Characterization of human adipose tissue derived adult multipotent precursor cells

i


CHARACTERIZATION OF HUMAN
ADIPOSE DERIVED ADULT
MULTIPOTENT PRECURSOR CELLS

















LEONG TAI WEI DAVID

NATIONAL UNIVERSITY OF SINGAPORE
2006



ii

CHARACTERIZATION OF HUMAN
ADIPOSE DERIVED ADULT
MULTIPOTENT PRECURSOR CELLS

LEONG TAI WEI DAVID
(Bachelor of Chemical Engineering, Hons)














A THESIS SUBMITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
DEPARTMENT OF BIOLOGICAL SCIENCES
NATIONAL UNIVERSITY OF SINGAPORE
2006

iii

PREFACE

This thesis is submitted for the degree of Doctorate of Philosophy in the

Department of Biological Sciences at the National University of Singapore. No
part of this thesis has been submitted for any other degree or equivalent to another
university or institution. All the work in this thesis is original unless references are
made to other works. Parts of this thesis had been published or presented in the
following:

International Refereed Journal Publications
Cover pages of some of the following papers are found in the appendix.

1. Leong TW, Chew FT, Hutmacher DW. Isolating bone marrow stem cells
using sieve technology. Stem Cells. 2004; 22:1123-1125.

2. *Ng KW, *Leong DT, Hutmacher DW. The challenge to measure cell
proliferation in three dimensions. Tissue Engineering. 2005; 11:182-191.

3. Leong DT, Hutmacher DW, Chew FT, Lim TC. Viability and adipogenic
potential of human adipose tissue processed cell population obtained from
pump-assisted and syringe-assisted liposuction. Journal of Dermatological
Science. J Dermatol Sci. 2005 37:169-176.

4. Leong DT, Khor WM, Chew FT, Lim TC, Hutmacher DW.
Characterization of osteogenically induced adipose tissue derived precursor
cells in 2-dimensional and 3-Dimensional environments. Cells Tissues
Organs. 2006; 182(1):1-11

5. Leong DT, Abraham MC, Rath SN, Lim T-C, Chew FT and Hutmacher
DW. Investigating the effects of preinduction on human adipose derived
precursor cells in an athymic rat model. Differentiation. In press.

iv

6. Leong DT et al. Absolute quantification of gene expression with real time
PCR. Biomaterials. 2007; 28(2):203-210.

7. *Gupta A, *Leong DT, Bai HF, SB Singh, Chew FT, Hutmacher DW. The
combined action of 1α, 25–dihydroxyvitamin D3, β–glycerophosphate and
ascorbic acid is essential for the osteo – maturation of adipose derived stem
cells. Manuscript under preparation.


8. Leong DT, Hutmacher DW, Chew FT. Genome wide gene expression
revealed activating transcription factor 5 (ATF5) to be responsible for
maintaining stemness in adipose derived stem cells. Manuscript under prep.
* contributed equally


Book Chapters
1. Hutmacher DW, Leong DT, Chen Fulin. Polysaccharides in Tissue
Engineering Applications, Handbook of Carbohydrate Engineering. In
press

Intellectual Competition
1. Finalist in Tan Kah Kee Young Inventors Awards 04. Making bone from
fat.


International and Local Conferences Presentations and Awards

1. Poster Presentation. Preliminary studies on human adipose derived stem

cells obtained from pump- assisted liposuction. 4th Sino-Singapore
Conference on Biotechnology, National University of Singapore,
Singapore, November 11-13th, 2003. Leong TW, Chew FT, Hutmacher
DW, Lim TC.

2. Oral Presentation. Preliminary studies on human adipose derived stem cells
obtained from pump- assisted liposuction. 2nd prize for 8th Biological
Sciences Graduate Congress, National University of Singapore,

v

Singapore, December 3-5th, 2003. Leong TW, Chew FT, Hutmacher DW,
Lim TC.

3. Oral Presentation. Osteogenesis of human adipose derived stem cells. The
Asian Surgical Association - 14th Biennial Congress & Scientific
Meeting, Kota Kinabalu, Malaysia. December 4-6 2003. Leong TW,
Chew FT, Hutmacher DW, Lim TC.

