characterisation of small leucine rich proteins gene and...
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Characterisation of Small Leucine Rich Proteins Gene
and Protein Expression in Mesenchymal Stem Cell
Differentiation into Osteoblasts, Adipocytes and
Chondrocytes
ANTHONY BUZZAI
Bachelor of Science (Biomedical Science)
School of Veterinary and Life Sciences
SUPERVISORS
Dr Joshua Lewis
Endocrinology and Diabetes
Sir Charles Gairdner Hospital
Dr Sarah Etherington
School of Veterinary and Life Sciences
Murdoch University
Professor Richard Prince
Endocrinology and Diabetes
Sir Charles Gairdner Hospital
This thesis is presented for the Honours Degree in Biomedical Science at
Murdoch University NOVEMBER 2013
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DECLARATION
I declare this thesis is my own account of my research and contains as its main content,
work which has not been previously submitted for a degree at any tertiary education
institution.
_______________________________ ____/____/____
Anthony Buzzai Date
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MANUSCRIPTS
Currently in submission
Jenny Z. Wang, Joshua R. Lewis, Lawrence J. Liew, Anthony C. Buzzai, Jeremy
Tan, Gerard Hardisty, Jeffrey M. Hamdorf, Minghao Zheng, Richard L. Prince.
Estradiol effects on cellular proliferation and extracellular calcification in
adipose tissue-derived stem cells during osteogenesis.
Currently in preparation
Anthony C. Buzzai, Jenny Z. Wang, Joshua R. Lewis, Sarah J. Etherington
Richard L. Prince. Characterisation of Small Leucine Rich Proteins Gene
and Protein Expression in Mesenchymal Stem Cell Differentiation into
Osteoblasts
Oral presentations
Combined Biological Sciences Meeting 2013. Perth, Australia. The gene
expression of Small Leucine Rich Proteins during the osteogenesis of
human mesenchymal stem cells.
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ABSTRACT
This thesis is directed to understanding the role of Small Leucine Rich Proteins (SLRPs)
in the cell biology of mesenchymal tissue in particular bone and cartilage. SLRPs are a
family of 17 biologically active macromolecules which form the extracellular matrix in
a variety of tissues and may play a role in bone and cartilage biology and diseases, in
particular osteoporosis. It was hypothesised that:
1) The gene and protein expression of specific SLRPs will be up-regulated during
the development of bone and cartilage.
2) During osteogenesis, the location of these SLRPs shows a pattern of distribution
within the extracellular matrix.
3) Osteogenesis related SLRPs are specific to the cell development of that tissue.
To investigate the first hypothesis, a bioinformatics study of a human osteosarcoma cell
was initially used to determine the gene expression on all 17 SLRP members. The six
highest expressed members Lumican, Epiphycan, Tskushi, Biglycan Decorin, and
Osteomodulin (OMD) were selected for further analysis. To investigate the second
hypothesis, the gene expression of these six selected members were analysed using real
time quantitative reverse transcriptase polymerase chain reaction in both long term (up
to 28 days) and short term (up to 7 days) osteogenesis of donor matched human adipose
and bone marrow mesenchymal stem cells. These results showed the increase in
expression of OMD in osteogenic stimulated media. As a result of these studies OMD
was selected for further study, as a potential biomarker of osteoblasts.
The gene expression of OMD was only increased significantly in osteoblast-like cells
compared to other mesenchymal stem cell lineages including cartilage and adipose
tissue. Protein expression of OMD was further investigated by western blotting. This
was followed by confocal microscopy to further understand the expression of this
protein. It was found through both methods that the protein expression of OMD was
increased during osteogenesis, reflecting the gene expression previously observed.
In conclusion, it was shown that the gene and protein expression of OMD was increased
specifically during osteogenesis, and therefore could be used as a marker of
osteogenesis of mesenchymal stem cells. Furthermore, its role in osteogenic
development should be further studied to understand its role in osteogenesis.
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TABLE OF CONTENTS
DECLARATION .......................................................................................................... 2
MANUSCRIPTS .......................................................................................................... 3
ABSTRACT ................................................................................................................. 4
TABLE OF CONTENTS .............................................................................................. 5
ACKNOWLEDGEMENTS .......................................................................................... 8
ABBREVIATIONS ...................................................................................................... 9
PART I: LITERATURE REVIEW.............................................................................. 11
1.1 Osteoporosis overview....................................................................................... 11
1.1.1 Clinical definition of osteoporosis by bone mineral density ......................... 11
1.1.2 Epidemiology of osteoporosis ..................................................................... 12
1.1.3 Falls and fractures associated with osteoporosis .......................................... 12
1.1.4 Burdens of osteoporosis .............................................................................. 14
1.2 Bone physiology ................................................................................................ 16
1.3 Pathogenesis of osteoporosis ............................................................................. 17
1.4 The bone matrix................................................................................................. 18
1.5 Proteoglycans in the ECM ................................................................................. 18
1.6 Small Leucine Rich Protein Family ................................................................... 19
1.6.1 Structure of SLRPs ...................................................................................... 21
1.7 The Functions of Small Leucine Rich Proteins in Mesenchymal Stem Cell
Lineages .................................................................................................................. 31
1.7.1 Definitions and characteristics of mesenchymal stem cells .......................... 31
1.7.2 Process by which Mesenchymal Stem Cells mature into Osteoblasts ........... 31
1.7.3 Process by which Mesenchymal Stem Cells mature in Adipocytes .............. 37
1.7.4 Process by which Mesenchymal Stem Cells mature into Chondrocytes ....... 39
1.8 Conclusion ........................................................................................................ 42
PART II: MATERIALS AND METHODS ................................................................. 43
2.1 Materials manufacturers .................................................................................... 43
2.2 Human adipose and bone marrow derived mesenchymal stem cell primary cell
culture procedure ..................................................................................................... 45
2.2.1 Isolation of mesenchymal stem cells (performed by Ms Jenny Wang) ......... 45
2.2.2 Cell resuscitation ......................................................................................... 46
2.2.3 Cell passage ................................................................................................ 46
2.2.4 Cell cryopreservation .................................................................................. 47
2.2.5 Cell counting assay ..................................................................................... 47
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2.2.6 Adipogenic and chondrogenic lineage differentiation assay ......................... 47
2.2.7 Osteogenic lineage differentiation assay ...................................................... 48
2.3 RNA isolation and qRT-PCR ............................................................................. 48
2.3.1 RNA isolation ............................................................................................. 48
2.3.2 Quantitative reverse transcriptase real time PCR ......................................... 49
2.3.3 Gel electrophoresis for amplification products ............................................. 50
2.3.4 Statistical analysis of gene expression ......................................................... 51
2.4 Protein isolation and western blotting ................................................................ 52
2.4.1 Protein isolation .......................................................................................... 52
2.4.2 Western blotting .......................................................................................... 53
2.5 Immunofluorescence staining ............................................................................ 56
2.5.1 Collagen coating of #1 glass coverslips ....................................................... 56
2.5.2 Immunofluorescence staining procedure...................................................... 56
PART III: RESULTS .................................................................................................. 58
3.1 Selection of Small Leucine Rich Proteins .......................................................... 58
3.2 Optimisation of selected SLRP genes for qRT-PCR in ADSCs (Figure 3.2 and
Figure 3.3) ............................................................................................................... 59
3.3 Optimisation of selected SLRP genes for qRT-PCR in BMSCs (Figure 3.4 and
Figure 3.5) ............................................................................................................... 60
3.4 Patient characteristics ........................................................................................ 61
3.5 Baseline gene expression of SLRPs in unstimulated ADSC and BMSC cultures 62
3.6 The gene expression of SLRPs during osteogenesis of human MSCs ................. 62
3.6.1 Short-term gene expression of SLRPs in ADSCs (Figure 3.8) ..................... 63
3.6.2 Long-term gene expression of SLRPs in ADSCs (Figure 3.9) ...................... 64
3.6.3 Short-term gene expression of SLRPs in BMSC (Figure 3.10)..................... 65
3.6.4 Long-term gene expression of SLRPs in BMSCs (Figure 3.11) ................... 66
3.7 Comparison of SLRP gene expression between tissue types ............................... 67
3.8 Osteomodulin .................................................................................................... 68
3.8.1 OMD gene expression during multi-lineage differentiation of human ADSCs
(Figure 3.12) ........................................................................................................ 68
3.8.2 Protein expression of OMD during osteogenesis ......................................... 69
3.9 Subcellular location of OMD during osteogenesis.............................................. 71
3.9.1 Distribution of OMD within the ECM during osteogenesis of ADSCs (Figure
3.15) .................................................................................................................... 71
3.9.2 Distribution of OMD within the ECM during osteogenesis of BMSCs (Figure
3.16 and 3.17) ...................................................................................................... 73
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PART IV: DISCUSSION ............................................................................................ 76
4.1 Principal findings .............................................................................................. 76
4.1.1 Expression of SLRP family members during osteogenesis ........................... 76
4.1.2 Comparison of SLRPs gene expression between the osteogenesis of ADSC
and BMSC ........................................................................................................... 81
4.1.3 The protein expression and subcellular localisation of OMD during
osteogenesis ......................................................................................................... 82
4.1.4 Osteomodulin as a marker of osteogenesis .................................................. 83
4.1.5 Advantages and disadvantages of this thesis ................................................ 85
4.1.6 Future directions and concluding statement ................................................. 85
PART VI: APPENDIX ............................................................................................... 87
PART VII: REFERENCES ...................................................................................... 101
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ACKNOWLEDGEMENTS
The image on the front cover of this thesis shows stem cells I had cultured undergoing cell
death. This image is not only a reminder of the tough moments throughout my honours year, but
a reminder of those who helped me get through them.
To my amazing supervisor Sarah, thank you for taking the time to check up on me and your
willingness to always go through my thesis, even when I was too scared to show you.
To my awesome supervisor Josh, thank you for patience with me this year. Your knowledge and
passion for research has inspired me to pursue a research career. Out of everyone I have worked with this year, I have learnt the most from you about research and I cannot thank you enough for
the amount time you have spent ensuring I was on track to finish.
To my brilliant supervisor Richard, thank you for all you have taught me about not only being a
researcher, but general life skills such as assertiveness. Thank you for giving the opportunity to work with you and your group. Although this year was frustrating at times, it has been fun of
course.
To my unofficial supervisor Jenny, thank you for taking the time to answer my questions every
five seconds and teach me everything I needed to know in the lab. You have been such great
company in the office and I hope to see you back in Australia soon.
To the Cellular Orthopaedics Laboratory at the Centre for Orthopaedic Research, in particular Nathan, Tak, Ying Hua and Audrey, I thank you for allowing me to use your facilities and
taking the time to teach me western blotting.
To Ben, Shelby, Marie, Cynthia, Simone, Felicia, Bee, Alexia, Lawrence, Helena and Kerry,
thank you for your company in the office this year and taking the time to help me out before I had a mental breakdown.
To my closest friends Ryan, Stephanie, Sarah, Michelle, Tamika, Andrew, Rhiannon, Natalie, Sheldon, Belinda, Elisa, Amber, Joe and Ben and my cousins Jess and Sebastian, thank you for
helping me not only procrastinate, but ensured that I made time to relax.
To my fellow honours friends Matheo, Lauren, Sam and Rachael, thank you for putting up with my non-stop whinging and overall rudeness this year. I wish you all the best with you future
studies.
To my siblings Michael, Elisa, Daniel and Andrew, I am sorry that you all had to deal with my bad moods the most, but I am grateful for your patience with me.
To my parents Tony and Angela, thank you for not only making sure that I ate and slept this year, but for your unconditional moral support which without I would not have made it through
this year.
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ABBREVIATIONS
Bone Mineral Density BMD
Adipose Derived Stromal Cells ADSCs
Asporin ASPN
Biglycan BGN
Bone Marrow Stromal Cells BMSCs
Bone Morphogenic Protein BMP
Chondroadherin CHAD
Decorin DCN
Epiphycan EPYC
Extracellular Matrix ECM
Extracellular Matrix Protein 2 ECM2
Glyceraldehyde-3-Phosphate Dehydrogenase GAPDH
Glycosaminoglycan GAG
Interleukin IL
Leucine Rich Repeats LRRs
Lumican LUM
Mesenchymal Stem Cells MSCs
Nyctalopin NYX
Opticin OPTC
Osteogenic media OSM
Osteoglycin OGN
Osteomodulin OMD
Phosphate Buffered Saline PBS
Podocan PODN
Podocan-Like Protein PODNL1
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Polymerase chain reaction PCR
Proline-Arginine-Rich End Leucine Rich Repeat Protein PRELP
Quantitative real time reverse transcriptase PCR qRT-PCR
Small Leucine Rich Proteins SLRPs
Sodium Dodecyl Sulphate SDS
Transforming Growth Factor Beta TGF-β
Tsukushi TSKU
Tumour Necrosis Factor Alpha TNFα
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PART I: LITERATURE REVIEW
1.1 Osteoporosis overview
Osteoporosis is a common complex metabolic disease of the bone characterised by a
loss of skeletal mass leading to inadequate mechanical support and greater susceptibility
to fractures during trauma (Raisz 2005). It is often referred to as the “silent epidemic”,
since no apparent symptoms from the progressive deterioration of bone are observed in
those affected (Osteoporosis Australia 2008). Since the condition is without any
observable symptoms, the majority of people with a higher risk of experiencing a
fracture are not being treated or have yet to be diagnosed with osteoporosis (Sandhu and
Hampson 2011). It is suggested that four in five people with osteoporosis are unaware
that they are at very high risk of an incident resulting in a fracture (Osteoporosis
Australia 2008). Minimal trauma fractures is the most common clinical sign that an
individual has an underlying osteoporotic condition (Iqbal 2000; Osteoporosis Australia
2008). Alternatively osteoporosis can be diagnosed through incidental findings when
measuring bone mineral density (BMD) or in x-ray films (Iqbal 2000).
1.1.1 Clinical definition of osteoporosis by bone mineral density
The gold standard for the diagnosis of osteoporosis is the measurement of BMD using
dual x-ray absorptiometry (Iqbal 2000; Sandhu and Hampson 2011). This type of scan
can survey the hip, wrist, heel, spine and/or entire body at once to determine the bone
density of the individual (Iqbal 2000). The dual x-ray absorptiometry method for
assessing bone health is able to report BMD as a T-score (Mulder et al. 2006;
Osteoporosis Australia 2008; Sandhu and Hampson 2011). The T-score predominantly
used for the assessment of BMD for men over the age of 50 and postmenopausal
women, and is reported as a comparison to a healthy adult of the same sex (Sandhu and
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Hampson 2011). The World Health Organisation has specified that an individual has
osteopenia if a T-score between -1.0 and -2.5 is reported, and an individual with a T-
score of less than -2.5 is to be diagnosed with osteoporosis (Osteoporosis Australia
2008; Sandhu and Hampson 2011) and pharmacological treatment is recommended
(Iqbal 2000).
