TOWARDS FEEDER-FREE AND SERUM- FREE GROWTH...

SEAN RICHARDS

TOWARDS FEEDER-FREE AND SERUM-FREE GROWTH OF CELLS 1

TOWARDS FEEDER-FREE AND SERUM-

FREE GROWTH OF CELLS

By

Sean D. Richards, Bachelor of Science. (Hons)

Faculty of Science - School of Life Sciences

A thesis submitted for the degree of Doctor of Philosophy of the

Queensland University of Technology, June 2007.

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DISCLOSURE STATEMENT:

The research described in this thesis was funded by Tissue Therapies limited a

Biotechnology spin off company from the Queensland University of Technology

(QUT). Tissue Therapies has a license to commercialise the intellectual property

described in this thesis. I also received a PhD scholarship stipend (top up) from

Tissue Therapies and have stock in this company.

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ABBREVIATIONS

Abbreviation Translation

ACN Acetonitrile ALP Alkaline PhosphataseANK Activin:Keratinocyte growth factor:NicotinamidebFGF Basic fibroblast growth factorBPE Bovine pituitary extractBSE Bovine spongiform encephalopathycDNA complementary deoxyribose nucleic acidCM Conditioned MediumCG Complete Green’s CHCA Matrix for MS protein spottingCJD Creutzfeldt-Jacob diseaseDED De-cellularised dermisDKM Defined keratinocyte mediumDKMF Defined keratinocyte medium + Feeder cellsDMEM Dulbecco’s modified eagle mediaECM Extra-cellular matrixEGF Epidermal growth factorFBS Foetal bovine serumFDA Food and Drug AdministrationgDNA genomic deoxyribose nucleic acidGMP Good manufacturing practiceGTP Good tissue practiceH&E Haemotoxylin and eosinHaCaT Keratinocyte cell lineHBD Heparin binding domainsHCL Hydrochloric acid hEC Human embryonic carcinoma cellhEG Human embryonic germ cellhES Human embryonic stem cellHGF Hepatocyte growth factorHPLC High performance liquid chromatographyHSA Human serum albuminhTERT Human telomerase reverse transcriptasei3t3 Irradiated mouse embryonic fibroblastsICM Inner cell mass IGF Insulin-like growth factorIGFBP Insulin-like growth factor binding proteinIVF In-vitro fertilisationKSR Knock-out serum replacementLC/MS Liquid chromatography/mass spectrometryLC/ESI Liquid chromatography/electrospray ionisationLC/MALDI Liquid chromatography/ Matrix assisted laser desorption ionisation LIF Leukaemia inhibitory factorMALDI Matrix assisted laser desorption ionisation

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MALDI-TOF Matrix assisted laser desorption ionization-Time of flight MEF Mouse embryonic fibroblastmES Mouse embryonic stem cellMTT Thiazolyl blue tetrazolium bromideNaCl Sodium ChlorideOct-4 Octamer binding protein 4PBS Phosphate buffered salineRT-PCR Reverse Transcriptase Polymerase Chain Reaction SCID Severe combined immunodeficiencySG Stripped Green’sSSEA Stage Specific Embryonic AntigensTFA Trifluro acetic acidTGA Therapeutic Goods AdministrationTGF Transforming growth factorVN VitronectinVN:GF (hES VN:IGFBP-3:IGF-I:bFGFVN:GF VN:IGFBP-3:IGF-I:EGF

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ABSTRACT

The in-vitro culture of human embryonic stem and keratinocyte cells has great

potential to revolutionise the therapeutics industry. Indeed it is hoped that these cells

will provide a superior alternative to current tissue and organ transplantation.

However, both of these cell types require animal and/or donor products for their

successful maintenance in-vitro. This requirement results in a significant risk of

cross contamination from the animal or donor products to either the primary

keratinocyte or hES cells. These potentially transplantable cells therefore need to be

cultured in an environment free from animal or donor products to remove the risk of

contamination to the patient.

The ideal growth conditions must comprise of two attributes; firstly they must be

free from animal or donor products, and secondly the culture system must be fully

defined. Recently, it was discovered that an extra-cellular matrix protein, vitronectin,

could be used in conjunction with growth factors and growth factor-binding proteins

(VN:GF combination), to promote enhanced cell migration and growth through the co-

activation of integrin and growth factor receptors. Given that growth factors and serum

are clearly important in supporting the in-vitro cultivation of mammalian cells, and that

vitronectin is an abundant protein in serum, I hypothesised that these VN:GF

combinations could be translated into a serum-free medium that would support the serial

propagation and self renewal of primary keratinocytes and hES cells. As reported in this

thesis I have developed a defined, serum-free media for the culture of these cells that

incorporates the VN:GF combinations. While the two media differ slightly in their

compositions, both support the serial, undifferentiated expansion of their respective

cells types.

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Together, this represents a significant advance that will ultimately facilitate the

therapeutic use of these cells. However, the in-vitro expansion of these cells in these

new media still required the presence of a feeder cell layer. In view of this I aimed to

explore the in-vitro micro-environment of primary keratinocytes using a novel

proteomic approach in an attempt to find candidate factors that could be used in

conjunction with the VN:GF media to replace both serum and the feeder cells. The

proteomic approach adopted examined the secretion of proteins into the defined,

minimal protein content VN:GF media when the feeder cells were cultured alone, as

well as in co-culture with primary keratinocytes. This strategy allowed assessment of

proteins/factors that are secreted in response to both autocrine and paracrine cellular

interactions and revealed a number of candidate factors that warrant further

investigation.

Ultimately this proteomic information and the associated new insights into the

keratinocyte in-vitro culture microenvironment may lead to the development of a culture

system for these cells that is not reliant on either a feeder cell layer or serum for their

successful propagation. Moreover, it is likely that this will also be relevant to the feeder

cell-free propagation of hES cells. This has obvious advantages for the culture of

primary keratinocytes and hES cells in that it will allow a safe defined culture system for

the undifferentiated propagation of these cells. This will facilitate the generation of cells

and tissues free from xenogeneic and allogeneic contaminants, thus ensuring any

therapeutics developed from these cell types are approved for therapeutic applications

and importantly, will minimise risks to patients.

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TABLE OF CONTENTS

PAGE

Title 1

Disclosure Statement 3

Abbreviations 4

Abstract 6

Table of contents 8

List of Figures 13

List of Tables 14

Statement of original authorship 15

Acknowledgements 16

Chapter 1: Introduction 19

1.1 Cells and the Micro-Environment 20

1.2 Introduction to Human Embryonic Stem Cells 21

1.2.1 Development and The Human Embryonic Stem Cell 23

1.2.2 Human Embryonic Stem Cell Culture 24

1.2.3 The Potential of Human Embryonic Stem Cells 27

1.2.4 Issues Associated With Human Embryonic Stem Cells 29

1.3 Skin 34

1.3.1 Keratinocytes 36

1.3.2 Keratinocyte Culture 37

1.4 Vitronectin 39

1.4.1 (VN:GF) technology 42

1.5 Conclusion 44

Chapter 2: The serum-free culture of human keratinocytes 47

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2.1 Introduction 48

2.2 Materials and Methods 52

2.2.1 Ethics and material collection 52

2.2.2 Isolation of primary kertinocytes 52

2.2.3 Standard culture 52

2.2.4 VN:GF culture 53

2.2.5 Defined keratinocyte media (DKM) culture 54

2.2.6 HaCaT culture 54

2.2.7 Proliferation assays 54

2.2.8 Immunohistochemistry 55

2.2.9 Preparation of dermal equivalent (de-epidermised

dermis)

56

2.2.10 Preparation and culture of skin equivalent 56

2.2.11 Immunohistochemistry and histology of skin

composites

57

2.2.12 Statistical analysis 58

2.3 Results 59

2.3.1 The culture of a keratinocyte cell line using VN:GF

medium

59

2.3.2 Proliferation of HaCaT cells in the presence of different

growth conditions

60

2.3.3 Establishment of primary keratinocyte cells using

VN:GF combination

62

2.3.4 Proliferation of primary keratinocyte cells in the

presence of different growth conditions

63

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2.3.5 Characterisation of primary keratinocyte cells

propagated under different growth conditions

66

2.3.6 The histology and staining of the reconstituted

epidermis

68

2.4 Discussion 71

Chapter 3: Serum-free growth of human embryonic stem cells 77

3.1 Introduction 78


3.2.1 Ethics and training 81

3.2.2 Cell culture 81


3.2.4 ANK culture 83

3.2.5 Immunofluorescence 83

3.2.6 Reverse transcriptase polymerase chain reaction (RT-

PCR) analysis

84

3.2.7 Proliferation assay 85

3.2.8 Karyotype analysis 85

3.3 Results 87

3.3.1 VN:GF medium for the propagation of hES cells 87

3.3.2 Morphology of hES cells grown using VN:GF medium

as a serum-free medium

87

3.3.3 Identification of markers expressed by undifferentiated

hES cells

89

3.3.4 Karyotype analysis of H1 cells grown using VN:GF

medium

90

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3.3.5 RT-PCR analysis of hES cells 91

3.3.6 Morphology of HUES-7 cells grown using the VN:GF

medium in conjunction with the ANK protocol

95

3.3.7 Identification of markers expressed by HUES-7 cells

grown using the VN:GF medium in conjunction with

the ANK protocol

95

3.4 Discussion 99

Chapter 4: The proteomic investigation of keratinocyte conditioned

medium

105

4.1 Introduction 106


4.2.1 Ethics and material collection 109

4.2.2 Isolation of primary keratinocytes 109


4.2.4 Two-dimensional proteomics 110

4.2.5 Sample Preparation and LC/MS using LC/ESI/MS and

LC- MALDI Analysis

112

4.2.6 Sample preparation and MALDI-TOF-TOF mass

spectrometry

113

4.2.7 Database analysis and interpretation 114

4.3 Results 115

4.3.1 Morphology and expression of cell surface markers on

passage-2 keratinocytes propagated using VN:GF

medium for proteomic analysis

115

4.3.2 Two dimensional separation of conditioned media 117

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collected from both feeder cells alone and feeder

cell:keratinocyte cultures

4.3.3 Proteins identified in the feeder cell and the feeder-

cell:keratinocyte conditioned media

119

4.3.4 Differences in expression of protein species found in

the feeder cell and the feeder cell:keratinocyte

conditioned media

125

4.4 Discussion 127

Chapter 5: General Discussion 137

5.0 General Discussion 138

5.1 Serum-free propagation of primary keratinocytes 138

5.2 Serum-free propagation of human embryonic stem cells 140

5.3 Proteomics of keratinocyte conditioned media 143

5.4 Conclusion 147

Chapter 6: 6.0 References 149

Appendix I 173

Appendix II 176

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LIST OF FIGURES PAGE

1.1 Schematic representation of the ECM proteins and their ECM

binding

21

1.2 Timeline and structures for development 24

1.3 Schematic representation of derivation and culturing methods of

hES cells

26

1.4 Schematic representation of the differentiation of hES cells 29

1.5 Anatomy of the skin 35

1.6 Strata of the epidermis 36

1.7 Schematic representation of vitronectin 42

2.1 The culture of a keratinocyte cell line using the VN:GF medium 60

2.2 Proliferation of HaCaT cells using the VN:GF medium 61

2.3 Establishment of primary keratinocyte cells using the VN:GF

medium

63

2.4 Proliferation of primary keratinocyte cells using the VN:GF

medium

65

2.5 Characterisation of primary keratinocyte cells using the VN:GF

medium

67

2.6-I The histology and P63 immuno-staining of the reconstituted

epidermis

69

2.6-II Immuno-staining for keratin 6 and 1/10/11 in the reconstituted

epidermis

70

3.1 VN:GF combinations for the propagation hES cells 88

3.2 Morphology of hES cells grown using the VN:GF medium 89

3.3 Markers expression of the hES cells grown using the VN:GF 93

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medium

3.4 Karyotype analysis of h1 ES cells grown in VG conditions 94

3.5 RT-PCR analysis of hES cells grown using VN:GF medium 94

3.6 Morphology of HUES-7 cells grown using the VN:GF medium in

conjunction with the ANK protocol

97

3.7 Markers expression of HUES-7 cells grown using the VN:GF

medium in conjunction with the ANK protocol

98

4.1 Passage 2 morphology and marker expression of keratinocytes

propagated using VN:GF medium for proteomic analysis

116

4.2 Two dimensional separations of conditioned media 118

LIST OF TABLES

4.1 Proteins identified from feeder cell conditioned media using

LC/ESI/MS system and LC-MALDI

120

4.2 Proteins identified from feeder cell:keratinocyte conditioned

media using LC/ESI/MS and LC-MALDI

122

4.3 Differences in protein species abundance between the feeder cell

and the feeder cell:keratinocyte conditioned media

126

AI.1 MALDI-TOF-TOF Feeder Cell Conditioned Medium 174

AI.2 MALDI-TOF-TOF Keratinocyte Conditioned Medium 174

AII.1 Proteins identified from the MEF conditioned media using

MALDI-TOF-TOF, LC/ESI/MS and LC-MALDI

178

AII.2 Proteins identified from the MEF:hES cells conditioned media

using MALDI-TOF-TOF, LC/ESI/MS and LC-MALDI

180

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STATEMENT OF ORGINIAL AUTHORSHIP

The work contained in this thesis has not been previously submitted for a degree or

diploma or any other higher degree institution. To the best of my knowledge and

belief, the thesis contains no material previously published or written by any other

person(s) except where due reference is made.

Sean D. Richards –

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ACKNOWLEDGEMENTS

This PhD was by no means a smooth ride, with constant problems arising at every

step. However, I believe this experience has helped me to develop into a better

scientist and a more determined person. I have many people who have helped me

through this 3 year journey. Initially, I would like to thank my supervisor for giving

me the opportunity to work on this project. Zee Upton allowed me the freedom to

think for myself, but also cracking the whip when I would get side tracked with

ideas, not necessarily in line with my project.

Furthermore, I would like to thank, Tissue Therapies Ltd. for providing this project

with the growth factors and the Australian Red Cross Blood Service for providing

the cell irradiation service. We would like to further acknowledge the Australian Red

Cross Bali Appeal for funding, as well as Dr Anthony Kane and Dr Phillip

Richardson for supplying us with the skin for this project. I would like to further

thank Gemma Topping for her assistance with setting up the DED studies and

Rebecca Dawson for staying on my case and her assistance in learning the skin

isolation.

Additionally, I would like to express my gratitude to Martin Pera for training me in

hES cell culture at the MIRD. Their guidance and support throughout the year has

been greatly appreciated. I would also like to thank Chris Joy and Sue White, from

QML, for undertaking the karyotype analysis on the hES cells. The technical advice

from Tony Parker, Steve Myers, and Levi Carroll was also of great value. To Gillian

Beattie, Alberto Hayek, and Ana Lopez at the Whittier Institute, thank you for the

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opportunity to travel and work in your lab, it was a great experience, and I believe

this collaboration holds great promise.

A special thanks to my fellow students and friends, especially, Louise Ainscough,

Brett Hollier, James Broadbant and Alun Jones (especially for their invaluable help

with the proteomics), and more recently my student Luke Cormack, for their support

in and out of the lab and their friendship.

Finally, to my friends and family their support was invaluable, their guidance and

strong work ethics set the foundation for my self-belief and discipline, this was

especially important during my first year, when most of my work was destroyed.

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CHAPTER 1

INTRODUCTION

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1.0 INTRODUCTION

1.1 Cells and the Micro-Environment

A cell is the smallest functional unit within the body capable of metabolism,

replication, and respiration. The body consists of several different cell types each

carrying out unique roles that allow us to function as a living unit. In the body, cells

are provided with a favourable environment, their ‘micro-environment’, which allow

them to survive, replicate and carry out their respective functions. This micro-

environment is the immediate environment that the cell is in contact with. The

micro-environment can include other cells, nutrients, growth factors and an intricate

array of extra-cellular matrix (ECM) proteins, (refer to Figure 1.1 A). The ECM

proteins allow the cells to anchor to their immediate environment via cell surface

proteins, primarily known as integrins, (refer to Figure 1.1 B). Furthermore, ECM

proteins can influence other cellular responses, including migration, proliferation and

cellular morphology.

Indeed, it was the replication of this micro-environment that allowed researchers to

grow cells ‘outside of the body’, (in-vitro). This is accomplished by providing the

cells with an appropriate platform on which they can attach, i.e. culture-ware; the

appropriate nutrients, which can be delivered through media; and a favourable

atmosphere, generally provided by an incubator. The ability to grow cells in-vitro

allows scientists to investigate single cell or mixed cell populations which can

provide insights into the cells physiology and metabolic activity. Moreover, cell

culture can lend itself to other facets of cell biology such as, drug design and the

generation of transplantable tissues (Docherty 2001; Guan et al. 2001; Bagutti et al.

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1996). In fact, the promise of transplantable tissues has led to the exploration of

more primitive cell types such as, pluripotent cells, i.e. human embryonic stem cells,

which have the ability to form several different cell types.

Figure 1.1: A) A variety of proteins, involved with attachment, migration, and proliferation,

of cells, that are commonly located in the ECM, B) A schematic representation of the cells

integrin proteins binding to the ECM. Modified from, www.glycoforum.gr.jp/.../

14/images/2.gif

1.2 Introduction to Human Embryonic Stem Cells

A stem cell has the ability to renew itself as well as give rise to more specialised cell

types. There are primarily two types of stem cells; adult stem cells and embryonic

stem cells. Adult stem cells are pluripotent, thus they are able to differentiate into a

number of cell and tissue types. Human embryonic stem (hES) cells on the other

hand are totipotent, possessing the potential to differentiate into all somatic cell and

tissue types within the body (Pera et al. 2000).

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Human embryonic stem cell research is viewed as a relatively new field of science

which has primarily developed over the last 7 years. However, this field of research

has stemmed from scientific work that is approximately 125 years old. The first step

in this research was in 1878 with the first reported attempt of fertilising mammalian

eggs outside the body (Trounson et al. 2000b). Another 81 years passed before the

first reported case of a successful in-vitro fertilization (IVF) with rabbits in the USA

in 1959 (Chang, 1959). In 1969 Edwards and Bavister performed the first reported

human egg fertilization in-vitro (Edwards and Bavister 1969). Ten years later, the

first IVF baby was born in England, with Australia following suit two years later

(Trounson 1982). The first embryonic stem cells explored in the field of science

were mouse embryonic stem (mES) cells. These were derived and cultured from the

inner cell mass of mouse blastocysts (Evans and Kaufman 1981). This in turn led to

the first attempt at culturing hES cells in 1994, when Bongso tried to propagate the

inner cell mass from blastocysts donated from patients within the IVF program.

These hES cell cultures could only be passaged twice before differentiating and no

longer demonstrating totipotential behavior (Bongso et al. 1994). Thomson et al.

(1998) were the first group to successfully establish a system for the culture of hES

cells. They demonstrated that these cells could be propagated for long periods of

time whilst still maintaining markers that are representative of an undifferentiated

hES cell (Thomson et al. 1998). This early work has now expanded such that many

groups all around the world are developing these cells as tools for the

pharmacological industry, using their totipotency to drive them into specific cell and

tissue types, and to develop improved culture technologies.

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1.2.1 Development and the Human Embryonic Stem Cell

The pathway for development of the human embryonic stem cell begins with the

human egg being fertilised by a sperm cell. Following this, a series of events occur

that lead to the formation of the blastocyst. The first step that occurs is at day two

post-fertilization, and involves the first cleavage. By day three, two more divisions

have occurred to give rise to an eight cell structure called the morula. After another

two days of development a structure known as the blastocyst is formed. This

structure is formed from approximately 200-250 cells resulting in the creation of two

distinct cell layers. The outer wall of the blastocyst is known as the trophectoderm,

which accounts for the majority of cells. The blastocyst also has a single polar cell

clump known as the inner cell mass, which contains approximately 30-34 totipotent

hES cells. It is from the inner cell mass that the first hES cell line was established

(Thomson et al. 1998).

In addition to hES cells, there are two other cell types of interest that are related to

this field of research, human embryonic germ (hEG) and human embryonic

carcinoma (hEC) cells. The hEG cells are derived from the primordial germ cells,

which exist in a region of the foetus known as the gonadal ridge. The hEC cells are a

cell type derived from teratocarcinomas which are germ cell tumours consisting of

multiple cell and tissue types (Iacovitti et al. 2001) (refer to Figure 1.2). Both these

cell types demonstrate high levels of similarity to the hES cells, morphologically and

biochemically, and thus are regularly used in this field as model systems for hES cell

studies.

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Figure 1.2: The timeline and structures for development and derivation of hES, human

embryonic germ (hEG), human embryonic carcinoma (hEC) cells. Taken from www.stem

cellresearch.org/testimonies/ prentice3.htm

1.2.2 Human Embryonic Stem Cell Culture

Human embryonic stem (hES) cells have great potential to revolutionise the

therapeutics industry due to this totipotential ability. Indeed some scientists believe

these cells will provide a superior alternative to tissue and organ transplantation.

Nevertheless, the field of hES cell research is relatively new when compared to the

field of mouse embryonic stem (mES) cell research. Mouse embryonic stem cell

research was also the direct source of the initial culturing techniques for hES cells.

As stated previously, Thomson et al. (1998) published the first successful method for

the long term culture of hES cells in an undifferentiated state. This culture

methodology involved the removal of the inner cell mass from the blastocyst stage

embryo and the seeding of the inner cell mass into specific culture conditions.

Thomson et al. (1998) discovered that successful propagation of the hES cells was

halla

This figure is not available online. Please consult the hardcopy thesis available from the QUT Library

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obtained when using a culture system containing a mitotically-inactivated feeder cell

layer and foetal bovine serum (FBS). It is not yet understood what the exact function

of the MEF feeder cell layer in the culture system is, but it has been demonstrated to

supply a range of extra-cellular matrix (ECM) proteins, growth factors and

cytokines, such as the IGFs, bFGF and leukaemia inhibitory factor (LIF), all of

which may be vital for maintaining the hES cells in an undifferentiated state

(Barreca et al. 1992). Furthermore, it has been demonstrated that mES cells express

many of the receptors for the above growth factors, including the IGF-I receptor and

αv integrins, thus suggesting that the same situation is likely to exist for hES cells

(Newman-Smith and Werb 1995). Nevertheless, further studies are required to

determine what receptors are present on hES cells, and to further identify the role

that the feeder cell layer and serum has in the maintenance of the hES cells.

Once hES cells have been grown in these culture conditions for approximately seven

days they reach a morphological state classed as confluent. At this stage the colonies

also begin to differentiate. Thus, the hES cell colonies are passaged into fresh culture

conditions. There are currently two methods for the passaging of hES cells, namely

mechanical dissection and chemical dissection. Mechanical dissection is the process

of cutting the undifferentiated portions of the confluent hES cell colonies into

smaller pieces for transfer to fresh culture. The second method, known as chemical

dissection, involves the use of enzymes such as trypsin/EDTA or collagenase to

disaggregate the hES cell colonies into smaller pieces for the transfer into fresh

culture conditions (refer to Figure 1.3).

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Figure 1.3: Schematic representation of derivation and culturing methods of hES cells. hES

cells are mechanically or chemically dissected on ~ day7 and transferred ~1:3 to new culture

plates.

Furthermore, the fragile nature of the hES cell requires particular freezing methods

for the long term storage of these cells. It has been demonstrated that the standard

methods of liquid nitrogen storage in FBS + dimethyl sulfoxide result in cell survival

rates of about 1 to 10 percent. However, Reubinoff et al. (2001) derived “the pulled

straw method of cryo-preservation”, which allowed for significantly enhanced cell

survival rates. This cryo-preservation technique involves a series of solutions in

which the hES cell colony pieces are incubated. The pieces are then transferred to a

cryo-straw and stored at -196 degrees celsius in liquid nitrogen. This technique has

allowed for the both the long term storage and upscaled provision/generation of

several of the hES cell lines available in the market. Nevertheless, it is clear that the

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methods used to culture and store hES cells to date are not optimal and there is a

need to develop novel culturing methods if hES cells are to prove useful for

therapeutic applications.

1.2.3 The Potential of Human Embryonic Stem Cells.

Scientists currently have the ability to culture hES cell colonies in relatively large

amounts whilst still maintaining them in an undifferentiated state. This has led to

studies which exploit the totipotential ability of hES cells including: generating new

tissues for donation (Docherty 2001; Guan et al. 2001; Bagutti et al. 1996);

investigating the complex events that occur during the early developmental stages

(Dinsmore et al. 1998); and the use of these cells to examine the toxicity or efficacy

of a new drug/treatment (Rohwedel et al. 2001). Dinsmore et al. (1998) discovered

components that trigger the hES cells to differentiate into dopamine neurons. It is

from these developmental discoveries that scientists will be able to understand and

map out the sequence of events that occur during the evolution of the embryonic

stem cell to the somatic cell. Another emerging use for human embryonic stem cells

is in the testing of new drugs and chemicals. Due to the potentially hazardous side

effects it is often hard to test experimental drugs and chemicals on humans.

Therefore, human embryonic stem cells can be manipulated to form specific tissue

types for testing these drugs and chemicals. This not only removes the risk of

adverse effects and/or injury to the patient but also provides scientists with a readily

available and relevant cell line to test their drugs (Rohwedel et al. 2001). It is hoped

that the hES cell developmental studies will reveal appropriate environmental, and

culture conditions, for each of the specific cell types within the body, thus providing

society with a viable alternative to current transplantation therapies.

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The totipotential behaviour of the hES cells provides a means of generating new

cells and tissue for therapeutics. These cells originate from a structure known as the

blastocyst. As the blastocyst progresses through it’s developmental stages, the inner

cell mass continues to differentiate resulting in gastrulation and the formation of the

three embryonic germ layers. These layers are commonly known as the ectoderm,

mesoderm, and endoderm. It is from these three germ layers that all the somatic cells

and tissues within the body are formed, thus demonstrating the potential value of

these cells for use as a therapeutic tool. Interestingly, these cells have recently been

shown to also differentiate into haploid cell types such as sperm and egg cells

(Geijsen et al. 2003). As development progresses the three embryonic germ layers

start to differentiate into their prospective tissues. For example, ectoderm derives

into the nervous tissue; the endoderm derives into the gut endothelium and the

mesoderm derives into the connective tissue such as striated muscle (refer to Figure

1.4).

As eluded to earlier, investigators have discovered that they can supply a range of

conditions and growth factors to the hES cells to drive them towards specific cell

lineages, thus creating a source of tissue that could potentially be used for

therapeutic applications. Lumelsky et al. (2001) have discovered a method for

driving embryonic stem cells into insulin-secreting structures similar to that of the

pancreatic islets. Furthermore, Boheler et al. (2002) have discovered ways to drive

embryonic stem cells into cardiomyocytes. Interestingly, these cardiomyocytes were

demonstrated to have the ability to spontaneously beat, thus representing beating

heart tissue. The applications for generating transplantable tissue from embryonic

stem cells are enormous, with scientists now able to create both haploid and diploid

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cells. However if these cells are to prove useful there are a number of hurdles to

overcome, i.e. specifically, the safe culture of these cells.

Figure 1.4: Schematic representation of the differentiation of hES cells into the three germ

layers. Adapted from en.wikipedia.org/wiki/User:Lexor/Temp/Cell_(NCBI)

1.2.4 Issues Associated With Human Embryonic Stem Cells.

There are several problems associated within the field of hES cell research. The

major disadvantages being: the moral implications of the scientific technologies and

procedures (McLaren 2002); the second disadvantage is that hES cells are

allogeneic; the third disadvantage is the lack of definitive assays for determining

whether the hES cells are differentiated; and the fourth being concerns with cross

contamination from the feeder cell layers and / or the serum during the culture

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process. Additionally, there are concerns with safety and resource allocation. The

harvesting of hES cells is carried out by culturing cells from the inner cell mass

(ICM) of a blastocyst, which are donated with consent, from people involved with

an IVF program. This process requires dissolving the blastocyst, which has led to

controversial debates focusing on the moral and legal status of the human embryo.

The Catholic and Anglican churches have taken the position that from the moment

of conception the human embryo/foetus constitutes an individualised human entity.

The question is, does life begin at conception or does it in fact start with the

development of consciousness? This question will be the main restriction for some

time as society tries to weigh the moral issues against the promise of a healthier

tomorrow that the human embryonic stem cells can give.

While ethical and moral considerations will always exist, other biological problems

affecting this area of science are the lack of definitive assays for recognising a hES

cell and the rejection of non-self tissue. For hES cells to be therapeutically

beneficial, immune rejection needs to be overcome. One way to combat this

problem is through therapeutic cloning, which is accomplished by removing the

nucleus of either a donor or self oocyte, and inserting the nucleus of a self somatic

cell into this oocyte. The cell is then stimulated to divide to produce a blastocyst for

the harvesting of embryonic stem cells, which can later be used for therapeutic

applications free from the risk of immune rejection. Furthermore, novel molecular

engineering approaches have also started to address potential problems such as the

adverse immunological responses elicited by allogeneic hES cell antigens (Rideout

et al. 2002). It has also been reported that cells from embryonic origins present

fewer immunological response stimuli, than for example, allogeneic adult

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stem/progenitor cells (McLaren 2002). Moreover, there are many problems

associated with the lack of knowledge on what a human embryonic stem cell is.

