expressing

Pdx-1 (Eto et al., 2007), expression of eGFP using fluorescent microscopy could not be detected at any point after the transduction initiation. Gene expression analysis using mRNA from a control non-transduced Min6 population confirmed that the Min6 cells used in our experiments did express Pdx-1 (Fig. 5.6).

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Fig. 5.5: Control transfection of HEK293T cells and control transduction of Min6 cells. HEK293T cells were transfected with the control vector pLenti-SFFV-eGFP. At 48 hr post transfection, the transfected HEK293T cells were observed under a fluorescence microscope and found to be expressing the brightest levels of GFP (A). The lentiviral particle-containing medium was applied to Min6 cells. Panel B shows the transduced Min6 cells observed under a fluorescent microscope at the GFP channel at 72 hr after the transduction initiation. The photographs were used together with a brightfield microscopy image of the same areas in order to approximately identify the transduction rate, by calculating the percentage of gfp positive cells in the total cell population. The pictures show a combined fluorescent- brightfield acquired image from an area of the transfected cells on the tissue culture flask. Scale bar at 50 m.

Fig. 5.6: Confirmation of Pdx-1 gene expression in Min6 cells. cDNA from non- transfected Min6 cells was used as a template for a PCR amplification reaction, with a primer against a region of the mouse Pdx-1 gene. The reaction product was run on an agarose gel, which confirmed the Pdx-1 expression in the Min6 cells used for our experiments. M: Hyperladder IV DNA Marker, P: PCR product. M P

At this time, all the Min6 cells were harvested and run through flow cytometric analysis by the Core Flow Cytometry Facility (School of Life Sciences, University of Manchester), in order to assess the eGFP expression levels in the control pLenti-SFFV-eGFP transduced cells and the eGFP expression levels in the pLNT-mPdx1-I-II-III-eGFP transduced cells.

144

The entire experiment was run three times on three separate occasions using freshly made virus each time. In one out of three repeats, a population of the pLenti-mPdx1-I-II-III- eGFP transduced Min6 cells (63.2%) showed low eGFP expression. In that same run, the majority of the control eGFP transduced cells (77.5%) showed high expression of eGFP, while the non-transduced negative control cells showed minimal levels of autofluorescence for the eGFP channel. In each one of the other two repeats, the Min6 cells that had been transduced with our construct did not show any fluorescence in the eGFP channel.

However, in these two repeats, the control eGFP lentivirus transduced cells presented much lower expression of eGFP. This indicated that we had a positive result in one out of three experiments. Table 5.1 summarises the percentages of expression in the eGFP channel for each of the three repeats.

Table 5.1: A summary of the percentages of positive eGFP cells in three runs of Flow Cytometry Analysis. Table 5.1 presents the proportion of live, single and non-apoptotic cells that showed eGFP expression at 72 hr after the transduction initiation during each run of the flow cytometric analysis. Min6-eGFP Ctrl: Cells that had been transduced with the control lentivirus pLNT-SFFV-eGFP; Min6-Pdx1: Cells that had been transduced with the lentiviral construct pLNT-mPdx1-I-II-III-eGFP. A total of 10,000 events were counted for each transduction in each run of the experiment.

Min6-eGFP Ctrl Min6-Pdx1

1st Run 77.5% 63.2%

2nd Run 19.1% -

3rd Run 37.0% -

Genomic DNA and cDNA from pLenti-mPdx1-I-II-III-eGFP transduced Min6 cells were used in PCR expression analysis using an eGFP primer set, in order to verify the status of the lentiviral genome integration into them. The results indicated that the lentivirus we had constructed had both integrated into the Min6 genome (since there was eGFP detection in the genomic DNA sample) and was being actively transcribed towards mRNA (since there was expression in the cDNA sample) (Fig. 5.7).

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Fig. 5.7: The lentiviral construct had been integrated into the Min6 cell's genome and is expressed into cDNA. Primers for eGFP were used for a PCR reaction analysis of the genomic DNA (gDNA) and the cDNA from the Min6 cells that had been transduced with our construct. Plasmid DNA of the lentiviral construct was used as template in the positive control reaction. A band of the appropriate size (452bp) was present in the genomic DNA and the cDNA template samples. M: DNA marker (Hyperladder IV, Bioline), showing two bands, at 400bp and 500bp from bottom to top; (+): pLNT-mPdx1-I-II-III-eGFP DNA used as a template in the PCR reaction; c: cDNA from transduced Min6 cells; g: gDNA from transduced Min6 cells; -RT: sample that underwent the cDNA conversion without the addition of the Reverse Transcriptase enzyme.

5.3.2 Testing of the pTiger-mPdx1 I-II-III-RFP/rIns2-eGFP vector

During the course of construction and testing of our own reporter lentiviral vector, a similar approach was taken by other researchers elsewhere and published (Szabat et al.,

2009). This vector was obtained and tested in our hands (kind gift from Dr Jim Johnson,

University of British Columbia, Vancouver, BC, Canada).

The DNA from this vector was amplified by transfecting Sure2® (Stratagene, UK) bacterial cells. Restriction analysis of the lentiviral DNA was performed, using the BamHI and NheI enzymes, in order to confirm the successful transformation and amplification of the vectors. The digested products were run on an agarose gel. The obtained band profile did not matching the vector maps provided by Dr Johnson’s group, according to which a single band at 13.215 kb would be expected from the NheI digest, while two bands at 11.120 and

2.095 kb would be expected from the BamHI digest (Fig. 5.8).

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Fig. 5.8: Restriction analysis of DNA extracted from pTIGER-mPdx1-RFP-rIns1-eGFP clones. Nine clones of pTIGER-mPdx1-RFP-rIns1-eGFP were picked from freshly streaked antibiotic selection agar plates. DNA was extracted from them and was subjected to digestion with either the BamHI (B) or the NheI (N) enzymes. The products were run on a 1% agarose gel containing ethidium bromide together with Hyperladder I (Bioline, UK) as a DNA marker (M) and viewed under a UV light transilluminator. The inset picture presents the Hyperladder I marker (Bioline, UK) and the arrow points at the 1 kb band. Expected band sizes were 11,120 and 2,095 bp for the BamHI digest and a single band at 13,215 bp for the NheI digest.

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The amplification of the DNA was repeated by transfecting a different bacterial strain,

MAX Efficiency® Stbl-2™ (Invitrogen, UK), which, as claimed by the manufacturer, should have a reduced chance of recombination events. Running a digest analysis on the amplified

DNA from the transformed bacteria provided similar bands to the previous experiment, suggesting that the possible recombination event had not been corrected. DNA extracted from transfected bacterial clones was sequenced. The DNA sequencing analysis revealed that all the important regions related to the lentiviral transduction efficiency and the areas related to the promoters and reporter sequences were intact.

However, repeat attempts to transduce Min6 cells with this lentiviral construct were unsuccessful (n=5 attempts). In fact, only after concentrating the packaged viral particles by centrifuging at a high g force via ultracentrifugation it was possible to see a few transduced cells where the control pTIGER-CMV-eGFP virus had actually transduced the cells (n=1/1 attempts). However, the level of transduction was still very low. This meant that the virus titration must have been too low in the initial experiments, so, further experiments would need to be performed in order to increase the viral titration. However, because of a lack of time, we did not manage to finish these experimental repeats.

