characterisation of novel cell surface-reactive antibodies for … · surface-reactive antibodies...

Characterisation of novel cell

surface-reactive antibodies for

identifying and isolating liver and

pancreas progenitor cells

Benjamin Dwyer, BSc. (Hons)

School of Medicine and Pharmacology and the

Harry Perkins Institute of Medical Research

This thesis is presented for the degree of Doctor of

Philosophy at the University of Western Australia

March 2015

Supervisors: Professor Grant Morahan (Co-ordinating supervisor)

Professor George Yeoh (Co-supervisor)

I

-Declaration-

The research contained in this thesis is my own unless otherwise stated. This

work was carried out at the Centre for Diabetes Research at the Harry

Perkins Institute of Medical Research and the School of Biomedical,

Biomolecular and Chemical Sciences at the University of Western Australia.

The material presented in this thesis has not been submitted for any other

degree.

Benjamin Dwyer

24th June 2014

II

-Acknowledgements -

This PhD has been an unexpected adventure from start to finish. Thank you

to the many people who helped make the journey possible.

Firstly, I would like to thank my supervisor, Professor Grant Morahan.

Thank you for taking me into your laboratory as a PhD student. I appreciate

the freedom given to explore ideas, the means and facilities to hone my skills

as a scientist, the critical evaluation of my work and the patience you have

shown in allowing me to finish this thesis. The scientific and personal lessons

of thinking critically, aiming high and working hard have significantly

moulded my approach to science and will stay with me for my entire career. I

am extremely grateful for the time spent in your laboratory.

Thank you to my co-supervisor Professor George Yeoh. In addition to being a

fantastic teacher, you have given me endless advice and support in science

and in life. Thank you for the time I have spent in your laboratory. To see you

still at the bench and excited about science is truly inspiring.

To the wonderful members of the Perkins Monoclonal Antibody Facility,

Kathy, Tammy and Jess. Thank you firstly for expertly producing the

reagents used in this study and secondly for the laughs, love and occasional

burst of karaoke. It’s been a pleasure!

Thank you to all the people who collaborated and contributed to this project.

Dr Manami Hara from the University of Chicago, and Dr Masahito

Matsumoto from Saitama Medical School, provided MIP-GFP and Ngn3-

GFP/RIP-dsRed transgenic mice that were vital to this study. Dr Belinda

Knight from Fremantle Hospital offered DDC mouse liver tissue. Dr Nina

Tirnitz-Parker and Dr Jasmine Low from the University of Western Australia

provided raw microarray data from CDE liver and liver progenitor cell lines

respectively. Dr Nadine Dudek from the University of Melbourne performed

III

two-dimensional Western blotting studies. Thank you for your contributions.

Your input was crucial to the outcomes of this thesis.

A very special thank you to my Morahan lab mates at the Centre for Diabetes

Research: Thank you to Emma for making the hours under the dissecting

microscope fun, it was a pleasure injecting foetal pancreata with you! To

Jemma, Lois, Erika, Lakshini, Anne, Sarah, Irma, Violet, EJ, Fang-Xu,

Munish, Quang, Baten and Kevin, I’ll fondly look back on the perfect mix of

banter, science, coffee, laughs and wine during my years at the CDR. I will

also never forget your initiation into the world of pancreas; a shirt

emblazoned with “The Liver is Evil and Must Be Punished!”

Thank you to past and present members of the Liver group at the University

of Western Australia: Nina, Conny, Robyn, Jo, Megan, Adam, Sarah, Ken,

Ros, Caryn and Bernard. I’ll miss the laughter, fantastic cooking skills, all

the coffees, cake and fun we had during my time in the Yeoh lab. You helped

to show me that the liver is interesting and must be studied!

Thank you to my friends, particularly those who inhabited Redfern Street

over the last two years. Your friendship, love, support, wisdom, spirit and

endless encouraging words helped to push me past the finishing line.

Finally, to my parents Peter and Megan, and the rest of my family Adam, Bec,

Bruno, Brayden, Sophie and Leo. I appreciate the love and support you have

provided through the highs and the lows, particularly the last few years. I am

eternally grateful for everything you have given to me.

IV

-Abstract-

The use of renewable stem cell populations to generate hepatocytes and β-

cells for therapy is a beguiling prospect to overcome the challenge of limited

supply of donor organs used to treat end-stage liver disease and Type 1

Diabetes. Although progress towards this goal has been significant,

generating mature cells in vitro, and overcoming the potential

tumourigenicity of stem cells, are problems which must be addressed before

the promise of stem-cell based therapies can be translated from the laboratory

to the clinic. Understanding the mechanisms involved in expansion,

differentiation and transformation are therefore vital. The liver and pancreas

share both a common origin and developmental mechanisms, so studying

their development in parallel should help make progress towards the goal of

cell based therapies.

The ability to study the biology of liver and pancreas progenitors is currently

hampered by a lack of markers to identify and isolate their progenitors. To

help overcome this problem, the goals of my project were to (i) develop novel

cell surface-reactive monoclonal antibodies marking liver and pancreas

progenitors; (ii) to characterise the ability of these antibodies for identifying

and isolating liver and pancreas progenitor cells; and (iii) to identify protein

targets of useful antibodies in order to facilitate translational and functional

studies. A panel of monoclonal antibodies, termed ‘Western Australian

Monoclonal’ (WAM) antibodies, was generated. These antibodies were specific

for antigens on the cell surface of immature liver and pancreas cells. From

this panel, two antibodies, WAM18 and WAM21, recognised antigens

common to immature liver and pancreas cells.

These antibodies were further characterised. First, their utility was tested in

studies of the biology of liver progenitor cells (LPCs) in cell lines and in diet-

induced mouse liver injury models. Double immunofluorescence with WAMs

and the ‘gold standard’ LPC marker, A6, demonstrated that both antibodies

marked A6-defined LPCs induced in the CDE and DDC chronic liver injury

models. Critically, unlike A6, the WAM antibodies were compatible with

V

enzyme-dissociation and antibody-based purification of liver cells from

normal and CDE-injured livers. The WAM antibodies facilitated purification

of a subset of cells from the PIL-4 LPC line which co-expressed hepatocyte

and ductal markers, a feature of bipotential progenitors. Interrogation of

microarray data from LPC lines differentially expressing WAM18 and

WAM21 antigens led to the definition of two novel candidate LPC surface

markers whose expression was induced in chronic liver injury.

Having shown that WAM18 and WAM21 were useful in studies of hepatic

progenitors, their capacity for isolating islet progenitor cells from the

developing mouse pancreas was assessed. Initial screening studies using

Ngn3-GFP/RIP-dsRed mice suggested that WAM21 would be most useful for

enriching Ngn3-GFP+ islet progenitors. Characterisation of gene expression

by RT-PCR confirmed that WAM21 defines immature pancreas cells with

islet progenitor characteristics. Under defined culture conditions, WAM21,

either alone or in combination with Cd49f, could be used for significant

enrichment of pancreatic precursors capable of forming β-like cells, as defined

by insulin promoter-driven fluorescent protein expression and RT-PCR

analysis of gene expression.

To fulfil the final aim of this thesis, an expression cloning approach was

employed to identify cDNAs encoding the WAM18 and WAM21 antigens. This

led to the discovery that a single cDNA encoded a protein bearing both

antigens. Inhibition of this transcript’s expression in an LPC line resulted in

its reduced proliferation. Finally, a commercial antibody against the human

WAM18/21 protein equivalent could enrich human foetal islet pancreas cells.

In summary, novel cell surface antibodies with demonstrated ability to

identify and isolate mouse liver and pancreatic islet progenitors were

developed. These new antibodies, WAM18 and WAM21, will be of great

benefit for researchers studying the biology of hepatic and pancreatic

progenitors for the purpose of developing methods to derive mature cells for

transplantation therapy.

VI

DECLARATION FOR THESES CONTAINING

PUBLISHED WORK AND/OR WORK

PREPARED FOR PUBLICATION

This thesis contains published work and/or work prepared for

publication, some of which has been co-authored. The

bibliographical details of the work and where it appears in the

thesis are outlined below.

1. Benjamin Dwyer, Emma Jamieson, Tamara Jacoby-Alner,

Jasmine Low, Janina Tirnitz-Parker, Kathleen Davern, George

Yeoh and Grant Morahan. Generation and characterisation of novel

cell surface antibodies to mouse liver progenitor cells.

Manuscript prepared for submission to Stem Cell Research.

Presented in Chapter 2.

This study describes the generation and characterisation of a panel of

monoclonal antibodies and the development of two of these antibodies, WAM18

and WAM21, for identifying and isolating liver progenitor cells from

progenitor cell lines and chronically injured liver.

Contribution statement: BD designed and carried out experiments (animal

studies, Immunostaining, flow cytometry, PCR and data mining) and drafted

the manuscript. EJ, BD and TJA performed antibody screening studies. JL

and JTP performed microarrays on progenitor cell lines and CDE tissue

respectively. TJA and KD produced and purified antibodies used in this

study. All authors read and approved the final manuscript. GY and GM

conceived the study and contributed to experimental design and critical

evaluation of the manuscript.

VII

2. Benjamin Dwyer, Emma Jamieson, Tamara Jacoby-Alner,

Kathleen Davern, Fang-Xu Jiang, Masahito Matsumoto, and

Grant Morahan. Definition, enrichment and development of

pancreatic beta cell progenitors using a novel monoclonal antibody.

Manuscript prepared for submission to Diabetologia.

Presented in Chapter 3.

This study describes the use of one of our antibodies, WAM21, for isolating

islet progenitor cells which can differentiate to insulin-producing cells from

the developing mouse pancreas as a model for studying beta cell development

in vitro.

Contribution statement: BD designed and carried out WAM21-based

experiments and drafted the manuscript. EJ performed antibody screening

studies. TJA and KD produced and purified antibodies used in this study. FXJ

developed the foetal pancreas differentiation protocol and contributed to

experimental design and critical evaluation of the manuscript. MM developed

and provided Ngn3-GFP/RIP-dsRed mice. GM conceived the study and

contributed to experimental design and critical evaluation of the manuscript.

All authors read and approved the final manuscript.

VIII

3. Benjamin Dwyer, Emma Jamieson, Erika Bosio, Nadine

Dudek, Tamara Jacoby-Alner, Kathleen Davern and Grant

Morahan. Expression cloning a single cDNA encoding the WAM18

and WAM21 antigens.

Manuscript in preparation. Presented in Chapter 4, Results 3

This study describes the identification of a cDNA encoding the WAM18 and

WAM21 antigens using an expression cloning approach and subsequent use of

a human equivalent antibody in isolating islet progenitor cells from

developing human pancreas.

Contribution statement: BD designed and carried out experiments

(expression cloning, Immunostaining, PCR, data mining, Western blotting,

isolation and FACS analysis of foetal tissues, glycosylation studies, RNAi

studies, cell proliferation analyses) and drafted the manuscript. EJ and EB

isolated human foetal pancreas cells. ND performed 2D Western blotting,

TJA and KD produced and purified antibodies used in this study. BD and GM

conceived the study. GM conceived the study and contributed to experimental

design and critical evaluation of the manuscript.

Benjamin Dwyer (Candidate)

Professor Grant Morahan (Co-ordinating supervisor)

IX

-Abbreviations-

A absorbance

AFP alpha-fetoprotein

Alb albumin

ALT alanine transaminase

ANOVA analysis of variance

αSMA alpha-smooth muscle actin

BMOL bipotential murine oval liver

BMP bone morphogenetic protein

bp base pairs

BSA bovine serum albumin

CD cluster of differentiation

CDE choline deficient, ethionine-supplemented

cDNA complimentary deoxyribonucleic acid

C/EBP CCAAT/enhancer-binding protein

CK cytokeratin

COS CV-1 (simian) in Origin, and carrying

SV40 genetic material

CX connexin

DAPI 4´,6-diamidino-2-phenylindole

DDC 3,5-diethoxycorbonyl-1,4-dihydrocollidine

DM diabetes mellitus

DMSO dimethylsulphoxide

DMEM Dulbecco’s modified eagle medium

DNA deoxyribonucleic acid

DNase deoxyribonuclease

E embryonic day

EDTA ethylene diamine tetraacetic acid

EGTA ethylene glycol tetraacetic acid

X

EGF epidermal growth factor

EGFR epidermal growth factor receptor

ESC embryonic stem cell

FBS foetal bovine serum

FGF fibroblast growth factor

FITC fluorescein isothiocyanate

Foxa forkhead box A

GAPDH glyceraldehyde-3-phosphate dehydrogenase

GGT gamma-glutamyl-transpeptidase

GLUT glucose transporter

GRE glucocorticoid response element

HCC hepatocellular carcinoma

H&E haematoxylin and eosin

Hex haematopoietically expressed homeobox

HGF hepatocyte growth factor

HNF hepatocyte nuclear factor

HSC hepatic stellate cell

IFN interferon

IgG Immunoglobulin G

IGF insulin-like growth factor

IL interleukin

i/p intraperitoneal

iPSC induced pluripotent cell

ITS insulin, transferrin, selenium

kDa kilodalton

KLF Kruppel-like factor

KO knockout

LPC liver progenitor cell

LT lymphotoxin

XI

MAPK mitogen-activated protein kinase

M2PK muscle pyruvate kinase 2

mRNA messenger-ribonucleic acid

MYT1 Myelin transcription factor 1

OCT4 octamer-binding transcription factor-4

OCT optimal cooling temperature medium

OD optical density

OSM oncostatin M

p passage

PBS phosphate buffered saline

PCR polymerase chain reaction

PIL p53-/- immortalised liver

RNAi RNA interference

RT room temperature

RT-PCR reverse transcription-polymerase chain reaction

Sca-1 stem cell antigen-1

SDS sodium dodecylsulphate

SDS-PAGE sodium dodecyl sulphate–polyacrylamide

gel electrophoresis

SOX SRY-box

T1DM Type 1 Diabetes Mellitus

T2DM Type 2 Diabetes Mellitus

TAT tyrosine aminotransferase

Thy-1 thymocyte differentiation antigen-1

TNF tumour necrosis factor

uPA urokinase-like plasminogen activator

UV ultraviolet

Wnt Wingless

XII

-Table of contents-

DECLARATION I

ACKNOWLEDGEMENTS II

ABSTRACT IV

PUBLICATION DECLARATION VI

ABBREVIATIONS IX

LIST OF FIGURES XVI

LIST OF TABLES XVIII

CHAPTER 1 General Introduction 1

1.0 Introduction 2

1.0.1 Liver and pancreas structure and function 2

1.0.2 Diseases of the liver and pancreas 5

1.0.2.1 Diabetes Mellitus 5

1.0.2.2 Liver disease 6

1.0.2.3 Progenitor-derived hepatocytes and β-cells 6

1.1 Liver and pancreas development 8

1.1.1 Shared origins of the liver and pancreas 8

1.1.2 Fate choices in liver and pancreas progenitor domains 9

1.1.3 Cell-type specific transcription factors 10

1.1.3.1 Gene expression networks for islet cell specification 10

1.1.3.2 Liver lineage determination 12

1.2 Liver and pancreas progenitors in regeneration 14

1.2.1 Liver progenitor cells 14

1.2.1.1 Progenitor cell mediated liver regeneration 14

1.2.1.2 LPCs as a tumour precursor 16

1.2.1.3 LPCs as an expandable source of hepatocytes 18

1.2.2 Models of β-cell regeneration in the adult pancreas 19

1.3 Liver and pancreas interconversions 20

1.3.1 Pancreas to liver conversions 20

1.3.2 Liver to pancreas conversions 21

1.3.3 Summary 23

XIII

1.4 Phenotyping hepatic and pancreatic progenitors 23

1.4.1 Surface markers of LPCs 23

1.4.2 Surface markers of islet progenitor cells 25

1.5 Summary and project aims 26

CHAPTER 2 Generation and characterisation of

novel cell surface antibodies to mouse

liver progenitor cells 27

2.1 Abstract 28

2.2 Introduction 29

2.3 Materials and Methods 32

2.3.1 Production and screening of monoclonal antibodies 32

2.3.2 Liver cell culture 32

2.3.3 CDE diet induction of liver damage 32

2.3.4 Histology and immunofluorescence 33

2.3.5 Liver non-parenchymal cell (NPC) isolation 34

2.3.6 Flow cytometry 34

2.3.7 RNA Isolation and Reverse Transcription PCR 35

2.3.8 Interrogation of microarray data 35

2.3.9 Statistical Analyses 36

2.4 Results 37


2.4.2 WAM18 and WAM21 mark the ductular reaction to

chronic liver injury 40

2.4.3 Cell sorting potential of WAM18 and WAM21 45

2.4.4 Identifying candidate genes encoding novel cell

surface markers of LPCs 47

2.5 Discussion 49

CHAPTER 3 Definition, enrichment and development of

pancreatic beta cell progenitors using a

novel monoclonal antibody 51

3.1 Abstract 52

3.2 Introduction 53



3.3.2 Foetal pancreas isolation 55

3.3.3 Cell sorting 55

XIV

3.3.4 Differentiation of WAM21+ cells 56

3.3.5 RNA Isolation and RT-PCR 56


3.3.7 Statistical Analyses 57

3.4 Results 58

3.4.1 Production and screening of hybridoma supernatants 58

3.4.2 e15.5 pancreas WAM21+ cells are enriched for cells

with islet progenitor traits 60

3.4.3 WAM21+ e15.5 pancreas differentiate to Ins+

cells in vitro 62

3.4.4 Further definition of WAM21+ progenitors with

antibodies to Cd49f 64

3.5 Discussion 69

CHAPTER 4 Expression cloning a single cDNA encoding

the WAM18 and WAM21 antigens 72

4.1 Abstract 73

4.2 Introduction 74



4.3.2 Mammalian cell culture 76

4.3.3 Expression cloning cDNA clones encoding

WAM antigens 77

4.3.4 DNA purification and amplification of cloned sequences 77

4.3.5 Foetal tissue isolation 78

4.3.6 Flow cytometry 79

4.3.7 Treatment of COS-7 cells with glycosylation inhibitors 79

4.3.8 Antibody inhibition assay 79

4.3.9 Protein isolation, quantitation and western blotting 80

4.3.10 RNA interference in BMOL cells 81

4.3.11 Assessment of cell growth 81

4.3.12 Analysis of gene expression 82

4.3.13 Statistical analysis 82

XV

4.4. Results 83

4.4.1 Expression cloning cDNA encoding WAM antigens 83

4.4.2 Establishing the identity of WAM18 and WAM21 85

4.4.3 Biochemical characterisation of WAM18 and WAM21 89

4.4.4 Utility of WAM18 and WAM21 for investigating

organ-specific CD24 biology 93

4.4.5 Inhibition of Cd24a mRNA expression

alters LPC growth 95

4.4.6 Human foetal islet progenitors are defined

by CD24 expression 97

4.5 Discussion 99

CHAPTER 5 General Discussion 103

5.1 Discussion 104

5.2 Summary and conclusions 110

REFERENCES 112

APPENDIX I 143

APPENDIX II 151

APPENDIX III 162

XVI

-List of Figures-

CHAPTER 1 Fig. 1.1. Spatial relationship and structural overview of the

liver and pancreas 4

Fig. 1.2. Lineage determination in the liver and pancreas 13

Fig. 1.3 Pathways to progenitor-derived

hepatocytes and β-cells. 22

CHAPTER 2 Fig. 2.1. WAM antibody labelling in 2 wk CDE liver 39

Fig. 2.2. Comparison of WAM and A6 antibody labelling in

normal and CDE liver 41

Fig. 2.3. Quantitation of LPC induction in the CDE model

using WAM antibodies 42

Fig. 2.4. Comparison of WAM and A6 antibody

labelling in DDC liver 43

Fig. 2.5. Comparison of WAM and CD45 antibody labelling

in chronic liver injury 44

Fig. 2.6. Defining liver progenitor cell subsets with

WAM antibodies 46

Fig. 2.7. Screening for candidate genes encoding WAM18

and WAM21 48

CHAPTER 3 Fig. 3.1. Identification of islet progenitors in

developing pancreas 61

Fig. 3.2. Differentiation of WAM21-defined e15.5

MIP-GFP pancreas populations 63

Fig. 3.3. Comparison of WAM21 and Cd49f staining 66

Fig. 3.4. Molecular characterisation of WAM21 and

Cd49f-defined e15.5 pancreas cells 67

Fig. 3.5. Differentiation of WAM21/Cd49f-defined

Ngn3-GFP/RIP-dsRed foetal pancreas populations 68

CHAPTER 4 Fig. 4.1. Expression cloning WAM18 and WAM21 antigens

From a cDNA library 84

Fig. 4.2. Isolation of a cDNA encoding WAM18 and WAM21 86

Fig. 4.3. Sequence alignment of Sp6-sequenced insert and the

muCd24a cDNA 87

Fig. 4.4. Confirmation of WAM18 and WAM21 identity 88

Fig. 4.5. WAM18 and WAM21 identify unique CD24 epitopes 90

Fig. 4.6. Effects of glycosylation inhibitors on WAM18 and

WAM21 antibody surface labelling 92

XVII

Fig. 4.7. WAM18 and WAM21 identify unique cell

populations in e15.5 Ngn3-GFP/RIP-dsRed

mouse neuroendocrine tissues 94

Fig. 4.8. Effects of stable Cd24a knockdown on liver progenitor cell

proliferation 96

Fig. 4.9. Human foetal islet progenitors are CD24-positive 98

APPENDIX I Fig. A1.1. Assessment of liver damage in CDE diet 146

Fig. A1.2. Immunofluorescence controls for dual rat

antibody labelling 147

Fig. A1.3. Magnetic bead enrichment of WAM18 and

WAM21-defined normal liver subsets 148

Fig. A1.4. Localisation of WAM antibody staining at the

cell surface of BMOL1.2 LPCs 149

Fig. A1.5. Comparison of WAM and α-Smooth Muscle

Actin (αSMA) antibody labelling in chronic

liver injury 150

APPENDIX II Fig. A2.1. Immunofluorescence controls for dual rat

antibody labelling 153

Fig. A2.2. Enrichment and differentiation of WAM18-defined

foetal pancreas cells 154

Fig. A2.3. Magnetic bead enrichment and differentiation

of foetal pancreas cells with WAM21 155

Fig. A2.4. Immunocytochemical staining of differentiated

WAM21+ MIP-GFP e15.5 foetal pancreas

with islet markers 156

Fig. A2.5. Developmental hierarchy of gene expression in

WAM21+ e15.5 foetal pancreas cells 160

APPENDIX III Fig. A3.1. Effect of stable Cd24a knockdown on

BMOL cell growth 165

Fig. A3.2. Assay precision of the Cellavista™ system

versus the MTT assay 166

XVIII

-List of Tables-

Table 2.1. Screening WAM antibody panel 38

Table 3.1. Screening WAM antibody panel 59

Table 3.2. Screening supernatants from hybridomas

numbered with a WAM prefix 60

APPENDIX TABLES

Table A1.1. Mouse PCR Primer sequences and annealing

temperatures (A. Temp.) used 145

Table A2.1. Mouse PCR Primer sequences and annealing

temperatures (A. Temp.) used 152

Table A2.2. Surface markers enriched in WAM21+ foetal

pancreas cells 161

Table A3.1. PCR Primer sequences and annealing

temperatures (A. Temp) used 164

CHAPTER 1 GENERAL INTRODUCTION

1

Chapter 1

General Introduction


2

1.0 Introduction

1.0.1 Liver and pancreas structure and function

In complex organisms, including mammals, the liver and pancreas co-

ordinately control metabolism, producing hormones and enzymes which

regulate blood glucose levels and support digestion. The liver is the primary

site in the body for xenobiotic detoxification and nutrient processing,

accepting blood via the portal vein laden with nutrients and secretions from

the gut which are processed by hepatocytes. Hepatocytes constitute around

80% of the liver mass and secrete plasma proteins, synthesise amino acids

and lipids into the liver sinusoids (the capillary system of the liver), and

produce bile which is drained into bile canaliculi. From the canaliculi, bile

drains into the terminal duct system, composed of cholangiocytes, and is

transported to the small intestine to aid in digestion or to the gall bladder for

storage (Figure 1.1). Uptake of glucose from the bloodstream into hepatocytes

and its storage (in the form of glycogen), or release is controlled by hormone

signalling from the endocrine pancreas.

The pancreas is composed of three distinct tissue types, exocrine, endocrine

and ductal. The exocrine pancreas is composed of acinar cells, which produce

nucleases, proteases, pancreatic amylase and lipases for digestion. Acinar

cells form lobular structures, termed acini, which line the ductal network,

giving the pancreas its alveolar-like appearance. The pancreatic duct system

carries these exocrine secretions and drains into the duodenum via the main

pancreatic duct, which interfaces with the ductal system exiting the liver.

Interspersed throughout the acinar tissue are the endocrine ‘Islets of

Langerhans’ (termed ‘islets’). Islets make up 1-2% of the total pancreatic

weight in healthy adults and form spherical structures composed of five major

cell types which produce specific endocrine hormones. These cell types are α-

cells (glucagon), β-cells (insulin), δ-cells (somatostatin), P.P.-cells (pancreatic

peptide) and ε-cells (ghrelin). The islets have a distinct structure with the

majority of β-cells contained at the centre of the islet and the other cell types


3

dispersed around the periphery (Figure 1.1). Despite their relatively small

volume, islets command up to 20% of the total pancreatic blood flow (Lifson

et al., 1980), allowing for effective islet-mediated metabolic control exerted by

hormone secretions of the five different islet cell types (Figure 1.1).

The β-cells constitute around 75% of the islet and produce the hormone

insulin in response to high glucose levels. Glucose sensing takes place via the

transport of glucose into the β-cell via the high affinity glucose transporter-2

(Glut-2). The α-cells comprise around 20% of the islet and release the hormone

glucagon. In response to low blood glucose levels, glucagon is released into the

bloodstream where it signals to the liver to upregulate

glycogenolytic/gluconeogenic pathways to raise blood glucose levels.

(Reviewed in Korc, 1993). Delta cells comprise up to 5% of the islet and are

characterised by their production of the hormone somatostatin, which acts on

the β-cells to inhibit insulin production. Making up less than 1% of islet cells

are P.P-cells and ε-cells that make pancreatic polypeptide and ghrelin

respectively. Pancreatic polypeptide is a paracrine hormone which inhibits

acinar secretions whilst ghrelin exerts a positive effect on β-cell proliferation,

survival and insulin release (Reviewed in Granata et al., 2010).


