Development and Characterization of a Controlled Expression System for Osteogenic … ·...

Development and Characterization of a Controlled Expression

System for Osteogenic Genes

by

Hyun Woo Albert Kim, H. B. Sc.

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Dentistry

University of Toronto

© Copyright by Hyun Woo Albert Kim, 2011

ii

Development and Characterization of a Controlled Expression System

for Osteogenic Genes

Hyun Woo Albert Kim

Master of Science Candidate

Graduate Department of Dentistry


2011

Abstract

Current treatment methods for non-union bone defects present problems. The objective of this

study was to genetically engineer primary and immortalized cell types to express osteogenic

molecules BMP2, RUNX2, OSX, or VEGF in a doxycycline dose-dependent manner for tissue

regeneration. Coding cDNA sequences for all four factors were sub-cloned into the pRTS-1

expression plasmid and transfected into HUCPVCs, RBMCs, ROS cells. Electroporation was the

most effective method of transfection for all cells but stably transfected cells could only be

established for RBMCs and ROS cells. Cells achieved maximum expression within 72hours of

induction and returned to basal levels after 18 days. Enhanced osteogenic bioactivity was only

observed upon activation of BMP-2. The tight regulation of the pRTS-1 system allowed for a

controlled gene expression. Future transplantation experiments using these engineered RBMC

and ROS cells in vivo will evaluate the usefulness of the dox-inducible gene expression system

in bone defects.

iii

Acknowledgments

For the successful completion of this thesis work, I wish to sincerely thank my professors Dr.

Bernhard Ganss and Dr. John E. Davies. Thank you for your continuous guidance,

encouragement, and most of all, a life-changing opportunity reflected not only by this work, but

also by the way I think as a person. I owe a debt of gratitude for your tireless efforts in correcting

and revising this thesis.

I also wish to thank Mr. James Holcroft for all of his teaching and assistance through the

duration of my graduate program. Your witty personality made our lab into the lively place that I

will never forget. Thank you for always putting a positive spin to my negative results, and also

for keeping my lab bench messy so that I would have something to clean.

Lastly, I would like to acknowledge Dr. Dena Taylor for her efforts in revising and guiding me

through my thesis. Dr. Sharon Zikman for your years of continuous care and support. Thank you

to the members of my committee: Dr. Lidan You, Dr. Seal Peel and Dr. Jane Mitchell for your

suggestions. Many thanks to all the members of the Matrix Dynamics Group for their continuous

support, and to my close friends and family, who have motivated me towards my goal.

This has been a long, but life-defining journey. This opportunity turned some of the most

challenging times of my life into something memorable and rewarding. Looking back, I am very

fortunate to have so many amazing people and positive influences in my life. From the bottom of

my heart, thank you; these lifelong lessons will never be forgotten.

iv

Table of Contents Abstract ii

Acknowledgments iii

Table of Contents iv

List of Abbreviations vii

List of Tables ix

List of Figures x

Chapter

1.0 Introduction 1

1.1 Non-Union Bone Defects and their Treatment 4

1.2 Tissue Engineering Triad 6

1.2.1 rhBMP-2 Treatment and their Limitations 8

1.3 Methods to Control BMP2 Release 10

1.3.1 rhBMP-2-CBD 10

1.3.2 rhBMP-2-Cell System 10

1.3.3 The Control Mechanism: The Tet-Off and Tet-On Systems 11

1.3.4 pRTS-1: The Enhanced Tet-On System 13

1.3.5 The Cells 16

1.4 Transfection Methods 18

1.5 Rationale 20

1.6 Hypothesis 21

1.7 Objectives 21

2.0 Materials 22

2.1 General Materials 22

2.2 Cloning Materials for pRTS-1-X Constructs 22

2.3 Cell Culture 25

2.4 Kill Curve 28

2.5 Transfections 28

2.6 Transfection Efficiency Analyses 28

2.7 Transcriptional Analyses 28

2.8 Translational Analyses (Western Blots) 29

2.9 Alkaline Phosphatase Activity Assay 31

v

3.0 Methods 33

3.1 Cloning of pRTS-1-RUNX2/SP7/VEGF-A Constructs 33

3.1.1 Preparation of pRTS-1 Plasmid Backbone 34

3.1.2 Amplification of the Gene Inserts 37

3.1.3 Ligation of the pRTS-1-X Plasmids 39

3.1.4 Transformation of the pRTS-1-X Plasmids 39

3.1.5 pRTS-1-X Orientation Diagnostic Digest 40

3.2 Cell Culture – General Procedures 41

3.3 HUCPVC Characterization 42

3.4 Hygromycin B Kill Curve of HUCPVC, RBMC, ROS cells 43

3.5 Transfection 44

3.5.1 Amaxa Nucleofector II 44

3.5.2 Invitrogen Lipofectamine 2000 46

3.5.3 Qiagen SuperFect 46

3.5.4 Clonetech Xfect 47

3.5.5 Bio-Rad Gene Pulser MX Cell Electroporation System 47

3.6 Transfection Efficiency Analyses 49

3.7 Single Clone Isolation 49

3.8 mRNA Expression 50

3.9 Western Blot Analyses 51

3.10 Doxycycline Dose-Reponse 52

3.11 Induction Kinetics 52

3.12 Bioactivity Assay 53

4.0 Results 54

4.1 pRTS-1-X Construct Verification 54


4.3 Hygromycin B Kill Curve of HUCPVC, RBMC, and ROS cells 56

4.4 Transfection 58

4.4.1 Amaxa Nucleofector II 58

4.4.2 Invitrogen Lipofectamine 2000 66

4.4.3 Qiagen SuperFect 66

4.4.4 Clonetech Xfect 67

4.4.5 Bio-Rad Gene Pulser MX Cell Electroporation System 70

vi

4.5 Transient vs. Stable Expression and Production of Transfected Clones 72

4.6 Rate of Proliferation 73

4.7 mRNA Expression 74

4.8 Western Blot Analyses 74

4.9 Doxycycline Dose-Response 75

4.10 Induction Kinetics 77

4.11 Alkaline Phosphatase Activity Assay 79

5.0 Discussion 82

5.1 Development of Stable pRTS-1-BMP-2 Cell System is Only Achieved with

Electroporation Combined with RBMCs and ROS Cells

83

5.1.1 Chemical Transfection Methods 85

5.1.2 Electrical Transfection Methods 87

5.1.2.1 Nucleofection 87

5.1.2.2 Electroporation 89

5.2 pRTS-1-BMP-2, Low Background and Doxycycline Dose-Dependent

Activation

90

5.3 pRTS-1 Controlled Kinetics 90

5.4 pRTS-1 Bioactivity 91

5.5 Experimental Limitations 92

Conclusions 95

Future Directions 96

References 97

vii

List of Abbreviations

Abbreviation Full Description

AB Antibiotics

BM Bone Marrow cells

BMP Bone Morphogenetic Protein

BMP-2 Bone Morphogenetic Protein 2

CBD Collagen type I Binding Domain

Cbfa-1 (also known as RUNX2) Core-binding factor alpha - 1

CMV CytoMegalovirus (promoter)

Dox Doxycycline

EBV Epstein-Barr Virus

ECM Extracellular Matrix

FACS Fluorescence Activated Cell Sorting

eGFP Enhanced Green Fluorescence Protein

GBR Guided Bone Regeneration

GFP Green Fluorescence Protein

GTR Guided Tissue Regeneration

HA Hydroxyapatite

hBM Human Bone Marrow Cells

HIF Hypoxia-Inducible Factor

HUCPVC Human Umbilical Cord Perivascular Cells

MSC Mesenchymal Stem Cell

NEB New England Biolabs

Osx (also known as SP7) Osterix

PCMV CytoMegalovirus promoter

PTet Tetracycline induced Promoter

PTet min. CMV Tetracycline induced cytomegalovirus minimal promoter

pRTS-1-BMP2 pRTS-1 plasmid with the rhBMP-2 gene

pRTS-1-Luc pRTS-1 plasmid with the Luciferase gene

pRTS-1-SP7 pRTS-1 plasmid with the Osterix (sp7) gene

pRTS-1-RUNX2 pRTS-1 plasmid with the RUNX2 gene

pRTS-1-VEGFA pRTS-1 plasmid with the VEGFA gene

pRTS-1-X pRTS-1 plasmid with any of the genes used in this thesis research

viii

PMT Photomultiplier Tube

RBMC Rat Bone Marrow Cells

rcf Relative Centrifugal Force

ROS Rat Osteosarcoma Cells

rhBMP-2-CBD Recombinant Human Bone Morphogenetic Protein-2-Collagen Type I

Binding Domain

rpm Revolutions Per Minute

RBMC-BMP2 RBMC transfected with pRTS-1-BMP2 construct

RBMC-RUNX2 RBMC transfected with pRTS-1-RUNX2 construct

RBMC-SP7 RBMC transfected with pRTS-1-SP7 construct

RBMC-VEGF-A RBMC transfected with pRTS-1-VEGF-A construct

RE Restriction Enzyme

ROS-BMP2 ROS transfected with pRTS-1-BMP2 construct

ROS -RUNX2 ROS transfected with pRTS-1-RUNX2 construct

ROS -SP7 ROS transfected with pRTS-1-SP7 construct

ROS -VEGF-A ROS transfected with pRTS-1-VEGF-A construct

rTetR Reverse tetracycline repressor

rtTA Reverse tetracycline-controlled transactivator

RUNX2 (also known as Cbfa-1) Runt-related transcription factor 2

SP7 (also known as Osx) Osterix

Tc or Tet Tetracycline

Tet-Off Tetracycline-based gene repressor system

Tet-On Tetracycline-based gene inducible system

TetO Tetracycline Operator

TetR Tetracycline Repressor

TetRKRAB

Tetracycline Repressor (Kruppel-associated box)

TRE Tetracycline Response Element

tTA Tetracycline-controlled Transactivator

tTS Tetracycline Transcriptional Silencer

tTSKRAB

Tetracycline Transcriptional Silencer modified with Kruppel-associated box

UCB Umbilical Cord Blood

USSC Unrestricted Somatic Stem Cells

ix

List of Tables

Table

Number

Full Description Page

3.1.1.1 Restriction Enzyme Diagnostic Digest Protocol 34

3.1.1.2 Enzymatic Activity Temperature Guideline for Restriction Enzymes 34

3.1.1.3 Restriction Enzyme Digestion Protocol 36

3.1.2.1 PCR Gene Amplification Protocol 38

3.1.2.2 PCR Amplification Program – KPTOUCH Protocol 38

3.1.3 Ligation Protocol 39

3.1.4 DH5α Transformation Protocol 39

3.1.5 pRTS-1-X Diagnostic Digest – Orientation Confirmation of Gene Insert 41

3.2.1 Cell Thawing Protocol 42

3.2.2 Trypan Blue Cell Viability Protocol 42

3.4 Hygromycin B Concentration Levels Use for Kill Curve Experiment 43

3.5.3 SuperFect Optimization Experiment (DNA and DNA:SuperFect Ratio) 47

4.4.1 Summary of Nucleofection Experiments 65

4.4.5.1 Bio-Rad Electroporation Voltage Gradient Results 70

4.4.5.2 Bio-Rad Electroporation Duration Gradient Results 70

5.1 Summary of All Transfection Experiments in Chronological Order 84

x

List of Figures

Figure

Number

Full Description Page

1.2 Venn diagram of Tissue Engineering Triad 8

1.3.3.1 Cartoon depiction of the Tet-Off system 12

1.3.3.2 Cartoon depiction of the Tet-On system 12

1.3.4.1 Cartoon depiction of the enhanced Tet-On system (Silencer) 14

1.3.4.2 Cartoon depiction of the fully enhanced Tet-On system found in the pRTS-1

Plasmid

15

1.3.4.3 Schematic map of the pRTS-1 plasmid 16

1.3.5 HUCPVC and the SEM of an excised umbilical artery indicating the location

of HUCPVCs

17

3.1 Schematic map of the pRTS-1 plasmid 33

3.1.2 Primer details of gene inserts 37

3.5.5 Diagram of the V-grad/D-grad Program 48

4.1.1 Sfi I Restriction Sequence and its Unique 5‟ Overhang 54

4.1.2 Confirmation of Insert Orientation through Diagnostic Digest 55


4.3.1 HUCPVC Kill Curve 56

4.3.2 RBMC Kill Curve 57

4.3.3 ROS Kill Curve 57

4.4.1.1 Nucleofection Optimization Kit Solution L and V Transfection (24 Hours) 59

4.4.1.2 Cell viability after four days of selection 60

4.4.1.3 Nucleofection Optimization Kit Solution L and V GFP expression (X-001) 61

4.4.1.4 Diminishing GFP expression of Nucleofection transfected HUCPVCs 61

4.4.1.5[A] MSC Kit Nucleofection (A-020) 62

4.4.1.5[B] MSC Kit Nucleofection (C-017) 63

4.4.1.5[C] MSC Kit Nucleofection (P-016) 63

4.4.1.5[D] MSC Kit Nucleofection (X-001) 64

4.4.2 Lipofectamine 2000 transfection (HUCPVC) 66

4.4.3.1 Qiagen SuperFect transfection (HUCPVC) 67

4.4.3.2 Qiagen SuperFect transfection (RBMC) 67

4.4.4.1 Clontech Xfect transfection (HUCPVC, RBMC, ROS Cell) 68

4.4.4.2 Long Term decrease in GFP expression (RBMC, ROS Cell) 68

4.4.4.3 Formation of large complexes and cell disintegration caused by confluence 69

4.4.4.4 Xfect Transfection with 50% Confluence 70

4.4.5.1 Bio-Rad Electroporated RBMCs and ROS Cells express GFP after 12 hours 71

4.4.5.2 Bio-Rad Electroporation (DNA optimization) 71

4.5.1 Single Clone Expansion of ROS-pRTS-1-BMP-2 after 13 days 72

xi

4.5.2 Xfect Transfected and Electroporated RBMC - Single Clone Expansion 73

4.7 mRNA Expression of all genes from transfected RBMC and ROS cells 74

4.8 Western Blot of all transfected RBMC and ROS cells 75

4.9 Doxycycline Dose-Response after 24 Hours (RBMC and ROS Cell) 76

4.10.1 Induction Kinetics of RBMC-RUNX2 and ROS-RUNX2 78

4.10.2 pRTS-1 Transfected RBMC Doxcycline Kinetics 79

4.11.1 ALP activity Assay (Conditioned Media) 80

4.12.2 ALP activity Assay (Co-Culture) 81

5.5 Vector map of pRTS-1 highlighting the region with the Tet-On enhancement 93

1

Chapter 1 – Introduction

Human bone is a specialized connective tissue with major functions that include: being a

primary mechanical support for locomotion, a physical barrier for vital organ protection, and a

reservoir for ions, particularly calcium, and growth factors.32, 74

However, like all tissues in the

body, injury or disruption to the bone tissue can occur and depending on the severity of the latter;

smaller injuries may be repaired through active remodeling while more extensive injuries may

require therapeutic intervention to regenerate bone.