4. Poster Presentation: Viability of human adipose tissue stem cells from
pump- vs syringe-assisted liposuction. The Asian Surgical Association -
14th Biennial Congress & Scientific Meeting, Kota Kinabalu, Malaysia.
December 4-6 2003. Leong TW, Chew FT, Hutmacher DW, Lim TC.

5. Poster Presentation. The behavior of human adipose tissue derived
precursor cells seeded on polycaprolactone-tricalcium phosphate
scaffolds. Tissue Engineering Society International, 6
th
Annual
International Conference and Exposition, Orlando Florida, United States

of America. December 10-13, 2003. Leong TW, Khor WM, Chew FT,
Hutmacher DW.

6. Poster Presentation. Osteoprogenitor Cells induced from Adipose derived
Mesenchymal Cells. 8
th
National University of Singapore-National
University Hospital Annual Meeting, October 7-8, 2004. Lim TC, Leong
TW, Chew FT, Hutmacher DW.

8. Poster Presentation. In vivo characterization of adipose derived
osteoprogenitors within polycaprolactone-tricalcium phosphate scaffolds.
Regenerate 2005, June 1-3 2005, Atlanta Georgia. Leong TW, Lim TC,
Chew FT, Hutmacher DW.


vi

Acknowledgements
“Therefore, since we are surrounded by such a great cloud of witnesses, let us throw off
everything that hinders …, and let us run with perseverance the race marked out for us.”
Hebrews Chapter 12, verse 1, The Holy Bible.

This thesis and its corresponding research work was a race, a race that would have been
impossible for me to finish if not for the people that God had placed in my life. I would
like to thank them with my deepest gratitude.

Prof Chew Fook Tim, I will always remember how you believed in a stranger to the field
of biology. I hope that the many life lessons you have passed down to me had been and
would continue to be useful to me. Thank you for teaching me some skills that would

ensure that I survived in this competitive arena. Thank you for these many years of
patience and huge funding for my experiments. I hope that I would be as good as you, or
even better, in research work, as you have once so expressed. Thank you for making this
experience fondly memorable.

Prof Dietmar Hutmacher, you are a well-spring of innovative ideas. I hope that some of
your creativity had rubbed off on me. Thank you for looking out for me and nurturing me.
Thank you for your encouraging support to listen to my crazy ideas and actually funding
some of them. You have taught me so much over these four years. I know that I will be
able to give to my students in the future just as you have given to me. Thank you.

Prof Lim Thiam Chye, many thanks for providing us with the adipose tissues. Without
which, it would be impossible for me to work. Many thanks for giving your clinical inputs
in my project.

To my good friends in TE laboratory, Kee Woei, Mohan, Suman and Amy. Thanks for the
many hours of tea sessions discussing our ideas and aspirations. I will always treasure
those times we had. Kee Woei thank you for teaching me at the beginning of my thesis
work. It would not be as smooth if not for your help.

To my colleagues in TE laboratory, many thanks for your help over these four years.

Last but certainly not least, to the dearest people in my life,
my wife, Shirley Ting – you are my light at the end of the long tunnel. Thank you for your
long-suffering patience. I would never have made it without your constant encouragement.
Thank you love.

and my mom, Teo Siok Lew who never failed to support me in what I love to do. Thank
you for teaching me perseverance in my growing up years. Thank you mom.

Both of you share the degree with me. Thank you for your support, sacrifice, patience and
love during this period. I love you.

This thesis is dedicated to the memory of my late father, Leong Fook Wing.

Above all, to God be the glory.



David Leong
July 31, 2006

vii

Table of Contents

Page
Preface
iii
Acknowledgements
vi
Table of Contents
vii
Summary
x
List of Tables
xii
List of Figures
xiii

Chapter 1 – Introduction
1.1 Background 1-1
1.1.1 Clinical need requiring bone healing or regenerative
intervention
1-1
1.1.2 The use of stem cells in regeneration of bone 1-2