4

Figure 1.1. Spatial relationship and structural overview of the liver

and pancreas. Exocrine secretions from the liver and pancreas drain via a

shared ductal system into the duodenum to aid digestion. The liver is

composed of hepatocytes which secrete bile into the bile canaliculi and process

nutrients and secrete hormones into the adjacent sinusoidal blood vessels.

Blood from the gut enters the liver via the portal vein (PV) and oxygenated

blood via the hepatic artery (HA). Together, with the terminal bile ducts,

these three structures form the ‘portal triad.’ Blood exits the liver via the

central vein (CV). Liver-resident Stellate and Kupffer (macrophage) cells

store fat and clear debris respectively. Pancreatic enzymes are produced by

acinar cells that surround the ductal system and feed into the common bile

duct connecting the liver, pancreas and duodenum. Endocrine hormones are

produced by ‘Islet of Langerhans’ cells and are secreted into a rich network of

islet-associated capillaries. Figure adapted from Burke and Tosh (2012).


5

1.0.2 Diseases of the liver and pancreas

1.0.2.1 Diabetes Mellitus

Diabetes mellitus (DM) is the term which describes metabolic disorders where

hyperglycaemia is observed due to inadequate insulin production, or impaired

tissue responses to insulin signalling. Worldwide, ~285 million adults (6.4%

of people aged 20 to 79) were affected by DM in 2010, and this number is

projected to reach 439 million (7.7%) by 2030 (Shaw et al., 2010). DM can be

broadly divided into two subtypes; Type 1 (T1DM), results from autoimmune

destruction of islet β-cells and subsequent insulin-deficiency, whilst Type 2

(T2DM) results from inadequate insulin production from β-cells and tissue

insulin resistance. T2DM is the major subtype of DM, affecting ~90% of DM

patients. It is typically diagnosed during middle age (>40 years) and is

associated with risk factors such as age, obesity and genetic predisposition.

Conversely, T1DM onset typically occurs during puberty and affects 5-10% of

all diabetes patients (Harjutsalo et al., 2008). Recent efforts to investigate

genetic risk factors associated with T1DM have demonstrated the complexity

of this disease, identifying over 50 risk-associated genetic regions via genetic

linkage analyses (Barrett et al., 2009, Morahan et al., 2011).

Overall, DM is the 9th leading cause of global deaths (Lozano et al., 2012).

Conventional treatment for T1DM involves administration of exogenous

insulin and blood glucose monitoring, which does not achieve complete

glucoregulation. Complications arising from inadequate blood glucose

homeostasis include diseases such as retinopathy, nephropathy, neuropathy,

cardiovascular disease and stroke, which result in significant reduction in

lifespan and quality of life (extensively reviewed in Forbes and Cooper, 2013).

Even with exogenous insulin treatment, half of all patients will develop

diabetic complications after 30 years with the disease (Nathan et al., 2009).

Since insulin release is glucose-dependent in β-cells, these outcomes may be

improved with islet transplantation from cadaveric donors (Shapiro et al.,

2000). Unfortunately, demand for transplant tissue far outweighs its supply.


6

1.0.2.2 Liver disease

Liver disease can also be divided into two major subtypes, acute and chronic,

depending on the duration and progression of injury. Acute liver disease is

most commonly caused by agents such as acetaminophen toxicity, drug-

induced hepatotoxicity, acute viral hepatitis infection or autoimmune liver

disease (Polson et al., 2005). The survival rates for such diseases are 48%

prior-to, and 74% following liver transplantation (Bernal et al., 2013). In

chronic disease, liver fibrosis develops during sustained injury and can

progress to cirrhosis and hepatocellular carcinoma (HCC) and/or liver failure.

Examples of fibrosis-causing agents include chronic hepatitis B and C

infection, non-alcoholic fatty liver disease and genetic hemochromatosis.

Liver cirrhosis and liver cancer are the 12th and 16th leading causes of global

deaths respectively (Lozano et al., 2012), with most HCC cases occurring in

the setting of established cirrhosis, with a median survival of 6-16 months if

untreated (Bruix et al., 2005). Although treatment options such as antiviral

therapy and weight reduction strategies can be effective, significant numbers

of patients progress to end-stage liver disease and require orthotopic liver

transplantation.

Although T1DM and liver disease are treatable through organ transplant,

limited availability of donor organs and religious and/or economic reasons

may restrict access to transplantation surgery (Johnson et al., 2014). Given

the predicted rise in prevalence in liver disease and T1DM, alternative

cellular sources are needed to meet the demand for transplant tissue.

Stem/progenitor cells which can be expanded in vitro to clinically useful

proportions, and induced to form hepatocytes or β-cells represent a promising

renewable source of cells for transplantation.

1.0.2.3 Progenitor-derived hepatocytes and β-cells

Because of their capacity to proliferate and form virtually any tissue type,

stem/progenitor cells represent ideal cellular sources of surrogate hepatocytes

and β-cells. Pluripotent embryonic stem cells (ESCs) can be derived from the


7

inner cell mass of blastocysts from pre-implantation embryos (Thomson et al.,

1998). Alternatively, somatic cells can be reprogrammed to form induced

pluripotent cells (iPSCs) via forced upregulation of the ‘pluripotency factors’

c-MYC, SOX2 OCT4 and KLF4 (Takahashi et al., 2007). Methods to derive

liver and pancreas cells from pluripotent cell sources have involved attempts

to recapitulate patterning signals of embryonic development in vitro to

differentiate cells into hepatocytes or β-cells (Kroon et al., 2008, Si-Tayeb et

al., 2010). Although much progress has been made towards producing

clinically viable cells in vitro, concerns over both the potential

tumourigenicity of pluripotent-derived cells (reviewed in Lee et al., 2013) and

the ability to derive fully functional cells in vitro, still limit the translation of

these technologies to the clinic.

Understanding the signals involved in patterning liver and pancreas

domains, as well as promoting their proliferation and differentiation into

functional tissue, is vitally important to develop efficient protocols to derive

therapeutically viable cells. Additionally, since stem/progenitor cells and

cancer cells share common pathways in activation and differentiation, the

study of these processes also has application in chemotherapeutic targeting,

particularly in the liver.

The ability to purify and culture organ-specific progenitor cell domains is

fundamental in achieving this outcome. This introduction will explore what

is currently known about the developmental programs of each organ, and how

these programs are recapitulated during progenitor cell activation in disease

and tumorigenesis. Finally, it will assess the current state and limitations in

phenotyping liver and pancreas progenitor cells using monoclonal antibodies,

and how these limitations may be overcome.


8

1.1 Liver and pancreas development

Because of their shared origins, the liver and pancreas are ideal organs to

study in parallel. The following section of this review will examine the

fundamental concepts of liver and pancreas development from primitive

endoderm cells and is summarised in Figure 1.2.

1.1.1 Shared origins of the liver and pancreas

The liver and pancreas develop from a common pool of endodermal

hepatopancreatic precursor cells which upregulate organ-specific gene

expression cascades in response to Fibroblast Growth Factor (FGF) signals

from the surrounding mesenchyme at E8.0-E8.5 in the mouse (Deutsch et al.,

2001, Rossi et al., 2001, Serls et al., 2005). During this period, expression of

key transcription factors such as the CAAT-enhancer binding protein family

of transcription factors (C/EBPα, C/EBPβ), in concert with modulators of

hepatic gene expression, Hepatocyte Nuclear Factor (Hnf)-4 and alpha

fetoprotein (AFP), define the hepatic rudiment (Westmacott et al., 2006).

Concurrent with C/EBP family expression in the liver primordium, pancreas

development is initiated by the expression of the Pancreatic and Duodenal

Homeobox 1 transcription factor (Pdx1) in the absence of FGF signalling

(Gualdi et al., 1996), which initiates pancreas-specific gene expression

(Jonsson et al., 1994, Offield et al., 1996). Organ-specific gene expression is

further reinforced by the actions of Bone Morphogenetic Proteins (BMP),

which suppress pancreatic gene expression (Rossi et al., 2001), and Retinoic

acid (RA) signals that promote pancreatic specification but do not affect the

liver (Chen et al., 2004, Martin et al., 2005, Molotkov et al., 2005).

Suppression of Sonic Hedgehog (Shh) signalling by Activin-βB and FGF2

signalling from the notochord permits the formation of the dorsal pancreatic

domain by promoting pancreas over stomach/duodenal fate (Apelqvist et al.,

1997, Hebrok et al., 1998).

Following organ specification, the liver and pancreas undergo a period of

expansion and morphogenesis. In the pancreas, this period is known as the


9

‘primary transition’ and involves the development of the pancreatic bud into

a branched epithelium via a process of ‘branching morphogenesis’. Initially,

the pancreatic primordium becomes stratified, before invading the

surrounding mesenchyme and forming a ‘tree-like’ network of epithelial tubes

(Villasenor et al., 2010). This process is driven by proliferative multipotent

Carboxypeptidase-A1(Cpa1)+/Pdx1+ progenitor cells present at the distal tips

of the branching epithelium (Zhou et al., 2007). In the liver, newly specified

hepatoblasts delaminate and migrate through the basement membrane and

invade the surrounding mesenchyme. The hepatoblasts continue to

proliferate, forming distinct cords interspersed with mesenchyme which

forms the hepatic sinusoids (Reviewed in Lemaigre, 2009). This process

occurs under the control of the Hex homeobox protein and Prox1 (Sosa-Pineda

et al., 2000), which along with Gata-6 promote the expression of albumin

throughout the hepatic primordium (Gualdi et al., 1996, Bossard and Zaret,

1998). During this early expansion period, the transient upregulation of the

Wingless (Wnt)/β-catenin pathway in the liver (Suksaweang et al., 2004, Tan

et al., 2008) and Notch signalling pathway in the pancreas (Apelqvist et al.,

1999, Jensen et al., 2000, Hald et al., 2003) promotes proliferation of organ-

specific progenitors, whilst inhibiting premature differentiation.

1.1.2 Fate choices in liver and pancreas progenitor domains

As well as controlling expansion, Notch and Wnt/β-catenin signalling regulate

cell lineage choices in the multipotent progenitors of the liver and pancreas.

Notch signalling promotes ductal over non-ductal differentiation in both

organs. If Notch signalling is activated in liver progenitors, ductal-specific

genes are upregulated and hepatocytic gene expression is repressed

(Tanimizu and Miyajima, 2004). During liver development, cells positive for

the Notch ligand, Jagged-1, surround portal veins in close proximity to

Notch2+ hepatoblasts that form cholangiocytes (Kodama et al., 2004,

Tanimizu and Miyajima, 2004). Accordingly, duct formation is severely

impaired in Notch-2 knockout mice but hepatocyte differentiation is not

affected (Geisler et al., 2008).


10

Similarly, endocrine and ductal fates are determined in the pancreas by the

interplay between Notch signalling and the islet-specific transcription factor

Neurogenin-3 (Ngn3). This period from e13.5 to e17.5 is termed the ‘secondary

transition’, and marks the time when islet progenitors are patterned into

hormone-specific adult islet cell subtypes (Gradwohl et al., 2000,

Schwitzgebel et al., 2000, Desgraz and Herrera, 2009). If Notch signalling is

activated, endocrine-specific gene expression is suppressed and ductal cells

form (Ahnfelt-Ronne et al., 2007). Conversely, upregulation of Ngn3 in islet

progenitors promotes islet cell fate and reduction of competitive-inhibition of

Notch signalling in adjacent ductal cells (Magenheim et al., 2011). Ngn3+ cells

originate from Pdx1+ epithelium at e9.0 in the dorsal mouse pancreas and

e10.5 in the ventral pancreas (Apelqvist et al., 1999, Gu et al., 2002). Ngn3

expression is transient during the secondary transition, peaking at e15.5

during islet progenitor expansion, then decreasing to be at low levels by e17.5

in the mouse, during which time islet lineages are determined (Apelqvist et

al., 1999).

Hepatocyte and acinar cell differentiation are promoted by active Wnt/β-

catenin signalling in the liver and pancreas. Targeted inactivation of Apc

following hepatic specification leads to hypoplastic liver growth and

embryonic lethality due to failure of hepatocyte differentiation. Meanwhile,

cholangiocyte differentiation is unaffected and these cells form bile ducts

when transplanted into normal recipients (Decaens et al., 2008). Knocking

out β-catenin in the embryonic pancreas results in exocrine hypoplasia (Wells

et al., 2007) and reduced acinar differentiation (Dessimoz et al., 2005, Wells

et al., 2007).

1.1.3 Cell-type specific transcription factors

1.1.3.1 Gene expression networks for islet cell specification

The initiation of Ngn3 expression in islet progenitors promotes the

upregulation of the islet developmental program (summarised in Figure 1.2),

leading to the differentiation of functional adult islet cells (Juhl et al., 2008).


11

Pax4 and Pax6 are essential for endocrine development in duodenal, stomach

and pancreatic cells (Larsson et al., 1998) and function to specify the β/δ-cell

(Pax4) or α/PP-cell (Pax6) lineages in the pancreas. Evidence for this role

comes from transgenic mouse studies. Pax4 mutant mice do not develop β- or

δ-cells (Sosa-Pineda et al., 1997) and Pax4 knockout mice produce α-cells at

the expense of the β/δ-cell lineage (Dohrmann et al., 2000). Pax6 knockout

mice do not develop α-cells and display a perturbed islet structure (St-Onge

et al., 1997). Additionally, Pax4/Pax6 double knockout mice do not develop

any mature endocrine cells (Larsson et al., 1998). Nkx2.2 is regulated by

Pax4, Ngn3 and Hnf1β (Wilson et al., 2003) and its inactivation leads to

maturation arrest of β-cells (Sussel et al., 1998). Nkx6.1 expression is

downstream of Nkx2.2 and is restricted to β-cells in neonatal development.

Knockout of Nkx2.2/Nkx6.1 blocks β-cell differentiation (Sander et al., 2000).

In the adult pancreas, Pdx1 is restricted to endocrine cells and its

downregulation is critical for ductal maturation and function (Deramaudt et

al., 2006). In the islets, Pdx1 protein activates the downstream signalling

cascade for proper endocrine development (Oliver-Krasinski et al., 2009) and

in adult β-cells, is necessary for insulin gene transcription and β-cell function

(Melloul, 2004, Babu et al., 2007). Islet-specific Pdx1 transcription is

controlled by a combination of transcription factors including; Hnf1 (Gerrish

et al., 2001), FoxA2 (Wu et al., 1997, Gerrish et al., 2000, Marshak et al.,

2000), Pax6 (Samaras et al., 2002), Nkx2.2 (Van Velkinburgh et al., 2005),

Hnf6 (Jacquemin et al., 2003), MafA (Vanhoose et al., 2008) and Pdx1 protein

itself (Gerrish et al., 2001). MafA is a β-cell enriched transcription factor

which acts to control Insulin (Zhao et al., 2005, Artner et al., 2008) and Pdx1

(Vanhoose et al., 2008) gene transcription, maintaining β-cell specific gene

expression and function. Though initial studies found that Ngn3 is not

expressed in adult pancreas (Apelqvist et al., 1999, Gradwohl et al., 2000),

more recent studies have shown by RT-PCR, western blotting and fluorescent

imaging that Ngn3 is both expressed and required for adult β-cell function

(Wang et al., 2009). Furthermore, transgenic studies have demonstrated


12

Insulin and Ngn3 promoter driven fluorescent protein co-expression in the

developing pancreas (Mellitzer et al., 2004, Hara et al., 2006).

1.1.3.2 Liver lineage determination

In addition to C/EBPα, Hnf4 is a key regulator of adult hepatic genes (Parviz

et al., 2003), binding to nearly half of transcriptionally active chromatin in

human hepatocytes (Odom et al., 2004), and regulating a number of genes

controlling hepatocyte maturation (Li et al., 2000). Furthermore, Hnf4

overexpression in undifferentiated hepatoma cells promotes hepatocyte

differentiation (Spath and Weiss, 1997). Hnf6 is expressed in hepatoblasts

and biliary epithelial cells, driving the cholangiocyte differentiation program

following its upregulation in response to C/EBPα suppression (Yamasaki et

al., 2006). Hnf6 regulates the correct timing and positioning of cholangiocyte

differentiation, restricting bile duct formation to areas around the portal

mesenchyme. Downstream of Hnf6, Hnf1β promotes biliary differentiation

(Clotman et al., 2002). It is not until around e15.5 that the ductal plate starts

to form in proximity to the hepatic sinusoids, which promote bile duct fate via

Transforming Growth Factors (TGF’s), BMP’s and FGF signalling. Studies of

hepatic explant cultures have shown BMP4 enhances FGF signalling to

promote bile duct formation. FGF receptors are expressed in portal

hepatoblasts and differentiate into cholangiocytes under the influence of

mesenchymal bFGF and FGF7. Differentiation is enhanced by BMP4 which

is also produced by liver mesenchymal cells (Yanai et al., 2008). Between

e15.5 and e18.5, hepatocyte and cholangiocyte maturation occurs and a

distinct ductular network forms (Reviewed in Lemaigre, 2009; Figure 1.2).


13

Figure 1.2. Lineage determination in the liver and pancreas.

Endodermal precursors are patterned into liver or pancreas fates under the

control of FGF and BMP signals from the mesenchyme. Liver and pancreas

proliferation and fate choice is controlled by Wnt and Notch signalling, which

divert the organ primordium away from ductal and towards

parenchymal/islet lineages.


14

1.2 Liver and pancreas progenitors in regeneration

Under certain circumstances, progenitor cells in the liver and pancreas

proliferate and differentiate to regenerate lost tissue. Understanding the

mechanisms underlying these processes is important for identifying factors

that could be used to derive functional cells for transplantation as well as

mechanisms leading to progenitor cell tumorigenesis. In this section, we will

explore the role of adult-derived stem cells in regenerative models in the liver

and pancreas.

1.2.1 Liver progenitor cells

1.2.1.1 Progenitor cell mediated liver regeneration

The liver possesses a remarkable ability to regenerate via the division of

remaining hepatocytes if acute injury, such as tissue resection or hepatocyte

necrosis occurs. However, hepatocyte proliferation is impaired by chronic

liver injuries, necessitating alternative pathways for liver regeneration. In

response to signals from dying hepatocytes and immunomodulatory

molecules associated with liver inflammation, a progenitor cell compartment

is activated. Unlike hepatocytes, liver progenitor cells (LPCs) retain

proliferative capacity and can differentiate to restore hepatocytes under

severe injury conditions.

LPCs are a liver-resident population residing in the ‘Canals of Hering’, the

interface between the hepatocytes canaliculi and biliary tree (Theise et al.,

1999), and are characterised by their co-expression of hepatocyte and ductal

markers (Germain et al., 1985, Jelnes et al., 2007) and ability to form both of

these tissues (Alison, 2003, Wang et al., 2003). Proof of the regenerative

capacity of LPCs during liver injury has come from lineage tracing studies in

mouse models of chronic liver injury, which have demonstrated that LPCs

arise from a subset of Sox9+ ductal cells that proliferate and differentiate to

replace damaged hepatocytes (Furuyama et al., 2011, Espanol-Suner et al.,


15

2012). This process of proliferation and regeneration is initiated by the

inflammatory response to injury.

Early chronic liver injury is characterised by infiltration of inflammatory

leukocytes which produce cytokines such as Tumour Necrosis Factor (TNF)

(Knight et al., 2000, Viebahn et al., 2006) and Interferon (IFN)-γ (Akhurst et

al., 2005) that promote LPC proliferation at the expense of hepatocyte

division (Brooling et al., 2005). Macrophages, which function to clear cellular

debris from the sites of injury, secrete TNF-like Weak Inducer of Apoptosis

(TWEAK) (Viebahn et al., 2010), which signals via its receptor Fn14, present

on LPCs, to promote their proliferation (Tirnitz-Parker et al., 2010).

Transforming Growth Factor (TGF)-β is a suppressor of cell division.

However, LPC’s are significantly less sensitive to TGFβ-mediated growth

inhibition compared to hepatocytes (Nguyen et al., 2007). Dying hepatocytes

also support regeneration by releasing Hedgehog ligand (Jung et al., 2010)

which signals via canonical Smoothened-dependent signalling to promote

proliferation of primary-cilium positive LPCs (Grzelak et al., 2014). Thus,

following hepatocyte injury and liver inflammation, a microenvironment

develops which promotes LPC-mediated regeneration whilst supressing

hepatocyte proliferation.

The inflammatory reaction to chronic liver injury also initiates the fibrogenic

wound healing response. This response is mediated by liver resident

perisinusoidal Ito cells, also termed hepatic stellate cells (HSCs). In response

to inflammatory signals, HSCs transition to an ‘activated’ state characterised

by their expression of α-smooth muscle actin (αSMA) (Mederacke et al., 2013).

Activated HSCs deposit extracellular matrix (ECM) proteins which act to

shield the surrounding liver from diseased tissue, and modulate LPC biology.

They also reinforce the wound healing and repair responses by recruiting

inflammatory and progenitor cells to the site of injury via the expression of

chemotactic factors such as intercellular adhesion molecule 1 (ICAM-1) and


16

regulated upon activation, normal T-cell expressed and secreted (RANTES)

(Ruddell et al., 2009).

If the hepatic insult is removed, fibrosis recedes and the structural and

functional integrity of the liver is restored. Conversely, continuing hepatic

insult reinforces the regenerative and fibrotic responses which become

pathological, progressing to liver cirrhosis and failure. Persistent stimulation

of LPCs by pro-proliferative/survival signals from inflammatory cells can give

rise to genetic and epigenetic mutations and subsequent neoplastic

transformation of LPCs resulting in the formation of hepatocellular

carcinoma (HCC) associated with chronic liver injury.

1.2.1.2 LPCs as a tumour precursor

Observations in chronic liver disease patients and rodent models of liver

disease have established LPCs as a potential HCC precursor cell. LPCs are

observed at all stages during chronic liver disease progression, regardless of

the underlying pathology, including chronic hepatitis B (HBV) and C virus

(HCV) infection (Lowes et al., 1999, Sobaniec-Lotowska et al., 2007), non-

alcoholic fatty liver disease (Nobili et al., 2012), alcoholic liver disease and

genetic hemochromatosis (Lowes et al., 1999). LPC numbers increase with

disease severity in a number of different liver chronic liver injury pathologies

(Lowes et al., 1999, Clouston et al., 2005) and are observed in association with

human liver tumorigenic progression from preneoplastic lesions (Roskams et

al., 1996) to well-developed HCCs (Lee et al., 2005, Lee et al., 2006, Haybaeck

et al., 2009). Although these studies associate LPCs with the progression of

liver damage to tumorigenesis, several types of evidence from cell culture and

rodent modelling of chronic liver disease support the hypothesis that LPCs

represent a direct cellular precursor of HCC.

The mouse choline deficient, ethionine-supplemented (CDE) diet is a well-

established regime to induce hepatocyte damage, liver inflammation, fibrosis


17

and LPC activation in short-term studies (Akhurst et al., 2001) which

progresses to neoplasia and HCC development in long-term studies (Knight

et al., 2000). In this model, tumour incidence is reduced by inhibiting of LPCs

by cytokine receptor knockout (Knight et al., 2000) or chemical inhibition

(Knight et al., 2008) suggesting a contribution of LPCs to

hepatocarcinogenesis. In transgenic mice with liver-specific doxycycline-

controlled MYC oncogene expression, induction of MYC expression in liver

cells gives rise to tumours in LPC-like cells which are dormant in non-induced

tissue. Following MYC-induced carcinogenesis, these tumour cells

differentiate and/or undergo apoptosis if inducible MYC oncogene expression

is inactivated, providing evidence of a precursor cell-type within the liver that

has tumour-forming potential (Shachaf et al., 2004).

In vitro modelling of HCC supports a progenitor cell origin of liver cancer.

LPC lines have been isolated from p53-null mice fed a CDE diet which differ

in their tumorigenic potential when injected into nude mice (Dumble et al.,

2002) providing evidence that subpopulations of LPCs have the potential to

give rise to tumours. Recently, it was demonstrated that cell lines can be

generated from resected liver tissue surrounding human HCC containing

cells positive for LPC markers. The cell lines derived were phenotypically

similar to LPCs observed in vivo and gave rise to tumours in nude mice

suggesting a precursor-product relationship between the LPCs observed in

vivo and susceptibility to neoplastic transformation following culture

immortalisation (Zhang et al., 2010).

Altogether, these studies provide a compelling argument that subpopulations

of LPCs are capable of neoplastic transformation to form HCC in chronic liver

disease and as such, represent an important chemotherapeutic target for

HCC treatment.


18

1.2.1.3 LPCs as an expandable source of hepatocytes

Studies of LPCs in vitro have provided valuable information as to the

mechanism underlying the conditions required for LPC expansion and

differentiation. Many different liver progenitor cell lines have been derived

from both adult and foetal sources of liver from human (Malhi et al., 2002),

rat (Radaeva and Steinberg, 1995) and mouse (Dumble et al., 2002, Strick-

Marchand and Weiss, 2002, Fougere-Deschatrette et al., 2006, Tirnitz-Parker

et al., 2007). Studies utilising these cell lines have helped define conditions

for hepatocyte differentiation of liver progenitors. Hepatocyte-like cells can

be induced in foetal-derived cell lines through formation of cell clusters by

culture on plates treated to prevent attachment, inducing cell to cell contact

and upregulating hepatocyte genes (Strick-Marchand and Weiss, 2002). To

date, the mechanisms underlying this differentiation have not been probed.

However, the initiation of cell-to-cell contact and contact inhibition of mitosis

has been shown to induce hepatocyte differentiation in adult-derived LPC

lines, in combination with the glucocorticoid dexamethasone and factors

supporting hepatocyte function such as insulin, transferrin and selenium

(Tirnitz-Parker et al., 2007). Dexamethasone promotes hepatocyte

differentiation of liver progenitors by upregulating hepatocyte-specific genes

such as tyrosine aminotransferase (Yeoh et al., 1979a, Yeoh et al., 1979b,

Shelly and Yeoh, 1991) and supports the expression and secretion of

hepatocyte-specific proteins such as transferrin (Kraemer et al., 1986) and

albumin (Yeoh et al., 1985).