Due to the infancy of this field of research there are currently no definitive testing

methods to prove that the cultured hES cells are healthy and have retained their

undifferentiated state. Nevertheless, certain tests are commonly applied in this field,

albeit with some limitations, to identify that the cells have maintained their

embryonic-like nature. For example, karyotyping is routinely used to determine if

abnormalities such as chromosome exchange have occurred in cultured hES cells

(Amit et al. 2000). Furthermore, other tests can be applied to hES cells to identify

whether they express several genes/markers such as stage specific embryonic

antigens (SSEAs) (Draper et al. 2002). Most of the testing revolves around the use of

PCR and biochemical methods for the detection of undifferentiated hES cells (zur

Nieden et al. 2001). In addition to marker detection, alkaline phosphatase and

telomerase are two enzymes commonly expressed by hES cells and thus are used to

categorise these cells. The presence of these two enzymes indicates the degree of

differentiation within these cells (Lanzendorf et al. 2001). Currently, the most

definitive test for truly totipotent hES cells is the formation of teratomas (a complex

tumor containing several different tissue types) following the injection of hES cells

into severe combined immunodeficiency (SCID) mice (Richards et al. 2002).

The previously mentioned methods for propagating the hES cells have proved to be

effective. However, a significant problem exists in that they require xenogeneic or

allogeneic products such as human or animal serum, and require the presence of

allogeneic feeder cells such as mouse embryonic fibroblasts (MEF) (Henderson et al.

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2002; Schick et al. 2003). The presence of these components presents a significant

risk to patients that may ultimately be treated with these cells. For example, it is

possible that patients may inadvertently be infected with diseases such as “new

variant CJD”, which may be present in these poorly-defined animal products. More

recently, Dr. Ajit Varki demonstrated that hES cell lines were starting to express a

non-human sialic acid (Neu5Gc), which was thought to have come from either the

serum or the mouse embryonic fibroblast feeder-layer (Martin et al. 2005). This

finding demonstrates that the hES cells are vulnerable to what is present in their in-

vitro micro-environment.

In view of this, several investigators have attempted to address the problems

associated with the use of animal products in the culture of these cells by replacing

these animal derived feeder cell layers with human-derived feeder cell layers (Amit

et al. 2000; Richards et al. 2002; Cheng et al. 2003). While this has proven

successful it still does not remove the risk of cross-contamination from diseases that

could be potentially carried by the human derived feeder cell layer. Alternatively,

other researchers have adopted the approach of removing the feeder cell layer totally

and replacing it with an extra-cellular matrix (ECM) protein. For example, laminin

and matrigel™ (a solubilised basement membrane preparation extracted from the

Engelbreth-Holm-Swarm (EHS) mouse sarcoma), were demonstrated to replace the

need for a feeder cell layer (Xu et al. 2001). Moreover, it was revealed that the hES

cells could survive for ~130 population doublings whilst still maintaining an

undifferentiated state. However, this system only proved functional with the addition

of MEF conditioned medium, which still carries the risk of transmitting diseases to

the hES cells. Moreover, due to the fact that matrigel™ is derived from xenogeneic

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origins this also poses the risk of contaminating the hES cells. Furthermore, Richards

et al. (2002), Cheng et al. (2003), Amit et al. (2000), as well as our own group

(unpublished data), have found it difficult to duplicate the results reported by Xu et

al. (2001). These difficulties in reproducing Xu’s work could be attributed to hES

cell variability between labs.

More recently, Amit et al. (2004) discovered a method to propagate these cells using

serum replacement medium with a range of growth factors such as, transforming

growth factor β1 (TGF β1), leukaemia inhibitory factor (LIF), basic fibroblast

growth factor (bFGF), and a fibronectin matrix. In this study they demonstrated that

the hES cells could be propagated for over 20 passages by replacing both the serum

and feeder cell layer with an ECM protein and a range of growth factors.

Nevertheless, there was still a requirement for Knockout Serum Replacement (KSR)

(Invitrogen), which is a commercial serum product from Invitrogen and is still not

fully defined. Perhaps one of the more promising advances in the removal of the

feeder-layer was established by Beattie et al. (2005). They demonstrated that hES

cells could be serially propagated for greater than 20 passages by substituting the

feeder-layer with a laminin coated culture vessel and by supplementing the media

with activin A, nicotinamide and keratinocyte growth factor. While this is an

important step forward, this protocol still had a dependence on Knockout serum

replacement.

Recently, scientists discovered that they can maintain the hES cells in a pluripotent

state by triggering certain pathways in development such as the Wnt signaling

pathway (Sato et al. 2004). Thus, Sato et al. (2004) demonstrated that a protein found

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in mollusks known as 6-bromoindirubin-3`-oxime (BIO) is a critical activator of the

Wnt signaling pathway. They demonstrated that BIO has the ability to stimulate

feeder-free self-renewal of the hES cells and that this reaction was reversible through

the removal of the BIO compound. However, this approach has not yet been

demonstrated to support long-term, serial, undifferentiated passage of hES cells, and

hence the use of BIO as an alternative to feeder cells is far from established. It is of

vital importance that a serum-free and feeder-free methodology, for the

establishment and bulk culture of these cells, be developed so that we can take

advantage of the human embryonic stem cell potential.

1.3 Skin

The early techniques developed for culturing hES cells were originally based on

those developed for the ex-vivo expansion of skin cells. In view of this I will also

review the literature with respect to skin and more specifically, keratinocyte cell

culture.

Skin is the largest and one of the most complex organs of the human body. This vital

sensory organ carries out many functions, which include: acting as a physical barrier

from our external environment; immune surveillance; aiding the production of

hormones/vitamins; and facilitating various homeostatic functions such as

temperature regulation and the maintenance of fluid levels. The skin is a multilayer

tissue made up of an epidermis, which is separated from the dermis by a basement

membrane composed of several extra-cellular matrix (ECM) proteins (refer to Figure

1.5).

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Figure 1.5: Anatomy of the skin. Taken from Wikipedia:- This file is licensed under

Creative Commons Attribution 2.5 License. Attribution: http://www.3dscience.com.

The epidermis is an epithelial cell layer primarily composed of keratinocytes. These

cells are involved in the self-renewal, maintenance and formation of the skin’s outer

layer (Leary et al. 1992). Keratinocytes are thought to arise from a more primitive

‘keratinocyte stem cell’, which exists within the basal layer of skin, the stratum

basale, (Kaur et al. 2004; Li and Kaur 2004; Li et al. 2004). As these cells divide and

mature they migrate to the outer surface of the skin forming 4 distinct layers in the

process, the stratum spinosum, stratum granulosum, stratum lucidium, and the

stratum corneum (refer to Figure 1.6). Whilst these cells are migrating through the

strata they undergo a process named keratinisation. The keratinisation process results

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in a change in the keratinocyte phenotype and function resulting in the formation of

the protective outer layer of skin.

Figure 1.6: Strata of the epidermis.

1.3.1 Keratinocytes

Due to the critical role that keratinocytes have in the maintenance and formation of

skin, researchers worldwide have been exploiting these cells for use in a range of

skin defects such as the treatments of burns and skin ulcers (Green 1991; Mean et al.

1998; Wright et al. 1998). It was in 1975 that Rheinwald and Green developed the

first viable methodology for the culture of human epidermal keratinocytes in-vitro

(Rheinwald and Green 1975). In this study they demonstrated that keratinocyte cells

could be obtained from patient skin biopsies and co-cultured with irradiated murine

i3T3 cells in the presence of serum to produce a system that supported the serial

propagation of human keratinocyte cells. However, as alluded to earlier, a significant

Dermis Stratum Basale

Stratum Spinosum

Stratum Corneum

Stratum Lucidium Stratum Granulosum

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problem arises with culturing keratinocyte cells in the presence of human and/or

animal products such as serum and mouse fibroblasts as “feeder” cells. The

introduction of these foreign products into the culture presents a risk in that patients

may inadvertently be infected with diseases such as “new variant Creutzfeldt-Jacob

disease (CJD)”, thought to be derived from Bovine Spongiform Encephalopathy

(BSE), which may be present in these ill-defined animal products (Rolleston 1999).

The risk of infection, albeit small, will eventually make it difficult, if not impossible,

for keratinocyte cell-based treatments to gain broad approval as therapeutics by the

Food and Drug Administration (FDA) and the Australian Therapeutic Goods

Administration (TGA). As is the case with hES cells, if these skin cells are to

maintain their usefulness in an ever changing regulatory environment, improved cell

culture technologies need to be developed to eliminate the risk of pathogens

contaminating the cultured cells, whilst at the same time providing the necessary

conditions for their in-vitro expansion.

1.3.2 Keratinocyte Culture

Due to the need to remove both the serum and the feeder cell layer from skin culture,

research efforts have been trying to address the problems associated with the use of

donor and animal-derived components in this system. Thus far, the main focus has

been the removal of serum from the culture. Currently, serum-free alternatives for

the growth of keratinocytes are commercially available. Defined keratinocyte

medium (Invitrogen, Mulgrave, VIC, Australia) and MCDB 153 (Sigma, St. Louis,

MO) are examples of these products, all of which have been demonstrated to support

the propagation of keratinocytes. However, these products all require the inclusion of

human and/or animal products, such as purified human serum albumin (HSA) or

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bovine pituitary extract (BPE), for the long-term survival of keratinocytes and also

generally require high cell seeding densities.

Perhaps one of the most difficult components to remove from the culture of

keratinocytes is the feeder cell layer. Indeed, Sun et al. (2006) demonstrated that the

fibroblasts could normally migrate into fibrin gels when cultured alone. However,

when co-cultured with the keratinocytes this fibroblast migration was reduced,

suggesting that the keratinocytes require the fibroblasts to be in close proximity.

Certain groups are currently focused on examining whether extra-cellular matrix

(ECM) proteins can provide the solution to serum-free and feeder-free culture

techniques. One such approach involves the use of laminin 10/11, a common ECM

protein found in adult skin (Pouliot et al. 2002). While it has been demonstrated that

laminin can provide the cells with an environment favourable for attachment,

proliferation and migration, the use of a keratinocyte growth medium containing

BPE was still required for the establishment and growth of these cells. Whilst these

novel approaches address the potential problems of pathogen transfer that exist

through the use of serum, they do not avoid the problems associated with poorly-

defined and uncharacterised compounds such as BPE and HSA found in the serum-

free media.

Our laboratory has been investigating alternative culture technologies comprising of

vitronectin (VN), Insulin-like growth factor-I (IGF-I) and Insulin-like growth factor

binding protein-3 (IGFBP-3) (VN:GF medium), suitable for adult and embryonic

stem cells and it is thought that this culture methodology may prove useful for the

establishment and in-vitro expansion of keratinocytes for skin grafting applications.

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The VN:GF medium, which combines the ECM protein VN, a major component of

human serum, with IGFBP-3, IGF-I, may provide a substitute for both the serum and

the feeder cell layer. In view of this, we hypothesised those in-vitro culture

technologies incorporating the VN:GF combination, may be useful for developing a

fully defined serum-free medium suitable for in-vitro expansion of human

keratinocytes for clinical applications such as cultured epithelial autografts for

patients.

1.4 Vitronectin

Vitronectin (VN) is an extra-cellular matrix ECM glycoprotein with an open reading

frame of 459 amino acids including a 19 amino acid signal peptide resulting in a 75

kDa mature protein. Proteases can cleave this 75 kDa protein to yield 65 kDa and 11

kDa disulfide-linked fragments (Kitagaki-Ogawa et al. 1990; Gibson and Peterson

2001). The VN protein exists in both monomeric and multimeric forms within the

body and possess domains and binding sites which are differentially revealed

depending on the conformation of the protein. For example, the denatured form of

VN has binding sites for collagen, glycosylaminoglycans (GAGs) and urokinase

receptor urokinase complex (Seiffert 1997; Francois et al. 1999) (refer to Figure 1.7

A). An Arginine, Glycine and Aspartate (RGD) sequence exists within the VN

protein towards the N-terminus (residues 45-47); the primary function of the RGD

sequence being to mediate cell attachment and spreading (Morris et al. 1994; Seiffert

and Smith 1997), (refer to Figure 1.7 B). The cell attachment and spreading function

of VN arises when the RGD sequence interacts with certain cell-surface receptors

called integrins (αvβ3, αvβ5 αvβ1 αIIbβ3 αvβ6 αvβ8) (Schvartz et al. 1999; Nam et al.

2002). When VN binds to its integrin receptors it activates intracellular signaling

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pathways that regulate cytoskeletal reorganization, intracellular ion transport, lipid

metabolism and gene expression (Schvartz et al. 1999).

Vitronectin is predominantly synthesised in the smooth muscle cells of the liver but

is also expressed in other tissues throughout the body (Schvartz et al. 1999).

Expression has also been demonstrated at high levels by certain tumours, raising the

possibility that this protein may play a role in malignancy (Schvartz et al. 1999). The

concentrations of VN in human plasma are high, at approximately 200-400 μg/mL.

Interestingly, the plasma concentrations are up-regulated after vascular injury,

especially during the formation of new blood vessel layers (Dufourcq et al. 2002). In

addition, it has been demonstrated that VN can be deposited within the ECM of

endothelial cells and is localised at other extra-vascular sites (Zhuang et al. 1996 part

I; Schvartz et al. 1999). Taken together, this clearly demonstrates that VN has

important roles in the process of wound healing. Vitronectin’s role in the process of

wound healing has been demonstrated in studies utilizing VN knockout mice in

which the VN gene was inactivated. The knockout mice exhibited a delayed wound

healing response and an imbalance in the fibrinolytic pathway (Jang et al. 2000). In

addition, many proteases have been found to degrade VN. Thrombin, elastase, and

plasmin, which are present with VN at the wound-healing site, have all been shown

to cleave VN at its basic amino acid cluster (Gechtman et al. 1997).

Many of VN’s biological responses are mediated through specific interactions of

other proteins with the various structural domains within VN. These interactions

mediate multiple physiological functions within the ECM and the circulation

including; blood coagulation; fibrinolysis; pericellular proteolysis; complement-

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dependent immune responses; cell attachment; and cell migration (Zhuang et al.

1996 part I; Zhuang et al. 1996 part II; Chavakis et al. 1998). These activities result

in VN having roles in several diseases such as cancer, atherosclerosis and

degenerative central nervous system disorders. Relevant to this project, VN has also

been suggested to play a role in cellular differentiation during embryonic

development (Pons and Marti 2000).

The biological significance of the interactions between ECM proteins such as VN

and growth factors is becoming increasingly appreciated. Of interest to this project,

VN has been demonstrated to specifically bind IGF-II (Upton et al. 1999). While

IGF-I does not directly bind to VN, it can form a trimeric complex with VN in the

presence of select IGFBPs such as IGFBP-5 (Nam et al. 2002; Kricker et al. 2003).

Other heparin-binding growth factors have also been examined and there are

suggestions that these too have the ability to bind to VN. These growth factors

include: transforming growth factor-β (TGF-β); vascular endothelial growth factor

(VEGF); epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF)

(Schoppet et al. 2002).

Thus multimeric VN, the predominant form of VN found in the ECM, has the

potential to bind several different growth factors at the one time. Taken together,

these findings suggest that this protein, in conjunction with other binding proteins,

may be a potential mechanism for the delivery and concentration of growth factors at

their cell surface receptors. Indeed, this will be the foundation from which I propose

to develop a serum-free and feeder-cell free culture technology, specifically focusing

on hES and primary keratinocyte cells.

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Figure 1.7: Schematic representation of the vitronectin protein and its binding domains;

for plasminogen activator inhibitor-1 (PAI-1), urokinase receptor (uPAR), integrins,

thrombin–antithrombin III complex (TAT) and collagen are located in the N-terminus of the

molecule, while the binding domains for plasminogen, heparin and PAI-1 are located in the

carboxyl terminal edge (Scharvtz et al. 1999).

1.4.1 (VN:GF) technology

The VN:GF technology being developed at QUT relies on elucidating optimal

complexes of VN, growth factors and binding proteins to support the ex-vivo

survival and growth of particular cells. Initial studies performed within the Tissue

Regeneration and Repair program have examined the ability of different

combinations and concentrations of IGF-I, IGF-II, IGFBPs, EGF, FGF and VN to

support the short-term serum-free, feeder-cell free expansion of primary adult skin

and corneal-derived keratinocytes. The initial research in our program has focused

on complexes comprised of VN and IGFs.


halla

This figure is not available online. Please consult the hardcopy thesis available from the QUT Library

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IGF-I and IGF-II have been demonstrated to stimulate DNA synthesis, cell cycle

progression, angiogenesis and differentiation. Although IGF-II is considered to be

less potent than IGF-I (Clemmons 1998; Marinaro et al. 1999) it is the pre-dominant

IGF expressed during embryogenesis in humans (Nonoshita et al. 1994). The IGF

proteins primarily act through the type-1 IGF receptor (IGFI-R) and their ability to

interact with this receptor is modulated by at least six IGFBPs (Marinaro et al.

1999). It has recently been demonstrated that IGFBP-3 and -5 bind to VN via the

heparin binding domain (HBD) present in many of the IGFBPs (Nam et al. 2002;

Kricker et al. 2003). Moreover, it has been found that IGFBP-5 enhances the effects

of IGF-I in the presence of VN (Rees and Clemmons 1998; Nam et al. 2002). More

recently, our laboratory has also demonstrated that IGFBP-2, -3, -4 and -5 enhance

the proliferative and migratory effects of IGF-I in the presence of VN (Upton and

Kricker 2002; Kricker et al. 2003; Noble et al. 2003). These data have stimulated us

to analyse the effects of trimeric complexes, consisting of VN, IGF-I and IGFBPs,

and dimeric complexes, consisting of IGF-II and VN, on the attachment,

proliferation and migration of a range of cell lines including short-term studies in

hES cells. Furthermore it has been demonstrated that these enhanced effects require

activation of not only the IGFI-R, but also require VN to bind its cell-surface

receptors, the αv integrins.

Interestingly, prior studies examining combinations of IGF-I and EGF have been

shown to increase growth of keratinocytes beyond the responses that either of these

mitogens elicits alone (Vardy et al. 1995). It has also been demonstrated that IGF-I

increases the levels of the EGF receptor (EGFR) in keratinocytes (Krane et al. 1991)

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and that EGF can enhance the growth of keratinocytes in media and prevent

senescence (Rheinwald and Green 1977). Thus our laboratory hypothesised that EGF

can be used with IGF in VN:GF complexes to further enhance cell proliferation and

migration of cells. Furthermore, FGFs are potent inducers of cell attachment and

proliferation (Heun Rho et al. 2001; Tanghetti et al. 2002) and certain FGFs, in

particular FGF-4 and FGF-8, have been demonstrated to be important during

embryonic development (Wilder et al. 1997; Valve et al. 2000). Hence FGF-4 and

FGF-8 are candidate mitogens that are being considered for incorporation into novel

VN:GF complexes designed to support growth of cells, especially hES cells. Indeed

it has recently been demonstrated that IGF-I enhances DNA synthesis in

oligodendrocyte stem cells to a greater degree when in the presence of FGF-2

(bFGF) (Jiang et al. 2001). In addition, the incorporation of bFGF into the hES cell

culture media enhances the undifferentiated growth of these cells (Cowan et al.

2004). Of importance to this project, we have demonstrated that incorporating bFGF

into the VN:GF complexes enhances the proliferation of keratinocyte cells (Hollier

et al. 2005). In addition to exploring various VN:GF combinations as a serum

substitute for the growth of keratinocytes and hES cells, it is also of great importance

to study what critical factors the feeder cells supply to the micro-environment of

these two cell types.

1.5 Conclusion

Clearly in-vitro cell culture research is in its infancy and many questions remain to

be answered i.e. how can we propagate cells without the need for serum? Thus, I

hypothesised that the VN:GF combinations can be translated into a serum-free media

for the serial propagation of primary keratinocytes and hES cells. Additionally, I

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believed that the investigation of the in-vitro micro-environment of primary

keratinocytes would reveal potential candidate proteins that will aid in the removal

of fibroblast feeder cells from the keratinocyte culture system. To this end the aim of

this PhD project was to specifically examine whether VN:GF complexes can be

developed to support serum-free and feeder-cell free culture of both primary

keratinocytes and hES cells. The specific aims were to: 1) develop a serum-free

medium for primary keratinocytes; 2) develop a serum-free medium for hES

cells; and 3) explore the in-vitro micro-environment of primary keratinocytes

using proteomic approaches to reveal novel candidate factors that may

ultimately lead to the replacement of feeder cells. The development of synthetic

culture methods, such as those potentially encompassed by the VN:GF complexes

technology, will be vital for eliminating the need for animal and semi-defined

products for the propagation of these cells. Furthermore, the cultivation of hES cells

is still in it’s infancy with many of the technologies currently used with these cells

being ‘bucket science technologies’. Thus, the third aim of my PhD project was to

help fill this gap in the knowledge by conducting proteomic studies. This approach

would provide insights into the several important pathways that are active during the

development and differentiation of cells. Hence, the information derived from

addressing these three aims may lead to developing not only an animal free culture

system for these cells, but will also be the foundation for creating viable cell types

that may provide clinicians with a readily available source of tissue for

transplantation therapies. To this end the investigations reported in this thesis

demonstrated: the serum free isolation, establishment and serial passaging of primary

human keratinocytes using a VN:GF combination specific for keratinocytes; the

establishment and serial passaging of hES cells using a second VN:GF combination

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specific for hES cells; and finally the identification of factors secreted by feeder cells

alone as well as feeder cells co-cultured with keratinocytes using a novel proteomics

approach. Together this data provides an enhanced understanding of the factors

required by these cells for their successful in-vitro expansion.

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CHAPTER 2

The serum-free culture of human keratinocytes

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2.1 INTRODUCTION

Human skin is primarily composed of keratinocytes, which are involved in the self-

renewal, maintenance, and formation of the skin’s epidermal layer (Leary et al.

1992). There are several different types keratinocytes ranging from the more

primitive, or stem cell-like keratinocytes (Kaur et al. 2004; Li and Kaur 2004; Li et

al. 2004), which are located near the basement membrane, through to the more

differentiated keratinocytes, located at the outermost layer of the skin. The cells

closer to the external environment phenotypically change via a keratinisation process

that acts as our natural barrier to external elements.

As stated in Chapter 1 the first successful propagation of keratinocytes in-vitro was

achieved in 1975 (Rheinwald and Green 1975). They discovered that an inactivated

feeder cell layer and animal serum could provide an in-vitro micro-environment

favourable for keratinocyte growth. The ability to grow large quantities of

keratinocytes in-vitro has provided clinicians and scientists with a useful research

tool to develop techniques for the repair of skin defects such as, burns and skin

ulcers (Green 1991; Meana et al. 1998; Wright et al. 1998). However, using

xenogeneic products such as serum and mouse fibroblasts can lead to the

introduction of contaminating products, such as Bovine Spongiform Encephalopathy

(BSE) (Rolleston 1999), to the keratinocytes. Clearly, improved cell culture

technologies need to be developed to eliminate the risk of pathogens contaminating

the cultured cells, whilst at the same time providing the necessary conditions for

their in-vitro expansion.

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Currently, serum-free alternatives for the growth of keratinocytes are commercially

available. Defined keratinocyte medium (Invitrogen, Mulgrave, VIC, Australia) and

MCDB 153 (Sigma, St. Louis, MO) are examples of these products, all of which

have been demonstrated to support the serum-free and feeder cell-free propagation of

keratinocytes. However, these products require the inclusion of undefined human

and/or animal products, such as purified human serum albumin (HSA) or bovine

pituitary extract (BPE), for the long-term survival of keratinocytes and also generally

require isolation using serum and high cell seeding densities. The problem associated

with high seeding cell densities is that this scenario may not be possible in a clinical

setting if patients are suffering from large surface area damage. Furthermore, if the

keratinocytes are originally isolated using serum, they can still be potentially

contaminated at this stage, thereby removing any advantage gained through the

subsequent expansion using serum-free or feeder cell-free technologies.

From studies reported thus far, it would appear that the most difficult component to

remove from the culture of keratinocytes is the feeder cell layer and scientists are

now looking to the extra-cellular environment for the answer. Indeed, extra-cellular

matrix (ECM) proteins, such as laminin 10/11, have been used to provided

keratinocytes with a favourable environment for their attachment, proliferation and

migration (Pouliot et al. 2002). Whilst this technology provided a serum-free and

feeder cell-free culture environment, bovine pituitary extract (BPE) was still required

for the establishment and growth of these cells. Whilst these novel approaches

address the potential problems of pathogen transfer that exist through the use of

serum, they do not avoid the problems associated with poorly defined and

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uncharacterised compounds such as BPE and human serum albumin (HAS) found in

the serum-free media.

The removal of a feeder cell layer from the culture of keratinocytes may not be

possible, thus Bullock et al. (2006) demonstrated the successful propagation and re-

epithelisation using a human-derived feeder cell layer and serum free conditions.

Nevertheless, this methodology still involves the use of serum for the initial

keratinocyte isolation and trypsin neutralistion, thus, resulting in the carry over of

serum components to the keratinocytes. Furthermore, human derived pathogens

could be easily transferred from the human feeder cell layer to the primary

keratinocytes.

Therefore our laboratory has been investigating alternative culture technologies

suitable for adult stem cells and has recently discovered a technique that may lead to

a fully defined serum-replacement method for the establishment and in-vitro

expansion of keratinocytes for skin grafting applications. This new technology is

based on the finding that a synergistic effect occurs between growth factors and a

specific extra-cellular matrix (ECM) protein called vitronectin (VN) (Hollier et al

2005; Kricker et al. 2003; Upton and Kricker 2002). This has led to the development

of novel dimeric, trimeric and multimeric growth-promoting combinations

incorporating growth factors such as insulin-like growth factors (IGFs) and insulin-

like growth factor binding proteins (IGFBPs) in conjunction with VN (VN:GF).

The addition of these VN:GF combinations to defined media has been demonstrated

to stimulate short-term migration and proliferation in a range of cells, including adult

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skin and corneal-derived keratinocytes (Ainscough et al. 2006; Hollier et al 2005;

Hyde et al. 2004). In view of this, I hypothesised those in-vitro culture technologies

incorporating ECM proteins and growth factors, such as those encompassed by these

VN:GF medium, may be useful for developing animal-product free media suitable

for in-vitro expansion of human keratinocytes for clinical applications such as

cultured epithelial autografts for burns patients. In the study reported here, I

examined the long-term survival and biological responses of a continuous

keratinocyte cell line (HaCaT), as well as primary keratinocyte cells derived from

adult human skin when grown in the presence of media containing the VN:GF

medium.

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2.2 MATERIALS AND METHODS

2.2.1 Ethics and material collection

Ethics for this project was approved by the Human Research Ethics Committee (ID:

3673H) (Queensland University of Technology) and the St. Andrews and Wesley

Hospitals, Brisbane, Australia. Skin was obtained from consenting patients

undergoing breast reductions and abdominoplasties.

2.2.2 Isolation of primary keratinocytes

Primary keratinocytes were isolated from split thickness skin biopsies obtained from

breast reductions and abdominoplasties as described by (Goberdhan et al. 1993).

Briefly, this method involved dissecting the skin biopsy into 0.5 cm2 pieces followed

by a series of antibiotic wash steps. The skin was then incubated in 0.125% trypsin

in PBS (Invitrogen, Mulgrave, VIC, Australia) overnight at 4°C. The isolation step

differed significantly from Goberdhan’s method in that all steps were conducted

serum-free. The trypsinised skin pieces were removed from the trypsin and

suspended in 50 mLs of Dulbecco’s modified eagle media (DMEM) (Invitrogen).

Epidermal and dermal layers were separated and keratinocytes removed via gentle

scraping. Keratinocyte cells were then suspended in DMEM (Invitrogen), filtered

(100 µm), and pelleted via a 500-600 g centrifugation step for 5 minutes.

2.2.3 Standard culture

The freshly isolated keratinocytes were then cultured on serum starved gamma-

irradiated (two doses of 25 Gy) (Australian Red Cross Blood Service, Brisbane,

QLD, Australia) mouse i3T3 cells (ATCC# CCL-92) using Complete Green’s (CG)

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media, which incorporated, DMEM/HAMS medium (Invitrogen); 0.4 μg/mL

hydrocortisone; 10 μg/mL EGF (Sigma-Aldrich, Castle Hill, NSW, Australia); 0.1

nM cholera toxin; 1.8x10-4 M adenine; 2x10-7 M triiodo-l-thyronine; 5 µg/mL

insulin; 5 μg/mL transferrin; 2x10-3 M glutamine (Invitrogen); 1000 IU/mL penicillin

/ 1000 μg/mL streptomycin (Invitrogen); and 10% foetal bovine serum (Trace

Scientific, Noble Park, VIC, Australia). The cultures were established in 25 cm2

flasks at a density of 1x106 cells and incubated at 37°C in 5% carbon dioxide, with

media changes every third day. The cells were seeded at 2.5 x 105 cells per 25 cm2

flask for subsequent passages.