Chapter 2 – Literature Review
2.1 Description of the adipose tissue 2-1
2.2 Adipose tissue – a possible alternative to bone marrow as a source of
adult mesenchymal stem cells
2-3
2.3 Adult mesenchymal stem cells or precursor cell populations 2-4
2.3.1 ADSC characteristics 2-5
2.3.2 Cell Surface Characterization 2-6
2.3.3 General information on ADSC 2-7
2.3.4 Specific differentiation capacity of ADSC 2-8
2.4 Scaffolds based bone engineering 2-12
2.5 Other potential applications in regenerative medicine 2-16
2.6 Gene delivery vehicles 2-17

viii

Chapter 3 – Research Program

3.1 Overview 3-1
3.2 Four study phases 3-2
3.2.1 Phase I: Procurement of adipose tissue and their viability
(Chapter 5)
3-2
3.2.2 Phase II: Characterization of ADSC in culture plastic and
polycaprolactone environments (Chapter 6 and Chapter 7)
3-3
3.2.3 Phase III: Characterization of ADSC in in vivo environments
(Chapter 8)
3-3
3.2.4 Phase IV: Genome-wide transcriptome analysis of ADSC under
induction gave insights into differentiation behavior of ADSC.
(Chapter 9)
3-5

Chapter 4 – General Materials and Methods


Chapter 5 – Viability and adipogenic potential of human adipose
tissue derived stem cells isolated from tissue aspirated with a vacuum
pump or syringe

5.1 Chapter Abstract 5-1
5.2 Introduction 5-2
5.3 Specific Materials And Methods 5-4
5.4 Results 5-6
5.5 Discussion 5-12
5.6 Conclusions 5-15


Chapter 6 – The combined action of 1,25–dihydroxyvitamin D3, β–
glycerophosphate and ascorbic acid was essential for the osteogenic
induction of ADSC

6.1 Chapter Abstract 6-1
6.2 Introduction 6-3
6.3 Specific Materials And Methods 6-6
6.4 Results 6-7
6.5 Discussion 6-16
6.6 Conclusions 6-18







ix

Chapter 7 – Characterization of osteogenically induced adipose tissue
derived precursor cells in 2D and 3D environments

7.1 Chapter Abstract 7-1
7.2 Introduction 7-2
7.3 Specific Materials And Methods 7-4
7.4 Results 7-8
7.5 Discussion 7-17
7.6 Conclusion 7-23


Chapter 8 – Investigating the effects of preinduction on human
adipose derived precursor cells in an athymic rat model

8.1 Chapter Abstract 8-1
8.2 Introduction 8-2
8.3 Specific Materials And Methods 8-5
8.4 Results 8-9
8.5 Discussion 8-21
8.6 Conclusion 8-24


Chapter 9 – Genome wide analysis revealed ATF5 as potential
transcription factor for downstream expression of a stemness gene in
adipose derived stem cells

9.1 Chapter Abstract 9-1
9.2 Introduction 9-2
9.3 Specific Materials And Methods 9-7
9.4 Results And Discussion 9-13
9.5 Conclusion 9-29

Chapter 10 – Conclusions and Future Work

10.1 Summary Of Work 10-1
10.2 Further Work 10-5


Chapter 11 – References

Chapter 12 – Appendix


x

Summary of Thesis

This thesis was motivated by the hypothesis that there is a subpopulation of cells in
human adipose tissue which possesses multipotential differentiation properties for
applications in regenerative medicine applications.

A sub-population of cells termed by Zuk et al (2002) as “adipose derived stem
cells” (ADSC) adhered to tissue culture plastics and was able to proliferate well in
standard tissue culture conditions. Further characterizations were carried out on
several fronts, from their responses to osteogenic induction on tissue culture
conditions to 3-D environments in an athymic mouse model. The characterization
of ADSC for this thesis ended with the eludication of a potential stemness marker
based on robust global gene expression array analysis from an in-house depository
of donors’ adipose derived stem cells.