Transplant studies with foetal and adult-derived LPCs have demonstrated

their ability to functionally repopulate injured liver. Transplanted foetal

LPCs expand in vivo before slowing their proliferation, differentiating to

hepatocytes after four to six weeks (Cantz et al., 2003). Adult LPCs isolated

from DDC-injured livers also repopulate the livers of fumarylacetoacetate

hydrolase (FAH)-deficient mice with equal efficiency to hepatocytes (Wang et

al., 2003). As well as primary LPCs, murine cell lines transplanted into

Albumin-uroplasminogen (Alb-uPA) mice, in which hepatocytes undergo


19

repeated necrosis, are able to differentiate into hepatocytes and repair the

damaged liver parenchyma (Strick-Marchand et al., 2004). LPCs derived from

human foetal liver are also expandable in vitro and functionally repopulate

the livers of immune deficient mice (Malhi et al., 2002), providing proof-of-

principle for potential cell-based therapy for human liver disease using LPCs.

1.2.2 Models of β-cell regeneration in the adult pancreas

Cell tracing studies have revealed that in normal adult pancreas, β-cell

turnover is very slow, and is mediated by endogenous β-cells rather than a

progenitor cell. Furthermore, the expansion of β-cell mass between three and

twelve months of age in the mouse is mediated by pre-existing β-cells (Dor et

al., 2004). β-cells not only derive from pre-existing cells during neonatal

development, but regenerate after partial pancreatectomy (Dor et al., 2004,

Teta et al., 2007) and expansion of β-cell mass during pregnancy or after

exendin-4 administration is also predominantly accomplished by the

endogenous β-cells in the mouse and human (Teta et al., 2007, Meier et al.,

2008). Under certain conditions, the pancreatic duct epithelium may serve as

a progenitor cell compartment from which β-cells may be derived (Trivedi et

al., 2001, Bonner-Weir et al., 2004, Lin et al., 2006, Oshima et al., 2007). In

humans, CK19 cells co-expressing insulin and the proliferation marker Ki-67

have been observed in biopsies of pancreas tissue from T1D patients receiving

pancreas transplants (Martin-Pagola et al., 2008) suggesting ductal to islet

differentiation is not merely an in vitro phenomenon. Similarly, during

partial ductal ligation in the mouse, pancreatic β-cell regeneration is partially

mediated by a resident duct-derived progenitor cell expressing Ngn3 which

can be expanded in vitro, and give rise to all islet cell types including glucose-

responsive β-cells (Xu et al., 2008).


20

1.3 Liver and pancreas interconversions

1.3.1 Pancreas to liver conversions

Pancreas to liver transdifferentiation was first observed in rats fed a copper

depleted diet for 8-10 weeks, after which copper deficiency was restored. This

regime induced almost complete loss of pancreatic acinar tissue and

concomitant pancreatic to hepatocytic metaplasia, with up to 60% of cells

expressing albumin (Rao et al., 1988). Cells transdifferentiated using this

regime exhibit a functional hepatocyte phenotype, making proteins including

carbamyl phosphate synthetase-1, urate oxidase and activate β-oxidation

pathways (Usuda et al., 1988). In vitro treatment of mouse and human

pancreatic explants reveal that dexamethasone and/or FGF2 can initiate

hepatic differentiation in these tissues (Horb et al., 2003, Sumitran-

Holgersson et al., 2009). These studies indicate that the ‘default’ pancreatic

program can still be overridden by signals conducive to hepatic differentiation

to produce functional hepatocytes from pancreatic tissue well after the

pancreatic lineage has been specified. In vitro studies of pancreatic to hepatic

transdifferentiation reveal some of the molecular mechanisms underlying

this phenomenon.

The pancreatic tumour cell line AR42J-B13 (B13) has been extensively

studied for its ability to transdifferentiate to the hepatic lineage in response

to dexamethasone treatment (Shen et al., 2000) and represents an ideal in

vitro model to study this phenomenon. Dexamethasone treatment induces

hepatic gene expression at the expense of Pdx1 in B13 cells and mouse foetal

pancreatic explants (Shen et al., 2000, Shen et al., 2003). This phenotype is

recapitulated when C/EBPβ is overexpressed in B13 cells, suggesting that

promotion of hepatic over pancreatic fate is controlled by this transcription

factor (Shen et al., 2000, Shen et al., 2003). Transdifferentiated cells are

functional, expressing key enzymes that perform the detoxification functions

of mature hepatocytes. However, the transdifferentiated phenotype is not


21

permanent, lasting up to 14 days after dexamethasone is removed from the

culture medium (Tosh et al., 2002).

1.3.2 Liver to pancreas conversions

Because LPCs are readily expanded in vitro, and are therefore theoretically

scalable to therapeutically beneficial numbers, they represent an exciting

prospect for cell therapy applications. There is some evidence to suggest that

under certain conditions, LPCs may be induced to give rise to insulin

producing cells. Extrapancreatic insulin-positive cells, including liver cells,

are a phenomenon observed in mouse models of Type 1 diabetes (Kojima et

al., 2004, Vorobeychik et al., 2008). In the liver, these Insulin+ cells co-localise

with A6+ cells indicating they may be of ductular and/or LPC origin

(Vorobeychik et al., 2008). Indeed, if ductal/LPC expansion is induced in mice

by administration of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) prior to

streptozotocin-induced diabetes, functional, hepatic insulin producing cells

develop which reverse the diabetes phenotype (Kim et al., 2007). These data

suggest that LPCs can be diverted to pancreatic fates under high glucose

conditions. This phenomenon can also be achieved by reprogramming

hepatocytes using pancreatic transcription factors, whereby overexpression

of Pdx1 in the livers of diabetic mice gives rise to hepatic insulin-expressing

cells (Ferber et al., 2000, Shternhall-Ron et al., 2007). This pancreatic

phenotype is stable, even if Pdx1 overexpression is transient (Ber et al., 2003,

Horb et al., 2003) and can be recapitulated in vitro with LPC lines (Yang et

al., 2002). Further studies have shown that Ngn3 overexpression can also

induce hepatic insulin expression and reverse experimentally induced

diabetes through the emergence of periportal Insulin+ clusters which

morphologically resemble LPCs (Yechoor et al., 2009).


22

Figure 1.3. Pathways to progenitor-derived hepatocytes and β-cells.

Several pathways exist to derive hepatocytes and β-cells from progenitor cell

sources. Some types of liver or pancreas injury promote ductal-derived

progenitor mediated regeneration in the liver and pancreas. Under certain

conditions, such as high glucose, copper deficiency or by exogenous

overexpression of lineage-specific transcription factors, liver and pancreas

progenitors can be transdifferentiated to form mature cells of other organs.

The mechanisms underlying in vivo differentiation/transdifferentiation are

useful for informing protocols to develop mature liver and pancreas cells in

vitro (e.g.: from pluripotent cells or LPC lines).


23

1.3.3 Summary

The identification of expandable progenitor cell subtypes from the liver and

pancreas that are capable of differentiation to mature tissues is of vital

importance for developing renewable cell sources for therapy. One of the

biggest risks in transplantation of stem cell-derived tissues is the potential

for progenitors to give rise to tumours. Therefore the ability to define the

phenotypes of progenitor cells, including those with tumorigenic potential, is

critical to develop cells for therapy. This would aid diagnosis of liver

pathologies, and establishing a phenotype for tumorigenic cells would be

beneficial for developing targeted chemotherapeutic drugs, particularly for

HCC where LPCs have been shown to contribute to cancer formation. One

potential avenue to achieve this goal is to define the cell surface phenotype of

the progenitor cell system of the liver and pancreas with antibodies which

enables cell populations to be isolated using antibody-based sorting

techniques such as flow cytometry for further study. Current progress

towards this goal will be reviewed in the next section.

1.4 Phenotyping hepatic and pancreatic progenitors

1.4.1 Surface markers of LPCs

The A6 monoclonal antibody was produced by immunising rats with a liver

progenitor cell-enriched isolate from the livers of mice with induced chronic

liver injury and it has since become the ‘gold standard’ antibody for

identifying LPCs in mouse models of liver injury (Engelhardt et al., 1990,

Bennoun et al., 1993, Preisegger et al., 1999, Petersen et al., 2003, Akhurst

et al., 2005) and to characterise novel LPC lines (Fougere-Deschatrette et al.,

2006, Tirnitz-Parker et al., 2007). Although an excellent antibody for

immunoidentification of LPCs, the successful use of A6 for antibody-based

isolation of LPCs has not been reported, presumably because the antigen does

not survive the enzymatic digestion process used to disperse liver cells.

Because of this, more recent research efforts have focused on defining new

surface antigens on LPCs which are useful for identification and isolation of

LPC subsets.


24

The majority of surface-reactive antibodies that have recently been defined

on LPCs are co-expressed by ductal cells. These markers include EpCAM,

CD24 and CD133 and have been used to enrich LPCs using antibody-based

sorting techniques (Ochsner et al., 2007, Rountree et al., 2007, Okabe et al.,

2009). Studies utilizing EpCAM and CD24 have shown that expandable

bipotential progenitors are present in these preparations providing firm

evidence of a liver-resident progenitor cell (Okabe et al., 2009, Qiu et al.,

2011). Additionally, E-cadherin is strongly expressed on ducts and LPCs in

chronically injured liver and weakly on hepatocytes (Ueberham et al., 2007).

Few antibodies have been developed that recognize injured, but not normal

liver. Recently, the novel cell surface marker Trop-2, a member of the GA733

gene family along with EpCAM (Linnenbach et al., 1993), was shown to co-

stain EpCAM+ cells in DDC-injured livers, although the cell populations

recognized by Trop-2 and EpCAM are identical (Okabe et al., 2009). Other

surface markers of LPCs include the hematopoietic stem cell markers Sca-1

and CD34, which are expressed by a subset of A6+ cells in DDC-induced injury

(Petersen et al., 2003) and Dlk/Pref-1 which also marks ductal cell

subpopulations (Tanimizu et al., 2003, Tanimizu et al., 2004, Jelnes et al.,

2007). However, when compared to the total CK19+ cells, Dlk/Pref-1+ cells are

less proliferative, based on Ki67 immunoreactivity, suggesting that Dlk/Pref-

1 cells may mark a subset of differentiating LPCs (Tanimizu et al., 2004).

More recently, a panel of monoclonal antibodies was generated by

immunising rats with the liver non-parenchymal cells of mice fed a DDC diet.

A number of antibodies were generated, representing a diverse resource for

characterising LPCs. The ductal-specific MIC-1C3 antibody generated from

this panel has proved useful for isolating LPCs (Dorrell et al., 2008). In

combination with previously described antibodies, the use of MIC-1C3

antibody has helped progress towards distinguishing LPCs at the clonal level,

in a similar fashion to landmark studies in the hematopoietic system. So far,

significant enrichment of LPCs has been achieved by isolating a subset of duct

cells, firstly by excluding hematopoietic cells and enriching progenitors using


25

the MIC-1C3+/CD133+/CD26- subset of non-hematopoietic hepatic non-

parenchymal cells (CD45-/Cd11b-/CD31-) to purify self-renewing cells capable

of bipotential differentiation in vitro in up to 4% of isolated cells (Dorrell et

al., 2011). Although this represents significant progress in identifying LPCs,

there is still much to be done to define the surface phenotype of the liver stem

cell for the purpose of purification at the clonal level.

1.4.2 Surface markers of islet progenitor cells

In the pancreas, studies of mechanisms underlying Ngn3+ islet precursor cell

differentiation to β-cells have been carried out on primary tissue because of a

lack of adequate cell line models. This has proved difficult because of the

scarcity and transient nature of Ngn3+ islet progenitors in the developing

pancreas (Villasenor et al., 2008). In order to study islet development

properly, especially specific effects of factors, it would be optimal to have a

pure fraction of Ngn3+ islet precursor cells. Unfortunately no specific marker

or marker combination has been established to isolate Ngn3+ cells. CD133 is

expressed on islet progenitors and has been used in combination with Cd49f

or PDGFRβ to isolate these cells. Ngn3+ cells are enriched by sorting with

CD133+/Cd49flow population of e15.5 mouse pancreas of which 0.7% of

cultured cells can be differentiated into insulin-expressing cells in vitro

(Sugiyama et al., 2007). These markers can also be used to purify endocrine

progenitors from human foetal pancreas (Sugiyama et al., 2007). Combining

CD133 sorting with negative selection using PDGFRβ-antibodies on e13.5

pancreas achieves a significant enrichment of Ngn3+ cells which can

differentiate into insulin producing cells in vitro and in vivo (Hori et al., 2008).

These studies represent the current best efforts in enriching endocrine

progenitors without genetic modification. However further studies are needed

to uncover the antigenic phenotype of Ngn3+ cells distinct to ductal and

neural progenitor cells.


26

1.5 Summary and project aims

The ability of researchers studying liver and pancreas development and

regeneration has been hampered by a lack of antibodies to identify the

common and lineage distinct progenitor cells that re-populate the respective

tissues. Ideally, novel antibodies for this purpose would also be useful not only

for identifying these progenitors, but for their isolation using antibody-based

sorting techniques. Furthermore, identification of the antigens recognised by

any new antibodies would potentiate the translation of isolation techniques

of these cells from mouse models to human progenitors.

Accordingly, the aims of the research reported in this thesis are to:

1. Generate a panel of surface-reactive monoclonal antibodies against liver

and pancreas cell populations and to identify potential markers of

liver and pancreas progenitor cells.

2. Characterise the ability of novel antibodies to identify and isolate liver

progenitor cell populations.

3. Characterise the ability of novel antibodies to identify and isolate

pancreatic islet progenitor cell populations

4. Identify protein targets of putative markers and to determine whether

human-equivalent antibodies can be used to isolate human islet

and/or liver progenitor cells.

CHAPTER 2 RESULTS I

27

Chapter 2 Generation and characterisation of

novel cell surface antibodies to mouse

liver progenitor cells

CHAPTER 2 RESULTS I

28

2.1 ABSTRACT Liver progenitor cells (LPCs) represent a heterogeneous precursor

compartment that is enlarged during chronic liver injury and acts to restore

the liver mass and function when hepatocyte-mediated regeneration is

impaired. LPCs are implicated as cellular source of hepatocellular carcinoma

associated with chronic liver injury. Therefore, understanding the

mechanisms that underpin their differentiation and tumourigenicity is

important for regenerative and cancer medicine. Here, we report the

development of two new antibodies, WAM18 and WAM21, and describe their

application in detecting and isolating mouse LPCs that will enable their

characterisation. Cells recognized by WAM18 and WAM21 were significantly

induced in the liver following CDE-induced injury, and dual

immunofluorescent staining demonstrated that WAM18 and WAM21 marked

highly similar cell populations to the ductal/LPC marker A6. We also

observed overlapping staining with WAM18 and A6 in DDC-induced liver

injury. However, WAM21 marked A6+ cells in addition to cells adjacent to the

DDC-induced ductular reaction. An LPC line, PIL-4 was characterised with

respect to WAM18 and WAM21 expression. WAM18+ and WAM21+ subsets of

PIL-4 cells displayed features of bipotential progenitors, co-expressing

hepatocyte and ductal markers. We interrogated microarray data from liver

progenitor cell lines differentially expressing WAM18 and WAM21 antigens

and identified two novel candidate surface markers of LPCs whose expression

is induced in chronic liver injury. In this study, we show that our novel

antibodies represent an ideal alternative to the A6 antibody, with the

potential for use in antibody-based enrichment of LPCs.

CHAPTER 2 RESULTS I

29

2.2 INTRODUCTION

Conditions of severe and chronic liver injury inhibit hepatocyte-mediated

liver regeneration, and induce the liver progenitor cell (LPC) compartment to

proliferate and differentiate to address the hepatocyte deficit (Paku et al.,

2001, Forbes et al., 2002). LPCs are characterised by their heterogeneous co-

expression of markers associated with hepatocytes and biliary cells (Germain

et al., 1985), and their ability to mature into either lineage (Evarts et al.,

1989, Evarts et al., 1996, Strick-Marchand and Weiss, 2002, Strick-Marchand

et al., 2004). Compelling evidence for this role has come from recent lineage

tracing studies in mice using either the Sox9 or Osteopontin promoters to

show that ductal-derived LPCs give rise to hepatocytes in chronic liver injury

(Furuyama et al., 2011). Since the process of LPC proliferation and

hepatocyte differentiation can be recapitulated in vitro (Strick-Marchand and

Weiss, 2002, Tirnitz-Parker et al., 2007), and they can be transplanted and

replenish injured rodent liver (Wang et al., 2003), LPCs represent a promising

renewable cell-based alternative to organ transplantation to address some

liver pathologies. In addition to their regenerative function, LPCs are also

associated with liver carcinogenesis. Progenitors are observed at various

stages of human liver tumorigenesis; in chronic liver injuries (Roskams et al.,

1998, Lowes et al., 1999, Xiao et al., 2004), preneoplastic lesions (Roskams et

al., 1996) and in hepatocellular carcinomas (HCC) (Lee et al., 2005, Lee et al.,

2006). Furthermore, limiting LPC proliferation in mouse models of liver

regeneration reduces tumour formation (Knight et al., 2000, Haybaeck et al.,

2009). Thus, detailed studies of the processes regulating growth,

differentiation and transformation of LPCs are an imperative for cancer and

regenerative medicine.

Rodent models of diet-induced liver injury are useful to study the mechanisms

regulating LPC biology during chronic liver injury. Commonly used models

include the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) (Preisegger et

al., 1999) and choline deficient-ethionine supplemented (CDE) diets (Akhurst

CHAPTER 2 RESULTS I

30

et al., 2001), which induce diverse LPC populations defined by intracellular

marker expression (Jelnes et al., 2007). CDE-induced liver tumorigenesis is

attenuated by impairing the progenitor cell response to chronic liver injury

through cytokine-receptor knockout (Knight et al., 2000) or by inducing LPC

apoptosis (Davies et al., 2006), providing an ideal model to study LPC-derived

HCC. Availability of more markers of LPCs, especially of transformed LPCs,

would greatly benefit studies of LPC transformation to HCC. Thus,

generation of surface-reactive antibodies to purify and study different

progenitor populations from complex cellular fractions would advance this

cause.

Mouse LPCs are commonly identified by the antibody A6, which binds to an

as-yet unidentified cell surface determinant present on ducts in normal liver

(Faktor et al., 1990) and ductal cells as well as more centrally located LPCs

in diet-induced chronic liver injury models (Preisegger et al., 1999, Akhurst

et al., 2001). Subsets of A6+ cells can be defined using antibodies against

hematopoietic markers such as CD34, Sca-1 (Petersen et al., 2003) and the

pan-hematopoietic marker CD45 (Petersen et al., 2003, Rountree et al., 2007).

A6 is considered the ‘gold standard’ for immunohistological identification of

liver progenitors. However, since its efficacy for antibody-based cell isolation

has not been reported, new antibodies that stain A6+ cells which could also be

used for antibody-based isolation would be an invaluable tool for LPC

biologists.

Recent efforts have focused on defining the surface marker phenotype of LPCs

to permit their isolation and study. Some antibodies against surface antigens

have been characterised and include Epithelial Cell Adhesion Molecule

(EpCAM) and Trop2, which identify similar cell populations (Okabe et al.,

2009); CD24 which stains a subset of CK19+/CD45- cells (Ochsner et al., 2007);

and CD133 which identifies bipotential progenitor cells in DDC-injured liver

(Rountree et al., 2007). A recently described antibody panel generated against

non-parenchymal cells from DDC livers marks various cell populations

CHAPTER 2 RESULTS I

31

induced in chronic liver injury (Dorrell et al., 2008). This panel includes the

MIC-1C3 antibody, which can be used in combination with CD133 to isolate

LPCs (Dorrell et al., 2011). Although these antibodies stain liver progenitor

cell populations in DDC livers, they remain untested in other models of

chronic liver injury such as the CDE diet. The significance of this model is

underlined by the recent finding that only LPCs generated by feeding a CDE

diet can be traced into hepatocytes (Espanol-Suner et al., 2012). Additionally,

an antibody that can be used for cell purification and stains LPCs, that has

been compared to A6 in different liver injury models, is not available.

Since the liver and pancreas derive from a single precursor cell type (Deutsch

et al., 2001, Furuyama et al., 2011), share common developmental

mechanisms (Zaret and Grompe, 2008) and even the potential for

interconversions (Shen et al., 2000, Tosh et al., 2002), studying their

progenitor domains in parallel is useful for developing novel techniques for

deriving liver and pancreas tissue for transplantation. Thus, in this study we

have generated a panel of monoclonal antibodies against liver and pancreas

tissue. To overcome the aforementioned problems associated with identifying

and isolating LPCs, we herein focus on a subset of antibodies from this panel

which mark LPC surface antigens and describe their utility in identifying,

isolating and characterising LPC populations in the CDE and DDC diet

models of chronic liver injury and LPC lines.

CHAPTER 2 RESULTS I

32

2.3 MATERIALS AND METHODS

2.3.1 Production and screening of monoclonal antibodies

An eight week old Wistar rat was immunised with a mixture of foetal mouse

liver and pancreas cells in Complete Freund’s Adjuvant (Difco) intra-

peritoneally (i/p), followed by i/p boosts in Incomplete Freund’s Adjuvant at 4

and 8 weeks and an aqueous boost at 17 weeks. Spleen cells were collected

and fused with the mouse myeloma line, Sp2/O (Sigma-Aldrich, Australia),

using PEG 1500 (Roche, Australia) under conditions previously described

(Morahan, 1982). Hybridomas were selected in HAT-supplemented medium

(100 µM hypoxanthine, 0.4 µM aminopterin and 10 µM thymidine) and

supernatants were screened by immunofluorescence on a variety of cell lines

and tissues. These included the mouse embryonic stem cell line W9.5,

bipotential murine embryonic liver (BMEL) cells, e13.5 mouse embryonic

fibroblasts (MEF) and Sp2/0 cells. Supernatants recognising Sp2/0 cells were

excluded from further analyses.

2.3.2 Liver cell culture

p53 Immortalised Liver (PIL) cell lines were maintained as described by

Tirnitz-Parker and colleagues (Tirnitz-Parker et al., 2007). Primary cell

isolates were cultured on collagen coated dishes in growth medium as

described above. Cells were maintained in a 37 ºC, 5% CO2 incubator with

90% humidity.

2.3.3 CDE diet induction of liver damage

Mice were housed under specific pathogen free, temperature controlled

conditions, with alternating 12h light-dark cycles. All procedures were

performed according to guidelines set by the National Health and Medical

Research Council of Australia under ethics approval from the University of

Western Australia. Four week old male C57Bl/6 mice were fed choline

deficient chow, supplemented with 0.165% ethionine (w/v) in the drinking

water for two weeks as previously described (Akhurst et al., 2001, Tirnitz-

CHAPTER 2 RESULTS I

33

Parker et al., 2007). Age-matched control mice were fed normal laboratory

chow and water. Liver damage was confirmed histological assessment and

quantitation of serum alanine transaminase levels as previously described

(Tirnitz-Parker et al., 2007). ALT level were elevated in all CDE-treated mice

(Appendix figure A1.1).

2.3.4 Histology and immunofluorescence

Mice were anesthetised by i/p Avertin injection (2.5% 2,2,2 tribromoethanol,

Sigma-Aldrich, 0.015 mL/g body weight) and livers were perfused with 20 mL

PBS via the hepatic portal vein. Livers were embedded in OCT medium for

cryosectioning and stored at -80 °C. OCT-embedded liver tissue from mice fed

a DDC diet for 2, 4 or 6 weeks was kindly provided by Dr Belinda Knight

(Fremantle Hospital, Fremantle, Western Australia). Cryosections (7 µm)

were cut and fixed by exposure to RT acetone for 5 min. Sections were air

dried and stored at -20 °C. Fixed cryosections were rehydrated by incubation

in PBS for 5 mins and endogenous biotin was blocked using a biotin blocking

kit (Dako, Denmark). Primary antibody labelling was performed overnight at

4 °C. Primary antibodies A6 (rat; 1:80) or CD45 (rat, 1:50, BD Biosciences)

were revealed with FITC-conjugated goat anti-rat IgG (1:200, Vector

Laboratories). Following secondary antibody labelling, sections were

incubated with rat immunoglobulin for 1 h at RT to block non-specific binding

of biotinylated primary antibodies to anti-rat secondary antibody.

Biotinylated primary antibodies were incubated for 2 h at RT. Primary

antibodies WAM18 (1:100) and WAM21 (1:100), which are described in

section 2.4.1, were detected by incubation with Streptavidin-Cy3 (1:300,

Amersham). Specificity of staining was confirmed with appropriate isotype

control staining (Appendix figure A1.2). Slides were mounted with ProLong

Gold Antifade mounting medium containing the nuclear stain 4',6-diamidino-

2-phenylindole (DAPI; Invitrogen, Australia) and visualised by fluorescence

microscopy. Single colour fluorescent microscope images were merged using

ImageJ version 1.39u software for Windows (ImageJ Software, National

CHAPTER 2 RESULTS I

34

Institutes of Health, USA, available at: http://rsb.info.nih.gov/ij/). All colour

corrections were applied equally to experimental and control samples.

2.3.5 Liver non-parenchymal cell (NPC) isolation

The liver NPCs of mice fed a CDE for two weeks were isolated using a

modified version of a two-step collagenase digestion (Yaswen et al., 1984),

incorporating low speed centrifugation to separate hepatocytes as previously

described (Dorrell et al., 2008, Okabe et al., 2009), but not centrifugal

elutriation steps. Mice were anesthetised by i/p injection of Avertin and livers

were perfused with 20 mL PBS via the portal vein. Livers were diced and cells

were dispersed by incubation in a 0.05% collagenase type I in PBS (Sigma–

Aldrich; pH 7.6) at 37 ºC for 20 min with gentle agitation. Cells were further

dissociated by pipetting and filtered through a 40 μm cell strainer (BD

Biosciences). Filtered cells were pelleted by centrifugation and resuspended

in Williams’ E medium containing 2% FBS. The undigested clot on the

strainer was subjected to further dissociation by incubation in a solution

containing 0.1% collagenase type VIII (Sigma–Aldrich), 0.09% Pronase

(Roche Diagnostics), 0.025% trypsin/0.01% EDTA (Invitrogen), 0.004% DNase

(Sigma–Aldrich) in PBS for 30 min at 37 °C with gentle agitation. Cells were

freed by pipetting and an equal volume of cold Williams’ E medium containing

2% FBS was added. The cell suspension was filtered through a 40 μm BD

Falcon™ cell strainer (BD Biosciences) and washed three times in Williams’

E medium. Cells from the first and second digestions were combined and

hepatocytes were pelleted by repeated centrifugation at 50 X g for 10 min at

4 °C. The supernatant containing NPCs were transferred to a fresh tube and

cells were pelleted by centrifugation at 800 X g for 10 min at 4 °C. Cells were

resuspended in flow cytometry buffer (0.5% FBS, 1 mM EDTA in PBS) prior

to antibody labelling and flow cytometry.