2.2.4 VN:GF Culture

The serum-free culture of the freshly isolated keratinocytes involved the use of the

previously mentioned irradiated i3T3 cells with the incorporation of Stripped

Green’s (SG) medium. This medium is the CG medium described above but without

serum, EGF, and insulin. The VN:GF culture media was created by adding, 0.6

µg/mL VN (Promega, Annandale, NSW, Australia), 0.6 µg/mL IGFBP-3 (N109D

recombinant mutant) (Auspep, Parkville, VIC, Australia), 0.2 µg/mL IGF-I (GroPep,

Adelaide, SA, Australia) and 0.2 µg/mL EGF (Invitrogen) (VN:GF) to 5 mL of SG

media in a 25 cm2 flask. The keratinocytes isolated from skin as described in section

2.2.2 were seeded at an initial density of 1 x 106 cells and incubated at 37°C in 5%

carbon dioxide, and re-fed every third day with half the amount of the VN:GF media

as described above. The cells were seeded at 2.5 x 105 cells per 25 cm2 flask for the

subsequent passages.

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2.2.5 Defined Keratinocyte Media (DKM) culture

Cells were also grown in a commercially available serum-free keratinocyte medium

developed for the in-vitro propagation of keratinocyte cells. The DKM media

(Invitrogen) evaluated includes animal and human products; however these were not

clearly defined by the manufacturer. The DKM cultures were set up in both the

presence and absence of the irradiated i3T3s. The “no i3T3” cultures were

established due to the fact that this product states that it is feeder and serum-free. The

keratinocyte cultures were seeded at an initial density of 1 x 106 cells in 25 cm2

flasks and incubated at 37°C in 5% carbon dioxide, using 5 mLs of DKM media,

with media changes occurring every third day. The cells were seeded at 2.5 x 105

cells per 25 cm2 flask for the subsequent passages.

2.2.6 HaCaT culture

The HaCaT cells (human keratinocyte cell line), obtained from Professor Norbet

Fusenig (DFZ, Heidelberg, Germany), were cultured using CG, VN:GF media and

DKM medium in the absence of feeder cells. These cultures were seeded at 2 x 105

cells per 25 cm2 flask serially propagated at 37°C in 5% carbon dioxide, with media

changes every third day.

2.2.7 Proliferation Assays

Proliferation was measured using two methods; the first method involved monitoring

the metabolic activity of the mitochondria with Thiazolyl blue tetrazolium bromide

(MTT) (Sigma Aldrich). MTT assays were performed in 24-well plates that were

pre-seeded with 1 x 105 HaCaTs/well and grown for 72 hours. Cultures were then

washed twice in PBS and incubated with MTT for 1 hour. The MTT was removed

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from the wells, which were subsequently washed as previously described. Dimethyl

sulfoxide was then added to the wells and the absorbances of the resulting solutions

were measured at 540 nm – 630 nm (Ealey et al. 1988). The MTT assays were

conducted in triplicate and all experiments were replicated at least twice.

Keratinocytes were cultured in 25 cm2 flasks in parallel with the MTT assays to

enable cell counts to be conducted on cells grown in the various treatments. The

i3T3 cells were firstly removed as described above, followed by a 0.05%

trypsinisation step to remove the keratinocytes from the 25 cm2 flasks. Both the

HaCaT and primary keratinocyte cells were resuspended in CG and counted using a

haemocytometer. Both proliferation assays (MTT and cell count) were conducted in

triplicate and all experiments repeated through 4 passages (P0-P4). P0 cells were

keratinocytes freshly isolated from patient biopsies. Three different patient samples

were used to conduct this study.

2.2.8 Immunohistochemistry

Immunohistochemistry was performed at several different passages to ensure that the

keratinocytes had maintained their basal phenotype. Mouse antibodies to keratin 6

(present in hyper-proliferative skin), keratin 14 (present in basal cells), and keratin

1/10/11 (present in more differentiated, supra-basal cells) (Research Diagnostics

Inc., Flanders, Ca, USA) were used in this study. Cells grown in the various

treatments were incubated in their respective media treatments for 2 days following

seeding in 96-well plates. Media was aspirated from the plates, and cells washed

twice in PBS. All treatments were incubated in extraction buffer (0.5% triton X-100,

0.1 M pipes buffer, 5 mM MgCl2, and 1mM EGTA at pH 7.0) for two minutes. This

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was then followed by a 10-minute incubation in a fixation buffer (2%

paraformaldehyde in extraction buffer). Treatments were then blocked for one hour

in PBS/5% normal goat serum (NGS). The primary antibodies were incubated for an

hour in PBS/1% NGS. A series of three 1 minute washing steps were carried out

followed by one hour incubation with an alexa-488 goat anti-mouse IgG antibody

(Molecular Probes, Eugene, OR, USA). The plates were then washed 5 times and

viewed with a Nikon TE-2000 fluorescence microscope. Secondary antibody

controls were also examined in these experiments to ensure that no non-specific

binding had occurred.

2.2.9 Preparation of dermal equivalent (de-epidermised dermis)

The human skin off cuts were washed in PBS and cut into pieces measuring approx

1.38 cm X 1.38 cm such that they fitted into a 24-well culture plate. Off cuts were

then incubated in 1 M NaCl at 37°C for 18 hours after which forceps were used to

remove the epidermis, leaving behind the de-cellularised dermis (DED). Subsequent

incubations were performed every 24 hours with DMEM (Dawson et al. 2006).

2.2.10 Preparation and culture of skin equivalent

DED pieces were placed in 24-well cell culture plates with the papillary side up.

Sterile stainless steel rings (Aix Scientifics, Aachen, Germany) with a 7 mm silicone

washer base were placed in the culture wells on top of the DED pieces. P5

keratinocytes, cultured in either CG or VN:GF medium, were placed into the rings at

a concentration of 1.9 x 104 cells/ring. Subsequently, the rings were removed after 3

days of culture and the dermis plus cells (composite) were then placed onto stainless

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steel grids in 6 well culture plates. The composites were then cultured at the air-

liquid interface in either CG or VN:GF medium for 5 days.

2.2.11 Immunohistochemistry and histology of skin composites

The skin composites were washed in PBS three times then fixed using 4% formalin.

The composites were then subjected to a series of ethanol washes to dehydrate the

samples. The dehydrated composites were then embedded using paraffin and cut into

5 µm sections. A set of sectioned slides were stained using haemotoxylin and eosin

(H&E), (H&E staining conducted by Mr Don Geyer School of Life Sciences QUT).

Remaining composite sections were stained for keratin 1/10/11, keratin 6, Keratin

14, Collagen IV, and P63 (Research Diagnostics). Initially, sections were de-

paraffinised and rehydrated in 100% xylene for 10 minutes, 100% xylene for 5

minutes, 100% ethanol for 5 minutes, 100% ethanol for 5 minutes, 95% ethanol for 5

minutes, 70% ethanol for 5 minutes, and ddH20 for 10 minutes. This was followed

by two 5 minute washes in PBS. DAKO blocking reagent (Dakocytomation, Botany,

NSW, Australia) was added to the sections for 5 minutes and then rinsed off with

water. Sections were added to a PBS bath for 5 minutes then blocked using 2% BSA

for 30 minutes, this was followed by another 5 minute PBS bath. Primary antibodies

were then added to the sections and incubated for 2 hours. Primary antibodies were

then removed and sections were subjected to two x 10 minute PBS washes. The

labeled DAKO polymer was then added and incubated for 20 minutes. Sections were

then washed in PBS for 5 minutes and the DAB chromagen solution was added until

colour development is observed. Sections were then washed in PBS and

counterstained using haematoxylin for 35 seconds. The counterstain was washed for

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10 minutes under running water and the sections dehydrated using the reverse of the

above rehydration step.

2.2.12 Statistical analysis

The Tukey’s T test was used to analyse the proliferation data. A p-value of 0.05 was

determined to be statistically significant.

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2.3 RESULTS

2.3.1 The culture of a keratinocyte cell line using VN:GF medium.

Previous studies have demonstrated that HaCaT cells can attach, migrate and

proliferate in short term assays using VN:GF combinations (Hollier et al 2005; Hyde

et al. 2004). Thus we wished to examine whether the SG medium containing VN:GF

combinations (VN:GF medium) could support the serial propagation of these cells

and replace the need for serum for the long term propagation of this cell line.

Initially the HaCaT cells were grown using DMEM + 10% FCS and then later

transferred to, and serially propagated in either: CG (Figure 2.1 A and B), DKM

(Figure 2.1 C and D), or VN:GF medium (Figure 2.1 E and F). This experiment

revealed that the HaCaT cells could be successfully propagated for at least 5

passages using VN:GF medium whilst still displaying the expected cobble stone-like

morphology (Figure 2.1 F refer to arrow), whereas colonies grown using DKM

started to display a larger, more spindle-like cell phenotype representative of a more

differentiated cell (Figure 2.1 D refer to arrow). Interestingly other replicate

experiments demonstrated that the VN:GF medium could support the growth of

HaCaTs for up to 10 passages (data not shown). Therefore, it appears that the

VN:GF medium is able to support a normal morphology for the HaCaT cells through

5 passages, whilst maintaining a morphology similar to cells grown in the CG

medium (Figure 2.1 B).

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Figure 2.1: The culture of a keratinocyte cell line using the VN:GF medium. The

HaCaT cell line was subjected to a range of growth conditions as described in the materials

and methods section. The morphology of HaCaT cells grown in: A) CG medium (passage

2); B) CG medium (passage 5); C) DKM (passage 2); D) DKM (passage 5); E) VN:GF

medium (passage 2) and F) VN:GF medium (passage 5), are depicted. (Scale bar = 100µm)

(n=3).

2.3.2 Proliferation of HaCaT cells in the presence of different growth conditions.

To further assess the efficacy of these VN:GF media, HaCaT cell proliferation was

determined by MTT assay and by total cell count. We employed the cell count

method due to the fact that MTT is only an indication of metabolic activity thus may

not reflect accurately the actual proliferative response. The MTT assay revealed that

there were no significant differences between any of the treatments (Figure 2.2 A),

however, when the cells were manually counted the CG treatment significantly

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enhanced (p < 0.01 %) the proliferative response of these cells and was more

effective than either the SG + VN:GF or DKM treatments (Figure 2.2 B).

Figure 2.2: Proliferation of HaCaT cells using the VN:GF medium. Passage 1 HaCaT

cell proliferation in the presence of different media was assessed by; A) MTT and B) manual

cell counting. The following treatments were analysed: CG medium; DKM; and VN:GF

medium (SG + VN:GFs). Each treatment was replicated 3 times and in each experiment the

cells were passaged 5 times. The cells were assayed for MTT activity and cell counting at

the end of each passage. The data from these experiments was analysed using the Tukey’s

test and standard error of the mean (SEM) represented by the error bars. Significant

differences in proliferation (p < 0.05) from the Complete Green’s treatment are represented

by the asterisks (*) (n=3).

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2.3.3 Establishment of primary keratinocyte cells using VN:GF medium.

Having ascertained that HaCaT cells could be serially passaged with a defined

medium containing VN:GF medium, we then examined whether primary

keratinocyte cells derived from adult skin could be established and serially

propagated using this same method. This would determine whether the VN:GF

medium could provide a viable alternative for replacing the need for

xenogeneic/allogeneic serum for the culture of these cells.

Freshly isolated keratinocyte cells were cultured in CG + i3T3 feeder cells (Figure

2.3 A and B), DKM + i3T3 feeder cells (Figure 2.3 C and D), and VN:GF + i3T3

feeder cells (Figure 2.3 E and F). Similar to the results found with the HaCaT cells,

this experiment revealed that the primary keratinocyte cells could be isolated and

established using the VN:GF medium, thus in serum-free conditions (Figure 2.3 E).

Furthermore, these cells were successfully cultured to passage 4 using these serum-

free conditions (Figure 2.3 F refer to arrow), whereas cultures grown using DKM for

4 passages started to display a larger, more dysplastic phenotype representative of a

more differentiated cell (Figure 2.3 D refer to arrow). Therefore, VN:GF + i3T3

feeder cells is able to support the establishment and growth of primary keratinocyte

cells with a phenotype similar to those established and cultured using CG + i3T3

feeder cells (Figure 2.3 F and B respectively).

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Figure 2.3: Establishment of primary keratinocyte cells using the VN:GF medium. The

primary cells were subjected to a range of growth conditions as described in the materials

and methods section. The morphology of keratinocytes grown in: A) CG + i3T3 feeders

(passage 2); B) CG + i3T3 feeders (passage 4); C) DKM + i3T3 feeders (passage 2) and D)

DKM + i3T3 feeders (passage 4); E) VN:GF medium + i3T3 feeders (passage 2); F) VN:GF

medium + i3T3 feeders (passage 4), are depicted. (Scale bar = 100µm) (n=6).

2.3.4 Proliferation of primary keratinocyte cells in the presence of different growth

conditions.

Proliferation assays using the primary keratinocytes were undertaken to ascertain

whether the VN:GF medium was an efficient stimulator of cell proliferation. Once

again cell count was utilised, however, this time measurements were taken during all

4 passages of the cells in these treatments. For these assays we included DKM in the

presence (DKMF) and absence (DKM) of i3T3 feeder cells as shown in Figure 2.4.

Manual cell counting revealed that the DKM treatment resulted in a significantly less

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(p < 0.05) proliferative response when compared to all other treatments using the

Tukey’s test (Figure 2.4 #). Additionally, the CG treatment had a significantly

higher proliferative response than P1, P2 DKM and P3 DKMF treatments (Figure 2.4

*). All remaining treatments were equivalent in stimulating cell growth.

Interestingly, the same level of proliferation was observed through the four passages

in the SG + VN:GFs treatment. This suggests that the primary keratinocyte cells

were being maintained in a state of self-renewal. The cells shown in Figure 2.3 are

from one of the patient samples that were used in the proliferation studies.

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Figure 2.4: Proliferation of primary keratinocyte cells using the VN:GF medium.

Keratinocyte cell proliferation in the presence of different media was assessed by cell

counts. The following treatments were analysed: complete Green’s medium + i3T3 feeders

(CG); Defined Keratinocyte Medium + i3T3 feeders (DKMF); Defined Keratinocyte

Medium (DKM); and VN:GF medium + i3T3 feeders (SG + VN:GFs). Each treatment was

replicated twice for cell counts and the experiment was repeated through 4 passages. The

data from these experiments was analysed using the Tukey’s test to compare all treatments

across all passages with significant differences in proliferation (p < 0.05) being represented

by the (#). The Student T-test was utilised to compare between treatments within the same

passage with significant differences in proliferation (p < 0.05) being represented by the

asterisks (*) (n=2).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

P1 P2 P3 P4

Serum

VN:GF-KC

DKM+F

DKM

* * *

#

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2.3.5 Characterisation of primary keratinocyte cells propagated under different

conditions.

At present there are no definitive assays for determining whether cultured primary

keratinocyte cells have maintained a stem cell like state. However, keratin markers

can be used to provide useful information regarding the proliferative state of the cell,

and whether or not the cell is a basal keratinocyte (Watt 1998). Therefore antibodies

that recognise keratin 6 (present in hyper-proliferative skin), keratin 14 (present in

basal cells), and keratin 1/10/11 (present in more differentiated, supra-basal cells)

were used to assess the differentiation status of the cells cultured in these media.

Once again the cells pictured in Figure 2.3 were used to conduct these immuno-

fluorescence studies. Fluorescently-labeled secondary antibodies to the keratin

primary antibodies were used to establish whether these proteins were present.

Passage 4 keratinocyte cells propagated with CG, VN:GF media demonstrated high

expression levels of keratin 6 and 14 (Figure 2.5 C, D and F, G respectively). In

addition, low levels of K1/10/11 expression were observed in the CG and VN:GF

treatments (Figure 2.5 B and E). Similar keratin expression was also observed in the

DKMF treatments, however, passage 4 DKMF treatments appeared to have reduced

levels of keratin 6 and 14 and increased expression of keratins 1/10/11 (Figure 2.5

H-J). A “no primary antibody control”, was included by incubating the keratinocyte

cells with the secondary antibody only and this indicated minimal non-specific

binding between the secondary antibodies and the cells (Figure 2.5 A). Taken

together, these immunofluorescence studies suggest that the cells grown with these

combinations can be established and serially passaged whilst maintaining markers

that are representative of an undifferentiated state.

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Figure 2.5: Characterisation of primary keratinocyte cells using the VN:GF medium.

The primary cells were serially passaged in a range of growth conditions as described in the

materials and methods section. The expression of keratin 1/10/11, 6, and 14 expression in

cells grown in: B-D) CG + i3T3 feeders (passage 4); E-G) VN:GF medium + i3T3 feeders

(passage 4); and H-J) DKM + i3T3 feeders (DKMF) (passage 4), are depicted. (Scale bar =

100µm) (n=3).

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2.3.6 The histology and staining of the reconstituted epidermis

Having ascertained that the primary keratinocytes could be propagated for 5

passages using the VN:GF medium, I then sought to examine whether these cells

could reconstitute an epidermal layer. Histological staining demonstrated that

keratinocytes grown using VN:GF long term could in fact re-constitute the epidermis

(Figure 2.6-I A-C). However, there did seem to be a slightly different epidermal

morphology between the VN:GF (Figure 2.6-I B) and the serum-grown keratinocytes

(Figure 2.6-I C). Furthermore, these reconstituted epidermal constructs were

subjected to p63 staining to determine if there was still a population of primitive

keratinocyte cells. Clearly, the VN:GF (Figure 2.6-I E) and serum-grown (Figure

2.6-I F) epidermal constructs both expressed p63. Figures 2.6-I A and D are a

negative controls and represent cells that were transferred to the DED constructs in

the absence of either VN:GF or serum.

The ability for these cells to re-constitute the epidermis was further assessed using

keratin 6, a marker present in hyper-proliferative skin and keratin 1/10/11 a marker

present in more differentiated, supra-basal cells. Keratin 6 staining looked similar

between the VN:GF (Figure 2.6-II B) and serum (Figure 2.6-II C). However, there

seemed to be a marked increase in keratin 1/10/11 in the serum-grown treatment

(Figure 2.6-II F) over that of the VN:GF (Figure 2.6-II E). Figures 2.6-II A and D are

a negative controls and represent cells that were transferred to the DED constructs in

the absence of either VN:GF or serum.

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Figure 6-I

Figure 2.6-I: The histology and P63 immuno-staining of the reconstituted epidermis.

Primary keratinocytes were serially passaged 5 times in CG + i3T3 feeders and VN:GF

medium + i3T3 feeders. Cells were then added to the DED pieces and grown in, SG (A&D),

VN:GF medium (B&E), and CG (C&F) as described in the Materials and Methods section.

Haemotoxylin and Eosin staining was carried out on (A-C) and P63 staining on (D-F) as

described in the Materials and Methods section. All treatments were conducted in triplicate

and 3 patient samples were assessed. (Scale bar = 100µm).

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Figure 6-II

Figure 2.6-II: Immuno-staining for keratin 6 and 1/10/11 in the reconstituted

epidermis. Primary keratinocytes were serially passaged 5 times in CG media + i3T3

feeders and VN:GF medium + i3T3 feeders. Cells were then added to the DED pieces and

grown in, SG (A&D), VN:GF medium (B&E), and CG (C&F) as described in the Materials

and Methods section. Cells stained for keratin 6 are observed in panels (A-C) and keratin

1/10/11 staining in panels (D-F) as described in the Materials and Methods section. All

treatments were conducted in triplicate and 3 patient samples were assessed. (Scale bar =

100µm).

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2.4 DISCUSSION

While in-vitro cultured keratinocytes appear to have great potential to cure a wide

variety of diseases and injuries (Green 1991; Meana et al. 1998; Wright et al. 1998),

the fact that these culture systems require xenogeneic and poorly defined

components means that these cells are unlikely to be readily approved for broad

clinical use. Thus, there is a global move towards serum-free, animal product-free,

defined media for the culture of keratinocytes, and indeed other cell types. Many

researchers have investigated ways to minimise the risk of contamination from

animal products such as serum. One such example is the replacement of foetal

bovine serum with purified bovine serum albumin (Castro-Munozledo et al. 1997).

Whilst this method did prove successful in the propagation of keratinocytes, it does

not remove the risk of contamination by the BSA from the culture system. Nor does

it result in a fully defined media as the BSA used was purified from serum rather

than made recombinantly.

As irradiated feeder cells (i3T3) are known to secrete large quantities of IGFs and

ECM proteins (Barreca et al. 1992), and given that VN is a major component of

serum (Schvartz, et al. 1999), the hypothesis underlying this project was that

combinations comprised of IGFs and VN may trigger matricellular signaling events

that support the serum-free establishment and growth of human keratinocyte cells.

Moreover, keratinocytes have also been demonstrated to express the receptors for

both these proteins (Adams and Watt 1991; Haase et al. 2003; Rodeck et al. 1997;

Stoll and Elder 1997; Watt and Jones 1993; Watt et al. 1993). Indeed, other

laboratories have investigated the use of these proteins for the culture of

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keratinocytes. For example, Dawson et al. (1996) demonstrated that keratinocytes

can attach and proliferate in response to VN-coated surfaces.

Our studies here, differ, however, as they specifically exploit VN’s demonstrated

ability to bind to IGF-I through the mediation of IGFBP3 (Kricker et al. 2003). We

have previously demonstrated that this interaction can significantly enhance cell

attachment, migration and protein synthesis in short term assays in a range of tissues,

including skin, corneal and breast epithelial cell lines (Kricker at al. 2003; Noble et

al. 2003; Upton et al. 1999; Upton and Kricker 2002). Indeed, it was these results

that prompted us to investigate whether the VN:GF technology could be translated

for the undifferentiated, serial propagation of primary keratinocyte cells. Recently,

Dawson et al. (2006), demonstrated that primary keratinocytes could be serially

propagated using complexes of VN:IGFBP-3:IGF-I:EGF or VN:IGFBP-5:IGF-

I:EGF. The Dawson study revealed that the keratinocytes could be propagated

through to passage two and that the complex containing IGFBP-5 demonstrated a

morphologically better cell. However, this work still involved the use of serum in the

isolation of the primary keratinocyte cells, thus, introducing another potential source

of contamination. Therefore, I hypothesised that VN:GF combination could be

improved in two ways, firstly by incorporating the complex technology into a serum-

free medium rather than as a pre-bound substrate, and secondly using the complexes

to replace the need for serum in the isolation steps.

To examine this hypothesis we firstly examined the potential of the VN:GF medium

to support the long-term growth of the HaCaT keratinocyte cell line. We demonstrate

here, that the HaCaT cell line can be serially propagated using the VN:GF medium

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(Figure 2.1). However, these experiments revealed that the culture of HaCaTs in the

CG medium resulted in a significantly higher rate of proliferation above that found

with either DKM or the SG medium containing the VN:GF (Figure 2.2).

Nevertheless, the morphology of the cells grown using SG medium containing the

VN:GF more closely reflect the morphology of those grown in the CG medium. In

contrast the cells cultured in DKM appeared larger and more differentiated.

In view of this, we then examined the same culture conditions to establish whether a

serum-free medium could be developed for the in-vitro expansion of primary human

keratinocytes derived from adult skin. Our results indicate that this is indeed the

case, as the primary cells were successfully established and propagated using SG

with the VN:GF (Figure 2.3). The findings we report here demonstrate that not only

did this medium containing VN:GF result in similar levels of cellular proliferation to

that obtained with cells grown in the CG medium (Figure2.4), but in addition we

demonstrate that these cells maintain an undifferentiated phenotype (Figure 2.5).

This is significant as cells grown using the commercially available DKM + i3T3

demonstrate decreased expression of markers relevant for the undifferentiated state

at passage 4 (Figure 2.5). Furthermore, DKM used alone was not able to support the

establishment and serial propagation of the keratinocyte cells involved in this study.

These results suggest that the SG media with VN:GFs can support the establishment

and undifferentiated propagation of keratinocyte cells, and can also support the

prolonged growth of these cells. Future studies will further investigate and quantitate

the levels of markers expressed in undifferentiated keratinocyte cells. Flow

cytometry and Western blot analysis will be employed to evaluate the expression

differences between specific treatments. Additionally, the components that make up

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the VN:GF combination will be evaluated using dose response studies in order to

optimise this technology. Moreover, keratinocytes propagated using VN:GF media

were able to still form an epidermis, whilst maintaining markers similar to those

grown under normal culture conditions (Figures 2.6 I and II).

Whilst we have demonstrated that keratinocytes can be established and serially

propagated using this VN:GF technology, further investigation is required to confirm

whether these cells are therapeutically useful and whether a more optimal

concentration of VN:GF can be identified. In particular there is still a need to

investigate alternatives to using the i3T3 feeder cell layer. Recently, it was

demonstrated that pre-established normal human keratinocytes could be propagated

under xenobiotic-free conditions using a non-irradiated human skin-derived feeder

cell layer on plasma-coated surfaces (Sun et al. 2004a). While this represents an

improvement, the ideal scenario would be the development of culture conditions in

which feeder cells are not required, as the presence of feeder cells, whether of human

or animal origin, adds increased regulatory burden.

Since a paracrine relationship exists between the dermal fibroblasts and

keratinocytes (Maas-Szabowski 1999; Werner and Smola 2001), we propose that

genomic and proteomic approaches to analyse this paracrine relationship may reveal

other biological agents that could be used in conjunction with the VN:GF medium to

more closely mimic the micro-environment generated by the feeder cell layer.

Furthermore, other heparin-binding growth factors, such as transforming growth

factor-β (TGF- β), vascular endothelial growth factor (VEGF), epidermal growth

factor and bFGF (Hollier et al. 2005; Schoppet et al. 2002), also appear to be able to

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bind to VN. Thus, investigations into these additional growth factors may also

improve the culture system reported here. Moreover, this technology is likely to be

applicable to not only primary keratinocyte cells, but also a range of other cell and

tissue types important to the development of medical therapies. Importantly, any

culture system developed must contain synthetic and/or recombinant components. To

this end, recombinant VN has recently been produced in our laboratory and has been

shown to be functional when incorporated into VN:GF complexes (data not shown).

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CHAPTER 3

Serum-free growth of human embryonic stem cells

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3.1 INTRODUCTION

As stated in Chapter 1 human embryonic stem (hES) cells are a totipotent cell type

that has the therapeutic potential to generate new cells and tissues for donation.

However, methods to culture these cells rely on the use of animal-derived products

such as foetal bovine serum (FBS) and mouse embryonic fibroblasts (MEFs) for

their successful self renewal (Thomson et al. 1998; Pera et al. 2000; Henderson et al.

2002; Schick et al. 2003). While these products are successful in supporting in-vitro

hES cell growth, their non-human origin can potentially introduce pathogenic, toxic

and immunogenic agents (Martin et al. 2005; Heiskanen et al. 2007).

With this in mind, intensive research efforts worldwide are now starting to address

the problems associated with using animal-derived feeder cells in hES cell culture

systems. As discussed in Chapter 1 feeder-free growth of hES cells has been

examined in two general ways. The first approach involves the removal of feeder cell

layers. Thus, Lebkowski et al. (2001) and Xu et al. (2001) demonstrated a successful

feeder-free hES culture system that allowed undifferentiated cells to be maintained

for at least 130 population doublings. These techniques are based on the culture of

hES cells on either matrigel-coated or laminin-coated surfaces, in media conditioned

by MEF cells. However, Richards and co-workers (2002) reported an inability to

replicate this methodology and instead developed an approach utilising human foetal

and adult fibroblast feeder cells to support the prolonged undifferentiated growth of

hES cells (Richards et al. 2002). This method was also shown to be superior to

culture technologies that have been developed using cell-free matrices (collagen 1,

human extra-cellular matrix, Matrigel, and laminin) supplemented with conditioned

media from MEF feeder cells. While this novel approach addresses the potential

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problem of pathogen transfer that exists through the use of xenogeneic cells, it does

not avoid the problems associated with the use of poorly defined and uncharacterised

compounds found in serum, or purified from serum. Nor does it eliminate the risk of

pathogen contamination from the use of allogeneic feeder cells and/or serum.

More recently, Beattie et al. (2005) discovered that using a combination of activin-A,

nicotinamide and keratinocyte growth factor, in conjunction with a commercially

available undefined, human serum-derived serum replacement product termed

knock-out serum replacement (KSR), could remove the need for a feeder cell layer.

However, while this technology proved effective, more research needs to be

conducted in order to remove the need for KSR in this culture system. Interestingly,

Ludwig et al. (2006) created a culture system termed TeSR1 which was independent

of both a feeder cell layer and serum. This technology involved the use of large

quantities of purified human serum albumin (HSA) and several other reagents

purified from human serum. The TeSR1 culture system also required large doses of

recombinant insulin (23 μg/mL) for the successful propagation of the hES cells.

Whilst this technology was an exciting step forward for the culture of hES cells, the

high cost and poorly defined nature of TeSR1 suggests that this may not be the

answer. Furthermore, long term propagation of hES cells using the TeSR1

technology resulted in chromosomal abnormalities. Recently, Ludwig et al. (2007)

reported that bovine serum albumin and matrigel, two purified xeno-derived

components, needed to be re-introduced into the TeSR1 culture system (mTeSR1) to

make it commercially viable (Ludwig et al. 2007). Thus, there is clearly a need to

examine alternative serum-free and feeder-cell free culture technologies.