Bone tissue comprises a mineralized extracellular matrix (ECM), which consists of both

inorganic and organic components. The main inorganic component is the hydroxyapatite (HA)

while the main organic component is type I collagen. Synthesized by osteoblasts, type I collagen

is composed of two αI chains and one αII chain (transcribed from Col1a1 and Col1a2 genes,

respectively) in a coiled coil structural motif. This structure undergoes fibrillogenesis and the

rate of deposition results in either woven or lamellar bone formation.84

In the human adult both trabecular, or cancellous, and cortical, or compact, bone are

lamellar in structure, although the former defines interstices for marrow and possesses a large

surface area for increased endocrine function, while the latter is dense and packed with osteons,

remodels more slowly than cancellous bone, but provides resistance to torsional loading during

locomotion. 32

Arising from both mesoderm and neural crest, bone is formed through a process known

as osteogenesis, and constantly remodeled through the individual cellular activities of osteoblasts

and osteoclasts.51

Osteoblasts are derived from mesenchymal/ectomesenchymal cells and

synthesize type I collagen as well as other specialized bone-related proteins such as bone

sialoprotein, osteocalcin, osteonectin, and osteopontin; and mineralize this organic component

2

with hydroxyapatite (HA) microcrystallites. On the contrary, osteoclasts are multinucleated cells

that are derived from hematopoietic stem cells and function to resorb bone and maintain bone

homeostasis and active remodeling.

Although the molecular pathways resulting in osteogenesis have yet to be fully

understood, some proteins have been shown to be critically necessary for osteoblastic

differentiation. One major group of proteins is the Bone Morphogenetic Protein (BMP) family

which belongs to the TGF-β superfamily.10

In the 1960s, Urist et al. discovered auto-inductive

osteogenic activity when a decalcified bone matrix was placed into the belly of the rectus

abdominus muscle of a rabbit.114

This transplant resulted in ectopic bone formation and the

finding quickly became the basis of the field of bone induction research. However, is was not

until the 1980 that Wozney et al.126

first cloned and sequenced BMP, since which time BMPs

have been found to have multiple functions in embryonic development, bone induction, and

other growth and differentiation cascades, although this thesis will focus on the impact of BMP-

2 on bone induction.10

In fact, in this thesis, three osteoinductive genes and an angiogenic gene were chosen as

targets to be selectively expressed in a variety of cell types, to assess their potential effects on

bone induction for the purpose of tissue regeneration. The three osteoinductive genes were BMP-

2, Osterix (Osx or SP7), and Runt-related transcription factor 2 (RUNX2 or Cbfa-1). BMP-2, as

mentioned above, is a powerful cytokine selected for its ability to promote osteoblastic

differentiation.10

OSX has been reported to be an inhibitor of chondrogenesis75

, while an OSX

knockout mouse revealed complete absence of osteoblasts, which is indicative of the critical

importance of OSX in osteoblastogenesis.75

The third osteoinductive gene, RUNX2, has been

found to be up-regulated in hypertrophic chondrocytes and is another crucial gene for

osteoblastogenesis.37

3

Current literature suggests a complex signaling pathway associated with these three genes.

For example, the expression of both OSX and RUNX2 are up-regulated by BMP-2 through the

SMAD pathway.37

Early studies indicate OSX as a downstream gene of RUNX2 due to the lack

of OSX expression in RUNX2-/-

mice.24

Overexpression of RUNX2 induced expression of OSX

in MSCs, thus, the expression of OSX was controlled by BMP2 via RUNX2.75

However, it has

been recently demonstrated that transfection of OSX into RUNX2-/-

MSCs induced

osteoblastogenic activity.75

Furthermore, embryos deficient in either OSX or RUNX2 resulted in

the absence of bone8,24

but the phenotypes of the two null mice are different at birth, suggesting a

difference in function during bone formation.75

OSX deficient mice exhibited normal

chondrocyte differentiation while RUNX2 deficient mice presented neither osteoblasts nor

hypertrophic chondrocytes.24

Moreover, OSX and RUNX2 stimulate the expression of distinct

genes and thus, collectively, this suggests OSX functions as a downstream target of RUNX2 but

also functions independently of RUNX2 for osteoblastogenesis.75

Although the complete

signaling pathways are unclear, these three genes were selected for their involvement in

osteoblastogenesis.

The final gene selected for this thesis was the vascular endothelial growth factor A

(VEGF-A). All living tissues in the body are composed of cells and ECM. Cells require fresh

nutrients, oxygen, as well as a method of removing waste in order to function. Bone tissue is no

exception. Although the initial signal for the invasion of blood vessels into bone is not fully

known, it is believed that hypoxia triggers the signaling cascade.122

Hypoxic conditions trigger

cells to release hypoxia-inducible factor (HIF) and, as a result, activates VEGF, which in turn

promotes angiogenesis. Experimental blockade of angiogenesis resulted in decreased bone

density and disturbed bone mineralization.56, 122

Consequently, VEGF-A was included as part of

4

this research study to induce angiogenesis and potentially improve the gene therapy treatments of

large bone defects.

1.1 – Non-Union Bone Defects and their Treatment

Bone fractures may be generalized into two categories, union and non-union defects. Due

to active matrix remodeling, fractures normally heal or are said to have achieved union, but

complications including excessive movement could increase the complexity of the injury. In

these cases, the damage or defect to the bone is too severe or too large for the natural restoration

process to take place and thus, a non-union defect is generated. Current treatments for replacing

bone through bone grafting or cytokine delivery of superphysiological doses (1.7-3.4mg) of

rhBMP-2125

require multiple invasive surgeries, which translates into further increase in the

overall cost of non-union bone treatments.

Bone grafts are separated into three major categories: autografts, allografts or xenografts,

and biomaterials. In autografts, bone is harvested from one site of the patient and transferred to

the recipient site. This procedure is most successful because it avoids immunological issues and

provides the cells and bioactive molecules to induce and sustain the regenerative process. A

major drawback of autografts is the limited availability of bone in the patient.

In contrast, the availability of allograft and xenograft bone is greater. In allografts, bone

is transferred from a cadaveric donor to the recipient patient. Allografts are common but there is

a non-zero risk of immune response and also the possibility of pathogen transfer, both of which

detract from this approach. Xenograft utilizes bone from an animal and is used rarely, and only,

as an end-stage organ failure treatment where a lack of alternative treatment will compromise the

life of the patient. This procedure has similar complications to allografts with the addition of

disease transmission, non-matching biological molecules as well as a different lifespan of the

5

bone.124

In addition, graft tissues can first be demineralized to create a demineralized bone

matrix (DBM). DBMs are osteoconductive, can re-mineralize over a long period of time, but do

not provide mechanical strength for structural support until neo-osteogenesis has occurred.91

Finally, synthetic variants or biomaterials have been developed as an alternative source

for bone regeneration. These materials, such as hydroxyapatite and beta-tricalcium phosphates,

act as osteoconductive scaffolds for bone regeneration and can be supplemented with

osteoinductive agents such as BMPs, bone marrow aspirates53

, and cultured osteoblasts.11

For the

purpose of this thesis, osteoinductive refers to the ability to stimulate osteoprogenitor cells to

differentitate into osteoblasts to form new bone and osteoconductive refers to the growth of bone

on a surface. The two major benefits of this treatment are availability and biocompatibility, while

they are limited by not being osteoinductive (although some high surface area calcium

phosphates would appear to have osteoinductive properties, probably through the adsorption of

growth factors from the host blood stream64

); and they also exhibit slow degradation and high

radiodensity.53

In addition to the grafts and biomaterials briefly reviewed above, increasing the blood

supply to the bony wound site, can enhance the regenerative process. In 2004, Munk and Larsen

conducted a systematic review of 147 publications that included 5246 cases of scaphoid non-

unions. Their study included literature from 1928 to 2003 and focused on non-vascularized bone

grafting with or without internal fixation and vascularized bone grafting outcomes. The

consensus reached was that a vascularized regeneration process results in a significant increase

in the rate of union for bone grafting compared to non-vascularized processes.80

Other experimental techniques such as Guided Tissue Regeneration (GTR) and Guided

Bone Regeneration (GBR) have been explored with limited successs.34,95

Introduced in the 1980s,

6

the Guided Tissue Regeneration (GTR) principle states that the regeneration of a specific tissue

is achieved when the cells with the regenerative capacity are allowed to populate during

regeneration.95

In 1988, the Guided Bone Regeneration (GBR) protocol was developed and in

this treatment, the surface of the bone is coated with a cell-occlusive membrane that prevents soft

tissue invasion while permitting osteoprogenitor cells to be recruited to increase the overall

osteogenic activity.95

1.2 – Tissue Engineering Triad

The aim of tissue engineering is to generate new tissue which restores both structure and

function to the site of injury without adverse effects. To generate this tissue, three issues should

be considered: cells, cell signaling factors, and scaffolds.

First, the cells must be evaluated for their availability and immunophenotype. Specific

cell types are better suited for targeted environments and stem cells are often selected for their

capacity to differentiate into multiple lineages.95,101

One issue with stem cells is their availability;

in a study conducted by Horwitz et al., six patients with severe osteogenesis imperfecta received

two infusions of bone marrow-derived mesenchymal progenitor cells at a median dose of

4.68x106 cells/kg of body weight.

39 In order to obtain this large number of cells for each

therapeutic treatment, primary cells have to be expanded in vitro but the frequency of colony-

forming unit-fibroblast (CFU-F) of stem cells can vary greatly.102

For example, unrestricted

somatic stem cells (USSC) which are isolated from human umbilical cord blood (UCB)60

have a

CFU-F frequency of 1:200 million, and a cell yield only 4 times out of 10 attempts, while the

commonly used human bone marrow (BM) stem cells are found at a frequency of 1:10,000 at

birth, diminishing to 1:100,000 in adult marrow.102

More recently, an alternate source of

mesenchymal stem cells have been isolated from the umbilical cord tissue known as the Human

7

Umbilical Cord Perivascular Cells (HUCPVC – described in more detail in Section 1.3.5). These

cells have been shown to have a CFU-F frequency of 1:333 at harvest that increases to 1:3 after

two passages.102

Thus, HUCPVCs represent an interesting putative source of stem cells for future

therapies. In addition to the availability, cells must either possess the immunophenotype of the

host, or be immunoprivilieged, to prevent undesired immune response. Clinically, mesenchymal

stem cells (MSC) have shown to be immunoprivileged thus avoiding this issue, and making

MSCs a preferred choice for tissue engineering. 40,102

The second condition of the tissue engineering triad (TET) is to provide the proper cell

signaling factor(s) at the site of implantation to induce tissue regeneration. This requires either

the exogenous addition of growth factors, or the employment of well-characterized genes to

propagate and enhance the regeneration process. To achieve the latter, a therapeutic gene is

genetically modified into plasmids and delivered into cells through replication-deficient

recombinant viruses or DNA molecules/complexes.125

The genetically engineered cells are then

selected for transgene expression, thus, allowing for in situ production of cell signaling factors.

In bone, the BMP-2 gene has been known to have a critical role in initiating osteogenesis114

and

thus was selected as one of the target genes for this thesis.

The final condition of the TET is the tissue scaffold. In some approaches, cells or

recombinant genes are transplanted using biopolymers or collagen gels.78,120

These artificial

carriers must be biocompatible and biodegradable for the new tissue to be formed; in addition,

the extracellular matrix of the region should be unaffected by the foreign or artificial material, to

minimize disturbance to tissue regeneration.

8

Together, these requirements form the tissue regeneration triad and once all conditions

are satisfied, the newly generated tissue should restore the structure and function to the site of

injury.

1.2.1 – rhBMP-2 Treatment and their Limitations

The development and use of a recombinant human bone morphogenetic protein 2

(rhBMP-2) began in 1997 and in 2002, the rhBMP-2 was approved by the Food and Drug

Administration (FDA) for clinical interbody spinal fusion and to treat acute open tibial shaft

fractures.124

Since then, the application for this treatment was extended to oromaxillofacial

procedures, alveolar cleft repair, and a variety of orthopedic disorders.63

In clinical studies, the rhBMP-2 delivery utilizes superphysiological doses at orders of

magnitude greater (1.7-3.4mg) 125

than the serum physiological levels of pg/mL.86

Milligrams of

rhBMP-2 are absorbed in a collagen sponge, which is then placed into non-union bone defects.

Figure 1.2 Tissue Engineering Triad – Venn diagram depicting the three components and the necessary

combination required for a successful tissue regeneration (indicated by the yellow triangle).

9

Clinically, numerous successful cases of bone regeneration have been reported with this

treatment but, more importantly, the success of this treatment addressed two major factors:

morbidity associated with transplantation complications and limited availability of bone tissue

associated with bone grafts.120

However, there are limitations to this treatment and its drawbacks

lie with the rhBMP-2 itself.12

The first limitation of rhBMP-2 is its short systemic half-life of 7 to 16 minutes6 and the

second limitation is the rate of release from the carrier. Pharmacokinetic release profile of

rhBMP-2 showed an initial, “burst”, release of more than 80% of rhBMP-2 within 10 minutes,

followed by a secondary long-term release of 1 to 10 days. The duration of the secondary release

phase is determined by the biomaterial carrier used for delivery.12

These two limitations result in

inefficient tissue regeneration and, thus, large quantities of rhBMP-2 are used for this

treatment.12

Furthermore, the risks and side effects of delivering superphysiological levels of rhBMP-

2 have not been fully studied; nonetheless, there are reports of increased osteoclastic resorption48

and areas of bone-void formation in long-term regeneration that are suspected to be caused by

the high dose of rhBMP-2.106

Furthermore, it has been suggested that superphysiological doses

of rhBMP-2 may “leak” and stimulate ectopic bone growth. In the cervical spine, this can cause

soft-tissue swelling that could lead to detrimental effects such as airway compromise.88

Other

complications such as the development of renal insufficiency have also been reported.63

One goal

of the current thesis was to explore an alternative delivery platform for BMP, and other

osteogenic-related genes, that would minimize such negative side effects associated with

supraphysiological dosing.

10

1.3 – Methods to Control BMP2 Release

Different approaches have been reported in the literature that address issues associated

with the short half-life and burst release of rhBMP-2: genetically modifying rhBMP-2 and

inclusion of a cell system to express BMP-2 in vivo.

1.3.1 rhBMP-2-CBD

As discussed earlier, rhBMP-2 delivery is clinically used in conjunction with the only

FDA-approved carrier, collagen type I, to induce osteogenesis. One suggested method of

addressing the rapid release limitation is to prolong the release of rhBMP-2 from the collagen

carrier. To achieve this, Visser et al. genetically engineered rhBMP-2 to include a decapeptidic,

collagen type I binding domain (CBD) without disrupting its osteogenic function. This rhBMP-

2-CBD protein decreased the rate of efflux by binding to collagen fibres and, as a result,

prolonged the period of release by a week or longer.120

By extending the effective duration of this

protein, this modification could lower the quantity of rhBMP-2 used and subsequently reduce the

cost as well as the associated side effects. Although this modification addresses the burst release

of rhBMP-2 from the carrier, a direct delivery of the protein does not address the issue of half-

life as discussed in Section 1.2.1.