In this thesis, it was shown that ADSC isolated from adipose tissue aspirated with

the vacuum pump were viable for subsequent cellular studies. The osteogenic
differentiation was chosen as a proof of principle of the multipotential properties of
ADSC. It was shown that 1,25–dihydroxyvitamin D3 worked synergistically with
β-glycerophosphate and ascorbic acid to induce ADSC towards the osteogenic
lineage. ADSC were able to express bone matrix proteins and mineralization of its
matrix under such conditions. Progressing from culture plastics systems to 3-
dimensional studies using polycaprolactone-tricalcium phosphate scaffolds, ADSC
were still able to express bone characteristics when induced. Similar characteristics
of ADSC were confirmed in an in vivo model.


xi

After characterizing ADSC from the in vitro to in vivo systems, the study
progressed to the molecular level. Using microarray technologies, novel subgroups
of donors samples were discovered. Analyzing the downregulated genes and
looking for possible candidates of genes important to maintaining stemness,
activating transcription factor 5 (ATF5) was determined. Further validation using
real time reverse-transcription PCR showed a highly significant and consistent
downregulation of ATF5 in samples but no such downregulation of ATF5 in the
negative control cells samples. This placed ATF5 on the line-up as a candidate for
a gene important in maintaining stemness of ADSC or at least as a novel precursor
gene target involved in controlling osteo-differentiation of ADSC.

xii

List of Tables

Table 2.1
Differentiation potentials of ADSC

Table 4.1
Supplements in osteogenic and adipogenic induction cocktail

Table 6.1
Nomenclature of the various study groups in induction cocktail
study

Table 6.2
Comparison table between various groups and their
corresponding results

Table 7.1
Summary table listing details of R-phycoerythrin (R-PE) or
phycoerythrin-Cy5 (PE-cy5) conjugated primary antibodies used
for cell surface marker profiling.

Table 9.1
Primer sequences used for real time PCR.

Supplemental
Table 11.1
46 significantly expressed genes, comparing I
+
with I
-
with at
least p-value < 0.05.

xiii

List of Figures

Fig. 2.1
Microscopic image of the human adipose stroma.

Fig. 2.2
Illustration representing essential components of a tissue engineered
construct.

Fig. 5.1
Image showing the initial centrifugal separation of the different
layers of pump-aspirated adipose tissue in a 50-ml Falcon tube.

Fig. 5.2
Colonies of ADSC in a heterogeneous cell population.

Fig. 5.3
alamarBlue assay study indicating thatmetabolic activity of cell
population arising from both methods of aspiration

Fig. 5.4
Oil Red O staining for fat vacuoles present in adipogenically
induced ADSC

Fig. 5.5
Semi-quantitative measure of extent of Oil Red O Stain of
adipogenic induced cells over uninduced cells for the two methods

of aspiration

Fig. 6.1
Alizarin red S staining of ADSC cultures subjected to different
culture conditions

Fig. 6.2
Immunostaining of ADSC cultures for collagen type 1

Fig. 6.3
Immunostaining of ADSC cultures for osteonectin

Fig. 6.4
Immunostaining of ADSC cultures for osteopontin

Fig. 6.5
Immunostaining of ADSC cultures for osteocalcin

Fig. 7.1
Flow cytometric data: Grey histograms refer to isotypic controls.
Black histograms refer to various CD markers
Fig. 7.2
Light microscopy pictures of a heterogeneous cell population and
positive osteonectin staining

Fig. 7.3
Bridging cells and cell sheets formation in scaffolds

Fig. 7.4
Graphs showing DNA content, alamarBlue reduction and alkaline

phosphatase concentration changes over time

Fig. 7.5
Summary of RT-PCR

Fig. 8.1
Flow chart illustrating the experimental process for the scaffolds;
broadly divided into the in vitro 3D and in vivo 3D phases


xiv

Fig. 8.2
Microscope pictures of ADSC in culture

Fig. 8.3
Histology and immunohistochemical images of cells on culture
plastics and induced for 28 days with osteogenic media

Fig. 8.4
Scanning electron microscope images

Fig. 8.5
Histology and immunostaining images

Fig. 8.6:
Semi-quantitative stain counting data for Alizarin Red S and
Masson Trichrome stains

Fig. 8.7

Semi-quantitative stain counting data for Collagen Type I,
Osteopontin and Osteonectin staining

Fig. 8.8
Mechanical testing results

Fig. 9.1
Flow diagram describing the procedure underwent in analysing the
Genechip data