1.1.2 Epidemiology of osteoporosis
Between 2007 – 08, it was estimated that osteoporosis affected 692,000 Australians,
accounting for 3.4% of the population (Australian Institute of Health and Welfare
2011). This figure however only accounts for cases diagnosed by doctors, and due to the
absence of overt symptoms, it is considered an underestimate (Australian Institute of
Health and Welfare 2011). Of these diagnosed cases, the majority (84%) were in
individuals aged fifty five years and older, and women were accountable for 82% of all
cases (Australian Institute of Health and Welfare 2011).
1.1.3 Falls and fractures associated with osteoporosis
Individuals with osteoporosis are more susceptible to the consequences of falls, such as
minimal trauma fractures, due to a decrease in bone strength (Runge and Schacht 2005).
These types of fractures transpire from a fall occurring at standing height or lower
(Australian Institute of Health and Welfare 2011). It has been approximated that for
every three people aged sixty five years and older, one elderly person will experience a
minimum of one fall annually (Ganz et al. 2007; Kannus et al. 2005; Osteoporosis
Australia 2008; Runge and Schacht 2005). This fall rate increases in older populations
(Kannus et al. 2005) and it has been described to increase to one in two elderly people
experiencing at least one fall annually in nursing homes or places of residential care
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(Kannus et al. 2005). In 80% of these cases, the fall would occur while the individual is
still conscious and devoid of an external force, such as carrying out routine activities
(Runge and Schacht 2005).
Fracture of the pelvis and hip are the most type of osteoporotic fractures requiring
hospitalisation (Australian Institute of Health and Welfare 2011; Cole et al. 2008) and
cause the most significant debilitation and mortality (Cole et al. 2008; Osteoporosis
Australia 2008). Forty percent of fractures in this area can be attributed to a fall
(Osteoporosis Australia 2008). More than 40% of osteoporotic fractures were in the hip
and pelvic region in Australia during 2007 – 08, and 40% of these fractures occurred at
the femoral neck (Figure 1.1) (Australian Institute of Health and Welfare 2011).
Figure 1.1: Proportions of various hip and pelvic fracture sites following minimal
trauma fracture between 2007 – 08 in persons aged 40 years and over. Adapted from the
Australian Institute of Health and Welfare (2011).
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In those aged fifty years and older, 25% of people who fracture their hip will die within
a year, and in 50% of those who survive more than twelve months, will require long
term care and need walking aids (Osteoporosis Australia 2008). It has been estimated
that 16% of postmenopausal women will sustain a fracture of the hip in their life time
(Runge and Schacht 2005)
1.1.4 Burdens of osteoporosis
In Australia during 2000 – 01, osteoporosis accounted for $1.9 billion in direct costs in
particular; hospitalisation and nursing home fees but also therapy and rehabilitation
(Access Economics and Osteoporosis Australia 2001; Osteoporosis Australia 2008;
Parker 2013) (Figure 1.2).
Figure 1.2: The proportions of osteoporotic direct costs between 2000 – 01. Adapted
from Access Economics and Osteoporosis Austrlaia (2001).
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Indirect costs of having osteoporosis such as loss of earnings due to early retirement and
equipment, account for $5.7 billion in expenditure (Access Economics and Osteoporosis
Australia 2001). In 2001, the cost of fall related injuries in in those aged sixty five
years and older was estimated to account for 1.5% of health expenditure ($83 million)
in Western Australia (Hendrie et al. 2004). On average, $4,450 was the average cost for
those who presented at any emergency department in Western Australia with a fall
related injury (Hendrie et al. 2004). In the European Union, the combined expenditure
of osteoporotic fractures has been estimated at €30 billion and $20 million in the U.S
annually (Cole et al. 2008). In addition to the financial burden there is also a significant
impact of osteoporosis on the patient’s quality of life.
Osteoporosis has negative effect on an individual’s mobility, pain, phobia of
falling, mortality and further fracture risk (Australian Institute of Health and Welfare
2011; Sandhu and Hampson 2011). In those suffering with osteoporosis in Australia,
35.5% of people have reported limitations in essential daily activities (Australian
Institute of Health and Welfare 2011). In particular, hip fractures cause the most
significant impact on a person’s quality of life, with half of these patients becoming
disabled permanently and not regaining their independence (Osteoporosis Australia
2008; Sandhu and Hampson 2011). It has been reported that 80% of hip fracture
patients become restricted in activities such as shopping and driving and 40% are not
able to independently walk within twelve months (Osteoporosis Australia 2008).
Elderly people, who have suffered a fracture of the forearm such as in the wrist, have
issues with simple tasks such as preparing meals and writing (Osteoporosis Australia
2008). Overall, it is evident that osteoporosis is a major contributor to both the financial
and social burden of disease in Australia and the Western World.
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1.2 Bone physiology
In order to understand the pathogenesis of osteoporosis, the normal physiology of bone
should first be understood. Bones are a dynamic tissue and organ found in higher
vertebrates (Downey and Siegel 2006), which not only provide structural support for the
organism (Clarke 2008), but also assist with locomotion (Clarke 2008; Downey and
Siegel 2006), maintaining mineral homeostasis (Clarke 2008; Downey and Siegel
2006), providing protection of the other organs (Downey and Siegel 2006), and serving
as a reservoir for cytokines and growth factors (Clarke 2008). Approximately 80% of
the mature skeleton is corticol bone (Clarke 2008; Downey and Siegel 2006) which is
morphologically solid, compact and dense (Clarke 2008; Downey and Siegel 2006)
providing mechanical strength to the organ (Downey and Siegel 2006). This type of
bone is responsible for forming the diaphysis, which surrounds the marrow cavity of
long bones (Downey and Siegel 2006). The remaining 20% of the mature skeleton, is
composed of cancellous bone (also known as trabecular bone), which is spongy and
honeycomb-like in appearance (Clarke 2008; Downey and Siegel 2006). This type of
osseous tissue is responsible for the metabolic activity of the bone and is observed in the
epiphysis and metaphysis of bones (Clarke 2008; Downey and Siegel 2006).
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1.3 Pathogenesis of osteoporosis
Osteoporosis is a disease that represents a combination of disruptions in the bone
remodelling process leading to altered microarchitecture of the bone and gradual loss of
bone mass (Sandhu and Hampson 2011). Fragility of the skeleton has been attributed to
three main pathophysiological processes: 1) a reduction in bone formation during bone
remodelling, 2) microarchitecture deterioration due to excessive bone resorption and 3)
the failure to have achieved optimal skeletal strength and peak bone mass during growth
(Iqbal 2000; Raisz 2005). Based on the pathogenesis of the disease in the individual,
osteoporosis can be categorised as primary osteoporosis, further grouped as type I or II,
or can be categorised as secondary osteoporosis (Iqbal 2000).
At a cellular level, bone is composed of a number of different cell types, which
play unique roles in bone regulation. The regulation of bone is a tightly coupled
process, which involves the removal of old bone and the deposition of an equivalent
amount of new bone (Syed and Ng 2010). This constant renewal of bone occurs in the
Bone Multicellular Unit which comprises of osteoclasts and osteoblasts that remodel
bone to help the skeleton to adapt to biomechanical forces and maintain mineral
homeostasis (Clarke 2008). Osteoclasts are giant, multinuclear cells derived from
haemopeotic stem cells that resorb old and damaged bone by proteolytic digestion and
acidification (Manolagas 2000). This event is stimulated by T cells and osteoblasts that
produce Receptor Activator of Nuclear Factor Kappa-B Ligand, a cytokine that binds
to Receptor Activator of Nuclear Factor Kappa-B receptor on an osteoclast precursor,
causing it to mature and exert is resorptive functions (Becker 2008). It is critical to the
integrity of bone that the resoprtion process is balanced by bone formation from the
osteoblasts because an imbalance of this remodelling process can ultimately lead to
osteoporosis (Manolagas and Jilka 1995).
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1.4 The bone matrix
The extracellular matrix (ECM) is a dynamic, non-cellular component of all organs and
tissues that not only serve as scaffolding, but is also involved in the differentiation,
homeostasis and morphogenesis of the organs and tissues they compose (Frantz et al.
2010). The matrix of the bone is composed predominantly of mineral (50 – 70%) in
which hydroxyapatite is most abundant (Clarke 2008). Less than 3% of the bone matrix
is composed of lipids and water composes up to 10% of the ECM (Clarke 2008).
Organic material accounts for 20 – 40% of the matrix, in which 90% is collagen (mostly
type I collagen) (Sroga and Vashishth 2012; Young 2003). The remaining 10% of the
bone ECM is composed of growth factors, glycosylated proteins and proteoglycans
(Clarke 2008).
1.5 Proteoglycans in the ECM
Proteoglycans are large biological macromolecules that are composed of a protein core
covalently substituted with glycosaminoglycan (GAG) side chains (Dolan et al. 2007;
Schaefer and Schaefer 2010). Proteoglycans have a diverse range of functions within
the ECM, which is evident through the Small Leucine Rich Protein (SLRP) family
(Dolan et al. 2007).
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1.6 Small Leucine Rich Protein Family
The SLRP family are a group of ubiquitous and abundant (Iozzo and Schaefer 2010)
macromolecules that are related by their structures and functions (McEwan et al. 2006),
and operate as active components of the ECM (Merline et al. 2009). In vivo the family
of SLRPs are synthesised and secreted dynamically (S. Chen and Birk 2013) into the
pericellular matrix (Schaefer and Schaefer 2010), permitting them to interact with
growth factors, cell surface receptors, cytokines (Merline et al. 2009) and other protein
components of the ECM (Dellett et al. 2012; Merline et al. 2009), or alternatively they
can be incorporated into the basement membrane (Schaefer and Schaefer 2010). These
matricellular proteins tend to reside in the ECM since they do not diffuse readily
(Dellett et al. 2012), which promotes them to fully exert their functions on regulating
cell-matrix interactions indirectly by cytokines and growth factors and directly through
receptor mediated actions (Merline et al. 2009). To date, the SLRP family is comprised
of 17 genes (Dellett et al. 2012; Schaefer and Iozzo 2008) (Figure 1.3) which are
distributed over 7 chromosomes (S. Chen and Birk 2013; Schaefer and Iozzo 2008). It
has been implied that this arrangement provides functional redundancy for this family
of proteins (S. Chen and Birk 2013).
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Figure 1.3: Phylogenetic tree of the seventeen SLRP family members. Adapted from
Dellet et al. (2012).
All members of the SLRP family have two main structural features – a uniquely
conserved protein core with a varying number and types of GAG side chains (Dellett et
al. 2012; Matsushima et al. 2000; Ward and Ajuwon 2011). The conserved protein core
includes a hallmark motif of 11 amino acids with a consensus sequence, which is
repeated in tandem (S. Chen and Birk 2013; Dellett et al. 2012; Ikegawa 2008;
Matsushima et al. 2000; McEwan et al. 2006). The number and type of substituted GAG
side chains influences the function and properties of the macromolecule (Brown et al.
2012).
Osteomodulin (OMD)
ECM 2
Asporin (ASPN)
Biglycan (BGN)
Decorin (DCN)
Fibromodulin (FMOD)
Lumican (LUM)
PRELP
Keratocan (KERA)
Osteoglycin (OGN)
Epiphycan (EPYC)
Opticin (OPTC)
Tsukushi (TSKU)
Chondroadherin (CHAD)
Nyctalopin (NYX)
Podocan (PODN)
Podocan-like protein 1 (PODNL1)
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1.6.1 Structure of SLRPs
All family members of the SLRPs contain the tandem repeated, hall mark amino acid
sequence LxxLxLxxNxL where L represents a leucine which can be a substituted
isoleucine, valine or any other hydrophobic amino acid, N corresponds to a threonine,
asparagine or cysteine and X signifies any amino acid (S. Chen and Birk 2013; Dellett
et al. 2012; Ikegawa 2008; Matsushima et al. 2000; McEwan et al. 2006). The central
domain of leucine-rich repeats (LRRs) gives the proteins a curved, solenoid-like
structure (Ameye and Young 2002) giving the macromolecules concave and convex
faces (Figure 1.4) (S. Chen and Birk 2013).
Figure 1.4: The protein structure of SLRPs. The solenoid-like structure is a feature of
SLRP, providing the protein with a concave face to promote binding. Adapted from
McEwan et al. (2006).
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The number of tandemly repeated LRRs and number of amino acids that compose the
LRRs vary between different SLRPs (McEwan et al. 2006). SLRPs contain between 8 –
15 LRRs (Dellett et al. 2012) and the sizes of these repeats can vary between 20 - 30
amino acids (Ikegawa 2008). Most SLRPs have their LRRs arranged in a short – long –
long pattern (Figure 1.5) (McEwan et al. 2006).
Figure 1.5: The LRRs of five members of the SLRP family. The LRRs of most SLRPs
follow a short – long – long structure where each numbered box is an LRR and the
corresponding number of amino acids which make it up, the N-box represents an amino
terminal disulphide bond cap and the C-boxes represent a carboxy terminal cap.
Adapted from McEwan et al. (2006).
This LRR region is flanked on the amino-terminus side by four class conserved
cysteine residues and two cysteine residues on the carboxyl-terminus end (S. Chen and
Birk 2013; Dellett et al. 2012; Matsushima et al. 2000; McEwan et al. 2006).
Biglycan
Decorin
Lumican
Epiphycan
Osteomodulin
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Another common feature of SLRPs is the presence of GAG side chains which
are linear, sulphated and negatively charged disaccharide repeating units of uronic acid
and acetylated amino sugar moieties (Merline et al. 2009; Schaefer and Schaefer 2010).
Dermatan sulphate, chondroitin sulphate, and keratan sulphate (Dellett et al. 2012;
Matsushima et al. 2000; McEwan et al. 2006) are examples of possible GAGs that can
be linked covalently via serine residues to the protein core in the SLRP family members
(Merline et al. 2009). It is important to note that not all members of the SLRP family
contain GAG side chains (S. Chen and Birk 2013; Iozzo and Schaefer 2010), but these
proteins alternatively exhibit sites of sialylated O-linked glycosylation or a long
sequence of negatively charged amino acids, which act in a similar way to GAG side
chains (McEwan et al. 2006). The processed GAG side chains vary in sulphation,
number, size and epimerisation depending on the age and tissue the protein in which the
tissue is synthesised (S. Chen and Birk 2013).
1.6.1.1 Classes of Small Leucine Rich Proteins
The seventeen members that compose the family of SLRPs have been classified into
five different classes, proposed on the basis of their homology and conservation at the
genomic and protein level (S. Chen and Birk 2013; Merline et al. 2009; Schaefer and
Iozzo 2008; Schaefer and Schaefer 2010). This also reflects the protein’s functions and
bioactivity (Iozzo and Schaefer 2010). This classification system takes into account
several parameters such as their organisation within the chromosomes (Ameye and
Young 2002; Dellett et al. 2012; Iozzo and Schaefer 2010; Merline et al. 2009;
Nikitovic et al. 2012) (Figure 1.6), identical size and number of exons (Iozzo and
Schaefer 2010; Mochida et al. 2011), number of internal LRR motifs (Brown et al.