2.3.6 Flow cytometry

Liver progenitor cell lines were dispersed into single cells with trypsin (0.05%,

Gibco-BRL) and washed in FACS Buffer (Phosphate Buffered Saline/ 2%

http://rsb.info.nih.gov/ij/

CHAPTER 2 RESULTS I

35

FBS). Cells were incubated with 100 µL hybridoma supernatant for 20 min

on ice. Antibodies were detected by incubation with anti-rat IgG-FITC (1:200,

Vector labs, PIL cell lines) for 20 min on ice. Cells were washed and

resuspended in FACS buffer containing Propidium Iodide (1 µg/mL) and

analysed on a FACS-Canto flow cytometer (BD Biosciences). Positive

antibody staining was compared to negative antibody isotype control staining.

Liver NPCs were filtered through a 40 μm BD Falcon™ cell strainer (BD

Biosciences) and stained with primary antibodies. Antibodies used were, rat

IgG2a-biotin (1:200), rat IgG2a-Alexa647 (1:100, eBioscience), WAM 18-biotin

(1:200) or WAM 21-Alexa647 (1:100). Cells were washed twice and incubated

with Streptavidin-Alexa750 (1:200, Invitrogen) for 10 min on ice. Cells were

washed twice, counterstained with DAPI (1 µg/mL) and analysed on a FACS

AriaII flow cytometer (BD Biosciences).

2.3.7 RNA Isolation and Reverse Transcription PCR

Cells were lysed and RNA was extracted in 1 mL TRIzol reagent (Invitrogen).

RNA was treated with DNase I (Promega) to remove gDNA contamination

and first strand cDNA was synthesised with random hexamer primers using

Superscript III Reverse Transcriptase (Invitrogen). Negative control

reactions for gene expression analysis were generated by omitting RT

enzyme. Gene expression was analysed by RT-PCR amplification of cDNA

preparations for 30 cycles using gene specific primers with Platinum Taq

DNA polymerase (Invitrogen). All protocols were carried out according to

standard manufacturer instructions. Primer sequences and annealing

temperatures are listed in Appendix Table A1.1. RT-PCR products were

resolved by electrophoresis through a 1.5% agarose gel containing ethidium

bromide and visualised by UV illumination.

2.3.8 Interrogation of microarray data

Expression profiling of PIL-2 and PIL-4 cells, and CDE and control liver, was

carried out using Affymetrix Mouse Genome 430 2.0 GeneChips according to

manufacturer instructions (Affymetrix; http://www.affymetrix.com). Data

http://www.affymetrix.com/

CHAPTER 2 RESULTS I

36

normalisation and quality control analyses have been previously described

(Jellicoe et al., 2008). Heat maps of log-transformed data were generated

using the web-based Genesifter® program (PerkinElmer). To generate

candidate surface markers encoding WAM antigens, a ratio of PIL-2 to PIL-4

gene expression was generated by calculating log2 (PIL-2/PIL-4). A list of 476

genes which were expressed 1.5-fold higher in PIL-2 cells compared to PIL-4

cells was generated. This gene list was further refined by gene ontology (GO)

identification analysis using the Gene Ontology Enrichment Analysis

Software Toolkit (GOEAST) (Zheng and Wang, 2008). Those genes which

were deemed to be potential surface markers were chosen based on the

following GO identifiers; GO:0016020 (membrane), GO:0044425 (membrane

part), GO:00019867 (outer membrane).

2.3.9 Statistical Analyses

Plotted data represent the mean ± SEM of three separate samples. For each

sample, antibody-marked cells were counted in at least 5 fields (200X

magnification) of view per tissue and expressed as a percentage of DAPI+

cells. Unpaired t-tests were performed using GraphPad Prism version 5.00

for Windows (GraphPad Software, San Diego California USA,

www.graphpad.com). Values were considered significantly different when

p<0.05.

http://amigo.geneontology.org/cgi-bin/amigo/go.cgi?query=GO%3A0044425&search_constraint=terms&action=query&view=query

CHAPTER 2 RESULTS I

37

2.4 RESULTS


To develop novel antibodies that recognise mouse liver and pancreas

progenitors, we produced a panel of 54 rat monoclonal antibodies which were

raised against a mixture of BMEL cell lines and dispersed foetal pancreas

cells, which we termed ‘Western Australian Monoclonal’ (WAM) antibodies.

Screening to identify liver-specific antibodies are described herein. We

initially screened hybridoma supernatants for their cell-type specificity using

live, unfixed cells in vitro, to identify antibodies which would be useful for

isolating live-cell populations. These cell types included embryonic stem cells

(W9.5 cells), LPCs (BMEL), thymocytes and mouse embryonic fibroblasts

(MEF). Hybridoma supernatants were screened against Sp2/0 mouse

myeloma cells as a negative control. 16 WAM antibodies marked Sp2/0 cells

and were excluded from further characterisation (data not shown). Of the

remaining 38 WAM antibodies, 17 marked BMEL cells (Table 2.1). To find

antibodies which would be useful for identifying liver cell populations, we

assessed staining of these 17 antibodies on frozen sections of 2 wk CDE-

injured liver. Although these antibodies stained LPC lines, labelling of cells

in liver of CDE mice was inconsistent. WAM22 marked portal veins and

emanating sinusoidal endothelial cells. WAM45 labelled blood vessels in

addition to clusters of stellate-like cells extending from portal vasculature.

Nine supernatants failed to label any cell populations in CDE livers. WAM18

and WAM21 stained numerous groups of small epithelial-like cells which

extended from WAM+ portal areas into the liver parenchyma, suggesting that

they may recognise LPCs in vivo (Figure 2.1). Accordingly, WAM18 and

WAM21 were characterised further.

CHAPTER 2 RESULTS I

38

WAM # LPC CDE ESC Thym MEF Sp2/0

1 ++ - ++ - ++ -

4 + - ++ + ++ -

18 ++ +++ + - ++ -

21 + + + ++ ++ -

22 +++ +++ ++ + ++ -

30 + - ++ + ++ -

45 ++ +++ + + ++ -

121 + - - - - -

139 + - + - ++ -

163 + ++ - + + -

166 ++ - + ++ ++ -

203 + ++ - - - -

204 + ++ - - - -

224 + - - - - -

280 + + - + - -

285 + - + - ++ -

303 ++ - + +++ +++ -

Table 2.1 Screening WAM antibody panel. Strength of staining on

different cell types was visually determined. Strongly positive (+++), positive

(++), weakly positive (+), negative (-). Cell types are a liver progenitor cell line

BMEL (LPC), 2 wk CDE liver tissue (CDE), W9.5 embryonic stem cells (ESC),

thymocytes (Thym), mouse embryonic fibroblasts (MEF) and Sp2/0 mouse

myeloma cells.

CHAPTER 2 RESULTS I

39

Figure 2.1 WAM antibody labelling in 2 week CDE liver.

Immunofluorescent staining of cryosections (7 µm) of CDE diet-induced liver

with WAM antibodies (green). WAM18 and WAM21 identified small

epithelial-like cells around portal areas which formed clusters extending into

the liver parenchyma. WAM22 marked blood vessels and thin cells

resembling hepatic sinusoidal cells and WAM45 labelled stellate-like cells

which extended from WAM45+ vessels. Nuclei were counterstained with

DAPI (blue). Scale bars represent 100 µm.

CHAPTER 2 RESULTS I

40

2.4.2 WAM18 and WAM21 mark the ductular reaction to chronic liver

injury

We assessed the capability of WAM18 and WAM21 to mark LPCs in the CDE

and DDC diet-induced liver injury models. Figure 2.2 shows dual

immunofluorescence labelling of our new antibodies compared with the

established ductal/LPC marker A6 in normal and CDE-injured livers.

WAM18 and WAM21 labelled a subset of A6+ cells in normal livers (Figure

2.2A). In livers with CDE-induced LPC response, we observed both an

increase in the numbers of cells marked by WAM18 and WAM21 in addition

to a high degree of overlap with A6+ cell populations (Figure 2.2B). This

observation was supported by quantitative assessment of cell numbers that

overlap with A6 in normal and CDE-injured livers (Figure 2.3). Cells marked

by WAM18 and WAM21 were significantly induced in CDE-treated versus

control livers (11-fold and 8-fold respectively). Overlapping staining of

WAM18 or WAM21 with A6 was 40% and 45% in normal livers. Critically,

the degree of overlap of WAM18 or WAM21 with A6 increased to 98% and

96% respectively in LPC induced CDE-injured livers.

In DDC-injured livers, WAM18 marked a subset of A6+ cells after 2 weeks of

injury, and WAM21 staining was essentially similar to A6 immunoreactivity

(Figure 2.4A.). However, from 4 weeks of treatment, WAM18 and WAM21

marked distinct cell populations when compared with A6. WAM18 and A6

marked virtually identical cell populations in 4 and 6 week DDC liver, whilst

WAM21 identified A6+ cells as well as a subpopulation of cells that that were

A6- located adjacent to WAM21+/A6+ cells at 4 and 6 weeks of DDC treatment

(Figures 2.4B and 2.4C.). To test the specificity of WAM antibodies for LPCs,

we compared WAM antibody and CD45 immunospecificity in CDE and DDC

treated livers. WAM18 did not mark CD45+ inflammatory cells in CDE or

DDC-induced liver injury, confirming their specificity for LPCs induced by

these diets (Figure 2.5A&B.). However, a small number of WAM21+ cells were

also CD45+ in DDC-injured livers (Figure 2.5B.).

CHAPTER 2 RESULTS I

41

Figure 2.2 Comparison of WAM and A6 antibody labelling in normal

and CDE liver. Immunofluorescent staining with the liver progenitor

marker A6 (green) and WAMs (red) or WAM21 on cryosections (7 µm) of livers

of mice fed (A) a normal chow diet for 2 weeks or (B) a CDE diet for 2 weeks.

WAM18 and WAM21 localise to subpopulation of A6+ ductal cells in normal

livers and co-localise with A6+ in CDE-induced livers. Nuclei were

counterstained with DAPI (blue). Arrows indicate regions displayed by insets.

Scale bars represent 100 µm.

A

B

CHAPTER 2 RESULTS I

42

Figure 2.3 Quantitation of LPC induction in the CDE model using

WAM antibodies. The numbers of cells labelled with (A) WAM18 or (C)

WAM21 significantly increased during CDE-induced chronic liver injury. The

amount of co-staining of WAM antibodies with the LPC marker A6 was

quantitated during LPC induction. Pie charts show the relative proportion of

cells co-labelled with (B) WAM18 and A6, or (D) WAM21 and A6 increased in

CDE-injured versus control livers (yellow segment). Bars represent mean ±

SEM with n = 3. Statistical significance with respect to control diet is

represented as *p<0.05 or **p<0.01.

B A

C D

CHAPTER 2 RESULTS I

43

Figure 2.4 Comparison of WAM and A6 antibody labelling in DDC

liver. Immunofluorescent staining with the liver progenitor cell marker A6

(green) and WAMs (red) on cryosections (7 µm) of livers of mice fed a DDC

diet for (A) 2 weeks (B) 4 weeks or (C) 6 weeks. WAM18 and A6 stain similar

cell subsets at all timepoints. WAM21 stains a population of cells adjacent to

A6+ cells that are not marked by A6 at 4 and 6 weeks of DDC treatment.

Nuclei were counterstained with DAPI (blue). Arrows indicate regions

displayed by insets. Scale bars represent 100 µm.

A

B

C

CHAPTER 2 RESULTS I

44

Figure 2.5 Comparison of WAM and CD45 antibody labelling in

chronic liver injury. Immunofluorescent staining with the pan-

hematopoietic maker CD45 (green) and WAMs (red) on cryosections (7 µm) of

livers of mice fed a CDE diet for 2 weeks or a DDC diet for 6 weeks. (A) In 2

week CDE livers WAM18 and WAM21 positive cells are observed in close

association with CD45+ inflammatory cells, but co-localisation of markers is

not observed (B) In 6 week DDC livers WAM18+ cells are CD45-. A small

number of WAM21+ cells are CD45+ during DDC treatment. Nuclei were

counterstained with DAPI (blue). Diagonal arrows indicate regions displayed

by insets. Horizontal arrows mark WAM21+/CD45+ cell subsets. Scale bars

represent 100 µm.

A

B

CHAPTER 2 RESULTS I

45

2.4.3 Cell sorting potential of WAM18 and WAM21

The applicability of WAM18 and WAM21 for antibody-based sorting of live

cell populations was addressed by staining liver non-parenchymal cell (NPC)

preparations from 2 week CDE livers. WAM18 and WAM21 marked 10% and

31% of total liver NPC’s from 2 week CDE livers respectively. Dual antibody

staining of liver NPC’s with WAM18 and WAM21 showed that they defined

two cell subsets that were WAM18+/WAM21+ and WAM18-/WAM21+ (Figure

2.6A.). We purified these WAM-defined populations by FACS but were unable

to propagate them in culture. We also used magnetic bead sorting to enrich

WAM-defined cell subsets from normal livers, but were also unable to

propagate these cells in vitro (Appendix Figure A1.3). Hence, we undertook

further cell sorting studies to characterise subsets of WAM antibody-defined

liver progenitor cell lines. Staining was assessed by flow cytometry on five

p53 immortalised liver (PIL) cells. PIL-1, -2, -3 and -5 cells all expressed

single positive WAM18 and WAM21 populations. However, PIL-4 contained

two cell populations, positive and negative, defined by WAM18 or WAM21

staining (Figure 2.6B). We used this property to purify PIL-4 cell subsets

based on WAM antibody expression and characterised gene expression in

these populations. Expression of cytokeratins CK7 and CK18, the ductal gap

junction protein Cx43 and the hepatocyte/progenitor transcription factor

Hnf4α were enriched in WAM18 and WAM21-positive subpopulations of PIL-

4 cells. Hnf1β and CK19 expression was uniform between different WAM18

and WAM21-defined PIL-4 cells. The hepatocyte marker Albumin was not

expressed by PIL-4 cells (Figure 2.6C).

CHAPTER 2 RESULTS I

46

Figure 2.6 Defining liver progenitor cell subsets with WAM

antibodies. (A) Staining of non-parenchymal cells from freshly isolated 2

week CDE liver shows WAM18 marks a subset of WAM21+ non-parenchymal

cells. (B) Flow cytometric analysis of WAM antibody staining in PIL cell panel

(grey) versus isotype control staining (white). (C) RT-PCR analysis of

hepatocyte (Hep) and ductal (Duct) genes in WAM-defined PIL-4 cell

populations. Expression in WAM-defined populations was compared to

unsorted PIL-4 cells (US). E15 liver (e15) was used as a positive control for

gene expression.

A

B C

CHAPTER 2 RESULTS I

47

2.4.4 Identifying candidate genes encoding novel cell surface

markers of LPCs

Since flow cytometric analysis shows that PIL-2 and PIL-4 cells differentially

express WAM18 and WAM21 on their cell surface, we used this property to

identify potential new surface markers of LPCs by comparing global gene

expression between these two cell lines using a previously conducted

microarray experiment (performed by Jasmine Low, University of Western

Australia). We predicted that mRNA’s encoding WAM18 and WAM21

antigens would be expressed in PIL-2 cells in greater abundance than PIL-4

cells. RNA species that were expressed 1.5-fold higher in PIL-2 cells, and were

deemed to be expressed on the cell surface by gene ontology classification

were included in our analysis. Six putative LPC surface markers were

identified; BMP binding endothelial regulator (Bmper), Cadherin 11 (Cdh11),

Cdh17, Doublecortin-like and CAM kinase-like 1 (Dcamkl1), Insulin-like

growth factor receptor 2 (Igf2R) and Slit homolog 2 (Slit2). To confirm this

result, we assessed the expression of these markers, and the established LPC

surface markers Cd24a, Cd34, E-cadherin, Glypican-3 and Dlk in WAM18

and WAM21-defined PIL-4 cells by RT-PCR analysis.

Of previously described surface markers of LPC’s, Cd34 and E-cadherin

transcripts were enriched in WAM18+ and WAM21+ cells, whilst the

expression of Cd24a was uniform between WAM-defined populations. Dlk,

Glypican-3 and Slit-2 were not expressed in PIL-4 cells, and the expression of

Dcamkl1 and Igf2R were not specific to WAM+ populations. Of our new

candidate markers, Bmper, Cdh11 and Cdh17 expression was enriched in in

WAM18+ and WAM21+ versus WAM antibody-negative cells (Figure 2.7A). To

support cell line findings, we interrogated microarray data from CDE-induced

injury. Of the newly identified markers, Bmper and Cdh17 were expressed at

low levels in normal liver and were highly upregulated in 2 week CDE livers

(18.8-fold and 96.9-fold respectively), as were the established markers Cd24a

(14.7-fold), Glypican-3 (67.4-fold) and EpCAM (13.5-fold) (Figure 2.7B).

CHAPTER 2 RESULTS I

48

Figure 2.7 Screening for candidate genes encoding WAM18 and

WAM21. (A) RT-PCR analysis of established LPC surface markers and

putative surface genes encoding WAM18 and WAM21 antigens in WAM-

defined PIL-4 cells. (B) Heatmap representation of microarray analysis of

gene expression of candidate surface markers in 2 wk control versus CDE

livers. The intensity of each colour denotes the standardised ratio between

each value and the average expression of each gene across all samples. Red

pixels correspond to an increased abundance of mRNA, green pixels denote

decreased mRNA levels.

CHAPTER 2 RESULTS I

49

2.5 DISCUSSION

Development of antibodies against cell surface antigens of LPCs compatible

with antibody-based isolation techniques are vital for characterising the

regenerative and tumorigenic capacity of LPC subtypes in chronically injured

liver. To increase the limited repertoire of antibodies that meet this criterion,

we generated and characterised monoclonal antibodies recognising LPCs. We

have produced two antibodies, WAM18 and WAM21, with significant value

for in vitro and in vivo studies of LPCs.

The applicability of antibodies to enzyme-dissociation protocols for selecting

non-parenchymal cells from whole livers has been an issue with previously

described cell surface markers such as A6 (Engelhardt et al., 1990) and

glypican-3 (Grozdanov et al., 2006). Additionally, EpCAM is downregulated

in cultured LPCs (Okabe et al., 2009) preventing its use in LPC line model

studies. Our new antibodies, WAM18 and WAM21, mark highly similar cell

populations to the ‘gold standard’ LPC marker, A6, in CDE-induced livers,

and WAM18 and A6 display an overlapping staining pattern in DDC-induced

injury. Critically, we have shown that WAM18 and WAM21 are compatible

with enzyme dissociation protocols used to disperse whole liver and cultured

LPC lines. Thus these WAM antibodies are viable alternatives to A6 for

immunoidentification in multiple chronic liver injury models, and

additionally useful for isolation of chronic liver injury-induced and cultured

progenitors.

We used this property to characterise subsets of the PIL-4 cells, which had

previously been shown to heterogeneously express LPC markers including A6

(Dumble et al., 2002). We were able to purify a subset of PIL-4 cells which co-

expressed ductal (CK7, CK19, Hnf1β, Cx43) and hepatocytic (CK18 and

Hnf4α) mRNA’s as assessed by RT-PCR. Since the co-expression of bi-lineage

markers is a property of bipotential LPCs (Jelnes et al., 2007, Tirnitz-Parker

et al., 2007), these studies provide evidence of the utility of WAM18 and

CHAPTER 2 RESULTS I

50

WAM21 in isolating LPC subsets from heterogeneous starting populations.

Using WAM-antibody defined phenotypes in combination with previously

generated microarray data, we identified new candidate LPC surface markers

using LPC line models. Of these new markers present in PIL cell lines,

Cadherin-17 and Bmper are promising candidates for future study as their

mRNA’s are also upregulated in CDE-induced liver injury.

We have demonstrated that our novel antibodies, WAM18 and WAM21;

particularly WAM18 are useful reagents for identifying LPCs and they can

be used to generate enriched LPC populations from whole liver. They are

viable alternatives to A6 which has limited availability and cannot be used

for cell purification.

CHAPTER 3 RESULTS II

51

Chapter 3 Definition, enrichment and

development of pancreatic beta cell

progenitors using a novel monoclonal

antibody


52

3.1 ABSTRACT

Aims: Understanding the mechanisms regulating islet development is crucial

in manipulating stem cells to produce viable beta cell surrogates for Type 1

Diabetes therapy. Progress to this goal can be achieved by studying islet

progenitors isolated from the developing pancreas. However, few markers

currently exist to isolate islet progenitors. Accordingly, we produced

monoclonal antibodies that can define subsets of cells in the developing

pancreas.

Methods: Monoclonal antibodies were raised against foetal mouse islet tissue

and screened for their ability to bind to Ngn3-expressing pancreas cells from

e15.5 double transgenic Ngn3-GFP+/RIP-dsRed mice. One antibody, WAM21,

was characterised in detail for its ability to isolate putative islet progenitors

which could differentiate to insulin-expressing cells in vitro.

Results: Staining islet-specific transgenic reporter mouse lines and gene

expression analysis revealed developing islet progenitor cells are WAM21+.

Culture of WAM21-sorted transgenic insulin-reporter foetal pancreas cells

demonstrated the ability of WAM21+ islet progenitors to differentiate into

insulin-expressing cells.

Conclusions: Our novel antibody, WAM21, identifies a subset of developing

mouse pancreas cells which contains Ngn3+ beta cell progenitors capable of

differentiation to insulin expressing cells under defined culture conditions.

The ability of WAM21 to enrich for beta cell progenitors will greatly aid

studies of beta cell development for understanding islet development

mechanisms.


53

3.2 INTRODUCTION

Type 1 Diabetes arises from immune-mediated destruction of insulin

producing beta cells, resulting in chronic hyperglycaemia and risk of

complications including diabetic retinopathy, nephropathy and vascular

disease. Current therapies for Type 1 Diabetes include exogenous insulin

administration, which does not effect complete glucoregulation and is

associated with diabetic complications in the long-term, or transplant of

cadaveric islet tissue (Shapiro et al., 2000), for which the demand far

outweighs availability. Research efforts to alleviate this problem are focused

on deriving beta cells from diverse stem cell sources. These sources include

embryonic stem cells (Jiang et al., 2007, Kroon et al., 2008), pancreatic duct-

derived progenitors (Bonner-Weir et al., 2004, Xu et al., 2008), and non-

pancreatic tissue-derived stem cells such as liver progenitors (Yang et al.,

2002, Ber et al., 2003, Kim et al., 2007, Yechoor et al., 2009). Though stem

cells can be directed to produce insulin, current protocols are hampered by

inefficiency of beta cell derivation and incomplete maturation to glucose-

responsive cells. Consequently, in vitro studies of islet development are vital

to define the mechanisms underlying beta cell differentiation, maturation and

expansion for efficient derivation of clinically useful beta cell surrogates.

The pancreas develops from two endodermal buds; the ventral bud, which

contacts the cardiac mesoderm neighbouring the liver primordium, and the

dorsal bud which develops separately, adjacent to the septum transversum

mesenchyme (Slack, 1995, Field et al., 2003). Mesenchymal signals such as

fibroblast growth factors (Hebrok et al., 1998, Bhushan et al., 2001, Deutsch

et al., 2001, Hart et al., 2003), bone morphogenetic proteins (Rossi et al., 2001)

and retinoic acid (Kumar and Melton, 2003, Molotkov et al., 2005) induce the

pancreatic domain to express the gene Pancreatic and Duodenal Homeobox 1

(Pdx1) (Wright et al., 1989, Jorgensen et al., 2007). This gene drives

pancreatic-specific gene expression (Melloul, 2004, Babu et al., 2007, Oliver-

Krasinski et al., 2009). All epithelial cell types present in the pancreas derive

from the Pdx1+ epithelium (Wright et al., 1989, Burlison et al., 2008), which


54

undergoes branching morphogenesis and differentiates to form the ductal

tree, endocrine and exocrine cells (Habener et al., 2005). Downstream of Pdx1,

Neurogenin-3 (Ngn3) is transiently expressed in a subset of cells which form

all islet cell types (Gradwohl et al., 2000, Schwitzgebel et al., 2000, Desgraz

and Herrera, 2009). The Ngn3 transcription factor acts to inhibit Notch

signalling (Apelqvist et al., 1999) thereby facilitating islet gene expression.

Currently, few cell surface markers are available to identify and isolate islet

progenitor cells. One of the key problems is that candidate membrane

proteins such as E-cadherin (Jiang et al., 2002, Sugiyama et al., 2007), CD133

and Cd49f (Sugiyama et al., 2007) are common to progenitors of islets, ducts

and exocrine tissue. CD133 is expressed by Ngn3+ cells and carboxypeptidase-

A+ acinar cells but not mature hormone-expressing islet cells (Sugiyama et

al., 2007, Hori et al., 2008). Three distinct populations can be identified by

antibodies to Cd49f, with the low-positive population containing Ngn3+ islet

progenitors while the acinar cells are found in the Cd49f high-positive portion

(Sugiyama et al., 2007). Given the shared origin of these three lineages, it is

perhaps not surprising that no surface marker unique to Ngn3+ islet

precursor cells has been identified.

Development of additional surface markers defining islet precursor cells

would improve their isolation and aid in studies of beta cell differentiation.

Thus, we generated a panel of monoclonal antibodies against pancreas tissue.

In this study, we characterised the utility of antibodies in this panel to

identify precursors in developing mouse pancreas capable of differentiate to

insulin-producing cells under defined conditions in vitro.


55



Wistar rats were immunised with e15.5 foetal pancreas cells and liver

progenitor cells in Complete Freund’s Adjuvant (Difco) intra-peritoneally

(i/p), followed by i/p boosts in Incomplete Freund’s Adjuvant at 4 and 8 weeks

and an aqueous boost at 17 weeks. Spleen cells were collected and fused with

the mouse myeloma line, Sp2/O (Sigma), using PEG 1500 (Roche) under

conditions previously described (Morahan, 1982). Hybridomas were selected

in HAT medium and supernatants were screened by immunofluorescence on

foetal pancreas. Specificity for antigens expressed on foetal pancreas was

assessed by staining of other tissues, such as liver and thymus.