1.3.2 rhBMP-2-Cell System

As outlined above, successful tissue regeneration requires three conditions:

immunoprevileged cells expressing the correct cell signaling factors embedded in a

biocompatible scaffold. Thus, the second method reported in the literature to overcome the

problems of half-life and burst release is to genetically engineer cells to produce rhBMP-2 in

vivo. This modification could prolong the presence of rhBMP-2 in a non-union defect, thus

11

potentially shortening the regeneration process through active protein production. However, this

approach presents an additional problem: that of needing to control the amount and duration of

rhBMP-2 expression. Without a control switch this approach could also ultimately result in

overproduction of rhBMP-2 and cause similar complications to those discussed above.

Such a control switch has, in fact, been described (see below) and employs tetracycline to

either switch on, or off, a gene of interest and thus tightly regulate the expression level and the

duration of gene therapy.

1.3.3 The Control Mechanism: The Tet-Off and Tet-On Systems

In the past twenty years, experimental tools have been developed and enhanced to allow

conditional modulation of gene activity in eukaryotes with high specificity.29

One breakthrough

was the development of the Tet-On (i.e., tetracycline-on) system.29

This system is a modification

of the Tet repressor (TetR), tetracycline (Tc or Tet), and tetracycline operator (TetO) interaction

used in the Tet-Off system.

In the Tet-Off system, the Tc-controlled transactivator (tTA) protein is continuously

produced and functions differently in the presence or absence of Tc. In the absence of

doxycycline (dox), a member of the tetracycline antibiotic group, tTA binds to the TetO or the

Tet Response Element (TRE) located upstream of the promoter containing the gene of interest

and induces transcription. On the other hand, in the presence of dox, the tTA binds to dox and is

prevented from binding to the TetO. Thus, transcription of the gene of interest stops, as seen in

figure 1.3.3.1.29

12

The Tet-On system utilizes the same TetR-Tc-TetO interaction but with a modified,

reversed effect in the presence or absence of doxycycline. Gossen et al. randomly mutagenized

the TetR gene and screened for Tc dependence of repression in vivo. They found one mutant

with four amino acid changes (Glu71 Lys

71, Asp

9 5 Asn

95, Leu

101 Ser

101, Gly

102 Asp

102)

that produced a reverse phenotype and increased expression of the gene of interest 30-fold.30

This mutated TetR was renamed to reverseTetR (rTetR) for its opposite effects to presence or

absence of doxycycline and subsequently the tTA was renamed to rtTA.

Figure 1.3.3.2 The Tet-On System – The gene of interest is only transcribed in the presence of doxycycline.

Figure 1.3.3.1 The Tet-Off System – The gene of interest is only transcribed in the absence of doxycycline.29

13

As visualized in figure 1.3.3.2, in the presence of doxycycline, rtTA binds to TetO and

the gene of interest is expressed at a dose-dependent level.30

1.3.4 pRTS-1: The Enhanced Tet-On System

Constructed by Bornkamm et al., the pRTS-1 plasmid is an Epstein-Barr Virus (EBV)-

derived episomally replicating plasmid that includes all elements of the inducible gene

expression system.4 This one plasmid system advances the traditional Tet-On system by

improving the ease of use, suppressing the background activity, and simultaneously expressing

two genes.

The traditional Tet-On system consists of two plasmids: a regulator plasmid and a

response plasmid. The regulator plasmid contains the elements to produce the rtTA protein while

the response plasmid contains the rtTA binding element as well as the inducible gene of interest.

It has been reported that with the traditional system, the best results are obtained when the two

plasmids are transfected in a two-step procedure.4 In the first step, transfected cell lines with

proper rtTA expression are selected, followed by a secondary transfection and selection steps

with the response plasmid. In the pRTS-1 system, Bornkamm et al. joined the regulator and the

response plasmid into one. This reduces the need for multiple transfections, and it has been

suggested that this modification can be of great advantage for difficult transfections.

Furthermore, by reducing the number of steps, the time required to obtain the final cell product is

reduced, which is crucial for primary cells.

The second improvement to the traditional Tet-On system is the addition of a

transcriptional silencer, tTS. Normally, episomally replicating plasmids avoid the influence of

chromosomal integration; however, integration in appropriate chromosomal loci is possible.4 As

14

a result, significant rtTA-independent activity may be observed, but the addition of a tTS

prevents unspecific “leaky” expression by shielding TetO (refer to figure 1.3.4.1).

Furthermore, rtTA is replaced with point mutated rtTA2S-M2 (Asp

148 Glu

148 and His

179

Arg179

), which exhibits increased dox sensitivity with lower background activity116

, and the

tTS is replaced with the modified tTSKRAB

(Fusion of the repressor protein to a strong

transcriptional repressor, Kruppel-associated box “KRAB”), which binds downstream of the

TATA box to also lower the background activity.23

Simultaneous expressions of both regulators

create a stringent doxycycline-dose response system and it was reported that the background

activity was decreased by 1-2 orders of magnitude. As a result of this low background activity,

induction ranged from 1000- to 140, 000-fold whereas a traditional Tet-On system ranged from

30- to 100-fold increase.4

Figure 1.3.4.1 Enhanced Tet-On System (Silencer) – In the absence of doxycycline, tetracycline-dependent

transcriptional silencer (tTS) has a higher binding affinity to TetO and prevents unspecific expression (Left). In

the presence of dox, both tTS and rtTA bind with dox and reverse the binding affinity to TetO, resulting in gene

expression (Right).

15

The final enhancement to the traditional Tet-On system is found in the response element.

The regulator element of the pRTS-1 plasmid consists of a bicistronic expression cassette that is

driven by a chicken-beta-actin promoter, which is active in many eukaryotic cell types.4 The

pRTS-1plasmid contains a bidirectional promoter, PTetbi-1, which expresses two genes

simultaneously and at equivalent levels (refer to figure 1.3.5.2).36

The secondary position of the

bidirectional promoter may encode a secondary gene of interest or a reporter gene such as eGFP

to rapidly monitor expression of the gene of interest.4

Figure 1.3.4.2 pRTS-1: Enhanced Tet-On System – In the absence of doxycycline, tetracycline-

dependent transcriptional silencer (tTSKRAB

) has a higher binding affinity to the bidirectional promoter and

prevents unspecific expression of both the gene of interest and the reporter gene (Left). Conversely, in the

presence of dox, both tTSKRAB

and rtTA2s-M2 binds with dox and reverses the binding affinity to the

bidirectional promoter, resulting in 1000- to 140 000-fold increased gene expression (Right)

16

This pRTS-1 plasmid, developed by Bornkamm et al., is a technological improvement

that allows for a tightly regulated, doxycycline dose-dependent expression of the gene of interest,

and it was genetically modified to express the genes BMP-2, RUNX2, OSX, VEGF-A in this

thesis.

1.3.5 The Cells

Since the development of cell therapy treatment, bone marrow-derived mesenchymal

stem cells have been a common source of cells for cell-based therapy.125

The mesenchymal

population of the BM have the capacity to differentiate into a wide range of tissues including

tissues of the musculoskeletal systems.102

Thus, by genetically engineering cells with the

capacity to differentiate, the system aims to induce an autocrine and paracrine osteogenic

signaling at the site of implantation. According to the U.S. National Institute of Health

(www.clinicaltrials.gov), there are 165 clinical trials conducted with mesenchymal stem cells, of

which 100 studies are currently on-going.

Figure 1.3.4.3 Schematic map of pRTS-14 – Schematic map of the pRTS-1 plasmid. This system enhances the

traditional Tet-On system by incorporating both the regulator and response element into a single plasmid,

implementing a silencer and modifying the rtTA, and modifying the response element with a bidirectional

promoter.

17

For the purpose of this research thesis, three cell types were transfected to develop the

tightly regulated inducible system: Human Umbilical Cord Perivascular Cells (HUCPVC), Rat

Bone Marrow Cells (RBMC), and Rat Osteosarcoma cells (ROS). Cells were selected based on

their potential use as well as their abilities to overcome difficulties in transfections encountered

throughout this research.

HUCPVC is a mesenchymal stem cell derived from the umbilical cord. During the

growth of a fetus, the human umbilical cord reaches 48-60cm in only 40 weeks19

and it has been

postulated by Davies et al. that this rapid growth must be accompanied by rapidly proliferating

stem cells.102

These multipotent cells are found adjacent to the vasculature of the umbilical cord

and give rise to the connective tissue of Wharton‟s Jelly.102

To date, HUCPVC studies indicate

that they are similar to BM-MSC in terms of differentiation, gene expression, and profiling

markers. With their ability to evade allogeneic host immune recognition, these cells would be

optimal for tissue engineering treatments.19

Figure 1.3.5 Human Umbilical Cord Perivascular Cells and its location present in the umbilical cord102

– (A) Scanning electron microscopy of the umbilical artery excised from the umbilical cord. The dotted line

depicts the outer margin of the perivascular region where HUCPVCs are found. (B) HUCPVCs in vitro

presenting a fibroblastic morphology.102

18

In addition to HUCPVCs, Rat Bone Marrow Cells (RBMC) and Rat Osteosarcoma cells

(ROS) were employed herein to develop the pRTS-1-based controlled expression system as the

current state of HUCPVC characterization is not complete and it has been shown that primary

human cells are more refractory to transfections. In anticipation of such difficulties, RBMC and

ROS cells were transfected as alternative cell lines to overcome two major issues: transfection

difficulties and diminished proliferation post-transfection. Detailed discussions of these two

issues are found in Chapter 5. Original RBMC were obtained from the femoral bones of young

adult (120-130g) male Wistar rats. From those, non-transformed clonally derived RBMCs were

selected. RBMCs D8 (Materials # 46) are spontaneously immortalized stable population of cells

that upregulates RUNX2, OSX expression. Furthermore, these cells generate bone-like nodules

in the shortest time reported to date in supplemented culture media.50

Unlike the HUCPVC, these

cells are not immunoprevileged but these primary bone marrow cells have the capacity to

differentiate into osteoblasts under the correct conditions and begin osteogenesis.107

ROS,

specifically subclone ROS 17/2.8 was derived from a spontaneous tumor in an ACI rat by

Majeska et al. in 1980.71

This immortalized osteosarcoma cell line constitutively express

osteocalcin93

, and has increased alkaline phosphatase activity and type I collagen production.71

1.4 Transfection Methods

Transfection, which describes the process of incorporating an external gene into a cell,

can be broadly separated into three categories, chemical, electrical, and viral transfections.

Different transfection methods accomplish gene incorporation differently but the end results of

transfections must meet several criteria. First, cellular disruption such as membrane destruction

must not occur. Secondly, cell death post-transfection must be minimal and lastly, the

transfection efficiency must be high.

19

Viral transfections are a widely used method of transfection for gene therapy. There are

five main groups of viral vectors currently employed: adenovirus, lentivirus, retrovirus, adeno-

associated viruses (AAV), and herpes simplex virus-1 (HSV-1).111,118

Viral transfections have

shown success in implementing genes into the genome but the drawbacks such as insertional

mutagenesis and immune response in patients makes viral transfections less desirable. Thus,

alternative methods of gene delivery have been studied.

Amaxa Nucleofector II

The Amaxa nucleofector II is an electrical-based transfection device where the negatively

charged DNA is driven into the cells through a unique proprietary cell-type-specific electrical

parameter. Nucleofection was developed to allow efficient gene delivery directly into the nucleus

of the cell, thus, has been described as an efficient transfection method for primary cells.31,33

For

this research, two kits were used for nucleofection: Cell Line Optimization Nucleofector Kit

(Materials #76) and Human MSC Nucleofector Kit (Materials #78). It is important to note that

both kits require a unique buffer with proprietary composition that negatively affects cells during

prolonged exposure.

Invitrogen Lipofectamine 2000

Invitrogen Lipofectamine 2000 is a chemical transfection method that utilizes cationic

lipid molecules that bind to the negatively charged DNA to form a DNA-Cationic lipid complex.

This complex is then fused with the cell and the DNA is released to complete a successful

transfection. Zhao et al. demonstrated successful transfection to deliver siRNA into human

embryonic stem cells with high efficiency.133

20

Qiagen SuperFect

Qiagen SuperFect is a chemical transfection method whereby positively charged

nanoparticles interact with the negatively charged DNA to form a toroid-like structure. This

complex then binds to the surface of the cell for a nonspecific endocytosis.

Clontech Xfect

Clontech Xfect is a chemical transfection method that uses a biodegradable transfection

polymer that forms a nanoparticle complex with the DNA. Xfect has been developed with low

cytotoxicity to minimize cell death during transfection and this allows for higher quantities of

DNA complexes to be applied to each transfection, thus allowing for higher transfection

efficiency.

Bio-Rad Gene Pulser MXcell Electroporation System

Bio-Rad‟s Gene Pulser MXcell Electroporation System is an electrical-based transfection

system whereby the individual parameters of an electroporation can be modified, specifically the

following: time constant, voltage applied, pulse interval, pulse duration, and pulse type (square

or exponential). These parameters allow the system to be optimized for primary cells.

Furthermore, unlike the nucleofector, transfection for the Gene Pulser is completed in Opti-

MEM (Materials #52) which removes the cytotoxic effects associated with the proprietary

buffers of the nucleofector (Amaxa). This allows the user to optimize the transfection parameters.

1.5 Rationale

The current delivery of BMP-2 at super-physiological doses have achieved clinical

success in bone regeneration but a wasteful burst release of this expensive osteoinductive protein

21

warrants a need for effective method of delivering osteoinductive proteins. By genetically

engineering mesenchymal cells with osteogenic genes and regulating the expression, a

continuous presence of BMP-2 at the site of regeneration should be possible. This ability to

control the quantity as well as the duration of osteoinductive genes present during bone

regeneration should quicken the regenerative process and remove the need for multiple

expensive injections of BMP-2.

1.6 Hypothesis

It is hypothesized that:

1. Stably transfected pRTS-1 mesenchymal cells will continuously express the gene of interest

upon exposure to doxycycline

2. The dose and duration of gene expression can be tightly regulated by the concentration and

exposure length of doxycycline

1.7 Objectives

The specific objectives of this work are:

1. To generate stable clones of HUCPVCs, RBMCs, and ROS cells transfected with each of the pRTS-

1-X plasmids: optimal method of transfecting the large 18+kbp plasmid will be investigated.

2. To investigate the expression pattern of pRTS-1-X transfected cells: doxycycline dose-dependent

expression and the induction kinetics of the genes of interest will be investigated.

3. To investigate the osteogenic potentials of overexpressing BMP-2, RUNX2, and OSX:

overexpressing the necessary genes for osteogenesis and their effects on osteogenesis will be

investigated.