2012; Dellett et al. 2012; McEwan et al. 2006; Mochida et al. 2011) the structure and
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spacing of the cysteine rich residues at the amino terminal (Ameye and Young 2002;
Dellett et al. 2012; Ikegawa 2008; Iozzo and Schaefer 2010; McEwan et al. 2006;
Merline et al. 2009; Mochida et al. 2011; Nikitovic et al. 2012; Schaefer and Iozzo
2008; Schaefer and Schaefer 2010) and the configuration of the ear repeat and
disulphide bonds at both the amino and carboxyl terminal (S. Chen and Birk 2013;
Iozzo and Schaefer 2010; Nikitovic et al. 2012). It has been proposed that the ear repeat
is the true hallmark of which family the SLRP members is classified (Schaefer and
Iozzo 2008).
Figure 1.6: The chromosomal location of the SLRP family members. Adapted from
Schaefer and Iozzo (2008).
25
1.6.1.1.1 Class I Small Leucine Rich Proteins
Based on the above parameters, decorin (DCN) (Dellett et al. 2012; Ikegawa 2008;
Iozzo and Schaefer 2010; Matsushima et al. 2000; Schaefer and Iozzo 2008;
Waddington et al. 2003), biglycan (BGN) (Dellett et al. 2012; Ikegawa 2008; Iozzo and
Schaefer 2010; Matsushima et al. 2000; Schaefer and Iozzo 2008; Waddington et al.
2003), asporin (ASPN) (Dellett et al. 2012; Ikegawa 2008; Iozzo and Schaefer 2010;
Mochida et al. 2006; Schaefer and Iozzo 2008) and extracellular matrix protein 2
(ECM2) (Dellett et al. 2012; Schaefer and Iozzo 2008) have been classified as
canonical Class I SLRPs (Iozzo and Schaefer 2010; Nikitovic et al. 2012). These
proteins consist of twelve LRRs (Dellett et al. 2012; Matsushima et al. 2000; Schaefer
and Iozzo 2008) (except ECM2 which has fifteen LRRs (Dellett et al. 2012)) and can
contain chondroitin sulphate or dermatan sulphate GAG side chains (Ameye and Young
2002; Dellett et al. 2012; Iozzo and Schaefer 2010; Mochida et al. 2006; Nikitovic et al.
2012; Salgado et al. 2011; Schaefer and Iozzo 2008) depending on the tissue (Ameye
and Young 2002).
Asporin, however is not a classical proteoglycan (Ameye and Young 2002;
Mochida et al. 2006; Schaefer and Iozzo 2008) since does not contain any GAG side
chains (Dellett et al. 2012; Iozzo and Schaefer 2010; Mochida et al. 2006; Nikitovic et
al. 2012; Schaefer and Iozzo 2008), but N- and O- glycosylation sites exist on the
protein (Dellett et al. 2012; Ikegawa 2008).
26
Class I SLRPs have a signature cluster of cysteine residues at the amino terminal
with the consensus sequence of Cx3CxCx6 (Dellett et al. 2012; Mochida et al. 2006;
Schaefer and Iozzo 2008) and this region is the responsible for the formation of two
disulphide bonds (Nikitovic et al. 2012; Salgado et al. 2011; Schaefer and Iozzo 2008).
The ear repeat at the carboxy terminal is formed by two cysteine residues that are
located within LRR 11 and 12, which are linked by a disulphide bond (Schaefer and
Iozzo 2008).
Highly conserved exon/intron junctions also exist in this class of SLRPs and
they exhibit similar exon organisation since all are encoded by eight exons (Ameye and
Young 2002; Mochida et al. 2006; Schaefer and Iozzo 2008; Waddington et al. 2003) in
which the LRRs are encoded by exons III, IV, V, VI, VII and VIII (Matsushima et al.
2000). This class of SLRPs are spread over three different chromosomes with DCN
mapping to chromosome 12, ASPN and ECM2 physically linked to each other
(Nikitovic et al. 2012; Schaefer and Iozzo 2008) on chromosome 9 and BGN located on
chromosome X (Ameye and Young 2002; Schaefer and Iozzo 2008). The genes of the
class I SLRPs are orientated to lie 5’ to the class II SLRP genes (Ameye and Young
2002).
27
1.6.1.1.2 Class II Small Leucine Rich Proteins
Compared to the SLRPs of class I, the class II SLRPs are more tissue-specific in their
distribution (Ameye and Young 2002). This class encompasses the SLRPs fibromodulin
(FMOD), lumican (LUM), proline-arginine-rich end leucine rich repeat protein
(PRELP), keratocan (KERA) and osteomodulin (OMD), also known as osteoadherin
(OSAD) (Dellett et al. 2012; Matsushima et al. 2000; Schaefer and Iozzo 2008). The
SLRPs of canonical class II also are composed of twelve LRRs (Dellett et al. 2012;
Matsushima et al. 2000) which can carry keratan sulphate or polylactosamine (an
unsulphated version of keratan sulphate) side chains (Ameye and Young 2002; Dellett
et al. 2012; Iozzo and Schaefer 2010; Nikitovic et al. 2012; Salgado et al. 2011;
Schaefer and Iozzo 2008). At the amino terminal of these proteins, a cluster of sulphated
tyrosine residues are present (Ameye and Young 2002; Iozzo and Schaefer 2010;
Nikitovic et al. 2012; Salgado et al. 2011; Schaefer and Iozzo 2008) which is thought to
give a polyanionic characteristic to these class II SLRPs (Nikitovic et al. 2012; Schaefer
and Iozzo 2008). Similarly to class I SLRPs, the class II proteins also contain a cluster
of cysteine residues at their amino terminal with the consensus sequence Cx3CxCx9C
(Ameye and Young 2002; Dellett et al. 2012; Schaefer and Iozzo 2008), and also have
ear repeats present at both their termini (Dellett et al. 2012; Schaefer and Iozzo 2008).
The genes of the class II SLRPs have analogous exonic organisation, which is
evident due to the fact that all proteins in this class are encoded by three exons (Ameye
and Young 2002; Matsushima et al. 2000; Nikitovic et al. 2012; Schaefer and Iozzo
2008), in which exons II and III are responsible for most of the protein’s LRRs
(Schaefer and Iozzo 2008). Class II members are also dispersed over three
chromosomes with FMOD and PRELP being mapped to chromosome 1, LUM and
28
KERA encoded on chromosome 12 and chromosome 9 being responsible for the OMD
gene (Ameye and Young 2002; Schaefer and Iozzo 2008). The SLRPs of class II are
aligned 5’ to those of class III (Ameye and Young 2002).
1.6.1.1.3 Class III Small Leucine Rich Proteins
Epiphycan (EPYC) (Dellett et al. 2012; Iozzo and Schaefer 2010; Matsushima et al.
2000; Nikitovic et al. 2012; Schaefer and Iozzo 2008), osteoglycin (OGN) (Dellett et al.
2012; Iozzo and Schaefer 2010; Matsushima et al. 2000; Nikitovic et al. 2012; Schaefer
and Iozzo 2008) and opticin (OPTC) (Dellett et al. 2012; Iozzo and Schaefer 2010;
Nikitovic et al. 2012; Schaefer and Iozzo 2008) form this canonical class of SLRPs
which all are composed of eight LRRs (Dellett et al. 2012). Unlike class I and II SLRPs,
this class does not share the same short-long-long repeat sequence of their LRRs, owing
to the absence of a long repeat in the centre of the leucine-rich domain (McEwan et al.
2006). This class does not share any of the same GAG side chains as each other, with
EPYC expressing dermatan sulphate (Dellett et al. 2012; Iozzo and Schaefer 2010;
Nikitovic et al. 2012) or chondoitin sulphate side chains (Iozzo and Schaefer 2010;
Nikitovic et al. 2012), OGN containing keratan sulphate side chains (Dellett et al. 2012;
Iozzo and Schaefer 2010; Nikitovic et al. 2012) and OPTC, being a non-classical
proteoglycan (Mochida et al. 2006), has an absence of GAG side chains (Dellett et al.
2012; Iozzo and Schaefer 2010; Nikitovic et al. 2012). Compared to class I and class II
SLRPs, the proteins of class III show the most tissue specificity (Ameye and Young
2002).
29
Class III SLRPs also contain a class specific cysteine rich region at their amino
terminal, with the consensus sequence Cx2CxCx6C (Ameye and Young 2002; Dellett et
al. 2012; Schaefer and Iozzo 2008). Since some of these in the tissue exist as
glycoproteins, they also contain a consensus sequence for glycanation (Nikitovic et al.
2012; Schaefer and Iozzo 2008). The amino terminal end of the protein also includes
sulphated tyrosine residues (Ameye and Young 2002). The genes of SLRPs from class
III are encoded by seven exons (Ameye and Young 2002; Matsushima et al. 2000;
Nikitovic et al. 2012; Schaefer and Iozzo 2008) in which exons V, VI and VII encode
the LRRs (Ameye and Young 2002; Matsushima et al. 2000). This class has been
mapped three different chromosomes, with EPYC being mapped to chromosome 12,
OGN to chromosome 9 and OPTC being located on chromosome 1 (Ameye and Young
2002; Schaefer and Iozzo 2008).
1.6.1.1.4 Class IV Small Leucine Rich Proteins
The non-canonical (Schaefer and Iozzo 2008) class IV SLRPs encompass the proteins
chondroadherin (CHAD) (Ameye and Young 2002; Dellett et al. 2012; Iozzo and
Schaefer 2010; Matsushima et al. 2000; Nikitovic et al. 2012; Schaefer and Iozzo 2008),
tsukushi (TSKU) (Dellett et al. 2012; Nikitovic et al. 2012; Schaefer and Iozzo 2008)
and nyctalopin (NYX) (Ameye and Young 2002; Dellett et al. 2012; Nikitovic et al.
2012; Schaefer and Iozzo 2008).The SLRPs of class IV do not share some of the
common features of this family of SLRPs such ear repeats (Dellett et al. 2012) formed
by disulphide bonds and GAG side chains (Dellett et al. 2012; Iozzo and Schaefer 2010;
Nikitovic et al. 2012) (except for CHAD which has keratan sulphate substitutions)
(Iozzo and Schaefer 2010; Nikitovic et al. 2012), but do have prospective glycosylation
sites (Dellett et al. 2012).
30
Twelve LRRs have been reported in this class (Dellett et al. 2012) and these
motifs do not follow the typical short – long – long pattern that class I, II and III SLRPs
exhibit (Schaefer and Iozzo 2008). The class IV SLRPs are also flanked at the amino
terminal with a region rich in cysteine residues (Ameye and Young 2002; Nikitovic et
al. 2012; Schaefer and Iozzo 2008) (Cx3CxCx6-17) (Dellett et al. 2012; Schaefer and Iozzo
2008); however the proteins of this class do not contain a single consensus sequence,
since the number of intervening amino acids varies between the third and last cysteine
residue. These class have been mapped to chromosomes 11, 17 and X by TSKU, NYX
and CHAD respectively (Schaefer and Iozzo 2008).
1.6.1.1.5 Class V Small Leucine Rich Proteins
Podocan (PODN) and Podocan-like protein 1 (PODNL1) are two very homologous
proteins (Schaefer and Iozzo 2008) which make up the non-canonical class V of the
SLRP family (Dellett et al. 2012; Nikitovic et al. 2012; Schaefer and Iozzo 2008). Like
class IV SLRPs, PODN and PODNL1 do not contain any reported GAG side chains
(Dellett et al. 2012; Iozzo and Schaefer 2010) (but do contain potential glycosylation
sites) (Dellett et al. 2012) or ear repeats (Dellett et al. 2012) and are composed of twenty
and twenty one leucine-rich motifs respectively (Dellett et al. 2012). In contrast to class
I, II and III and in similarity to class IV SLRPs, they express different cysteine rich
clusters at both their carboxyl terminal (Nikitovic et al. 2012; Schaefer and Iozzo 2008)
and amino terminal (Cx3-4CxCx9C) (Dellett et al. 2012). PODN and PODNL1 have been
mapped to chromosome 1 and 19 respectively (Schaefer and Iozzo 2008). This thesis
will only focus on six members of the SLRP family.
31
1.7 The Functions of Small Leucine Rich Proteins in Mesenchymal Stem Cell
Lineages
1.7.1 Definitions and characteristics of mesenchymal stem cells
Mesenchymal stem cells (MSC) are multipotent cells of the stroma that display self-
renewal and are capable of multi-lineage differentiation (D. Ding et al. 2011). The
International Society for Cellular Therapy, has provided the minimal criteria for
defining MSCs as being able to show in vitro multi-lineage differentiation into
chondrocytes, adipocytes and osteoblasts, while exhibiting plastic adherence in standard
conditions, must express the surface molecules CD90, CD73 and CD105 and lack the
expression of HLA-DR, CD19 or CD79α, C11b or CD14, CD34 and CD45 surface
molecules (Dominici et al. 2006) . MSCs can be isolated from many tissues in humans
such as adipose tissue (ADSCs), bone marrow (BMSCs), umbilical cord, amniotic fluid,
placenta, synovial membrane, dental pulp, thymus, spleen, liver and skeletal muscle (D.
C. Ding et al. 2006; D. Ding et al. 2007; Liu et al. 2009; Ohishi and Schipani 2010).
1.7.2 Process by which Mesenchymal Stem Cells mature into Osteoblasts
The process of MSCs to mature into cells with the phenotypic characteristics of an
osteoblast is known as osteogenesis. The differentiation process involves an initial stage
of cellular proliferation, followed by maturation of the ECM with subsequent
mineralisation of the newly synthesised matrix (Frith and Genever 2008). This course of
differentiation involves the MSCs to developing into committed osteoprogenitor cells
then to pre-osteoblasts before completely maturing into an osteoblasts (Frith and
Genever 2008). The exposure of MSCs to β-glycerol phosphate, ascorbic acid and
dexamethasone is the most widely used method for promoting the formation of
osteoblast-like cells in vitro (Frith and Genever 2008; Zuk et al. 2002).
32
1.7.2.1 Osteoblasts
Osteoblasts are the bone forming cells of the skeleton (Nakamura 2007). They are
responsible for the deposition of osteoid (uncalcified bone matrix) (Nakamura 2007)
through the synthesis of an array of proteins such as type I collagen, osteocalcin,
osteopontin, bone sialoprotein, fibronectin, osteonectin (Guan et al. 2012; Nakamura
2007) and various proteoglycans (Nakamura 2007) that regulate hydroxyapatite and
calcium binding for calcification. Osteoblasts also express high levels of alkaline
phosphatase, which can be measured to indicate the activity of the cells (Sabokbar et al.
1994) and used as a marker of osteogenesis (Frith and Genever 2008). Other markers of
osteoblasts include increased expression of transcription factors such as Cbfa1 and
osteoblast related genes such as osteocalcin (Figure 1.7).