3.3.2 Foetal pancreas isolation

Mouse embryos at e15.5 were dissected from the uterus and placenta and

placed in a petri dish containing cold PBS prior to dissection of pancreata

under sterile conditions. Embryonic pancreata were removed and stored in

medium (high glucose DMEM (Invitrogen) with 10% FBS) until processing.

Pooled pancreata were centrifuged briefly and the medium aspirated. Cells

were dissociated as previously described (Jiang et al., 1999), using Dispase

(BD Biosciences) instead of trypsin for 1 h at 37 °C. Animal experiments were

performed according to the Australian Code of Practice for the care and use

of scientific animals and ethics approval was obtained from the Animal

Resources Centre, Perth and the University of Western Australia Animal

Ethics Committee.

3.3.3 Cell sorting

For flow cytometric sorting, cells were resuspended in FACS Buffer (PBS/ 2%

FBS) and stained with primary antibodies. Antibodies used were AlexaFluor

647-conjugated-WAM21 or rat IgG2a isotype control-AlexaFluor 647

(eBioscience). For dual antibody labelling, anti CD49f-PE (clone GoH3, BD

Biosciences) and biotin-conjugated WAM21 antibody were used. Biotinylated


56

antibody was detected with Streptavidin-AlexaFluor 750 (Invitrogen). Cells

were washed and resuspended in FACS buffer containing DAPI (1 µg/mL) and

analysed on a FACSAriaII flow cytometer (BD Biosciences). For magnetic

bead sorting (MACS), cells were resuspended in MACS Buffer (PBS/ 0.5%

FBS/ 1 mM EDTA) and stained with biotin-conjugated WAM21 antibody.

After washing, cells were stained with Anti-Rat IgG MicroBeads or Anti-

Biotin Multisort beads (Miltenyi Biotec). For fluorescent detection, cells were

stained with Streptavidin-AlexaFluor 750 (Invitrogen). After washing,

unlabelled cells were separated by passing the cells through an MS

MiniMACS Column according to manufacturer instructions (Miltenyi Biotec).

A small aliquot was stained with DAPI, and purity was assessed by flow

cytometry.

3.3.4 Differentiation of WAM21+ cells

For in vitro differentiation, sorted cells were differentiated by culture in high

glucose DMEM medium (Invitrogen) containing nicotinamide (10 mM,

Sigma-Aldrich), growth factor reduced Matrigel™ (200 µg/mL, BD

Biosciences) and B27 supplement (2X, Gibco). Cells were seeded at a density

of 104 cells/well of a 96-well plate in triplicate. Differentiation was assessed

by daily counting of cells that were MIP-GFP+ or RIP-dsRed+ (Ins-reporter+)

over 5 to 7 days in a 37 °C, 10% CO2 incubator with 90% humidity.

3.3.5 RNA Isolation and RT-PCR




Superscript III Reverse Transcriptase (Invitrogen). Gene expression was

analysed by RT-PCR amplification of cDNA preparations for 35 cycles using

gene specific primers with Platinum Taq DNA polymerase (Invitrogen). All

protocols were carried out according to manufacturer instructions. Primer

sequences and annealing temperatures are listed in Appendix Table A2.1.


57

RT-PCR products were resolved by electrophoresis through a 1.5% agarose

gel containing ethidium bromide and visualised UV light illumination.


Cryosections (7 µm) of OCT-embedded e15.5 foetal pancreas tissue were cut

and fixed in acetone (-20 °C, 5 mins), air-dried and stored at -20 °C until use.

Sections were rehydrated in PBS for 5 mins and non-specific binding was

blocked by incubation in blocking buffer (5% FBS diluted in PBS) for 1 hour

at room temperature. Labelling of primary antibodies was performed

overnight at 4 °C. Antibodies used were WAM21-Biotin or Cd49f (BD

Biosciences). Specificity of labelling was confirmed by a lack of labelling

following incubation with IgG2a-Biotin or IgG2a isotype control antibodies

(Appendix figure A2.1). Primary rat antibodies were detected with FITC-

conjugated goat anti rat IgG (1:200, Vector) and biotinylated antibodies with

Streptavidin-Cy3 (1:300, Amersham). Slides were mounted with ProLong

Gold Antifade mounting medium containing the nuclear stain DAPI

(Invitrogen) and visualised by fluorescence microscopy. Single colour

fluorescent microscope images were merged using ImageJ version 1.39u

software for Windows (ImageJ Software, National Institutes of Health, USA,

http://rsb.info.nih.gov/ij/).

3.3.7 Statistical Analyses

Plotted data represents the mean average ± SEM. Two-tailed, unpaired t-

tests were performed using GraphPad Prism version 6.00 for Windows

(GraphPad Software, San Diego California USA, www.graphpad.com). Values

were considered significantly different for p<0.05.



58

3.4 RESULTS

3.4.1 Production and screening of hybridoma supernatants

In order to develop novel antibodies that recognise mouse liver and pancreas

progenitors, we produced a panel of 54 rat monoclonal antibodies which were

raised against dispersed mouse foetal pancreas cells. Hybridoma

supernatants were initially screened to select appropriate specificities using

various cell sources. These included embryonic stem cells (W9.5 cells), NIT-1

insulinoma cells (representing insulin-producing cells), and cryosections of

e15.5 mouse pancreas. Supernatants were screened against thymocytes

mouse embryonic fibroblasts (MEF) and Sp2/0 myeloma cells as negative

controls. The selected hybridomas were designated with a ‘Western

Australian Monoclonal’ (WAM) number. A total of 33 WAM antibodies

marked pancreatic populations and were characterised further (Table 3.1). 16

antibodies were non-specific (data not shown) and were excluded from further

characterisation.

To identify antibodies useful for isolating islet progenitors, we screened

hybridoma supernatants on foetal pancreata isolated from dual transgenic

mice (Ngn3-GFP/RIP-dsRed) that express GFP and dsRed under the control

of the mouse Ngn3 and rat insulin promoters, respectively. By flow cytometry,

four antibodies were found to be useful as positive selectors of Ngn3-GFP+

cells. The best of these was WAM21, which stained 17% of total cells,

representing a potential 4.2-fold enrichment of Ngn3-GFP+ cells.

Additionally, four antibodies identified populations of Ngn3-GFP- cells and

were considered useful negative selectors of islet progenitors. The best of

these was WAM316 which stained 43% of cells, a 1.9-fold enrichment of Ngn3-

GFP+ cells by negative selection (Table 3.2). WAM21 was the most promising

antibody for isolating islet progenitors, so was selected for further

characterisation.


59

WAM # Foetal

panc. NIT-1 ESC Thym. MEF Sp2/0

1 + - ++ - ++ -

2 + - + - ++ -

4 + - ++ + ++ -

7 + - - - ++ -

9 + - - - - -

10 + - - - - -

18 + + + - ++ -

20 + - + - ++ -

21 +++ + + ++ ++ -

22 + - ++ + ++ -

30 + - ++ + ++ -

32 + - - - - -

36 + - + - ++ --

45 + - + + ++ -

85 + - + +++ ++ -

94 + - - - - -

107 + - - - - -

119 + - - - + -

132 + - - - + -

139 + + + - ++ -

159 + - + + - -

167 + + + - ++ -

185 ++ + + - ++ -

247 + - + + + -

251 + - - - + -

273 + - + ++ ++ -

280 + - - + - -

281 ++ + + + + -

285 + ++ + - ++ -

303 +++ ++ + +++ +++ -

316 ++ - +++ +++ ++ -

318 + - - - ++ -

354 + - - + + -

Table 3.1. Screening WAM antibody panel. Strength of staining on

different cell types was visually determined. Strongly positive (+++), positive

(++), weakly positive (+), negative (-). Cell types are e15.5 foetal pancreas

(Foetal panc.), NIT-1 insulinoma cells, W9.5 embryonic stem cells (ESC),

thymocytes (Thym.), mouse embryonic fibroblasts (MEF) and Sp2/0 mouse

myeloma cells.


60

Category WAM#

e15.5 cells

detected

(%)

Enrichment

of Ngn3-

GFP+ cells

(fold)

Positive Enriching 4 18 2.9

Abs 18 60 1.5

21 17 4.2

280 65 1.2

Negative 20 16 1.3

Enriching Abs 316 43 1.8

318 29 1.9

354 28 1.2

Table 3.2. Screening supernatants from WAM hybridomas.

Supernatants were evaluated for binding to cells from e15.5 pancreas of

Ngn3-GFP/RIP-dsRed transgenic mice.

3.4.2 e15.5 pancreas WAM21+ cells are enriched for cells with islet

progenitor traits

WAM21 was used to stain and sort cells from foetal pancreata from two

different transgenic reporter strains: the above-mentioned Ngn3-GFP/RIP-

dsRed mice and the MIP-GFP strain that expresses GFP under the control of

the mouse insulin I promoter (Hara et al., 2003). In the former, WAM21

stained 28% of e15.5 pancreas cells; these included virtually all the Ngn3-

GFP+ cells (Figure 3.1A, upper panel). The ability to mark foetal beta cells

was assessed by FACS analysis of MIP-GFP foetal pancreata. WAM21

stained 29% of MIP-GFP pancreas, including cells in which the insulin

promoter was active (i.e. GFP+ cells; Figure 3.1A, lower panel).


61

Figure 3.1. Identification of islet progenitors in developing pancreas.

The ability of WAM21 to identify islet precursors was tested by flow

cytometric analysis of WAM21 staining of cells from transgenic reporter

mouse foetal pancreas. (A) WAM21 marked Ngn3-GFP+ and MIP-GFP+

pancreatic cells from Ngn3-GFP/RIP-dsRed and MIP-GFP mice, respectively.

(B) RT-PCR analyses of genes expressed in islet cells, epithelial stem cells,

neural/mesenchymal cells and ductal cells in WAM21- and WAM21+ e15.5

pancreas populations.

Gene expression analysis of sorted foetal pancreas subpopulations showed

WAM21-defined populations demonstrated characteristics of islet progenitor

cells. By RT-PCR, the WAM21+ population expressed Ngn3 and was enriched

for cells expressing both insulin genes. Furthermore, WAM21+ cells contained

all cells expressing Pdx1, as well as the beta cell precursor genes Pax4 and

Nkx2.2, confirming that WAM21 could enrich for cells with an islet/beta cell

progenitor phenotype. The WAM21+ population also expressed the acinar

marker Ptf1a, progenitor cell markers E-cadherin and CD133. Cells

expressing Cd24a, CK19 and the neural/mesenchymal marker PDGFRβ were

not enriched in the WAM21+ population (Figure 3.1B).

A B


62

3.4.3 WAM21+ e15.5 pancreas cells differentiate to Ins+ cells in vitro

Since WAM21+ cells had molecular characteristics of beta cell progenitors, we

assessed the capacity of these putative progenitors to differentiate into

Insulin-expressing cells in vitro (Figure 3.2). Developing MIP-GFP pancreata

were separated into WAM21+ and WAM21- populations by FACS and seeded

in defined culture medium. The number of GFP+ cells formed was assessed

over 5 days. To calculate enrichment of islet progenitors, differentiation was

compared to non-antibody enriched controls. It should be noted that MIP-

GFP+ cells were excluded at the sorting stage, so that only those cells which

differentiated in culture and activated the Ins1 promoter were detected. No

MIP-GFP+ cells were observed on the day of plating. During differentiation,

only single MIP-GFP+ cells were observed during the differentiation process,

suggesting MIP-GFP+ cells did not arise by proliferation of progenitors

(Figure 3.2A). WAM21-sorting significantly enriched progenitors which gave

rise to MIP-GFP+ cells. Appearance of MIP-GFP+ cells peaked at 4-5 days

post-plating (Figure 3.2B). In three independent experiments, WAM21+ cells

differentiated at a frequency which was 3.2-fold higher than non-antibody

enriched cells (Figure 3.2C). Conversely, WAM21- cells yielded very few GFP+

cells.

Gene expression in differentiated WAM21-defined populations was analysed

by RT-PCR (Figure 3.2D). In addition to the production of MIP-GFP+ cells,

culture in differentiation medium resulted in down-regulation of the

progenitor cell transcripts Ngn3, Pax4 and Nkx2.2. Pdx1, which becomes

restricted to beta cells during their development, was also down-regulated.

Expression of both Insulin genes was increased. Loss of Ptf1a expression

indicated that growth and/or differentiation of acinar cells were not supported

by these culture conditions. No islet-specific gene expression was observed in

cultures of sorted WAM21-negative cells. Together, these results

demonstrated that WAM21-based sorting significantly enriched from islets

those progenitor cells that could differentiate into Insulin-expressing cells.


63

Figure 3.2. Differentiation of WAM21-defined e15.5 MIP-GFP

pancreas populations. Differentiation was assessed in MIP-GFP-, WAM21-

and WAM21+ cells by observing MIP-GFP+ cells. (A) MIP-GFP+ cells at day 0

and day 4 of differentiation. (B) Differentiation dynamics of WAM21-defined

populations compared to MIP-GFP- cells show differentiation peak at 4-5

days. (C) Ratio of MIP-GFP+ cells formed after differentiation in WAM-

defined foetal pancreas populations compared to MIP-GFP- cells. (D) RT-PCR

analyses of islet/β-cell markers post-differentiation in WAM21- and WAM21+

cells. Bars represent mean ± SEM (n=3; **p<0.01 compared to MIP-GFP-

cells). Scale bars represent 20 µm.

A B

C D B


64

3.4.4 Further definition of WAM21+ progenitors with antibodies to

Cd49f

As we had shown that WAM21+ foetal pancreas cells were of a mixed islet

progenitor/acinar lineage by RT-PCR analysis, dual antibody sorting with an

acinar cell marker could potentially be used to further purify islet progenitors

in the WAM21+ subset. Cd49f was previously shown to mark

Carboxypeptidase-A+ acinar cells (Sugiyama et al., 2007). We therefore

assessed the potential of further enriching WAM21+ islet progenitor cells in

combination with Cd49f. First, we compared the specificity of WAM21 and

Cd49f. Immunostaining of e15.5 wild-type pancreas illustrated the similar

but distinct staining pattern of these markers: WAM21 stained a

subpopulation of pancreatic cells, strongly localising to the apical membrane

of Cd49flow cells (Figure 3.3A). Cells which stained strongly for Cd49f also

stained strongly for WAM21, and flow cytometric analysis of Cd49f and

WAM21 staining of Ngn3-GFP/RIP-dsRed e15.5 pancreas confirmed that

Ngn3-GFP+ cells were enriched in the WAM21+/Cd49flow subpopulation

(Figure 3.3B). These results suggested that WAM21+ islet progenitors could

be further enriched by their low Cd49f expression.

We further characterised WAM21/Cd49f-defined foetal pancreas populations

by analysis of gene expression and in vitro differentiation to beta-like cells.

Islet progenitors were purified in a two-step process. Magnetic bead-enriched

WAM21+ progenitors were FACS-separated into Cd49flow and CD49fhigh

populations. RIP-dsRed+ cells were excluded at the sorting stage so that

subsequent analysis was restricted to newly formed beta cells (Figure 3.4A).

Ngn3-GFP+ cells were enriched up to 38% in WAM21+/Cd49flow cells; this

compared to 0.6% in the WAM21+/Cd49fhigh population (Figure 3.4B). The

identity of cell populations defined by WAM21 and Cd49f was assessed by RT-

PCR analysis of islet, ductal, acinar, neural and cell surface markers (Figure

3.4C). WAM21+/Cd49flow cells expressed a mixture of these markers including

Pdx1, Ngn3, Pax4, Nkx2.2 insulin-1 and insulin-2 (islet lineage markers),

CK19 (ductal/epithelial cells) and β3-tubulin (neural cells).


65

WAM21+/Cd49fhigh cells exclusively expressed Ptf1a (acinar), MAP2 and

PDGFRβ (neural), and contained cells expressing Pdx1, Ngn3, Pax4, Nkx2.2,

insulin-1, insulin-2 and CK19. Sorting WAM21+ cells with Cd49f also further

divided populations expressing mRNAs encoding the cell surface markers

CD133, Cd24a and E-cadherin.

When we assessed the differentiation potential of these populations, only

WAM21+/Cd49flow cells gave rise to significant numbers of RIP-dsRed+ cells.

We observed more Ngn3-GFP+ cells in cultures of WAM21+/Cd49flow cells

compared to those of non-antibody enriched cells. Over the first two days of

culture, Ngn3-GFP expression was reduced to near-background levels (Figure

3.5A). RIP-dsRed+ cells began to appear after 24h of culture and their

numbers gradually increased, peaking at 4 days (Figure 3.5B). Conversely,

WAM21+/Cd49fhigh foetal pancreas cells produced few RIP-dsRed+ cells

compared to non-antibody enriched cells (Figure 3.5B). At the peak of

differentiation, WAM21+/Cd49flow cells differentiated at a frequency 11.5-fold

higher than non-antibody enriched cells (Figure 3.5C).

RT-PCR analysis (Figure 3.5D) revealed that cultures of WAM21+/Cd49flow

cells down-regulated Ngn3, Pax4 and Nkx2.2 and upregulated ins1, ins2 and

Pdx1, confirming beta cell differentiation of enriched islet progenitors. These

cells maintained expression of the neural markers β3-tubulin and a small

amount of MAP2, indicating that neural cells were maintained in this culture.

Intriguingly, in WAM21+/Cd49fhigh cells, neural markers were also down-

regulated during culture but the neural-type insulin gene expression pattern

was maintained; ins2 predominated over ins1 but Pdx1 was not present,

indicative of a non-pancreatic epithelial phenotype (Figure 3.5D).


66

Figure 3.3. Comparison of WAM21 and Cd49f staining. (A)

Immunofluorescent staining with the islet progenitor marker Cd49f and

WAM21 on cryosections (7 µm) of wild-type e15.5 pancreata. Nuclei were

counterstained with DAPI (blue). Scale bars represent 20 µm. (B) FACS

analysis of populations of Cd49f and WAM21-defined e15.5 Ngn3-GFP/RIP-

dsRed pancreata. Numbers of Ngn3-GFP+ cells were assessed in Cd49flow,

WAM21+ and Cd49flow/WAM21+ populations.

A

B


67

Figure 3.4. Molecular characterisation of WAM21 and Cd49f-defined

e15.5 pancreas cells. (A) Islet progenitors from e15.5 Ngn3-GFP/RIP-dsRed

foetal pancreas were enriched by magnetic bead sorting with WAM21.

WAM21+/RIP-dsRed- cells were further separated into two Cd49f-defined

populations (high and low) by FACS for subsequent RT-PCR and

differentiation studies. (B) Ngn3-GFP+ islet precursor cells were enriched in

WAM21+/Cd49flow cells, and depleted in WAM21+/Cd49fhigh cells. (C) RT-PCR

analyses of genes expressed in islet cells, epithelial stem cells, mesenchymal,

neural and ductal cells in WAM21+/Cd49flow and WAM21+/Cd49fhigh defined

e15.5 pancreas.

B

A

C


68

Figure 3.5. Differentiation of WAM21/Cd49f-defined Ngn3-GFP/RIP-

dsRed foetal pancreas populations. Differentiation was assessed in RIP-

dsRed-, WAM21+/Cd49flow and WAM21+/Cd49fhigh cells by observing RIP-

dsRed+ cells. (A) Ngn3-GFP/RIP-dsRed cells at day 0, 2 and day 4 of

differentiation in Cd49f-defined WAM21+ cells. Images taken with GFP and

dsRed filters were merged. (B) Beta-cell differentiation dynamics of Cd49f-

defined WAM21+ populations. (C) Ratio of ins-reporter+ cells after

differentiation in Cd49f-defined WAM21+ cells compared to RIP-dsRed- cells.

(D) RT-PCR analyses of islet/β-cell and neural markers post-differentiation in

Cd49f-defined WAM21+ cells. Bars represent mean ± SEM (n=3; **p<0.01

compared to RIP-dsRed- cells). Scale bars represent 20 µm.

A

B C D


69

3.5 DISCUSSION

Antibody-based sorting is a powerful method to isolate rare stem cells from

heterogeneous populations for further study. Its usefulness for stem cell

research was first demonstrated for the isolation of progenitor cells from the

hematopoietic system (Spangrude et al., 1988) and more recently in the

generation of new antibodies against cell surface determinants of liver

progenitors (Dorrell et al., 2008b) and adult human pancreas (Dorrell et al.,

2008a). Like these tissues, the developing pancreas is composed of many cell

types of which progenitors comprise a small subset. A lack of surface markers

defining islet progenitors has hampered efforts to identify and isolate these

cells for further study. Although previous studies examined established

antibodies for defining the foetal pancreatic progenitor cell surface (Sugiyama

et al., 2007, Hori et al., 2008), we approached this problem differently. We

generated a panel of monoclonal antibodies against pancreas progenitors.

One marker, WAM21, proved most valuable for isolating and characterising

foetal beta cell progenitors.

We demonstrated the effectiveness of WAM21-based sorting for isolating islet

progenitors which gave rise to insulin-reporter+ cells in differentiation

medium. WAM21 identified a subpopulation of CD133+ cells and could be

used in combination with Cd49f to enrich islet progenitors significantly.

CD133 was shown previously to mark non-committed islet precursors,

excluding islet-hormone+ cells (Sugiyama et al., 2007). In contrast, we found

that WAM21 identified a subpopulation of Insulin-expressing cells. Our

studies suggest that both committed beta cell progenitors and primitive islet

precursors are WAM21+. By using transgenically-labelled beta cells for

visualising differentiation dynamics, we could exclude Ins-reporter+ cells at

the sorting stage study, allowing study of only the progenitor cells. We

achieved a differentiation rate of up to 5% ins-reporter+ cells from the dual

antibody-sorted subset. In this system, ins-reporter+ cells did not develop in

the absence of factors such as nicotinamide and glucose present in the

differentiation medium (F.X. Jiang, unpublished data) suggesting that


70

fluorescently tagged cells derived over the culture period are progenitor-cell

derived and dependent on specific factors for differentiation into Insulin+

cells. However, in order to understand the commitment and development of

beta cells effectively, further delineation of the surface phenotype of all

pancreatic lineages using monoclonal antibodies would be useful. In our

studies, we provide promising candidate markers for future lineage definition

studies in developing pancreas.

Unexpectedly, Ngn3 expression was observed in WAM21+/Cd49fhigh cells. A

small number of Ngn3+ cells have been previously shown to arise from

cultured Cd49fhigh cells (Sugiyama et al., 2007) though their differentiation to

beta cells was not reported. The Ngn3+ cells observed in our Cd49fhigh

population may be analogous to previously reported Ngn3+/Cd49fhigh cells as

they naturally occur in vivo. When these cells were cultured under

differentiation conditions, we observed Ins expression in the absence of Pdx1

expression. Although Pdx1 mRNA was present in this population prior to

differentiation, its down-regulation in conjunction with loss of Ptf1a

expression after culture suggests acinar-derived Pdx1 expression. Pdx1 is

expressed upstream of both Ngn3 and Ins during pancreatic development,

binding the Ngn3 promoter (Oliver-Krasinski et al., 2009) and is required for

beta cell-specific Ins transcription, as well as beta cell function (Melloul, 2004,

Babu et al., 2007) and becomes restricted to beta cells during development

(Deramaudt et al., 2006). However, Ngn3 is also expressed in neural

progenitors during brain development (Sommer et al., 1996). Ins genes are

also expressed during neuronal development, with Ins2 expression

predominating over Ins1 after e10.5 in the mouse (Deltour et al., 1993).

WAM21+/Cd49fhigh cells express the neural markers MAP2 and PDGFRβ and

exhibit a neural-type Ins2 over Ins1 expression pre- and post-differentiation.

The absence of Pdx1 in cultured WAM21+/Cd49fhigh cells indicates non-islet

transcription of neuroendocrine markers, and raises the potential for a neural

cell origin of these transcripts.


71

Using antibodies that define beta cell precursors in conjunction with efficient

models of differentiation will facilitate generation of better in vitro methods

for producing beta cells for therapy. Our novel antibody, WAM21, is a

versatile and effective surface-reactive antibody for identifying and isolating

foetal beta cell progenitors which can be differentiated into Ins+ cells in vitro.

In other experiments, we also showed that WAM21 could also enrich liver

precursor cells (MS in preparation, Chapter 2). This novel antibody will aid

in studies of beta cell development, which in turn will allow for improved

methods to derive beta cells from stem cells for Type 1 Diabetes treatment.

CHAPTER 4 RESULTS III

72

Chapter 4 Expression cloning a single cDNA

encoding the WAM18 and WAM21

antigens


73

4.1 ABSTRACT

The ability to purify progenitor cells from complex organs is vital for studies

of tissue regeneration and tumourigenesis. We have previously described two

antibodies, WAM18 and WAM21, which are useful for isolation and

characterisation of progenitor cell domains of the mouse liver and pancreas.

To facilitate functional and translational studies, we sought to identify the

antigens bound by these antibodies. A cDNA encoding the WAM18 and

WAM21 antigens was cloned by immunoselection of COS-7 cells transfected

with a cDNA library. Antibody inhibition studies demonstrated that WAM18

and WAM21 antibodies bind overlapping, but distinct, epitopes. Further

characterisation of these epitopes showed that expression of WAM18 and

WAM21 was cell type-specific in developing mouse brain and pancreas.

Having identified a cDNA encoding WAM18 and WAM21, we carried out

RNAi experiments, which demonstrated a role for this transcript in

controlling liver progenitor cell proliferation in vitro. Finally, we showed that

an antibody recognising a human WAM18/21 protein equivalent could be used

to enrich human foetal islet pancreas cells with features of islet progenitors.

These studies have enabled us to define a cDNA encoding WAM18 and

WAM21 and suggest a role for this cDNA in regulating liver progenitor cell

proliferation. Furthermore, we have shown the expression of the protein

recognised by our antibodies on the cell surface of islet progenitors is

conserved between mice and humans. This discovery of a novel marker of

human islet progenitors will benefit human islet developmental studies to

generate stem cell-derived beta cell surrogates for Type 1 Diabetes therapy.


74

4.2 INTRODUCTION

The liver and pancreas arise from a common bipotential precursor cell type

residing within the developing endoderm (Deutsch et al., 2001). Although

specific liver or pancreas-specific lineage gene cascades are initially induced

by relative exposure to fibroblast growth factor signals from the septum

transversum mesenchyme (Deutsch et al., 2001, Rossi et al., 2001, Serls et

al., 2005) studies of their respective progenitor cell domains reveal striking

similarities in mechanisms underlying their growth and differentiation.