5.4 Discussion

In order to create a system for monitoring the differentiation status of hESCs, a lentiviral construct was developed. In this construct, the Pdx-1 gene promoter regions termed “area I”, “area II” and “area III” were cloned into a lentiviral backbone vector in such a way so that they would control the expression of an eGFP fluorochrome. The generation of the lentiviral construct was successful. However, upon transduction of Min6 cells, a cell line expressing Pdx-1, with the packaged lentivirus and observing the transduced cells under a fluorescent microscope, no fluorescence could be detected in the eGFP channel. At the same time, use of a control lentiviral construct suggested that the HEK293T packaging and

Min6 transduction system was working. Further investigation revealed that the lentivirus had integrated in the genome of the Min6 cells. However, only in one out of three flow cytometric analysis experiments did we manage to see functional fluorochrome protein expression in the cells that were transduced with our construct. The fact that one out of three runs of flow

148 cytometric analysis gave a positive result for eGFP, also suggested that the extra sequence that had been revealed through the restriction analysis of our clones (fig. 5.2A) did not have a major effect on lentiviral integration. The 72 hr required for the transduced cells to develop maximum fluorescence was not unexpected, since earlier studies have shown that the VSV-

G pseudotyped lentiviral vectors can take up to 48 hr or longer to integrate into the host cell genome (Van Maele et al., 2003). However, our inability to detect fluorescence using flow cytometric analysis in the Min6 that were transduced with our construct in more than one experiment suggested either that variable functional vector titer (the number of viral particles that are needed in order to infect a cell) of the virus might have been responsible or that post-proviral integration events, such as epigenetic modifications, could be affecting the expression of our lentivirus. In a study of pigs as transgenic animals, Hofmann (Hofmann et al., 2006), suggested that the integration site(s) of the provirus of a lentiviral vector affected its expression levels. Their results showed that this might be attributed to epigenetic modifications, such as the hypermethylation of the integrated sequences. However, the chances of this phenomenon being associated with our lentivirus expression is small. This is because of two features of our lentiviral construct. The pLNT-Gateway® vector (Invitrogen,

UK) that was used as the backbone of our construct, is a HIV-1 based lentivirus. Previous studies have shown that HIV provirus integration shows a preference towards active transcription units, promoting the efficient transcription of the genes encoded by the lentivirus

(Wang et al., 2007). In their study, Wang (2007) demonstrated through computational analysis of the HIV integration sites that the virus showed a bias towards integrating into sites near transcription-associated histone modifications, which include H3 acetylation, H4 acetylation and H3-K4 methylation (Wang et al., 2007). At the same time, they showed that sites near regions of transcription inhibiting histone modifications, such as H3-K27 tri- methylation, were unfavourable. This study implies that our lentiviral construct had a higher possibility of avoiding the integration into areas of the genome that could be silenced. The second feature that offered an advantage to our lentiviral system against silencing, is the fact that it is a self-inactivating lentivirus. Indeed, the pLNT vector backbone contains a deletion in the U3 enhancer region of the 3' Long Terminal Repeat (LTR) sequence. This feature was originally introduced in the lentiviruses for safety purposes -without affecting the

149 lentiviral provirus integration, integration into the cell genome makes the virus incapable of producing infecting viral particles (Logan et al., 2004). Later studies proposed that the self inactivating lentiviruses have an advantage versus the ones that include wild type HIV LTRs

-they can avoid silencing by counteracting the activity of cellular transcriptional repressors

(Pfeifer et al., 2002). Therefore, the most likely explanation for the low transduction rate we observed with our lentivirus, is a variable titre. Lentiviral titre is defined as the number of viable vector particles in a given vector stock volume (Zhang et al., 2004). Many factors can affect viral titre, as demonstrated by Zhang (2004), including the vector inoculation volume, the number of target cells, the stability of the vector and the time length of the vector internalisation by the target cells. A viral titration analysis using a suitable assay could potentially improve our transduction rate. Such assays are available and they are usually

ELISA based, detecting viral envelope proteins. The final experiment with the pTIGER lentiviral construct, where a small number of Pdx-1 expressing transduced cells was observed under the fluorescent microscope after the viral stock had undergone ultracentrifugation, added credit to the idea that improved titration could lead to more transduced cells. However, finding the optimum titration could involve fine tuning, since a high concentration of lentiviral particles has been shown to be toxic to the cells, due to the cytotoxic properties of the VSV-G envelope (Burns et al., 1993).

Besides the viral titre, other factors might have affected the transduction efficiency of our Min6 cells. Even though the stability of the vector should not change and variants such as the inoculation volume and the number of target cells remained stable throughout our experiments, the time the lentiviral particles would need in order to enter the cells could be variable. This is associated with the nature of our transduction system. The envelope for our lentiviral particles originated from a plasmid encoding the VSV-G envelope, which had been co-transfected with our vector in the HEK293T cells. The VSV-G envelope allowed the binding of the lentiviral particles to the cell membrane phospholipids, in particular, phosphatidylserine and phosphatidylcholine (Zhang et al., 2004). The phospholipid content of the cell membrane is closely associated with the cell cycle, with variable speeds of biosynthesis and degradation (Zhang et al., 2004). While we tried to maintain the Min6 cells at a growth phase, by limiting the amount of cell coverage on the tissue culture surfaces

150 upon plating and not allowing the cells to become over-confluent, variations in the cell cycle of the cells cannot be ruled out. Therefore, further troubleshooting of the lentiviral construct could include, apart from a suitable viral titration assay, a method of monitoring the cell cycle of the Min6 cells or use of a suitable agent that would allow cell cycle synchronisation.

5.5 Conclusions and Future Experiments

In this part of our study, we managed to generate a reporter lentivirus that could allow the monitoring of the developmental stage of differentiating hESCs towards a pancreatic  cell phenotype. In addition, this lentivirus could be used by research companies to monitor the effectiveness of medicines targeting diabetes type 2, since the upregulation of Pdx-1 expression is a desirable effect of potential anti-diabetic medicines. The low GFP expression in the transduced Min6 cells in one of two of our experiments suggested that the titre of the packaged lentivirus budding out from the HEK293T cells might have been too low.

If that was the case, we would have to concentrate the lentivirus, using ultra-centrifugation, in a similar way to the procedure that was carried out with the pTIGER construct.

Furthermore, in order to optimise the virus production, several ways to check viral titration could be tested in the future, such as the detection of viral envelope proteins by western blot analysis in the packaging cells. A variety of tissue culture conditions can be changed in order to increase the viral titre, like a change in temperature during the packaging in the

HEK293T cells, or closer monitoring of the tissue culture medium pH, as well as the use of several additives that can enhance viral particle production (McTaggart et al., 2002).

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Chapter 6: The effects of modified glucose levels on monolayer differentiation of hESCs towards definitive endoderm

6.1 Introduction

The major biochemical processes of cells, like the active transport of some molecules/ions in and out of the cell, the production of proteins, cell division and movement require energy, where a carbon source is oxidised releasing energy. Therefore, it is not a surprise that cells have developed adaptation mechanisms that allow them to modify their behaviour in a way that reflects the amount of energy that is available in their environment.

This ability to develop adaptation mechanisms to environmental cues is called phenotypic plasticity (Nijhout, 2003). Even on relatively simple organisms, like the bacterium E. coli, the processes that respond to starvation are quite complex, involving a multitude of genes

(Ropers et al., 2006). Bioenergetics is the science that studies these processes (Wallace,

2010). In his review, Wallace, DC, suggests that the adaptation of animals to available energy resources occurs at three levels. In the long term, animal cells can use nuclear DNA mutations to evolve different anatomical forms that permit the exploitation of alternative energy reservoirs, resulting in new species. Alternatively, they can rely on the high mutation rate of mitochondrial DNA encoded bioenergetic genes to evolve different physiologies that permit the adjustment to regional energetic environments. However, acute and sort term environmental energetic fluctuations require a much faster response mechanism. This is where the third mode of adaptive mechanisms comes into play, as suggested by Wallace

DC, 2010, where the response to energy fluctuations is mediated through the epigenetic regulation of the thousands of bioenergetic genes found in cells or the modification of transcription factors through the action of high-energy intermediates.

Fluctuations in glucose levels have been documented to have effects on the transcription levels of several genes. In mammals, the effects of glucose on gene transcription can be directly attributed to glucose metabolism or can be the result of glucose- dependent hormonal modifications, such as the pancreatic stimulation of insulin secretion and inhibition of glucagon secretion (Vaulont et al., 2000). Similarly, the effects of glucose in

152 the liver, in the presence of insulin, include the induction of gene expression for genes encoding glucose transporters, glycolytic and lipogenic enzymes, such as the L-type pyruvate kinase (L-PK), acetyl-CoA carboxylase (ACC), and fatty acid synthase (Vaulont et al., 2000).