Fig. 9.2
Normalized data presented in a box plot for 54,675 genes of each
sample

Fig 9.3
Condition tree of groups based on significantly expressed genes

Fig. 9.4
Alizarin Red S staining for mineralization

Fig. 9.5
Immunostaining of various osteo-related proteins

Fig. 9.6
Graph summarizing gene expression of Ataxin 1, Muscleblind-like
1, EH1 domain and eukaryotic translation initiation factor 4E
binding protein 1

Fig. 9.7
Graphs summarizing normalized ATF5 mRNA data from

microarray analysis for each sample

Chapter 1 Introduction
1 – 1
CHAPTER 1
1 INTRODUCTION
1.1 Background
This section provides background information on the following two topics:
• The current clinical need shrouding bone repair and regeneration
• Adipose tissue as a potential depository of stem cells to answer this need

1.1.1 Clinical need requiring bone healing or regenerative intervention
The Healthcare Cost & Utilization Project stated that 12,700 craniotomies and
craniectomies were performed in 2001 and 20,616 procedures to correct facial
trauma defects. The total costs of these surgical procedures were estimated to be
US$949 million (Panagiotis M., 2005). These statistics however do not include
those from orthopedic fractures and bone tumor osteoectomies and excision. In
such cases, the in situ and surrounding bone could not provide good structural,
cosmetic and protective functions. It then becomes clear that the ability to repair
bone defects can help in the restoration of function to the affected area.

One possible treatment for non-unions of bone fractures is using autologous bone
grafts. Autologous bone grafts enhance osseous union by contributing
osteoconductive and possibly osteoinductive materials to the fracture site and
remains a common graft material (Panagiotis M., 2005). Often, bone grafting is
employed in patients’ bone defects where existing internal or external fixation was
ineffective and must be replaced. Vascularized bone autografts, had been used to
enhance the biologic environment and fill bone defects. Unfortunately, a finite
amount of bone graft was available from each individual and donor site morbidity
Chapter 1 Introduction

1 – 2
could be up to 30% (Younger EM 1989). This limitation was further aggravated in
post excised bone tumor cases where the amount of available autologous bone
would not be enough to fill large bone defects. Therefore there is a need to look for
alternative strategies for bone.

1.1.2 The use of adult stem cells in regeneration of bone
Stem cells showed much promise in curing diseases like diabetes, Parkinson’s
disease, neurological degeneration, and congenital heart disease. The existence of
stem cells is a matter of public discussion where religious, ethical, political, and
economic implications interest coincide. Concerns plaguing the field are
theoretical and religious, such as defining when human life begins, which reflect
beliefs and philosophies. These concerns and the technical problem of customizing
donor stem cells which would not elicit an immunological response from the host,
remained unsolved. Much of these impediments had made the progress of
embryonic stem cells applications from the bench to the bedside, difficult. These
concerns therefore had motivated more intensive research into characterization of
other potential sources of stem cells which might avoid these issues altogether.

1.1.2.1 Adult stem cells
Stem cells are loosely defined as self-renewing progenitor cells that can generate
one or more specialized cell type.

The edge that adult stem cells have over their embryonic counterparts in that the
ethical and immuno-incompatibility issues are sidestepped since the use of adult
stem cells can be autologous. With some of the spotlight shifted instead to adult
Chapter 1 Introduction
1 – 3
stem cells in recent years, especially the multipotent bone marrow mescenchymal
stem cells (BMSC) (Gundle R et al., 1995; Haynesworth SE et al., 1996). Several

elegant studies have documented the ability of BMSC to differentiate into non-
hematopoietic cell types, including brain (Brazelton TR et al., 2000; Zhao LR et al.,
2002), skeletal muscle (Ferrari G et al., 1998; Gussoni E et al., 1999), liver and
epithelium (Petersen BE, et al., 1999; Theise ND et al., 2000), heart (Tomita S et
al., 1999; Orlic D et al. 2001, Kucia M et al., 2004) and bone (Prockop DJ 1997).
There are clinical applications of stem cells which include the reconstitution of
blood lineage cells (Lagasse E et al., 2001, Morrison SJ et al.1994, Weissman IL et
al., 2000a) using hematopoietic stem cells, and bone reconstruction (Petite H et al.
2000, Krebsbach PH et al., 1998, Quarto R et al., 2001).