Figure 1.7: The expression of transcription factors and osteoblast-related genes
throughout the differentiation stages of osteogenesis. Adapted from Frith and Genever
(2008).
33
1.7.2.2 Small Leucine Rich Proteins expressed in osteoblasts
Several SLRPs have been shown to be involved with bone formation, in particular
BGN. BGN is the most characterised SLRP in bone, and has been shown to be a
positive regulator of bone formation (X. Chen et al. 2003). The importance in bone
formation of BGN was first observed by the age dependent osteoporosis-like phenotype
in BGN deficient mice (Xu et al. 1998). Reduced corticol bone mass and reduced
trabecular bone mass in the metaphysis and epiphysis of these mice was observed
(Figure 1.8), and this was attributed to reduced bone formation due to failure to achieve
peak bone mass (Xu et al. 1998).
Figure 1.8: Femur comparison of wild type and BGN deficient mice. Radiological
analysis of femurs from mice in wild type (+/0) and BGN deficient mice (-0) at (a)
three, (b) six and (c) nine months. Progressive decrease in trabecular bone mass
indicated by (*) with age, compared to wild type mice. Adapted from Xu et al. (1998).
34
Further studies in BGN deficient mice showed that this was the result of the
diminished ability of mice to produce BMSC, the precursor of cell of osteoblasts (X.-D.
Chen et al. 2002). This was further investigated in BGN-deficient osteoblasts, which
showed that the absence of BGN restricted Bone Morphogenic Protein (BMP) 4 binding
to the osteoblast, ultimately disrupting differentiation (Chen et al. 2004). In BGN and
DCN double knockout mice models, a severe osteoporosis-like phenotype has also been
observed and this was attributed to insufficient sequestration of TGF-β in the ECM,
leading to the excessive binding of TGF-β on BMSCs. The cells responded to this over
activation by inducing apoptosis, resulting in a decreased number of osteoblast
precursor cells and ultimately reduced bone formation (Bi et al. 2005). Collagen fibril
formation has also been assessed in BGN and DCN double knockout mice, and a more
severe osteoporosis-like phenotype was observed compared to a BGN only knockout
mice model, demonstrating a synergistic effect between the Class I SLRPs (Corsi et al.
2002). Although BGN appears to be critical for osteoblast differentiation, it has been
shown that in vitro the level of BGN expression does not change between control and
osteogenic media (Hashimoto 2012). However, other SLRPs such as OMD, DCN,
PRELP and ASPN were up-regulated during differentiation (Figure 1.9) (Hashimoto
2012).
35
Figure 1.9: A comparison of the gene expression level of seven SLRPs in control and
osteogenic media. qRT-PCR data showing the expression of the SLRPs ASPN, OMD,
PRELP, DCN, BGN, FMOD and LUM compared to the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A significant increase in
expression in osteogenic media (MSCOIM) compared to control media (MSCGM) was
observed in ASPN, OMD, PRELP and DCN in a human mesenchymal stem cell line.
Adapted from Hashimoto (2012).
36
The expression of LUM has been shown to increase during osteoblast
differentiation (Raouf et al. 2002). Three days after osteogenic induction of MSCs,
LUM was not expressed in proliferating pre-osteoblasts, however by day five a 4.1 fold
increase in LUM expression was shown, which further increased during the
mineralisation stage (Raouf et al. 2002). OMD is another SLRP that has been shown to
be involved in osteogenesis (Liu et al. 2007; Rehn et al. 2008). Studies comparing
BMSC and ADSC have shown that OMD expression was increased during early
osteogenesis in both types of MSCs, however during late osteogenesis OMD was
further increased in BMSCs (Liu et al. 2007). Over expression of OMD has also been
shown to increase osteoblast differentiation measured by increased ALPase and
mineralisation (Liu et al. 2007). The expression of the selected SLRPs in mice
osteoblasts have also been reported at varying time points (Figure 1.10).
Figure 1.10: The gene expression relative to an internal control of the six selected
SLRPs LUM, OMD, EPYC, BGN, DCN and TSKU in mouse cells during osteogenesis
at 5, 14 and 21 days. The relative gene expression of SLRPs. Adapted from The Scripps
Research Institute (2013).
1
10
100
1000
10000
100000
DAY
5
DAY
14
LUM
DAY
21
DAY
5
DAY
14
OMD
DAY
21
DAY
5
DAY
14
EPYC
DAY
21
DAY
5
DAY
14
BGN
DAY
21
DAY
5
DAY
14
DCN
DAY
21
DAY
5
DAY
14
TSKU
DAY
21
Gen
e e
xp
ress
ion
37
1.7.3 Process by which Mesenchymal Stem Cells mature in Adipocytes
Adipogenesis is the process of differentiation that occurs when MSCs matures into an
adipocyte (Frith and Genever 2008). Adipocyte development involves the accumulation
of lipid-rich vacuoles (Rosen and Spiegelman 2006; Zhu et al. 2009) within the cell and
an enlarged morphology (Zhu et al. 2009). Early adipogenesis is associated with the up-
regulation of genes involved in formation of the ECM, while late adipogenic
differentiation involves the increased expression of genes involved in the metabolism of
lipids including fatty acid binding protein and lipoprotein lipase (Frith and Genever
2008; Urs et al. 2004).
1.7.3.1 Adipocytes
Adipocytes are the predominant cells of adipose tissue which are responsible for the
storage of energy in the form of triglycerides (Urs et al. 2004). These particular cells
perform many endocrine functions involved in the regulation of systemic lipid and
glucose homeostasis (Christodoulides and Vidal-Puig 2010; Urs et al. 2004).
Adipocytes achieve these roles through the secretion of numerous cytokines and
hormones such as adiponectin and leptin (Christodoulides and Vidal-Puig 2010; Urs et
al. 2004). In vitro supplementation of the growth medium with insulin (Liu et al. 2009),
isobutyl-methylxanthine, indomethacin and dexamethasone promotes MSCs to
differentiate into adipocyte-like cells (D. Ding et al. 2011; Liu et al. 2009) which can be
confirmed by the staining of the lipid-rich vacuoles with Oil Red dye (Hung et al.
2004).
38
1.7.2.2 Small Leucine Rich Proteins expressed in adipocytes
Evidence of the expression of SLRPs involved in adipocytes is limited. To date BGN
and DCN have been the most studied SLRPs in adipose tissue, with the expression of
both SLRPs up-regulated in this depot during the development of type II diabetes and
obesity (Bolton et al. 2012). In obese murine models, BGN expression has been shown
to increase (Figure 1.11) (King et al. 2010) and furthermore, up-regulation of BGN has
been observed in human omental fat tissue in contrast to lean adipose tissue (Ward and
Ajuwon 2011).
Figure 1.11: The expression of BGN in lean and obese in murine models. The
expression of BGN normalised to the housekeeping gene 18S. A significant increase in
the expression of BGN is observed in obese mice compared to the lean mice.
* represents P < 0.05.Adapted from Ward and Ajuwon (2011).
Other studies have observed that DCN is expressed highly in pre-adipocytes (Urs et al.
2004) and that BGN and DCN can reduce the proliferation of pre-adipocytes (Ward and
Ajuwon 2011). In humans, the expression of SLRPs in adipocytes has been reported
(Figure 1.12).
*
39
Figure 1.12: The expression levels of the selected SLRPs members in human
adipocytes. Adapted from The Scripps Research Institute (2013).
1.7.4 Process by which Mesenchymal Stem Cells mature into Chondrocytes
The maturation of chondrocytes from MSCs is known as chondrogenesis. This
developmental process is accompanied by a stage of MSC condensation followed by
chondroblast proliferation (Frith and Genever 2008). The cells develop a flattened
morphology and arrange themselves in columns while progressively becoming larger
until they mature into hypertrophic chondrocytes (Frith and Genever 2008).
1.7.4.1 Chondrocytes
Chondrocytes are responsible for the maintenance, organisation and production of
cartilage (Poole 1997), which aids the smooth movement of joints (Martinez-Sanchez et
al. 2012). This is achieved by the synthesis of an ECM rich in type II collagen (Barry et
al. 2001; Mahmoudifar and Doran 2012), type X collagen (Frith and Genever 2008),
aggrecan (Barry et al. 2001; Frith and Genever 2008) and versican (Barry et al. 2001).
Chondrocyte-like cells can be stimulated in vitro by the addition of TGF-β2 and TGF-
β3 to MSCs (Liu et al. 2009).
1
10
100
1000
10000
LUM OMD EPYC BGN DCN TSKU
Gen
e ex
pre
ssio
n
40
1.7.4.2 Small Leucine Rich Proteins expressed in Chondrocytes
EPYC, BGN, DCN (Nuka et al. 2010), LUM (Nuka et al. 2010; Roughley 2006) and
OMD (Liu et al. 2007) have all been shown to be expressed in chondrocytes. The
expression of both chondroitin sulphate and dermatan sulphate substituted EPYC has
been observed during embryonic development in epiphyseal cartilage and has been
proven to be involved in joint maintenance (Nuka et al. 2010). In EPYC and BGN
double deficient murine models, osteoarthritis developed with age (Figure 1.13) and
shorter and lighter femurs were observed at nine months old (Nuka et al. 2010).
Figure 1.13: A comparison of the degree of osteoarthritis in wild type and SLRP
deficient mice displayed as the mean osteoarthritis severity scores in male wild type and
SLRP deficient mice. A significant increase (*** P < 0.005) is observed in EPN
(EPYC)/BGN double deficient mice compared to the wild type at the same age (shown
on x-axis in months). Adapted from Nuka (2010).
Ost
eoa
rth
riti
s G
rad
e
41
Interestingly, the expression of other SLRPs such as LUM, ASPN and FMOD were
increased in this particular model (Nuka et al. 2010). This study also demonstrated the
first synergistic effect of SLRPs from two different classes and this was described to be
due to either the exacerbation of the lost independent functions of EPYC and BGN or
due to the overlapping functions of cartilage these two SLRPs share (Nuka et al. 2010).
The expression of BGN during chondrogenesis appears to be absent after three
days of chondrogenic induction and only after day seven does it become readily
detectable (Barry et al. 2001). BGN and FMOD deficient MCCs have demonstrated that
due to the insufficient sequestration of TGF-β1, the overactive binding of TGF-β1
stimulates turnover of the ECM leading to the degradation of the matrix which
ultimately resulted in temporomandibular joint osteoarthritis in these mice (Embree et
al. 2010).
The expression of DCN in chondrogenesis is low during the first three days after
chondrogenic induction, but then is up-regulated rapidly in the following days (Barry et
al. 2001). There is evidence to show that the DCN in cartilage is involved in the
assembly and growth of type II collagen (Zhang et al. 2006).
42
1.8 Conclusion
As SLRP expression and localisation has not been characterised in human MSCs
undergoing osteogenesis, adipogenesis and chondrogenesis, this formed the basis for
my honours project. To do this I will use real time-PCR to semi-quantitatively analyse
the relative gene expression and fold change of the six selected SLRPs LUM, EPYC,
OMD, BGN, DCN and TSKU during the multipotent differentiation of BMSCs and
ADSCs. Additionally, the localisation and protein abundance of OMD will be
characterised using immunofluorescence and western blotting respectively.
Understanding the roles of SLRPs in MSC differentiation, may help in improving the
current understanding of the pathogenic mechanisms behind osteoporosis.
43
PART II: MATERIALS AND METHODS
2.1 Materials manufacturers
Plastics and machines
50 ml tubs Sarstedt
15ml tube Sarstedt
2 ml screw-capped tubes Sarstedt
Flasks (T25 and T75) Sarstedt
Plates (24,48 well plates) Sarstedt
Coverslips ProSciTech
96 well PCR plates Fisher Biotechnology
Heamacytometer Hirschmann
Filter tips Interpath Services
RNA concentration spectrometer
Nanodrop ND-1000
Nanodrop
Microscope Olympus IMX
Flow cytometer BD CantoII
Confocal microscope Nikon A1+
RT-PCR cycler Bio-Rad iQ5
Chemicals
Bovine Serum Albumin Sigma Aldrich
Trypsin Invitrogen
L-ascorbic acid Invitrogen
β-glycerol phosphate Sigma Aldrich
Dexamethasone Sigma Aldrich
Trizol®
Life Technologies
TGF-β Sigma Aldrich
Human insulin Sigma Aldrich
44
Rat Tail Type I Collagen BD bioscience
3-isobutyl-1-methylxanthine (IBMX) Sigma Aldrich
Indomethacin Sigma Aldrich
Analytical grade agarose Promega
Ethidium bromide Life Technologies
20 bp DNA Ladder Geneworks
Antibodies and primers
Primer assay for qRT-PCR Qiagen
Anti-OMD, antibody produced in rabbit,
purified immunoglobulin
Sigma Aldrich
Anti-Rabbit immunoglobulin -Peroxidase
antibody produced in goat
Sigma Aldrich
Hoechst 33342 nuclear staining Life Technology
Alexa Fluor® 647 Phalloidin (F-Actin) Life Technology
Culture media
Dulbecco's Modified Eagle
Medium/Nutrient Mixture F-12
Life Technologies
Antibiotic and antimyotic solution Sigma Aldrich
Kits
RNA DNA Protein extraction Allprep Kit Qiagen
Purelink RNA mini kit Life Technologies
Iscript One step PCR kit SYBR green Bio-Rad
Serum
Goat serum Sigma Aldrich
Data analysis software
Image J NIH image version 1.44
SPSS IBM SPSS Statistics 20
45
2.2 Human adipose and bone marrow derived mesenchymal stem cell primary cell
culture procedure
2.2.1 Isolation of mesenchymal stem cells (performed by Ms Jenny Wang)
Patients who underwent elective laparoscopic abdominal surgery or hip surgery
(including total hip replacements for osteoarthritis or hemiarthroplasty for hip fracture
were recruited) from Sir Charles Gairdner Hospital and Hollywood Hospital. All
participants were given written informed consent for the study according to the Human
Research Ethics Committee of Sir Charles Gairdner Hospital (2008-068) and the
University of Western Australia (RA/4/1/4907).
The isolation method for MSCs was adapted from previously published
protocols (Dubois et al. 2008; Wolfe et al. 2008). Briefly, after surgery tissue samples
were placed immediately in sterile jars with phosphate buffered saline (PBS) and were
transported for immediate processing and cell culture.
