Fiona Anastasia van Vollenstee

209 Pages·2015·10.08 MB·English

University of Pretoria
Faculty of Health Sciences
School of Medicine

Isolation and characterization of human adipose
derived mesenchymal stem cells and production
of GFP-labeled primary cells for in vivo tracking
following transplantation

Masters Degree in Immunology

Fiona Anastasia van Vollenstee

21104370

Department of Immunology and Institute for Cellular and Molecular Medicine

Supervisor: Prof MS Pepper
[email protected]

Co-Supervisor: Dr M Potgieter
[email protected]

Author Contact details
Physical: 1211 Dr Keyser str
Queenswood X1
Postal: PO Box 13570
Sinoville
0129
Email: [email protected]
Mobile: [phone]
Abstract

Abstract to dissertation
Hell is empty and all devils are here!
Shakespeare W, The Tempest (1610-1611), Act I, Scene 2, line 215.

Introduction
It is well known that resident adipose stem/stromal cells (ASCs) are a heterogeneous
population of multipotent cells characterized by (a) their ability to adhere to plastic; (b)
immunophenotypic expression of certain cell surface markers, while lacking others; and (c)
the capacity to differentiate into cells of mesodermal origin including osteocytes,
chondrocytes and adipocytes. Adipose derived stromal cells offer great therapeutic potential
in multiple medical fields, including, orthopedics, cardiology, oncology and degenerative
diseases, to name a few. Combining different disciplines of medicine and engineering, organ
and tissue repair can be achieved through tissue engineering and regenerative medicine.
Adipose derived stromal cells (ASCs) can be utilized as biological vehicles for vector-based
gene delivery systems, since they home to sites of inflammation and infection in vivo. In order
to reach the long-term aim of clinical translation of cell-based therapy, preclinical safety and
efficacy need to be shown in animal models. This has motivated the development of
standardized isolation, characterization and differentiation operating procedures as well as
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Abstract
Fiona Anastasia van Vollenstee

an in vivo tracking system for ASCs and lentiviral vector transduction for a vector-based gene
delivery system.
Methodology
Human ASCs were isolated from lipoaspirate, expanded in culture, immunophenotyped
using flow cytometery and induced to differentiate into adipogenic, osteogenic and
chondrogenic lineages. Tri-lineage differentiation was confirmed by microscopy. The ASCs
were then transduced with green fluorescent protein (GFP)-expressing lentiviral vectors in
vitro. The effect of the GFP lentiviral vector on ASCs was investigated by studying ASC
immunophenotypic expression of surface markers as well as their capacity to differentiate
into osteocytes, chondrocytes and adipocytes.
Results
The isolated and expanded cell population, from harvested lipoaspirate adhered to
recommended ASC identity criteria. The heterogeneity of ASCs was confirmed by the
presence of sub-populations. Transduction efficiency in ASC cultures of approximately 80%
was observed after introducing a total of 300 µl of concentrated lentiviral vector suspension
per 4.8 x 104 cells. No immunophenotypic differences were observed between GFP positive
and GFP negative cultures. Flow cytometric analysis revealed a progressive increase in GFP
expression following in vitro expansion of transduced ASCs. Both non-transduced and
transduced cultures successfully differentiated into osteocytes, chondrocytes and
adipocytes.
Conclusion
The isolated and expanded cell population conformed to the recommended characterization
criteria. Heterogeneity was demonstrated with the identification of immunophenotypic sub-
populations and semi-quantification of adipogenesis was performed. ASCs were efficiently
transduced using the GFP lentiviral vectors produced in our facility. In addition, transduced
ASCs maintained adherence to plastic, ASC immunophenotype and were able to differentiate
successfully into cells of the three lineages of mesodermal origin. This optimized GFP-ASC
transduction technique offers a feasible tracking system as well as a vector-based gene
delivery system for future preclinical studies.

Key words: mesenchymal stem cells, adipose derived stromal cells (ASCs), isolation,
expansion, characterization, ASC immunophenotype, differentiation, green fluorescent
protein (GFP), lipoaspirate
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Acknowledgements

Extending my appreciation to mentors and colleagues giving me the opportunity, guidance
and assistance to complete this study.
This was the noblest Roman of them all
Shakespeare W, Julius Caesar (1599), Act V, Scene 5, line 68.

 Prof Michael Sean Pepper (Supervisor)
 Prof Riana Cockeran (Head of Immunology Department) and the Department of
Immunology
 Dr Marnie Potgieter (Co-supervisor)
 Dr Danie Hoffmann (Collaborator, Plastic and Reconstruction Surgeon)
 Miss Karlien Kallmeyer (Colleague)
 Mr Carlo Jackson (Colleague)
 Dr Chrisna Durant (Colleague)
 Dr Eddie Silberbouer (Assistance with graphics)
 Dr AW Dreyer (Husband: Manuscript editing as well as all his love and support)
 Lucia van Vollenstee (Mommy: For motherly wisdom and support)

Dear Lord thank you for using me as your tool to reveal knowledge to mankind. Humbly I know that I
could not achieve anything if You do not will it so. I thank You.
Amen
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List of Abbreviations

A list containing all the abbreviations used throughout this dissertation.

Where words are scarce, they are seldom spent in vain,
Shakespeare W, King Richard II (1592), Act 2, Scene 1, line 7.