The potential of glucose to alter gene transcription prompted scientists to study its effects on embryo development. The need to address the effects of glucose in embryo development became possibly stronger after early studies suggested that mothers with insulin dependent diabetes mellitus and poor metabolic control had higher chances of giving birth to infants with various dysmorphic anomalies (Diamond et al., 1990). In addition, early investigations on the effects of hypoglycaemia on the developing mouse and rat embryos showed that even an acute exposure to low glucose can result in malformations (Sadler et al., 1987; Tanigawa et al., 1991).

The ability of glucose to affect embryo development by modifying gene transcription has been since documented in vitro. Kentaro Mizuno (Mizuno et al., 2006) suggested that a high glucose concentration (25 mM) promotes the differentiation of mESCs grown as EBs towards germ cells, through the action of three genes whose transcription is directly affected by glucose concentration in the medium. At the same time, physiological glucose concentration (5.6 mM) did not have the same effect. In a different study, Mochizuki H.,

(Mochizuki et al., 2011) showed that glucose concentration during EB formation from mESCs had an effect on the inclination of cells to differentiate towards different cell types in the attachment cultures of formed EBs. This fact was further supported in a different study, where it was suggested that a high concentration of reactive oxygen species, which can be provided in high and not in low glucose concentration conditions, was essential for the differentiation of mESCs into cardiomyocytes (Crespo et al., 2010). Tonack S (Tonack et al.,

2006) reported that low glucose delayed the differentiation of mESC generated EBs with an associated up-regulation of pluripotency markers Oct-4 and SSEA-1. They also claimed that the changes they observed in facilitative glucose transporter isoforms (GLUTs) expression during differentiation, suggest that glucose and glucose transporters play a functional role in

ES cell differentiation and embryonic development, observations that were later made with hESC generated EBs as well (Segev et al., 2012). A change in GLUTs expression was also

153 observed by Guillet-Deniau (Guillet-Deniau et al., 1994) during the differentiation of rat foetal myoblasts, which are functionally indistinguishable from myoblasts of the embryo, towards myotubes. In a different study, it was reported that hyperglycaemia affects the ability of preimplantation mouse embryos to utilise glucose, a result of reduced expression of the GLUT transporters (Moley et al., 1998).

Another good example of the effects of glucose in development can be obtained from the pancreatic organogenesis. Two transcription factors that control pancreas organogenesis, Pdx-1 and the carbohydrate-responsive element-binding protein (ChREBP) are activated in the presence of glucose (Hui et al., 2002; Soggia et al., 2012). Glucose is also essential for the expression of the transcription factor NeuroD (Guillemain et al., 2007).

NeuroD is a direct target of neurogenin3 (Ngn3) and is known to be important for the proper cell development of the pancreatic endocrine compartment.

Despite all this evidence that glucose can affect differentiation during development, differentiation protocols for the generation of functional pancreatic cells from hESCs use a medium with high concentration of glucose, as this is the normal environment for growing pluripotent hESCs.

The latest experimental evidence suggests that Pdx-1 expression in EBs generated from hESCs differs according to whether they have been grown in low or high glucose medium concentration, both in terms of spatial distribution and in terms of the number of positive cells expressing Pdx-1 within the EBs (Segev et al., 2012). In the protocol they used, they differentiated EBs in hESC medium with either low or high concentration of glucose.

However, in all published bibliography, there is little information regarding the effect of physiological glucose concentrations on the differentiation potential of hESCs after they have received an initial cue to differentiate towards definitive endoderm.

6.2 Aims

The aim of this set of experimental work was to study the effects of different glucose concentrations on the fate of hESCs, after having been differentiated towards definitive

154 endoderm. We studied the effects of high versus low glucose concentration on a variety of pluripotency and endoderm markers. For this study, the H7 hESC line was initially directed through monolayer differentiation towards definitive endoderm, using a modified protocol of the one published by D’Amour (D'Amour et al., 2006). This was the first stage of the differentiation. Once the definitive endoderm had been established, the cells were further differentiated in the presence of different concentrations of glucose and analysed for gene expression of important markers of the differentiation towards a pancreatic endocrine cell.

6.3 Materials and Methods

6.3.1 Differentiation of H7 hESCs

The human embryonic stem cell line H7 was cultured on 6 well plastic tissue culture plates in the presence of feeders (described in Chapter 2). Differentiation towards definitive endoderm was induced by using a modified version of the first stage of the protocol described by D’Amour et al. (D'Amour et al., 2006). In more detail, on the first day of differentiation, the H7 hESCs were exposed to 100 g/ml of Activin A (Promega, UK), in

RPMI medium (Invitrogen, UK) containing 11 mM of Glucose and 2 mM L-Glutamine

(Invitrogen, UK). On the second day of differentiation, the medium was changed to medium containing 100 g/ml of Activin A in RPMI medium with the addition of 20 g/ml Wnt-3a

(R&D Biosciences, UK), 11 mM of Glucose and 2 mM L-glutamine. The medium for the third day of differentiation, consisted of 100 g/ml of Activin A in RPMI containing 11 mM of

Glucose and 2 mM L-Glutamine (Invitrogen, UK), with the addition of 0.5% FBS (Invitrogen,

UK). This last medium was replaced with fresh on the fourth day of differentiation. This was considered the end of the first stage of the directed differentiation. Earlier experiments, in which we had differentiated H7 hESCs using the protocol mentioned above, confirmed that by the end of that first stage of differentiation, definitive endoderm had been formed

(Fig.6.1). From then on, the differentiating hESCs were split into three groups. One of them was cultured in RPMI medium (Invitrogen, UK) containing a physiological concentration of glucose (5.6 mM) with the addition of 10% FBS, 2 mM L-glutamine. The two other groups of differentiation medium contained either 11 mM or 25 mM of D-glucose. The cells were kept

o at 37 C, 5% CO2 for an additional 9 days, with the differentiation medium being changed

155 daily. The cells were observed daily for morphological changes and images of the cells were collected using an Olympus CKX41 microscope, connected to a CQ Imaging Micropublisher

5.0 camera, controlled by the QCapture Pro software (QImaging, UK). Total RNA was extracted from a batch of each of the groups of the differentiating cells at the end of the fourth and the end of the final day of differentiation and was converted to cDNA for subsequent PCR (described in Chapter 2).

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Fig 6.1: Differentiation of H7 hESCs towards definitive endoderm. In earlier experiments, we had differentiated H7 hESCs using a three day protocol. The cells were exposed for one day to activin A and Wnt-3, followed by Activin A and 0.5% FBS for two days. RNA was extracted from the differentiated cells and a control population of H7 hESCs that had not been exposed any differentiation signals. The experiment was repeated three times. The RNA was converted to cDNA and real-time Q-PCR analysis was performed with sets of primers for Oct4, Sox17 and FoxA2. Each sample from each condition was run in triplicate, along with primers for ATP5B and CyclinD1, which were used as internal control genes. A triplicate of samples that had undergone the cDNA conversion procedure omitting the reverse transcriptase was included for each set of primers. The results suggested that the three day protocol generated definitive endoderm, as indicated by the 32% reduction in relative Oct4 expression and the rise of Sox17 and FoxA2 relative expression by 230% and 55% respectively, when compared against the un-differentiated hESCs. The bar charts present the mean values from all the experiments normalised against the internal control genes. The error bars represent the SEM. Statistical analysis revealed that the differences in gene expression were statistically significant (p<0.05).

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6.3.2 Transfer of differentiating cells to coverslips

Undifferentiated H7 hESCs, as well as cells at the end of the fourth day of differentiation -when definitive endoderm was expected to have arisen- and at the end of the final day of glucose differentiation, were placed on glass coverslips. As the initial attempts to make the cells attach to coverslips after the fourth day of differentiation were unsuccessful, different conditions for permitting the cells to attach onto the coverslips were subsequently tested. In more detail, the coverslips were covered with one of the four following reagents:

1) Undiluted FBS (Invitrogen); 2) 10% bovine gelatine (Sigma Aldrich, UK) dissolved in water and autoclaved; 3) 0.01% poly-L-lysine (Sigma Aldrich, UK) in double distilled autoclaved water; or 4) a mix 30 g/ml Fibronectin (Millipore, UK) and 8 g/ml Laminin A from human placenta (Sigma Aldrich, UK).