For adipose tissue processing, the adipose tissue was cut into 3mm × 3mm
pieces with scissors and then further digested in 10 mL of digestion solution containing
1% bovine serum albumin (BSA) and 0.3% collagenase II (Life Technologies,
Australia) at 37°C on a shaking platform for up to 3 hours until fully digested. The
digest solution was then pressed over a 500 μm sterile pore-sized disposable nylon mesh
followed by centrifugation at 1, 000 x g for 10 minutes. The mature adipocytes (floating
on the upper layer) were then separated and the MSCs (pellet) were incubated with 3
mL of red cell lysis buffer (155 mM NH4Cl, 0.1 mM EDTA, 10 mM KHCO3) for 15
minutes at 37°C. Stem cells were then centrifuged at 1,000 x g for 5 minutes and
resuspended in 3 mL of complete media (Dulbecco's Modified Eagle Medium/Nutrient
Mixture F-12 (DMEM/F12) (Life Technologies, Australia) containing 10% foetal
46
bovine serum (a single batch of fetal bovine serum was used throughout the study
(Serana, Australia)), 1% antibiotic-antimycotic solution). Cell counting assay was then
performed to determine the cell number. 15 mL of complete media (was added and the
cells were then seeded into a 75 cm2 plastic flask and cells were left to incubate at 37°C
in a humidified atmosphere with 5% CO2/95% air.
For bone marrow tissue, samples were minced by scissors and followed by the
similar procedure as adipose tissue.
The following work was performed by me and formed the basis of my honours thesis
2.2.2 Cell resuscitation
Complete culture medium was pre-warmed to 37°C in a water bath before
cryopreserved cells were resuspended. The cell suspension was transferred into 15 mL
of complete media and pipette into a T75 cell culture flask (Sarstedt, Germany) which
was transferred into an incubator at 37°C with a humidified atmosphere and 5%
CO2/95% air. Medium change was then performed 24 hours after the resuscitation.
2.2.3 Cell passage
Medium was changed every 3 days until 85% confluence was reached. Subculture
procedures involved washing the cell monolayer twice with PBS after aspirating the old
media. Cells were then incubated with 0.25% trypsin-EDTA (Invitrogen, Australia) for
5 to 8 minutes at 37°C until cells detached from culturing surface. To stop
trypsinisation, 15 mL of complete media was added into each flask and then the cell
pellet was centrifuged at 1,000 x g. The cells were then re-suspended in complete
media.
47
2.2.4 Cell cryopreservation
Medium was aspirated and cells were harvested by trypsin. Cell number was then
determined by manual cell count. Cells were then centrifuged at 1,000 x g for 2 minutes
and resuspended in 1 mL cryopreservation media (90% foetal bovine serum, 10%
dimethyl sulfoxide). Cryopreservation tubes were then transferred to the -20°C freezer
for 20 minutes, -80°C freezer for 20 minutes and then a liquid nitrogen dewar.
2.2.5 Cell counting assay
Cell counting assay was performed to determine cell number before seeding cells in 24
or 48 well plates. Briefly, 10 μL of cell suspension was loaded onto a standard
heamacytometer (Hirschmann, Germany). Cells in the 1 mm square and the four corner
squares were counted. All cell counts were performed using biological triplicates.
2.2.6 Adipogenic and chondrogenic lineage differentiation assay
Cells were trypsinised harvested and seeded onto 48-well plates at a seeding density of
4 ×104/mL. After 24 hours, media was changed and the cells were divided into a control
group using complete media, adipogenic group using complete media with 500nM
dexamethasone (Sigma Aldrich, Australia), 5 μg/mL human recombinant insulin (Sigma
Aldrich, Australia), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma Aldrich, Australia)
and 50 μM indomethacin (Sigma Aldrich, Australia) or a chondrogenic group using
complete media supplemented with 50 μM ascorbic acid (Invitrogen, Australia), 100
nM dexamethasone, 10 ng/mL transforming growth factor beta (TGF-β) (Sigma
Aldrich, Australia) and 5 μg/mL human recombinant insulin (Sigma Aldrich, Australia).
Culture medium was changed every 3 days. RNA was extracted after 28 days of
differentiation.
48
2.2.7 Osteogenic lineage differentiation assay
Cells were trypsin harvested and seeded onto 24-well plates or 48-well plates at the
seeding density of 3×104/mL (75×10
2 /cm
2). After 24 hours, media was changed and the
cells were divided into control group using complete media; osteogenic media (OSM
containing 10 mM β-glycerol phosphate (Sigma Aldrich, Australia), 50 μM ascorbic
acid and 100 nM dexamethasone). All culture media was changed every 3 days.
Osteogenic stimulation continued for 7 days. RNA was extracted for the multipotent
study at day 28, for the for the long term osteogenesis study at baseline, 7 and 28 days,
and for the short term expression study at baseline, 3 and 7 days. Protein was extracted
at baseline, day 7 and 28 of osteogenic differentiation.
2.3 RNA isolation and qRT-PCR
2.3.1 RNA isolation
At each time point, cells were homogenised in TRIzol (Life Technologies, Australia).
The TRIzol method with the Purelink RNA mini kit (Life Technologies, Australia)
procedures were followed according to the manufacturer’s instructions. Briefly, 500uL
of TRIzol was added into cell pellets from each condition (pooled from biological six
biological replicates) and homogenisation was performed by mixing tubes for 15
seconds. Samples were then stored at -80°C and subject to RNA extraction. Briefly, the
homogenized samples were incubated for 5 minutes at room temperature followed by
adding 100 μL of chloroform. Tubes were then mixed vigorously for 15 seconds and
incubated at room temperature for 3 minutes. Cells were then centrifuged at 12,000 x g
for 15 minutes at 4°C. The mixture separates into a lower red phenol-chloroform phase,
an interphase and a colourless upper aqueous phase. To summarise, RNA remains
exclusively in the aqueous phase which is then separated by pipetting into a new tube.
The remaining content was then stored in a freezer at -80°C.
49
Up to 350 μL of 70% ethanol was added into each sample tube and mixed
vigorously. Samples were then transferred into a column with collection tube and
centrifuged at 12,000 x g for 15 seconds. Wash buffer I and II were subsequently added
into the column. 40 μL of RNase-free water was added to the tubes and incubated at
room temperature for 5 minutes which was eluted by centrifuging at 12,000 x g for 2
minutes. RNA concentrations and purity of each sample were then determined by
measuring the absorbance at 260 nm and the 260/280 ratio using Nanodrop 1,000
spectrophotometer and stored at -80°C.
2.3.2 Quantitative reverse transcriptase real time PCR
Quantitative reverse transcriptase real time PCR (qRT-PCR) were performed using
iScript one-step RT-PCR with SYBR Green (Bio-Rad, Australia) according to
manufacturer’s protocol. Briefly, all the reagents were brought to room temperature to
defrost meanwhile each master reaction tube (for one gene; 15 μL volume in each
reaction well) was set up. Each reaction was set up as presented in Table 2.1:
Table 2.1: Master mix reagents for each reaction
1X (μL)
2x SYBR® Green RT-PCR reaction mix 7.5
Primer (10X) 1.3
iScript reverse transcriptase for one-step RT-PCR 0.3
RNA template (5 ng/μL) 1
Nuclease-free water 4.9
Total volume 15
50
QuantiTect Primer Assays (Qaigen, Netherlands) for the genes LUM, OMD, EPYC,
BGN, DCN, TSKU, GAPDH and 18S were used in the above setup in a 96-well PCR
plate layout. Once the PCR plate was set up, it was mixed gently and centrifuged at
1,000 x g before being placed in the Bio-Rad iQ5 cycler. The reaction cycler was set up
as described in Table 2.2:
Table 2.2: Thermocycler reaction protocol
Cycle Temperature Time
Cycle 1: (1X)
Step 1: 50°C 10 minutes
Cycle 2: (1X)
Step 1: 95°C 5 minutes
Cycle 3: (45X)
Step 1: 95°C 10 seconds
Step 2: 60°C 30 seconds
To analyse qRT-PCR data, the following equation was used to calculate the relative
gene expression: Relative gene expression = CT housekeeping gene / CT gene of interest. All PCR
reactions were performed in biological triplicates with technical duplicates.
2.3.3 Gel electrophoresis for amplification products
To confirm the correct genes were being amplified and to optimise the RNA
concentration for the PCR reactions, amplification products of qRT-PCR were
confirmed using gel electrophoresis. Briefly, 2% agarose gel was made using 2 g
analytical grade agarose (Promega, Australia) in 100 mL of 1X TAE buffer. Agarose
solution was heated until clear which was followed by the addition of ethidium bromide
(Life Technologies, Australia) in a final concentration of 0.3 mg/mL in the gel. The
solution was then poured into a gel casting tray with a comb and left to set. Once set,
the gel in the gel casting tray was placed in a gel tank and filled with 1X TAE buffer
until the gel was submerged. 2 μL of 2X loading buffer was added to 4 μL of 20 base
51
pair DNA ladder and to 7 – 10 μL of amplification products from qRT-PCR. Samples
were run for 1 hour at 100 V. Gel was then removed from gel tank and imaged using the
Bio-Rad Molecular Image Gel Doc XR System. Comparing the bands of the
amplification product to the DNA ladder (Geneworks, Australia) showed the correct
amplicon sizes for each gene and that 5 ng/μL was the lowest ideal RNA concentration
to be used.
2.3.4 Statistical analysis of gene expression
Once CT values were made relative to GAPDH only (due to the instability of the 18S
housekeeping gene) the data was exported into IBM SPSS Statistics 20 software
package. Comparison of the gene expression between tissue types was analysed using a
paired samples t – test in which the relative expression of ADSC was matched with the
corresponding relative expression of the gene in BMSC samples. To compare the
difference in gene expression between ADSC and BMSC in control and OSM, a
univariate analysis of variance (ANOVA) was performed for each gene in both tissue
types. P values less than 0.05 were considered significant. To test the difference in
relative expression of OMD in control media, osteogenic media, adipogenic media and
chondrogenic media, a one way ANOVA was performed with a Bonferroni post-hoc
correction.
52
2.4 Protein isolation and western blotting
2.4.1 Protein isolation
At each time point, cells were homogenised in TRIzol. The method for extracting
protein with TRIzol was followed according to the manufacturer’s instructions. Briefly,
500 μL of TRIzol was added into cell pellet from each condition (pooled biological
triplicates) and homogenisation was performed by mixing tubes for 15 seconds. 100 µL
of chloroform was added to the homogenised samples followed by vigorous mixing
then left to incubate for at room temperature for 3 minutes. Samples were subsequently
centrifuged at 12,000 x g for 15 minutes at 4°C. The mixture separates into a lower red
phenol-chloroform phase, an interphase and a colourless upper aqueous phase. Protein
and DNA remain exclusively in the organic phase under these conditions.
150 μL of 100% ethanol was added to the organic phase and left to incubate at
room temperature for 3 minutes. The samples were centrifuged at 2,000 x g for 5
minutes at 4°C to sediment the DNA. The phenol-ethanol supernatant was separated
from the DNA pellet. To promote precipitation, the phenol-ethanol supernatant was
treated with 400 μL of isopropanol and left to incubate for 10 minutes at room
temperature. To sediment the protein, the samples were centrifuged at 12,000 x g for 10
minutes at 4°C.
53
The supernatant was separated from the pellet, and 0.3 M guanidine
hydrochloride in 95% ethanol was used to wash the protein. 150 μL of the wash solution
was added to the protein pellet and left to incubate for 20 minutes at room temperature
before centrifuging the samples at 7,500 x g for 5 minutes at 4°C. Once this process had
been completed three times, the protein pellet was washed with ethanol for 20 minutes
at room temperature and subsequently centrifuged at 7,500 x g at 4°C.
The ethanol was removed, and the protein pellet was left to dry for 10 minutes. 20 μL of
1% Sodium Dodecyl Sulphate (SDS) in water was used to dissolve the protein. The
protein solution was stored in a -20°C freezer until further test.
2.4.2 Western blotting
2.4.2.1 Sodium Dodecyl Sulphate Polyacrylamide gel electrophoresis (SDS – PAGE)
The protein expression of osteomodulin during 0, 7 and 28 days of osteogenic
differentiation was determined by western blotting. Briefly, protein samples were
quantified using the Bio-Rad Protein Assay according to manufactures instructions then
diluted to 0.063 mg/mL in 4X SDS loading buffer and heated at 95°C before being
centrifuged at 12,000 x g. Samples were then separated using electrophoresis on SDS –
PAGE gels. A 10% separating solution was prepared and poured into a gel cast for each
gel with 20% ethanol layered on top to prevent oxidation of the gel and left to set for 30
minutes at room temperature. Subsequently, the ethanol was removed and a stacking
solution was prepared and poured into the gel cast above the separating gel, and left to
set for 45 minutes at room temperature with a comb to create wells in the gel. Once the
stacking solution had set, the electrophoresis apparatus was prepared and placed into a
tank filled with 1X SDS-PAGE running buffer. 5 μL of the protein standard Precision
54
Plus Protein Prestained Standards (Bio-Rad, Australia) to use as a means of size
determination was loaded into the first well of each gel alongside each sample in the
order of day 0, day 7 control, day 7 OSM, day 28 control, day 28 OSM. Proteins were
electrophoresed through the stacking gel at 80 V for 25 minutes, then through the
separating gel at 100 V for 1 hour.
Once separation was completed, proteins were transferred from the gel to
Whatman Protran nitrocellulose blotting membranes. This was performed by inserting
the gel and membrane between three layers of Whatman 3MM Chr Paper and an
absorbent page on both sides, enclosed in a cassette. The cassette was placed in a
transfer tank with an ice pack and filled with transfer buffer. The transfer was
performed overnight at a constant amp of 0.03 A.
2.4.2.2 Protein transfer
Once the proteins had transferred, the membrane was removed and blocked with 5%
skim milk/water. After 1 hour of blocking, the skim milk was removed and the
membrane washed with 1X TBS-T three times for 5 minutes. Once the membrane was
washed, anti-OMD antibody produced in rabbit purified immunoglobulin was added in
a 1:4,000 dilution to 10 mL of 1% skim milk/1X TBS-T solution and used to wash the
membrane overnight at 4°C while rocking. Once complete, the primary antibody
solution was removed and the membrane was washed in 1X TBS-T three times for 5
minutes. Subsequently, the membrane was washed for an hour at room temperature with
10 mL of anti-rabbit immunoglobulin (whole molecule)-Peroxidase antibody produced
in goat (1:5,000) in 1% skim milk/1X TBS-T solution. Once completed, the secondary
antibody solution was removed and the membrane was washed with 1X TBS-T twice
followed by two rinses with 1X TBS to reduce the background when the membrane was
imaged.
55
2.4.2.3 Membrane imaging
To observe the immunostained protein, the membrane was incubated with enhanced
chemiluminescence reagent prepared by mixing enhanced luminol reagent and oxidising
agent in 1:1 ratio. After 1 minute of incubation, excess the enhanced
chemiluminescence reagent was removed and the membrane was loaded into the
FujiFilm LAS-3,000. A digital image was taken to observe the ladder, followed by a
chemoluminescence image taken in increments of 30 seconds until a satisfactory signal
was achieved. Protein densitometry was performed using ImageJ software.
2.4.2.4 Stripping membranes
For detection of another protein on the membrane that has been previously probed, the
primary antibody must be removed. In this case, the primary antibody for detecting
OMD was stripped from the membrane to allow the housekeeping protein β-actin (Cell
Signalling Technology, United States of America) (1:2,000) to be probed. The
membrane was washed with 1X TBS-T for 5 minutes, then wash solution was removed
and stripping buffer was added to the membrane and left to incubate for 30 minutes at
55°C. Once the membrane was ready to be probed with the next protein of interest, the
stripping buffer was removed and the protocol re-commenced from blocking the
membrane (Refer to section 2.4.22 Protein transfer).