Specifically in repairing and regenerating bone, the osteogenic capacity of bone
marrow has been demonstrated (Bruder SP et al., 1994). Bone marrow aspirated
from the iliac crest contains progenitor cells that can be used to augment the
osteogenic response of the implanted allografts or to heal a non-union when
injected percutaneously into the non-union fracture.

Though BMSC is well characterized, the amount of bone marrow that can be
safely procured from the patient is limited. In addition, it is estimated that the
concentration of stem cell is in the range of 0.001 to 0.01% of the total population
(Pittenger MF et al., 1999). This meant that extensive culture is necessary to
achieve therapeutic relevant cell numbers. In addition, the effects of long term
culture on the differentiation potential of these cells remained largely unknown.
Therefore there is a need to find an alternative source of adult stem cells.
Chapter 1 Introduction
1 – 4
1.1.2.2 Adipose tissue as a potential depot of stem cells
Studies have shown the differentiation of ADSC along mesodermal lineages
(osteogenic, chondrogenic, adipogenic and myogenic lineages) (Mizuno H et al.,
2002; Zuk PA et al., 2002).

Phenotypically, ADSC under specific induction conditions, expressed osteogenic
genes like osteonectin, osteopontin, osteocalcin, alkaline phosphatase (Zuk PA et
al., 2002) and chondrogenic markers like collagen II, chondroitin-4-sulphate and
keratan sulphate, collagen type X and aggrecan (Huang JI et al., 2004; Ogawa R et
al., 2004)

and histological resemblance of hyaline cartilage (Dragoo JL et al.,
2003a).

ADSC seeded on apatite coated poly-lactic-co-glycolic acid scaffolds were used to
repair critical size murine calvarial defects which otherwise would be impossible
for the calvarium to heal itself (Cowan CM et al., 2004). There was also one
reported clinical case where iliac crest bone was mixed with isolated ADSC in an
autologous fibrin glue delivery system to repair a critical sized calvarial defect in a
7 year old girl (Lendeckel S et al., 2004). However, in the above two cases, the
isolated in vivo osteogenic properties of ADSC was not confirmed because the
chosen site of ADSC implant was in a bone region. It is possible that ADSC
merely assisted in bone healing rather than a direct differentiation from ADSC to
osteoblasts in vivo.

Chapter 1 Introduction
1 – 5
ADSC when induced towards the adipogenic lineage also expressed adipogenic
genes like leptin, peroxisome-proliferating activated receptor γ, aP2 and
lipoprotein lipase (Zuk PA et al., 2002; Bernlohr DA

et al., 1985).

ADSC cells also expressed neuron-specific proteins, neuronal nuclei protein and
neuron specific enolase. Late markers neuron-specific enolase and neurofilament

were expressed by 60-85% of the cell population (Yang L et al., 2003).

With these phenotypic evidences of specific tissue commitment, ADSC might
make a good candidate for stem cell intervention in reconstruction of bone,
cartilage and adipose tissue and even neuropathlogic diseases.


Raising hope for genetic diseases, genetically engineered autologous stem cells
might be used to replace dysfunctional cells. Genetic bone diseases like
osteogenesis imperfecta and fibrous dysplasia of bone may be improved with the
implantation of engineered stem cells at the site of clinical trauma caused by the
diseases (Bianco P, Robey PG. 2001). Bone morphogenetic protein 2 transfected
ADSC have increased bone precursors with a faster onset of calcified extracellular
matrix than transfected bone marrow derived stem cells (Dragoo JL

et al., 2003b).
since usually transfected with retroviral delivery systems, ADSC was able to
maintain transgenic expression even after differentiation (Morizono K

et al., 2003).

Chapter 2 Literature Review

2– 1
CHAPTER 2
LITERATURE REVIEW
2.1 Description of the adipose tissue
Adipose tissue consists of adipocytes embedded in a vascular loose connective
tissue, which is divided into lobules by stronger fibrous septa carrying the larger
blood vessels, in order that each lobule receives an independent blood supply.