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As documented in Chapter 2, I have been investigating alternative culture

technologies suitable for adult stem cells and this has led to the development of a

technique that may lead to the first defined serum-free media for the in-vitro

expansion of keratinocytes for skin grafting applications. Briefly, this technology

involves the use of vitronectin (VN), insulin-like growth factors (IGFs) and insulin-

like growth factor binding proteins (IGFBPs) (Upton and Kricker 2002; Kricker et

al. 2003). This technology has been demonstrated to support the serum-free isolation

and serial propagation of primary keratinocytes (Refer to Chapter 2). Furthermore,

earlier work I conducted as a part of my honours project, demonstrated that

complexes of VN:IGF-I or –II:IGFBP-3 could support the short term growth (9 days)

of hES cells. In addition, via these studies I proved that the incorporation of IGF-I

was more successful than IGF-II for the expansion of hES cells (unpublished data).

Taking the data I obtained during honours, combined with the fact that hES cell

culture has similar culture requirements to keratinocytes i.e. a need for feeder cells

and serum, I hypothesised that the VN:GF medium incorporating IGF-I would be

useful for developing a fully defined, serum-free medium suitable for in-vitro

expansion of hES cells. In the study reported herein, the biological responses of hES

cells to this serum-free media were examined. This involved the establishment of a

hES cell line and associated culture technologies at Queensland University of

Technology (QUT). Having established the hES cells, I subsequently examined

whether these cells could be propagated in an undifferentiated state using the VN:GF

medium in the place of serum-containing media. In this chapter I report my progress

towards the translation of this new culture media for the in-vitro expansion of hES

cells.

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3.2 MATERIALS AND METHODS

3.2.1 Ethics and Training

Specialist training in hES cell culture was undertaken at the Monash Institute of

Reproduction and Development (MIRD, Melbourne, Victoria) and Embryonic Stem

Cell International (ESI, Melbourne, Victoria). Ethics approval to use the human

embryonic stem cell lines was received from the QUT Human Research Ethics

Committee (ID: 2943H) with strict adherence to the state, federal and NHMRC

guidelines regarding the conduct of research using hES cells. Additionally, informed

donor consent was obtained by the investigators who initially derived the hES cell

lines utilised.

3.2.2 Cell Culture

Mouse embryonic fibroblasts (MEF) cells (ESI, Melbourne, Australia) were cultured

in 80 cm2 culture flasks in 85% Dulbecco’s Modified Eagle Medium (DMEM)

(Invitrogen, Mulgrave, VIC, Australia), 10% foetal bovine serum (FBS) (Gibco,

Mulgrave, VIC, Australia), 1 mM L-glutamine, 0.5% penicillin/streptomycin and

0.01% gentamycin. Passage 7 MEFs were used as the feeder cell layer for the hES

cells. Surfaces to be seeded with the MEFs were coated in 0.1% gelatin for a

minimum of 1 hour before addition of the cells. Mitomycin-C was subsequently

added to the flasks containing the MEFs and incubated at 37ºC, 5% CO2 for 2.5 to 3

hours to mitotically inactivate the MEF cells. The MEFs were seeded into 6-well

plates (NUNC) (10 cm2) at a density of 2 x 104 cells/cm2.

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The HUES-9 and H1 cells (Harvard University, Boston, Chicago, U.S.A. and

WiCell, Madison, Wisconsin, U.S.A. respectively) were cultured on passage 7

mitomycin-C inactivated MEFs in hES medium containing: Dulbecco’s Modified

Eagle Medium (DMEM) (Invitrogen); 20% Knock-out Serum Replacement (KSR)

(Invitrogen); 1000 IU/mL penicillin/1000 μg/mL streptomycin (Invitrogen); 1 mM

glutamax (Invitrogen); 1% non-essential amino acids; 0.1 mM β-mercaptoethanol;

10 ng/mL basic fibroblast growth factor (bFGF) (Chemicon); and 12 ng/mL

leukaemia inhibitory factor (LIF) (Chemicon). Following the hES cell colony

obtaining confluence, the cells were washed in 2 mL PBS-/well (Invitrogen, PBS-)

and exposed to 0.05% trypsin/EDTA (Invitrogen) for a 1-2 minute incubation (37ºC

5% CO2). The cells were then re-suspended in hES media and spun at 500-600 g for

5 minutes. The cells were then transferred to fresh inactivated MEFs and the medium

was changed daily 48 hours post transfer.

3.2.3 VN:GF Culture

The serum-free culture of the hES cells involved the use of the previously mentioned

inactivated MEF cells being pre-plated 24 hours before use and then serum starved

for four hours. Human embryonic stem cells were then plated onto the serum starved

MEFs in 2.5 mL of serum free medium containing: Dulbecco’s Modified Eagle

Medium (DMEM) (Invitrogen); 1000 IU/mL penicillin/1000 μg/mL streptomycin

(Invitrogen); 1 mM glutamax (Invitrogen); 1% non-essential amino acids; 0.1 mM

β-mercaptoethanol; 0.6 µg/mL VN (Promega, Annandale, NSW, Australia); 0.6

µg/mL IGFBP-3 (N109D recombinant mutant) (Auspep, Parkville, VIC, Australia);

0.2 µg/mL IGF-I (GroPep, Adelaide, SA, Australia); 12 ng/mL LIF; and 0.02 µg/mL

bFGF (Chemicon). The cultures were then grown at 37°C in 5% CO2 and re-fed

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every day 48 hours after the initial transfer. The hES cells were then split, using

previously described methods (section 3.2.2), 1:3 to 1:4 depending on their rate of

growth and confluence.

3.2.4 ANK Culture

Briefly HUES-7 cells were cultured on plates coated with 20 µg/mL of laminin

(Chemicon) and were grown in Dulbecco’s Modified Eagle Medium (DMEM)

containing: 20 % knock-out serum replacement (Invitrogen); 1000 IU/mL

penicillin/1000 μg/mL streptomycin (Invitrogen); 1 mM glutamax (Invitrogen); 1%

non-essential amino acids; 0.1 mM β-mercaptoethanol; 50 ng/mL of activin-A

(PreproTech); 50 ng/mL keratinocyte growth factor (PreproTech), and 10 mM

nicotinamide (Sigma) (ANK culture) (Beattie et al. 2005). ANK/VN:GF culture,

both in the presence and absence of KSR, involved various combinations of 0.6

µg/mL VN (Promega, Annandale, NSW, Australia); 0.6 µg/mL IGFBP-3 (N109D

recombinant mutant) (Auspep, Parkville, VIC, Australia); 0.2 µg/mL IGF-I (GroPep,

Adelaide, SA, Australia); 0.02 µg/mL bFGF (Chemicon); 50 ng/mL of activin-A

(PreproTech); 50 ng/mL keratinocyte growth factor (PreproTech); and 10 mM

nicotinamide (Sigma).

3.2.5 Immunofluorescence

Stage specific embryonic antigen-1 (SSEA–1), stage specific embryonic antigen-4

(SSEA–4), tumour repressor antigen 1-81 (Tra 1-81) and octamer binding

transcription factor-4 Oct-4 are expressed by undifferentiated hES cells (Thomson et

al. 1998; Reubinoff et al. 2000). The presence of these proteins was monitored using

mouse monoclonal antibodies raised against the proteins. The cultures were fixed

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using 4% paraformaldehyde/phosphate buffered saline (PBS) for 15 minutes, or in

100% methanol for 2 minutes. The fixing agent was removed and the cultures were

washed twice, for 15 minutes per wash in 20 mM Tris-HCl, 0.15 M NaCl, 0.05%

Tween-20, pH 7.4 (TBST). The cells were then permeabilised with 0.1% Triton X-

100/PBS for 10 minutes prior to a further washing step. The cultures were then

blocked in 4% goat serum for 30 minutes at room temperature. The blocking solution

was removed and primary antibodies against SSEA-1, SSEA-4, Tra 1-81, and Oct-4

(Chemicon, Boronia, Victoria, Australia) were diluted to 1:50 in 4% goat serum and

incubated on the cultures for 1 hour. The primary antibodies were removed and the

wash steps repeated. The anti-mouse secondary antibodies (Chemicon) were diluted

in PBS at 1:100 and incubated for 1 hour. The secondary antibodies were removed,

the wash steps were repeated and the colonies were viewed with a Nikon TE-2000

fluorescence microscope.

3.2.6 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Analysis

Oct-4, human telomerase reverse transcriptase (hTERT) and alkaline phosphatase

(AP) have been shown to be expressed in the undifferentiated human embryonic

stem cells (Xu et al. 2001). Hence, RT-PCR was applied to these markers to

determine whether the hES cell colonies maintained an undifferentiated state. RNA

was isolated from the colony pieces using tri-reagent and its accompanying protocol

(Sigma). The RNA samples were applied to oligo-dT 18mers to create cDNA. The

Oct-4 primers were: sense, 5’-CTTGCTGCAGAAGTGGGTG-GAGGAA-3’; and

antisense, 5’-CTGCAGTGTGGGTT-TCGGG-CA-3’. The hTERT primers were:

sense, 5’-CGGAAGAGTGTCTGGAGCAA-3’; and antisense, 5’-GGATGA-

AGCGGAGTCTGGA-3’. The alkaline phosphatase primers were: sense, 5’–

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CGTGGCTAAGAATGTCATCATGTT-3’; and antisense, 5’-TGGTGGAGCT-

GACCCTTGA-3’. The 18sRNA internal standard primers were: sense, 5’-TT-

CGGAACTGAGGCCATGAT-3’; and antisense, 5’-CGAACCTCCGACTTTCGT-

TCT-3’. One µg of cDNA was added to each of the four primer sets and subjected to

an initial denaturation step of 94˚C for 5 minutes, followed by 30 cycles of

denaturation at 94˚C for 30 seconds, annealing at 55˚C for 30 seconds and extension

at 72˚C for 30 seconds, followed by a final extension at 72˚C for 5 minutes.

3.2.7 Proliferation Assays

Proliferation of the hES cells was measured by assessing the metabolic activity of

the mitochondria with thiazolyl blue tetrazolium bromide (MTT) (Sigma Aldrich).

MTT assays were performed in 24-well plates that were pre-seeded with 4 x 104

MEFs/well and ~ 1 x 104 hES cells (value determined by cell count of 20 colony

pieces). The cultures were grown for 4 days and then washed twice in PBS and

incubated with MTT for 1 hour. The MTT was removed from the wells and was

subsequently washed as previously described. Dimethyl sulfoxide was then added to

the wells and the absorbances of the resulting solutions were measured at 540 nm –

630 nm. The MTT assays were conducted in triplicate and all experiments were

replicated twice. Data from these experiments were statistically analysed using the

Tukey’s test and a p < 0.05 was determined to be statistically significant.

3.2.8 Karyotype Analysis

Queensland Medical Laboratories (QML, Brisbane, Queensland, Australia)

performed karyotype analysis on hES cells that had been passaged 10 times. The

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Giemsa staining method was used to analyse 20 cells from 2 different cultures to

assure that the karyotype revealed was accurate.

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3.3 RESULTS

3.3.1 VN:GF medium for the propagation hES cells.

Having previously established that hES cell colonies could be propagated short-term

using pre-bound VN/growth factor complexes (data not shown, results from my

honours thesis), I wished to assess whether the proteins involved in these complexes

could be translated into a VN:GF medium. Due to the fact that these cells need to be

grown in colonies for their optimal growth, it was difficult to quantify exactly how

many cells were added to the treatments, however, we estimated that ~1 x 104 hES

cells per well were used. The proliferation of hES cells on the treatments and control

were assessed utilising the MTT assay. This assay revealed that whilst there were no

significant differences between treatments, the VN:GF medium, demonstrated by the

last bar Figure 3.1, showed a trend towards an increase in proliferation above both

the DMEM control and the growth factors alone (Figure 3.1). Treatments were

assessed independently in triplicate using the HUES-9 cell line and the experiment

repeated 3 times.

3.3.2 Morphology of hES cells grown using the VN:GF combinations as a serum-

free medium.

Having demonstrated that the VN:GF combination would induce the propagation of

hES cells, I then proceeded to examine whether this new VN:GF medium could be

used for the long term serial propagation of hES cells. This needed to be established

to determine whether the VN:GF medium could effectively replace the need for

serum in the culture of hES cells. Human embryonic stem cell lines, H1 (WiCell)

and HUES-9 (Harvard), were grown for 10 passages in culture medium containing

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serum and a feeder cell layer, and also in VN:GF medium in the presence of a feeder

cell layer (Figure 3.2 A & C and Figure 3.2 B & D respectively). This experiment

revealed that both the VN:GF propagated H1 and HUES-9 cell lines (Figure 3.2 B &

D) looked similar in morphology to those propagated using serum containing media

(Figure 3.2 A & C). Interestingly, the hES cells grown in the serum-free medium

appeared morphologically less differentiated than those grown for extended passages

in medium containing serum. Therefore, the VN:GF medium appears to be a viable

method for the serum-free culture of hES cells.

Figure 3.1: VN:GF combinations for the propagation hES cells (HUES-9). hES cell

proliferation in the presence of DMEM, VN:GF medium and its various components were

assessed by MTT assay, as described in the materials and methods. The following treatments

were analysed: DMEM; DMEM + VN; DMEM + IGFBP3; DMEM + IGF-I; DMEM +

bFGF; DMEM + VN:IGFBP3:IGF-I:bFGF. Each of the above treatments were conducted in

wells containing 4 x 104 MEFs and 1 x 104 hES cells. Treatments were assessed in triplicate

and the experiment conducted 3 times, with the data from the 3 experiments pooled and

averaged. The bars on the graph represent the standard error of the mean. The data from

these experiments was analysed using the Tukey’s test (p < 0.05).

0

0.5

1

1.5

2

2.5

3

DMEM VNBP3

IGF

bFGF

VN+IGF+bF

GF+BP3

Treatment

(ABS

540

-630

nm)

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Figure 3.2: Morphology of hES cells grown using the VN:GF medium. Colonies were

propagated using: A) H1 cells (WiCell) grown in serum with MEFs; B) H1 cells grown in

VN:GF medium with MEFs; C) HUES-9 cells (Harvard) grown in serum with MEFs; and

D) HUES-9 cells grown in VN:GF medium with MEFs. Experiments were conducted in

triplicate and repeated in two distinct hES cells lines. (Scale bar = 500 µm).

3.3.3 Identification of markers expressed by undifferentiated hES cells.

At present there are no definitive assays for determining whether cultured hES cells

have maintained an undifferentiated state. However, hES cells have been shown to

express several markers, such as, stage specific embryonic antigens (SSEAs). These

markers can be used to assess the differentiation status of hES cells (zur Nieden et al.

2001; Draper et al. 2002), therefore antibodies that recognise SSEA-1 (expressed on

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differentiated cells) and SSEA-4 (expressed on undifferentiated cells) were selected

to analyse the differentiation status of the hES cell colonies grown for 10 passages

using the VN:GF medium. Fluorescently labeled, secondary antibodies to the SSEA-

1 (red) and SSEA-4 (green) primary antibodies were used to establish whether these

proteins were present on the hES cells. Interestingly, hES cells propagated using the

VN:GF serum-free medium demonstrated high levels of SSEA-4 expression and next

to no levels of SSEA-1 expression (Figures 3.3). The “no primary antibody control”

results were obtained by incubating the hES cell colonies with the secondary

antibody only and no non-specific binding of primary antibody was observed (data

not shown).

3.3.4 Karyotype analysis of H1 cells grown using VN:GF medium..

Karyotype analysis involves the identification of all the morphological

characteristics of a single cell’s chromosomes. It relies on staining the cell during the

period of metaphase with Giemsa staining reagent. The stained cell is then pressed

firmly onto a microscope slide to analyse the banding patterns of each of the

chromosomes. This analysis can reveal a variety of chromosomal aberrations such as

aneuploidy events, deletions, insertions, and translocations (Schrock et al. 1996;

Amit et al. 2000). Thus, karyotype analysis of the hES cell lines was performed to

ascertain whether the hES cells grown using the VN:GF medium for multiple

passages had retained a normal karyotype. The karyotype analysis represented in

Figure 3.4 was performed by Queensland Medical Laboratories (QML) on hES cell

colonies that have been passaged 10 times. This analysis revealed a 46XX normal

karyotype. No chromosomal aberrations were noted from the karyotype analysis

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suggesting that the culture method has no adverse effect on the propagation of these

cells.

3.3.5 RT-PCR analysis of hES cells.

Due to the fact that expression of SSEA-4 alone is not definitive for identifying an

undifferentiated hES cell colony, RT-PCR analysis was also used to analyse the

expression of 3 genes present in undifferentiated hES cell colonies. The genes

analysed encode Oct4 (a transcription factor), hTERT (human telomerase reverse

transcriptase) and AP (alkaline phosphatase). Combined, the expression of these

genes is reported to result in a more accurate assessment of the differentiation status

of the hES cell colonies (Xu et al. 2001).

In order to establish that the samples were not contaminated with complementary

deoxyribose nucleic acid (cDNA) or genomic deoxyribose nucleic acid (gDNA), the

template was omitted in the series of negative controls (data not shown). The

primers were designed such that they annealed to different exons within the gene,

therefore any contaminating genomic deoxyribose nucleic acid (gDNA) present in

the PCR reaction would result in a larger molecular weight band than the cDNA.

This analysis revealed that hES cell colonies grown under normal conditions and

using the VN:GF medium expressed Oct4, AP, and 18sRNA (included as an internal

standard control) (Figure 3.5). No hTERT expression was observed in either of the

cell lines tested nor in either the normal or VN:GF medium treatments. Furthermore,

I believe that this is a result of extremely low expression levels which did not affect

the pluripotent nature of the cells, as confirmed by the presence of the other markers

for the hES cell undifferentiated state. This experiment further verifies that the

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VN:GF medium is maintaining the hES cells in a similar condition to those

propagated using normal culture conditions.

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Figure 3.3: Expression of cell-surface markers on the hES cells grown using the VN:GF

medium. H1 hES cell colonies (A-C) and HUES-9 hES cell colonies (G-I) were grown

using KSR + feeder cells. H1 hES cell colonies (D-F) and HUES-9 hES cell colonies (J-L)

were also grown using the VN:GF medium, as described in the materials and methods

section. Cultures were subsequently incubated with mouse anti-SSEA1 (red) antibodies

(B,E,H,K) and mouse anti-SSEA-4 antibodies (green) (C,F,I,L). Colonies were also

incubated with secondary antibodies only as a control against non-specific binding (M-O).

Experiments were conducted in triplicate and repeated in two distinct hES cells lines. (Scale

bar = 250 µm).

M N O

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Figure 3.4: Karyotype analysis of H1 hES cells grown in VN:GF medium. The

karyotype was performed using the Giemsa banding method of chromosome analysis. The

H1 cell line was 46XX normal (n=2, representative experiment depicted).

Figure 3.5: RT-PCR analysis of hES cells grown using VN:GF medium. Colonies were

grown in either serum (Normal) or VN:GF medium (VN:GF) in conjunction with a MEF

feeder cell layer. Two cell lines were analysed, A) H1 and B) HUES-9. 18sRNA (151 bp

band), Oct-4 (169 bp band), TERT (145 bp band) and ALP (90 bp band) transcripts were

used to analyse the differentiation status of the hES cells. Negative controls involved no

addition of the template to ensure no contamination occurred in the PCR reaction (data not

shown) and C) a TERT positive control to demonstrate that the primers were working. The

molecular weight marker used was a 100 bp DNA ladder (Roche).

C

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3.3.6 Morphology of HUES-7 cells grown using the VN:GF medium in

conjunction with the ANK protocol.

Recently, Beattie et al. (2005) demonstrated that hES cells could be propagated

feeder-free using Activin-A, nicotinamide and keratinocyte growth factor (ANK).

Therefore, we used ANK in conjunction with our optimum serum-free VN:GF

medium for the propagation of hES cells. The hES cells propagated using ANK +

VG:GF combinations for 6 days (Figure 3.6 D) demonstrated a cellular morphology

similar to hES cells grown using KO-serum + feeder cells, VG:GF combinations +

feeder cells and ANK (Figures 3.6 A, B, and C respectively). However, the colonies

looked less differentiated in the first three treatments than they did in the ANK +

VG:GF combinations. Consequently, cells grown in AN + VN:GF medium, A +

VN:GF medium, ANK + VN:GF medium – bFGF, ANK + VN:GF medium – VN,

and ANK – laminin + VN:GF medium (Figures 3.6 E, F, G, and H respectively)

treatments were analysed to determine if there was a more efficient culture

combination for the hES cells. Interestingly, the A + VN:GF medium and the ANK +

VN:GF medium – VN (Figures 3.6 F, and H respectively) treatments demonstrated

both cell and colony morphologies similar to the KO-serum + feeder culture (Figure

3.6 A).

3.3.7 Identification of markers expressed by HUES-7 cells grown using the

VN:GF medium in conjunction with the ANK protocol.

The differentiation status of hES cells grown using combinations of ANK and

VN:GF medium was assessed using Oct-4 and TRA 1-81 primary antibodies. Both

of these markers are expressed on undifferentiated hES cells. Fluorescently labeled,

secondary antibodies to TRA 1-81 (red) and Oct-4 (green) primary antibodies were

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used to establish whether these proteins were present on the hES cells. Interestingly,

hES cells propagated in all treatments demonstrated high levels of TRA 1-81 and

Oct-4 (Figures 3.7). High immuno-reactivity of the two markers was observed in the

feeder + KO-Serum, feeder + VN:GF medium, ANK + KO-Serum, ANK + VN:GF

medium (Figures 3.7 A, B, C, and D respectively). The treatments of ANK + VN:GF

medium – VN and ANK – laminin + VN:GF medium also demonstrated marker

expression, however, the cells and colonies appeared to be more differentiated when

compared to the feeder + serum treatment. The “no primary antibody control” results

were obtained by incubating the hES cell colonies with the secondary antibody only

and no non-specific binding between primary antibody and hES cell was observed

(data not shown).

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Figure 3.6: Morphology of HUES-7 cells grown using the VN:GF medium in

conjunction with the ANK protocol. Colonies were propagated using: A) Feeder + KO-

Serum; B) Feeder + VN:GF medium; C) ANK + KO-Serum; D) ANK + VN:GF; E) AN +

VN:GF; F) A + VN:GF; G) ANK + VN:GF – bFGF; H) ANK + VN:GF – VN; and I) ANK

– laminin + VN:GF, refer to Materials and Methods. (Scale bar = 250 µm) (n=2,

representative experiment depicted).

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Figure 3.7: Expression of cellular markers in HUES-7 cells grown using the VN:GF

medium in conjunction with the ANK protocol. Colonies were propagated using: A)

Feeder + KO-Serum; B) Feeder + VN:GF medium; C) ANK + KO-Serum; D) ANK +

VN:GF; E) AN + VN:GF; F) A + VN:GF; G) ANK + VN:GF – bFGF; H) ANK + VN:GF –

VN; and I) ANK – laminin + VN:GF. All colonies were stained with Oct-4 (Green) and

TRA 1-81 (Red), refer to Materials and Methods. (Scale bar = 250 µm) (n=2, representative

experiment depicted).

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3.4 DISCUSSION

While hES cells appear to have great potential to cure a wide variety of diseases and

injuries (Docherty 2001; Guan et al. 2001; Boheler et al. 2002), the fact that their

culture systems require poorly defined components and/or xenogeneic products

means that hES cells may inherently be exposed to pathogens, resulting in unsafe

material for transplantation. This has prompted investigators to attempt to develop

culture systems that are fully defined and xenogeneic-free. For example, many

researchers have focused on replacing animal-derived feeder cell layers with human-

derived feeder cell layers (Amit et al. 2000; Richards et al. 2002; Cheng et al. 2003).

While this creates a favourable micro-environment there is still a risk of cross-

contamination from the human-derived feeder cell layer, hence this method of

culture is far from ideal.

The use of extra-cellular matrix (ECM) proteins in place of a feeder cell layer has

also been demonstrated to successfully provide a self renewing culture system for

hES cells. Thus, Xu et al. (2001) used laminin and matrigel to replace the feeder cell

layer (Xu et al. 2001). However, this system only proved functional with the addition

of MEF conditioned medium, which still carries the risk, albeit small, of

transmission of pathogens to the hES cells grown in these conditions. In fact a range

of ECM proteins such as laminin (LN), fibronectin (FN), and vitronectin (VN)

(Bagutti et al. 1996; Bradshaw et al. 1995) have all been investigated as potential

factors to replace feeder cells. It is important to also note that in all cases the ECM

proteins used for these studies were purified from plasma or tissues hence are not

completely defined, nor synthetic; leading to another potential source of

contamination.

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Due to the demonstrated potential of ECM proteins to potentially support the growth

and self-renewal of hES cells, along with VN’s demonstrated ability to interact with

IGF-I, IGFBP-3 and bFGF (VN:GF) (Clemmons 1998; Rees and Clemmons 1998;

Nam et al. 2002; Kricker et al. 2003; Upton et al. 1999; Schoppet et al. 2002), I

hypothesised that the technology developed in Chapter 2 for the primary

keratinocytes could be translated to hES cells. Initially, the most robust VN:GF

combination was determined by assessing the proliferation of the hES cells in short

term assays and it was demonstrated that the VN:IGFBP-3:IGF-I:bFGF combination

was the most successful treatment for the propagation of hES cells (Figure 3.1).

Additionally, the components that make up the VN:GF combination will be

evaluated using dose response studies in order to optimise this technology for the

hES cells.

To examine this VN:GF medium further, I established cultures of both the H1 and

HUES-9 hES cell lines using MEF cells and KO serum. I then proceeded to transfer

the hES cells from the serum-containing medium to the VN:GF serum-free media.

Encouragingly, the hES cell lines established in the serum free conditions

demonstrated an undifferentiated morphology typical of cells grown using a MEF

feeder cell layer and serum (Figure 3.2). This was further verified by subjecting the

hES cells to a range of assays examining the presence of specific markers, such as

SSEA-4, SSEA-1 (Figure 3.3), alkaline phosphatase, Oct-4, and hTERT (Figure 3.5)

(Thomson et al. 1998; Lanzendorf et al. 2001; Lebkowski et al. 2001; Xu et al. 2001;

Richards et al. 2002). Additionally, the hES cells were examined to determine if they

had maintained a normal karyotype after being cultured over a long time frame (10

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passages). The chromosomal analysis revealed that the H1 (Figure 3.4) and HUES-9

(data not shown) cells had indeed maintained their original karyotypes.

Having demonstrated that two independent hES cell lines could be established in a

fully defined serum-free culture system, I then decided to incorporate a feeder-free

technology with our VN:GF culture in an attempt to create the first synthetic, fully

defined culture system. Beattie et al. (2005) discovered that the feeder cell layer

could be removed from the culture of hES cells by replacing it with a combination of

activin-A, keratinocyte growth factor, and nicotinamide (ANK). However, KSR was

still required within this culture system. Therefore, I conducted studies at the

Whittier Institute San Diego CA, USA, in conjunction with Beattie et al. (2005) to

determine if it was feasible to combine the ANK technology with the VN:GF

medium and remove the need for both feeder cells and serum for the culture of hES

cells. Initially the cultures were established using MEF cells and KO-serum, prior to

transferring the hES cells to a variety of conditions to evaluate the possibility that the

feeder-free and serum-free technologies combined, could replace the reliance of hES

cells on feeder cells and serum. In this chapter I demonstrated that the HUES-7 cell

line could be established, and that the cells remained undifferentiated when cultured

using ANK and the VN:GF medium (Figure 3.6 D). This was further verified by

examining the expression of Tra 1-81 and Oct-4 markers present in undifferentiated

hES cells (Figure 3.7 D). However, this analysis revealed that hES cells grown in

these novel conditions were not as undifferentiated as those grown using KO serum

and feeder cells. Consequently, hES cell cultures were grown in media containing

various combinations of the ANK and VN:GF components (Figures 3.6 & 3.7).

Interestingly, combinations where keratinocyte growth factor and nicotinamide were

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removed seemed more robust (Figures 3.6 F and 3.7 F). However, only short term

studies were conducted and further examination is required to explore the potential

of this system.

Recently, other serum-free and feeder-free culture systems have been developed for

hES cell growth. For example, Ludwig et al. (2006) utilised a combination of growth

factors, insulin (23µg/ml) and HSA (13 mg/ml), termed TeSR1, for the culture of

hES cells. Interestingly, the karyotype analysis conducted on the hES cells that were

serially propagated using the TeSR1 technology revealed a 47 XXY chromosomal

aberration. Perhaps the inclusion of supra-physiological concentrations of growth

factors in the TeSR1 media led to the coincidental selection of cells with

tumourigenic potential. Additionally, the large amounts of protein required in this

TeSR1 technology result in this product being excessively expensive. Similarly,

another serum-free, feeder-cell free protocol was reported by Lu et al. (2006). This

technology also included high concentrations of other factors purified from serum

such as HSA and growth factors, as well as the addition of insulin. Thus, none of

these recent “advances” in hES culture media are commercially viable from a cost

perspective, nor are they readily regulatory compliant.

The results from the experiments reported in this chapter are clearly very

encouraging. I have demonstrated that the VN:GF medium can replace the need for

serum in hES culture. Furthermore, the short term studies examining the ANK

components with the VN:GF medium warrants additional investigation. In particular,

studies examining whether hES cells can be serially passaged long term both serum-

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free and feeder-free using the ANK/VN:GF medium and still retain their desired

undifferentiated state are necessary.