56
2.5 Immunofluorescence staining
2.5.1 Collagen coating of #1 glass coverslips
Coating solution of 500 μL Rat Tail Type I Collagen in acetic acid was added into 24-
well plate, with coverslips at the bottom. The plate was then swung gently until the
coating solution was evenly distributed. Coating was performed for an hour at room
temperature. Excessive solution was then removed and the plate was rinsed with PBS
twice. The plate was stored at 4°C after dehydration at room temperature with lid off.
Before the cells were seeded at 3×104/mL (75×10
2 /cm
2), each plate was heated in an
incubator to 37°C and washed by warm PBS twice.
2.5.2 Immunofluorescence staining procedure
Human adipose tissue-derived and bone marrow-derived stem cells were prepared as
previously described and treated with control media and OSM on glass coverslip (as
prepared before) for 7 days and 28 days (performed by Ms Jenny Wang). 4% PFA was
used to fix the coverslips for 10 minutes at room temperature followed by 3 washes of
PBS for 5 minutes. Serum blocking was then performed with serum blocking buffer
(5% goat serum, 1% bovine serum albumin, 0.1% Triton X-100, 0.05% Tween-20,
0.05% sodium azide in PBS) for 30 minutes in room temperature. Cells were then
incubated with primary antibody Anti-OMD produced in rabbit buffer overnight at 4°C.
Cells were then washed in PBST for 5 minutes × 3 times and then incubated in
secondary antibody buffer for 1 hour at room temperature followed by washing with
PBST 3 times for 5 minutes.
57
The plate was placed in a draw to avoid light exposure during the secondary
antibody incubation. Cells were then counterstained with Actin probe (1:300) for 30
minutes and then stained with Hoechst 33342 (1:5,000) for 5 minutes. After washing
with PBST for 3 times for 5 minutes, coverslips were picked up and flipped using fine
forceps and immersed in anti-fade buffer onto glass microscopic slides. For long-term
storage, coverslips were sealed using nail polish and stored at 4°C. Coverslips were
visualised using the Nikon A1
+ Confocal Laser Microscope System. Images were
merged and adjusted using ImageJ software.
58
PART III: RESULTS
3.1 Selection of Small Leucine Rich Proteins
I obtained previous microarray data from our group (from Dr Ben Mullin) on human
osteosarcoma cell line SAOS 2 measured the expression levels of the 17 SLRP family
members (Figure 3.1). Using this data the six SLRPs members LUM, OMD, EPYC,
BGN, DCN and TSKU were characterised as they showed the highest level of
expression with-in this cell line and therefore we hypothesised they may serve as robust
expression markers of osteogenesis.
Figure 3.1: The gene expression of the SLRP family members in the human
osteosarcoma cell line SAOS 2. The red line represents highly expressed SLRP genes
relative to an internal control for further investigation. Data was supplied by Dr Ben
Mullin.
1
10
100
1000
10000
Gen
e ex
pre
ssio
n
59
3.2 Optimisation of selected SLRP genes for qRT-PCR in ADSCs (Figure 3.2 and
Figure 3.3)
The gene expression of all 6 SLRPs was observed using 5 ng RNA for each PCR
reaction at both baseline and 28 days after osteogenic stimulated in ADSCs cultures
(Figure 3.2 and Figure 3.3). At 2.5 ng and 1 ng of RNA the gene expression of OMD
(the gene with the lowest expression) before osteogenic stimulation was not observed.
Based on this data, 5 ng of RNA was determined to be used for each individual PCR
reaction.
Figure 3.2: Representative gel image of ADSCs undergoing osteogenic stimulation
(lanes 1-3) and after 28 days of osteogenic stimulation (lanes 4-6) with 1 ng of RNA
(lanes 3 and 6), 2.5 ng of RNA (lane 2 and 5) and 5 ng of RNA (lanes 1 and 4).
Figure 3.3: Representative gel image of ADSCs before osteogenic stimulation (lanes 1-
2) and after 28 days of osteogenic stimulation (lanes 3-4) with 10 ng of RNA (lanes 1
and 3) and 5 ng of RNA (lanes 2 and 4).
Lane 1 2 3 4 5 6
OMD (150 bp)
18S (149 bp)
GAPDH (119 bp)
Lane 1 2 3 4
LUM (148 bp)
EPYC (148 bp)
DCN (87 bp)
TSKU (150 bp)
BGN (94 bp)
18S (149 bp)
GAPDH (119 bp)
60
3.3 Optimisation of selected SLRP genes for qRT-PCR in BMSCs (Figure 3.4 and
Figure 3.5)
The gene expression of the 6 selected SLRPs was observed using 5 ng RNA for each
PCR reaction at both time-points for unstimulated and osteogenic stimulated BMSC
cultures (Figure 3.4 and Figure 3.5). Similar to the ADSCs at 2.5 ng and 1 ng of RNA,
the gene expression of OMD before osteogenic stimulation was not observed.
Figure 3.4: Representative gel image of BMSCs undergoing osteogenic stimulation
(lanes 1-3) and after 28 days of osteogenic stimulation (lanes 4-6) with 1 ng of RNA
(lanes 3 and 6), 2.5 ng of RNA (lane 2 and 5) and 5 ng of RNA (lanes 1 and 4).
Figure 3.5: Representative gel image of BMSCs undergoing osteogenic stimulation
(lanes 1-2) and after 28 days of osteogenic stimulation (lanes 3-4) with 10 ng of RNA
(lanes 1 and 3) and 5 ng of RNA (lanes 2 and 4).
Lane 1 2 3 4 5 6
OMD (150 bp)
18S (149 bp)
GAPDH (119 bp)
Lane 1 2 3 4
LUM (148 bp)
EPYC (148 bp)
DCN (87 bp)
TSKU (150 bp)
BGN (94 bp)
18S (149 bp)
GAPDH (119 bp)
61
3.4 Patient characteristics
Bone marrow and adipose tissue samples collected from ten patients during surgery
were used throughout this study (Table 3.1). Donor matched ADSCs and BMSCs
derived from patients JAM207, IS223 and DM221 were analysed for the short-term
gene expression studies. Donor matched ADSCs and BMSCs patients MJ219, LS191,
MS209, VS216 and EJB200 were used for the long-term gene expression study.
Additionally donor matched MSCs samples of patients LS191, MS209, VS216, DM221
were also used to examine protein expression and subcellular localisation of OMD
while due to time constraints only ADSCs from patients, CE317, KD327 and DM221
were used to examine the multi-lineage gene expression of OMD.
Table 3.1: Demographic data for patients used in the gene and protein expression
studies
ADID Gender Age Reason for surgery
JAM207 F 69 Osteoarthritis left hip
IS223 F 79 Osteoarthritis left hip
DM221 F 48 Osteoarthritis left hip
MJ219 F 68 Osteoarthritis left hip
LS191 F 68 Fracture right neck of femur
MS209 F 83 Fracture right neck of femur
VS216 F 69 Osteoarthritis right hip
EJB200 F 77 Osteoarthritis right hip
CE317 F 38 Lap banding
KD327 F 43 Lap banding
62
3.5 Baseline gene expression of SLRPs in unstimulated ADSC and BMSC cultures
All the six SLRPs were highly expressed prior to osteogenic stimulation in both ADSCs
and BMSCs (Figure 3.6). In both tissue types, LUM had the highest levels of expression
(relative expression to GAPDH 0.96 ± 0.11 in BMSC and 1.04 ± 0.3 in ADSC) while
OMD had the lowest gene expression in both tissue types (relative expression to
GAPDH 0.68 ± 0.11 in BMSC and 0.66 ± 0.11 in ADSCs). Higher levels of DCN gene
expression were observed in ADSC (relative expression to GAPDH 0.99 ± 0.07) than
BMSC (relative expression to GAPDH 0.93 ± 0.08). The gene expression of BGN,
EPYC and TSKU did not vary between tissues.
Figure 3.6: The gene expression of the six SLRPs in unstimulated ADSC and BMSC
cultures represented as mean gene expression relative to GAPDH ± standard error of the
mean (SEM) (n=8).
3.6 The gene expression of SLRPs during osteogenesis of human MSCs
To investigate if gene expression of specific SLRPs was up-regulated during the early
and late development of bone, the gene expression of LUM, OMD, EPYC, BGN, DCN
and TSKU was analysed by qRT-PCR. qRT-PCR from samples collected at day 0, 3
and 7 day in 3 patients an at 0, 7 and 28 days of osteogenic stimulation of human donor
matched ADSC and BMSC in 5 patients.
0
0.2
0.4
0.6
0.8
1
1.2
LUM OMD EPYC BGN DCN TSKU
Gen
e ex
pre
ssio
n r
elati
ve
to G
AP
DH
ADSC
BMSC
63
3.6.1 Short-term gene expression of SLRPs in ADSCs (Figure 3.8)
All 6 of the SLRPs examined were expressed at all-time points of osteogenesis in
ADSCs similar to the data derived from the SAOS2 cell line (Figure 3.8). The gene
expression of LUM, TSKU, EPYC, DCN, and BGN did not significantly change
between control and osteogenic stimulated cultures at the same time points; however
OMD gene expression was significantly higher at day 3 and 7 in osteogenic stimulated
cultures compared to control media (P < 0.001 respectively).
Figure 3.8: The gene expression of SLRPs relative to GAPDH during early
osteogenesis of human donor matched ADSCs (n = 3). Unadjusted ANOVA was
performed comparing control against OSM cultures at the same time point. Data is
presented as mean gene expression relative to GAPDH ± SEM. *** represents P <
0.001.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
LUM OMD EPYC BGN DCN TSKU
Gen
e e
xp
ress
ion
rela
tive t
o
GA
PD
H
Day 0
Day 3 Control
Day 3 OSM
Day 7 Control
Day 7 OSM
*** ***
64
3.6.2 Long-term gene expression of SLRPs in ADSCs (Figure 3.9)
Similarly all 6 of the SLRPs examined were expressed at day 7 and day 28 of
osteogenesis in ADSCs (Figure 3.9). The gene expression of TSKU, EPYC and DCN,
did not significantly change between control and osteogenic stimulated cultures,
however both LUM, BGN were significantly down-regulated at day 28 in osteogenic
stimulated cultures (P < 0.05 respectively) whilst OMD gene expression was
significantly higher at day 7 and 28 in osteogenic stimulated cultures (P < 0.01 and P <
0.05 respectively).
Figure 3.9: The gene expression of SLRPs during late osteogenesis of donor matched
human ADSCs (n = 5). Unadjusted ANOVA was performed by comparing control
against OSM at the same time point. Data is presented as mean gene expression relative
to GAPDH ± SEM. * represents P < 0.05; ** represents P < 0.01.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
LUM OMD EPYC BGN DCN TSKU
Rel
ati
ve
exp
ress
ion
to
GA
PD
H Day 0
Day 7 Control
Day 7 OSM
Day 28 Control
Day 28 OSM
** *
*
*
65
3.6.3 Short-term gene expression of SLRPs in BMSC (Figure 3.10)
All six the SLRPs examined were expressed at both 7 and 28 days of osteogenesis in
BMSCs (Figure 3.10). The gene expression of LUM, TSKU, EPYC, DCN, and BGN
did not significantly change between control and osteogenic stimulated cultures at the
same time points. Similar to the observations in ADSCs, OMD gene expression was
significantly higher at day 7 in osteogenic stimulated cultures (P < 0.01).
Figure 3.10: The gene expression of six SLRPs during the early osteogenesis of human
donor matched human BMSC (n = 3). Unadjusted ANOVA was performed comparing
control against OSM at the same time point. Data is presented as mean gene expression
relative to GAPDH ± SEM. ** represents P < 0.01.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
LUM OMD EPYC BGN DCN TSKU
Rel
ati
ve
exp
ress
ion
to
GA
PD
H Day 0
Day 3 Control
Day 3 OSM
Day 7 Control
Day 7 OSM
**
66
3.6.4 Long-term gene expression of SLRPs in BMSCs (Figure 3.11)
All six of SLRPs examined were expressed at all-time points of osteogenesis in BMSCs
(Figure 3.11). Similar to the findings in ADSC, the gene expression of TSKU, EPYC,
DCN, and BGN did not significantly change between control and osteogenic stimulated
cultures at the same time points, however, LUM was significantly down regulated at
day 28 (P < 0.05) whilst OMD gene expression was significantly higher at day 7 and 28
in osteogenic stimulated cultures (P < 0.01 respectively).
Figure 3.11: The gene expression of SLRPs during the late osteogenesis of donor
matched human BMSC (n = 5). Unadjusted ANOVA performed comparing control
against OSM at the same time points. Data is presented as mean gene expression
relative to GAPDH ± SEM. * represents P < 0.05 and ** represents P < 0.01.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
LUM OMD EPYC BGN DCN TSKU
Rel
ati
ve
exp
ress
ion
to
GA
PD
H Day 0
Day 7 Control
Day 7 OSM
Day 28 Control
Day 28 OSM
** **
*
67
3.7 Comparison of SLRP gene expression between tissue types
Although broadly similar patterns of gene expression were noted in both ADSC and
BMSC, investigation revealed the gene expression of OMD compared to the
housekeeping gene GAPDH was significantly higher in BMSC cultures than ADSC
cultures (relative gene expression to GAPDH in BMSCs 0.84 ± 0.06; relative gene
expression to GAPDH in ADSCs 0.79 ± 0.02) (P<0.05) after 7 days of osteogenic
stimulation. Conversely when the relative gene expression of LUM was compared in
ADSC and BMSC, the expression in BMSC cultures was lower than in ADSC cultures
after 28 days of osteogenic stimulation (relative gene expression to GAPDH in ADSCs
0.94 ± 0.05; relative gene expression to GAPDH in BMSCs 0.84 ± 0.04) (P < 0.05).
68
3.8 Osteomodulin
3.8.1 OMD gene expression during multi-lineage differentiation of human ADSCs
(Figure 3.12)
OMD gene expression was significantly increased in osteogenic stimulated cells (P <
0.05) but not in chondrogenic (P = 0.109) or adipogenic (P = 1.000) stimulated cells
compared to cells treated with control media (Figure 3.12).
Figure 3.12: The gene expression of OMD after 28 days of osteogenic, adipogenic and
chondrogenic stimulation of human ADSCs (n = 3). A one way ANOVA was perfomed
comparing control media, OSM, ADM and CHM against each other using. Data is
presented as mean gene relative expression to GAPDH ± SEM. * Represents P < 0.05.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Control media Osteogenic
stimulation
Adipogenic
stimulation
Chondrogenic
stimulation
Rela
tiv
e e
xp
ress
ion
of
OM
D t
o
GA
PD
H
*
69
3.8.2 Protein expression of OMD during osteogenesis
Given the increased gene expression of OMD in cells undergoing osteogenesis I sought
to further investigate whether OMD protein expression was similarly up-regulated
during osteogenesis by western blotting of protein extracted from 3 patients at 0, 7 and
28 days of osteogenic stimulation of human donor matched ADSC and BMSC.