The 15 mm glass coverslips (Scientific Laboratory Supplies, UK) were sterilised in

100% ethanol, were positioned on 24 well tissue culture plates (Corning, UK) and covered with one of the solutions named above for 2 hr at RT. They were then washed twice with

PBS (Sigma Aldrich, UK). The poly-L-lysine covered coverslips were allowed to dry before use, as poly-L-lysine is cytotoxic and any traces of it in the medium would possibly affect the viability of the growing cells. All the rest of the coverslips were stored in the culture plates covered in PBS at 4oC until further use.

Plating the differentiated cells onto the glass coverslips involved using a 19G needle

(BD Biosciences, UK) to cut and remove all the morphologically different types of cells from the culture plates and manually transferring them on to the coated glass coverslips. The cells recovered on the coverslips from the end of the fourth day of differentiation, were

o allowed to stick on the coverslips overnight at 37 C, 5% CO2, in medium that consisted of

RPMI containing 11 mM of Glucose, 2 mM L-Glutamine and 0.5% FBS (all from Invitrogen,

UK), which was their basal medium, without the Activin A. Similarly, the cells that were recovered after the last day of differentiation were grown overnight on the coverslips at 37oC,

5% CO2, in medium that contained the same concentration of glucose to that which they had been previously grown in, with 10% FBS and 2 mM L-glutamine. After the cells had been allowed to attach to the coverslips overnight, the cells from a set of coverslips from each

158 glucose concentration condition were fixed and used for immunofluorescent staining

(methods described in Chapter 2).

The cells that were not fixed were either used for total RNA isolation prior to RT-PCR

(described in Chapter 2), or allowed to continue growing in their respective medium.

6.3.3 Passaging differentiating cells on coverslips

Two days later, the cells that had been grown in medium containing 25 mM glucose were over-confluent on the coverslips, while the cells that were grown at 5.6 mM glucose were much slower in proliferating. The cells that had been grown in medium containing 11 mM glucose seemed to be the healthiest of all, both morphologically and in terms of their estimated proliferation rate. These 11 mM glucose grown cells were split onto new coverslips by being trypsinised by applying 0.05% Trypsin-EDTA (Invitrogen, UK) on them after they had been washed with PBS (Sigma Aldrich, UK). The trypsin was inactivated by resuspending the trypsinised cells in their FBS containing medium. This was followed by pelleting the cells by spinning them at 400xg for 5 min. They were then resuspended in fresh medium containing 11 mM glucose and transferred to new pre-coated glass coverslips placed in 24 well. The cells were allowed to grow for an additional six days after having been passaged onto the new coverslips. They were finally either fixed with 4% paraformaldehyde or frozen down in a freezing mix containing 10% DMSO (Sigma Aldrich,

UK) in 90% FBS (Invitrogen, UK).

6.3.4 Immunofluorescent staining

The cells that had been fixed on glass coverslips at the various stages of differentiation were immunostained with either goat anti-human Nanog antibody (R&D

Systems, UK) or rabbit anti-human Oct-3/4 antibody (Santa Cruz, UK).

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6.3.5 PCR Gene Expression Analysis

The total RNA samples that had been converted into cDNA, after having been extracted out of the undifferentiated and differentiated cells at various stages of the differentiation protocol, were amplified using primers for the housekeeping genes Cyclin-D1,

ATP5B (one of the ATP synthase subunits), GAPDH and 18s RNA, as well as for a variety of pluripotency and differentiation markers, namely, Nanog, Oct-4, Pdx-1, Sox-17, Cxcr-4,

FoxA2 and the Glut-2 expressing gene Slc2a2. The inclusion of the housekeeping genes in the study of the expression of pluripotency and differentiation-associated genes would allow the comparison of observable changes in expression to be compared to the expression of the housekeeping genes, which, theoretically, should not change too much during differentiation. This principle provided a semi-quantitative method of assessing gene expression, since it enabled us to compare the band intensities and get an estimation of any gene expression changes during the different steps of differentiation. The densitometric analysis of band intensities was carried out by using the ImageJ software. The steps involved in the analysis included the subtraction of background from the obtained images.

This was followed by defining the equally sized parts of the lanes containing the band of interest. Finally, the band intensities were plotted by the software and the wand tool was used to select and measure the area representing the associated band.

6.4 RESULTS

6.4.1 Gene and protein expression of pluripotency markers

In order to assess the pluripotency status of the hESCs during the various stages of the differentiation protocol, the gene expression of Oct-4 and Nanog was studied by semi- quantitative RT-PCR analysis. The results show a reduction in both Oct-4 and NANOG expression as the differentiation advances towards later stages (Fig. 6.2A, B). In more detail, at the end of the first stage of differentiation, Oct-4 expression had been reduced by

20%, compared to the undifferentiated hESCs that were used as the starting material. The reduction of Nanog gene expression was less pronounced, as it fell only by 7% by the end of the first stage of differentiation. At the end of the second stage of differentiation, the gene

160 expression of both Oct-4 and Nanog was further reduced in the cells that had been subjected to all different glucose concentrations. However, the fact that this is a result from a single experiment did not allow us to calculate the statistical significance of this decrease.

The statistical analysis would be particularly interesting in the case of Oct-4, which, under the 25 mM high glucose concentration cell culture conditions, was ~24% less expressed, compared to the cells cultured in 11 mM and 5.5 mM glucose concentrations.

As gene expression changes do not always correspond to changes in protein expression levels, we also immunostained the cells at different stages of the differentiation protocol. In order to study the protein expression of the pluripotency markers, the cells were stained with polyclonal antibodies against Oct-4 or Nanog (Fig 6.2C). In addition, separate coverslips were set aside, which were only incubated with the secondary antibodies used for each of the pluripotency markers immunofluorescent staining. Both Oct-4 and Nanog showed strong protein expression in the hESCs, however, by the end of the first stage of differentiation, their protein expression had been lost. The secondary antibody controls showed absence of any staining, confirming that the immunofluorescent staining results corresponded to true variations in protein expression levels (results not shown).

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Fig. 6.2: Expression of pluripotency markers Oct-4 and Nanog. Gene expression of pluripotency markers Oct-4 and Nanog (A) in hESCs cells at the last day of the first stage of differentiation (D4), at the end of the second stage of differentiation (D12), and at the end of the experiment (D18). (M) = 100 bp DNA Marker. The (-) labels represent the -RT samples. Presentation of normalised data relative to the levels of ATP5B and 18s RNA (B), where the relative expression of the pluripotency markers compared to hESCs can be seen. Presentation of immunofluorescent data, showing the staining of Oct-4 (green) and Nanog (red) in combination with DAPI on undifferentiated H7 hESCs (C). Scale bar at 50 m.

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6.4.2 Gene expression of definitive endoderm associated markers

In order to assess the status of the differentiating cells in relation to the definitive endoderm differentiation markers, the gene expression of two markers, FoxA2 and Sox17, was studied by semi-quantitative RT-PCR. Results from a single experiment showed that the FoxA2 expression rose by 460% at the end of the first stage of differentiation, compared to the undifferentiated hESCs (Fig 6.3B). By the end of the full differentiation protocol, the levels of FoxA2 had fallen back to about twice the hESC levels for the cells that had been grown in 5.5 mM and 11 mM of glucose concentration. However, the FoxA2 gene expression of the cells that had been grown in 25 mM of glucose was just 20% higher than the hESC value.

The Sox17 gene expression presented a modest rise of 26% at the end of the first stage of differentiation, compared to the hESCs. However, the Sox17 gene expression levels were already quite high in the undifferentiated hESCs (Fig. 6.3B). For Sox17, the 5.5 mM glucose and 11 mM glucose concentration grown cells presented values of gene expression 12% and 4% higher than the control hESC levels respectively. However, there was no Sox17 gene expression in cells that had been grown in 25 mM of glucose concentration. Both FoxA2 and Sox17 were not expressed at the gene level in the - differentiating cells that had been passaged onto coverslips at the end of the experiment.