Within the lobules the cells are round or polygonal (Fig. 2.1). Fat deposits serve as
energy stores, sources of metabolic lipids, thermal insulation (subcutaneous fat),
mechanical shock-absorbers (soles of feet, palms of hands, gluteal fat, synovial
membrances). It occurs in abundance in subcutaneous tissue, around the kidneys,
in the mesenteries and omenta, in the female breast, in the orbit behind the eyeball,
in the marrow of bones to the plantar skin of the foot, and as localized pads in the
synovial membrane of many joints.

The adipose tissue is capable of considerable changes in volume during the course
of life of mammals. Although relatively small increases in volume can be
accommodated by changes in the amount of lipid stored in hypertrophic
adipocytes, larger changes are mediated by the hyperplasic growth of new
adipocytes accompanied by coordinated remodeling of the adipose vasculature
(Rupnick MA et al., 2002; Hausman DB et al., 2001). It can be thought that the
dynamism is mediated by resident stem cells. These stem cells can be
enzymatically isolated from the stroma of the adipose tissue and separated from the
buoyant lipid filled adipocytes (Fig. 2.1) by centrifugation. A more homogeneous
population emerges in culture under conditions supportive of MSC growth.

Chapter 2 Literature Review

2– 2
The most important features of adipose tissue as a cell source might be the easy
expendability of its MSC and the consequent ease of procurement of large
quantities with minimal risk to the patient. Liposuction is a common surgical
procedure with 478 251 elective liposuction surgeries performed in the USA
during 2004 (American Society for Aesthetic Plastic Surgery 2005). It is also safe:
an American Society for Dermatologic Surgery study of outpatient cosmetic
liposuction performed between 1994 and 2000 showed zero deaths on 66 570
procedures and a serious adverse event rate of 0.68 per 1000 cases (Housman TS et

al., 2002).











Fig. 2.1: Microscopic picture of cells within the stroma of adipose tissue. Adipocytes were filled
with lipids and adopted globular or polygonal morphologies. Stem cells lie within this stroma.
Chapter 2 Literature Review

2– 3
2.2 Adipose tissue – a possible alternative to bone marrow as a source of adult
mesenchymal stem cells
Using bone marrow mescenchymal stem cell (BMSC) is one of the most promising
strategies in regenerative medicine applications. However, the actual volume of
bone marrow that can be procured from the patient is limited. Therefore, another
potential source of stem cells that could be procured in large amounts and had a
reasonably high concentration of stem cells is adipose tissue. These multipotent
stem cells were capable of differentiating into the adipogenic, chondrogenic and
osteogenic lineages when subjected to appropriate induction stimulants (Zuk PA et
al., 2002). Donor site morbidity also limited the amount of marrow that can be
obtained and thereby extended the time in culture required to generate a
therapeutic cell dose. Thus, the volume of human marrow taken under local
anaesthesia is generally limited to no more than 40ml and yielded approximately

2×10
9
nucleated cells (Bacigalupo A et al., 1992). Obtaining a larger volume of
bone marrow would necessitate the use of general anaesthesia, increase donor site
morbidity (Auquier P et al., 1995; Nishimori M et al., 2002) and further dilute the
stem cell fraction with stem cell-free blood ((Bacigalupo A et al., 1992). By
contrast, a typical harvest of adipose tissue, under local anaesthesia, could easily
exceed 1,000 ml and yield at 2×10
8
nucleated cells per 100 ml of lipoaspirate (Aust
L et al., 2004).

There were attempts to enrich the “true” stem cell population from amongst a
heterogeneous adult stem cell population. Friedenstein et al. were the first to
identify in the adult bone marrow a cell population with strong osteogenic potential
(Friedenstein AJ 1995). They purified a subpopulation of BMSC from the rest of
Chapter 2 Literature Review

2– 4
the stromal cells of the bone marrow by the BMSC adherence to culture plastics.
Subsequent studies by other groups on these adherent cells showed that these cells
were able to differentiate into the mesenchymal lineages. Even within the adherent
cell population, it was still heterogeneous in cell types. Therefore more
sophisticated methods like cell sorting were utilized to purify the true BMSC from
amongst this adherent cell population. Gronthos S et al showed that cells with
surface expression of STRO-1 and CD106 had clonogenic and differentiation
potential to osteoblastic, adipogenic and chondrogenic lineage (Gronthos S et al.,
2003). Another novel method was purification based on cellular size (Hung SC et
al., 2002). This proposed method of “sieving” out cells was an interesting idea but
the results were inconclusive (Leong TW et al., 2005). Nonetheless, the question of

whether cells should be purified from their niche cells remained unanswered.