22

Chapter 2 – Materials

This chapter lists all chemical, cell culture reagents, and additional materials used in my

experiments. They are organized into corresponding experiments with complete details of origin.

# Materials Vendor Catalog Number

Additional Information

2.1 General Materials

1 Round-Bottom Tubes (14.0 ml)

BD Falcon 352057

2 Conical Tubes (15

ml) BD Falcon 352095

3 Conical Tubes

(50 ml) BD Falcon 352070

4 Cell Scraper, 25cm

(1.7cm blade) Sarstedt 83.1830

5 Glass Pasteur

Pipette 9" Fisherbrand

2559 - 136786H

6 Serological pipette

(5ml, 10ml, 25ml) Sarstedt

86.1253.001 86.1254.001 86.1685.001

7 Microtubes

(1.5 ml) Axygen 311-08-051

8 PCR Strip Tubes

(0.2 ml) VWR 53509-304

2.2 Cloning Materials for pRTS-1-X Constructs

Chemicals & Reagents

9 Agar BioShop AGR 001.1

10 Agarose BioShop AGA 001.500

1% Agarose Gel:

- 2mL of TAE buffer

- 98mL of dH2O

- 1g of Agarose

- EtBr Concentration 10 mg/mL

11 Ampicillin BioShop AMP 201 1000x Concentration

of 50 mg/mL

12 Competent DH5α

Cells Invitrogen 18265-017

23

13 DNA Ladder

(100bp)

New England Biolabs

N3231S

14 DNA Ladder

(1kbp)

New England Biolabs

N3232S

15 DNA Ladder

(1kbp Plus) Invitrogen 10787018

16 EDTA BioShop EDT 001.500

EDTA Solution Composition (1L) :

- 186.1g of EDTA in 700mL H2O

- Adjust pH to 8.0 with 10M NaOH

- Add H2O to 1 L

17 Ethidium Bromide BioShop ETB 333.1

18 Gel Loading Dye

(6x) New England

Biolabs B70221S

19 Glycerol BioShop GLY 002.1

Glycerol Solution Composition (per 100ml) :

- 65% Glycerol (v/v)

- 0.1M MgSO4

- 0.025M Tris pH 8.0

20 Kanamycin Sulfate BioShop KAN 201.5 1000x Concentration

of 30 mg/mL

21 LB Broth Lennox BioShop LBL 405.1

22 Platinum Taq DNA

Polymerase Invitrogen 10966018

100rxns

23 T4 DNA Ligase New England

Biolabs M0202S

24

TAE (Tris/Acetate EDTA)

Electrophoresis buffer

TAE Electrophoresis Buffer Composition (50x Stock Solution) :

- 242g Tris base

- 57.1ml acetic acid

- 37.2g EDTA

- Add dH2O to 1 L

- pH 8.0

25 Tris BioShop TRS 001.1

26a Restriction Enzyme

(Bgl II)

New England Biolabs

R0144S

24

26b Restriction Enzyme

(Hind III)

New England Biolabs

R0104S

26c Restriction Enzyme

(Not I)

New England Biolabs

R0189S

26d Restriction Enzyme

(Sfi-I)

New England Biolabs

R0123S

27 QIAprep Spin

Miniprep Kit (250) Qiagen 27106

28 EndoFree Plasmid

Maxi Kit (10) Qiagen 12362

29 QIAquick Gel

Extraction Kit (50) Qiagen 28704

Plasmids

30 pRTS-1-Luciferase pRTS-1 Base

Plasmid

Received from Dr. Georg W. Bornkamm at GSF-Institut fur Kilnische Molekularbiologie und Tumorgenetik, Marchioninistrasse 25, D-81377

Munchen, Germany4

31 pRTS-1-BMP2

plasmid Described in section 3.1

32 RUNX2 Human

plasmid Origene SC302270

33 SP7 Human

plasmid Open

Biosystems 8069055

Accession #BC065522

34 VEGF-A Human

plasmid Open

Biosystems 6006890

Accession #BC101549

35 Primers ACGT Corp. www.acgtcorp.com

Apparatus

36 Sequencing TCAG

Sequencing Server

http://tcag-sequencing.ccb.sickkids.ca/

37 PCR

Eppendorf 97017 Mastercycler gradient

Perkin Elmer GeneAmp PCR System 2400

38 Gel Doc XR

System Bio-Rad 170-8170

25

39 Gel Electrophoresis Life

Technologies

Model H5 Series 1087

40 Max Q 5000

Shaking Incubator Thermo Scientific

SHKA5000

41 Nanodrop 1000 Thermo Scientific

Nanodrop 1000

42 Table Top Centrifuge

Eppendorf

5415D Non-refrigerated centrifuge for 1.5mL eppendorf tubes

5810R Refrigerated centrifuge for 15mL and 50mL Falcon tubes

2.3 Cell Culture

Cells

43

Human Umbilical Cord Perivascular

Cells

(HUCPVC)

Tissue Regeneration Therapeutics

www.verypowerfulbiology.com

Cord Numbers:

0408003, 0408004, 0508001, 0508002, 0508003,

44 Human Bone

Marrow Cells (hBM)

Tissue Regeneration Therapeutics

www.verypowerfulbiology.com

45 Rat Osteogenic Sarcoma Cells

(ROS) ATCC CRL-1663

www.atcc.org

46

Rat Bone-marrow derived

Mesenchymal stem Cells (RBMC),

clone D8

Generated in Dr. Sodek’s

lab

Reference # 50

General Reagents

47 Antibiotics (10X)

See Below for the

Individual Antibiotics

Antibiotics Composition:

- 2.5 µg/ml Fungizone

- 0.5mg/ml Gentamicin Sulphate

- 1650 units/ml Penicillin

48

Dulbecco’s Modified Eagle

Medium

(D-MEM) 1X

Gibco – Invitrogen

11995-065

26

49

Minimum Essential Medium Alpha

(α-MEM) 1X

Gibco - Invitrogen

12561-056

50 α-MEM Freezing

Solution

α-MEM Freezing Solution Composition :

- 80% α-MEM

- 10% FBS

- 10% DMSO

51 D-MEM Freezing

Solution

D-MEM Freezing Solution Composition :

- 80% D-MEM

- 10% FBS

- 10% DMSO

52 Dimethyl Sulfoxide

(DMSO) Sigma-Aldrich 276855

53

Dulbecco’s Phosphate Buffered

Saline

(PBS) 1X


14190-144

54 Opti-MEM

Reduced-Serum Medium (1X)


11058-021

55 Fetal Bovine Serum Hyclone SH30397.03 Lot# K4M25240

and KPJ22093

56 Fungizone Gibco –

Invitrogen 15290-018

57 Gentamicin

Sulphate Gibco –


58 Parafilm University of

Toronto Medstore

2099-1337410 www.uoftmedstore.com

59 Penicillin G Sodium

Salt Sigma-Aldrich P3032-100MU

60 Trypan Blue Stain

0.4% Gibco –

Invitrogen 15250061

61 Trypsin Gibco –


27

Plasticware

62

96-well Microplate, tissue-culture

treated, flat-bottom with lid

BD Falcon 353075

63

48-well Cell Culture Plates, tissue-culture treated

polystyrene, flat-bottom with lid

BD Falcon 353230

64



BD Falcon 353226

65



BD Falcon 353225

66



BD Falcon 353224

67 Dish 35X10mm TC BD Falcon 353001

68 Dish 60X15mm TC BD Falcon 353002

69 Dish 100x20mm TC BD Falcon 353003

70 Cell Culture Flask

(25 cm²) BD Falcon 353808

71 Cell Culture Flask

(75 cm²) BD Falcon 353136

Apparatus

72 Bright Field Microscope

Nikon 802162

73 Imaging Camera PixeLINK PL-A642

74 Cell Counter Beckman Coulter

Z1 Coulter Particle Counter

28

75 Hemacytometer American

Optical

Bright line Hemacytometer

improved neubauer

0.1mm deep

76 Incubator Forma

Scientific

5% CO2, 37oC

2.4 Kill Curve

Additional Reagents

77 Hygromycin B Invitrogen 10687-010

2.5 Transfections

Additional Reagents

78 Cell Line

Optimization Nucleofector Kit

Amaxa-Lonza VCO-1001N Includes pmaxGFP control plasmid (3.4kbp)

79 Doxycycline,

Hydrochloride Calbiochem 324385

[200ng/ml] used unless specified otherwise

80 Human MSC

Nucleofector Kit Amaxa-Lonza VPE-1001

Includes pmaxGFP control plasmid (3.4kbp)

81 Lipofectamine 2000

Reagent Invitrogen 11668-027

82 SuperFect

Transfection Reagent (1.2 ml)

Qiagen 301305

83 Xfect Clontech 631317

Apparatus

84 Gene Pulser MXcell

Electroporation System

Bio-Rad 165-2670 96-well Plate Chamber :

165-2672

85 Fluorescence Microscope

Zeiss AXIOVERT

135M

86 Imaging Camera Hamamatsu C10600-10B

87 Nucleofector II Amaxa-Lonza

2.6 Transfections Efficiency Analyses

Apparatus

88 Fluorescence Activated Cell Sorter (FACS)

Beckman Coulter

Epics Altra

2.7 mRNA Expression Analyses

Additional Reagents

29

89 CelLytic-M, Cell

Lysis Reagent, 250 ml

Sigma-Aldrich C2978-250ML

90 GenElute Direct

mRNA Miniprep kit Sigma-Aldrich DMN10

91

High Capacity RNA-to-cDNA

Master Mix with ‘No RT Control’, 50

rxns

Applied Biosystems

4390711

92

Protease Inhibitor Cocktail for use with mammalian cell and tissue extracts, 5 ml

Sigma-Aldrich P8340-5ML

Recommended Dosage:

100µl/ml at cell density

of 108 cells/ml

93 RNase-Free DNase

Set Qiagen 79254

2.8 Western Blot Analyses

Additional Reagents

94 Acrylamide/Bis-

Acrylamide (37.5:1) 30% Solution

BioShop ACR 010.500

95 Acrylamide/Bis-

Acrylamide (19:1) 40% Solution

BioShop ACR 003

96

Amersham ECL Advance Western Blotting Detection

Kit

GE Healthcare

RPN2135

97 Amicon Ultra

Centrifugal Filters – Ultracel – 30K

Millipore UFC803024

98 Ammonium Persulfate

Bioshop AMP 001.25

99 Anhydrous Ethyl

Alcohol


Medstore

39752-PO16-EAAN

www.uoftmedstore.com

100

BioFlex MRI Single Emulsion X-Ray

Film 8 x 10" 100 sheets

CLMR810(for Radiology)

Clonex 2316-

CLMR810

30

101 Bio-Rad Protein

Assay Bio-Rad 500-0006

102 DL-Dithiothreitol

(DTT) Caledon 27565-41-9

103 Ethyl Alcohol 95% University of

Toronto Medstore

39753-PO16-EA95

www.uoftmedstore.com

104 Immun-Star

Western C Kit Bio-Rad 170-5070

105 Isopropyl Alcohol EMD PX1835-5

106 Glycine BioShop GLN 001.1

107 Goat Anti-Mouse

IgG HRP Conjugate Bio-Rad 170-5047

Secondary AB used

at 1/25 000 dilution

108 Goat Anti-Rabbit IgG (H+L)-HRP

Conjugate Bio-Rad 170-6515

Secondary AB used

at 1/100 000 dilution

109 Immobilon – P Millipore IPVH00010

110 Methanol Caledon 67-56-1

111 PageRuler

Prestained Protein Ladder

Fermentas SM0671

112 Primary Antibodies Abcam

BMP2:ab17885

GFP:ab290

RUNX2:ab48811

SP7:ab57335

VEGF:ab9570

[BMP2]: 0.5 µg/ml

[GFP]: 1 µg/ml

[RUNX2]: 1.25 µg/ml

[SP7]: 2.5 µg/ml

[VEGF]: 10 µg/ml

113 SDS BioShop SDS 001.1

114 SDS

Electrophoresis Buffer (5x)

SDS Buffer Components :

- 15.1 g Tris base

- 72.0 g Glycine

- 5.0 g SDS

- H2O to 1 L

- pH 8.8

115 Skim Milk Powder BioShop SKI 400.500

116

N,N,N’N’ – Tetramethylethyl-

enediamine (TEMED)

BioShop TEM 001.25

31

117

Tris-Buffered Saline-T

(TBS-T)

TBS-T Components :

- 100mM Tris-Cl, pH 7.5

- 0.9% (150mM) NaCl

- 0.1% Tween

- pH 7.4

118 Tween 20 BioShop TWN 510.500

Apparatus

119 ImageJ Software National

Institutes of Health

http://rsbweb.nih.gov/ij/index.html

120 PowerPac Universal

Bio-Rad 164-5097

121 Western Blot Cell Bio-Rad Mini-

PROTEAN II Tube Cell

122 Gel Transfer Apparatus

LKB 2197 Power

Supply 100mA per 0.75mm thick polyacrylamide gel

2.9 Alkaline Phosphatase Activity Assay

123 ALP solution

Suppliers of individual reagents

See below

ALP solution Components :

- 20 µl of 50mg/ml Naphthol AS-MX Phosphate dissolved in N,N - DMF

- 120 µl of 50mg/ml Fast Red TR or Fast Blue BB salt dissolved in dH2O

- 10 ml TM buffer (100mM Tris, pH 8.6, 10mM MgCl2)

124 Fast Blue BB Salt Sigma-Aldrich F-3378

125 Fast Red TR Sigma-Aldrich F-8764

126 Formaldehyde

Solution BDH UN1198

127 L-Ascorbic Acid Sigma-Aldrich A-5960

128 Magnesium

Chloride Sigma-Aldrich M9272-500G

129 Naphthol AS-MX

Phosphate Sigma-Aldrich N-4875

32

130 N,N-

Dimethylformamide Sigma-Aldrich D4551-250ML

131 Paraformaldehyde Sigma-Aldrich P6148-1KG

132 rhBMP-2 Obtained

from Dr. Sean Peel’s lab

University of Toronto: Faculty of Dentistry

133 C2C12 Obtained

from Dr. Sean Peel’s lab

University of Toronto: Faculty of Dentistry

33

Chapter 3 – Methods

Please refer to Chapter 2: Materials for details on all reagents and apparatus.

3.1 Cloning of pRTS-1-RUNX2/SP7/VEGF-A Constructs

The sub-cloning of the human genes of interest into the pRTS-1 plasmid (Materials #30)

was first designed. The pRTS-1-BMP2 plasmid was already available from Dr. Bernhard

Ganss‟s lab and was not created but used in this thesis.

The Tet-dependent bidirectional promoter, indicated by a red box in figure 3.1.1,

transcribes two genes simultaneously. For the purpose of this research, the portion containing the

luciferase gene was replaced with the gene of interest using the flanking Sfi-I restriction sites.