% percent/percentage
˚C degrees Celsius
˚C/min degrees Celsius per minute
< less than
> more than
≤ less than and equal to
µg/ml micrograms per millilitre
10x ten times magnification
293T cells human embryonic kidney 293 cell/ HEK 293 cells
ADAS adipose derived adult stem
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List of Abbreviations
Fiona Anastasia van Vollenstee

AD-SVF adipose derived stromal vascular fraction
ANOVA analysis of variance
ASAPS American Society for Aesthetic Plastic Surgery
ASCs adipose derived stem cells/adipose derived stromal cells
AT-CFU-F adipose derived fibroblastic like colonies
BM bone marrow
BMI body mass index
BMSC bone marrow stromal cell
BSA bovine serum albumin
BSC-II type II biosafety cabinet
Ca + Calcium ions
2
CaCl calcium chloride
2
CAL calibration factor
Cat# catalogue number
cc cubic centimetre
CCR5 C-C chemokine receptor type 5
CD105 cluster of differentiation 105
CD34 cluster of differentiation 34
CD4 cluster of differentiation 4
CD45 cluster of differentiation 45
CD73 cluster of differentiation 73
CD90 cluster of differentiation 90
cDNA complementary deoxyribonucleic acid
cells/cm2 cells per square centimetre/density

cells/ml cell per millilitres
CFU-F fibroblastic like colonies
CFU-Fs colony-forming unit fibroblasts
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List of Abbreviations
Fiona Anastasia van Vollenstee

GMP current good manufacturing practices
cm centimetre
cm2 centimetre squared
CO carbon dioxide
2
CP cryopreserved cultures
CXCR4 C-X-C chemokine receptor type 4
DAPI 4’,6-Diamidino-2-phenylindole
ddH O doubled distilled water
2
dH O distilled water
2
DH5α cloning strain of E. Coli bacteria
DMEM Dulbecco’s Modified Eagle Medium
DMSO dimethyl sulfoxide
DNA Deoxyribonucleic acid
e.g. example
ECD electron coupled dye
EDTA ethylene diamine tetraacetic acid
et al. and others
EU European Union
FBS fetal bovine serum
FDA United States Food and Drug Administration
FFA free fatty acids
FGF-2 fibroblast growth factor 2
FITC fluorescein isothiocyanate
FS lin forward scatter linear
F-value random variable with an f distribution
g gram
g gravitational force/ centrifugal force
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List of Abbreviations
Fiona Anastasia van Vollenstee

G quiescent state
0
G committed to advance to S phase within the cell cycle
1b
GFP- green fluorescent protein negative (lacking expression)
GFP green fluorescent protein
GFP+ green fluorescent protein positive (expression)
Gp120 envelope glycoprotein
GVHD graft versus host disease
HBS HANKS Ca2+Mg2+ solution
HBV hepatitis B virus
HCl hydrochloric acid
HCV hepatitis C virus
HIV human immunodeficiency virus
HIV-1 human immunodeficiency virus type 1
HIV-2 human immunodeficiency virus type 2
HLA human leukocyte antigen
HLA-DR class II human leukocyte antigen
hrs hours
HSCs haematopoietic stem cells
hUCMSCs human umbilical cord blood mesenchymal stem cells
i.e. in other words
IA intra-arterial
IC intra-cardiac
ICAM-1 intercellular adhesion molecule-1
IDO indoleamine 2, 3-dioxygenase
IFATS International Fat Applied Technology Society
IFN-β- hUCMSCs interferon-beta transduced gene human umbilical cord blood
mesenchymal stem cells
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List of Abbreviations
Fiona Anastasia van Vollenstee

IFN-β interferon-beta
IFN-γ interferon-gamma
in vitro in glass/not within the living
in vivo within the living
IP intra-peritoneal
ISCT International Society for Cellular Therapy
ISO Internation Organization for Standardization
IV intravenous
JNK c-jun N-terminal kinase
L liter
LV lentiviral stock solution
mg milligram
Mg2+ Magnesium ions
MgCl magnesium chloride
2
MHC major histocompatibility complex
min minutes
ml millilitres
mM milimolar
mm millimetre
mm/Hg millimetres mercury
MSCs mesenchymal stem cell/ mesenchymal stromal cells
NaCl sodium chloride
NaCO sodium carbonate
3
NC non-cryopreserved cultures
nm nanometer
O Oxygen
2
PAS Periodic Acid Schiff
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List of Abbreviations
Fiona Anastasia van Vollenstee

PBS phosphate buffer solution
PC5 phycoerythrin-cyanine 5.1
PC7 phycoerythrin-cyanine 7
PE phycoerythrin
pen/strep penicillin and streptomycin
pH potential of hydrogen
PLA processed lipoaspirate
PPAR-γ peroxisome proliferator-activated receptor gamma
P-value probability of testing a statistical significance
RCF relative centrifugal force
RCRs replication competent recombinants
RNA ribonucleic acid
ROS reactive oxygen species
rpm revolutions per minute
s seconds
SCID severe combined immunodeficiency
SS lin side scatter linear
STD Dev standard deviation
Students T-test statistical hypothesis test
SVF stromal vascular fraction
T-cells T-lymphocytes
TEM transmission electron microscope
TGF-β3 transforming growth factor beta-3
TZDs thiazolidinediones
UCB umbilical cord blood
USA United States of America
VCAM-1 vascular adhesion molecule-1
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