2.3 Adult mesenchymal stem cells or precursor cell populations
Caplan was among the first who introduced the term “mesenchymal stem cells”
widely into the scientific community (Caplan AI 1994). Jiang Y et al (2002)
showed at the single cell level, that 0.1% of CD45
-
TER119
-
murine bone marrow
monocular cells differentiated not only into mesenchymal cells, but also into cells
with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro.
However, that work was based on the murine model and has yet to be proven to be
correspondingly true in humans. There is a possibility of a difference within the
human and mouse stem cell systems, as seen in the ability of murine leukaemia
inhibitory factor (LIF) in maintaining stemness in mouse embryonic stem cells but
human LIF did not have similar maintenance effect (Sato N et al., 2004).

Chapter 2 Literature Review

2– 5
Therefore, it could be argued that in reality there was no true MSC in human
marrow. The multipotential phenotype observed was due to various lineage-
specific precursor populations, creating an illusion that the bone marrow cell
population possessed multipotent phenotypes.

Zuk et al (2002) working with human adipose derived stromal cells showed with
clonal studies that there existed a subpopulation of cells capable of differentiating
into at least three lineages. Guilak et al (2006) also reported similar observations in
a recent clonal study. However, there was a lack of conclusive function evidences

using cloned ADSC to confirm in vitro observations. Therefore whether MSC
really exist in adipose tissue currently remained controversial.

Not hampered by the controversy, this thesis used precursor and stem cells terms
interchangeably.

2.3.1 ADSC characteristics
A general misconception associated with ADSC is that the donor must be
overweight or obese to have sufficient adipose tissue available for harvest.
Comparing adipose tissue harvested from obese BMI patients with that from
normal BMI patients however, did not show any significant increased amounts of
stem cells (Morizono K et al., 2003). In addition, there were no significant
differences between ADSCs from patients with and without type II diabetes in
terms of their mesenchymal stem cell characteristics and osteogenic and
adipogenic potential (Strem BM and Hedrick M, 2005). Besides, assays performed
Chapter 2 Literature Review

2– 6
on the factors secreted by ADSC also revealed the presence of multiple angiogenic
and anti-apoptotic cytokines (Rehman J et al., 2004).

2.3.2 Cell Surface Characterization
The cell surface phenotype of human ADSC was quite similar to BMSC (Strem
BM et al., 2005a). Both stem cell types were positive for CD105, STRO-1 and
CD166. CD117 (the stem cell factor receptor), also expressed by embryonic stem
cells, hematopoietic stem cells, MSCs and ADSCs (Aye MT et al., 1992; Lemoli
RM et al., 1993; Ogawa M et al., 1993). In addition to these multipotent markers,
ADSCs and MSCs, both expressed numerous other molecules including CD29
(beta-1 integrin, which plays a critical role in therapeutic angiogenesis (Li TS et
al., 2005), CD44 (a hyaluronate receptor) and CD49e (important for cell adhesion

to fibronectin). ADSCs also expressed high levels of CD54 (ICAM-1) when
compared with BM-MSCs (De Ugarte DA et al., 2003a). ICAM-1 is a member of
the immunoglobulin supergene family and can be up-regulated in response to
numerous inflammatory mediators and cytokines (Roebuck KA and Finnegan A
1999). ADSC did not express the HLA-DR protein and the majority express MHC
Class I molecules (Aust L et al., 2004) suggesting their potential for allogeneic
transplantation (Lee RH et al., 2004). One difference in the surface marker
expression appears to be the reciprocal expression of VLA-4 (CD49d/CD29) and
its cognate receptor VCAM-1 (CD106). It was observed that there was expression
of VLA-4 but not VCAM-1 by ADSC from the majority of donors (Strem BM et
al., 2005a) but the trend is reversed in BMSC (De Ugarte DA et al., 2003a). Since
these molecules were involved in hematopoietic stem and progenitor cell homing
to and mobilization from the bone marrow (Simmons PJ et al., 1992; Sudhoff T et