This exchange does not affect the doxycycline dose-dependent control mechanism and expressed

both the gene of interest and the eGFP reporter gene.

Figure 3.1 Plasmid map of pRTS-1 - Genes of interest were sub-cloned into the plasmid via the Sfi-I restriction

sites, indicated by arrows. The red box indicates the doxycycline-dependent bidirectional promoter.

34

3.1.1 Preparation of pRTS-1 Plasmid Backbone

First, a sufficient amount of pRTS-1 plasmid was obtained. A stab culture of the glycerol

stock of pRTS-1-Luciferase plasmid (Materials #30) was placed in 3mL of LB broth culture

media (Materials #21) with 3µL of 1000x ampicillin (Materials #11) and incubated for

approximately 16 hours in a 37oC shaking incubator (Materials #40), set at 300 rpm. The LB

culture media (1 mL) was transferred into a 1.5mL eppendorf tube and centrifuged at 15 700 rcf

for 10 minutes in a tabletop centrifuge. The supernatant was carefully decanted from the

eppendorf tube and the replicated pRTS-1 plasmid was purified from the pelleted D5Hα cells

using the Qiagen Miniprep Kit (Materials #27). A Sfi I diagnostic digest was conducted on 5µL

of [5 µg/µL] eluted plasmid using the following protocol (Table 3.1.2.1):

Table 3.1.1.1 Restriction Enzyme Digest Protocol

Volume (µL) Item

5 µL DNA [5 µg/µL]

2 µL Restriction Enzyme Buffer Associated with Each Restriction Enzyme (10x

Concentrate)

2 µL BSA (Final Concentration 0.1mg/mL)

1 µL Restriction Enzyme (Table 3.1.2.2)

10 µL dH2O

20 µL Total Final Volume

Table 3.1.1.2 Enzymatic Activity Temperature Guideline for Restriction Enzymes

Restriction Enzyme Incubation Temperature (oC) Inactivation Temperature (

oC) Materials #

Bgl II 37oC N/A 26a

Hind III 37oC 65

oC 26b

Not I 37oC 65

oC 26c

Sfi I 50oC N/A 26d

Each restriction digests were conducted at the required incubation temperature in a PCR machine

(Materials #37) for 2 hours.

The digested and undigested (control) pRTS-1 plasmids were fractionated in a 1%

agarose gel (Materials #10) containing 50µg/mL of EtBr (Materials #17) and 5µL of 1kbp DNA

35

ladder (Materials #14). Desired resolution was obtained through electrophoresis conducted in

TAE buffer (Materials #24) with 20µL of DNA and 5µL of loading dye (Materials #18). The gel

was then exposed to UV light and imaged in a gel dock (Materials #38).

Once the pRTS-1-Luc plasmid digest was confirmed, 3µL from the stab culture was

transferred into 100mL LB broth culture media containing 100µL of 1000x ampicillin (Materials

#11) and incubated for approximately 16 hours in a 37oC shaking incubator, set at 300 rpm. The

culture was then transferred into two 50mL falcon tubes (Materials #3) and centrifuged at 13 257

rcf for 30 minutes. The supernatant was carefully decanted and the replicated plasmids were

purified using the Qiagen Maxiprep Kit (Materials #28). The purified plasmids were eluted into

100 µL of elution buffer. The concentration and the quality of the plasmids were verified using a

Nanodrop (Materials #41).

Nanodrop 1000 Instructions

Prepare 100µL of dH2O, 50µL of DNA elution buffer, and 2µL of each construct. First,

run the application and set the scan for nucleic acids. Open the Nanodrop (Materials # 41)

apparatus and clean the loading area with dH2O and dry with Kimwipes. Load 1µL of dH2O and

begin calibration. Dry and load 1µL of the DNA elution buffer to calibrate the baseline. Clean

the loading area with dH2O and load the sample. Measure the quality and quantity of the DNA

using the provided software. Rinse with dH2O between each sample.

Lastly, to create the pRTS-1 plasmid without the luciferase gene, 45µL of the pRTS-1-

Luc plasmid was digested using Sfi I using the following digestion protocol (table 3.1.1.3).

36

Table 3.1.1.3 Restriction Enzyme Digest Protocol

Volume (µL) Item

45 µL DNA [3 µg/µL]

6 µL Restriction Enzyme Buffer Associated with Each Restriction Enzyme (10x

Concentrate)

6 µL BSA (Final Concentration 0.1mg/mL)

3 µL Sfi I Restriction Enzyme (Table 3.1.2.2)


This digestion was conducted for 2 hours at 37oC and fractionated against an undigested pRTS-1

plasmid in a 1% agarose gel. The large band (approximately 17kbp) from the digested pRTS-1

lane was excised and purified using the Qiagen Gel Extraction Kit (Materials #29) and stored in

-20oC freezer until later use.

37

3.1.2 Amplification of the Gene Inserts

The OSX, RUNX2, and VEGF-A gene inserts were created by PCR amplification using

custom made primers, designed specifically to amplify genes with flanking Sfi I restriction sites.

The primer design of each gene is as follows (figure 3.1.2):

Legend - Restriction Site (Sfi I)

- Start/Stop codon

- Gene of Interest

Name: VegP

VEGF-A

Forward Primer

5’-GTTC-GGCC-TCACT-GGCC-ATG-GCA-GAA-GGA-GGA-GGG-3’

Reverse Primer

5’-GTTC-GGCC-TCACT-GGCC-TCA-CCG-CCT-CGG-CTT-GTC-3’

Name: OsxP

Osx

Forward Primer

5’-GTTC-GGCC-TCACT-GGCC-ATG-GCG-TCC-TCC-CTG-CTT-3’

Reverse Primer

5’-GTCC-GGCC-TCACT-GGCC-TCA-GAT-CTC-CAG-CAA-GTT-3’

Name: RunP

RUNX2

Forward Primer

5’-GTTC-GGCC-TCACT-GGCC-ATG-GCA-TCA-AAC-AGC-CTC-3’

Reverse Primer

5’-GTTC-GGCC-TCACT-GGCC-TCA-ATA-TGG-TCG-CCA-AAC-3’

As described in figure 3.1.2, the forward primer contains the Sfi I restriction site attached

to the start codon for the genes of interest. The reverse primer contains the Sfi I restriction site as

well as the reverse complementary of the genes of interest. The designed primers were ordered

Figure 3.1.2 Primer Details of Gene Inserts - Primer details of genes to be amplified using the KP touch

protocol.

38

from ACGT Corporation (www.acgtcorp.com). Lyophilized primers were hydrated with dH2O

(1µg/µL) and used in the PCR amplification protocol found in table 3.1.2.1 and 3.1.2.2:

Table 3.1.2.1 PCR Gene Amplification Protocol

Volume (µL) Item

2 µL ⁄ Diluted template plasmid including gene of interest

1 µL 0.1 µg/µL of forward primer

1 µL 0.1 µg/µL of reverse primer

2.5 µL 10x pfu buffer

2.5 µL 10mM dNTP mix

1 µL 50mM MgSO4

1 µL pfu Platinum Taq DNA Polymerase (Materials #22)

14 µL dH2O


Table 3.1.2.2 PCR Amplification Protocol - KPTOUCH Program

(Eppendorf Mastercycler Gradient PCR – Materials #37)

Step Number Temperature (oC) Duration (h:m:s)

1 94.0

0:04:00

2 94.0 0:00:30

3 60.0 0:00:30

4 72.0 0:00:30

5 94.0 0:00:30

6 59.0 0:00:30

7 72.0 0:00:30

8 94.0 0:00:30

9 58.0 0:00:30

10 72.0 0:00:30

11 94.0 0:00:30

12 57.0 0:00:30

13 72.0 0:00:30

14 94.0 0:00:30

15 56.0 0:00:30

16 72.0 0:00:30

17 94.0 0:00:30

18 55.0 0:00:30

19 72.0 0:00:30

20 Back to step 17 Repeat 35 times

21 72.0 0:10:00

22 4.0 Hold

39

The amplified PCR products were digested with Sfi I using the digestion protocol in table

3.1.1.1. The digested products were fractionated on 1% agarose gel with 100bp and 1kbp DNA

ladders (Materials #13, 14). Gene inserts were excised and purified using the QIAquick Gel

Extraction Kit (Materials #29) and stored in -20oC freezer until further use.

3.1.3 Ligation of the pRTS-1-X Plasmids

The purified Sfi I digested empty pRTS-1 plasmid and the PCR-amplified inserts were

ligated using the protocol described below (Table 3.1.3) for 2 hours at 25oC:

Table 3.1.3 Ligation Protocol

Volume (µL) Item

1 µL Sfi I digested empty pRTS-1 plasmid

5 µL Sfi I digested gene insert

1 µL T4 DNA Ligase (Materials # 23)

2 µL T4 DNA Ligase Buffer

11 µL dH2O


3.1.4 Transformation of the pRTS-1-X Plasmids

10 µL of the respective ligation mixes were transformed into 50µL of competent DH5α

cells (Materials #12) using the following transformation protocol (Table 3.1.4).

Table 3.1.4 DH5α Transformation Protocol

Step Description

1 Mix 5µL of the ligated pRTS-1-X plasmid with 50µLof competent cells

2 Incubate on ice for 30 minutes

3 Heat shock for 20 seconds in a 42oC water bath without shaking

4 Incubate on ice for 2 minutes

5 Add 945µL of pre-warmed LB Broth culture media

The transformed cell suspensions were incubated for one hour in a shaking incubator

(Materials #40) at 37oC and 300 rpm. Cells were then centrifuged at 15 700 rcf for 5 minutes in a

40

tabletop centrifuge. After 800µL of the supernatant was removed, the cells were resuspended in

the remaining 200 µL of supernatant by pipetting gently. The cell suspension was plated onto

prepared LB-Agar plates with 50µg/mL of ampicillin (Materials #11).

Ampicillin LB-Agar Plate Preparation

Mix 20g/L of LB broth (Materials #21) with 16g/L of Agar (Materials #9) and add dH2O

to the desired volume. Autoclave for 15 minutes then cool the bottle down below 55oC.

Ampicillin is heat sensitive and needs be added to the liquid LB-Agar mix below 55oC. Add

ampicillin and shake to mix. Distribute 15mL into 100mm culture dish and cool the plates until it

solidifies and store in 4oC fridge for future use.

Plates were incubated for 16 hours in a 37oC incubator (Materials #40) and 10 isolated

colonies were selected for screening. Stab cultures of the colonies were incubated for 16 hours

(37oC, 300 rpm) in 3mL of amp-added culture media. Plasmid isolation from each stab culture

was then conducted using the same method explained in section 3.1.2.

3.1.5 pRTS-1-X Orientation Diagnostic Digest

Due to the nature of Sfi I digests, gene inserts may be ligated in either direction into the

empty pRTS-1 plasmid, thus, diagnostic digest was performed on each pRTS-1-X construct to

confirm the orientation of the gene insert. The restriction enzymes (RE) were carefully selected

to cut once within the insert and once or twice in the plasmid. These DNA fragments were

fractionated using 2% agarose gel and the size of DNA fragments assessed. Table 3.1.5 presents

the RE used for each construct and their expected respective fragments released post-digestion:

41

Table 3.1.5 pRTS-1-X Diagnostic Digest - Orientation Confirmation of Gene Insert

Construct Restriction

Enzyme Correct Orientation Incorrect Orientation

pRTS-1-RUNX2

Not I

(Materials #26c) 7500bp, 10 800bp 6400bp, 12 000bp

Hind III

(Materials #26b) 700bp, 5540bp, 12 000bp 800bp, 5430bp, 12 000bp

pRTS-1-OSX Bgl II

(Materials #26a) 2600bp, 15 600bp 1450bp, 16 750bp

After orientation was confirmed, the quality and the quantity of the plasmids were

reviewed with a Nanodrop (Materials #41).

Next, plasmids were sequenced to screen for any mutations. 7µL (1-4µg/µL) of each

plasmid was combined with 0.7µL of 0.04 µg/µL forward or reverse sequencing primer and sent

to TCAG sequencing facilities (Materials #36) at Sick Kids Hospital.

Sequencing Primers

pRTS-1-seq-forward-1

5‟-TGA CCT CCA TAG AAG ACC G-3‟

pRTS-1-seq-forward-2

5‟-CTA TCA GTG ATA GAG AAA AG-3‟

pRTS-1-seq-reverse

5‟- ATG GCC TCA CTG GCC ATT-3‟

3.2 Cell Culture – General Procedures

α-MEM (Materials # 49) and D-MEM (Materials #48) culture media were prepared in

100mL aliquots with 10% FBS (Materials #53) and 10% antibiotics cocktail (Materials #45)

unless specified otherwise. Prior to use, all media were pre-warmed in a 37oC water bath. Cells

were plated at a density of 1x105 cells/mL in T75 cell culture flasks (Materials #69) unless

specified otherwise and culture media was replaced every Monday, Wednesday, and Friday of

42

the week. Cells were passaged upon 80% confluence and a 1:3 split ratio was used for

distribution.

Table 3.2.1 Cell Thawing Protocol

Step Description

1 Thaw 1mL cell vial in a 37oC water bath

2 Add 1mL of pre-warmed culture media and wait 2 minutes

3 Transfer cells to a 14mL round-bottom tubes (Materials #1)

4 Add 2mL of pre-warmed culture media and wait 2 minutes

5 Add 4mL of pre-warmed culture media (total of 8mL) and wait 2 minutes

6 Centrifuge for 5 minutes at 1500 rpm

7 Aspirate the supernatant

8 Resuspend the cell pellet with 8mL of culture media

9 Transfer 500µLof cell suspension into a cell counter isotonic solution (Materials #72) and determine cell

count

Table 3.2.2 Trypan Blue Cell Viability Protocol

Step Description

1 Prepare a 0.4% solution of trypan blue (Materials #58) in buffered isotonic salt solution, pH 7.2 to 7.3

2 Add 0.1 mL of trypan blue stock solution to 1 mL of cells

3 Load a hemacytometer (Materials #73) and examine immediately under a microscope at low

magnification

4 Count the number of blue staining cells and the number of total cells

5 % viable cells = [1.00 – (Number of blue cells ÷ Number of total cells)] × 100

3.3 HUCPVC Characterization

HUCPVCs (Materials #43) characterization was conducted with two different α-MEM

culture media and they were prepared with 10% fetal bovine serum (Materials # 53) from two

separate lots: Hyclone (Cat# SH30397.03, Lot# K4M25240) and Hyclone (Cat# SH30396.03,

Lot# KPJ22093). HUCPVCs from cords 0408003, 0508001, 0508002, 0508003 were used and

plated onto 6-well culture plates (Materials #66) at a cell density of 10 000 cells/well. Culture

media was replaced every Monday, Wednesday, and Friday. Cells were passaged at 80%

confluence and the dates were recorded and compared between cords.