Given the diverse approaches reported in the literature to replace feeder cells, it

would appear that there is also a need to investigate the hES cell in-vitro micro-

environment using a more systematic approach, for example through proteomic

analysis. Such an approach may well identify molecules that could be added in

conjunction with the VN:GF medium to further develop and optimise a synthetic,

fully defined, culture system. Nevertheless, the data reported in this chapter have

demonstrated that hES cells can attach, expand long term and survive in an

undifferentiated state when using the VN:GF medium as a serum-free media; this

represents a significant advance in the field of hES cell culture. The results reported

herein also indicate that technologies developed for improved culture of primary

keratinocytes are highly applicable to hES cells.

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CHAPTER 4

Proteomic Analysis of Media Conditioned by Keratinocytes

Cultured In-Vitro

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4.1 INTRODUCTION

As discussed in Chapter 1 human skin is made up of an epithelial cell layer which is

primarily composed of keratinocytes. These cells are involved in the self-renewal,

maintenance and formation of the skin’s outer layer (Leary et al. 1992). The natural

in-vivo micro-environment provides the keratinocytes with an array of growth

factors, extra-cellular matrix (ECM) proteins and other nutrients important for their

survival and renewal. In 1975 Rheinwald and Green discovered a way to recapitulate

this micro-environment using an irradiated feeder cell layer and animal serum. This

revolutionised research into the skin as the newly established methods for the in-

vitro culture of keratinocytes provided researchers with a tool to examine methods to

regenerate and repair skin defects (Green 1991; Meana et al. 1998; Wright et al.

1998). However, this culture method uses xenogeneic components, hence carries the

risk of contaminating keratinocytes with infectious and pathogenic agents. More

recently, the addition of these animal components has also been demonstrated to

introduce immunogenic agents (Martin et al. 2005; Heiskanen et al. 2007) suggesting

that the cells grown in these conditions can be phenotypically manipulated by their

micro-environment.

As described in Chapter 2, I have developed a fully defined serum-replacement

method for the isolation, establishment and in-vitro expansion of keratinocytes for

skin grafting applications. Whilst this technology has proved to be successful in

removing serum from the culture of keratinocytes, there is still a need for a feeder

cell layer in the culture system. Interestingly, other groups have demonstrated that

human embryonic stem cells which are cultured using a similar approach, can be

propagated by using medium that has been conditioned by feeder cells (Lebkowski et

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al. 2001; Xu et al. 2001). This suggests that a soluble factor/s secreted by the feeder

cells may be critical for the propagation of hES cells.

The complexity and importance of the feeder cell layer to the hES micro-

environment is unquestionable and has led to the proteomic profiling of culture

medium conditioned by feeder cells (Wee Eng Lim and Bodnar 2002; Prowse et al.

2006). These studies analysed animal and human-derived feeder cell layers to

determine what proteins were expressed by the cells, and in turn, facilitated

identification of factors which may be important for the self renewal of hES cells.

However, while these studies examined the proteome of the conditioned media and

the feeder cells, they have left out one important aspect; namely, what do the hES

cells contribute to their micro-environment? That is, what proteins important to the

survival of feeder-dependent cells are secreted by either the feeder cells, or the

feeder-dependent cells in response to their paracrine interactions? Similarly, no

proteomic investigation into the paracrine interactions has been conducted with

primary keratinocytes, another feeder-dependent cell type.

In the chapter reported here, I aimed to rectify this situation by undertaking a

comprehensive examination of the keratinocyte in-vitro micro-environment. In

particular, I adopted a proteomic approach to identify the critical factors produced by

the feeder cells that are required for keratinocyte growth. Furthermore, I utilised the

serum-free media developed in Chapter 2, which is fully defined and has minimal

protein content. The minimal protein content of this serum-free media provides a

significant advantage in that it will not “mask” the critical factors secreted by the

feeder cells which may be important for supporting keratinocyte cell growth.

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Additionally, serum-containing medium normally requires a pre-processing step

before proteomic analysis, such as the "Multiple Affinity Removal System" (MARS)

(Agilent Technologies). This MARS immuno-depletion technology involves the

removal of high abundant proteins from serum-containing media, which could result

in a loss of candidate factors important for the self renewal of primary keratinocytes.

Similarly, there is no need to grow the cells in serum-free basal media, an approach

routinely adopted in the collection of “conditioned” media. Instead, the media to be

analysed was collected from cells cultured in their normal “growth” media; hence

they were actively growing, rather than nutrient starved and in a stressed state. Taken

together, this provided us with a useful tool to identify the critical factors produced

in the in-vitro keratinocyte culture microenvironment.

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4.2 METHODS

4.2.1 Ethics and material collection

Ethics for this project was approved by the Human Research Ethics Committee (ID:

3673H) (Queensland University of Technology) and the St. Andrews and Wesley

Hospitals, Brisbane, Australia. Skin was obtained from informed, consenting patients

undergoing breast reductions and abdominoplasties.

4.2.2 Isolation of primary keratinocytes

Primary keratinocytes were isolated from split thickness skin biopsies obtained from

breast reductions and abdominoplasties as described by Goberdhan et al. (1993).

Briefly, this method involved dissecting the skin biopsy into 0.5 cm2 pieces followed

by a series of antibiotic wash steps. The skin was then incubated in 0.125% trypsin

(Invitrogen, Mulgrave, VIC, Australia) overnight at 4 °C. The epidermis was then

separated from the dermal layer and the keratinocytes isolated. Keratinocyte cells

were then suspended in DMEM (Invitrogen), filtered (100 µm) and pelleted.

4.2.3 VN:GF Culture

Freshly isolated keratinocytes were initially cultured in 75 cm2 flasks at a density of

2 x 106 cells and were then seeded at 2 x 105 cells per 75 cm2 flask for subsequent

passages. Prior to seeding the keratinocytes, a gamma-irradiated (two doses of 25

Gy) (Australian Red Cross Blood Service, Brisbane, QLD, Australia) mouse i3T3

cell feeder cell layer was pre-seeded for four hours at 2 x 106 cells. The feeder cell

layer was then serum-starved for three hours following seeding. The keratinocytes

were propagated in VN:GF medium containing: phenol red-free DMEM/HAMS

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medium (Invitrogen); 0.4 μg/mL hydrocortisone; 0.1 nM cholera toxin; 1.8 x 10-4 M

adenine; 2 x 10-7 M triiodo-l-thyronine; 5 μg/mL transferrin; 2 x 10-3 M glutamine

(Invitrogen); 1000 IU/mL penicillin/1000 μg/mL streptomycin (Invitrogen); 0.6

µg/mL VN (Promega, Annandale, NSW, Australia); 0.6 µg/mL IGFBP-3 (N109D

recombinant mutant) (Auspep, Parkville, VIC, Australia); 0.2 µg/mL IGF-I (GroPep,

Adelaide, SA, Australia); and 0.2 µg/mL EGF (Invitrogen) (VN:GFs). The

keratinocyte cultures were incubated at 37°C in 5% carbon dioxide and re-fed with

VN:GF medium every two days. Morphology and marker expression were used to

ensure that the keratinocytes used in this experiment were phenotypically similar to

those grown using serum. Briefly, this involved probing the cultures with antibodies

against keratins 6 and 14, a marker expressed by undifferentiated keratinocytes,

(refer to Chapter 2, and section 2.2.8).

4.2.4 Two-dimensional proteomics.

Two-dimensional liquid chromatography was used to fractionate conditioned media

samples and employed a BioLogic Duo-flow high performance liquid

chromatography (HPLC) (Bio-Rad, Hercules, California, USA) for the first

dimension separation, while the second stage of the Beckman Coulter’s

ProteomeLab™ PF 2D platform was utilised for the second dimension separation.

Initially, 15 mL of conditioned media was collected from the feeder cell alone or

feeder cell:keratinocyte cultures every 2 days and pre-processed and concentrated

using bulk-phase SPE phenyl-silica sorbant (Alltech- Australia, Dandenong South,

VIC, AUS). Briefly, matrix was prepared in 100% methanol and poured into a 10

cm3 column; the packed column was then equilibrated using ultrapure water

containing 0.1% tri-fluro acetic acid (TFA). Following this, the samples were loaded

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onto the column and the column was then washed in 0.1% TFA. Bound protein was

eluted from the column using 80% acetonitrile in ultrapure water containing 0.1%

TFA and the fraction collected was lyophilised using an eppendorf concentrator

5301 (Eppendorf South Pacific, North Ryde, NSW, AUS) to concentrate the protein.

The protein samples were then reconstituted using 20 mM Tris-HCl and resolved in

the first dimension using a UNO-Q (Bio-Rad) anion-exchange chromatography

column attached to the BioLogic DuoFlow HPLC. Several NaCl gradients were

tested in order to optimise the resolution of the proteins/peptides in the samples. The

first dimension separation involved fractionating the proteins and peptides present in

the conditioned media using a salt gradient (20 mM TRIS-HCL through to 20 mM

TRIS-HCL containing 500 mM NaCl, pH 8.8) and collecting 1 mL fractions at 2 min

intervals using a flow rate of 0.5 mL/min.

These fractions were then further separated using the second dimension platform of

the ProteomeLab PF2D (Beckman Coulter, Gladesville, NSW, AUS) which

employed high performance, reversed-phase liquid chromatography. The second

dimension separation was performed at 50 oC at a flow rate of 0.75 mL/min. Two

hundred microlitres from each first dimension fraction were injected and further

fractionated independently using a 0-100% Acetonitrile/0.1%TFA gradient over 30

min. Two-dimensional images were generated based on protein absorbances

generated by the 2nd dimension separation for both the feeder cell and feeder

cell:keratinocyte conditioned media samples using the ProteoVue software

(Beckman Coulter).

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4.2.5 Sample Preparation and LC/MS using LC/ESI/MS and LC-MALDI

Analysis.

To identify proteins present in both the feeder cell and feeder cell:keratinocyte

conditioned media samples, two liquid chromatography/mass spectrometry (LC/MS)

procedures were used, liquid chromatography/electrospray ionization (LC/ESI) and

liquid chromatography/matrix assisted laser desorption ionisation (LC-MALDI).

Initially, the samples were lyophilised using an eppendorf concentrator 5301

(Eppendorf South Pacific, North Ryde, NSW, AUS), then reduced, alkylated and

digested with trypsin. The reduction involved resuspending the lyophilised protein in

100 µL of 0.1 M NH4CO3/20 mM DTT pH 7.9 and incubating the sample at 56°C

for 1 h. Samples were then alkylated using iodoacetamide (Sigma Aldrich) to a final

concentration of 50 mM and incubated at 37°C for 30 minutes in the dark. Finally, a

trypsin digestion using 2.2 µL of (0.5 µg/mL) sequencing grade modified trypsin

(Promega; Madison, WI, USA) was added and by incubated at 37°C for 4 h. The

samples for Liquid Chromatography (LC) were then lyophilised and dissolved in

50/50 solvent A/B (solvent A 0.1% Formic acid) (solvent B 90% acetonitrile in 0.1%

Formic acid). Samples were loaded onto a C18 300A column (150 mm x 0.5 mm x 5

µm particle size) (Vydac, Hesperia, California, USA) with 40/60 solvent A/B at a

flow rate of 300 µL/min. Solvent delivery was achieved by using an Agilent 1100

Binary HPLC system (Agilent, Inc Santa Clara, California, USA).

Column elutes were analysed using the 4000 QTRAP ESI-QqLIT analyser (Applied

Biosystems, Foster City, CA, USA) at the Institute of Molecular Biosciences, at The

University of Queensland (St Lucia, QLD, Australia). Data was analysed using the

Analyst 1.4.1 software. The protein analysis was conducted using the MASCOT

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database GPS Explorer™ software (version 4.0) with the mass/ion peak information

obtained from both the MS and the MS/MS spectra. Alternatively, samples collected

from the LC phase were spotted onto MS plates using 1:1 volume of 5 mg/mL of α-

cyano-4-hydroxycinnamic acid (CHCA): protein sample (Sigma-Aldrich) for LC-

MALDI analysis. Plates were then analysed using the 4700 Proteomics Analyser

(Applied Biosystems) at the Institute for Molecular Bioscience. A plate-wide

calibration for MS and MS/MS data was performed using mass standards contained

in the MS/MS Mass Standards kit (Sigma-Aldrich). Potential protein matches were

then identified from automated searching of the MASCOT database using GPS

Explorer™ protein analysis software (version 4.0) with the mass/ion peak

information obtained from both the MS and the MS/MS spectra.

4.2.6 Sample Preparation and MALDI-TOF-TOF Mass Spectrometry.

Protein peaks for matrix assisted laser desorption ionization-time of flight-time of

flight (MALDI-TOF-TOF) analysis were selected using the ProteoVue software.

Briefly, 400 µL of samples, selected based on their respective protein peaks, were

lyophilised as previously described (section 4.2.5). The lyophilised samples were

then digested as previously described and desalted using Eppendorf C18 PerfectPure

desalting tips (Millipore; Milford, MA, USA) following the manufacturer’s

instructions. De-salted fractions were then spotted onto a MS plate with a 1:1 ratio of

protein:CHCA matrix. Mass spectrometry was performed using the 4700

Proteomics Analyser (Applied Biosystems) at the Institute for Molecular Bioscience.

A plate-wide calibration for MS and MS/MS data was performed using mass

standards contained in the MS/MS Mass Standards kit (Sigma-Aldrich). Potential

protein matches were then identified from automated searching of the MASCOT

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database using GPS Explorer™ protein analysis software (version 4.0) with the

mass/ion peak information obtained from both the MS and the MS/MS spectra.

4.2.7 Database Analysis and Interpretation

The protein score, protein score confidence interval, total ion score (TIS) and total

ion score confidence intervals obtained from MS and MS/MS database analysis were

used to rank proteins from a list of potential matches. Potential proteins for each

spot were firstly ranked by TIS, reflecting how well the proteins were matched to

sequence data obtained from MS/MS analysis, where scores of ≥ 38 were considered

significant (p <0.05 that protein sequence data was matched randomly). However,

select proteins with scores less than 38 are also reported in this chapter due to their

potential as candidate factors for serum-free and feeder-free growth. When MS/MS

data were not able to return total ion scores of ≥ 38, potential matches were ranked

based on protein score. The protein score reflects how well peptide masses matched

those from predicted trypsin cleaved peptide sequences, where scores equal to or

greater than 60 are considered significant (p <0.05 that masses were matched

randomly). Proteins were selected based on the highest TIS and/or the highest

protein score. In this study the TOF-TOF analysis revealed only protein scores of

less than 60. Furthermore, the proteins were identified with their respective

functions using Swiss-Prot (http://au.expasy.org/sprot/), PubMed

(http://www.ncbi.nlm.nih.gov/sites/entrez), and Online Medelian Inheritance in Man

(OMIM) searches (http://www.ncbi.nlm.nih.gov/sites/entrez?db=OMIM).

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4.3 RESULTS

4.3.1 Morphology and expression of cell surface markers present on the passage 2

keratinocytes propagated using VN:GF medium for proteomic analysis.

Morphology and marker expression were used to ensure that the conditioned media

to be analysed was collected from undifferentiated primary keratinocytes. Presently,

there are no definitive assays for determining whether cultured primary keratinocyte

cells have maintained an undifferentiated state. However, keratin markers can be

used to provide useful information regarding the proliferative state of the cell and

whether or not the cell is a basal keratinocyte. Therefore antibodies that recognise

keratin 6 (present in hyper-proliferative keratinocytes), keratin 14 (present in basal

cells), and keratin 1/10/11 (present in more differentiated, supra-basal cells, data not

shown) were used to assess the differentiation status of the cells cultured for the

proteomics study. This analysis revealed that cells propagated using the VN:GF

medium had maintained a normal morphology compared to those grown using serum

(Figure 4.1 B and A, respectively). Additionally, keratinocytes cultured in the

VN:GF medium continued to express keratin 6 and 14 (Figure 4.1 C and D,

respectively), thus suggesting these cells have maintained their undifferentiated

primary keratinocyte morphology.

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Figure 4.1: Morphology and expression of cell surface markers on the passage 2

keratinocytes propagated using VN:GF medium for proteomic analysis. Primary

keratinocytes were isolated serum-free and then propagated using: (A) medium

containing serum and a feeder cell layer, or (B) propagated serum-free using the

VN:GF medium in conjunction with a feeder cell layer. Day 4 keratinocytes were

probed with antibodies against (C) keratin 6, and (D) keratin 14 to assess whether the

primary keratinocytes propagated using the VN:GF remained undifferentiated.

Conditioned media was collected from the cultures every two days from three

different patient samples. (Scale bar = 100 µm) (n=3, images are of a representative

culture of one of the 3 separate patients samples analysed).

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4.3.2 Two dimensional separation of conditioned media collected from both feeder

cells alone and feeder cell:keratinocyte cultures.

Proteins present in the conditioned media of feeder cells alone and from feeder

cell:keratinocyte co-cultures (Figures 4.2 A and B, respectively) were separated

using a novel form of two-dimensional liquid chromatography separation. This

involved separating proteins via a salt gradient in the first dimension, followed by a

second dimension separation using an acetonitrile gradient. The first dimension of

the standard Beckman Coulter ProteomeLab was replaced with Bio-Rad’s Duo-flow

HPLC due to poor first dimension resolution of the platform. The first dimension

HPLC fractions were then applied to the second dimension of the ProteomeLab and

proteins were visualised based on their absorbance values using the ProteoView

software. Clearly, there is an increase in the number of distinct protein spots

(fractions) expressed in the feeder cell:keratinocyte culture conditioned media

(Figure 4.2 B), above that found with the feeder cell alone conditioned media.

Furthermore, there appear to be observable changes in abundance of

proteins/peptides between the feeder cells alone conditioned media (Figure 4.2 A)

and that obtained from the feeder cell:keratinocyte cultures (Figure 2B).

Subsequently, 187 protein fractions represented in Figure 4.2 A and 238 protein

fractions represented in figure 4.2 B were isolated, digested and analysed using

MALDI TOF-TOF.

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Figure 4.2: Two dimensional separation of conditioned media. Media was

collected from (A) feeder cells alone and (B) feeder cell:keratinocyte cultures. First

dimension separation involved injecting 1 mg of protein, concentrated from the

conditioned media, onto a 0 - 500 mM NaCl gradient. Subsequently, fractions were

collected and applied to a second dimension separation which involved using a 0-

100% acetonitrile gradient as per the material and methods section. (Conditioned

medium from 3 separate patient cultures were pooled). The first and second

dimension separation were depicted on the X and Y axes and are represented by

increasing colour intensity (from blue to red). These colour outputs represent protein

fractions and were generated from absorbance readings.

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4.3.3 Proteins identified in the feeder cell and the feeder cell:keratinocyte

conditioned media.

Initially, MALDI-TOF-TOF analysis was performed on the protein fractions and did

not reveal significant ion scores for the feeder cell alone or the feeder

cell:keratinocyte conditioned media (CM). Consequently, the CM was analysed

using two liquid chromatography methods; the first involved the QTRAP MS/MS

system (LC/ESI/MS) (conducted on fractions from the first dimension separation),

while the second utilised LC-MALDI (conducted on the concentrated CM sample)

(Table 4.1 and 4.2). The Mascot database was then employed to analyse the proteins

present in the conditioned media. The LC/ESI/MS and LC-MALDI results were

organised into seven major groups; extra-cellular matrix (ECM), membrane, nuclear,

secreted, serum-derived and miscellaneous proteins/factors. Additionally, the

proteins were categorised using accession number, molecular weight, total score and

peptide count. All proteins identified in Tables 4.1 and 4.2 are either identified or

exhibit extensive homology as determined by ion score. The feeder cell alone results

revealed; 12 ECM, 2 growth factors, 17 miscellaneous, 14 membrane, 10 nuclear, 5

secreted, and 3 serum-derived proteins (Table 4.1). The feeder cell:keratinocyte

results revealed; 3 cytoplasmic, 22 ECM, 30 miscellaneous, 21 membrane, 19

nuclear, 9 secreted, and 4 serum-derived proteins. LCMS results were organised by

their total ion score (Table 4.2).

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Table 4.1

Protein Accession Number

MW (kDa)

Total Ion Score

(LC/ESI/MS)

Ion Score LC-

MALDI Extra-Cellular Matrix Cartilage intermediate layer protein 1 O75339 135 42 Collagen type 1, alpha-2 P08123 130 40 Collagen type 4, alpha-4 Q9QZR9 166 63 Collagen type 5, alpha-1 P20908 184 50 Collagen type 6, alpha-3 P12111 345 44 Collagen type 7 Q63870 296 52 19 Collagen type 19, alpha-1 Q14993 116 48 Collagen, type 27, alpha-1 Q8IZC6 187 40 Fibronectin precursor P11276 276 31 Laminin subunit alpha-5 Q61001 416 40 18 Stretch-responsive fibronectin protein type 3 Q70X91 399 41 Tenascin-X O18977 454 38 Growth Factors Insulin-like growth factor-I Q13429 15 143 Insulin-like growth factor-II P09535 20 25 Miscellaneous CaM Kinase ID Q8IU85 43 21 Catalase P04040 60 38 Complement C4 [Precursor] AAN72415 193 51 Discs large homolog 5 Q8TDM6 203 43 Fucosyltransferase 8 Q543F5 67 41 Metastasis suppressor protein 1 Q8R1S4 74 28 Myosin-9 P35579 146 44 Neuronal apoptosis inhibitory protein 5 Q8BG68 162 23 Neutral alpha-glucosidase C type 3 Q8TET4 105 28 Peroxiredoxin 1 Q9BGI4 22 42 Plectin-1 Q9QXS1 535 40 Poly [ADP-ribose] polymerase 14 Q460N5 172 48 Transglutaminase y Q6YCI4 80 40 Tuberin CAA56563 276 41 Tyrosine-protein phosphatase non-receptor type 13

Q64727 117 38

Uncharacterised progenitor cells protein Q9NZ47 9 25 Vinculin Q64727 117 38 Membrane Activin receptor type-2B Q13705 58 39 EGF-like domain-containing protein 4 Q7Z7M0 265 38 Fat3 Q8R508 505 41 Hepatocyte growth factor receptor Q9QW10 30 21 Insulin-like growth factor 1 receptor Q60751 158 20 Integrin alpha-7 Q13683 130 47 Intercellular adhesion molecule 1 Q95132 60 41 Chondroitin sulfate proteoglycan 4 Q6UVK1 251 40 Mucin-4 Q8JZM8 367 44 Neurexin-2-alpha Q9P2S2 180 38 Protein patched homolog 2 O35595 130 24 Serine/threonine-protein kinase MARK2 O08679 81 48 Tumor-associated hydroquinone oxidase Q16206 71 48 Ubiquitin thioesterase T30850 293 49

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Nuclear Antigen KI-67 P46013 321 55 34 PPAR-binding protein Q925J9 105 40 Scapinin Q8BYK5 63 38 Sentrin-specific protease 2 Q91ZX6 67 22 SON protein P18583 260 41 STAT5a Q3UZ79 32 19 Telomerase-binding protein EST1A P61406 162 38 Tra1 homolog Q80YV3 294 48 Zinc finger protein HRX P55200 425 46 Zinc finger protein spalt-3 [Fragment] Q9EPW7 136 40 Secreted Alpha-fetoprotein P49066 68 78 Insulin P01317 11 22 Kininogen P01044 69 61 Latent-transforming growth factor beta-binding protein 2

Q14767 204 39

Transferrin

P02787 79 64

Serum-Derived Bovine Serum Albumin AAN17824 71 198 524 Fetuin S22394 39 147 124 Hemiferrin Q64599 25 91

Table 4.1: Proteins identified from feeder cell conditioned media using

LC/ESI/MS system and LC-MALDI. Conditioned media was collected every two

days, pooled concentrated, isolated and separated in the first dimension as per the

material and methods section. Fractions collected from the first dimension separation

were processed and applied to the LC/ESI/MS. Data was analysed using the Analyst

1.4.1 software and proteins were later identified using the Mascot database search

engine. Individual ions scores > 37 indicate identity or extensive homology (p<0.05).

Alternatively, LCMS-MALDI was conducted on protein samples as a secondary

analysis. Potential proteins matches were then identified from automated searching

of the MASCOT database using GPS Explorer™ protein analysis software (version

4.0) with the mass/ion peak information obtained from both the MS and the MS/MS

spectra.

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Table 4.2


MW (kDa)

Total Ion Score

(LC/ESI/MS)

Ion Score LC-

MALDI Extra-Cellular Matrix Cartilage intermediate layer protein 1 O75339 135 42 Collagen type 1, alpha-2 P08123 130 40 Collagen type 2 alpha-1 P02458 142 40 Collagen type 4, alpha-1 Q9QZR9 166 63 Collagen type 4, alpha-3 Q9QZS0 163 39 Collagen type 7 P12111 345 44 Collagen type 7, alpha-1 Q63870 295 56 Collagen type 11, alpha-2 P13942 172 54 Collagen type 12, alpha-1 Q99715 334 55 Collagen, type 27, alpha-1 Q8IZC6 187 40 25 Fibronectin 1 Q3UHL6 260 22 Hypothetical fibronectin type III Q8BKM5 82 38 Lamb3 protein Q91V90 132 41 Laminin subunit alpha-1 CAA41418 297 69 Laminin subunit alpha-2 Q59H37 204 27 Laminin subunit alpha-5 Q61001 416 40 Laminin alpha 3b chain Q76E14 376 61 Laminin subunit beta-2 [Precursor] Q61292 203 43 Laminin subunit gamma-3 [Precursor] Q9Y6N6 177 39 Laminin 5 WO0066731 132 37 Stretch-responsive fibronectin protein type 3 Q70X91 399 41 Tenascin-X O18977 454 38 Cytoplasm Liprin-alpha-2 Q8BSS9 143 63 Liprin-alpha-3 O75145 133 40 Serine/threonine-protein kinase TAO1 Q7L7X3 116 42 Growth Factors

Transforming growth factor alpha P01135 18 31 Miscellaneous Actin alpha 2 P62736 42 81 Actin, beta [Fragment] Q96HG5 41 78 Ankyrin-3 Q12955 482 37 Carbamoyl-phosphate synthetase I P31327 165 38 Catalase P04040 60 38 CDNA FLJ11753 fis, clone HEMBA1005583 Q9HAE5 32 37 Complement C4 [Precursor] AAN72415 193 51 Discs large homolog 5 Q8TDM6 203 43 Dystrophin P11531 427 48 Exostosin-1 Q16394 87 48 Fucosyltransferase 8 Q543F5 67 41 Granulocyte inhibitory protein II homolog Q9UD48 2 31 Hypothetical protein Q8C7W2 55 40 Kinesin-like protein KIF13A Q9H1H9 200 46 Myosin-9 P35579 146 44 myosin-IXb Q14788 230 45 Myosin-XVIIIa Q9JMH9 117 42 Neuron navigator 3 Q8NFW7 245 58 Peroxiredoxin 1 Q9BGI4 22 42 Plectin-1 Q9QXS1 535 40

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Poly [ADP-ribose] polymerase 14 Q460N5 172 48 Protein diaphanous homolog 2 O70566 125 46 Protein disulfide-isomerase P04785 30 37 Protein piccolo Q9Y6V0 568 59 Sacsin Q9NZJ4 441 39 Serine protease inhibitor EIC Q8K3Y1 42 41 Transglutaminase y Q6YCI4 80 40 Tuberin CAA56563 276 41 Tyrosine-protein phosphatase non-receptor type 13

Q64727 117 38

Ubiquitin specific protease 1 Q8BJQ2 88 58 Membrane Acetyl-CoA carboxylase 2 O00763 281 39 Cadherin EGF LAG seven-pass G-type receptor 3

Q91ZI0 363 40

Cation-independent mannose-6-phosphate receptor

P11717 281 41

Chondroitin sulfate proteoglycan 4 Q6UVK1 251 40 Cytokeratin-1 P04264 66 68 Cytokeratin-9 P35527 62 127 EGF-like domain-containing protein 4 Q7Z7M0 265 38 EMR1 hormone receptor Q14246 101 40 Fat3 Q8R508 505 41 Integrin beta-4 P16144 211 43 Integrin alpha-7 Q13683 130 47 Intercellular adhesion molecule 1 Q95132 60 41 Mucin-4 Q8JZM8 367 44 Mucin-16 Q8WXI7 747 49 Neurexin-2-alpha Q9P2S2 180 38 RIM ABC transporter P78363 258 58 Serine/threonine-protein kinase MARK2 O08679 81 48 Talin-1 Q9Y490 273 43 Talin-2 Q9Y4G6 274 40 Tumor-associated hydroquinone oxidase Q16206 71 48 Ubiquitin thioesterase T30850 293 49 Nuclear DNA-binding protein SMUBP-2 Q60560 109 43 Antigen KI-67 CAA46520 321 42 Lipin-3 Q7TNN8 95 38 Nesprin-2 AAL33548 801 53 Nef-associated factor 1 Q15025 35 46 NFX1-type zinc finger-containing protein 1 Q9P2E3 225 46 Periaxin AAK19279 155 52 PPAR-binding protein Q925J9 105 40 Putative rRNA methyltransferase 3 Q9DBE9 95 48 Scapinin Q8BYK5 63 38 SET-binding factor 1 O95248 210 41 SON protein P18583 233 42 Telomerase-binding protein EST1A P61406 161 49 Tra1 homolog Q80YV3 294 48 Transcription factor 7-like 2 Q924A0 52 34 TTF-I-interacting protein 5 Q9UIF9 210 57 Zinc finger protein HRX P55200 425 46 Zinc finger protein spalt-3 [Fragment] Q9EPW7 136 40 Zinc finger protein 40 P15822 299 57 Secreted Apolipoprotein A-II P81644 8 56

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Follistatin-related protein 5 Q8BFR2 95 27 Latent-transforming growth factor beta-binding prot-2

Q28019 208 38

Matrix-remodeling-associated protein 5 Q9NR99 314 39 Nidogen P10493 139 26 Platelet glycoprotein V Q9QZU3 64 39 Proteoglycan-4 [Precursor] Q9JM99 117 37 SCO-spondin [Precursor] P98167 575 38 Transferrin P02787 79 64 Serum-Derived Bovine Serum Albumin AAN17824 71 198 335 Fetuin S22394 39 147 126 Hemiferrin A39684 24 50 Human Serum Albumin CAA23753 71 64

Table 4.2: Proteins identified from feeder cell:keratinocyte conditioned media

using LC/ESI/MS and LC-MALDI. Conditioned media was collected every two

days, from cell cultures derived from skin isolated from 3 different patients. The

conditioned media was pooled, concentrated, isolated and separated in the first

dimension as per the material and methods section. Fractions collected from the first

dimension separation were processed and applied to the QTRAP MS/MS system.