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Fig. 6.3: Expression of definitive endoderm markers. The endodermal marker expression profile of hESCs differentiated towards definitive endoderm, monitored by RT-PCR (A). FoxA2 maximum levels appeared at D4 of differentiation. Sox17 gene expression presented a rise of 26% at the end of the first stage of differentiation (D4), compared to the hESCs. Increased concentrations of glucose caused a reduction in both FoxA2 and Sox17 expression, with Sox17 expression being completely lost at 25 mM [Glucose]. The expression of both FoxA2 and Sox7 was lost in the definitive endoderm-like cell population that was recovered at the end of the experiment (D18) after having been grown in 11 mM glucose and passaged twice for a total of 18 days post the initiation of the differentiation protocol. The data presented in (B) represent the relative expression values, when compared to the gene expression in hESCs (ES), normalised against the expression values of two endogenous control genes, ATP5B and 18s RNA.

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6.4.3 Gene expression of pancreatic endoderm and pancreatic progenitor associated cell markers

The pancreatic differentiation status of the differentiating cells was assessed by the study of gene expression for Pdx-1 (a transcription factor associated with pancreatic endoderm) and Slc2a2 –the gene encoding the GLUT-2 glucose transporter protein. As with the previous gene expression analyses, these were carried out by using semi-quantitative

RT-PCR. Pdx-1 gene expression was not present in hESCs and in cells recovered at the end of the first stage of differentiation (day 4; Fig. 6.4A). The expression of Pdx-1 first appeared at the end of the second stage of differentiation (day 12), showing minor differences between the cells that had been cultured in 5.5 mM or 25 mM glucose, while cells that had been cultured in 11 mM of glucose showed ~14% higher gene expression compared to the other two glucose concentrations, which is a difference that could not be verified statistically from a single experiment. Finally, the Pdx-1 expression almost doubled in the epithelial like cells that were recovered at the end of the experiment (day 18) after being grown and passaged in medium containing 11 mM glucose, compared to the cells recovered at the end of the second stage of the differentiation, which had been growing at the same glucose concentration.

Expression of the Slc2a2 gene first appeared at the end of the first stage of differentiation (day 4; Fig. 6.4B). At the end of the second stage of differentiation (day 12),

Slc2a2 was highly expressed in the cells that had been cultured in 5.5 mM glucose, whereas it showed a smaller rise of expression in cells that had been grown in 11 mM glucose, compared to the cells that were recovered at the end of the first stage of differentiation. The

Slc2a2 gene expression of the cells that had been grown in 25 mM of glucose was almost equal to the expression in the cells that resulted from the first stage of differentiation.

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Fig. 6.4: Expression of pancreatic markers. The pancreatic commitment marker expression profile of hESCs differentiated towards definitive endoderm and furthered by culture in media containing different concentrations of glucose. Gene expression was monitored by RT-PCR (A). Pdx-1 expression first appeared at D12 of the differentiation protocol. The cells that had been cultured and passaged for 18 days in 11 mM glucose presented increased levels of Pdx-1 expression compared to all the other time points and conditions. Slc2A2 gene expression first appeared at day 4 of the differentiation, after the end of the first differentiation stage. Furthermore, the Slc2A2 gene expression presented a peak at D12 of the cells that had been subjected to 5.5 mM of glucose during the differentiation protocol. The data presented in (B) represent the relative expression values, when compared to the gene expression in hESCs (ES). Before being plotted on the bar charts, these values were normalised against the expression values of two endogenous control genes, ATP5B and 18s RNA.

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6.4.4 Morphological and physiological changes of the differentiating cells

The hESCs that were used as the starting material for this experiment presented the classic characteristics of hESC cells, with a high nucleus to cytoplasm ratio and large visible nucleoli (Fig. 6.5A). At the end of the first stage of differentiation (day 4), the culture presented a mixture of different types of cells that looked like fibroblasts or nerve cells (Fig

6.5 B, C). However, most cells looked like definitive endoderm, which is characterised by epithelial-like morphology with clear cell-cell boundaries (Fig. 6.5D) (Tada et al., 2005).

Attempts to make the epithelial-like cells attach on glass coverslips using either gelatine or

Fibronectin showed little success, with only a small number of recovered cells.

The end of the second stage of differentiation (day 12) came after the differentiating cells had been subjected to different concentrations of glucose for a total of seven days.

These cells had lost the characteristic morphology of definitive endoderm. They had a cobblestone morphology and formed colonies (Fig. 6.5E). As these colonies grew in size, they formed 3D structures with smooth edges (Fig. 6.5F), which were higher in number with increasing concentrations of glucose.

The structures from the cells that were grown in 11 mM glucose were mechanically dissociated and plated on fresh plastic culture dishes. These cells were passaged once with trypsin/EDTA and after six days of being cultured, they resembled endothelial-like cells and created colonies with smooth edges, as the hESCs do (Fig. 6.5 G, H).

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Fig. 6.5: The morphological characteristics of differentiating hESCs. An undifferentiated colony of H7 hESCs, cleared from random differentiation areas, ready to be subjected to the differentiation protocol (A). After four days of differentiation towards definitive endoderm, the culture appeared to consist of a mix of various cell types (B,C). However, the most commonly observed one was having the characteristics of definitive endoderm-epithelial-like cells with clearly defined and visible cell-cell limits (D). Subjecting the differentiated cells to medium containing modified levels of glucose concentration, caused the loss of the definitive endoderm cell morphology (E) and led to the formation of 3D structures on the culture dish (F), which were higher in numbers in higher glucose concentration media. Recovery of these structures and growth on plastic tissue culture surfaces led to the formation of colonies containing endothelial-like cells (G,H). Arrows in images D and F point at definitive endoderm like cells and the 3D structures mentioned above respectively. Scale bars at 50 mm (A, D, E, F, G, H) or at 100 mm (B, C).

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6.5 Discussion

6.5.1 Expression of pluripotency markers during differentiation

Our results show that the pluripotency marker Oct-4 was down-regulated during initial hESC differentiation towards definitive endoderm. However, its expression persisted throughout the differentiation process. Oct-4 expression was completely lost only after the selection and passaging of the newly formed cell population at 18 days after the initiation of the differentiation protocol. Persistent expression of Oct-4 has been demonstrated before in monolayer directed differentiation protocols (D'Amour et al., 2005; D'Amour et al., 2006;

Norrman et al., 2012). The slow down-regulation of Oct-4 has also been observed in protocols directing the differentiation through the formation of EBs (Cao et al., 2008;

Sundberg et al., 2009).

This effect could be partially explained by the double role of Activin A in promoting pluripotency, but also in driving differentiation. Several studies have reported that Activin A is one of the factors that contribute to the preservation of pluripotency in hESCs by inducing the expression of pluripotency associated factors and inhibiting differentiation associated signalling (Beattie et al., 2005; James et al., 2005; Vallier et al., 2005; Xiao et al., 2006).

However, under the right conditions, including inhibition of PI3K signalling, which prevents the formation of definitive endoderm (DE), exposure of hESCs to high concentrations of

Activin A leads to the differentiation of hESCs towards definitive endoderm (D'Amour et al.,

2006; McLean et al., 2007; Sulzbacher et al., 2009).

Therefore, one would expect that exposure of hESCs to such conditions, would lead to the differentiation of all hESCs towards definitive endoderm. Yet, this is not the case –and this is one of the major concerns of using hESCs for therapeutic purposes. There have been reports on the differences in different hESC lines on their capability to differentiate and their capacity for self-renewal (Tavakoli et al., 2009). However, more importantly, regional differences in gene expression profiles can be observed within the colonies of a single tissue culture dish (Laursen et al., 2007). These differences could be the result of the fact that individual cells in hESC colonies accumulate epigenetic variation, becoming more distant epigenetically to each other while in culture (Tanasijevic et al., 2009). As a result, hESCs

169 cultured even on the same tissue culture plate can behave differently, showing varied responses to differentiation signals (Annab et al., 2012; Gibson et al., 2009).