43

3.4 Hygromycin B Kill Curve of HUCPVC, RBMC, ROS cells

A kill curve determines the lowest possible concentration of antibiotic as well as the time

it takes to kill all untransfected cells. In order to separate transfected cells from the untransfected

cells, hygromycin B antibiotic (Materials #77) was used as the selection pressure. Hygromycin B

is an antibiotic that inhibits the growth of eukaryotic cells by interfering with protein synthesis.

HUCPVCs, RBMCs (Materials #46), and ROS cells (Materials #45) do not naturally carry the

hygromycin B resistance gene and fail to survive under its selection pressure. The pRTS-1

plasmid contains the hygromycin B resistance gene and it encodes the hygromycin B

phosphotransferase (HpH). Cells transfected with the pRTS-1 plasmid have a selective advantage

and thus, survives under selection pressure.

Three 6-well plates (Materials #66) were labeled as HUCPVCs (cord #0508003), RBMCs

or ROS cells and a 3 mm x 3 mm area was marked below each well. These areas were marked to

ensure consistent daily data collection. Ten thousand cells were seeded into each well of their

respective plates and cultured to 80% confluence in the absence of hygromycin B. Upon 80%

confluence, corresponding hygromycin B concentrations were added to the culture media and

replaced daily. The marked region was photographed daily and cell counts of live cells were

gathered over ten days or until all cells in the region were lost.

Table 3.4 Hygromycin B Concentration Levels for Kill Curve

Well Number HUCPVC (µg/mL) RBMC (µg/mL) ROS (µg/mL)

1 0 0 0

2 5 25 25

3 10 50 50

4 25 100 100

5 50 200 200

6 1000 1000 1000

44

3.5 Transfection

Five different methods were used to transfect the pRTS-1-X constructs into three cell

types and they are listed in the chronological order followed in this thesis:

3.5.1 Amaxa Nucleofector II

Nucleofection is an electrical transfection method where the negatively charged

DNA is delivered into the cell nucleus by an electrical current.64

All nucleofections were

conducted in accordance to the protocols supplied by the nucleofection kit. Each

transfection was completed with trypsinzed 1x106 cells in Nucleofection cuvette unless

specified otherwise.

First sets of nucleofection experiments were conducted with the Cell Line

Optimization Nucleofector Kit (Materials #78). All optimizations were completed with

either 2µg of pmaxGFP (3.4kbp) control plasmid (Materials #78) or 2-5µg of pRTS-1-

Luc (18.5kbp) plasmid (Materials #30). Optimization programs were tested with both

solution L and V transfection reagents. Transfected cells were transferred into 6-well

plates containing culture media and incubated for 24 hours at 37oC and 5% CO2 to allow

cell recovery. After 24 hours of incubation, the culture media was replaced with media

containing the optimized hygromycin B concentration of 50µg/mL. At this point,

transfected cells were treated with doxycycline (Materials #79) at 500ng/mL. Cells were

observed daily using a fluorescence microscope (Materials #85) to check cell viability

and GFP expression. Finally, transfection efficiencies were visually approximated using

GFP expression 24 and 144 hours after addition of dox. Furthermore, upon confluence,

activated cells were sorted using Fluorescence Activated Cell Sorting (FACS)

(Materials #88) and only the GFP expressing cells were passaged.

45

Fluorescence Activated Cell Sorting (FACS)

In this research, cells were sorted using GFP, which is expressed only in pRTS-1

transfected cells. GFP sorting uses an Argon laser at 488nm excitation which

fluorescence GFP and is captured using a 525nm band pass filter of the photomultiplier

tube (PMT).

First, the passaging cells and negative, untransfected control cells were diluted to

1x106 cells/mL in α-MEM. Then the cell debris was excluded by gating 80-90% of cell

suspension based on granularity and size. Subsequently, a 1% baseline for GFP

expression was set using the negative control and using this baseline, each transfected

cell suspension was sorted from non-GFP expressing cells.

The second set of nucleofections was completed using the Human Mesenchymal

Stem Cell Nucleofector Kit (Materials #78). This kit used a different nucleofection

buffer solution and applied four different programs (A-020, C-017, P-016, and X-001).

In addition to these differences, the following suggestions were made by an Amaxa‟s

technical support to increase transfection efficiency:

a. Increase the number of cells to be transfected from 1x106 to 3x10

6 cells.

b. Increase the amount of DNA to 10 or 20 µg per reaction.

c. Perform a recovery step post-nucleofection – Transfer transfected cells

into an eppendorf tube immediately and incubate for 10 minutes prior to

plating.

d. Activate and apply selection pressure after 6 hours instead of 24 hours.

e. Check cell viability and transfection efficiency at 6/24/48 hour mark.

Cells were observed for 72 hours using fluorescence microscopy to check cell

viability and level of GFP expression.

46

3.5.2 Invitrogen Lipofectamine 2000

Lipofectamine is a chemical transfection reagent that utilizes cationic lipids to deliver

DNA into the cytosol. Lipofectamine 2000 (Materials #81) protocol for adherent mammalian

cells was followed and scaled up to a 35-mm culture dish. In this protocol, HUCPVCs were first

cultured to 80% confluence in the absence of any antibiotics. Next, the lipofectamine reagent

was combined with 2µg of pRTS-1-Luc plasmid and incubated for 45 minutes at room

temperature prior to transfection. The cultured media was then replaced with the lipofectamine-

DNA complex and incubated for 5 hours. Transfected cells were induced and placed under

selection pressure after 5 hours and observed daily.

3.5.3 Qiagen SuperFect

SuperFect is a chemical transfection reagent that generates a DNA-nanoparticle complex

which is then taken into the cell through nonspecific endocytosis. Qiagen SuperFect‟s (Materials

#82) standard transfection optimization protocol for adherent cells was conducted on HUCPVCs

and RBMCs in a 24-well culture plate (Materials #64). Four different parameters were studied

for this optimization process: (1) initial number of cells, (2) amount of DNA, (3) DNA to

SuperFect reagent ratio, and (4) the incubation period. Transfected cells were induced and placed

under selection pressure immediately after transfection.

47

(1) 2x104/4x10

4/6x10

4/8x10

4 cells were transfected with 1µg of DNA at 1:5

DNA to SuperFect ratio and incubated for 4 hours.

(2) & (3) 6x104 cells were transfected using the following amount of DNA and

DNA to SuperFect ratio (Table 3.5.4) and incubated for 4 hours:

Table 3.5.3 SuperFect Optimization Experiment (DNA and DNA:SuperFect Ratio)

Amount of DNA 1:2 Ratio 1:5 Ratio 1:10 Ratio

1µg DNA - 1 µg

SF - 2 µg

DNA - 1 µg

SF - 5 µg

DNA - 1 µg

SF - 10 µg

2µg DNA - 2 µg

SF - 4 µg

DNA - 2 µg

SF - 10 µg

DNA - 2 µg

SF - 20 µg

4µg DNA - 4 µg

SF - 8 µg

DNA - 4 µg

SF - 20 µg

DNA - 4 µg

SF - 40 µg

(4) 6x104 cells were transfected using 1µg of DNA at 1:5 ratio and incubated for

1/2/4/8/16 hours.

3.5.4 Clonetech Xfect

Similar to SuperFect, Xfect (Materials #83) was the final chemical transfection

reagent that was used in this research thesis and it functions by generating a DNA-

Nanoparticle complex, which is taken up by the cell. HUCPVCs were first transfected

for 4 hours using the optimization protocol at 80% confluence with 5µg of pRTS-1-X

plasmid. A second transfection was conducted on HUCPVCs, RBMCs, and ROS cells at

<50% confluence with 5µg of pRTS-1-X plasmid. Transfected cells were induced and

placed under selection pressure immediately after transfection.

3.5.5 Bio-Rad Gene Pulser MX Cell Electroporation System

The MX Cell electroporation system utilizes an electrical current to force the

negatively charged DNA into the cell. The first optimization protocol was performed on

four different cell types: human Bone Marrow cells (Materials #44), HUCPVCs,

RBMCs, and ROS cells. The optimization protocol used a preset 96-well program with a

square wave pulse and performed both voltage gradient and duration gradient

48

experiments in triplicates (Figure 3.5.6). The set parameters of V-grad (100-400V) were

1000Ω resistance, 2000µf (capacitance), with a 20ms pulse duration. The set parameters

of the D-grad (10-30ms) were 1000Ω resistance, 2000µF (capacitance), and 250 volts.

This program was applied to 100µL of 1x106cells/mL with 20 µg/mL of DNA

suspended in Opti-MEM (Materials #54).

The second optimization experiment was conducted to determine the optimal

DNA concentration for transfection. The set parameters were 250 volts, 15ms, 2000µf,

1000Ω, with a square wave at a cell density of 1.5x106cells/mL. Eight different plasmid

concentrations were tested (0.5, 1, 2, 4, 8, 10, 20, 40 µg/mL) with RBMCs.

The final transfection experiment was performed to generate RBMC- and ROS-

pRTS-1-X cell systems. Twelve repeats of both cell types with each construct were

Figure 3.5.5 Diagram of the V-grad/D-grad Program – Diagram depicting the optimization protocol

experimental parameters. Rows A & E – hBM // B & F – HUCPVC // C & G – RBMC // D & H - ROS

V-grad

(100Volts)

V-grad

(200Volts)

V-grad

(300Volts)

V-grad

(400Volts)

D-grad

(10ms)

D-grad

(15ms)

D-grad

(20ms)

D-grad

(30ms)

49

completed. The set parameters were 250 volts, 15ms, 2000µF, 1000Ω, with a square

wave at a cell density of 1.5x106cells/mL. Immediately following transfection, cells

were transferred to a 96-well culture plate. Cells were activated with dox 500ng/mL

(Materials #79) and placed under hygromycin B 50µg/mL (Materials #77) selection

pressure. Transfection efficiency and cell viability was observed after 24 hours.

3.6 Transfection Efficiency Analyses

Transfection efficiencies were analyzed in two ways. The first and more commonly used

method was a visual approximation of the GFP expression under a fluorescence microscope. As

stated earlier, transfected cells were activated immediately or between 4 to 24 hours post-

transfection. Cells were observed on a regular 24 hour interval unless specified otherwise. Three

positions of each well of each transfection were marked and photographed in visible and blue uv

light (FITC – 488nm). Images were manually quantified to approximate transfection efficiency.

Second and more accurate transfection efficiency were conducted between passages using FACS

as described in section 3.5.1.

3.7 Single clone Isolation

Single clones were isolated from cells transfected with the Bio-Rad electroporator

(Materials #84). Transfected RBMCs and ROS cells were selected for 7 days and 12 clones were

isolated into a 96-well plate (Materials #62) through limited dilution. Clones were cultured to

confluence under selection and visually inspected for GFP expression. Three clones with the

brightest GFP expression were selected and cultured for all subsequent experiments.

50

3.8 mRNA Expression

Clones of each cell-construct combination were visually inspected to isolate clones that

retained GFP expression over time. Viable clones were cultured to 80% confluence in T75

culture flasks. The three most active clones of each construct were selected and lysed using 4mL

of cell lysis reagent (Materials #89). mRNA was then extracted using a Sigma-Aldrich GenElute

Direct mRNA Miniprep Kit (Materials #90). The extracted mRNA was converted into cDNA

using High Capacity RNA-to-cDNA Master Mix with „No RT Control‟ (Materials #91).

Constructed RT-cDNAs and No RT-cDNAs were PCR amplified using primers designed

specifically to contain a part of our gene as well as the 3‟ UTR within the plasmid. The amplified

product is approximately 150-300bp and this specific design ensured that, if present, the

amplified product was a result of our cDNA not a contamination by genomic DNA. At the same

time as the RT and No RT control, β-actin was replicated as a positive control. The following are

the sequences of the primers designed for confirmation.

BMP2-cDNA

Forward: CAT GCC ATT GTT CAG ACG TT

Reverse: CAG GTC GAG GGA TCT CCA TA

RUNX2-cDNA

Forward: CCA GAA TGA TGG TGT TGA CG

Reverse: CAG GCG TAC GGG ATC TTC

SP7-cDNA

Forward: GGA AGA GGA GGC CAG TCA G

Reverse: AGG CGT ACG GGA TCT TCC

VEGF-A-cDNA

Forward: CAG CGG AGA AAG CAT TTG TT

Reverse: CAG GTC GAG GGA TCT CCA TA

51


To detect the expression of our gene at a protein level, the conditioned media of BMP2

and VEGF-A were collected and whole cell lysates of SP7 and RUNX2 were collected using

4mL of cell lysis reagent (Materials #89) with 25µL of Protease Inhibitor Cocktail (Materials

#92).

The protein extracts were separated on a SDS-polyacrylamide gel using a western blot.

The proteins were separated on a 12% PAGE (BMP2, OSX, RUNX2) or 15% PAGE (VEGFA)

for 1 hour and 10 minutes at 120V, then blotted onto an Immobilon-P membrane (Materials

#109) using a semi-dry transfer apparatus (Materials #121) at 100mA per gel for 1 hour.

Once transferred, the membrane was blocked with 10% skim milk powder (Materials

#115) in TBS-T (Materials #117) at 4oC, overnight, to prevent non-specific binding. The

membrane was rinsed twice with TBS-T, and then incubated with the corresponding primary

antibody (Materials #112) for 1 hour with 25% skim milk powder in TBS-T. Unbound primary

antibody was removed from the membrane and washed six times with TBS-T for 5 minutes each

time. The membrane was then incubated with the corresponding secondary antibody (Materials

#107/108) for 1 hour with 25% skim milk powder in TBS-T. Unbound secondary antibody was

removed from the membrane and washed six times with TBS-T for 5 minutes each time. The

HRP signal was activated using an ECL western blot detection kit (Materials #96/104) and the

chemiluminescent signal was captured on an 8” x 10” X-ray film (Materials #100). The X-rays

were developed and scanned into the computer. For dose-response and kinetic studies, westerns

blots were quantified using the ImageJ software (Materials #119) and plotted in Excel.

52

3.10 Doxycycline Dose-Response

To determine the dose-response, stable clones were cultured without doxycycline for

more than 18 days to stop GFP expression. These clones were then plated into 6-well plates at a

density of 1x104 cells/well and cultured to 80% confluence and activated with 0, 50, 100, 250,

500, and 1000ng/mL of doxycycline. The conditioned media (BMP-2) or the cell lysates

(RUNX2 and SP7) were collected after 24 hours. Using the methods described in section 3.9,

western blots were completed and quantified through imageJ to analyze the dose-dependent

effects of doxycycline concentration.

3.11 Induction Kinetics

For short term induction analysis, non-induced transfected cells were plated into a 6-well

plate and cultured to 80% confluence. Transcription of all constructs was activated using

500ng/mL of doxycycline. Either conditioned media (BMP-2) or cell lysates (OSX and RUNX2)

were obtained at 0, 1, 2, 4, 8, 24, 72, and 144 hour time points. Samples at each time points were

collected and preserved in the -80oC freezer until all time points were obtained. Using the

methods described in section 3.9, western blots were completed. Each western blot contains

samples from all six time points and one sample of cells that have always been exposed to

doxycycline as the positive control lane. This blot was then quantified through imageJ and the

induction kinetics was studied.