Data was analysed using the Analyst 1.4.1 software and proteins were later identified

using the Mascot database search engine. Individual ions scores > 37 indicate

identity or extensive homology (p<0.05). Alternatively, LCMS-MALDI was

conducted on the protein samples as a secondary analysis. Potential proteins matches

were then identified from automated searching of the MASCOT database using GPS



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4.3.4 Differences in abundance of protein species found in the feeder cell and the

feeder cell:keratinocyte conditioned media.

Relevant proteins and protein differences for the culture of keratinocyte cells were

tabulated from the LC-ESI, LC-MALDI (Table 4.1 and 4.2) and MALDI-TOF-TOF

(Table AI.1 & 2) analysis. These candidate proteins were then separated into their

respective categories including; Extra-cellular Matrix, Growth Factors and

Cytokines, Secreted and Intracellular proteins. There was overlap between proteins

in both treatments including: Collagens I, IV and VII; fibronectin I; Laminin V;

TGFs alpha and beta; VEGF; Interleukins 1, 10 and 15; Telomerase-binding protein

EST1A; and Tra1 homolog. However, unique proteins were also observed in the

feeder cell alone treatment including: Collagens V and VI; Bone Morphogenic

protein 1 (BMP 1); bFGF; human growth hormone (hGH); FGF 3; Insulin; IGF-I and

-II; Interleukin-8; Leukemia inhibitory factor and Megakaryocyte-CSF. Furthermore,

unique proteins were also observed in the feeder cell:keratinocyte treatment

including: Fibronectin III; Laminin I and III; nerve growth factor (NGF); PC cell-

derived growth factor; platelet-derived growth factor beta (PDGF); Interleukin 4 and

6; PDGF-inducible JE glycoprotein; Follistatin-related protein 5; growth inhibitory

factor; Growth differentiation factor 9 and telomerase reverse transcriptase.

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Table 4.3

Feeder Cell Alone Score Ion (I) Protein

(P)

Feeder Cell:Keratinocyte Score Ion (I) Protein

(P) Extra-cellular Matrix Extra-cellular Matrix Collagen I I-40 Collagen I I-40 Collagen IV I-63 Collagen IV I-63 Collagen V I-50 Collagen VII I-44 Collagen VI I-44 Fibronectin I I-22 Collagen VII I-52 Fibronectin III I-38 Fibronectin I I-31 Laminin I I-69 Laminin V I-40 Laminin III I-61 Laminin V I-37 Growth Factors and Cytokines BMP 1 P-25 Growth Factors and Cytokines bFGF P-32 FGF-2 associated protein 3 P-36 FGF homologous factor 3 P-20 NGF homolog 1 P-40 Growth hormone P-34 PC cell-derived growth factor P-36 Insulin I-22 PDGF bb P-16 Insulin-like growth factor 1 I-143 TGF alpha I-31 Insulin-like growth factor 2 I-25 TGF beta I P-18 TGF alpha P-14 VEGF P-20 TGF beta 2 P-34 Interleukin 1 alpha P-21 VEGF P-20 Interleukin 4 P-20 Interleukin 1 beta P-39 Interleukin 10 P-21 interleukin-8 P-32 Interleukin-6 P-37 Interleukin 10 P-32 Shorter isoform of interleukin 15 P-19 Isoform of interleukin 15 P-25 PDGF-inducible JE glycoprotein P-43 Leukemia inhibitory factor P-33 Secreted Secreted Follistatin-related protein 5 I-27 Megakaryocyte- CSF P-22 growth inhibitory factor P-15 Growth differentiation factor 9 P-31 Intracellular Telomerase-binding protein EST1A I-38 Intracellular Tra1 homolog I-48 Telomerase reverse transcriptase P-31 Telomerase-binding protein

EST1A I-49

Tra1 homolog I-48

Table 4.3: Differences in abundance of protein species found in the feeder cell

and the feeder cell:keratinocyte conditioned media. Mass spectrometry analysis

was performed on the liquid fractions obtained from the feeder cell alone or the

feeder cell:keratinocyte conditioned media (CM) and candidate and or proteins of

interest were categorised by functional class of the protein and ion score (I) or

protein score (P).

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4.4 DISCUSSION

Many novel technologies involving primary keratinocytes are being developed for

the therapeutics industry to aid in the regeneration and healing of skin defects

(Harkin et al. 2006; Sun et al. 2007). However, technologies used to propagate these

cells ex-vivo still require undefined components, such as serum and/or feeder cells,

and generally utilise a poorly defined culture system. As outlined in Chapter 2, I

have developed a fully defined serum-free technology (VN:GF) that can support the

isolation, establishment and serial propagation of undifferentiated keratinocytes.

Whilst this discovery was a step forward, the culture approach still required the use

of an irradiated i3T3 feeder cell layer for successful serial propagation and in-vitro

expansion.

It has been demonstrated that irradiated i3T3 feeder cells secrete large quantities of

IGFs and ECM proteins (Barreca et al. 1992), as well as a variety of other proteins.

Moreover, keratinocytes have also been demonstrated to express the receptors for

many growth factors and ECM proteins (Adams. and Watt 1991; Haase et al. 2003;

Rodeck et al. 1997; Stoll, Garner, and Elder 1997; Watt and Jones 1993; Watt et al.

1993). Indeed, other laboratories have investigated the use of these proteins for the

culture of keratinocytes. For example, Dawson et al. (1996) demonstrated that

keratinocytes can attach and proliferate in response to VN-coated surfaces.

Nevertheless, the most robust culture systems for keratinocytes still require the use

of a feeder cell layer (Huang et al. 2006). This requirement for a feeder cell layer

highlights the importance that the feeder cells have in the culture system. More

recently, other groups have demonstrated that other cell types, such as human

embryonic stem cells, can be propagated feeder-free using ECM proteins when the

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culture system is supplemented with conditioned media obtained from MEFs, thus

suggesting that the critical component provided by the feeder cells is a soluble factor

secreted by the feeder cell layer (Lebkowski et al. 2001; Xu et al. 2001) .

Therefore, I hypothesised that novel proteins in conditioned media may be able to be

identified using proteomic techniques and that these proteins could potentially be

used in conjunction with the VN:GF medium to support serum-free and feeder cell-

free propagation of keratinocytes. Furthermore, given that the VN:GF media does

not contain serum or high abundance proteins such as albumin i.e. it is a low protein

content media, I was in a unique position to identify critical factors important to

keratinocyte survival. These factors may normally be masked by the high abundant

proteins traditionally incorporated into serum-containing or high protein content

media.

To date, most proteomic analysis in this area and related fields, has been conducted

on the feeder cell layer alone (Prowse et al. 2005; Boraldi et al. 2003; Wee Eng Lim

and Bodnar 2002), providing insight into what fibroblasts secrete into the media.

However, my research takes this one step further by establishing a system in which

secretions triggered by paracrine interactions of the feeder cells with the

keratinocytes could also be analysed. To examine this hypothesis I examined what

the feeder cell alone and feeder cell:keratinocyte cultures were secreting into the

media. The study of both of these treatments provides a more complete picture of the

secreted factors in response to not only the autocrine interactions, but also the

paracrine interactions, and gives a greater insight into the optimal in-vitro micro-

environment for keratinocytes. In an ideal situation, I would have also examined a

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keratinocyte alone treatment and realise that this is a shortcoming of my approach,

however, the VN:GF medium would not allow the serial propagation of

keratinocytes feeder-free. Indeed, this is what prompted me to conduct the proteomic

analysis reported here. Serum- and feeder-free mediums do exist for the propagation

of keratinocytes and could have been employed for my studies, however, using one

of these media would have altered the proteomic profiles due to the fact that these

media are undefined and contain many compounds purified from animal and human

sources.

In the scope of this study I observed 187 protein spots from the feeder cell alone

conditioned medium (CM) represented in Figure 4.2 A and 238 protein fractions

from the feeder cell:keratinocyte CM represented in figure 4.2 B. Following the mass

spectrometry analyses 63 proteins from the feeder cell alone CM and 108 proteins

from the feeder cell:keratinocyte CM were further prioritised with respect to their

potential use in generating a serum-free feeder-free culture system for primary

keratinocytes (Table 4.3). The higher number of proteins identified in the feeder

cell:keratinocyte CM (Table 4.2) was expected and is likely to arise from the fact

that there were two cell types present within this culture system. However, of the

proteins reported in Table 4.3 only 25 proteins in the feeder cell alone CM and 27

proteins in the feeder cell:keratinocyte CM were observed as having a possible

relevance for future studies. Briefly, these proteins were prioritised based on the

functional class of the protein, previous characterisation in the published literature,

reagents available for testing. Interestingly, many of the candidate proteins were

present in the two CM treatments, with the major exceptions being: Collagens V and

VI, Bone Morphogenic protein 1 (BMP 1), bFGF, human growth hormone (HGH),

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FGF 3, Insulin, IGF-I and -II, Interleukin-8, Leukemia inhibitory factor and

Megakaryocyte-CSF in the feeder cell alone CM; and Fibronectin III; Laminin I and

III; nerve growth factor (NGF); PC cell-derived growth factor; platelet-derived

growth factor beta (PDGF); Interleukin 4 and 6; PDGF-inducible JE glycoprotein;

Follistatin-related protein 5; growth inhibitory factor; Growth differentiation factor 9

and telomerase reverse transcriptase in the feeder cell:keratinocyte CM (Table 4.3).

Whilst the system employed here utilised the serum-free VN:GF medium described

in Chapter 2, several bovine and human serum-derived proteins were identified in

the feeder cell alone and feeder cell:keratinocyte treatments; namely, bovine and

human serum albumin, fetuin, and members of the transferrin family. This was not

unexpected as the fibroblast cells were cultivated in, and exposed to bovine serum-

containing medium prior to transferring cells to VN:GF medium. Likewise, the

human serum-derived proteins were carried over from the donor patient’s skin

during the keratinocyte isolation, despite the fact that the keratinocytes themselves

were isolated and cultured entirely serum-free. Additionally, high abundant serum

proteins, such as albumin are often adhesive and associate with extra-cellular

surfaces and culture vessels, thus making them difficult to completely remove

through washing steps alone. Indeed, this serum carry over is commonly observed

when conducting proteomic studies and indeed Prowse et al. (2005) and Lim and

Bondar (2002) also observed these serum-derived proteins even though they

employed a series of wash steps and an incubation of the cells in serum-free

medium. Nevertheless, it is clear that the series of washes and serum-starvation steps

employed in both their study and ours, prior to transfer to serum-free conditions

significantly reduced the presence of these serum-derived components in the CM.

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Therefore, these serum-derived proteins did not markedly interfere with the

resolution of the proteomic profiles (Figure 4.2 A & B). Future studies will involve

developing a serum-free media for the propagation of fibroblast feeder cells. This

will aid in the removal of the foetal bovine serum-derived proteins that are

commonly carried over with the serum-cultivated fibroblast cells.

Several intra-cellular proteins were observed within both CM treatments of this

study. The presence of these proteins is most likely due to the cells lysing, hence

leaking their intracellular contents into the culture system. Whilst these intracellular

proteins were not the prime focus of this study, some of the proteins identified

warrant further investigation such as telomerase reverse transcriptase, telomerase

binding protein, c-myc, and Tra1. It is also important to note here that several

proteins were omitted from tables 4.1 and 4.2 due to the fact that they could not be

identified; that is, they are classified on the database as being i.e. hypothetical

proteins, unknown proteins, and proteins with no known function. While these may

well provide to be critical factors, the discussion that follows focuses on proteins

whose functions and characteristics are known, at least to some extent.

The analysis of the feeder cell alone conditioned medium (Figure 4.2A, Table 4.1

and Table AI.1) and the feeder cell:keratinocyte culture conditioned medium (Figure

4.2B, Table 4.2 and Table AI.2), revealed several proteins important for the survival

of primary keratinocytes. Several ECM proteins were identified and include;

Collagen I, IV, V, VI, and VII, Fibronectin 1 and 3, Lamb3, Laminin alpha 1, 3, 5,

and Tenascin X (Tables 4.1 and 4.2). Indeed, many of these ECM proteins were also

identified in the studies conducted by Prowse et al. (2005) and Lim and Bondar

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(2002). The ECM proteins generally exist associated with cellular surfaces rather

than as secreted products, however, they may have been present in the CM due to the

cells lysing and the presences of proteases in the conditioned medium. All the same,

these ECM proteins are found in-vivo in the extra-cellular matrix of the epidermis

and dermis (Marionnet et al. 2006). Furthermore, these proteins are commonly

involved in the attachment, migration and or proliferation of keratinocytes and have

also been proposed to have roles in wound healing (Rho et al. 2006; Schneider et al.

2006; Spichkina et al. 2006; Hartwig et al. 2007).

Research groups involved in the development of serum-free and feeder cell-free

culture methods for hES cells have recently commenced exploring the use of ECM

proteins, such as those mentioned, in their culture systems. For example, laminin was

demonstrated to replace the need for a feeder cell layer when grown in the presence

of mouse embryonic fibroblast (MEF) conditioned medium (Xu et al. 2001) or with

knock-out serum replacement (KSR) + Activin-A (Beattie et al. 2005). Moreover,

Amit et al. (2004) discovered a method to propagate these cells using a fibronectin

matrix in conjunction with a range of growth factors including, transforming growth

factor β1 (TGF β1), leukaemia inhibitory factor (LIF) and basic fibroblast growth

factor (bFGF). Due to the fact that the culture of primary keratinocytes is analogous

to hES cell culture, it is therefore likely that these ECM protein-based technologies

can be translated to the culture of keratinocytes. Interestingly, all the ECM

technologies developed for the propagation of keratinocyte and hES cells thus far,

also involve the use of some form of mitogen.

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The results reported herein demonstrated that several growth factors and mitogens

were present in the conditioned medium including IGF-I, IGF-II, insulin,

transforming growth factors (TGF) α and β, platelet-derived growth factor (PDGF)

and bFGF, all of these being present in the conditioned media of the two treatments

(Tables 4.1, 4.2 and Appendix I). Interestingly, neither Prowse et al. (2005) or Lim

and Bondar (2002) identified any of these growth factors in their studies examining

at the proteomic profiles of fibroblast cells. I believe this is due to the previously

described advantages that I gained through using a minimal protein content medium.

Alternatively, the secretion of these growth factors may have been induced via the

use of the serum-free medium.

One of the growth factors identified, insulin is a critical component in many

mammalian cell culture media and has been incorporated into the culture of

keratinocytes for some time now. Usually insulin is present in these keratinocyte

culture media at high concentrations, however, we recently demonstrated that low

concentrations of IGF-I can replace the need for insulin (Hollier et al. 2005; Hyde et

al. 2004). Indeed, it has been reported that when insulin is present at high

concentrations its growth stimulating effects are in fact mediated by the IGF-I

receptor (Dupont and leRoith 2001), hence the ability to replace insulin with IGF-I in

media used to culture keratinocytes is not surprising. Similarly, studies conducted in

my own honours project revealed that IGF-I and IGF-II when used in conjunction

with VN caused mitogenic effects in hES cells. Moreover, bFGF, TGF-beta and

PDGF, are heparin binding growth factors that have been demonstrated to enhance

the proliferation and self renewal of feeder cell dependent hES cells (Wang et al.

2005; Liu et al. 2006; James et al., 2005; Pebay et al., 2005). Furthermore, the TGF

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proteins have been demonstrated to enhance migration (Li et al. 2006) and

proliferation of epidermal and keratinocyte cells (Fortunel et al. 2003; Sawamura et

al. 2004) and thus have been proposed as potentially being effective in mediating

wound healing events (Sun et al. 2004b). Interestingly, these heparin-binding growth

factors appear to be able to bind to VN through its heparin binding domain (Hollier

et al. 2005; Schoppet et al. 2002). Thus, the growth factors identified in this

proteomic analysis all have roles related to keratinocyte growth and may well prove

to be useful in conjunction with the VN:GF medium in providing a serum-free,

feeder-free media for the in-vitro expansion of transplantable cells for use in clinical

therapies.

In addition to the proteins discussed above, telomerase reverse transcriptase (TERT)

telomerase-binding protein (EST1A), Follistatin-like 5, and tumor rejection antigen1

(Tra1) homolog, were also expressed in the conditioned media of both treatments

(Tables 4.1, 4.2 and Appendix I). The telomerase-binding protein is involved in

telomere replication in-vitro via human telomerase reverse transcriptase.

Interestingly, a down regulation in hTERT or telomerase abundance is linked to

embryonic stem cell differentiation (Wang et al. 2007). Therefore, if this protein can

be induced, directly or indirectly, in the culture of keratinocytes, it may facilitate the

long term propagation of primary keratinocytes. Another nuclear protein that is of

interest is the Tra1 homolog which has a central role in c-Myc transcription

activation, and also participates in cell transformation. Furthermore, c-Myc has been

demonstrated to be important in the activation and regulation of hTERT (Chen et al.

2006). The secreted protein, follistatin-like 5, was also present in the conditioned

media examined. Notably, the follistatin-like domain present in this protein has been

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implicated in the inactivation of activin-A and TGF-β (Schneyer et al. 2003;

Schneyer et al. 2004), two proteins which have been demonstrated to be important

for the self renewal of human embryonic stem cells (James et al. 2005). Taken

together, these data suggests that these proteins may also play an important role in

maintaining the undifferentiated status of other primitive cells, such as primary

keratinocytes.

In summary, the proteomic study reported here has revealed the abundance of many

proteins from both the feeder cells alone and the feeder cell:keratinocyte culture

treatments. In light of the paracrine relationship which exists between the dermal

fibroblasts and keratinocytes (Maas-Szabowskiet al. 1999; Werner and Smola 2001),

the study here identified not only what the feeder cells are secreting in isolation, but

what they and the keratinocytes secrete due to their paracrine interactions. Ideally, it

would have been of great benefit to also examine media conditioned by keratinocytes

alone to determine what these cells secrete when cultivated without feeder cells.

However, this highlights the key point of this investigation, i.e. keratinocyte cells

grow poorly in the absence of a feeder cell layer.

While this chapter reports a preliminary investigation into the feeder

cell:keratinocyte paracrine interaction, future studies will also adopt a more

quantitative approach and will also compare and contrast the differences in

abundance between similar proteins expressed in both the culture conditions. This

will provide insights into not only which candidate proteins/factors warrant further

investigation, but also provide information into the concentrations of candidate

proteins to add in conjunction with the VN:GF medium. Indeed, the most obvious

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candidates to apply to the VN:GF medium would be the ECM proteins and HSA as

these have been demonstrated to be promising candidate factors in the feeder- and

serum-free culture of hES cells (Ludwig et al. 2006). More specifically, the VN:GF

medium will initially be examined in keratinocytes in conjunction with a laminin

bed, HSA and bFGF as these have all been shown to provide an appropriate micro-

environment for hES cells. Additionally, leukaemia inhibitory factor, which has been

demonstrated to support the self renewal of mouse embryonic stem but not human

stem cells, may also provide a keratinocyte culture environment free from fibroblast

feeder cells.

Nevertheless, whilst extensive validation and testing of these candidates is still

required, the data reported in this chapter has provided intriguing preliminary

insights into the in-vitro micro-environment of primary keratinocytes and has

provided useful initial information on candidate proteins that may hold potential if

used in conjunction with the serum-free medium. The proteins reported in Table

4.3.4 may provide an important first step towards developing the first fully defined,

synthetic culture system for primary keratinocyte cells, and may ultimately provide

researchers and clinicians with a realistic culture approach to generate

therapeutically viable tissue source for transplantation.

.

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CHAPTER 5

DISCUSSION AND

CONCLUSION

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5.0 GENERAL DISCUSSION

Tissue engineering is the construction of tissues, such as skin, bone, and cartilage, in

an in-vitro micro-environment. Currently, the tissue engineering market is worth an

estimated $US120 billion worldwide (Urban 2005) and carries great promise for the

treatment of cell, tissue and organ damage, through the generation of transplantable

material. However, the in-vitro culture of cells relies heavily on the use of undefined

foreign components for their successful cultivation. Examples of these foreign

components include fibroblasts and serum, sourced from allogeneic or xenogeneic

origins. These two components have been demonstrated to support the cells of

interest, such as keratinocytes and hES cells, by providing them with a suitable

microenvironment that promotes their self renewal. Particularly important to the

growth of cells are ECM proteins and growth factors, which aid in their attachment,

migration and proliferation. Indeed, irradiated 3T3 (i3T3) feeder cells are known to

secrete large quantities of IGFs and ECM proteins (Barreca et al. 1992), suggesting

that this could be a key role of feeder cells in tissue culture systems. Furthermore,

serum has also been demonstrated to contain large quantities of ECM proteins e.g.

VN (Schvartz, et al. 1999), fibronectin, albumin, as well as various other growth

promoting components such as growth factors.

5.1 Serum-free propagation of primary human keratinocytes.

Human keratinocytes are a cell type which demonstrates the most immediate

potential in the cell-based therapeutics industry (Green 1991; Meana et al. 1998;

Wright et al. 1998). The first successful propagation of these cells was in 1975

(Rheinwald and Green 1975). However, this cultivation method required both serum

and fibroblasts for their undifferentiated growth, thus exposing potentially

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transplantable material to diseases and pathogens that could be present within either

the serum or feeder cell layer. In view of this there has been a concerted effort by

scientists world-wide to develop ways to reduce the risk of contamination from the

culture system, and in particular by developing methods that allow the removal of

serum and/or the feeder cell layer.

Castro-Munozledo et al. (1997) first hypothesised that they could replace foetal

bovine serum with purified bovine serum albumin. Whilst this method did prove

successful in the propagation of keratinocytes, it did not remove the risk of potential

contamination of the cells by the bovine serum-derived albumin, nor did it result in a

fully defined media, as the BSA, which acts as a carrier protein, was purified from

serum, thus other serum-derived proteins could be transferred with it into the media.

More recently, Sun et al. (2004a) developed a technique whereby normal human

keratinocytes, pre-established on fibroblasts using serum-containing media, could be

propagated under xenobiotic-free conditions. They demonstrated that a non-

irradiated human skin-derived feeder cell layer, used at the appropriate density,

would support the serum-free propagation of human keratinocytes. However, whilst

this proved successful for the propagation of keratinocytes, this technology could not

be used for the isolation and the initial establishment of primary keratinocytes from

skin, as serum was still required for these steps. Indeed, all primary keratinocyte cell

lines isolated and established have been reported to use either allogeneic or

xenogeneic serum for these initial steps, which once again carries the risk of

contaminating the cells that are ultimately to be used for transplantation.

Additionally, Bullock et al. (2006) reported a method to substitute xenogeic feeder

cell layers for human-derived feeder cell layers sourced from the fibroblast cells of

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the dermis. They demonstrated successful propagation and subsequent re-

epithelisation of cells using the human feeder cell layer under serum-free conditions.

Nevertheless, this methodology still required the use of serum for the initial isolation

of the keratinocytes and for trypsin neutralistion, thus resulting in the potential carry

over of serum components to the serum-free culture conditions subsequently used.

As described in this thesis, I have demonstrated that primary keratinocytes can be

isolated and serially propagated long term, in a fully defined serum-free medium.

Furthermore these cells expressed markers demonstrating that they remained in the

undifferentiated state and were able to re-epithelialise 3D dermal skin equivalent

models (Figures 2.3, 2.4, 2.5, 2.6 Chapter 2). The successful propagation of primary

keratinocytes using the VN:GF serum-free medium is supported to some extent by

the fact that human keratinocytes express the receptors for these proteins (Adams

and Watt 1991; Haase et al. 2003; Rodeck et al. 1997; Stoll and Elder 1997; Watt and

Jones 1993; Watt et al. 1993). However, while this new serum-free media represents

an important step forward, the culture approach still relies on the presence of a

feeder cell layer for the undifferentiated propagation of keratinocytes. Nevertheless,

its potential as a fully defined serum-free alternative has now caught the attention of

biotechnology companies, and the media is currently being externally tested as a

potential product for keratinocyte culture by Invitrogen Corporation.

5.2 Serum-free propagation of primary human embryonic stem cells.

Human embryonic stem cells on the other hand were successfully passaged in-vitro

for the first time by using donated embryos that were discarded from in-vitro

fertilisation (IVF) programs (Thomson et al. 1998). These cells demonstrated a

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unique pluripotent state when propagated on a MEF feeder cell layer in media

containing foetal bovine serum (FBS) (Thomson et al. 1998; Reubinoff et al. 2000).

This pluripotency allows hES cells to transform into all the cell types within the

body, thereby underpinning the potential of hES cells in the regenerative medicine

therapeutics industry (Docherty 2001; Guan et al. 2001; Boheler et al. 2002).

However, like their keratinocyte counterparts, hES cells rely on undefined and

xenogeneic culture components, and carry the same risks of introducing pathogens

and immunogenic agents (e.g. N-glycolylneuraminic acid, Neu5Ac) (Martin et al.

2005; Heiskanen et al. 2007). Thus, there is a global move towards creating a fully

defined synthetic medium for the undifferentiated growth of these cells.

To this end, many investigators have attempted to replace xeno-derived feeder cells

with human-derived feeder cell layers (Amit et al. 2000; Richards et al. 2002; Cheng

et al. 2003). However, this is still not an ideal situation as hES cells could just as

easily be infected from pathogens within the human-derived feeder cell layer. Due to

the feeder cell layer’s support role as a source of ECM proteins and growth factors,

Xu et al. (2001) hypothesised that an ECM bed, such as laminin or matrigel, could

replace the need for a feeder cell layer. However, this technology was only

successful with the addition of MEF conditioned medium, and hence any benefit

obtained through the removal of the MEF feeder cell layer was abrogated. More

recently, Ludwig et al. (2006) demonstrated a methodology which allowed hES cells

to be propagated both feeder-free and serum-free. This technology involved the use

of large quantities of purified human serum albumin, as well as a range of other

growth factors and proteins. Through this innovation they were able to serially

passage hES cells for several months. However, chromosomal aberrations ultimately

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appeared in their cell lines, suggesting that while this was a significant step forward,

it was still not the answer. Thus, with the project reported herein, I sought to

determine whether a fully defined serum-free VN:GF medium could be developed

for hES cells. Similar to the media developed in Chapter 2 for keratinocytes, the

VN:GF medium for hES cells included; VN, IGF-I and IGFBP-3, with, bFGF being

used instead of EGF. Using this media, karyotypically normal hES cells could be

serially propagated long term, whilst still maintaining markers for their

undifferentiated state (Figures 1-5 Chapter 3).

Having demonstrated that two independent hES cell lines could be serially

propagated in a fully defined serum-free culture system (Chapter 3), I then decided

to explore whether the feeder-free technology developed by Beattie et al. (2005)

could be combined with the VN:GF media. The technology developed by Beattie et

al. (2005) removed the need for a MEF feeder cell layer and involved the use of

activin-A, keratinocyte growth factor and nicotinamide (ANK). Beattie’s studies

demonstrated that hES cells can be serially propagated feeder-free long term, whilst

still maintaining an undifferentiated state. However, their protocol still required

KSR, a serum-replacement component which is undefined and uncharacterised.

Therefore, I conducted studies at the Whittier Institute (La Jolla, CA, USA), in

conjunction with Beattie et al. (2005) examining whether ANK could be used in

conjunction with the VN:GF medium. As reported in this thesis, the HUES-7 cell

line could be grown in an undifferentiated state using ANK and the VN:GF medium

(Figures 6 D and 7D Chapter 3). However, hES cells cultured in these conditions

were not optimal as determined by the morphology of the hES colonies. Moreover,

only short-term studies exploring this approach were conducted, so clearly further

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investigation is required to determine unequivocally whether hES cells can be

established and propagated long term using the ANK/VN:GF medium.