Signals such Wnt/β-catenin and Notch share common functions in both

organs, controlling tissue-specific progenitor cell expansion and ductal versus

non-ductal fate choices. Furthermore, under certain injury or tissue culture

conditions, liver to pancreas (Ferber et al., 2000, Cao et al., 2004, Kojima et

al., 2004, Kim et al., 2007, Shternhall-Ron et al., 2007) and pancreas to liver

conversions are observed (Tosh et al., 2002, Shen et al., 2003, Westmacott et

al., 2006, Tosh et al., 2007). Since the liver and pancreas share a common

origin, develop under the control of similar pathways and have the potential

to interconvert under appropriate conditions, they are ideal organs to study

in parallel. Consequently, it is unsurprising that they also share cell surface

markers defining their progenitor domains.

The majority of studies to develop new surface markers of liver and pancreas

progenitors have focused on known antigens using established antibodies.

Markers such as CD133 (Rountree et al., 2007, Sugiyama et al., 2007, Hori et

al., 2008), Cd49f (Suzuki et al., 2000, Sugiyama et al., 2007) and E-cadherin

(Jiang et al., 2002, Ueberham et al., 2007) are common to both hepatic and

pancreatic progenitor cells. An approach that has been particularly useful in

developing new antibodies for progenitor cell isolation is to raise antibodies

against cell types of interest rather than known antigens. As opposed to

targeting antibodies against known proteins or peptides, this approach

generates antibodies against undefined cell surface proteins. Because

relatively few tools exist for antibody based sorting of liver/pancreas


75

progenitors, more recent studies have focused on developing reagents which

are useful for antibody-based enrichment of cells induced by chronic liver

injury (Dorrell et al., 2008) and adult mouse pancreas cell subsets (Dorrell et

al., 2011b). Using a similar approach, we have also described a panel of

antibodies generated against developing liver and pancreas cells which mark

progenitor cell domains from these organs (Chapter 2). This has led to the

development of two antibodies, WAM18 and WAM21, which recognise liver

progenitor cell lines and the ductular reaction induced by two unique chronic

liver injury diets (Chapter 2). We have also shown WAM21-based cell sorting

effectively enriches Ngn3-expressing islet precursors from foetal pancreas,

which can be differentiated to insulin producing cells in vitro using defined

conditions. This system represents a convenient in vitro model for studying

beta cell development (Chapter 3). Critically, the antigens recognised by

WAM18 and WAM21 remain stable following enzyme-mediated tissue

dissociation protocols making them ideal tools for isolating progenitor cells

from tissue isolates, a property not described for the ‘gold standard’ liver

progenitor cell surface marker A6 (Faktor et al., 1990, Factor et al., 1994)

Although our newly developed WAM antibodies have demonstrated value in

cell isolation and histological identification of hepatic and pancreatic

progenitors, the determinants they recognise remain unknown. Knowledge of

the antigens they mark would enable functional and translational studies.

Accordingly, we have undertaken experiments in this study to establish the

identity of WAM18 and WAM21, facilitating functional studies in a mouse

liver progenitor cell line. Furthermore, we describe the use of a commercially

available antibody recognising the equivalent human WAM18/WAM21

protein to purify putative islet progenitor cells from the developing human

pancreas.


76



Cryosections (7 µm) of OCT-embedded e15.5 foetal liver tissue were cut and

fixed in acetone (-20 °C, 5 min), air-dried and stored at -20 °C until use.

Sections were rehydrated in PBS for 5 min and endogenous biotin was blocked

using a biotin blocking kit (Dako, Denmark). Non-specific binding was

blocked by incubation in blocking buffer (5% FBS diluted in PBS) for 1 h at

RT. Labelling of primary antibodies was performed overnight at 4 °C.

Antibodies used were WAM18 or rat IgG2a isotype control. Primary rat

antibodies were detected with FITC-conjugated goat anti rat IgG (1:200,

Vector). After secondary antibody labelling, sections were incubated with rat

immunoglobulin for 1 h at RT to block non-specific binding of biotinylated

primary rat antibodies to anti-rat secondary antibody. Biotinylated primary

antibodies were incubated for 2 h at RT. Antibodies used were biotin-

conjugated WAM21 or IgG2a which were detected by incubation with

Streptavidin-Cy3 (1:300, Amersham). Slides were mounted with ProLong

Gold Antifade mounting medium containing the nuclear stain DAPI

(Invitrogen) and visualised by fluorescence microscopy. Single colour

fluorescent microscope images were merged using ImageJ version 1.39u

software for Windows (ImageJ Software, National Institutes of Health, USA,

http://rsb.info.nih.gov/ij/).

4.3.2 Mammalian cell culture

COS-7 cells were maintained in high glucose DMEM (Invitrogen)

supplemented with glutamine, penicillin/streptomycin (Gibco-BRL) and 10%

FBS as previously described (Gluzman, 1981). NIT-1 insulinoma cells were

maintained in high glucose DMEM supplemented with glutamine,

penicillin/streptomycin, 0.02% bovine serum albumin (BSA) and 10% FBS as

previously described (Hamaguchi et al., 1991). Bipotential Murine Oval Liver

(BMOL) cells were maintained as previously described (Tirnitz-Parker et al.,

2007). All cells were grown in a 37 ºC incubator with 90% humidity. COS-7



77

and NIT-1 cells were maintained with a 10% CO2 atmosphere. BMOL cells

were maintained in a 5% CO2 atmosphere.

4.3.3 Expression cloning cDNA clones encoding WAM antigens

Superscript® Mouse 13.5 Day Embryos cDNA was prepared according to

manufacturer instructions (Invitrogen, Australia; cat. 10666-014). Library

DNA was transfected into COS-7 cells using Lipofectamine 2000 (Invitrogen,

Australia). For expression cloning of WAM antigens from cDNA library

plasmids, WAM18/21+ cells were selected by FACS 72 h post-transfection, re-

plated and allowed to grow to confluence for 15 days to allow for stable

integration of plasmids. After this period, cells were trypsinised and sorted

with WAM18 and WAM21 a second time. WAM+ stably transfected cells were

regrown to confluence prior to DNA extraction.

4.3.4 DNA purification and amplification of cloned sequences

WAM18+/WAM21+ COS-7 cells were lysed and gDNA purified using the

QIAamp DNA Mini Kit according the manufacturers’ instructions (Qiagen).

100 ng of DNA was used for subsequent PCR amplification. Stably

incorporated e13.5 cDNA library sequences were amplified using Platinum

Taq DNA polymerase for 40 cycles, according to manufacturer instructions.

Commercial primers specific for T7- and Sp6-RNA polymerase sequences

flanking cDNA inserts were used (Promega, Australia). Prepared e13.5 cDNA

library plasmid DNA was used as a positive control and adult mouse liver

DNA was used as a negative control. PCR products were resolved by

electrophoresis through a 1.5% agarose gel and visualised by ethidium

bromide staining under ultraviolet light.

Specific PCR products were excised from the gel and purified using the

QIAquick Gel Extraction Kit according to the manufacturers’ instructions

(Qiagen, USA). Products were sequenced using labelled dideoxy-nucleotides

by the Australian Genome Research Facility (AGRF, Perth, Western

Australia) with Sp6 primers. Derived sequences were compared against


78

known sequences using the Basic Local Alignment Search Tool (BLAST)

algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences alignments

were generated using the Clustal Omega Multiple Sequence Alignment

Program (http://www.ebi.ac.uk/Tools/msa/clustalo/) and edited using the

Jalview program available at http://www.jalview.org/ (Waterhouse et al.,

2009).

4.3.5 Foetal tissue isolation

Mouse embryos at e15.5 were removed from the uterus and dissected free of

their placenta and placed in a petri dish containing cold PBS prior to

dissection of pancreata, liver, brain or small intestine under sterile

conditions. Tissues were stored in medium (high glucose DMEM (Invitrogen)

with 10% FBS) until processing commenced. Tissues were incubated in 600

µL medium and 300 µL of Dispase (BD Biosciences) for 1 h at 37 °C with

shaking at 400 rpm. The reaction was stopped by the addition of 500 µL of

12% BSA in PBS. Cells were dispersed by pipetting and pelleted by

centrifugation at 800 x g for 5 min. Cells were incubated in 500 µL medium

containing 1 µL DNase (Promega) for 10 min at room temperature. 1 mL of

medium was added and the cells were centrifuged at 800 x g for 5 min at RT.

Pelleted cells were resuspended in 10 mL medium and stored on ice until use.

Human foetal pancreata (14 to 18 weeks old) were dissociated by fine mincing

with a scalpel blade and incubating with 500 µL Liberase TL (2.5 mg/mL;

Roche, Australia) for 12 min at 37 °C. The reaction was stopped by the

addition of 9 mL of neutralising buffer (HBSS with 10% FBS, 10 mM HEPES,

10 µg/mL DNase I). Cells were centrifuged at 300 X g for 5 min at RT, washed

twice in neutralising buffer and filtered through a 70 µm cell strainer prior to

antibody labelling. Human pancreata were prepared by Dr Erika Bosio and

Emma Jamieson at the Centre for Diabetes Research, Western Australian

Institute for Medical Research. Animal experiments were performed

according to the Australian Code of Practice for the care and use of scientific

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://www.ebi.ac.uk/Tools/msa/clustalo/

http://www.jalview.org/


79

animals and ethics approval from the Animal Resources Centre, Perth.

Human tissue was acquired with patient consent from King Edward

Memorial Hospital (Perth, Western Australia) under ethics approval from the

University of Western Australia.

4.3.6 Flow cytometry

Cells were resuspended in FACS Buffer (PBS/ 2% FBS) and stained with

primary antibodies. Antibodies used were rat IgG2a WAM21-AlexaFluor-647,

WAM18-biotin, rat M1/69 antibody (unconjugated; 1/100, eBioscience) rat

IgG2a isotype control-AlexaFluor 647 (eBioscience), rat IgG2a WAM21-

biotin, rat anti CD49f-PE (clone GoH3, BD Biosciences). Cells were washed

and resuspended in FACS buffer containing DAPI (1 µg/mL) and analysed on

a FACSAriaII flow cytometer (BD Biosciences). FACS plots were generated

using FlowJo version 7.6 (TreeStar).

4.3.7 Treatment of COS-7 cells with glycosylation inhibitors

COS-7 cells overexpressing mouse Cd24a (pCMV-Cd24a; Origene, USA) were

trypsinised and split into 6-well plates 24 h post-transfection. Growth

medium was changed after 16 h and cells were treated with glycosylation

inhibitors according to previously published protocols (Sohail et al., 2011).

Tunicamycin (5 µg/mL; Sigma-Aldrich) and/or Benzyl 2-acetamido-2-deoxy-α-

D-galactopyranoside (BGalNAc, 2 mM; Sigma-Aldrich). Control cells were

treated with DMSO (final concentration 0.2%). Cells were incubated in

glycosylation inhibitors after which time WAM18 and WAM21 staining was

assessed by flow cytometry.

4.3.8 Antibody inhibition assay

Antibody blocking experiments were carried at similarly to previously

described protocols (Alterman et al., 1990). NIT-1 cells were incubated with

2.5 µg/mL of unconjugated blocking antibodies for 30 min. After washing in

FACS buffer, cells were incubated in 2.5 µg/mL Alexa647-conjugated rat

IgG2a isotype control, WAM18 or WAM21 for 30 min. Cells were washed and

resuspended in FACS wash buffer containing 1 µg/mL DAPI. The median


80

fluorescence intensity (MFI) was assessed and specific inhibition calculated

using the following equation:

% 𝑺𝒑𝒆𝒄𝒊𝒇𝒊𝒄 𝒊𝒏𝒉𝒊𝒃𝒊𝒕𝒊𝒐𝒏 =𝑪𝒐𝒏𝒕𝒓𝒐𝒍 𝒃𝒍𝒐𝒄𝒌 𝑴𝑭𝑰−𝑨𝒏𝒕𝒊𝒃𝒐𝒅𝒚 𝒃𝒍𝒐𝒄𝒌 𝑴𝑭𝑰

𝑪𝒐𝒏𝒕𝒓𝒐𝒍 𝒃𝒍𝒐𝒄𝒌 𝑴𝑭𝑰𝑿 𝟏𝟎𝟎.

4.3.9 Protein isolation, quantitation and Western blotting

Snap-frozen mouse thymus was pulverised using a mortar and pestle and

resuspended in a lysis buffer containing Tris-HCl (20 mM, pH 8.0), 137 mM

NaCl, 2 mM EDTA, 10% (v/v) glycerol, 1% (v/v) IGEPAL® CA-630 with a

protease inhibitor cocktail (Promega, Australia). Lysates were freeze-thawed

in liquid nitrogen three times, dispersed by pipetting and insoluble material

removed by centrifugation at 16,000 X g at 4 °C for 15 min. Protein

concentration was determined by Bio-Rad assay (Bio-Rad) against a BSA

standard curve. For one-dimensional western blotting, 20 µg of protein was

separated on a 12% SDS-polyacrylamide gel in running buffer containing SDS

and β-mercaptoethanol. For non-denaturing conditions, SDS and β-

mercaptoethanol were excluded. For two-dimensional Western blotting,

proteins were first separated by isoelectric focussing then separated by SDS-

PAGE and transferred to a PVDF membrane. Two-dimensional Western

blotting analysis was carried out by Dr Nadine Dudek at the University of

Melbourne, Australia.

Blotted proteins were washed in PBS/0.1%-Tween (PBS-T), blocked in 5%

non-fat skim milk diluted in PBS-T for 1 h at RT. Primary rat antibodies were

diluted 1:2000 in blocking buffer and incubated overnight at 4 °C. Following

overnight incubation, membranes were washed once in PBS-T for 15 min,

then three times for 5 min in PBS-T. Membranes were incubated with HRP-

conjugated goat anti-rat IgG (1:5000, Abcam) for one hour at room

temperature, washed once in PBS-T for 15 min, then three times for 5 min.

Antibodies were detected with ECL plus reagent according to the


81

manufacturers’ instructions (Amersham) and visualised by film exposure

(Kodak, Australia).

4.3.10 RNA interference in BMOL cells

BMOL cells were seeded at 105 cells per well in a 6 well plate in growth

medium and allowed to adhere overnight. The following day, medium was

aspirated and 1 mL of OptiMEM medium (Invitrogen) containing 2% FBS

without antibiotics was added to the cells. Plasmid shRNA constructs

(Origene USA) were transfected using Lipofectamine 2000 according to the

manufacturers’ instructions (Invitrogen, Australia). 24 hours after

transfection, cells were trypsinised and split 1:5 into 3 wells of a 12 well plate

in oval cell growth medium to generate 3 pools of transfected cells. After an

additional 24 hours, medium was changed to oval cell growth medium

containing Puromycin (1 µg/mL; Sigma Aldrich, Australia) for selection of

stable transfectants. Cells were cultured in selection medium for 15 days after

which time 2-3 colonies remained in each well. Cells in individual wells were

expanded, mRNA assessed by qPCR to confirm gene knockdown and used for

cell growth studies (Appendix figure A3.1).

4.3.11 Assessment of cell growth

BMOL cell growth was measured using the Cellavista™ system (Roche). Prior

to performing experiments, a standard curve was generated to correlate cell

numbers to measured well confluence (Appendix figure A3.2). To further

validate the Cellavista™ system, Cellavista™ measurements were directly

compared to the MTT assay (Appendix figure A3.2) which is commonly used

to measure cell proliferation (Mosmann, 1983). BMOL cells were seeded at a

density of 2000 cells per well in a 96 well plate in quadruplicate and incubated

overnight to adhere. Plates were read in the Cellavista™ system twice daily

for 6 days. Population doubling times were calculated in the log growth phase

according to previously published protocols (Viebahn et al., 2006). Growth

curves generated using Cellavista™ were fitted to an exponential curve of the


82

equation y=a egx, R2>0.96 (a: constant; g: growth rate, R2: correlation

coefficient). Doubling time was defined as: doubling time=ln(2)/g and was

calculated using GraphPad Prism (GraphPad Software, San Diego California

USA, www.graphpad.com).

4.3.12 Analysis of gene expression




Superscript III Reverse Transcriptase (Invitrogen). For semi-quantitative

analysis of gene expression, RT-PCR amplification of cDNA preparations for

35 cycles using gene specific primers with Platinum Taq. DNA polymerase

(Invitrogen) was carried out. All protocols were performed according to

standard manufacturer instructions. Primer sequences and annealing

temperatures are listed in Appendix table A3.1. RT-PCR products were

resolved by electrophoresis through a 1.5% agarose gel containing ethidium

bromide and visualised by ultraviolet light illumination.

4.3.13 Statistical analysis

Plotted data represent the mean ± SEM of three experiments. One way

analysis of variance (ANOVA) with Tukey’s multiple comparison test, or

paired t-test was performed using GraphPad Prism version 6.00 for Windows

(GraphPad Software, San Diego California USA, www.graphpad.com). Values

were considered significantly different when p<0.05.

http://www.graphpad.com/


83

4.4. RESULTS

4.4.1 Expression cloning cDNA encoding WAM antigens

We had previously shown that WAM18 and WAM21 are expressed at the cell

surface of liver (Chapter 2) and developing pancreas cells (Chapter 3),

permitting antibody-based enrichment of live cell populations. Accordingly,

we applied this property of our antibodies to expression clone potential

cDNA’s from a mixed cDNA library encoding WAM18 and WAM21 antigens.

To expedite this process, we utilised a commercially available e13.5 whole

mouse cDNA library. Prior to our expression cloning experiments, we

conducted immunofluorescence staining on e13.5 liver and found that both

WAM18 and WAM21 identified similar populations of e13.5 liver cells (Figure

4.1A). Next, we overexpressed cDNA’s with flanking T7 and Sp6 sequences

from a commercially available e13.5 whole mouse cDNA library in COS-7 cells

and selected cells on the basis of WAM18 and WAM21 expression by FACS.

To increase the initial pool of WAM-selected cells, we sorted with WAM18 and

WAM21 antibodies at the same time. Seventy-two hours after cDNA library

transfection, 3.1% of COS-7 cells were WAM18/21+ compared to isotype

control staining (Figure 4.1B). These cells were purified, replated and

expanded for 15 days to allow for stable integration of plasmid DNA. At the

end of this period, 0.1% of cells were WAM18/21+ (Figure 4.1C). These cells

were replated and expanded for DNA analysis of incorporated plasmids.


84

Figure 4.1. Expression cloning WAM18 and WAM21 antigens from a

cDNA library. (A) Expression of WAM18 (green) and WAM21 (red) at e13.5

in mouse development was confirmed by immunofluorescent staining of e13.5

frozen liver sections. Merged image shows both antibodies recognise similar

cell populations. Nuclei were counterstained with DAPI (blue). Scale bars

represent 50 µm. An e13.5 cDNA library was overexpressed in COS-7 cells

and WAM18/21+ cells were purified (B) 72 h post-transfection. These cells

were replated and expanded for 15 days to allow stable incorporation of

plasmids. (C) WAM18/21+ stably-transfected cells were re-purified. All

staining was compared to rat IgG2a isotype control staining (control).

A

B

C


85

4.4.2 Establishing the identity of WAM18 and WAM21

After expansion and passage into two separate wells (‘pool 1’ and ‘pool 2’),

cells were trypsinised and stained with either WAM18 or WAM21 antibodies

prior to DNA extraction. WAM18 and WAM21 identified almost all cells

compared to isotype control staining (Figure 4.2A). Genomic DNA was

extracted from these cells, and a PCR was carried out to amplify stably

transfected library DNA using T7 and Sp6 primers (which flanked cDNA

inserts). Library plasmid DNA and normal mouse liver were used as positive

and negative controls respectively. PCR amplification of e13.5 cDNA library

DNA displayed multiple products shown by a ‘smear’ by agarose gel

separation. A 2 kB insert was amplified from pool 1 and pool 2 DNA, which

was not present in control liver DNA. (Figure 4.2B). These fragments were

excised from the gel, purified and dideoxy-sequenced using Sp6 primer. A

sequence which was 800 bp long was derived. A BLAST search revealed the

identity of the cDNA encoding the WAM18 and WAM21 antigens as Cd24a.

A comparison of the sequenced fragment and Cd24a demonstrated 100%

identity between the two sequences (Figure 4.3).

To confirm this result, we amplified DNA using primers specific for muCd24a

cDNA from the 2 kB T7/Sp6 PCR product from stably transfected COS-7 cells

(Figure 4.4A). Additionally, we overexpressed a pCMV6-Cd24a expression

cassette in COS-7 cells to confirm that WAM18 and WAM21 stained CD24

surface protein. A CD24 antibody (M1/69) was used as a positive control for

CD24 protein expression. None of the antibodies assessed identified

untransfected COS-7 cells. Cd24a-overexpressing COS-7 cells were M1/69+,

WAM18+ and WAM21+ (Figure 4.4B). Altogether, these data confirmed the

identity of the gene encoding WAM18 and WAM21 antigens is mouse Cd24a.


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Figure 4.2. Isolation of a cDNA encoding WAM18 and WAM21. (A)

Immunofluorescent staining of live cell populations of WAM-purified COS-7

cells immediately prior to DNA extraction shows WAM18 and WAM21

antigens are present on virtually all cells. DNA was extracted from these cells

and inserts amplified using flanking T7 and Sp6 primer sequences. (B)

Agarose gel electrophoresis and ethidium bromide staining of PCR using

flanking primers shows a unique 2 kB fragment present in WAM18/21+ COS-

7 cells which was extracted and purified from the agarose gel for further

analysis (red box). Gel lanes are molecular weight marker (M), WAM18/21+

COS-7 cell DNA from pool 1 (1) and pool 2 (2), e13.5 mouse cDNA library (L),

normal liver control (N) and water control (C).

A B


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Figure 4.3. Sequence alignment of Sp6-sequenced insert and the

muCd24a cDNA. Gel purified 2 kB insert amplified with T7 and Sp6 primers from

WAM18/21+ was sequenced using Sp6 primers and di-deoxy nucleotides. The

derived sequence was compared to the mouse Cd24a gene. Sequence alignment

shows 100% identity between our WAM antibody-cloned product and muCd24a from

bases 3 to 800 of Sp6-primed sequencing reaction.


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Figure 4.4. Confirmation of WAM18 and WAM21 identity. (A) PCR using

Cd24a-specific primers was carried out on T7/Sp6-amplified fragment from

WAM18/21-selected COS-7 cells transfected with e13.5 whole mouse cDNA library.

Lanes are molecular weight marker (M), pool 1 (1), pool 2 (2), cDNA library (L) and

no template control (C). To confirm WAM18 and WAM21 identity, Cd24a was

overexpressed in COS-7 cells. (B) WAM18 and WAM21 mark pCMV-Cd24a-

transfected (black), but not non-transfected COS-7 cells (red). CD24 antibody M1/69

was used as a positive control. Rat isotype control antibody did mark transfected or

non-transfected cells.

A

B


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4.4.3 Biochemical characterisation of WAM18 and WAM21

Although WAM18 and WAM21 both recognise CD24 proteins, we had

previously observed heterogeneity in the cells recognised by these antibodies

in developing mouse pancreas and DDC-induced liver injury. Hence, we

carried out studies to further clarify WAM-antibody epitope specificity. To

determine whether WAM18 and WAM21 mark identical CD24 epitopes, we

assessed the capacity of WAM18 and WAM21 to block the binding of one

another in NIT-1 insulinoma cells. The blocking ability of the M1/69 antibody

was also tested. Mean fluorescence intensity (MFI) was reduced when

antibodies were self’-blocked. M1/69 was able to block WAM18 binding and

partially blocked WAM21 binding. However, when the ability of WAM18 to

block WAM21, and vice-versa, was assessed, little change was observed in

MFI of detection antibody staining suggesting that our WAM antibodies

recognise distinct CD24 epitopes. (Figure 4.5A).

Immunoblotting of e15.5 liver and brain protein lysates revealed tissue-

specific differences in protein sizes recognised by our antibodies. Proteins

detected following denaturing or non-denaturing SDS-PAGE were of similar

size, though bands were more intense under denaturing conditions. In liver,

WAM21 appeared to mark a group of proteins whose sizes ranged from 30-37

kDa. Three faint WAM18+ bands could be visualised under denaturing

conditions of comparable size in foetal liver. Interestingly, WAM18 and

WAM21 appeared to differ in developing brain extracts. WAM21 marked a

protein of approximately 32 kDa, whilst an additional smaller band of

approximately 28 kDa was observed when WAM18 was used to probe the

membrane (Figure 4.5B). We also assessed staining in adult thymus.

Separation of proteins by size and isoelectric point revealed that WAM18

marked two proteins at 30 kDa and 60 kDa, whilst WAM21 preferentially

identified the 30 kDa protein (Figure 4.5C).


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Figure 4.5. WAM18 and WAM21 identify unique CD24 epitopes. (A) The

ability of CD24 antibodies to block each other was assessed by flow cytometry. NIT-

1 cells were pre-treated with isotype control or CD24 antibodies, then WAM antibody

staining was assessed. Antibody specific inhibition was calculated by comparing

antibody-specific blocking (‘antibody block’; blue) to isotype antibody blocking

(‘control block’; red) and expressed as a percentage. WAM antibodies show

differential blocking capacity. (B) Western blot analysis of WAM antibodies in e15.5

liver (L) and brain (B). Protein lysates were separated by non-denaturing (ND) or

denaturing (D) SDS-PAGE. (C) Two-dimensional Western blot analysis of WAM18

and WAM21 in mouse thymus. Red arrows denote different spots (red boxes)

identified by WAM antibodies.

A

B C


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The CD24 protein has been previously shown to be a heavily glycosylated

molecule, containing multiple sites for N-linked and O-linked glycosylation

(Ohl et al., 2003, Bleckmann et al., 2009). Since our antibodies marked

distinct epitopes as characterised by antibody blocking and Western blotting

experiments, we examined whether this distinction could be glycosylation

dependent. We assayed WAM staining by flow cytometry following treatment

of Cd24a-overexpressing COS-7 cells with inhibitors of N-linked

(tunicamycin) or O-linked (BGalNAc) glycosylation. Treatment of COS-7 cells

with tunicamycin significantly reduced cell surface binding of WAM18 and

WAM21 compared to treatment with DMSO vehicle. Inhibition of O-linked

glycosylation with BGalNAc did not affect WAM18 or WAM21 binding to the

cell surface (Figure 4.6). These data suggest that the size and specificity of

WAM18 and WAM21 on the cell surface may be due to N-linked glycosylation.