The apparent loss of Oct-4 and Nanog protein expression after the initiation of the differentiation protocol could suggest the presence of post-translational regulation, in a similar manner to the high rate of Oct-4 proteasomal protein degradation that has been observed in the P19 embryonal carcinoma cells (Saxe et al., 2009). This mechanism could lead to the destruction of all Oct-4 and Nanog protein constructs or lead to the preservation of only a limited number of protein constructs that would be at too low a level to be detected by immunofluorescense. The semi-quantitative RT-PCR method we used to quantify levels of gene expression is much more sensitive than the immunofluorescent protein detection, as it relies on the amplification of the template mRNA. This reality, combined with the fact that not all mRNA is translated into protein –i.e. it is subjected to post-transcriptional modification- may suggest that any protein that might have been expressed in our cells was not detectable.

6.5.2 Expression of differentiation markers during differentiation

Attempts to differentiate hESCs towards pancreatic beta cells have focused on trying to copy the developmental processes. One of the first steps on the pathway to functional pancreatic beta cells is the formation of definitive endoderm . In our study, hESCs were differentiated towards definitive endoderm by application of Activin A for one day, followed by

Activin A in combination with Wnt-3a. FoxA2 (or Hnf3b, as it is alternatively known) and

Sox17 are two of the genes the expression of which is linked to the formation of DE (Hallonet et al., 2002; Kanai-Azuma et al., 2002; Wang et al., 2011). FoxA2 has been found to play a role not only in the initiation, but also in the maintenance of the endodermal lineage (Ang et al., 1993). In mouse embryo development, FoxA2 first appears at the anterior region of the primitive streak, while at the later stages of gastrulation, its expression is observed in the invaginating foregut endoderm, to end up being expressed highly in the foregut and hindgut

(Sasaki et al., 1993). It has been suggested to assist in the regionalisation of the mouse endoderm, but also of the regionalisation of the embryo as a whole by defining the embryonal axis formation (Monaghan et al., 1993; Sasaki et al., 1993). At later stages,

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FoxA2 fully controls pancreas development as a regulator of Pdx1 expression by occupying enhancer regions of the Pdx1 promoter (Gao et al., 2008). Finally, in the adult mouse pancreas, FoxA2 controls Pdx1 expression (Lee et al., 2002), which contradicts the absence of any effects of FoxA2 on Pdx1 expression in the rat pancreatic insulinoma cell line Ins-1

(Wang et al., 2002a). FoxA2 also regulates the transcription of genes in response to alterations in glucose levels by controlling the transcription of the carbohydrate response element-binding protein (CREBP) in beta cells (Gao et al., 2010). The rise in the expression of FoxA2 we observed after the first stage of our differentiation protocol is in line with previous reports of similar experiments (D'Amour et al., 2006; Schulz et al., 2012).

Sox17 is required for the formation of definitive endoderm. In Xenopus embryos, it has been demonstrated that Sox17 needs -catenin, a downstream signalling molecule of the Wnt pathway, as a co-factor and that together they target and activate FoxA2 (Sinner et al., 2004). In mouse embryos, loss of Sox17 results in reduced numbers of definitive endoderm cells in the foregut, reduced expression of FoxA2 in definitive endoderm cells and is also lethal (Kanai-Azuma et al., 2002). Our experiment suggests that Sox17 levels of expression rise after the first stage of our differentiation protocol, which has been shown in previous experiments (D'Amour et al., 2006; Schulz et al., 2012), however, strangely, the expression of Sox17 in our undifferentiated population of H7 hESCs was quite high. Sox17 has been detected in the ICM of mouse blastocysts (Guo et al., 2010), but also in some undifferentiated hESC lines (International Stem Cell et al., 2007). In previous unpublished results of our group, it has also been demonstrated that the undifferentiated HUES7 hESCs can express Sox17 (Wright, 2009). However, to the writer’s knowledge, there are no other publicised experimental pieces of work to support a similar result. The rise in Sox17 expression that our result suggests at the end of the first stage of differentiation is a similar result to previous reports after directed differentiation of hESCs towards DE with Activin A,

Wnt-3a and FBS (Cho et al., 2008; D'Amour et al., 2006).

GLUT-2 is the major glucose transporter across the membrane of cells in the pancreas of mice and rats, but not in humans (McCulloch et al., 2011). Its partial inactivation or complete absence in mouse  cells results in effects similar to type 2 diabetes mellitus

(Guillam et al., 1997; Valera, A. et al., 1994). In human pancreatic  cells, the main glucose

171 transporter is GLUT-1 (De Vos et al., 1995). Early studies on Slc2a2, the gene encoding

GLUT-2 in humans, had suggested that its gene expression remains unchanged in human pancreatic islets from patients with type 2 diabetes mellitus (Ferrer et al., 1995). However, recent studies have debated this fact. Indeed, a 2011 study suggested that the expression of both GLUT-1 and GLUT-2 decreased by 70-90% in pancreatic islets of NIDDM (Ohtsubo et al., 2011). The same study showed that the knockout of both the Slc2a1 (the gene encoding GLUT-1 in humans) and the Slc2a2 gene was needed in order to cause significant effects in glucose stimulated insulin secretion (GSIS). Their results indicated that endogenous expression of Slc2a2 alone was sufficient to prevent the disruption of normal

GSIS in the human beta cells (Ohtsubo et al., 2011). Further to that, GLUT-2 expression in the developing human pancreas appears earlier than GLUT-1 expression (Richardson et al.,

2007). More studies have confirmed the GLUT-2 expression in the periphery of the early developing human pancreatic islets -at 12 weeks post conception (w.p.c.) co-localising with glucagon, with the central part of human islets showing co-localisation of GLUT-2 with insulin up to term time (Piper et al., 2004). In the rat pancreas,  cells arise from an endodermal population of GLUT-2 expressing cells (Pang et al., 1994). Similarly, it has been recently suggested that the pancreatic progenitor cells produced from the differentiation of hESCs arise from a pool of cells expressing GLUT-2 (Segev et al., 2012). We therefore, decided to study the expression of this marker in our differentiating hESCs. In our hands, the Slc2a2 gene expression appeared for the first time at the end of the first stage of differentiation. No other reports of Slc2a2 expression at this early stage of hESC differentiation have been published. This result, considered together with the abnormal levels of Sox17 in the undifferentiated H7 hESCs in our culture system, suggest the possibility of a randomly differentiated progeny of cells amongst our hESC colonies. Even though the cultured cells looked like stem cells, they might have been going through early stages of differentiation.

Even though the cells used for these experiments were subjected to karyotypic analysis and they came back with a normal karyotype, we should not forget the fact that each hESC is a dynamic cell, which can, depending on many factors, be closer or more distant to a lineage commitment fate (Enver et al., 2005). It has already been shown that the cells comprising a hESC colony can accumulate epigenetic heterogeneity progressively while cultured

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(Tanasijevic et al., 2009) and that the cells acquire a cell fate potential through these epigenetic modifications (Hong et al., 2011). All these factors can have an effect on hESC colonies, which could explain the variable results we got.

6.5.3 The effects of modified glucose concentrations on the differentiation of hESC after the establishment of definitive endoderm

Even though these results came out of a single experiment, in this experiment, it is suggested that Oct-4 gene expression levels seemed to stay relatively constant for the differentiated cells that had been cultured at the 5.5 mM and 11 mM concentrations of glucose. However, there seems to be a 30% drop in Oct-4 expression for the cells that had been cultured in high glucose concentration (25 mM). If this result were confirmed with more repeats of the experiment, it would be contradicting previously published results, which have suggested that a 25 mM concentration of glucose does not have a significant effect on the expression of pluripotency markers (Khoo et al., 2005).