3.8.2.1 Long-term protein expression of SLRPs in ADSCs (Figure 3.13)
Figure 3.13 shows the western blot for OMD protein expression in ADSCs. OMD was
expressed at both time points after commencing exposure to osteogenic media. Three
bands were evident at 49, 65 and 89 kDa, in which the former corresponds to
unmodified OMD according to the manufacturer of the antibody. Densitometric analysis
of the 49 kDa band demonstrated that after 28 days of osteogenic stimulation (lane 5), a
more intense band was present compared to the band in control at the same time point
(lane 4), indicating higher OMD expression.
Figure 3.13: Protein expression of OMD in human ADSC from representative western
blot. Protein expression at baseline (lane 1), 7 days and 28 days after stimulation (lanes
2-3 and lanes 4-5 respectively) in control media (lanes 2 and 4) and OSM (lanes 3 and
5). Incubated with primary anti-OMD produced in rabbit (1:4,000) and secondary
antibody anti-rabbit immunoglobulin (whole molecule)-Peroxidase antibody produced
in goat (1:5,000). Values below image correspond to the density of the 49 kDa band in
relative units.
Lane 1 2 3 4 5
100 kDA
75 kDA
50 kDA
37 kDA
89 kDA
65 kDA
49 kDA
269 4278 2455 1653 3331
70
3.8.2.2 Long-term protein expression of SLRPs in BMSCs (Figure 3.14)
Figure 3.14 shows the western blot for OMD protein expression in BMSCs. OMD was
expressed to some extent baseline and at 7 and 28 days after commencing exposure to
osteogeneic media. Three bands were also evident at 89, 65 and 49 kDA. Densitometric
analysis of the 49 kDa band showed more intense bands in osteogenic stimulated
cultures at both day 7 (lane 3) and day 28 (lane 5) compared to their respective controls
(lane 2 and 4 respectively) indicating higher OMD protein expression in osteogenic
stimulated cultures.
Figure 3.14: Protein expression of OMD in human BMSC from representative western
blot. Protein expression at baseline (lane 1), 7 days and 28 days after stimulation (lanes
2-3 and lanes 4-5 respectively) in control media (lanes 2 and 4) and OSM (lanes 3 and
5). Incubated with primary anti-OMD produced in rabbit (1:4,000) and secondary
antibody anti-rabbit immunoglobulin (whole molecule)-Peroxidase antibody produced
in goat (1:5,000). Values below image correspond to the density of the 49 kDa band in
relative units.
100 kDA
75 kDA
50 kDA
37 kDA
89 kDa
65 kDa
49 kDa
Lane 1 2 3 4 5
2113 167 4188 1273 6338
71
3.9 Subcellular location of OMD during osteogenesis
3.9.1 Distribution of OMD within the ECM during osteogenesis of ADSCs (Figure
3.15)
The localisation of OMD during the osteogenic differentiation of human donor matched
ADSCs was analysed using confocal microscopy (Figure 3.15) using 3 stains for the
cell nucleus (column 1), OMD (column 2) and F-Actin (column 3). Similar to the
Western Blot results protein expression of OMD was higher in the osteogenic
stimulated cultures compared to control with intracellular localisation observed.
72
Figure 3.15: Confocal microscopy of human ADSCs in control media (Row A) and osteogenic stimulated media
(Row B) during early osteogenesis (day 7) at 40X magnification. White line represents 50 μm. Nuclei (row 1) were
stained with Hoechst 33342, OMD (row2) was stained with Anti-OMD produced in rabbit purified immunoglobulin
(1:2,000) and F-actin (row 3) was stained using an actin probe (1:300). Green arrow shows area of extracellular OMD.
For larger images of column 4, please refer to appendix.
A
B
1 2 3 4
Nuclei OMD F - Actin Merge
d
Nuclei OMD F - Actin Merge
d
Merge
d
73
3.9.2 Distribution of OMD within the ECM during osteogenesis of BMSCs (Figure
3.16 and 3.17)
The localisation of OMD during the osteogenic differentiation of human donor matched
BMSCs was analysed using confocal microscopy. Protein expression of OMD was
increased increase in the osteogenic stimulated cultures compared to control and
appeared to have both intracellular and extracellular localisation (Figure 3.16).
Previous data from our group also shows the distribution of OMD after 28 days of
osteogenic stimulation of BMSCs (Figure 3.17). The protein expression of OMD was
also shown to be more abundant and distributed compared to control.
74
Figure 3.16: Confocal microscopy of human BMSCs in control media (Row A) and osteogenic stimulated media
(Row B) during early osteogenesis (day 7) at 40X magnification. White line represents 50 μm. Nuclei (row 1) were
stained with Hoechst 33342, OMD (row 2) was stained with Anti-OMD produced in rabbit purified immunoglobulin
(1:2,000) and F-actin (row 3) was stained using an actin probe (1:300). Green arrow demonstrates an area of
extracellular OMD. For larger images of column 4, please refer to appendix.
A
B
1 2 3 4
Nuclei OMD F-Actin Merged
Nuclei OMD F-Actin Merged
75
A
B
1 2 3 4
Nuclei OMD F-Actin Merged
Nuclei OMD F-Actin Merged
Figure 3.17: Confocal microscopy of human BMSCs in control media (Row A) and osteogenic stimulated media
(Row B) during late osteogenesis (day 28) at 40X magnification performed by Ms Jenny Wang. White line represents
50 μm. Nuclei (row 1) were stained with Hoechst 33342, OMD (row 2) was stained with Anti-OMD produced in
rabbit purified immunoglobulin (1:2,000) and F-actin (row 3) was stained using an actin probe (1:300). Green arrow
shows area of extracellular OMD. For larger images of column 4, please refer to appendix.
76
PART IV: DISCUSSION
4.1 Principal findings
4.1.1 Expression of SLRP family members during osteogenesis
LUM has been shown to be involved in the assembly of collagen in the skin
(Chakravarti et al. 1998) and to be expressed highly in the ECM of adult articular
cartilage (Grover et al. 1995), however its role in bone remains unclear. Throughout the
short-term osteogenesis of ADSCs and BMSCs LUM was highly expressed without any
significant variation in both control and osteogenic stimulated conditions (Figure 3.8
and 3.10 respectively). After 28 days of osteogenic stimulation of both ADSCs and
BMSCs, the gene expression of LUM was significantly down-regulated compared to
control. These findings are in contrast to the gene expression of LUM observed in
mouse progenitor cells (Figure 1.10) (The Scripps Research Institute 2013), where the
gene expression of LUM was up-regulated after 21 days osteogenesis. Similar findings
in mouse calvaria cell lines have also shown the up-regulation of LUM during
osteogenesis and have suggested this SLRP as a marker to discriminate between pre-
osteoblasts in the proliferating stage of osteogenesis and mature osteoblasts (Raouf et al.
2002). These findings are supported by the increase in LUM gene expression in the
mature human osteoblast-like cell line SAOS2 compared to the osteoprogenitor-like cell
line MG-63 (Nikitovic et al. 2008). However similar to our findings, another study
found in a human MSC cell line, the gene expression of LUM was not altered after 10
days of osteogenic stimulation compared to control (Figure 1.9) (Hashimoto 2012),
supporting the concept that supporting the concept that in humans LUM may not be a
specific marker of osteogenesis using these models (Jilka 2013).
77
EPYC was consistently expressed at high levels during osteogenesis (Figure 3.8
– 3.11). Despite this no differences in the gene expression of EPYC was observed
between baseline and 28 days after osteogenic stimulation. To date, the gene expression
of EPYC has not been investigated in human osteoblasts, hence its role in bone remains
unclear, however our findings are surprising since an increase in the gene expression of
EPYC was observed in mouse cells between 5 and 21 days of osteogenesis (Refer to
Figure 1.10) (The Scripps Research Institute 2013). EPYC has been characterised to be
expressed in epiphyseal cartilage of developing chick limbs, particularly in the zone of
flattened chondrocytes (Shinomura and Kimata 1992) which becomes replaced by bone
during maturation of the skeleton (Johnson et al. 1999). EPYC also shares 49% of its
LRR with ostoeglycin, a SLRP which is known to be located in the ECM of developing
bone (Johnson et al. 1999). EPYC has been considered as a marker for intermediate
chondrogenesis and immuofluorescence histochemistry has shown to be found at sites
of the ECM near proliferating hypertrophic chondrocytes, suggesting it has a role in
cartilage maturation (Johnson et al. 1999). Our findings suggest that despite high levels
of gene expression EPYC does not appear to be specific for cells undergoing
osteogenesis.
78
There was no statistically significant variation in the gene expression of BGN
during early osteogenesis, however after 28 days of osteogenic stimulation in ADSCs,
the gene expression of BGN was significantly lower than control (P < 0.05). Although
BGN has been characterised in bone more extensively than other members of the SLRP
family, it is not up-regulated after osteogenic stimulation. Similar findings have been
reported by others that gene expression of BGN is not significantly increased when
human MSC cell lines are treated with osteogenic stimulation (Figure 1.9) (Hashimoto
2012). These findings are different to the mouse progenitor cell data available that
demonstrates higher BGN expression during late osteogenesis (Figure 1.10) (The
Scripps Research Institute 2013).
The consistently high expression of BGN throughout the early and late
osteogenic differentiation course of MSCs reflects the diverse role of the protein in
osteoblasts. In early osteogenesis, BGN is responsible for the sequestration of TGF-β to
ultimately prevent over-activation and subsequent apoptosis of osteoprogenitor cells (Bi
et al. 2005). Furthermore, BGN can stimulate pathways such as BMP (Chen et al.
2004), TGF-β (Bi et al. 2005) and Wnt/β-catenin (Inkson et al. 2008) signalling that
stimulates the transcription of osteoblast-related genes, ultimately leading to osteoblast
differentiation. During the intermediate to late phase of osteogenesis, BGN has a
structural role of organising collagen fibrils (Corsi et al. 2002). This has been
highlighted by BGN deficient mice where the abnormal collagen fibril shape and size
was observed compared to wild type mice in bone (Corsi et al. 2002).
In the late osteogenesis, BGN has also been shown to possibly play a role in
matrix mineralisation (Parisuthiman et al. 2005). In mouse cell derived clones which
have been modified to express higher levels of BGN, osteogenesis was significantly
79
accelerated and enhanced mineralisation of the matrix was observed (Parisuthiman et al.
2005). In the same study, mouse cell derived clones were also made to express lower
levels of BGN, which results in impaired mineralisation and suppressed osteogenic
differentiation (Parisuthiman et al. 2005).
Compared to the other SLRPs after 28 days of osteogenic stimulation the gene
expression of DCN was the highest. Like BGN, DCN has also been well characterised
as a SLRP involved in bone tissues, yet at all time-points the gene expression of DCN
was not increased in OSM compared to control in both ADSCs and BMSCs. This data
is similar to osteogenic time course experiments in mouse cells (Figure 1.10) (The
Scripps Research Institute 2013). In contrast to human ADSCs and BMSCs undergoing
osteogenesis, the gene expression of DCN is lower than the other SLRPs studied.
Similar to BGN, DCN is also involved in the assembly of collagen fibrils as shown by
the irregular size and shape of individual collagen fibrils in the skin of mice with a
disrupted DCN gene (Danielson et al. 1997). However in contrast to BGN, the collagen
organisation in bone was not affected in this model (Danielson et al. 1997). Although
the collagen fibres were not affected in this in vivo model, an irregular shape of collagen
fibrils was observed in mouse cell cloned to express higher levels of DCN (Mochida et
al. 2009). In this study a lower quality of mineralisation was observed, suggesting that
DCN regulates mineralisation of the matrix in late osteogenesis though organisation of
collagen (Mochida et al. 2009).
The gene expression of TSKU was highly expressed during both short- and
long-term osteogenesis in both ADSCs and BMSCs which is consistent with the
original data from the human SAOS2 cell line (Figure 3.1) and mouse cell data (Figure
1.10). The gene expression of TSKU did not change between control and osteogenic
stimulated cells. TSKU is one of least studied members of the SLRP family and has
80
only been reported as a BMP inhibitor during the embryogenesis (Morris et al. 2007;
Ohta et al. 2004) and a Wnt inhibitor in peripheral eye formation of chicks (Ohta 2011).
Since the gene expression of TSKU is high throughout the entire time-course of this
study, it is proposed that TSKU may have a role in osteoblast development through
interactions with BMPs in a similar way to other SLRPs such as BGN (Chen et al.
2004).
After 28 days of osteogenic differentiation of both ADSCs and BMSCs, the only
SLRP to show increased gene expression in osteogenic stimulated cells compared to
control in both early and late osteogenesis was OMD. OMD was also shown to increase
during the differentiation of mice cells (Figure 1.10) (Hashimoto 2012) and was one of
the most highly expressed SLRPs out of the 17 SLRP family members in the human
SAOS2 cell line (Figure 3.1). The gene expression of OMD has been shown to increase
after 10 days of osteogenic stimulation compared to control in human MSC cell lines
(Figure 1.9) (Hashimoto 2012), OMD over-expression studies of mouse cell lines have
shown an increase in osteoblast differentiation markers such as mineralisation, ALP
activity and up-regulation of osteocalcin while OMD repression models using the same
cell line showed decrease ALP activity (Rehn et al. 2008). Furthermore, OMD has also
been shown to have a high affinity for hydroxyapatite (Wendel et al. 1995) and has the
ability to bind osteoblasts through the intergrin αvβ3 (Jilka 2013). The data presented in
this thesis suggests that OMD has a role throughout early and late stages of
osteogenesis.
81
4.1.2 Comparison of SLRPs gene expression between the osteogenesis of ADSC and
BMSC
OMD gene expression was only significantly up-regulated during the
osteogenesis of ADSCs and BMSCs
No differences in the gene expression of the SLRPs; EPYC, BGN, DCN, and TSKU
were observed between during the short- and long-term osteogenesis of human donor
matched ADSCs or BMSCs. It was interesting to note that the gene expression of OMD
was significantly higher in BMSCs undergoing osteogenesis for 7 days compared to
ADSCs. A similar study showed that OMD was significantly increased in human
BMSCs compared to ADSCs after 14 days of osteogenic stimulation by about 70-fold
(Liu et al. 2007). This study also compared the early expression of OMD during 3 days
of osteogenic stimulation and although up-regulated in both ADSCs and BMSCs, there
was no significant increase of OMD expression was observed between tissue types (Liu
et al. 2007). A reason for this increased OMD expression in BMSCs compared to
ADSCs is that cultures of BMSCs could be dominated by chondrogenic and osteogenic
precursor cells, while ADSC culture are dominated by adipogenic progenitor cells (Liu
et al. 2007). LUM was significantly lower in cells undergoing osteogenic differentiation
in BMSCs compared to ADSCs after 28 days (Figure 3.11 and 3.9 respectively) which
may reflect tissue specific differences in the role of the protein.