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Figure 4.6. Effects of glycosylation inhibitors on WAM18 and WAM21

antibody surface labelling. COS-7 cells were transfected with pCMV-Cd24a

and treated with glycosylation inhibitor of N-linked (Tunicamycin) and/or O-linked

(BGalNAc) glycosylation for 72 h. (A) Flow cytometric analysis of WAM antibody

staining on inhibitor-treated cells (blue) versus DMSO vehicle control (red). WAM

staining was compared to isotype control (black). (B) Quantitation of glycosylation

inhibitor effects on surface labelling of WAM18 and WAM21; Bars = mean ± SEM;

n=3; *p<0.05, **p<0.01 compared to vehicle (DMSO).

A

B


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4.4.4 Utility of WAM18 and WAM21 for investigating organ-specific

CD24 biology

Although differences in CD24 expression have been noted in the brain,

cellular subpopulations with respect to multiple CD24 antibody staining have

not been assessed. Brain, pancreas, liver and intestine from e15.5 Ngn3-

GFP/RIP-dsRed mice were stained with WAM18 and WAM21 antibodies and

cell populations identified by these antibodies were compared by flow

cytometric analysis. WAM18 identified virtually all cells in all tissue types

assessed. Similarly, WAM21 also identified WAM18+ cells in foetal liver and

intestine, but marked a subset of WAM18+ cells in foetal pancreas and brain.

In contrast, WAM18 and WAM21 did not identify distinct populations of cells

in foetal liver or intestine (Figure 4.7A). Since we had previously shown

specificity of WAM21 for Ngn3-expressing islet progenitors (Chapter 3), we

assessed the dual specificity of WAM18 and WAM21 for Ngn3-GFP+

neuroendocrine cells in developing pancreas, intestine and brain by flow

cytometry. The Ngn3-GFP+ cell subset was gated and cellular distribution

with respect to WAM18 and WAM21 immunoreactivity assessed. Ngn3-GFP+

cells in the developing intestine and pancreas were enriched in the

WAM18+/WAM21+ cellular subset. However, most Ngn3-GFP+ cells in the

developing brain were WAM18+/WAM21- (Figure 4.7B).

Having identified the cDNA encoding our WAM antibodies as Cd24a, we used

this information to carry out functional and translational studies. Firstly,

since WAM18 and WAM21 mark LPCs, we assessed whether knockdown of

Cd24a mRNA could modify the growth of an LPC cell line BMOL. Secondly,

since WAM18 and WAM21 mark mouse islet progenitor cells in developing

pancreas, we assessed the suitability of an antibody against human CD24 to

isolate islet progenitor cells from developing human pancreas.


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Figure 4.7. WAM18 and WAM21 identify unique cell populations in

e15.5 Ngn3-GFP/RIP-dsRed mouse neuroendocrine tissues. WAM18

and WAM21 staining was compared in developing mouse tissues. (A) WAM18 and

WAM21 identify virtually identical populations of e15.5 liver and intestine. WAM21

marks a subpopulation of WAM18+ developing brain and pancreas. (B) Distribution

of WAM+ cells was assessed in Ngn3-GFP+ neuroendocrine cells in e15.5 intestine,

brain and pancreas. Intestinal and pancreatic Ngn3-GFP+ cells are

WAM18+/WAM21+. Most brain Ngn3-GFP+ cells are WAM18+/WAM21-.

B

A


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4.4.5 Inhibition of Cd24a mRNA expression alters LPC growth

Since WAM18 and WAM21 mark LPCs (Chapter 2) and we had shown they

recognise CD24 protein, we investigated whether modulation of Cd24a

expression could affect LPC biology. Specifically, we assessed whether RNAi

of Cd24a could affect the cell growth properties of a liver progenitor cell line

BMOL using the novel Cellavista™ system. Initially, to assess the precision

of the Cellavista™ system for measuring cell growth, we compared

measurements of a standard curve of BMOL cells at varying densities to that

of the routinely used MTT assay on the same set of cells. We found a high

degree of correlation between cell numbers and absorbance readings derived

using the MTT assay (R2>0.97) as well as cell numbers and cell density

measurements generated by Cellavista™ in untransfected BMOL cells, or

BMOL cells transfected with scrambled or Cd24a-specific shRNA constructs

(R2>0.98; Appendix figure A3.2).

To determine whether inhibition of Cd24a mRNA expression could affect the

growth of BMOL cells, we assessed cell growth over 142 h in non-transfected

(‘Parental’), and stably-transfected scrambled control shRNA (‘shScram’) or

Cd24a-targeting shRNA (shCd24a) plasmids using the Cellavista™ system.

Visual assessment of well confluence after 78 h showed that Parental and

shScram BMOL cells were actively expanding, whilst shCd24a BMOL’s did

not appear to have proliferated significantly (Figure 4.8A). Growth curves

generated over 142 h showed that proliferation of shCd24-transfected cells

was significantly decreased compared to both Parental and scrambled control

shRNA transfected cells (Figure 4.8B). We achieved similar results using an

MTT assay (Appendix figure A3.1B). Using the logarithmic growth phase of

each cell condition, we calculated the population doubling time. Doubling

time was significantly increased in shCd24a-transfected cells (76±13.4 h)

compared to shScram (38±1.7 h) and Parental BMOL cells (29±1.6 h; Figure

4.8C).


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Figure 4.8. Effects of stable Cd24a knockdown on liver progenitor cell

proliferation. Cell proliferation in BMOL cells that were untransfected

(parental), or stably transfected with plasmids harbouring short hairpin

sequences that were targeted against Cd24a (shCd24a) or a non-specific

scrambled control sequence (shScram) was measured using Cellavista.(A)

Thresholded microscope images used by Cellavista software for measuring

cell confluence. (B) Cell confluence measurements over time in BMOL-T cells

transfected with shRNA plasmid constructs. (C) Calculated cell doubling time

of BMOL-T cells transfected with different shRNA plasmid constructs. Data

represent mean ± SEM, n=3 Statistical significance of one way ANOVA with

Tukey’s post-test is represented as *p<0.05 or not statistically significant (ns).

A

B C


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4.4.6 Human foetal islet progenitors are defined by CD24 expression

In Chapter 3, we showed that WAM21 could be used to significantly enrich

mouse beta cell progenitors capable of differentiating into insulin reporter-

positive beta-like cells. Flow cytometric assessment of a human liver cell line

(BHAL; Zhang et al., 2010), and a human embryonic stem cell line (performed

by Erika Bosio, Centre for Diabetes Research), demonstrated that our

antibodies did not recognise an epitope on human cells. Hence, for studies on

developing pancreas, we used a commercially available antibody recognising

the human CD24 protein.

Since human islet progenitors had previously been shown to be enriched in a

CD133+/CD49Flow population (Sugiyama et al., 2007), we carried out dual

immunolabelling with these markers and CD24 to determine whether our

novel marker is redundant. FACS analysis of double antibody-labelled human

foetal pancreas cell isolates demonstrated that CD24 marks unique

subpopulations of CD49Flow and CD133+ cells (Figure 4.9A) demonstrating

uniqueness of the populations recognised by CD24 compared to previously

described antibodies. We then assessed key markers of islet cell development

in CD24-defined populations of developing human pancreas. CD24+ cells

comprised a small subset of total foetal pancreas cells in two separate

pancreata, recognising 2% and 5% of cells respectively. CD24-defined

populations were purified by FACS and analysis of gene expression in these

populations by RT-PCR showed that PDX1 and SOX9 mRNA expression were

enriched in CD24+ populations. CD24+ populations contained virtually all of

the cells expressing the islet progenitor transcripts NGN3, MYT1 and PAX4.

Insulin-expression cells were enriched in the CD24+ cell population. Lower

levels of Insulin transcripts were detectable in CD24- cells (Figure 4.9B).


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Figure 4.9. Human foetal islet progenitors are CD24-positive. (A)

Comparison of CD24 staining of dispersed foetal human pancreas cells with

established islet progenitor markers CD133 and CD49F demonstrates uniqueness of

cellular populations recognised by CD24. (B) RT-PCR analysis of pancreatic/islet

progenitor gene expression in CD24-defined populations of human foetal pancreas

shows markers of islet progenitors are enriched in CD24+ cells in two separate

experiments.

A

B


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4.5 DISCUSSION

The generation of new antibodies recognising surface proteins is an important

step in defining the phenotype of progenitor cell domains in the liver and

pancreas in a way that permits their isolation. Consequently, recent research

efforts have focussed on this goal leading to the development of novel

antibodies against LPC subsets (Dorrell et al., 2008, Dorrell et al., 2011a) and

pancreatic endocrine, acinar and ductal lineages (Dorrell et al., 2011b).

Likewise, we have developed two novel monoclonal antibodies against liver

and pancreatic progenitors, WAM18 and WAM21, which we have shown

previously to identify liver and pancreatic beta cell progenitors. As these

antibodies bind hitherto undefined antigens their application is limited to a

tool for the isolation of mouse progenitor cells. To overcome this limitation,

we used an expression cloning approach to show that the antigens recognised

by WAM18 and WAM21 are encoded by the mouse Cd24a gene. Using this

information, we have extended our studies of liver and pancreas progenitors

to assess the role of Cd24a in LPC growth, and to establish the suitability of

CD24 as a marker of human foetal islet progenitors.

The mouse CD24 protein is a small, 30 amino-acid protein (Kay et al., 1990)

for which a number of monoclonal antibodies have been developed and mark

a variety of proteins ranging from 28 to 55 kDa in developing mouse brain

(Shirasawa et al., 1993), adult thymus (Rougon et al., 1991) and

hematopoietic lineages (Kay et al., 1991, Hubbe and Altevogt, 1994). This

difference in size is due to variations in N-linked and O-linked glycosylation

which has been observed most notably in the mouse brain (Ohl et al., 2003,

Bleckmann et al., 2009). Although these differences have been shown by

Western blotting using the M1/69 antibody, which marks the core CD24

peptide (Alterman et al., 1990), the antibodies to determine whether these

differences exist in different cell lineages have not been available since

established CD24-specific monoclonal antibodies M1/69, J11d and B2A2


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mark an identical antigenic determinant (Alterman et al., 1990). Similarly,

our antibody blocking studies suggest that WAM18 marks a similar epitope

to M1/69. We had previously shown that WAM21 marked a unique

populations of A6- and CD45+ cells in chronic liver injury which is not

observed with ductal cells marked by WAM18 (Chapter 2), or the M1/69

antibody in DDC-induced injury (Ochsner et al., 2007). Additionally, we have

observed that WAM21 marks a subset of e15.5 foetal mouse pancreas cells

containing all beta cell progenitors (Chapter 3). In the current study, we have

shown for the first time using dual antibody staining and flow cytometric

analysis that CD24 heterogeneity is cell type-specific in developing brain and

pancreas. Although different CD24 protein species have been known to exist,

the development of WAM21 will enable more detailed studies of the role of

CD24 cell populations of the foetal brain and pancreas, since cell subtypes of

these organs can now be separated based on differential expression of CD24

antigens defined by WAM18 and WAM21.

Although CD24 has been shown to mark ductal cells in normal (Qiu et al.,

2011) and DDC-injured livers (Ochsner et al., 2007), its biological function in

LPCs is yet to be probed. We had previously shown that WAM18 and WAM21

are expressed in virtually all LPC lines and on A6+ cells induced in mouse

models of chronic liver injury (Chapter 2). These observations suggest an

important biological function for CD24 in regulating the progenitor cell

response to liver injury. In the current study, we have shown reduced

proliferation in BMOL cells following Cd24a knockdown using a stably

expressed shRNA vector, providing evidence of a role for Cd24a in regulating

LPC proliferation. Current research suggests that CD24 may be a potential

target for anticancer therapies in liver tumours since it has been associated

with poor prognosis in HCC (Yang et al., 2009) and cholangiocarcinomas in

humans (Su et al., 2006, Agrawal et al., 2007), has been functionally

associated with tumour characteristics such as invasion, migration and


101

adhesion in HCC cells (Zheng et al., 2011) and promotes HCC establishment

via regulation of Nanog and STAT activation (Lee et al., 2011). Since LPCs

are implicated as a potential tumorigenic source in hepatocellular carcinoma

(HCC) associated with chronic liver injury, further studies on the role of CD24

in LPCs may provide valuable insight for hepatocellular carcinomas derived

from progenitor cells during chronic liver injury.

To date, CD24 expression has not been examined in developing human

pancreas. Currently, the only indication of CD24 as a marker of pancreatic

epithelium in human cells is where it has been used as a selection marker for

PDX1+ cells differentiated from human embryonic stem (hES) cell lines (Jiang

et al., 2011). However, a more recent study found that CD24 protein was

expressed at all stages of mouse and hES pancreatic differentiation in vitro

(Naujok and Lenzen, 2012). Although the utility of CD24-based purification

of committed pancreatic precursors in cell line studies is disputed, we have

shown for the first time that CD24-based cell sorting can be used to enrich

potential islet progenitor cells expressing NGN3, PDX-1 and SOX9, from

primary human foetal pancreas preparations. Expression data from this

study, and previously generated by assessment of WAM21-defined developing

mouse pancreas (Chapter 3), are consistent with the notion that CD24 marks

PDX1-expressing epithelium. Examination of the dynamics of CD24

expression throughout pancreatic development in human and mouse tissue,

particularly with respect to the different antigens marked by WAM18 and

WAM21 in the mouse, would provide further insight into the role of this

protein in defining the three major epithelial lineages of the pancreas.

Importantly, CD24-based sorting of human foetal pancreas is compatible with

previously described markers CD133 and CD49F (Sugiyama et al., 2007), and

is valuable in defining cells at the NGN3+ islet precursor stage. Since one of

the major hurdles of islet progenitor cell research is the definition of a

definitive islet progenitor cell surface phenotype, this discovery will enable


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further purification and more detailed study of human beta cell precursor

cells.

CD24 is a complex molecule which exhibits multiple functions most likely due

to its potential for multiple glycosylation patterns. Creation of antibodies

recognising different epitopes may give further insight into biology of

different cell types and the role of CD24 in diverse cellular processes. We have

described two such antibodies which will be useful for researchers studying

cellular subtypes defined by variations in CD24 epitopes from pancreas,

brain, bone marrow, liver and thymus. We have shown a role for Cd24a in

LPC expansion, and in doing so provided the first data validating the use of

the CellavistaTM system to non-invasively measure cell growth of LPCs in

vitro. Finally, we have demonstrated that the expression of CD24 on the cell

surface of foetal islet progenitors is conserved between mice and humans and

as will be beneficial for studies of human pancreatic islet development.

CHAPTER 5 GENERAL DISCUSSION

103

Chapter 5

General Discussion


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5.1 General discussion

Replacement of dysfunctional liver and pancreas tissue with exogenous

sources has been a clinical reality since the first successful human

transplants of these organs were performed in 1963 and 1967 respectively

(Starzl et al., 1963, Kelly et al., 1967). In the last two decades, the

establishment of techniques to isolate hepatocytes and islets from cadaveric

tissue has enabled transplantation of cells as an alternative to orthotopic

organ transplants. Hepatocyte transplants have been beneficial for patients

suffering inherited metabolic diseases (Fox et al., 1998) and islet

transplantation is currently the most effective strategy for limiting morbidity

associated with T1DM, as glucose-sensitive insulin release from transplanted

β-cells overcomes the blood glucose fluctuations associated with injectable

insulin (Shapiro et al., 2000). The advent of liver transplantation has enabled

significant extension of life for people with end-stage liver disease and liver

failure. Although these treatments are successful, transplant of liver and

pancreas tissue requires donor tissue. Unfortunately, the current shortage of

donor organs dictates the need for alternative cellular sources for therapy

(Johnson et al., 2014). Stem cells represent a promising and renewable

resource to provide liver and islet cells for transplantation. As protocols to

reprogram somatic cells into pluripotent cells that have the potential to form

any virtually cell type have developed, autologous cell transplants are a

foreseeable reality if certain hurdles can be overcome (Takahashi et al., 2007).

A major problem for liver biologists is the inability to form cells that are

functionally equivalent to primary hepatocytes from pluripotent cell sources

in vitro. Although these cells are extremely valuable for drug screening and

disease modelling purposes, their translation to a clinically viable cell source

is currently limited (Reviewed in Schwartz et al., 2014). Great progress has

been made in deriving insulin producing cells (IPCs) from stem cells

suggesting the goal of a stem cell-derived cell therapy for Type 1 Diabetes

may be within reach. Recently, Pagliuca and colleagues developed a novel

protocol for the differentiation of glucose responsive IPCs from one hES, and

two hiPS cell lines with a differentiation efficiency of approximately 45%.


105

These cells function to a level that is similar to isolated human adult islets

when transplanted into diabetic mice (Pagliuca et al., 2014). Although these

studies represent excellent progress in translating the promise of stem cells

to a clinical therapy, much work remains to improve efficiency of

differentiation and in meeting safety requirements for therapeutic use. Since

gene expression networks are conserved between stem/progenitor cells and

tumours, the safety of stem cell-derived liver or pancreas cells for

transplantation is a concern (Lee et al., 2013). To overcome these problems, a

thorough understanding of liver and pancreas progenitor cell biology is an

imperative for translating the promise of stem cell therapies in the laboratory

to beneficial therapeutic outcomes for patients.

The phenotypic definition of liver and pancreas stem/progenitor cell

compartments remain in its infancy. Currently, the benchmark approach for

defining and isolating stem cells is the method applied in defining

hematopoietic stem cells. Several decades of research have culminated in the

definition of bone marrow-derived cell lineages using monoclonal antibodies

recognising cell surface antigens (Spangrude et al., 1988, Baum et al., 1992).

This has ultimately led to the definition of single cells capable of

reconstituting the entire hematopoietic system (Krause et al., 2001). The liver

progenitor and pancreatic islet progenitor compartments encompass a

heterogeneous subset of cells. To date, the definitive stem/progenitor cell

subtypes of each organ are yet to be defined at the single cell level (Jelnes et

al., 2007, Desgraz and Herrera, 2009). A major limitation to these studies is

the lack of cell surface markers defining liver and pancreas progenitor cell

populations.

The availability of new markers would facilitate the isolation and

characterisation and eventual generation of therapeutically functional cells

from expandable stem cell sources. In light of these limitations, the overall


106

aim of the research described in this thesis was to generate and characterise

monoclonal antibodies that recognise liver and pancreas progenitors.

Since the liver and pancreas share a common endodermal precursor,

transcription factors networks for cell-type development and epithelial cell

surface antigens, new surface markers developed for the liver may be useful

in isolating progenitors from the pancreas and vice-versa. Hence, we

produced a panel of monoclonal antibodies against liver and pancreas cells of

foetal origin in order to characterise progenitor domains of these organs

together (Chapter 2). This approach to generating cell surface antibodies

against heterogeneous cell populations has previously been used successfully

in developing surface antibodies such as the widely used LPC marker, A6

(Faktor et al., 1990) and more recently liver non-parenchymal cells from

chronically injured liver (Dorrell et al., 2008) and adult mouse pancreas

(Dorrell et al., 2011). Prior to the studies described in this thesis, this

approach had not been used with developing liver or pancreas cells as an

immunogenic source. Although antibodies against adult pancreas also mark

developing pancreas populations (Dorrell et al., 2011), we reasoned that the

use of foetal pancreas to produce antibodies would be more successful in

producing antibodies against islet progenitors, since Ngn3+ cells are

transiently present during pancreatic development (Schwitzgebel et al.,

2000). Several antibodies from our panel co-identified developing liver and

pancreas cells, reinforcing the notion that these progenitor domains share a

similar phenotype and supporting our undertaking to study these organs in

parallel (Chapter 2). After a rigorous screening process, two antibodies,

WAM18 and WAM21 were further characterised.

WAM18 and WAM21 displayed a ductal/progenitor phenotype in liver

(Chapter 2), and WAM21 a mixture of ductal/acinar/islet progenitor cells in

developing pancreas (Chapter 3). This is similar to some established

antibodies which display a ductal/progenitor cell specificity and mark

hepatic/pancreatic populations such as CD133 (Hori et al., 2008, Rountree et

https://mail.google.com/mail/u/0/?shva=1#_ENREF_6



107

al., 2007, Sugiyama et al., 2007), Cd49f (Sugiyama et al., 2007, Suzuki et al.,

2000, Tsuchiya et al., 2007) and E-cadherin (Jiang et al., 2002, Ueberham et

al., 2007). Interestingly, none of these antibodies have specifically been raised

against liver or pancreas tissue. For example, EpCAM (G8.8), which has been

used to isolate LPCs (Okabe et al., 2009), was originally developed against

thymus (Farr et al., 1991). The CD133 (13A4) marks liver (Rountree et al.,

2007) and pancreas progenitors (Sugiyama et al., 2007, Hori et al., 2008), and

was raised against mouse embryonic neural tissue (Weigmann et al., 1997).

Cd49f (GoH3), which identifies pancreas progenitors (Sugiyama et al., 2007),

was raised against mammary tissue (Sonnenberg et al., 1986). Likewise, the

M1/69 antibody to CD24, which has been used to purify LPCs (Ochsner et al.,

2007), was generated against spleen cells (Springer et al., 1978). Although

these antibodies have been useful in isolating progenitors from the liver and

pancreas, antibodies against tissue-specific epitopes of these glycoproteins

may provide greater specificity of progenitors of these tissues.

The glycosylation of proteins, including those at the cell surface, have been

shown to be cell type specific (An et al., 2012), and change during cell

differentiation (Satomaa et al., 2009) and neoplastic transformation (Reis et

al., 2010). Therefore, the development of antibodies against cell-type specific

glycoproteins may prove beneficial in developing new tools for cell isolation.

In this study we were able to develop WAM18 and WAM21 which mark

overlapping, but distinct epitopes on the CD24 protein which are

glycosylation-dependent (Chapter 4). Using these antibodies, we were able to

demonstrate novel CD24+ cell types induced by chronic liver injury (Chapter

2) and present in developing pancreas (Chapter 3), developing brain and adult

thymus (Chapter 4). This property was especially useful since WAM21

marked a subpopulation of Cd24a-epxressing foetal pancreas cells which

contained all β-cell precursors as assessed by Ngn3-GFP transgene

expression and an in vitro differentiation assay (Chapter 3). These studies

highlighted the specificity of the WAM21-CD24 antigen for the islet

progenitor cell compartment which has enabled us to develop this antibody











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as a useful tool for studies of islet development. Although we have shown our

antibodies mark novel CD24 antigens, the biological reasons for their

differences in cell type specificity remain unknown. In-depth epitope mapping

of the glycodeterminants marked by WAM18 and WAM21 may provide

further insight into the biological roles of this diverse protein and will be the

subject of future studies.

One of the limitations of the work carried out in this thesis was the lack of

significant numbers of antibodies which marked epithelial liver populations

in vivo. Although we initially produced 54 antibody-producing hybridomas

which marked LPC lines, most did not stain liver tissue, or marked non-

epithelial cell populations (Chapter 2). Comparatively, six of our antibodies

marked transgenically-labelled islet progenitor cells and an additional four

antibodies could be used as negative selection markers (Chapter 3). One

explanation for this observation may the lack of cell surface antigen

complexity of the initial liver-specific immunogenic source (immortalised

BMEL cells), limiting the ability to produce antibodies marking rare cell

populations induced by early chronic liver injury. This problem is paralleled

in a previous study where expression profiling comparing undifferentiated

and differentiated BMEL cells produced Cd24a as a single candidate cell

surface marker (Ochsner et al., 2007). Conversely, studies utilising primary

liver cells have produced many antibodies against rare cell types (Dorrell et

al., 2008). Our observations in the liver may be due to a combination of

decreasing population complexity in addition to changes in gene expression

associated with the process of cell line generation which will be discussed

further in the following paragraph.

Although immortalised cell lines have been extremely valuable in studies of

tumourigenicity (Dumble et al., 2002, Davies et al., 2006, Jellicoe et al., 2008),

differentiation (Strick-Marchand and Weiss, 2002, Tirnitz-Parker et al.,

2007) and in studying the biological effects of inflammatory cytokines on

LPCs (Matthews et al., 2004, Akhurst et al., 2005, Yeoh et al., 2007, Tirnitz-


109

Parker et al., 2010), the process of cell line generation involves selection of a

subpopulation of cells that are not entirely reflective of the complexity or

nature of those seen in vivo. The ‘plate and wait’ method of spontaneous

immortalisation used to generate LPC cell lines utilised in this study involves

selecting for cell subpopulations which are able to bypass senescence and

continue to proliferate indefinitely (Strick-Marchand and Weiss, 2002).

Furthermore, cultured LPCs have been shown to downregulate expression of

liver-specific markers such as albumin, transferrin, α-fetoprotein and tyrosine

aminotransferase as cells are passaged (Dumble et al., 2002). Hence, the

process of generating LPC lines excludes a significant proportion of the initial

progenitor cell compartment through senescence, and the remaining cells

become dissimilar to their in vivo counterparts as they are cultured. Despite

this, antibodies marking LPC lines but not liver tissue may still prove useful

in identifying novel liver cell types, since the process of cell line generation

parallels some aspects of cellular transformation, an aspect of chronic liver

injury not explored in this thesis. We will explore this idea further in the

following paragraphs with a view to future studies using our antibodies.

The current framework for understanding the development of a cancer

phenotype involves the acquisition of mutations which enable evasion of

mechanisms regulating cell growth, such as reliance on growth factor

stimulation, and sensitivity to regulators of cell proliferation and death

(Hanahan and Weinberg, 2011). Pioneering work in a Retinoblastoma model

has demonstrated the requirement of at least two aberrations in these control

mechanisms for neoplastic transformation, termed the ‘two hit hypothesis’ of

cancer (Knudson, 1971). This hypothesis holds true in liver cancer formation.

Disruption of two tumour suppressor genes (TSGs) in combination, such as

mTert and p53 (Farazi et al., 2006) and more recently the phosphatase and

tensin homolog (Pten) with glucose-regulated protein 94 (Grp94) (Chen et al.,

2014) accelerate liver tumorigenesis. Furthermore, studies in which

overexpression of oncogenes or inhibition of TSGs in LPCs show that a

combination of these genetic ‘hits’ results in acquisition of genetic


110

abnormalities consistent with HCC formation in resulting tumours (Zender

et al., 2005).