Concerning the expression of definitive endoderm differentiation markers, a high concentration of glucose seemed to have an effect on reducing the expression of both Sox17 and FoxA2, with Sox17 expression being completely lost. This is not supportive of the results presented by Khoo et al. (Khoo et al., 2005), which could come down to the different hESC line they used and the differences between cell lines in their ability to respond to differentiation cues (Nairn et al., 2010). However, our result has been supported by a more recent research study which showed that a high glucose concentration can downregulate the

ESCs endoderm formation potential (Segev et al., 2012).

A suggestion of a slight rise in Pdx-1 expression would need a higher number of repeats to prove that it is a significant change in gene expression. The same applies to the down-regulation of the Slc2a2 gene expression with increasing concentrations of glucose.

However, based on published results, we would expect that the expression of both genes would be reduced with the increasing concentrations of glucose(Segev et al., 2012).

However, the Segev (2012) differentiation system is different – they used a culture adapted cell line, H9.2 and they used an EB differentiation system without any other differentiation

173 cues. Each of these factors can create differences in the results obtained; however, we would only be sure about the validity of our results by producing multiple repeat experiments.

6.5.4 Impaired ability of cells obtained at the end of the definitive endoderm differentiation protocol to attach on a range of different matrices.

The fact that very few cells were attached to any of the matrices used to attach hESCs on coverslips was something we did not expect to observe. However, it was later discovered that the integrin receptor expression profile of hESCs has been reported to change during differentiation towards definitive endoderm (Wong et al., 2010). Indeed, it was demonstrated that the hESCs express the integrins , all of which are associated with pluripotency. Upon differentiation towards definitive endoderm, the pluripotency associated integrins expression is down-regulated, while the V and 5 integrins are up-regulated

(Wong et al., 2010). This change has been shown to be enough to alter the ability of the cells to attach to different matrices, and it can be used to explain the tendency of the hESCs to prefer attachment to laminin, while the hESC derived definitive endoderm cells can better attach on vitronectin. Even though the integrins and  are not the only ones that are expressed in the pluripotent hESCs (Prowse et al., 2011), this observed change in expression levels could also possibly explain the morphological differences of the colonies of the epithelial cell-like population that was recovered at the end of the experiment.

6.5.5 Further experiments

It would be firstly most important to repeat the experiments, in order to validate our results statistically. A further improvement could be the addition of studies for the expression of more definitive endoderm related genes, such as the cell surface marker

CXCR4. In addition, a more sensitive method to detect changes in gene expression would involve real time Q-PCR, in order to compare the amplified products during the exponential phase of amplification.

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It would also be essential to make a full genetic profile analysis on the resulting cell population we recovered at the end of the experiment, in order to establish their differentiation status –identify which stage of development they might resemble, if any. In addition, it would be interesting to investigate whether this epithelial-cell-like cell population can have any effects on the induction and maturation of pancreatic progenitors from hESC, as it has been demonstrated recently (Jaramillo et al., 2012). Finally, if these cells still express Pdx-1 after been thawed and with repeated passaging, they could be used as a control cell population for the development of the lentivirus encoding eGFP under the control of the Pdx-1 promoter.

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Chapter 7: General Discussion

This study attempted to answer questions relating to the physiology and differentiation of hESCs. This is an essential step in the search for cell replacement therapies from hESCs.

A deep understanding of the hESC physiology and differentiation can lead to the generation of protocols leading to higher yields of mature phenotype cells. It would also help scientists address one of the major concerns in hESC associated cell replacement therapy, the risk of tumour development from any undifferentiated hESCs residing in the hESC-derived cell populations (Fong et al., 2009). Extension of our knowledge of how stem cells communicate with each other and their environment will lead to a better understanding of the processes of differentiation. Therefore, by studying the purinergic signalling potential of the hESCs, we added scientific knowledge to the early embryo development, since the differentiation of hESCs presents a close relationship to the differentiation of tissues in the early embryogenesis.

During the study of purinergic signalling in hESCs and their randomly differentiated counterparts, several challenges had to be faced. The first was the attachment of hESC colonies on glass coverslips in a way which would preserve their pluripotency in the absence of feeder cells. The reason we tried to attach the hESC colonies on glass coverslips in the absence of feeders, was the fact that the presence of feeders could potentially make the gathering and interpretation of the results difficult. The problems we faced were related both to the low percentage of attached colonies on the coverslips after overnight incubation in a tissue culture oven, as well as the high percentage of randomly differentiating cells in any colonies that attached onto the coverslips. There are published studies on different feeder free culture conditions for hESCs. However, these usually require long term culture and adaptation of the hESCs to the feeder free culture conditions. In addition, most of these methods of tissue culture require the addition of a number of growth factors, a fact that makes such methods quite expensive. This is especially true, since our experimental setup only required the overnight attachment of hESC colonies onto the glass coverslips, but also since a high number of throughput was essential. We addressed this problem by developing

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a protocol for the attachment of pluripotent hESC colonies onto glass coverslips in the absence of feeders.

A further challenge we faced related to the recognition of areas of pluripotency and differentiation of the hESC colonies we studied in the calcium microfluorimetry. The process of collecting the coverslips for immunofluorescent staining was tedious and, especially in the first few months, while the technique was being established, a high percentage of broken coverslips prevented us from conducting proper studies on the purinergic signalling differences between pluripotent and hESCs and their randomly differentiated counterparts.

In addition, an issue that needed to be addressed was the culture and maintenance of hESCs themselves. It has been shown before that different hESC lines present differences in genotypic characteristics, as well as differentiation potential. Indeed, since 2004, when a study on the gene expression profile of different hESC lines first appeared (Abeyta et al.,

2004), there have been a number of publications on this subject. These studies have revealed differences not only in the gene expression profile of undifferentiated hESC lines

(International Stem Cell et al., 2007), but have also demonstrated a bias of hESC lines to differentiate towards different cell tissues (Chang et al., 2008; Kim et al., 2007; Melichar et al., 2011; Tavakoli et al., 2009). The subject of hESC genetic variability and lineage differentiation bias is even more interesting, considering the fact that hESC lines that had been derived within the same groups have been shown to possess such characteristics

(Chang et al., 2008; Pal et al., 2009; Wu et al., 2007). The genetic variability, as well as the lineage differentiation bias could be partly attributed to epigenetic differences, such as those reported by Rugg-Gunn (Rugg-Gunn et al., 2007). In fact, epigenetic variability has been shown to occur even within different hESC colonies in a single cell culture, making the culture a mosaic of different subtypes of the hESC line (Tanasijevic et al., 2009). The effects of such differences were observed in our hESC tissue culture, where the two hESC lines used in our studies, HUES-1 and H7, presented differences in culture characteristics. One of the most obvious differences, was the fact that when thawing a new vial of H7 cells on

MEF feeders, addition of ROCK inhibitor (Pakzad et al., 2010) was essential for the survival of any colonies, a step that was not required in the HUES-1 tissue culture. We used the

HUES-1 cell line for the study of purinergic signalling because it was available at the time

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and our study was a build up on previous studies on a similar hESC line, HUES-7 (Wright,

Elli Alexander et al., 2009). The differentiation towards definitive endoderm in the presence of variable glucose concentrations was conducted using another cell line, H7. The reason behind this decision to change the cell line used, was the fact that the culture of HUES-1 was difficult. The HUES-1 cell line seemed to have a high tendency to randomly differentiate in vitro. In addition, it was observed during our studies that this tendency was at least partly attributed to MEF feeder batches that were used, an effect that has been studied and reported before and is one of the reasons behind the search for xeno-free media compositions for the culture of hESCs (Mallon et al., 2006). The observed tendency of

HUES-1 hESCs to randomly differentiate, in addition to the fact that hESC culture and passaging is on its own a laborious and time consuming technique (Oh et al., 2005), requiring up to twelve days after thawing a new batch before any usable colonies are available (Kent, 2009) and needing constant attention in order to mechanically dissociate and remove any differentiated parts of the hESC colonies, exposed us to time limitations in view of the number of experiments that could be carried out during the experimental part of the current study. Even though both the HUES-1 and H7 cells used were seemingly karyotypically normal, the H7 cells seemed to be growing better and differentiating less.