For long term induction study, a preliminary study was completed. Doxycycline was first

removed from the culture media of pRTS-1transfected RBMC cells. The GFP expression was

observed daily until the signal diminished and cells were re-exposed to doxycycline.

53

3.12 Alkaline Phosphatase Assay

To verify the osteogenic effects the pRTS-1-X constructs should induce, an alkaline

phosphatase activity assay in C2C12 (Materials #133) cells. Two types of ALP assay were

completed. The first method transferred a concentrated conditioned media of the activated cells

to an 80% confluent C2C12 plate. The second experiment was to co-culture the activated cells

along with the C2C12 cells. For all C2C12 assays, the media of the transfected cells were

changed to remove hygromycin B as it would kill the C2C12 cells upon transfer.

10,000 C2C12 cells were first plated and cultured to 80% confluence. Next, 4mL of

conditioned media of all cell-construct combination was placed into an Amicon Ultra Centrifugal

Filter (30k) device (Materials #97) and centrifuged for 10 minutes at 13, 257 rcf. The

concentrated conditioned media was then transferred to the C2C12 cells with 50 µg/mL of L-

Ascorbic Acid (Materials #126). Additionally, rhBMP2 200ng/mL (Materials #132), conditioned

media from pRTS-1-Luc transfected cells, and conditioned media from untransfected cells were

tested on C2C12 cells as controls.

After 3 days of incubation period the cells were rinsed twice with PBS and fixed twice

with 10% formalin (Materials #123) and twice with 4% Paraformaldehyde (Materials #128) for

10 minutes each. Cells were then rinsed again with PBS and dH20 and incubated in ALP solution

(Materials #123/124) for 30 minutes. The colour development was stopped by rinsing the cells

with PBS and dH20.

In the co-culture experiment, 1x105 cells of each C2C12 and stably transfected cells were

plated into 6-well plates and cultured for 3 days in culture media without hygromycin B. After 3

days of incubation, the same fixation and development protocol was conducted.

54

Chapter 4 – Results

This chapter reports all of the results from the pRTS-1-X plasmid transfections. First, the

construct verification is reported followed by characterization of the three cell types used in this

research. The variation between the different cords of HUCPVCs is first characterized followed

by the hygromycin B kill curve of all three cell lines. Next, the results of all five transfection

methods are presented. This includes the difference between transient and stable transfection as

well as the affected proliferation rate due to transfection. Expression of transfected genes in

stable isolated clones is then analyzed at mRNA and protein levels. Finally, results of the

induction kinetics as well as the doxycycline dose-response studies are reported and the section

concludes with an analysis of alkaline phosphatase activity in the transfected cells.

4.1 pRTS-1-X Construct Verification

pRTS-1-X constructs were generated using the methods stated in section 3.1. As seen in

the figure 4.1.1, the 3‟ overhang that exists after removal of the luciferase gene by digestion with

Sfi I consist of three bases, CAC, on both ends of the pRTS-1 plasmid and do not complement

each other. This ensures that the plasmid cannot be religated without an insert that complements

the 3‟ overhang.

>>>>>>

5‟---GGCCTCAC TGGCC------luciferase-------GGCCAGTG AGGCC---3‟

3‟---CCGGA GTGACCGG--------------------------CCGGT CACTCCGG---5‟

<<<<<<

GGCCTCAC AGGCC

GGCCA CACTCCGG

Figure 4.1.1 Sfi I Restriction Sequence in the pRTS-1-Luc Plasmid and its Unique 3’ Overhang – Sfi I

restriction sites are indicated in red; the resulting 3‟ overhang (Blue) prevents self relegation of the linearized

plasmid.

3‟

3‟

55

The disadvantage of having a Sfi I restriction site is that the gene of interest can be

inserted in either direction and as a result, the correct orientation must be confirmed through

diagnostic digests and sequencing (See section 3.1.5).

g

Cloning was accomplished using the techniques mentioned in section 3.1. The pRTS-1-

RUNX2 and pRTS-1-SP7 plasmids were confirmed to have the correct size and orientation of

insert (figure 4.1.2). Plasmids were further verified for point mutations through sequencing. The

quality of sequencing data for the pRTS-1-VEGF-A plasmid returned below threshold and thus

could not be confirmed.

4.2 HUCPVC Characterization

HUCPVCs from four different cords were tested to see if there is variability between

cords. Cells exhibited “fibroblast-like” appearance with stellate shape and long cytoplasmic

processes. After culturing HUCPVCs for over 10 passages, the only notable difference found

Figure 4.1.2 Confirmation of Insert Orientation by Diagnostic Digest – (A) Uncut pRTS-1-Luc, (B) pRTS-1

digested with Sfi I, (C) Sense orientation of pRTS-1-RUNX2 digested with Not I, (D) Antisense orientation of

pRTS-1-RUNX2 digested with Not I, (E) Sense orientation of pRTS-1-RUNX2 digested with Hind III, (F)

Antisense orientation of pRTS-1-RUNX2 digested with Hind III, (G) Sense orientation of pRTS-1-OSX digested

with Bgl II, (H) Antisense orientation of pRTS-1-OSX digested with Bgl II, (I) Sense sized insert of VEGF-A

digested with Sfi I.

H G

1450bp

2600bp

1600bp

2000bp

1500bp

1000bp

500bp

3000bp 4000bp 5000bp 6000bp 8000bp

10000bp

A B

2000bp

1500bp

1000bp

500bp

3000bp 4000bp 5000bp 6000bp 8000bp

10000bp

C D

7500bp

6400bp

2000bp

1500bp

1000bp

3000bp

4000bp

5000bp

6000bp 8000bp

10000bp

F E

5540bp 5430bp

700bp

800bp

pRTS-1 RUNX2 OSX VEGF-A

200bp

300bp

400bp

500bp

650bp

850bp

1000bp

1650bp

2000bp

5000bp

12000bp

100bp

500bp

I

56

was the number of passages the cells survived. Cells from cords 0508001 proliferated past 10

passages while cells from cord 0408003 did not proliferate past the 6th

passage.

4.3 Hygromycin B Kill Curve of HUCPVC, RBMC, and ROS cells

The lowest concentration of hygromycin B to kill all non-transfected cells was

determined as described in section 3.4. The results are as follows:

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

Ap

pro

xim

ate

Pe

rce

tage

of

Via

ble

Ce

lls

HUCPVC Kill Curve (Hygromycin B)

0 µg/mL

5 µg/mL

10 µg/mL

25 µg/mL

50 µg/mL

1000 µg/mL

Figure 4.3.1 HUCPVC Kill Curve - The minimal hygromycin B concentration to kill all cells within 7

days was determined to be 50 µg/mL.

Figure 4.2 HUCPVC characterization – (A) Cord 0408003, (B) Cord 0508001, (C)

Cord 0508002, (D) Cord 0508003. Minimal variability in cell proliferation was

observed between cords.

57

All HUCPVCs were killed within four days of period at 50µg/mL while RBMCs and

ROS cells were killed in seven days using 25µg/mL and 50µg/mL respectively. As a result, all

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

Ap

pro

xim

ate

Pe

rce

tage

of

Via

ble

Ce

lls

RBMC Kill Curve (Hygromycin B)

0 µg/mL

25 µg/mL

50 µg/mL

100 µg/mL

200 µg/mL

1000 µg/mL

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

Ap

pro

xim

ate

Pe

rce

tage

of

Via

ble

Ce

lls

ROS Kill Curve (Hygromycin B)

0 µg/mL

25 µg/mL

50 µg/mL

100 µg/mL

200 µg/mL

1000 µg/mL

Figure 4.3.2 RBMC Kill Curve – The minimal hygromycin B concentration to kill all cells within 7

days was determined to be 25 µg/mL.

Figure 4.3.3 ROS Kill Curve - The minimal Hygromycin B concentration to kill all cells within 7 days

was determined to be 50 µg/mL.

58

selection processes for transfection experiments were completed with 50µg/mL of hygromycin B

to select for cells transfected with the pRTS-1 vectors.

4.4 Transfection

Three critical factors were studied for each transfection method: cell survival post-

transfection, transfection efficiency, duration of expression after transfections (i.e., transient vs.

stable transfections).

4.4.1 Amaxa Nucleofector II

Initial transfections of HUCPVCs with the pRTS-1 plasmid using the optimization kit

showed that the program used for each transfection has an impact on cell survival. Programs A-

020, T-020, T-030, X-001, X-005, L-029, and D-023 were tested with both Nucleofection

solutions (L and V). Cell survival post-transfection with the pRTS-1 Vector is as follows (figure

4.4.1.1):

59

Figure 4.4.1.1 Phase Contrast of Cells 24 Hours Post-Transfection and Fluorescence after 24

Hours of Doxycycline Induction – HUCPVCs transfected using Nucleofection with the large pRTS-

1 vector (18.5kbp) kills large number of cells in both solution L and V.

Nucleofection Solution V

Program Phase-Contrast (24 hours post transfection)

Fluorescent (24 hours post dox induction)

Phase-Contrast (24 hours post transfection)

Fluorescent (24 hours post dox induction)

Nucleofection Solution L

A-020

D-023

L-029

T-020

T-030

X-001

X-005

No Pulse

60

In comparison to the no pulse control, HUCPVC viability was compromised by all

programs suggested in the Optimization Kit (Materials #78). Minimal discrepancy was found

between solution L and V. GFP expression was not observed in any of the transfected cells after

24 hours of dox induction (figure 4.4.1.1). Cultures of all programs with the exception of X-001

were killed after four days of selection (figure 4.4.1.2).

Program X-001 with solution V produced marginally higher cell viability than solution L.

GFP fluorescence was observed after four days of exposure to doxycycline in both solutions L

and V cultures (figure 4.4.1.3). Cells from solution V did not proliferate and died after 9 days of

selection, indicative of low, but transient transfection efficiency.

Program

Phase-Contrast (4 Days post-selection)


A-020

D-023

L-029

T-020


Phase-Contrast (4 Days post-selection)



Program

Figure 4.4.1.2 Phase Contrast of HUCPVCs after Four Days of Selection – Programs A-020, D-023, L-029, T-

020, T-030, and X-005 transfected cells did not produce viable cells after four days of selection. X-001 and the no

pulse control cells proliferated post-selection.

X-005

T-030

X-001

No Pulse

61

HUCPVCs transfected in solution L with program X-001 began expansion after 20 days

of selection but GFP expression decreased over every passage (figure 4.4.1.4). The first passage

of cells was from a 10cm culture dish to a T75 flask and was excluded from FACS. In

subsequent passages, cells were sorted through FACS upon confluence to enrich the GFP

expressing population of cells and cultured in T75 flasks.

Figure 4.4.1.4 Diminishing GFP Expression of Nucleofected HUCPVCs – Cells transfected with the pRTS-

1-Luc plasmid in solution L with program X-001 proliferated after 20 days but FACS indicates a diminished

GFP expression at every passage.


X-001


Phase-Contrast Fluorescent Phase-Contrast Fluorescent Program

Figure 4.4.1.3 HUCPVCs transfected (Program X-001) with the pRTS-1 Plasmid Expressing

GFP – GFP is observed from transfected cells after being exposed to doxycycline for four days.

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8

Pe

rce

nta

ge o

f G

ate

d G

FP E

xpre

ssin

g C

ells

(%

)

Passage Number

Diminishing GFP Expression of HUCPVCs Transfected with pRTS-1 Plasmid in Solution L Using Program X-001

HUCPVC(X-001,Solution L)

Passage #1: - 25 days after transfection. - Cells passaged from 10cm culture dish into T75 flasks

Passage #2: - 31 days after transfection. - Cells passaged into new T75 flasks

36 day

40 days

44 days

49 days

55 days

59 days

62

A second set of Nucleofection was completed with the Human Mesenchymal Stem Cell

Kit. As discussed in section 3.5.1, modifications were made to the protocol in attempts to

increase cell viability and transfection efficiency. Nucleofection results of the MSC kit (figure

4.4.1.5[A-D]) using the pRTS-1-Luc plasmid were similar to the results of the optimization kit

(figure 4.4.1.1). Cell viability post-transfection was negligible even with the increased number of

initial cells. Conversely, transfection with the control plasmid (3kbp) produced high transfection

efficiency.

Figure 4.4.1.5[A] Modified MSC Kit Nucleofection Protocol with Program A-020 – Despite a three-

fold increase in the initial number of transfected cells, cell viability and transfection efficiency of the

pRTS-1 plasmid is low.

24 Hours Post-Nucleofection

Control Plasmid


Phase-Contrast Fluorescent Phase-Contrast Fluorescent Plasmid

10µg of pRTS-1 Plasmid


[A] MSC Kit – Program A-020

63


Control Plasmid





[B] MSC Kit – Program C-017

Control Plasmid



[C] MSC Kit – Program P-016

Figure 4.4.1.5[B-C] Modified MSC Kit Nucleofection Protocol with Program C-017 [B] and P-016 [C]

– Despite a three-fold increase in the initial number of transfected cells, cell viability and transfection

efficiency of the pRTS-1 plasmid is low.

64

In figures 4.4.1.5[A-D], the cell viability after 24 hours appears to be higher in

comparison to the optimization kit (figure 4.4.1.1) but this may be the result of a three-fold

increase in the number of cells transfected. Thus the images presented in figure 4.4.1.5 [A-D] do

not give a correct visual representation of cell viability. It is also important to note the brightness

of GFP between the control plasmid and the pRTS-1-Luc plasmid. HUCPVCs transfected with

the control plasmid exhibit bright GFP fluorescence for all programs but the pRTS-1-Luc

transfected cells have minimal GFP fluorescence. Table 4.4.1 is a summary of the transfections

completed with Amaxa‟s Nucleofector.


Control Plasmid





[D] MSC Kit – Program X-001

Figure 4.4.1.5[D] Modified MSC Kit Nucleofection Protocol with Program X-001 – Despite a three-

fold increase in the initial number of transfected cells, cell viability and transfection efficiency of the

pRTS-1 plasmid is low.