The ability of LPC lines to bypass senescence, and proliferate indefinitely in

culture, represents the acquisition of one of the two required traits for cell

transformation. Since WAM antibodies were generated against immortalised

LPCs, some of the antigens they recognise may be associated with

tumorigenic progression associated with the development of an immortal

phenotype. Accordingly, these antigens would not be present after two weeks

of CDE diet-induced liver injury, as this timepoint represents the early stages

of LPC expansion (Chapter 2). Since chronic liver injury-associated HCC

arises after prolonged LPC activation which is supported by the inflammatory

niche responding to liver injury (Roskams, 2006), antigens marked by WAM

antibodies present on immortal LPC lines are more likely to be expressed as

LPC transformation progresses during long term chronic injury. Thus, our

WAM antibody panel may prove valuable in defining novel cell types induced

during HCC formation in chronic liver injury in future studies.

5.2 Summary and conclusions

In this study, we have produced a panel of monoclonal antibodies which

recognise progenitor cells of the liver and pancreas in addition to subsets of

cells from diverse sources including thymus, brain and embryonic stem cells.

Whilst the use of this antibody panel may have broader benefits for studying

progenitor cells of these organs, we have focused on two antibodies, WAM18

and WAM21, which mark unique CD24 antigens on the cell surface of liver

and pancreas progenitor cells. In further developing these antibodies we have

demonstrated the following:


111

1. Our newly developed antibodies are effective alternative markers

to A6, which is limited in supply, for identifying LPCs induced in

mouse chronic liver injury models, with the additional benefit of

applicability in antibody-based sorting of enzyme-dissociated

tissues and cell lines.

2. WAM21 marks a subset of foetal pancreas cells which contains beta

cell progenitors and is extremely useful for purifying these

progenitors for studying islet development in vitro.

3. Our expression cloning approach using FACS selection to produce

stably-expressing cells, rather than viral or serial transfection

methods, provides a methodological framework that is technically

simple and relatively fast in identifying the cDNA encoding

antigens recognised by our antibody.

4. Identification of Cd24a as the gene encoding the WAM18 and

WAM21 antigens has enabled us to advance CD24 as a novel

marker of putative human islet progenitor cells.

The markers we have developed in this thesis are useful tools for researchers

studying progenitor cells from the liver and pancreas in mouse models. Their

use for isolation and characterisation of liver and pancreas progenitor cells

will aid studies of the biology of liver and pancreas progenitors for

understanding liver and pancreas disease, regeneration and in developing

methods to derive mature cells for transplantation therapy.

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Appendix I

143

Appendix I Supporting and additional data from

Chapter 2

Appendix I

144

A1.1 Appendix I Methods

A1.1.1 Cytopreparation of BMOL 1.2 cells

Cytospins were prepared by diluting 1 X 104 cells in 100 L of ice cold PBS.

Cells were centrifuged at 500 rpm for 5 min at RT onto microscope slides and

fixed with methanol:acetone (1:1, -20° C) for 5 min. Slides were air-dried and

stored at -20° C until use.

A1.1.2 Immunofluorescence

Liver cryosections (7 µm) were cut and fixed by exposure to RT acetone for 5

min. Sections were air dried and stored at -20 °C until use. Fixed cryosections

or cytospins were rehydrated by incubation in PBS for 5 min. Primary

antibodies labelling was performed overnight at 4 °C. Antibodies used were

WAM18, WAM21 (rat; 1:200) and/or FITC-conjugated mouse anti-α-Smooth

Muscle Actin (1:1000, Sigma). Rat antibodies were detected which were

detected with Alexa 594-conjugated goat anti-rat IgG (1:200, Invitrogen)

following incubation at room temperature for 1 h. Slides were mounted with

ProLong Gold Antifade mounting medium containing the nuclear stain DAPI

(Invitrogen, Australia) and visualised by fluorescence microscopy. Single

colour fluorescent microscope images were merged using ImageJ version

1.39u software for Windows (ImageJ Software, National Institutes of Health,

USA, available at: http://rsb.info.nih.gov/ij/). All colour corrections were

applied equally to experimental and control samples.


Appendix I

145

A1.2 Supplementary material

Target Primer sequences A. Temp.

Alb Fwd: 5’-GCTACGGCACAGTGCTTG-3’

Rev: 5’-CAGGATTGCAGACAGATAGTC-3’ 61 ⁰C

Bmper Fwd: 5'-CTGCCTGTTTCGCAGTGAT-3'

Rev: 5'-CTTGCTACCGAACTTGCACA-3' 54 ⁰C

Cd133 Fwd: 5'-ACGGAGTTGGAAAACCTGAA-3'

Rev: 5'-TTCCAGGGTAGAGGCAAATG-3' 58 ⁰C

Cd24a Fwd: 5'-CTTCTGGCACTGCTCCTACC-3'

Rev: 5'-TACTTGGATTTGGGGAAGCA-3' 53 ⁰C

Cd34 Fwd: 5'-CTGGAATCCGAGAAGTGAGG-3'

Rev: 5'-AGCAGACACTAGCACCAGCA-3' 54 ⁰C

Cdh11 Fwd: 5'-GTGCCTGAGAGGTCCAATGT-3'

Rev: 5'-TCCACCGAGAAATAGGGTTG-3' 54 ⁰C

Cdh17 Fwd: 5'-CCTCACCCCATCAAAATCAC-3'

Rev: 5'-CCATTCTCATCCTTGGCAGT-3' 54 ⁰C

Ck18 Fwd: 5’-GACGCTGAGACCACACT-3’

Rev: 5’- TCCATCTGTGCCTTGTAT-3’ 58 ⁰C

Ck19 Fwd: 5'-GCTGCAGATGACTTCAGAACC-3'

Rev: 5'-CATGGTTCTTCTTCAGGTAGGC-3' 60 ⁰C

Ck7 Fwd: 5’-GCAGGATGTGGTGGAAGATT-3’

Rev: 5’-CGTGAAGGGTCTTGAGGAAG-3’ 58 ⁰C

Cx43 Fwd: 5’-GAACACGGCAAGGTGAAGAT-3’

Rev: 5’-GAGCGAGAGACACCAAGGAC-3’ 60 ⁰C

Dcamkl1 Fwd: 5'-AGGGTGGAGACCTTTTCGAT-3'

Rev: 5'-CAGGCTGTGCAGGTATTTGA-3' 54 ⁰C

E-cad Fwd: 5'-AGTTTACCCAGCCGGTCTTT-3'

Rev: 5'-AGCCTGAACCACCAGAGTGT-3' 58 ⁰C

GAPDH Fwd: 5’- AATCCCATCACCATCTTCCA-

Rev: 5’- GGCAGTGATGGCATGGACTG-3’ 58 ⁰C

Gpc3 Fwd: 5'-AGCCGAAGAAGGGAACTGAT-3'

Rev: 5'-CATGGTTCTCAGGAGCTGGT-3' 58 ⁰C

Hnf1β Fwd: 5'-GACCAGCCAGTCGGTTTTAC-3'

Rev: 5'-GGAGAGGCTGTGGATATTCG-3' 58 ⁰C

Hnf4α Fwd: 5'-TTCTTCAGGAGGAGCGTGAG-3'

Rev: 5'-GCTGTCCTCGTAGCTTGACC-3' 59 ⁰C

Igf2R Fwd: 5'-GGACAGGCTCGTTCTGACTT-3'

Rev: 5'- GGCATACTCGGTGATCCACT-3' 54 ⁰C

Slit2 Fwd: 5'-CTGCCTGAGACCATCACAGA-3'

Rev: 5'-GAAGGCATCTGGTGCAAGTT-3' 54 ⁰C

Appendix Table A1.1 Mouse PCR Primer sequences and annealing

temperatures (A. Temp.) used.

Appendix I

146

Appendix Figure A1.1 Assessment of liver damage in CDE diet.

Haematoxylin and eosin staining of livers from mice fed a (A) control or (B)

CDE diet for 2 weeks. Control livers display normal architecture. CDE livers

display fat accumulation in hepatocytes and ductal expansion. (C)

Assessment of serum ALT levels in control versus CDE fed mice. Serum ALT

was increased in all CDE fed mice compared to control mice.

A

B

Appendix I

147

Appendix Figure A1.2 Immunofluorescence controls for dual rat

antibody labelling. Specificity of antibody labelling on liver tissues was

confirmed by incubating cryosections (7 µm) of 4 wk DDC-injured liver tissue

with appropriate isotype control antibodies. WAM21-Biotin and WAM18

antibodies were used as positive controls.

Appendix I

148

Appendix Figure A1.3 Magnetic bead enrichment of WAM18 and

WAM21-defined normal liver subsets. Magnetic bead sorting of normal

liver non-parenchymal cells (NPC’s) with WAM antibodies. NPC’s from

normal liver were stained with WAM18 or WAM21 and enriched using

magnetic beads. Purity of fractions was assessed by flow cytometry. Cells

were gated by size using forward scatter (FSC-A) and side scatter (SSC-A)

profile, and live cells assessed by gating propidium iodide-negative cells.

WAM-staining was then assessed in these live cell populations. Histogram

overlays show that WAM-negative (black) and WAM-positive fractions were

highly pure prior to culturing in liver progenitor cell growth medium.

Appendix I

149

Appendix Figure A1.4. Localisation of WAM antibody staining at the

cell surface of BMOL1.2 LPCs. (A) Immunofluorescent staining with

WAM18 or WAM21 (red) on BMOL1.2 compared to isotype control staining.

(B) Confocal microscopy shows surface localisation of WAM antibody staining

on BMOL1.2 cells. Nuclei were counterstained with DAPI (blue).

A

B

Appendix I

150

Appendix Figure A1.5. Comparison of WAM and α-Smooth Muscle

Actin (αSMA) antibody labelling in chronic liver injury.

Immunofluorescent staining with the activated stellate cell marker αSMA

(green) and WAMs (red) on cryosections (7 µm) of livers of mice fed a DDC

diet for 2 weeks. WAM18 and WAM21 positive cells are observed in close

association with αSMA+ stellate cells, but co-localisation of markers is not

observed. Nuclei were counterstained with DAPI (blue). Diagonal arrows

indicate regions displayed by insets. Horizontal arrows mark WAM21+/CD45+

cell subsets. Scale bars represent 50 µm.

Appendix II

151

Appendix II Supporting and additional data from

Chapter 3

Appendix II

152


Target Primer sequences A. Temp.

Cd133 Fwd: 5'-ACGGAGTTGGAAAACCTGAA-3'

Rev: 5'-TTCCAGGGTAGAGGCAAATG-3' 58 ⁰C

Cd24a Fwd: 5'-CTTCTGGCACTGCTCCTACC-3'


Ck19 Fwd: 5'-GCTGCAGATGACTTCAGAACC-3'

Rev: 5'-CATGGTTCTTCTTCAGGTAGGC-3' 60 ⁰C

E-cad Fwd: 5'-AGTTTACCCAGCCGGTCTTT-3'

Rev: 5'-AGCCTGAACCACCAGAGTGT-3' 58 ⁰C

GAPDH Fwd: 5’-AATCCCATCACCATCTTCCA-3’

Rev: 5’-GGCAGTGATGGCATGGACTG-3’ 58 ⁰C

Ins1+2 Fwd: 5'-GTGAAGTGGAGGACCCACAA-3'

Rev: 5'-GTTGCAGTAGTTCTCCAGCTG-3' 58 ⁰C

Ins1 Fwd: 5’-CTGTTGGTGCACTTCTACC-3’

Rev: 5’-GCAGTAGTTCTCCAGCTGGT-3’ 56 ⁰C

Ins2 Fwd: 5'-TCAAGCAGCACCTTTGTGGTT-3'

Rev: 5'-GTTGCAGTAGTTCTCCAGCTG-3' 56 ⁰C

Map2 Fwd: 5'-AAGGCCAAGAACACACGATTC-3'

Rev: 5'-ACCAAGCCCTAAGCTTCGACTAA-3' 60 ⁰C

Ngn3 Fwd: 5'-CAGTCACCCACTTCTGCTTC-3'

Rev: 5'-GAGTCGGGAGAACTAGGATG-3' 58 ⁰C

Nkx2.2 Fwd: 5'-CCGAGTGCTCTTCTCCAAAG-3'

Rev: 5'-TGTACTGGGCGTTGTACTGC-3' 60 ⁰C

Pax4 Fwd: 5'-CCACCTCTCTGCCTGAAGAC-3'

Rev: 5'-CCCACAGCATAGCTGACAGA -3' 60 ⁰C

PdgfRβ Fwd: 5'-CATCATCCCCTTACCTGACC-3'

Rev: 5'-CTTGCTGTGGCTCTTCTTGG-3' 58 ⁰C

Pdx1 Fwd: 5'-GAGCTGGCAGTGATGTTGAA-3'

Rev: 5'-TCTCCGGCTATACCCAACTG -3' 60 ⁰C

Ptf1a Fwd: 5'-CACGCTACCCTACGAAAAGC-3'

Rev: 5'-CCTCTGGGGTCCACACTTTA-3' 60 ⁰C

β3-tub Fwd: 5'-CTCAGTCCTAGATGTCGTGCG-3'

Rev: 5'-GCGGAAGCAGATGTCGTAGA-3' 58 ⁰C

Appendix Table A2.1. Mouse PCR Primer sequences and annealing

temperatures (A. Temp.) used.

Appendix II

153

Appendix Figure A2.1 Immunofluorescence controls for dual rat

antibody labelling. Specificity of antibody labelling on foetal pancreas

tissue was confirmed by incubating cryosections (7 µm) of e15.5 foetal

pancreas tissue with appropriate isotype control antibodies. WAM21-Biotin

and Cd49f antibodies were used as positive controls.

Appendix II

154

Appendix Figure A2.2. Enrichment and differentiation of WAM18-

defined foetal pancreas cells. In vitro differentiation of WAM18-defined

e15.5 MIP-GFP foetal pancreas was assessed. WAM18+ cells were separated

by size by gating into FSC-low (low) and FSC-high (high) populations (FACS

plots), plated into differentiation medium and the numbers of newly formed

MIP-GFP+ cells were quantitated. (A) In the first experiment, WAM18+/FSC-

low cells were enriched for beta cell progenitors. (B) In the second experiment

WAM18+/FSC-high cells were enriched for beta cell progenitors.

A

B

Appendix II

155

Appendix Figure A2.3. Magnetic bead enrichment and differentiation

of foetal pancreas cells with WAM21. (A) Efficiency of WAM21

enrichment assessed by FACS analysis after separation with (A) one or (B)

two MiniMACS columns. (C) Beta cell differentiation dynamics of WAM21-

defined populations compared to unsorted control. (D) Ratio of insulin-

reporter+ cells formed after differentiation in WAM21-defined foetal pancreas

populations compared to unsorted cells. Bars represent mean ± SEM (n=3;

*p<0.05 compared to unsorted cells).

A B

C D

Appendix II

156

Appendix Figure A2.4. Immunocytochemical staining of

differentiated WAM21+ MIP-GFP e15.5 foetal pancreas with islet

markers. Comparison of MIP-GFP fluorescence and islet markers

demonstrates that WAM21+ cells differentiated to MIP-GFP+ cells in vitro (A)

do not co-express Glucagon (α-cells), and (B) express nuclear Pdx1 (islet

progenitor/β-cells). Scales bars represent 10 µm.

A

B

Appendix II

157

A2.2 Expression profiling WAM21+ foetal pancreas cells

A2.2.1 Methods

A2.2.1.1 In vivo incubation of WAM21+ foetal pancreas cells

WAM21+ e15.5 foetal pancreas cells were MACS-enriched as previously

described (Chapter 3). 100 000 WAM21+ e15.5 foetal pancreas cells were

embedded in 20 µL Matrigel™(BD Biosciences) containing Nicotinamide (10

mM, Sigma-Aldrich) and B27 supplement (2X, Gibco) and pipetted into 1 mm3

gelfoam to create an insert for transplant. Inserts were incubated at 37 °C for

20 min to allow Matrigel to solidify. Inserts containing cells were covered in

high glucose DMEM medium (Invitrogen) and incubated overnight in a 37 °C,

10% CO2 incubator with 90% humidity.

The next day, gelfoam inserts containing WAM21+ foetal pancreas cells were

transplanted into the left renal subcapsule of eight week old female NOD-

SCID mice. Mice were housed under specific pathogen free, temperature

controlled conditions, with alternating 12h light-dark cycles. All procedures

were performed according to guidelines set by the National Health and

Medical Research Council of Australia under ethics approval from the

University of Western Australia. Transplant was performed by Dr Erika

Bosio from the Centre for Diabetes Research at the Western Australian

Institute for Medical Research. A surgical plane of anaesthesia was induced

by i/p injection of Ketamine (80 mg/kg) and Xylazine (10 mg/kg). After shaving

the left flank of the mouse, a small incision was made in the left back of the

animal and the kidney was exposed on the outside of the body by applying

pressure around the incision. A small cut to the kidney capsule was made by

scraping a 23 gauge needle across the upper pole of the kidney. Gelfoam

inserts containing foetal pancreas cells were inserted under the renal capsule,

which was then heat-cauterised. The kidney was returned to the body and all

incisions were sutured.

Appendix II

158

After 3 weeks, mice were anesthetized by i/p injection of Ketamine/Xylazine

and euthanized by cervical dislocation. The left kidney was removed and the

graft was dissected away from the kidney capsule and placed into 1 mL TRIzol

(Invitrogen) for RNA extraction. For microarray analysis of gene expression,

RNA was extracted from freshly isolated and in vivo incubated WAM21+ e15.5

foetal pancreas cells in 1mL TRIzol reagent (Invitrogen) and prepared for

hybridization to Illumina Ref-8 arrays according to the manufacturer’s

instructions (Illumina, San Diego, CA).

A2.2.2 Results

A2.2.2.1 Expression profiling WAM21+ foetal pancreas cells

In an attempt to define pathways regulating in vivo differentiation of islet

progenitors, we transplanted enriched WAM21+ cells by magnetic bead

sorting, and incubated these cells under the kidney capsule of NOD/SCID

mice for a period of 3 weeks. We then extracted the in vivo incubated tissue

and carried out microarray expression profiling to ascertain potential new

pathways involved in islet development. Since we did not observe evidence of

islet progenitor differentiation, we instead used this experiment to create a

gene expression profile of WAM21-defined e15.5 foetal pancreas cells to

determine which developmental lineages are enriched by WAM21-sorting.

The in vivo incubated tissue was used as a baseline for non-islet gene

expression.

384 microarray probes were greater than 5-fold higher in freshly isolated

versus in vivo incubated tissue. We found many of these genes were acinar

cell-specific enzymes including proteases and lipases, and also acinar-specific

transcription factors Ptf1a and Mist1. Additionally, a number of transcription

factors involved in islet development were also highly expressed in WAM21+

e15.5 foetal pancreas cells including Pdx1, and Ngn3, which we had

previously observed by RT-PCR analysis, and more recently defined

Appendix II

159

transcription factors that are crucial for islet development Sox9, Rfx6, FoxA3

and Myt1. We also observed markers of committed islet progenitors of the

beta/delta cell lineage Pax4, Nkx2.2 and Nkx6.1 supporting our

differentiation studies showing that WAM21+ cells differentiate into beta-like

cells. We did not observe enrichment of pathways associated with ductal

differentiation, particularly upregulation of Notch pathway signalling

(Appendix Figure A2.5).

Since there are limited cell surface markers available for enriching islet

precursors, we carried out gene ontology (GO) profiling to determine if novel

potential surface markers of pancreatic lineages were present in WAM21+

foetal pancreas cells. Three GO identifiers associated with cellular

components, GO:0005911 (cell-cell junction; 11 transcripts), GO:00016324

(apical plasma membrane; 6 transcripts) and GO:00016323 (basolateral

plasma membrane; 6 transcripts) defined transcripts encoding proteins

expressed on the cell surface of WAM21+ cells. Of these markers, several

genes encoding proteins involved in cell-to-cell contacts were enriched in

WAM21+ cells including members of the claudin family of proteins, the

transcript encoding the desmosome-associated protein desmoplakin and the

gap junction protein connexin 36. Additionally, markers involved in cell-

extracellular matrix interactions (β4-integrin, Cxadr), ion transport (Slc12a2)

the glycoprotein mucin 1 and the previously identified surface marker of liver

progenitor cells Dlk were all enriched in WAM21+ cells (Appendix Table A2.2).

Appendix II

160

Appendix Figure A2.5. Developmental hierarchy of gene expression

in WAM21+ e15.5 foetal pancreas cells. Microarray analysis was carried

out on WAM21+ e15.5 foetal pancreas. WAM21+ cells are enriched for acinar-

specific transcription factors and enzymes, and islet precursor cells of the

beta/delta cell lineage according to the schema of pancreatic development.

Colour-coding represent fold upregulation of lineage-specific genes in freshly

isolated versus in vivo incubated WAM21+ e15.5 foetal pancreas cells.

Appendix II

161

Probe_ID Definition Gene

Symbol Accession

ILMN_2634167 Claudin 3 Cldn3 NM_009902.3





ILMN_3144575 Integrin beta 4 Itgb4 NM_133663.2

ILMN_3018017 Coxsackie virus and

adenovirus receptor Cxadr NM_001025192.2

ILMN_1244618 Delta-like homolog 1 Dlk1 NM_001190703.1

ILMN_1250138 Mucin 1 Muc1 NM_013605.1

ILMN_2654997 Desmoplakin 1 Dsp XM_621314.3

ILMN_2721734 Gap junction protein,

delta 2/ Connexin 36 Gjd2 NM_010290.2

ILMN_3146420 Solute carrier family

12, member 2 Slc12a2 NM_009194.2

Table A2.2. Surface markers enriched in WAM21+ foetal pancreas

cells.

Appendix III

162

Appendix III Supporting and additional data from

Chapter 4

Appendix III

163

A3.1 Appendix III methods

A3.1.1 Relative quantitation of gene expression

RNA from BMOL cells was extracted and reverse transcribed according to

previously described methods (4.3.12). Cd24a and β-actin gene expression

was assessed using SensiMix qPCR mastermix according to manufacturer

instructions (Bioline, Australia) in a Rotor Gene 2000 real-time PCR thermal

cycler (Corbett). Primers and annealing temperatures are listed in

supplementary table S4.1. No template controls (no reverse transcriptase and

water) were used to control for DNA contaminants. Standard curves were

generated using serial 10-fold dilutions of RT-PCR products for each gene

assessed and used to calculate reaction efficiency (>90%). Relative gene

expression was calculated using the comparative CT method (Schmittgen and

Livak, 2008). Data was normalised to β-actin expression and expressed as fold

change over shScrambled control cells.

A3.1.2 MTT assay of cell proliferation

The MTT assay was performed as previously described (Mosmann, 1983). Cell

viability is determined by the conversion of 3-(4,5-Dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT; 5 mg/mL: Sigma, USA) to formazan, a

purple precipitate. 10 L of MTT solution was added to each well of a 96-well

plate containing BMOL cells in 100 L of growth medium, and incubated at

37 C for 4 hrs. Formazan, produced by cell mitochondria, was solubilised by

the addition of 100 L of solubilisation buffer (26.6% (w/v) SDS in 46.8%

dimethylformamide; pH 4.7) and incubation for 2 h at 37 C. Optical density

(O.D.) was measured at 595 nm and directly compared to well confluence

measurements generated by the Cellavista™ system (Appendix figure A3.2).

Appendix III

164


Target Primer sequences A. Temp

mu β-actin Fwd: 5’- GCCAACACAGTGCTGTCTGG-3’

Rev: 5’- TACTCCTGCTTGCTGATCCA-3’ 60 ⁰C

mu Cd24a Fwd: 5'-CTTCTGGCACTGCTCCTACC-3'


hu GAPDH Fwd: 5’-GAGTCAACGGATTTGGTCGT-3’

Rev: 5’-TTGATTTTGGAGGGATCTCG-3’ 58 ⁰C

hu INS Fwd: 5’-CTACCTAGTGTGCGGGGAAC-3’

Rev: 5’-GCTGGTAGAGGGAGCAGATG-3’ 58 ⁰C

hu MYT1 Fwd: 5'-ACCCTGGTGGAAGAGGACTT-3'

Rev: 5'-GACTCAGGATGCCTTTCTGC-3' 60 ⁰C

hu NGN3 Fwd: 5’-CCCTCTACTCCCCAGTCTCC-3’

Rev: 5’-CCTTACCCTTAGCACCCACA-3’ 58 ⁰C

hu PAX4 Fwd: 5'-GTGAGGGTCTGGTTTTCCAA-3'

Rev: 5'-AGGTGGGGTGTCACTCAGAC-3' 60 ⁰C

hu PDX1 Fwd: 5'-GTTCCGAGGTAGAGGCTGTG-3'

Rev: 5'-AACATAACCCGAGCACAAGG-3' 60 ⁰C

hu SOX9 Fwd: 5'-AGGAAGCTCGCGGACCAGTAC-3'

Rev: 5'-GGTGGTCCTTCTTGTGCTGCAC-3' 60 ⁰C

Appendix Table A3.1. PCR Primer sequences and annealing

temperatures (A. Temp) used.

Appendix III

165

Appendix figure A3.1. Effect of stable Cd24a knockdown on BMOL

cell growth. Cell proliferation in BMOL cells that were untransfected

(Parental), or stably transfected with plasmids harbouring short hairpin

sequences that were targeted against Cd24a (shCd24a) or a non-specific

scrambled control sequence (shScrambled) was measured using MTT assay.

(A) Stable knockdown of Cd24a mRNA was confirmed by qPCR. (B) BMOL

cell numbers were indirectly measured using an MTT assay over 6 days. Cell

numbers were significantly reduced in shCd24a-expressing BMOL cells after

5 days in culture. Data represent mean ± SEM, n=3 Statistical significance of

paired t-test is represented as *p<0.05 or **p<0.01.

A B

Appendix III

166

Appendix figure A3.2. Assay precision of the Cellavista™ system

versus the MTT assay. BMOL cells were seeded at densities from 25000

cells to 500 cells per well, allowed to adhere for 16 h and well confluence was

measured using Cellavista™. A strong linear correlation (R2>0.98) between

cell number and measured well confluence was observed in (A) untransfected

BMOL cells or BMOL cells stably expressing (B) shScrambled or (C) shCd24a

constructs. Measured wells were then measured using an MTT assay. A

strong linear correlation (R2>0.97) between well confluence measurements

using the Cellavista™ and the established MTT assay was observed in (B)

untransfected BMOL cells or BMOL cells stably expressing (D) shScrambled

or (F) shCd24a constructs.

A B

C D

E F

R2=0.9946 R2=0.9752

R2=0.9884 R2=0.9805

R2=0.9949 R2=0.9900

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