However, the possibility of a minor karyotypic change offering growth advantage to the H7 cells that was not detectable by conventional karyotyping techniques cannot be excluded.

This is the reason why, were there no time limitation, a study like the current should include as many different hESC lines as possible.

On the study of purinergic signalling in hESCs, we assessed the functional profile of the purinergic receptors expressed in HUES1 hESCs and concluded that the P2Y6 purinoreceptor is highly involved in purinergic signalling in HUES1 hESCs and that ATP might be involved in the differentiation of hESCs. The next step on this study, would be the investigation of the changes in the purinergic expression profile during differentiation of hESCs towards definitive endoderm. There have been no reports of purinergic receptor expression in the definitive endoderm, however, the expression of purinergic receptors seems to be closely related to development. Both the gene and protein expression of purinergic receptors has been shown to change during the differentiation of P19 embryonic

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carcinoma cells (Resende et al., 2007). In more detail, the expression of the P2X2, P2X6,

P2Y2, and P2Y6 receptors was shown to increase during the differentiation of the cells. At the same time, other purinergic receptors, namely the P2X3, P2X4, P2Y1, and P2Y4 receptors are high in pluripotent P19 cells, but are down-regulated upon the initiation of differentiation

(Resende et al., 2007). The rise in the expression of the P2Y6 receptor during the differentiation of the P19 cells contradicts our results, which suggest that the role of P2Y6 in randomly differentiated hESCs is reduced. However, there have been many examples where signalling mechanisms in mouse models present differences from human models.

Such studies have been conducted for example for the comparison of gene expression profiles of mESCs vs. hESCs through microarray research analysis (Ginis et al., 2004).

These studies have suggested that gene expression levels, as well as pathways that control basic cell functions, such as the cycle regulation and the control of apoptosis differ between hESCs and mESCs (Ginis et al., 2004).

Other examples of purinergic signalling affecting embryo development come from studies on the foetal astrocytes where ATP analogues stimulated DNA synthesis, as well as the activation of the ERK mitogen-activated protein kinase (Neary et al., 1998). ERK is a signal transduction component that is involved in proliferation and differentiation of several different tissues (Mullenbrock et al., 2011; Saleem et al., 2009), including the differentiation of hESCs (Yang et al., 2012). The importance of cell-cell contact in the development of the pancreas has been demonstrated by the significance of the endothelial cells of blood vessels that arise from the mesoderm in promoting early pancreas development (Lammert et al.,

2001), which induce the dorsal Pdx-1+ endoderm region to express the pancreatic transcription factor Ptf1a and maintain Pdx-1 expression (Yoshitomi et al., 2004). In addition, the laminin  chains 4 and 5, which are produced by the vascular endothelial cells of the islets up-regulate insulin gene expression (Nikolova et al., 2006). Furthermore, the absence of vascular endothelial cells prevents the induction of glucagon expression

(Yoshitomi et al., 2004), confirming the importance of endothelial cells in the pancreatic development. However, there are still questions about the possible contribution of the nervous system in the development of the pancreas. Even though it is known that the autonomic nervous system nerves can release ATP as a co-transmitter to noradrenaline

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(Mortani Barbosa et al., 2000), there is still a gap in our knowledge of how this could affect the early differentiation in the developing embryo. Our studies suggest that purinergic signalling may play a role in hESC differentiation. Further steps into the purinergic signalling research should investigate the role of purinergic signalling in the developing pancreas, starting by the analysis of the timing of purinergic receptor expression during the various stages of hESC differentiation towards a pancreatic  cell phenotype.

In order to conduct studies on the differentiation of hESCs towards pancreatic progenitors, the rate of generation of Pdx-1 positive cells has to be improved. For this reason, we have developed a lentivirus encoding a reporter gene (eGFP), whose expression is guided by the mouse Pdx-1 promoter regions. Even though our results suggested that the lentivirus was potentially effective in transducing the Min6 cells, there are some issues that still need to be addressed before the lentivirus can be used in studies of the differentiation of hESCs. Initially, the un-differentiated hESCs would have to be transduced with the lentiviral vector. The mouse version of the Pdx-1 promoter we used in our construct has a high homology to the human version of the promoter (Gerrish et al., 2000). However, in humans, an additional distal enhancer element controls Pdx-1 expression, which binds transcription factors SP1 and SP3 (Melloul et al., 2002). Our hypothesis when starting this part of the project was that the sequence of the areas I, II and III of the mouse promoter would be enough to drive the expression of the eGFP reporter gene. Earlier studies by the group that provided us with the pTIGER vector have revealed that the mouse promoter sequences are enough to drive gene expression in human islets (Szabat et al., 2009). However, limitations on the size of the DNA sequence we wanted to introduce in our lentiviral vector, meant that the distance between the promoter regions and the origin of translation of the eGFP was not the same as in an in vivo environment. could be associated with the functioning of the promoter regions. It has been shown before that the DNA of promoter regions that may be distal to the initiation site of a gene can fold on itself, so that the transcription factors that bind the promoter region create a bridge to other promoter sites closer to the initiation site.

Models of such models of transcriptional regulation have been suggested before (Wiebe et al., 2007). These models imply that the distance of a promoter region from the actual initiation site of a gene might be important, as the distal promoter regions of a gene may

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create loops to come into close contact with other promoter regions of the same or other genes and employ more transcription factors. Further experiments would be needed in order to prove that this disparity will not have an effect on the efficacy of our lentivirus in a human developmental model, such as one that would use hESCs. If such experiments proved that the lentivirus can work in human embryonic stem cells, this would add an important tool in the part of this project where the role of glucose concentration in hESC differentiation was assessed. Our preliminary results suggested that glucose does affect the differentiation of hESCs. This was not an unexpected result, since it has been supported by earlier studies. Soria et al. (2000) has shown that using non-stimulatory glucose concentrations in the culture medium during the late stages of differentiation is as a crucial event in obtaining cell clones with a high yield of insulin production (Soria et al., 2000). This observation was supported by the fact that glucose seems to be a principal modulator of gene expression in  cells and that sustained hyperglycaemia is able to cause an alteration in the pattern of insulin secretion from  cells (Soria et al., 2000). Further to that, there has been a report indicating that chronic hyperglycaemia results in the loss of  cell differentiation in a hyperglycaemic rat model (Soria et al., 2000). These facts can possibly explain the importance of low glucose concentrations in ESC differentiation towards insulin secreting cells (Soria et al., 2000). Mizuno et al. (Mizuno et al., 2006) have suggested a possible relationship between glucose metabolism and germ-cell development, by using mESC as a developmental model.

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Chapter 8: Conclusions

In conclusion, this study developed and tested the methods that will allow the investigation of the hESCs potential to differentiate towards definitive endoderm and other tissues. During our study we demonstrated that the hESCs can be attached on glass coverslips and retain their pluripotency using conditioned medium from mitotically inactivated feeder cells, in combination with an extracellular matrix scaffold made out of the proteins fibronectin and laminin in specific concentrations. We also demonstrated that the HUES1 hESCs do not express functional P2X1 receptors, but express functional P2Y6 receptors and our early results suggested that signalling through these receptors might be linked to the hESC pluripotency. In addition, we generated a lentiviral reporter where the mouse Pdx-1 promoter guided the expression of a fluorochrome, eGFP. This construct, with some further testing and fine-tuning could be used in the developmental studies involving hESCs in an attempt to produce functional pancreatic  cells. In addition, our reporter lentivirus could be used by commercial drug screening companies that are involved in the treatment of type 2 diabetes. Finally, we investigated the role of glucose concentration in the growth medium in regards to the differentiation potential of hESCs towards definitive endoderm. Our preliminary results suggested that glucose concentration could affect the differentiation of hESCs, in the same way as it does to mESCs. Finally, our study on the effects of glucose on hESC differentiation has potentially yielded a hESC derived cell line which expresses

Pdx-1. Further studies would be needed in order to assess if that Pdx-1 expression would be continued upon thawing and re-establishing this cell line in tissue culture.

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