82
4.1.3 The protein expression and subcellular localisation of OMD during
osteogenesis
All three isoforms of OMD were also observed in BMSCs (and to a lesser extent
in ADSCs) unlike other cells which only express a post-translational version of
the protein.
The protein expression of OMD was studied in the long-term osteogenesis of human
donor matched ADSCs and BMSCs after 7 and 28 days of osteogenic stimulation
(Figure 3.13 and 3.14 respectively). The strongest band at 49 kDa represents the core
protein of OMD prior to post-translational modifications, which when compared
between groups is shown to be higher at day 7 and day 28 in BMSCs and only higher
after 28 days osteogenic stimulation of ADSCs based on densitometry of the bands.
These comparisons appear to reflect the findings of the gene expression of OMD
observed in the long-term osteogenesis study (Figure 3.9 and 3.11).
The western blot images in Figures 3.13 and 3.14 also show two weaker bands
at 65 and 89 kDa. These bands are likely to represent a partially GAG substituted OMD
and a fully GAG substituted version of OMD respectively that have been identified in
other cell types. In bovine bone and dentin, similar observations have been made where
immunoblotting has showed two bands corresponding to a fully glycolsylated OMD and
the core protein absent of these post translational modifications (Petersson et al. 2003).
Similar protein expression studies have also found different pools of glycosylated OMD
in developing bone (Sugars et al. 2013). In ADSCs undergoing differentiation (Figure
3.13) the 89 kDa is not evident in lanes 1, 3, 4 and 5 however is evident in all lanes in
BMSCs samples (Figure 3.14) suggesting that different post translational modifications
of OMD may exist between osteoblasts derived from different sources.
83
Visualisation of the protein expression of OMD after 7 days of osteogenic
induction showed a similar pattern of expression to both the western blots and gene
expression. At day 7 in both ADSCs and BMSCs, there is an increase in red
fluorescence (OMD antibody binding) in the osteogenic stimulated cells compared to
control despite similar numbers of nuclei (blue fluorescence) (Figure 3.15 and 3.16
respectively). The ADSCs and BMSCs in control media both show low levels of
intracellular OMD compared to high levels of intracellular and extracellular OMD in
osteogenic stimulated conditions. Comparing the localisation of OMD in BMSCs
undergoing osteogenesis for 7 days (Figure 3.16) and 28 days (Figure 3.17) shows more
distributed OMD throughout the extracellular matrix in the later stages of osteogenesis.
4.1.4 Osteomodulin as a marker of osteogenesis
As described above, the gene and protein expression of OMD is significantly increased
during osteogenesis, and as such could be used as a robust marker for osteogenesis.
Comparison of the gene expression of OMD was investigated during 28 days of
osteogenic, chondrogenic and adipogenic stimulation of ADSCs (Figure 3.12).
Although OMD was significantly increased during osteogenesis, the gene expression of
OMD was also increased during chondrogenesis however this did not reach statistical
significance, possibly due to the small sample size and as such OMD may be a marker
of both osteogenesis and chondrogenesis however further experiments are needed to
confirm this. The results observed here are similar to those found in a previous study in
which ADSCs and BMSCs taken from different donors that were only stimulated for 3
days in osteogenic, adipogenic and chondrogenic stimulation and OMD gene expression
was monitored (Liu et al. 2007). Like the results observed in this study, OMD was
84
highly up-regulated during osteogenesis, however OMD was equally up-regulated
during chondrogenesis with minimal change in OMD gene expression in adipogenesis
(Liu et al. 2007).
In contrast, BMSC stimulated cultures in this study showed that OMD gene
expression increased more during adipogenesis than chondrogenesis, but this was not as
high compared to osteogenic stimulated cells (Liu et al. 2007). Taken together with the
results presented in this thesis, OMD seems like a robust marker for both early and late
osteogenesis of MSCs compared to other MSC lineages. Currently recognised markers
of early osteogenesis are the up-regulation of the transcription factor Cbfa-1 and the
expression alkaline phosphatase (Figure 1.7), while later markers of ostoegnesis include
up-regulation of the osteoblast-related genes osteocalcin and ostoenectin. Although
these markers have become the gold-standard of determining osteogenic differentiation
they are only highly during early osteogenesis (Frith and Genever 2008). These results
show that OMD gene expression is increased from the early stages of differentiation in
committed osteoprogenitor cells until the later stages of differentiation when the cells
have become matured and therefore offer a promising marker of osteogenisis during all
stages of differentiation of osteoblasts.
85
4.1.5 Advantages and disadvantages of this thesis
A unique strength in the study presented in this thesis compared to similar studies, was
the access to donor matched ADSCs and BMSCs. Variability between patients is a
troublesome issue in basic science and can negatively impact the consistency between
results. With donor matched ADSCs and BMSCs, the variability between results was
lowered since the ADSCs and BMSCs from the patients these cells were derived had
the exact same clinical phenotypes. Variability was still observed between patients,
however this could be resolved by performing the same analyses in more samples to
increase the power of the study.
4.1.6 Future directions and concluding statement
Due to technical issues in differentiating BMSCs for 28 days, the gene expression of
OMD was not able to be compared between the osteogenic, adipogenic and
chondrogenic MSC lineages. As observed in previous studies this is a difference
between the expression of OMD in different MSCs lineages from different tissues (Liu
et al. 2007). This study did not have access to donor matched ADSC and BMSC
samples, which may have explained the variations in the OMD gene expression
observed. Furthermore, analysis of the protein expression of OMD in the different MSC
lineages such as chondrocytes and adipocytes may show differences in post translational
modifications which may further our understanding of the OMD’s role. OMD null mice
would be a useful model to study to further understand the roles that OMD has in the
development of osteoblasts, and to explore redundancy between the other SLRPs and
OMD. This may also help understand if deficiency of OMD plays a role in the
pathogenesis of osteoporosis, like the class I SLRPs BGN and DCN (Xu et al. 1997).
86
In conclusion, this research has shown that SLRPs may be involved in the
development of bone as demonstrated by high levels of gene expression of the SLRPs
LUM, OMD, EPYC, BGN, DCN and TSKU throughout the early and late stages of
osteogenesis of both ADSCs and BMSCs. However, OMD was the only SLRP which
was up-regulated during osteogenesis, which was reinforced by protein and localisation
studies. Further analysis of OMD gene expression in other MCS lineages did not show a
significant up-regulation, indicating that OMD could be used as a robust marker of both
early and late MSCs. These findings offer important insights into the potential role
OMD may play in the pathogenesis of osteoporosis like other SLRPs. Ultimately, to
establish whether OMD may prevent or even ameliorate osteoporosis, further basic and
clinical trials are necessary.
87
PART VI: APPENDIX
Cell culture reagents and solution
10X PBS
NaCl 80.0 g
KCl 2.0 g
Na2HPO4 14.4 g
KH2PO4 2.4 g
ddH2O 1000 mL
Mix to dissolve, adjust pH to 7.4. Store this solution at room temperature. Dilute 1:10
with distilled water and autoclave before use.
Complete culture media
Dulbecco's Modified Eagle
Medium/Nutrient Mixture F-12
500 mL
Foetal bovine serum 55 mL
100X Penicillin/Streptomycin 5.5 mL
Day 28 OSM
Day 28 control
88
10X Trypsin
NaCl 10 g
KCl 0.5 g
NaHCO3 0.75 g
Glucose 1.25 g
EDTA 0.25 g
Trypsin 0.625 g
ddH2O 125 mL
Mix to dissolve. Filter through a 0.45 μm filter, store aliquots in -20°C freezer. Dilute
1:10 with ddH2O and filter through a 0.45 μm filter again before use.
Digest Solution
Hepes 2.38 g
D-Glucose 0.36 g
NaCl 2.81 g
KCl 1.49 g
CaCl2 0.044 g
ddH2O 400 mL
Mix to dissolve. Filter through a 0.45 μm filter, store 8 mL aliquots in 4°C fridge.
89
Red cell lysis buffer
NH4Cl 0.824 g
KHCO3 0.10 g
EDTA 0.003 g
ddH2O 100 mL
Mix to dissolve. Filter through a 0.45 μm filter, store 12 mL aliquots in 4°C fridge.
Collagenase II stock solution
Type II collagenase 0.75 g
ddH2O 25 mL
Filter through a 0.45 μm filter, store 1 mL aliquots in -20°C freezer.
1X Bovine Serum Albumin
Bovine Serum Albumin 3.0 g
PBS 25 mL
Filter through a 0.45 μm filter, store 1.5 mL aliquots in -20°C freezer.
90
Cryopreservation media (1 mL)
Osteogenic induction reagents and solutions
Stock beta-glycerol phosphate solution (1 M)
Β-glycerol phosphate 2.16 g
Complete media 10 mL
Filter through a 0.45 μm filter. Store aliquots in -20°C freezer. Add to osteogenic
induction media in a 1:100 ratio (10 mM).
L-Ascorbic acid stock solution of (10 mM)
L-Ascorbic Acid 0.017 g
ddH2O 1 mL
Filter through a 0.45 μm filter. Add to osteogenic induction media in a 1:2000 ratio (50
μM) for every media change.
Dexamethasone stock solution (10-5
M)
10-3
M Dexamethasone solution 450 μL
Complete Media 45 mL
Filter through a 0.45 μm filter. Store aliquots at -20°C freezer. Add to osteogenic media
in a 1:100 (10-7
M) or 1:1000 ratio (10-8
M).
Foetal bovine serum 900 μL
Dimethyl sulfoxide 100 μL
91
Chondrogenic induction reagents and solutions
Human insulin stock solution (5 μg/ml)
Human recombinant insulin 0.02 g
ddH2O 2 mL
Store at 4°C freezer. Add to chondrogenic media in a 1:1000 (5 ng/mL) ratio.
Human transform growth factor beta (TGF-β) stock solution (2.5 μg/mL)
TGF-β 5 μg
4mM Sterile HCl 2 mL
Aliquot to 100 μL per tube and store at -20°C freezer. Add into chondrogenic media in a
1:250 (10 ng/mL) ratio.
Adipogenic induction reagents and solution
Isobutylmethylxanthine (IBMX) stock solution (0.5 M)
Isobutylmethylxanthine 1.1 g
Dimethyl sulfoxide 10 mL
Heat it up to 55°C in water bath until dissolved. Cool down and store at -20°C freezer.
Add to adipogenic media in a 1:1000 (0.5 mM) ratio.
92
Indomethacin stock solution (50 mM)
Indomethacin 0.018 g
Dimethyl sulfoxide 10 mL
Heat it up to 55°C in water bath until dissolved. Cool down and store at -20°C freezer.
Add to adipogenic media in a 1:1000 (50 μM) ratio.
1X TAE Buffer
Cellular staining reagents and solution
PBST buffer
1X PBS 500 mL
Tween-20 2.5 mL
Mix well and store at room temperature.
4% Paraformaldehyde
PBS 100 mL
Paraformaldehyde 4 g
10M NaOH 10 μL
Dissolve paraformaldehyde with constant stirring under 60°C in the fume cupboard.
Keep stirring until it is cooled. Stored it in aliquots at -20°C. Heat it up to 37°C when
use.
50X TAE Buffer 10 mL
ddH2O 490 mL
93
Rat tail type I collagen coating solution
Rat tail type I collagen 60 μL
0.02 M acetic acid 40 mL
Mix well, filter it through 0.45 μm filter and keep it under 4°C.
Serum blocking buffer
Goat serum 500 μL
Bovine serum albumin 0.1 g
Triton-X-100 10 μL
Tween-20 5 μL
Sodium azide 0.005 g
PBS 10 mL
Make fresh each time store at 4°C until use.
Primary antibody dilution buffer
Bovine serum albumin 2 g
Triton-X-100 1 mL
Sodium azide 0.1 g
PBS 200 mL
Mix well and adjust pH value to 7.2 to 7.4. Store it at 4°C.
94
Secondary antibody dilution buffer
Tween-20 100 μL
PBS 200 mL
Mix well and store it at 4°C.
Western blotting reagents
10% Sodium dodecyl sulphate
SDS (Sodium dodecyl sulphate) 10 g
MilliQ ddH2O 100 mL
Slow heat to ~50°C to help in dissolving SDS
1.5 M Tris, pH = 8.8
Trizma base 90.9 g
MilliQ ddH2O 500 mL
Adjust pH to pH = 8.8 with concentrated HCl
1.0 M Tris, pH = 6.8
Trizma base 60.6 g
MilliQ ddH2O 500 mL
95
Adjust pH to pH = 6.8 with Conc. HCl
10X SDS-PAGE Running Buffer
Trizma base 30.2 g
Glycine 144.1 g
10% SDS 100 mL
MilliQ ddH2O 1 L
1X SDS-PAGE buffer
10X SDS-PAGE 200 mL
MilliQ ddH2O 1.8 L
2X SDS Gel Loading buffer
Tris-HCl, pH 6.8 3.1 mL
SDS 10 mL
Glycerol 5 mL
Bromophenol Blue 0.1 g
2- Mercaptoethanol 2.5 mL
MilliQ ddH2O 50 mL
96
1X Western Blot Transfer Buffer
Trizma base 6.06 g
Glycine 28.8 g
100% Methanol 200 mL
MilliQ ddH2O 2 L
10X TBS (Tris Buffered Saline) pH = 7.4
Trizma base 60.57 g
NaCl 87.66 g
MilliQ ddH2O 1 L
Adjust pH to pH = 7.4 with concentrated HCl
1X TBS
10X TBS 50 mL
MilliQ ddH2O 450 mL
1X TBS-Tween
10X TBS 50 mL
MilliQ ddH2O 450 mL
0.1% Tween-20 0.5 mL
97
Stripping Buffer
Tris-HCl, pH = 6.7 3.79 g
SDS 10 g
2- Mercaptoethanol 3.5 mL
MilliQ ddH2O 500 mL
98
Short term osteogenesis of ADSCs
ADSCs after 7 days in control
media.
White bar represents 50 μm.
(Figure 3.15, row A, column 4)
ADSCs after 7 days in OSM.
White bar represents 50 μm.
(Figure 3.15, row B, column 4).
Green arrows show areas of
extracellular OMD.
99
Short term osteogenesis of BMSCs
BMSCs after 7 days in control
media.
White bar represents 50 μm.
(Figure 3.16, row A, column 4).
BMSCs after 7 days in OSM.
White bar represents 50 μm.
(Figure 3.16, row B, column 4).
Green arrow shows areas of
extracellular OMD.
100
Long term osteogenesis of BMSCs
BMSCs after 28 days in OSM.
White bar represents 50 μm.
(Figure 3.17, row B, column 4).
Green arrows shows area of
extracellular OMD.
BMSCs after 28 days in control
media.
White bar represents 50 μm.
(Figure 3.17, row A, column 4)
101
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