As with the primary keratinocytes, the VN:GF medium I developed for hES cells

still requires the MEF feeder cell layer for their successful propagation. Therefore

proteomic studies into the hES cell in-vitro micro-environment are required to

investigate what factors may better recapitulate a favourable culture system for the

ex vivo expansion of these cells. Whilst the technology I developed still requires a

MEF feeder cell layer for successful propagation of hES cells, the potential of

VN:GF medium has been recognised by many biotechnology companies in the

sector. Indeed, Invitrogen Corporation, recently independently validated the

technology I developed and have now acquired an exclusive international license to

sell and distribute the VN:GF media for stem cells. This new media will be launched

at the upcoming International Society for Stem Cell Research meeting in Cairns in

June 2007. The media to be distributed by Invitrogen will contain recombinant

vitronectin and growth factors, manufactured to GMP standards by Tissue Therapies

Limited, a biotechnology company spun out of QUT to commercialise the VN:GF

technology. Thus, this media will be the first fully defined, synthetic GMP-grade

media for the culture of hES cells.

5.3 Proteomics of Keratinocyte Conditioned Media

Since a paracrine relationship exists in-vivo between dermal fibroblasts and

keratinocytes (Maas-Szabowski 1999; Werner and Smola 2001), I proposed that

proteomic approaches may shed insights into this relationship. Furthermore, I

hypothesised that novel compounds important for the self-renewal of cells may be

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identified through this approach and thus may be able to be used in conjunction with

the VN:GF medium to generate a fully defined synthetic culture system for primary

keratinocytes. Moreover, these studies may also reveal potential candidates/factors

which may be of use to generate a similar system for hES cells. To date most

proteomic analyses in this field have been conducted on the feeder cell layer

(fibroblasts) alone (Boraldi et al. 2003; Prowse et al. 2005; Lim and Bodnar 2002),

providing insight therefore into only what the fibroblasts secrete. However, this is a

very limited method of analysis due to the fact that this type of study does not take

into account the paracrine relationship that exists between fibroblasts and

keratinocytes. Thus factors secreted by either cell type when they are co-cultured

would not be revealed.

I therefore decided to develop a two pronged proteomic approach in which firstly, I

identified what the fibroblasts secrete into the conditioned media, and secondly, I

analysed the conditioned media of co-cultured fibroblasts and primary keratinocytes.

The conditioned media was studied, rather than cell-surface associated proteins,

based on previous reports demonstrating that hES cells could be propagated using

media conditioned by mouse embryonic fibroblast cells, thus suggesting that the

critical component for feeder-free growth was a soluble factor secreted by the

fibroblast feeder cell layer (Lebkowski et al. 2001; Xu et al. 2001). Furthermore, my

strategy also involved the use of the VN:GF media instead of serum. The importance

of this is that this media has minimal protein content, thus will not suffer from the

“masking” effect of highly abundant proteins commonly seen when conducting

proteomics on samples that have been grown using serum. An additional benefit of

using this serum-free media approach was that no immuno-depletion step to remove

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highly-abundant serum-proteins from media was required, which in turn reduces the

chances of important or novel protein species being inadvertently lost through this

processing step.

The studies reported in Chapter 4 of this thesis revealed several proteins which may

be important for the survival of primary keratinocytes (Tables 1, 2 and Appendix I).

The most obvious candidate proteins for future studies included: the ECM proteins,

collagen (Rho et al. 2006), fibronectin (Spichkina et al. 2006) and laminin

(Schneider et al. 2006); the growth factors, IGF-I (Hollier et al. 2005; Chapter 2),

IGF-II, insulin (Ludwig et al. 2006), TGF α and β (Li et al. 2006; Fortunel et al.

2003; Sawamura et al. 2004), PDGF (Pebay et al. 2005), and bFGF (Liu et al. 2006;

Wang 2005). Additionally, other secreted proteins of interest observed in this study

were cytokines, particularly; interleukin 6 and LIF both of which have been

demonstrated to maintain feeder-dependent cells in a self renewing state (Hernandez-

Quintero et al. 2006; Hao et el. 2006). Interestingly, I supervised a concurrent

investigation into the hES cell in-vitro micro-environment conducted by the QUT

honours student, Luke Cormack. This study revealed a similar proteomic profile to

that herein. Thus, there was significant similarity in the proteins identified as being

secreted into the conditioned media by both the primary keratinocyte cells and hES

cells (refer to Appendix II) (Cormack et al. 2007). The results of both studies have

highlighted the above mentioned proteins as potential candidates and clearly warrant

future investigations in both the keratinocyte and hES culture systems. These future

studies will analyse the individual effects of these candidate proteins, and will also

analyse their effects when added in conjunction with the serum-free VN:GF medium.

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Many other potentially interesting proteins were also identified in the feeder cell

alone and feeder cell:keratinocyte cultures. For example, Telomerase reverse

transcriptase (TERT), Telomerase-binding protein EST1A, Tra1 homolog and

follistatin-like 5. Significantly, the abundance of Telomerase-binding protein EST1A

and telomerase reverse transcriptase has been linked to cells that are in a state of

self-renewal (Wang et al. 2007). This may suggest that the feeder cell layer is

secreting or directly interacting with the keratinocytes to trigger the expression of

this protein. Surprisingly, neither the Telomerase reverse transcriptase (TERT) nor

Telomerase-binding proteins (EST1A), two proteins commonly expressed in hES

cells, were discovered in the studies examining the hES cell in-vitro micro-

environment (refer to Appendix II).

Interestingly, both Tra1 homolog and follistatin-like 5 have roles in the regulation of

human telomerase reverse transcriptase (hTERT) and thus stem self-renewal. Tra1

homolog appears to elicit this function through regulating c-myc (Chen et al. 2006),

while follistatin-like 5 achieves this through its inactivation of TGF-β (Schneyer et

al. 2003; Schneyer et al. 2004), which has been demonstrated to suppresses human

telomerase reverse transcriptase activity (Li et al. 2006). Conversely, the

activin/TGF-β/nodal branch has been demonstrated to induce hES cell self-renewal

(James et al. 2005). The above studies suggest that while TGF-β inhibits hTERT,

thereby inducing differentiation, it also acts in conjunction with activin to promote

pluripotency in stem cells. Together this data highlights the interconnectedness and

tight regulation of these pathways. This also suggests that future proteomic studies

examining the intricate signaling pathways involved in the self-renewal of cells may

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reveal a component/s that will replace the need for a feeder cell layer in the

keratinocyte culture system.

Whilst this proteomic approach revealed interesting insights, this study was a

preliminary investigation into the in-vitro micro-environment of primary

keratinocytes and clearly further research is required. For example, studies

examining cell surface, cytoplasmic and nuclear proteins are also required to provide

a better understanding of the in-vitro micro-environment of the primary keratinocyte.

Whilst important components maybe secreted into the media, cell surface,

cytoplasmic, and nuclear proteins may also provide us with methods with which to

create a xeno-free culture environment for these cells. For example, these studies

may highlight relevant pathways for self renewal which we can activate by adding

exogenous factors to the culture system.

5.4 Conclusion

Clearly, research examining the ex vivo recapitulation of the in vivo

microenvironment is in its infancy and many questions remain to be answered.

However, there is a pressing need to eliminate animal products from the culture of

these cells and to develop a fully defined system. Synthetic alternatives, such as the

VN:GF medium I developed, are required and represent a major advance, as has

been tangibly recognised by the uptake of this technology by Invitrogen Corporation.

The research that I document in this thesis has comprehensively demonstrated that

both primary keratinocytes and hES cells can attach, expand and survive in an

undifferentiated state when using the VN:GF medium as a serum-free media.

Furthermore, the preliminary studies reported here, together with future proteomic

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and validation studies, may reveal important components that can be added in

conjunction with the VN:GF media to create a fully defined, synthetic culture system

that will facilitate the safe culture of transplantable cells and tissues. This may have a

profound impact on the translation and realisation of diverse regenerative medicine

therapies.

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CHAPTER 6

REFERENCES

SEAN RICHARDS


Adams, J. C. and F. M. Watt (1991). "Expression of beta 1, beta 3, beta 4, and beta 5

integrins by human epidermal keratinocytes and non-differentiating keratinocytes." J

Cell Biol 115(3): 829-41.

Ainscough, S. L., Z. Barnard, Z. Upton and D. G. Harkin (2006). "Vitronectin

supports migratory responses of corneal epithelial cells to substrate bound IGF-I and

HGF, and facilitates serum-free cultivation." Exp Eye Res 83(6): 1505-14.

Amit, M., M. K. Carpenter, M. S. Inokuma, C. P. Chiu, C. P. Harris, M. A. Waknitz,

J. Itskovitz-Eldor and J. A. Thomson (2000). "Clonally derived human embryonic

stem cell lines maintain pluripotency and proliferative potential for prolonged

periods of culture." Dev Biol 227(2): 271-8.

Amit, M., C. Shariki, V. Margulets and J. Itskovitz-Eldor (2004). "Feeder cell layer-

free and serum-free culture of human embryonic stem cells." Biol Reprod 70(3):

837-45.

Bagutti, C., A. M. Wobus, R. Fassler and F. M. Watt (1996). "Differentiation of

embryonal stem cells into keratinocytes: comparison of wild-type and beta 1

integrin-deficient cells." Dev Biol 179(1): 184-96.

Barreca, A., M. De Luca, P. Del Monte, S. Bondanza, G. Damonte, G. Cariola, E. Di

Marco, G. Giordano, R. Cancedda and F. Minuto (1992). "In vitro paracrine

regulation of human keratinocyte growth by fibroblast-derived insulin-like growth

factors." J Cell Physiol 151(2): 262-8.

SEAN RICHARDS


Beattie, G. M., A. D. Lopez, N. Bucay, A. Hinton, M. T. Firpo, C. C. King and A.

Hayek (2005). "Activin A maintains pluripotency of human embryonic stem cells in

the absence of feeder layers." Stem Cells 23(4): 489-95.

Boheler, K. R., J. Czyz, D. Tweedie, H. T. Yang, S. V. Anisimov and A. M. Wobus

(2002). "Differentiation of pluripotent embryonic stem cells into cardiomyocytes."

Circ Res 91(3): 189-201.

Bongso, A., C. Y. Fong, S. C. Ng and S. Ratnam (1994). "Isolation and culture of

inner cell mass cells from human blastocysts." Hum Reprod 9(11): 2110-7.

Boraldi, F., L. Bini, S. Liberatori, A. Armini, V. Pallini, R. Tiozzo, I. Pasquali-

Ronchetti and D. Quaglino (2003). "Proteome analysis of dermal fibroblasts cultured

in vitro from human healthy subjects of different ages." Proteomics 3(6): 917-29.

Bradshaw, A. D., K. M. McNagny, D. B. Gervin, G. M. Cann, T. Graf and D. O.

Clegg (1995). "Integrin alpha 2 beta 1 mediates interactions between developing

embryonic retinal cells and collagen." Development 121(11): 3593-602.

Bullock, A. J., M. C. Higham and S. MacNeil (2006). "Use of human fibroblasts in

the development of a xenobiotic-free culture and delivery system for human

keratinocytes." Tissue Eng 12(2): 245-55.

SEAN RICHARDS


Burger, H., K. Nooter, G. J. Zaman, P. Sonneveld, K. E. van Wingerden, R. G.

Oostrum and G. Stoter (1994). "Expression of the multidrug resistance-associated

protein (MRP) in acute and chronic leukemias." Leukemia 8(6): 990-7.

Carpenter, M. K., E. S. Rosler, G. J. Fisk, R. Brandenberger, X. Ares, T. Miura, M.

Lucero and M. S. Rao (2004). "Properties of four human embryonic stem cell lines

maintained in a feeder-free culture system." Dev Dyn 229(2): 243-58.

Castro-Munozledo, F., M. Hernandez-Quintero, M. Marsch-Moreno and W. Kuri-

Harcuch (1997). "Cultivation, serial transfer, and differentiation of epidermal

keratinocytes in serum-free medium." Biochem Biophys Res Commun 236(1): 167-

72.

Chang, M. C. (1959). "Fertilization of rabbit ova in vitro." Nature 184(Suppl 7):

466-7.

Chen, Z., Z. Wu, X. Guo and M. Jiang (2006). "[Expression of telomerase reverse

transcriptase and its relationship with expression of c-myc in laryngeal squamous

cell carcinomas]." Lin Chuang Er Bi Yan Hou Ke Za Zhi 20(21): 963-6.

Cheng, L., H. Hammond, Z. Ye, X. Zhan and G. Dravid (2003). "Human adult

marrow cells support prolonged expansion of human embryonic stem cells in

culture." Stem Cells 21(2): 131-42.

SEAN RICHARDS


Clemmons, D. R. (1998). "Role of insulin-like growth factor binding proteins in

controlling IGF actions." Mol Cell Endocrinol 140(1-2): 19-24.

Cole, S. P., K. E. Sparks, K. Fraser, D. W. Loe, C. E. Grant, G. M. Wilson and R. G.

Deeley (1994). "Pharmacological characterization of multidrug resistant MRP-

transfected human tumor cells." Cancer Res 54(22): 5902-10.

Cormack, L., S. Richards, D. Leavesley and Z. Upton (2007). Analysis of the Human

Embryonic Stem Cell In-vitro Micro-Environment. School of Life Sciences.

Brisbane, Queensland University of Technology: 47.

Cowan, C. A., I. Klimanskaya, J. McMahon, J. Atienza, J. Witmyer, J. P. Zucker, S.

Wang, C. C. Morton, A. P. McMahon, D. Powers and D. A. Melton (2004).

"Derivation of embryonic stem-cell lines from human blastocysts." N Engl J Med

350(13): 1353-6.

Dawson, R. A., N. J. Goberdhan, E. Freedlander and S. MacNeil (1996). "Influence

of extracellular matrix proteins on human keratinocyte attachment, proliferation and

transfer to a dermal wound model." Burns 22(2): 93-100.

Dawson, R. A., Z. Upton, J. Malda and D. G. Harkin (2006). "Preparation of

cultured skin for transplantation using insulin-like growth factor I in conjunction

with insulin-like growth factor binding protein 5, epidermal growth factor, and

vitronectin." Transplantation 81(12): 1668-76.

SEAN RICHARDS


Dietrich, J., M. Cella, M. Seiffert, H. J. Buhring and M. Colonna (2000). "Cutting

edge: signal-regulatory protein beta 1 is a DAP12-associated activating receptor

expressed in myeloid cells." J Immunol 164(1): 9-12.

Dinsmore, J., J. Ratliff, D. Jacoby, M. Wunderlich and C. Lindberg (1998).

"Embryonic stem cells as a model for studying regulation of cellular differentiation."

Theriogenology 49(1): 145-51.

Docherty, K. (2001). "Growth and development of the islets of Langerhans:

implications for the treatment of diabetes mellitus." Curr Opin Pharmacol 1(6): 641-

50.

Draper, J. S., C. Pigott, J. A. Thomson and P. W. Andrews (2002). "Surface antigens

of human embryonic stem cells: changes upon differentiation in culture." J Anat

200(Pt 3): 249-58.

Dupont, J. and D. LeRoith (2001). "Insulin and insulin-like growth factor I receptors:

similarities and differences in signal transduction." Horm Res 55 Suppl 2: 22-6.

Ealey, P. A., M. E. Yateman, S. J. Holt and N. J. Marshall (1988). "ESTA: a

bioassay system for the determination of the potencies of hormones and antibodies

which mimic their action." J Mol Endocrinol 1(2): R1-4.

Edwards, R. G., B. D. Bavister and P. C. Steptoe (1969). "Early stages of

fertilization in vitro of human oocytes matured in vitro." Nature 221(5181): 632-5.

SEAN RICHARDS


Eiges, R., M. Schuldiner, M. Drukker, O. Yanuka, J. Itskovitz-Eldor and N.

Benvenisty (2001). "Establishment of human embryonic stem cell-transfected clones

carrying a marker for undifferentiated cells." Curr Biol 11(7): 514-8.

Evans, M. J. and M. H. Kaufman (1981). "Establishment in culture of pluripotential

cells from mouse embryos." Nature 292(5819): 154-6.

Fortunel, N. O., J. A. Hatzfeld, P. A. Rosemary, C. Ferraris, M. N. Monier, V.

Haydont, J. Longuet, B. Brethon, B. Lim, I. Castiel, R. Schmidt and A. Hatzfeld

(2003). "Long-term expansion of human functional epidermal precursor cells:

promotion of extensive amplification by low TGF-beta1 concentrations." J Cell Sci

116(Pt 19): 4043-52.

Geijsen, N., M. Horoschak, K. Kim, J. Gribnau, K. Eggan and G. Q. Daley (2004).

"Derivation of embryonic germ cells and male gametes from embryonic stem cells."

Nature 427(6970): 148-54.

Goberdhan, N. J., R. A. Dawson, E. Freedlander and S. Mac Neil (1993). "A

calmodulin-like protein as an extracellular mitogen for the keratinocyte." Br J

Dermatol 129(6): 678-88.

Green, H. (1991). "Cultured cells for the treatment of disease." Sci Am 265(5): 96-

102.

SEAN RICHARDS


Guan, K., H. Chang, A. Rolletschek and A. M. Wobus (2001). "Embryonic stem

cell-derived neurogenesis. Retinoic acid induction and lineage selection of neuronal

cells." Cell Tissue Res 305(2): 171-6.

Haase, I., R. Evans, R. Pofahl and F. M. Watt (2003). "Regulation of keratinocyte

shape, migration and wound epithelialization by IGF-1- and EGF-dependent

signalling pathways." J Cell Sci 116(Pt 15): 3227-38.

Harkin, D. G., R. A. Dawson and Z. Upton (2006). "Optimized delivery of skin

keratinocytes by aerosolization and suspension in fibrin tissue adhesive." Wound

Repair Regen 14(3): 354-63.

Hartwig, B., B. Borm, H. Schneider, M. J. Arin, G. Kirfel and V. Herzog (2007).

"Laminin-5-deficient human keratinocytes: defective adhesion results in a saltatory

and inefficient mode of migration." Exp Cell Res 313(8): 1575-87.

Heiskanen, A., T. Satomaa, S. Tiitinen, A. Laitinen, S. Mannelin, U. Impola, M.

Mikkola, C. Olsson, H. Miller-Podraza, M. Blomqvist, A. Olonen, H. Salo, P.

Lehenkari, T. Tuuri, T. Otonkoski, J. Natunen, J. Saarinen and J. Laine (2007). "N-

glycolylneuraminic acid xenoantigen contamination of human embryonic and

mesenchymal stem cells is substantially reversible." Stem Cells 25(1): 197-202.

Henderson, J. K., J. S. Draper, H. S. Baillie, S. Fishel, J. A. Thomson, H. Moore and

P. W. Andrews (2002). "Preimplantation human embryos and embryonic stem cells

SEAN RICHARDS


show comparable expression of stage-specific embryonic antigens." Stem Cells

20(4): 329-37.

Hollier, B., D. G. Harkin, D. Leavesley and Z. Upton (2005). "Responses of

keratinocytes to substrate-bound vitronectin: growth factor complexes." Exp Cell

Res 305(1): 221-32.

Huang, Y. C., T. W. Wang, J. S. Sun and F. H. Lin (2006). "Investigation of

mitomycin-C-treated fibroblasts in 3-D collagen gel and conditioned medium for

keratinocyte proliferation." Artif Organs 30(3): 150-9.

Hyde, C., B. Hollier, A. Anderson, D. Harkin and Z. Upton (2004). "Insulin-like

growth factors (IGF) and IGF-binding proteins bound to vitronectin enhance

keratinocyte protein synthesis and migration." J Invest Dermatol 122(5): 1198-206.

Iacovitti, L., N. D. Stull and H. Jin (2001). "Differentiation of human dopamine

neurons from an embryonic carcinomal stem cell line." Brain Res 912(1): 99-104.

James, D., A. J. Levine, D. Besser and A. Hemmati-Brivanlou (2005).

"TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in

human embryonic stem cells." Development 132(6): 1273-82.

Jiang, F., T. J. Frederick and T. L. Wood (2001). "IGF-I synergizes with FGF-2 to

stimulate oligodendrocyte progenitor entry into the cell cycle." Dev Biol 232(2):

414-23.

SEAN RICHARDS


Kaur, P., A. Li, R. Redvers and I. Bertoncello (2004). "Keratinocyte stem cell

assays: an evolving science." J Investig Dermatol Symp Proc 9(3): 238-47.

Kitagaki-Ogawa, H., T. Yatohgo, M. Izumi, M. Hayashi, H. Kashiwagi, I.

Matsumoto and N. Seno (1990). "Diversities in animal vitronectins. Differences in

molecular weight, immunoreactivity and carbohydrate chains." Biochim Biophys

Acta 1033(1): 49-56.

Krane, J. F., D. P. Murphy, D. M. Carter and J. G. Krueger (1991). "Synergistic

effects of epidermal growth factor (EGF) and insulin-like growth factor

I/somatomedin C (IGF-I) on keratinocyte proliferation may be mediated by IGF-I

transmodulation of the EGF receptor." J Invest Dermatol 96(4): 419-24.

Kricker, J. A., C. L. Towne, S. M. Firth, A. C. Herington and Z. Upton (2003).

"Structural and functional evidence for the interaction of insulin-like growth factors

(IGFs) and IGF binding proteins with vitronectin." Endocrinology 144(7): 2807-15.

Kunugiza, Y., N. Tomita, Y. Taniyama, T. Tomita, M. K. Osako, K. Tamai, T.

Tanabe, Y. Kaneda, H. Yoshikawa and R. Morishita (2006). "Acceleration of wound

healing by combined gene transfer of hepatocyte growth factor and prostacyclin

synthase with Shima Jet." Gene Ther 13(15): 1143-52.

Lanzendorf, S. E., C. A. Boyd, D. L. Wright, S. Muasher, S. Oehninger and G. D.

Hodgen (2001). "Use of human gametes obtained from anonymous donors for the

production of human embryonic stem cell lines." Fertil Steril 76(1): 132-7.

SEAN RICHARDS


Leary, T., P. L. Jones, M. Appleby, A. Blight, K. Parkinson and M. Stanley (1992).

"Epidermal keratinocyte self-renewal is dependent upon dermal integrity." J Invest

Dermatol 99(4): 422-30.

Lebkowski, J. S., J. Gold, C. Xu, W. Funk, C. P. Chiu and M. K. Carpenter (2001).

"Human embryonic stem cells: culture, differentiation, and genetic modification for

regenerative medicine applications." Cancer J 7 Suppl 2: S83-93.

Li, A. and P. Kaur (2005). "FACS enrichment of human keratinocyte stem cells."

Methods Mol Biol 289: 87-96.

Li, A., N. Pouliot, R. Redvers and P. Kaur (2004). "Extensive tissue-regenerative

capacity of neonatal human keratinocyte stem cells and their progeny." J Clin Invest

113(3): 390-400.

Li, H., D. Xu, J. Li, M. C. Berndt and J. P. Liu (2006). "Transforming growth factor

beta suppresses human telomerase reverse transcriptase (hTERT) by Smad3

interactions with c-Myc and the hTERT gene." J Biol Chem 281(35): 25588-600.

Li, Y., J. Fan, M. Chen, W. Li and D. T. Woodley (2006). "Transforming growth

factor-alpha: a major human serum factor that promotes human keratinocyte

migration." J Invest Dermatol 126(9): 2096-105.

SEAN RICHARDS


Lim, J. W. and A. Bodnar (2002). "Proteome analysis of conditioned medium from

mouse embryonic fibroblast feeder layers which support the growth of human

embryonic stem cells." Proteomics 2(9): 1187-203.

Liu, Y., Z. Song, Y. Zhao, H. Qin, J. Cai, H. Zhang, T. Yu, S. Jiang, G. Wang, M.

Ding and H. Deng (2006). "A novel chemical-defined medium with bFGF and

N2B27 supplements supports undifferentiated growth in human embryonic stem

cells." Biochem Biophys Res Commun 346(1): 131-9.

Lu, J., R. Hou, C. J. Booth, S. H. Yang and M. Snyder (2006). "Defined culture

conditions of human embryonic stem cells." Proc Natl Acad Sci U S A 103(15):

5688-93.

Ludwig, T. E., M. E. Levenstein, J. M. Jones, W. T. Berggren, E. R. Mitchen, J. L.

Frane, L. J. Crandall, C. A. Daigh, K. R. Conard, M. S. Piekarczyk, R. A. Llanas and

J. A. Thomson (2006). "Derivation of human embryonic stem cells in defined

conditions." Nat Biotechnol 24(2): 185-7.

Ludwig, T. E. (2007). Long-term stability and differentiation potential of human

embryonic stem cells cultured in defined medium. 5th ISSCR Annual Meeting,

Cairns Convention Centre, Cairns, Queensland, Australia.

Lumelsky, N., O. Blondel, P. Laeng, I. Velasco, R. Ravin and R. McKay (2001).

"Differentiation of embryonic stem cells to insulin-secreting structures similar to

pancreatic islets." Science 292(5520): 1389-94.

SEAN RICHARDS


Maas-Szabowski, N., A. Shimotoyodome and N. E. Fusenig (1999). "Keratinocyte

growth regulation in fibroblast cocultures via a double paracrine mechanism." J Cell

Sci 112 ( Pt 12): 1843-53.

Marinaro, J. A., E. C. Hendrich, K. S. Leeding and L. A. Bach (1999). "HaCaT

human keratinocytes express IGF-II, IGFBP-6, and an acid-activated protease with

activity against IGFBP-6." Am J Physiol 276(3 Pt 1): E536-42.

Marionnet, C., C. Pierrard, C. Vioux-Chagnoleau, J. Sok, D. Asselineau and F.

Bernerd (2006). "Interactions between fibroblasts and keratinocytes in

morphogenesis of dermal epidermal junction in a model of reconstructed skin." J

Invest Dermatol 126(5): 971-9.

Martin, M. J., A. Muotri, F. Gage and A. Varki (2005). "Human embryonic stem

cells express an immunogenic nonhuman sialic acid." Nat Med 11(2): 228-32.

McLaren, A. (2002). "Human embryonic stem cell lines: socio-legal concerns and

therapeutic promise." C R Biol 325(10): 1009-12.

Meana, A., J. Iglesias, M. Del Rio, F. Larcher, B. Madrigal, M. F. Fresno, C. Martin,

F. San Roman and F. Tevar (1998). "Large surface of cultured human epithelium

obtained on a dermal matrix based on live fibroblast-containing fibrin gels." Burns

24(7): 621-30.

SEAN RICHARDS


Morris, C. A., P. A. Underwood, P. A. Bean, M. Sheehan and J. A. Charlesworth

(1994). "Relative topography of biologically active domains of human vitronectin.

Evidence from monoclonal antibody epitope and denaturation studies." J Biol Chem

269(38): 23845-52.

Nam, T., A. Moralez and D. Clemmons (2002). "Vitronectin binding to IGF binding

protein-5 (IGFBP-5) alters IGFBP-5 modulation of IGF-I actions." Endocrinology

143(1): 30-6.

Newman-Smith, E. D. and Z. Werb (1995). "Stem cell defects in parthenogenetic

peri-implantation embryos." Development 121(7): 2069-77.

Noble, A., C. Towne, L. Chopin, D. Leavesley and Z. Upton (2003). "Insulin-like

growth factor-II bound to vitronectin enhances MCF-7 breast cancer cell migration."

Endocrinology 144(6): 2417-24.

Nonoshita, L. D., N. C. Wathen, B. A. Dsupin, T. Chard and L. C. Giudice (1994).

"Insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and proteolyzed

IGFBP-3 in embryonic cavities in early human pregnancy: their potential relevance

to maternal-embryonic and fetal interactions." J Clin Endocrinol Metab 79(5): 1249-

55.

Pebay, A., R. C. Wong, S. M. Pitson, E. J. Wolvetang, G. S. Peh, A. Filipczyk, K. L.

Koh, I. Tellis, L. T. Nguyen and M. F. Pera (2005). "Essential roles of sphingosine-

SEAN RICHARDS


1-phosphate and platelet-derived growth factor in the maintenance of human

embryonic stem cells." Stem Cells 23(10): 1541-8.

Pei, Y., J. Ma, X. Zhang and W. Ji (2003). "Serum-free culture of rhesus monkey

embryonic stem cells." Arch Androl 49(5): 331-42.

Pera, M. F., B. Reubinoff and A. Trounson (2000). "Human embryonic stem cells." J

Cell Sci 113 ( Pt 1): 5-10.

Pons, S. and E. Marti (2000). "Sonic hedgehog synergizes with the extracellular

matrix protein vitronectin to induce spinal motor neuron differentiation."

Development 127(2): 333-42.

Pouliot, N., N. A. Saunders and P. Kaur (2002). "Laminin 10/11: an alternative

adhesive ligand for epidermal keratinocytes with a functional role in promoting

proliferation and migration." Exp Dermatol 11(5): 387-97.

Prowse, A. B., L. R. McQuade, K. J. Bryant, D. D. Van Dyk, B. E. Tuch and P. P.

Gray (2005). "A proteome analysis of conditioned media from human neonatal

fibroblasts used in the maintenance of human embryonic stem cells." Proteomics

5(4): 978-89.

Rees, C. and D. R. Clemmons (1998). "Inhibition of IGFBP-5 binding to

extracellular matrix and IGF-I-stimulated DNA synthesis by a peptide fragment of

IGFBP-5." J Cell Biochem 71(3): 375-81.

SEAN RICHARDS


Reubinoff, B. E., M. F. Pera, C. Y. Fong, A. Trounson and A. Bongso (2000).

"Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro."

Nat Biotechnol 18(4): 399-404.

Reubinoff, B. E., M. F. Pera, G. Vajta and A. O. Trounson (2001). "Effective

cryopreservation of human embryonic stem cells by the open pulled straw

vitrification method." Hum Reprod 16(10): 2187-94.

Reya, T. and H. Clevers (2005). "Wnt signalling in stem cells and cancer." Nature

434(7035): 843-50.

Rheinwald, J. G. and H. Green (1975). "Serial cultivation of strains of human

epidermal keratinocytes: the formation of keratinizing colonies from single cells."

Cell 6(3): 331-43.

Rho, K. S., L. Jeong, G. Lee, B. M. Seo, Y. J. Park, S. D. Hong, S. Roh, J. J. Cho,

W. H. Park and B. M. Min (2006). "Electrospinning of collagen nanofibers: effects

on the behavior of normal human keratinocytes and early-stage wound healing."

Biomaterials 27(8): 1452-61.

Richards, M., C. Y. Fong, W. K. Chan, P. C. Wong and A. Bongso (2002). "Human

feeders support prolonged undifferentiated growth of human inner cell masses and

embryonic stem cells." Nat Biotechnol 20(9): 933-6.

SEAN RICHARDS


Rideout, W. M., 3rd, K. Hochedlinger, M. Kyba, G. Q. Daley and R. Jaenisch

(2002). "Correction of a genetic defect by nuclear transplantation and combined cell

and gene therapy." Cell 109(1): 17-27.

Rodeck, U., M. Jost, J. DuHadaway, C. Kari, P. J. Jensen, B. Risse and D. L. Ewert

(1997). "Regulation of Bcl-xL expression in human keratinocytes by cell-substratum

adhesion and the epidermal growth factor receptor." Proc Natl Acad Sci U S A

94(10): 5067-72.

Rohwedel, J., K. Guan, C. Hegert and A. M. Wobus (2001). "Embryonic stem cells

as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies:

present state and future prospects." Toxicol In Vitro 15(6): 741-53.

Rolleston, W. B. (1999). "Bovine serum: reducing the variables through the use of

donor herds." Dev Biol Stand 99: 79-86.

Sato, N., L. Meijer, L. Skaltsounis, P. Greengard and A. H. Brivanlou (2004).

"Maintenance of pluripotency in human and mouse embryonic stem cells through

activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor." Nat

Med 10(1): 55-63.

Sawamura, D., S. Ina, M. Goto, M. Akiyama and H. Shimizu (2004). "In vivo

transfer of TGF-alpha and beta genes to keratinocytes." J Dermatol Sci 34(3): 234-6.

SEAN RICHARDS


Schick, B. P., H. C. Ho, K. C. Brodbeck, C. W. Wrigley and J. Klimas (2003).

"Serglycin proteoglycan expression and synthesis in embryonic stem cells." Biochim

Biophys Acta 1593(2-3): 259-67.

Schneider, H., C. Muhle and F. Pacho (2006). "Biological function of laminin-5 and

pathogenic impact of its deficiency." Eur J Cell Biol.

Schneyer, A., A. Schoen, A. Quigg and Y. Sidis (2003). "Differential binding and

neutralization of activins A and B by follistatin and follistatin like-3 (FSTL-

3/FSRP/FLRG)." Endocrinology 144(5): 1671-4.

Schneyer, A., Y. Sidis, Y. Xia, S. Saito, E. del Re, H. Y. Lin and H. Keutmann

(2004). "Differential actions of follistatin and follistatin-like 3." Mol Cell Endocrinol

225(1-2): 25-8.

Schoppet, M., T. Chavakis, N. Al-Fakhri, S. M. Kanse and K. T. Preissner (2002).

"Molecular interactions and functional interference between vitronectin and

transforming growth factor-beta." Lab Invest 82(1): 37-46.

Schrock, E., S. du Manoir, T. Veldman, B. Schoell, J. Wienberg, M. A. Ferguson-

Smith, Y. Ning, D. H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini and T. Ried

(1996). "Multicolor spectral karyotyping of human chromosomes." Science

273(5274): 494-7.

SEAN RICHARDS


Schuldiner, M., O. Yanuka, J. Itskovitz-Eldor, D. A. Melton and N. Benvenisty

(2000). "Effects of eight growth factors on the differentiation of cells derived from

human embryonic stem cells." Proc Natl Acad Sci U S A 97(21): 11307-12.

Schvartz, I., D. Seger and S. Shaltiel (1999). "Vitronectin." Int J Biochem Cell Biol

31(5): 539-44.

Schwarzenbach, H. (2002). "Expression of MDR1/P-glycoprotein, the multidrug

resistance protein MRP, and the lung-resistance protein LRP in multiple myeloma."

Med Oncol 19(2): 87-104.

Seiffert, D. (1997). "The glycosaminoglycan binding site governs ligand binding to

the somatomedin B domain of vitronectin." J Biol Chem 272(15): 9971-8.

Seiffert, D. and J. W. Smith (1997). "The cell adhesion domain in plasma vitronectin

is cryptic." J Biol Chem 272(21): 13705-10.

Spichkina, O. G., N. V. Kalmykova, L. V. Kukhareva, I. V. Voronkina, M. I.

Blinova and G. P. Pinaev (2006). "[Isolation of human basal keratinocytes by

selective adhesion to extracellular matrix proteins]." Tsitologiia 48(10): 841-7.

Stoll, S., W. Garner and J. Elder (1997). "Heparin-binding ligands mediate autocrine

epidermal growth factor receptor activation In skin organ culture." J Clin Invest

100(5): 1271-81.

SEAN RICHARDS


Sun, T., J. Haycock and S. Macneil (2006). "In situ image analysis of interactions

between normal human keratinocytes and fibroblasts cultured in three-dimensional

fibrin gels." Biomaterials 27(18): 3459-65.

Sun, T., M. Higham, C. Layton, J. Haycock, R. Short and S. MacNeil (2004a).

"Developments in xenobiotic-free culture of human keratinocytes for clinical use."

Wound Repair Regen 12(6): 626-34.

Sun, T., D. Norton, A. J. Ryan, S. MacNeil and J. W. Haycock (2007). "Investigation

of fibroblast and keratinocyte cell-scaffold interactions using a novel 3D cell culture

system." J Mater Sci Mater Med 18(2): 321-8.

Sun, X. Q., X. B. Fu, W. Chen and T. Z. Sun (2004b). "[Effects of fibroblast growth

factor-10 on the secretions of transforming growth factor-alpha, platelet-derived

growth factor-AB and vascular endothelial growth factor by normal adult human

keratinocytes in culture]." Zhongguo Wei Zhong Bing Ji Jiu Yi Xue 16(1): 13-8.

Tanghetti, E., R. Ria, P. Dell'Era, C. Urbinati, M. Rusnati, M. G. Ennas and M.

Presta (2002). "Biological activity of substrate-bound basic fibroblast growth factor

(FGF2): recruitment of FGF receptor-1 in endothelial cell adhesion contacts."

Oncogene 21(24): 3889-97.

Thomson, J. A., J. Itskovitz-Eldor, S. S. Shapiro, M. A. Waknitz, J. J. Swiergiel, V.

S. Marshall and J. M. Jones (1998). "Embryonic stem cell lines derived from human

blastocysts." Science 282(5391): 1145-7.

SEAN RICHARDS


Trojan, J., T. R. Johnson, S. D. Rudin, B. K. Blossey, K. M. Kelley, A. Shevelev, F.

W. Abdul-Karim, D. D. Anthony, M. L. Tykocinski, J. Ilan and et al. (1994). "Gene

therapy of murine teratocarcinoma: separate functions for insulin-like growth factors

I and II in immunogenicity and differentiation." Proc Natl Acad Sci U S A 91(13):

6088-92.

Trounson, A.O., Gardner, D.K., Baker, G., Barnes, F.L., Bongso, A., Bourne, H.,

Calderon, I., Cohen, J., Dawson, K., Eldar-Geve, T., Gardner, D.K., Graves, G.,

Healy, D., Lane, M., Leese, H.J., Leeton, J., Levron, J., Liu, D.Y., MacLachlan, V.,

Munné, S., Oranratnachai, A., Rogers, P., Rombauts, L., Sakkas, D., Sathananthan,

A.H., Schimmel, T., Shaw, J., Trounson, A.O., Van Steirteghem, A., Willadsen, S.,

and Wood, C. (2000b). Handbook of in vitro fertilization, (Boca Raton, London,

New York, Washington, D.C.: CRC Press).

Trounson, A. (1982). "Current perspectives of in vitro fertilization and embryo

transfer." Clin Reprod Fertil 1(1): 55-65.

Upton, Z. and J. A. Kricker (2002). International Patent. WO 02/24219 A1.

Upton, Z., H. Webb, K. Hale, C. A. Yandell, J. P. McMurtry, G. L. Francis and F. J.

Ballard (1999). "Identification of vitronectin as a novel insulin-like growth factor-II

binding protein." Endocrinology 140(6): 2928-31.

Urban, R. (2005). Tissue, when they all fall down. The Age, Fairfax Digital.

SEAN RICHARDS


Valve, E., P. Martikainen, J. Seppanen, S. Oksjoki, S. Hinkka, L. Anttila, S.

Grenman, P. Klemi and P. Harkonen (2000). "Expression of fibroblast growth factor

(FGF)-8 isoforms and FGF receptors in human ovarian tumors." Int J Cancer 88(5):

718-25.

Vardy, D. A., C. Kari, G. S. Lazarus, P. J. Jensen, A. Zilberstein, G. D. Plowman and

U. Rodeck (1995). "Induction of autocrine epidermal growth factor receptor ligands

in human keratinocytes by insulin/insulin-like growth factor-1." J Cell Physiol

163(2): 257-65.

Wang, G., H. Zhang, Y. Zhao, J. Li, J. Cai, P. Wang, S. Meng, J. Feng, C. Miao, M.

Ding, D. Li and H. Deng (2005). "Noggin and bFGF cooperate to maintain the

pluripotency of human embryonic stem cells in the absence of feeder layers."

Biochem Biophys Res Commun 330(3): 934-42.

Wang, S., C. Hu and J. Zhu (2007). "Transcriptional silencing of a novel hTERT

reporter locus during in vitro differentiation of mouse embryonic stem cells." Mol

Biol Cell 18(2): 669-77.

Watt, F. M. (1998). "Epidermal stem cells: markers, patterning and the control of

stem cell fate." Philos Trans R Soc Lond B Biol Sci 353(1370): 831-7.

Watt, F. M. and P. H. Jones (1993). "Expression and function of the keratinocyte

integrins." Dev Suppl: 185-92.

SEAN RICHARDS


Watt, F. M., M. D. Kubler, N. A. Hotchin, L. J. Nicholson and J. C. Adams (1993).

"Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix

interactions." J Cell Sci 106 ( Pt 1): 175-82.

Werner, S. and H. Smola (2001). "Paracrine regulation of keratinocyte proliferation

and differentiation." Trends Cell Biol 11(4): 143-6.

Wilder, P. J., D. Kelly, K. Brigman, C. L. Peterson, T. Nowling, Q. S. Gao, R. D.

McComb, M. R. Capecchi and A. Rizzino (1997). "Inactivation of the FGF-4 gene in

embryonic stem cells alters the growth and/or the survival of their early

differentiated progeny." Dev Biol 192(2): 614-29.

Wright, K. A., K. B. Nadire, P. Busto, R. Tubo, J. M. McPherson and B. M.

Wentworth (1998). "Alternative delivery of keratinocytes using a polyurethane

membrane and the implications for its use in the treatment of full-thickness burn

injury." Burns 24(1): 7-17.

Xu, C., M. S. Inokuma, J. Denham, K. Golds, P. Kundu, J. D. Gold and M. K.

Carpenter (2001). "Feeder-free growth of undifferentiated human embryonic stem

cells." Nat Biotechnol 19(10): 971-4.

Zhuang, P., M. N. Blackburn and C. B. Peterson (1996). "Characterization of the

denaturation and renaturation of human plasma vitronectin. I. Biophysical

characterization of protein unfolding and multimerization." J Biol Chem 271(24):

14323-32.

SEAN RICHARDS


Zhuang, P., H. Li, J. G. Williams, N. V. Wagner, D. Seiffert and C. B. Peterson

(1996). "Characterization of the denaturation and renaturation of human plasma

vitronectin. II. Investigation into the mechanism of formation of multimers." J Biol

Chem 271(24): 14333-43.

zur Nieden, N. I., L. J. Ruf, G. Kempka, H. Hildebrand and H. J. Ahr (2001).

"Molecular markers in embryonic stem cells." Toxicol In Vitro 15(4-5): 455-61.

SEAN RICHARDS


APPENDIX I

RESULTS

Proteins identified from feeder cell and feeder cell:keratinocyte culture

conditioned media.

MALDI TOF-TOF analysis was conducted on the protein spots transferred from the

second dimension separation as described in section 4.2.4. Several proteins were

observed in this study; however, these results were reduced to a few potential

candidate proteins that may be relevant for the culture of keratinocytes. Results were

organised into: cytokines, growth factors, secreted, and intracellular for the feeder

treatment; and, cytokines, growth factors, hormones, secreted, and intracellular for

the feeder:keratinocyte treatment. Moreover, proteins were categorised based on,

accession number, molecular weight, and protein score. The results for Table A.1

revealed; 5 cytokines, 7 growth factors, 2 secreted and 1 nuclear. Additionally, the

results for Table A.2 revealed; 6 cytokines, 8 growth factors, 2 hormones, 5 secreted

and 4 nuclear proteins.

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Table AI.1 MALDI-TOF-TOF Feeder Cell Conditioned Medium.


Molecular Weight

Protein Score

Cytokines Interleukin 1 beta (Fragment) Q6PUJ4 7928.9 39 interleukin-8 precursor [similarity] S42496 11575 32 Interleukin 10 (Fragment) Q4VHD7 8081 32 Shorter isoform of interleukin 15 (IL-15A precursor) Q9Z0G5 1501.7 25 Leukemia inhibitory factor Q8SPP1 8695 33 Growth Factors Growth hormone (Fragment) O19033 6253 34 Transforming growth factor alpha (Fragment) Q9UQ91 9882.9 14 Fibroblast growth factor homologous factor 3 BQ9JLA7 1869 20 bFGF (Human) E969488 16918 32 Vascular endothelial growth factor Q91ZE2 2836 20 Transforming growth factor beta 2 (Fragment) Q19KA8 5972 34 bone morphogenetic protein 1 C58788 4339 25 Secreted Megakaryocyte colony-stimulating factor P40225 33957 22 Secreted frizzled-related sequence protein 1 Q505A2 36317 20 Intracellular Stat4 Q3U098 54189 23

Table AI.2 MALDI-TOF-TOF Feeder Cell:Keratinocyte Conditioned Medium.


Molecular Weight

Protein Score

Cytokines Shorter isoform of interleukin 15 (precursor) Q9Z0G5 1501.7 19 Interleukin 1 alpha (Fragment).- Ovis aries Q9TU76 1518.7 21 Interleukin 4 (Fragment).- Capra hircus Q1WD38 2230.2 20 Interleukin 10 (Fragment).- Homo sapiens Q9BXR7 1616.8 21 Interleukin-6.- Sus scrofa (Pig). Q8MKE5 24034.9 37 Interleukin-1 alpha (Fragment).- Q71VD0 1841.9 19 PDGF-inducible JE glycoprotein precursor A30209 16543.6 43 Growth Factors FGF-2 activity-associated protein 3 Q96PS2 5633.9 36 PC cell-derived growth factor, PCDGF=EPITHELIN

Q9QWB4 5755.7 36

nerve growth factor homolog 1 - rat A49023 28150.9 40 Platelet-derived growth factor bb, chain B, fragment 2 - human

1PDGB2 7685 16

Platelet-derived growth factor bb, chain C, fragment 2 - human

1PDGC2 8032.1 15

Platelet-derived growth factor bb, chain A, fragment 2 - human

1PDGA2 8174.2 15

TGFBI (Fragment).- Mus musculus Q19PV3 4892.6 18 VEGF (Fragment).- Homo sapiens (Human). CAC60171 1356.8 20 Hormones OVINE GROWTH HORMONE AA 122-138.- Ovis aries

E963408 1911 18

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PREPROPARATHYROID hormone (Fragment).- Q9UD38 3505 18 Secreted growth inhibitory factor - mouse I67866 8144.1 15 Growth differentiation factor 9 (Fragment).- Q38G65 39567.1 31 secreted frizzled-related 1 Q6ZSL4 20923.1 23 Wnt-12 O00744 13581.7 30 Intracellular Telomerase reverse transcriptase Q5I1Y3 8126.1 31 insulin gene enhancer protein isl-1 - rat 1BW5 7985.2 35 Janus kinase 1 (Fragment).- Macaca Q4G3Y7 21692.7 26 STAT- 2 type a S63681 7419.7 35 SUMO-2 P61956 10920 28

Table AI.1 & AI.2: Proteins identified from feeder and feeder:keratinocyte cell

conditioned media using MALDI-TOF-TOF. Conditioned media was collected

every two days, from 3 different patients’ samples, concentrated, isolated, and

separated in two dimensions as per section (4.2.4). Spots from the 2nd dimension

were then lyophilised and tryptically digested as per section (4.2.5). Data was

analysed using the MASCOT database using GPS Explorer™ protein analysis

software (version 4.0) with the mass/ion peak information obtained from both the

MS and the MS/MS spectra refer to section (4.2.6).

SEAN RICHARDS


APPENDIX II

This work was submitted by Luke Cormack for his honours thesis at the

Faculty of Science - School of Life Sciences.

Queensland University of Technology, June 2007.

Analysis of the Human Embryonic Stem Cell In-vitro Micro-Environment

Mr Luke Cormack, Mr Sean Richards, Dr David Leavesley and Prof Zee Upton

Running Title: Analysis of the stem cell micro-environment

Key Words: human embryonic stem cells, mouse embryonic fibroblasts, serum-free,

undifferentiated, proteomic analysis, conditioned media.

Luke Cormack (n5021928)

Tissue Regeneration and Repair Program

School of Life Science

Institute of Health and Biomedical Innovation

60 Musk Ave

Kelvin Grove, QLD, 4059

Australia

Tel. +61-7-38657656

Fax. +61-7-38641534

Email: [email protected]

SEAN RICHARDS


Results

Identified Proteins from the MEF Cells Alone and the MEF:hES Cell Conditioned

Media. Proteins in the MEF CM and the MEF:hES cell CM was analysed using three

methods, MALDI-TOF-TOF, LC/ESI/MS and LC-MALDI. The Mascot database

was employed to analyse proteins present within the CM and the results were

organised into seven protein species; ECM, membrane, nuclear, secreted,

differentiation and growth factors, and serum-derived. Additionally, the proteins

were categorised using accession number, molecular weight, protein score and ion

score. The MALDI-TOF-TOF results were related to the protein score. The MALDI-

TOF-TOF results for the MEF CM media revealed 3 ECM, 3 membrane, 3 nuclear, 1

cytoplasmic, 4 secreted and 7 differentiation and growth factor proteins (Table 1).

Furthermore, the MALDI-TOF-TOF results for the MEF:hES cells CM revealed 4

membrane, 4 nuclear, and 6 secreted proteins (Table 2). All MALDI-TOF-TOF

results, except for the nuclear protein heterogeneous nuclear ribonucleoprotein M

(Table 2), were unconfirmed as determined by their protein scores. The LC/ESI/MS

results are related to the ion scores. The LC/ESI/MS results for the MEF CM

revealed 11 ECM, 4 membrane, 5 nuclear, 1 cytoplasmic, 3 secreted, and 3 serum-

derived proteins (Table 1). Additionally, the LC/ESI/MS results for the MEF:hES

cell CM revealed 12 ECM, 4 membrane, 6 nuclear, 6 cytoplasmic, 1 secreted, and 2

serum-derived proteins (Table 2). The LC-MALDI results are also related to the ion

score. The LC-MALDI results for the MEF CM revealed 1 ECM, 1 membrane, 2

cytoplasmic and 1 secreted protein (Table 1). Furthermore, the LC-MALDI results

for the MEF:hES cell CM revealed 1 cytoplasmic and 2 secreted proteins (Table 2).

All proteins revealed via the LC/ESI/MS analysis were either confirmed or exhibit

extensive homology as determined by their ion scores. All three analyses described

above were conducted in order to increase the legitimacy of the returned results.

SEAN RICHARDS


Table AII.1 Feeder Cell Conditioned Medium. Protein Accession Number MW

(kDa) MALDI-

TOF-TOF Protein score

LC/ESI/MS Ion

score

LC-MALDI Ion

score Extra Cellular Matrix Collagen alpha-1(I) chain Q63079 138 64 Collagen alpha-1(V) chain BAA14323 184 41 Collagen alpha-3(VI) chain CAB60731 549 52 Collagen alpha-1(VII) chain AAA58965 293 59 Collagen alpha-1(XII) chain AAC51244 334 52 Fibronectin S14428 275 44 Laminin alpha-1 chain MMMSA 347 40 Laminin alpha-4 chain LMA4_HUMAN 204 52 Laminin alpha-5 chain LMA5_MOUSE Laminin, gamma 1 Q5VYE7_HUMAN 30 Laminin, gamma-3 AAD36991 177 38 Laminin M I54245 16 43 Proteoglycan link protein A29165 11 35 Tenascin-X T42629 454 41 Thrombospondin 1 Q80YQ1 133 39 Vitronectin Q2Y097_9CARN 7 32 Membrane FGF receptor C44775 3 24 IGF-II mRNA-binding protein 2 AAD31596 66 34 IGF-II receptor Q95MI9 263 41 32 JAK1 protein AAA36527 133 40 JAK2 protein Q7TQD0 132 49 Mast/stem cell growth factor receptor AAA37420 2 43 Membrane-type matrix metalloproteinase-1

Q9XSP0 66 41

Nuclear Cell proliferation antigen Ki-67 T30249 325 40 p53 tetramerization domain 1AIE 3 41 MAP kinase kinase 7 Q8BSP1 47 40 MAPK/ERK kinase kinase 4 T03022 183 47 Protein inhibitor of activated STAT2 AAF12825 64 40 T-box transcription factor TBX20 CAC04520 33 25 Tra1 homolog TRAP_MOUSE 294 39 Ubiquitin carboxyl-terminal hydrolase 43

Q8N2C5 69 59

Probable E3 ubiquitin-protein ligase MYCBP2

O75592 518 49

Cytoplasmic Growth factor receptor-bound protein 14

AAH53559 60 23

Peroxiredoxin-1 BAB27120 22 39 Phospholipase C-epsilon Q8K4S1 258 42 Casein kinase I (isoform alpha) Q5U46 37 25 Secreted Follistatin-related protein 1 S38251 35 64 Interleukin-1 receptor antagonist [Precursor]

AAO24703 20 37

Interleukin-8 Q6LAA1_CANFA 7 37 Matrix-remodelling-associated protein 5

Q9NR99 314 39 23

Protein Wnt-2b AAC25397 45 41 Secreted frizzled-related protein 2 Q9BG86_RABIT 33 36 Suppression of Tumorigenicity 5 Q924W7 TGF-beta-induced protein ig-h3 AAB88697 4 22

SEAN RICHARDS


Serum Derived Alpha-2-HS-glycoprotein S22394 39 72 Serotransferrin] AAA96735 79 55 Serum albumin AAN17824 71 452 Differentiation and growth factor Bone morphogenetic protein 15 Q8MII6_BOVIN 12 37 Hepatocyte growth factor BAA01065 84 45 IGF-1 CAA01955 13 59 IGF-II protein CAA04657 8 34 Platelet-derived growth factor B chain

AAH53430 27 47

Pro-epidermal growth factor CAA24116 4 22 TGF beta 2 Q9MYZ1_CAPHI 10 32

Table AII.1: Proteins identified from the MEF conditioned media using

MALDI-TOF-TOF, LC/ESI/MS and LC-MALDI. Media conditioned by the MEF

cells alone was collected daily, concentrated, isolated, and separated in the first

dimension and second dimension as per the materials and methods. This was

repeated for three separate experiments. Fractions collected from the second

dimension were processed and applied to MALDI-TOF-TOF mass spectrometry.

Database searching of non-interpreted TOF-MS and TOF-TOF MS/MS data was

carried out using the GPS Explorer™ automated interrogation of the MASCOT

database using protein score, protein score confidence interval, total ion score and

total ion score confidence interval. Alternatively, LC/ESI/MS was conducted on

protein fractions as a secondary analysis. Fractions collected from the first dimension

separation were processed and applied to the LC/ESI/MS system. Data was analysed

using the Analyst 1.4.1 software and proteins were later identified using the

MASCOT database search engine. The probability Mowse score was used to

determine homology. Briefly, this score is -10*Log(P), where P is the probability

that the observed match is a random event. Individual ions scores > 38 indicate

identity or extensive homology (p<0.05). Further analysis was conducted on

processed “raw” CM samples using LC-MALDI. Potential protein matches were

then identified from automated searching of the MASCOT database using GPS



SEAN RICHARDS


Table AII.2 Feeder Cell:hES Cell Conditioned Medium. Protein Accession

Number MW

(kDa) MALDI-

TOF-TOF Protein score

LC/ESI/MS Ion

score

LC-MALDI Ion

score Extra Cellular Matrix Collagen alpha-2(I) chain AAC64485 129 50 Collagen alpha 1(IV) chain CGHU4B 161 38 Collagen alpha-6(IV) chain BAA04809 163 41 Collagen alpha-1(V) chain BAA14323 184 41 Collagen alpha-2(V) chain Q7TMS0 145 41 Collagen alpha-1(XI) chain BAA07367 181 63 Collagen alpha-1(XII) chain AAC51244 334 42 Collagen alpha-1(XV) chain Q9EQD9 140 38 Laminin alpha-2 chain S53868 351 42 Laminin subunit alpha-5 LMA5_MOUSE 416 43 Tenascin X T09070 442 39 Versican core protein T42389 371 43 Membrane Cadherin-20 AAG23739 89 39 Collagen alpha-2(VI) chain AAB20836 33 44 Catenin alpha-2 I49499 101 39 Insulin receptor AAB61414 1 20 Myelin-oligodendrocyte glycoprotein

Q29ZP9_CALJA 5 17

Tensin-1 Q9HBL0 186 41 Tumor-associated calcium signal transducer 1

CAA32870 35 26

Zeta-sarcoglycan AAK21962 33 55 Nuclear Cell proliferation antigen Ki-67 T30249 325 42 E3 SUMO AAC41758 362 65 Fos-related antigen 2 CAA58804 3 20 Myc-binding protein 2 O75592 518 43 Mitogen-activated protein kinase 14 AAC50329 34 44 Progesterone receptor Q9GLW0 99 50 T-box transcription factor TBX3 BAC34999 79 46 TGF-beta-inducible nuclear protein 1 BAB31689 6 49 Transcription factor Dp-2 TDP2_HUMAN 49 38 E3 ubiquitin-protein ligase UHRF1 O7TPK1 94 39 Cytoplasm Casein kinase I isoform alpha Q9GLY1 37 43 Dishevelled DVL3_HUMAN 78 40 MAP kinase kinase kinase 4 T03022 183 42 23 Peroxiredoxin AAH68135 147 49 Protein deltex-4 AAH58647 67 48 Triple functional domain protein AAC34245 326 40 Secreted Collagenase 3 AAC24596 45 22 Follistatin-related protein 1 S38251 35 47 Galanin-like peptide AAF19724 12 44 Interleukin-2 CAA42722 15 47 Interleukin-4 CAA28874 3 38 Interleukin-13 Q4VB53 9 40 Metalloproteinase-disintegrin domain containing protein

Q71U12_MOUSE 74 48

Prostaglandin-H2 D-isomerase BAA21769 21 23 Suppression of tumorigenicity 5 AAH36655 127 44 Serum Derived Serotransferrin AAA96735 79 94 Serum albumin AAN17824 71 452

SEAN RICHARDS


Table 2: Proteins identified from the MEF:hES cells conditioned media using

MALDI-TOF-TOF, LC/ESI/MS and LC-MALDI. Media conditioned by the

MEF: hES cells was collected daily, concentrated, isolated, and separated in the first

dimension and second dimension as per the material and methods. This was repeated

for three separate experiments. Fractions collected from the second dimension were

processed and applied to MALDI-TOF-TOF mass spectrometry. Database searching

of non-interpreted TOF-MS and TOF-TOF MS/MS data was undertaken using the

GPS Explorer™ automated interrogation of the MASCOT database using protein

score, protein score confidence interval, total ion score and total ion score confidence

interval. Alternatively, LC/ESI/MS was conducted on protein fractions as a

secondary analysis. Fractions collected from peaks in the first dimension separation

were processed and applied to the LC/ESI/MS system. Data was analysed using the

Analyst 1.4.1 software and proteins were later identified using the MASCOT

database search engine. The probability Mowse score was used to determine

homology. Briefly, score is -10*Log(P) where P is the probability that the observed

match is a random event. Individual ions scores > 38 indicate identity or extensive

homology (p<0.05). Further analysis was conducted on processed “raw” CM

samples using LC-MALDI. Potential protein matches were then identified from

automated searching of the MASCOT database using GPS Explorer™ protein

analysis software (version 4.0) with the mass/ion peak information obtained from

both the MS and the MS/MS spectra.

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