65

Table 4.4.1 Summary of Nucleofection Experiments

Date Parameters Result

March 3rd,

2009

Optimization Kit

Program: X-001

Cell Type: HUCPVC

# of Cells: 1.5x106

Plasmid Used: 2µg of pRTS-1

Nucleofection Solution: L and V

No surviving cells for solution L

Solution V shows low transfection

efficiency with massive cell death after 3

days

All cells are dead within 1 week with no

expansion

March 10th,

2009

Optimization Kit

Program: X-001

Cell Type: HUCPVC

# of Cells: 1.0x106


and 2µg of Amaxa Plasmid


Solution V – transient transfection (death

after 9 days)

Solution L – Proliferated but no GFP

expression seen until 4th day of induction

Transfection efficiency drops from 6% to

1% between 6 passages

Amaxa plasmid (3kb) shows high

efficiency

March 23rd,

2009

Optimization Kit

Program: A-020, T-020, T-030,

X-005, L-029, D-023

Cell Type: HUCPVC

# of Cells: 1.0x106



Massive cell death during transfection

No GFP expression

Surviving cells killed off during selection

period

May 28th,

2009

MSC Kit

Program: X-001

Cell Type: HUCPVC

# of Cells: 1.0x106


Low cell survivability post-transfection

No stable transfected cells found

August

17th, 2009

MSC Kit

Program: U-23, C-17, P-016, X-

001

Cell Type: HUCPVC

# of Cells: 1.0x106

Plasmid Used: 3µg of pRTS-1,

2µg of control plasmid(Amaxa)

Control plasmid has high survivability and

high efficiency through all programs

C-17, P-016 fails to have any viable cells

U-23 low cell survivability with single cells

expressing GFP after 5 days of selection

U-23 plate does not proliferate

March 29th,

2010

MSC Kit

Program: U-23, C-17, P-016, X-

001

Cell Type: HUCPVC

# of Cells: 3.0x106

Plasmid Used: 10µg, 20µg of

pRTS-1, 10µg of control

plasmid(Amaxa)

Control plasmid has high survivability and

high efficiency through all programs

Low cell survivability with all programs

using the pRTS-1 plasmid

No visible GFP expression

66

4.4.2 Invitrogen Lipofectamine 2000

Invitrogen‟s liposomal-based transfection method failed to achieve any transfection with

HUCPVCs. Cell viability was unaffected by the transfection but the cells were killed during

antibiotic selection. Furthermore, no visible GFP expression was observed after 24 hours of

exposure to doxycycline (figure 4.4.2). These results indicate that no transfection took place.

4.4.3 Qiagen Superfect

As described in section 3.5.3, four parameters were optimized: initial number of cells,

amount of DNA, DNA to SuperFect reagent ratio, and the transfection period. The cell density at

the time of transfection did not affect the results. Increasing the amount of DNA and the

Superfect-to-DNA ratio as well as the incubation period, all increased the transfection efficiency

without having an adverse effect on cell survivability. Superfect produced the highest

transfection efficiency with RBMCs of all methods used in this research and GFP expression was

observed as early as 12 hours post-activation (figure 4.4.3.2) while HUCPVCs showed minimal

transfection efficiency (figure 4.4.3.1). Under selection, HUCPVCs did not survive past 4 days.

RBMCs survived for seven days, which is approximately three days longer than selection, thus,

indicative of a transient transfection.

Figure 4.4.2 Lipofectamine 2000 Transfection of HUCPVCs – (A) HUCPVC is killed over four

days of selection pressure. (B) GFP fluorescence is absent post-transfection. The lack of GFP and

the rate of cell death suggest that no transfection was achieved.

Day 0

Day 0

(A)P

hase

-Contr

ast

(B)F

luore

scent

Day 1

Day 1

Day 2

Day 2

Day 3

Day 3

Day 4

Day 4

67

HUCPVC

RBMC

4.4.4 Clonetech Xfect

Initial transfections with the pRTS-1 plasmids were conducted at 80% confluence and

cells were placed under selection after four hours. GFP expression was observed in the first 12

hours of activation in RBMCs and ROS cells but not in HUCPVCs. Proliferation of ROS cells

were unaffected by Xfect while RBMCs resumed proliferation after two weeks and HUCPVCs

after three weeks post-transfection (figure 4.4.4.1). No GFP expression was found from Xfect

transfected HUCPVCs.

Figure 4.4.3.2 Qiagen SuperFect Transfection of RBMCs with Selection Pressure – (A) RBMCs are killed over seven days of selection pressure. (B) GFP fluorescence is visible as early as 12 hours post-activation but the overall number of cells decreases due to selection pressure. This data suggests a transient-only transfection was achieved.

12 Hours

12 Hours

Day 1

Day 1

Day 3

Day 3

Day 5

Day 5

Day 7

Day 7

(A)P

hase

-Contr

ast

(B)F

luore

scent

12 Hours

12 Hours

Day 1

Day 1

Day 2

Day 2

Day 3

Day 3

Day 4

Day 4

(A)P

hase-C

ontr

ast

(B)F

luore

scent

Figure 4.4.3.1 Qiagen SuperFect Transfection of HUCPVCs with Selection Pressure – (A) HUCPVCs are killed over four days of selection pressure. (B) GFP fluorescence is absent post-transfection. The lack of GFP and the rate of cell death suggest that no transfection was achieved.

68

g

Transfections Completed at 80% Confluent Culture Dish

(24 Hours Post-Transfection)

HUCVPC

Transfections Completed at <50% Confluent Culture Dish

(Three Weeks Post-Selection)

Phase-Contrast Fluorescent Phase-Contrast Fluorescent

Cell

RBMC

ROS

Figure 4.4.4.1 Xfect Transfected Cells Post-Selection – HUCPVCs do not proliferate but survive

past selection without GFP expression. RBMC and ROS cells express GFP post-selection and

continue to proliferate.

Figure 4.4.4.2 Long-Term Decrease in GFP Expression of Xfect Transfected RBMC and ROS –

Once the selection pressure is removed, both RBMCs and ROS cells lose GFP Expression over time.

One week Post-Transfection Five Months Post-Transfection

Phase-Contrast Fluorescent Phase-Contrast Fluorescent

Cell

RBMC

ROS

69

Cell viability was unaffected by the reagent but the level of confluence at the time of

transfection did affect the overall viability. Cells transfected at 80% confluence formed large

complexes that look like cell disintegration (figure 4.4.4.3). Under higher magnification, visible

changes to the cell morphology were observed. Transfected cells expressed GFP but massive cell

death was observed and cells began to peel off the culture dish. A secondary repeat of the

experiment confirmed this phenomenon for all cell types.

Repetition of the Xfect transfection using 50% confluence presented a different long-term

result for RBMC and ROS cells. The viability was unaffected by the chemical reagent and the

formation of large complexes and cellular disintegration was not observed (figure 4.4.4.2).

Transfection efficiency was approximately 10% after 24 hours of activation (figure 4.4.4.4).

Proliferation of ROS cells were unaffected by transfection but RBMCs began proliferation after

2 weeks of delay under selection. GFP expressing cells beyond the selection period were isolated

into single clones. These clones proliferated for a 5 month period under constant hygromycin B

Figure 4.4.4.3 Formation of Large Complexes and Cell Disintegration Caused by High

Cell Density during Transfection – Xfect transfection at high (80%) cell confluence cause

major changes to the cell.

(A)P

hase

-Contr

ast

(B)F

luore

scent

70

selection but once the selection was removed, the cells‟ GFP expression level diminished (figure

4.4.4.2).

4.4.5 Bio-Rad Gene Pulser MX Cell Electroporation System

The first optimization process using the preset 96-well program provided a wide range of

results between the different cell types. HUCPVC and human Bone Marrow (hBM) cells had no

cell attachment after transfection through all voltage and duration gradients while RBMC and

ROS cells produced viable cells. Transfected RBMC and ROS cells expressed GFP after 12

hours (figure 4.4.5.1) and proliferated within one week of transfection. From these cells the

following optimization data were obtained:

Table 4.4.5.1 – Bio-Rad Electroporation Voltage Gradient Results

Voltage (V) Results

100 Cells survived transfection, but, no live cells found after 8 days of selection

200 GFP expression found for ROS and RBMCs and passaged after 8 days of selection

300 GFP expression found for ROS and RBMCs and passaged after 8 days of selection

400 All cells were killed during transfection

Table 4.4.5.2 – Bio-Rad Electroporation Pulse Length Gradient Results

Duration (ms) Results

10 Low cell survivability (<1%)

15 High cell survivability (>50%)



(A)Phase-Contrast (B)Fluorescent

Figure 4.4.4.4 RBMCs Transfected with Xfect at 50% Confluence

Results in Low Transfection Efficiency – Transfection efficiency after 24

hours of dox induction is approximately 10%.

71

The result of the second transfection experiment indicated that 4 µg/mL of pRTS-1

plasmid is optimal for electroporation and no significant improvement was observed beyond this

concentration (figure 4.4.5.2).

Repetition of these experiments confirmed the optimum transfection condition to be a

square wave pulse at 250V for 15ms with 4 µg/mL of DNA in 1.5x106cells/mL. Using this

optimized condition, all pRTS-1-X constructs were transfected in both RBMC and ROS cells.

12 Hours Post-Transfection

Phase-Contrast Fluorescent

Cell

RBMC

ROS

Figure 4.4.5.1 Bio-Rad Transfected RBMCs and ROS Cells Express GFP after 12 Hours

0.5 µg/mL 1 µg/mL [DNA]

RBMC

2 µg/mL 4 µg/mL

8 µg/mL 10 µg/mL 20 µg/mL 40 µg/mL

Figure 4.4.5.2 Bio-Rad DNA Concentration Optimization Transfection – Transfection efficiency is

increased by increasing the transfecting DNA concentration to 4 µg/mL. Further increase appears to reduce

transfection efficiency.

72

These cells were isolated into stable clones and used for all analyses found in the following

sections.

4.5 Transient vs. Stable Expression and Production of Transfected Clones

All transfection methods mentioned in the previous sections achieved a certain degree of

transfection with the exception of lipofectamine 2000. In the electrical transfection method,

Amaxa‟s Nucleofector produced transient-only transfected HUCPVCs that did not proliferate

while Bio-Rad‟s electroporator produced stably transfected RBMC and ROS cells for all pRTS-

1-X constructs. From these stably transfected cells, individual clones were isolated through

limited dilution (figure 4.5.1 and figure 4.5.2 C&D) and they retained the GFP expression for 8

months in the absence of a selection pressure, thus, indicative of a stable transfection.

The two chemical transfection methods (excluding Lipofectamine) achieved higher

transient transfection efficiencies than electroporation methods. Xfect transfected RBMC (figure

4.5.2 A&B) and ROS cells proliferated and single clones were isolated. These clones were

believed to be stable but when the selection pressure was removed after 5 months, cells rapidly

lost GFP expression in the presence of doxycycline. Qiagen Superfect produced the highest

Figure 4.5.1 Single Clone Expansion of ROS-pRTS-1-BMP-2 After 13

Days – Image of a single ROS-pRTS-1-BMP-2 clone after 13 days of

expansion


73

transient transfection efficiency with RBMCs but was unable to proliferate. As a result, stable

single clone were isolated only from RBMC and ROS cells transfected by the Bio-Rad

electroporator.

4.6 Rate of Proliferation

The rate of proliferation of each cell type was affected by the plasmid and the method of

transfection. Transfections were performed with the pRTS-1 plasmid (~18.5kbp) and the GFP

control plasmid (3kbp). Following transfections, dates of cell passages were compared. The

control plasmid did not affect proliferation in any cell type for all transfection methods while

HUCPVCs transfected with pRTS-1 using Nucleofection (program X-001 in solution L) began

proliferation after 20 days of culture. HUCPVCs transfected with the pRTS-1 plasmid using

Xfect proliferated after 21 days while RBMCs proliferated after 14 days. RBMCs and ROS cells

transfected with electroporation showed no delay in proliferation post-transfection.

Figure 4.5.2 Xfect Transfected and Electroporated RBMC - Single Clone

Expansion – Xfect transfected RBMCs single clone isolation (A&B). Single clone

expansion of Bio-Rad transfected RBMCs (C&D).

A B

C D


74

4.7 mRNA Expression

It is important to note that all analyses from this point were conducted with RBMC and

ROS cells only as expansion of transfected HUCPVCs was not possible.

The PCR amplified 150-300bp fragments generated from cDNA of each clone are shown

in figure 4.7. At the mRNA level, transfected genes are expressed in both RBMC and ROS cells

upon induction. The quantity of expression may vary between individual clones but the specific

primer design ensures that the PCR products are from the pRTS-1-X plasmid and not from an

endogenous gene expression (figure 4.7).


Correct protein expression of BMP2 (45kDa), RUNX2 (57kDa), and SP7 (46kDa) was

obtained from dox exposed RBMC and ROS clones. Furthermore, clones not exposed to Dox

(after 18+ days) did not express the genes of interest at a detectable level (figure 4.8). Transcripts

of pRTS-1-VEGF-A transfected constructs were detected at an mRNA level but failed to be

detected at a protein level for both cell types.

Figure 4.7 mRNA Expression of All Genes from RBMC and ROS Clones – The expression level of

each gene of interest is low in comparison to the β-actin control. Visible bands in the no RT controls

indicate genomic contamination.

RT

-BM

P2

No R

T-B

MP

2

RT

-β-a

ctin

No R

T-β

-actin

RT

-RU

NX

2

No R

T-R

UN

X2

RT

-β-a

ctin

No R

T-β

-actin

RT

-β-a

ctin

No R

T-β

-actin

RT

-β-a

ctin

No R

T-β

-actin

RT

-SP

7

No R

T-S

P7

RT

-VE

GF

-A

No R

T-V

EG

F-A

ROS

RBMC

75

4.9 Doxycycline Dose-Response

Six different doxycycline concentrations were used to induce to RBMC-pRTS-1-X and

ROS-pRTS-1-X cells that have been cultured in the absence of doxycycline for more than 18

days. Cells were exposed to dox for 24 hours and the conditioned media or the cell lysates were

collected for western blot analyses to measure the dose-response (figure 4.9).

130

95

72

55

43

34

26

72 95

55

43

34

26

17

130 170

Figure 4.8 RBMC-pRTS-1-X and ROS-pRTS-1-X Western Blot

(cell lysates) -

(“+”, “-” indicates presence or absence of dox 500ng/mL)

A – ROS-pRTS-1-BMP2 (Size: 45 kDa)

B – RBMC-pRTS-1-BMP2 (Size: 45 kDa)

C – ROS- pRTS-1-RUNX2 (Size: 57 kDa)

D – RBMC-pRTS-1-RUNX2 (Size: 57 kDa)

E – ROS-pRTS-1-SP7 (Size: 46 kDa)

F – RBMC-pRTS-1-SP7 (Size: 46 kDa)

A C 130

95

72

55

43

34

26

B

72 95

55

43

34

26

17

130 170

170 130

95 72 55 43

34

26

D

E F

+

+ +

+ + +

ROS-BMP2 RBMC-BMP2 ROS-RUNX2 RBMC-RUNX2

ROS-SP7 RBMC-SP7

76

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6No

rmal

ize

d p

rote

in E

xpre

ssio

n (

%)

Doxycycline Concentration

ROS Cells Doxycycline Dose-Response (24 Hours)

BMP-2

RUNX2

SP7

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6No

rmal

ize

d p

rote

in E

xpre

ssio

n (

%)

Doxycycline Concentration

RBMC Doxycycline Dose-Response (24 Hours)

BMP-2

RUNX2

SP7

Figure 4.9 RBMC and ROS Cells Doxycycline Dose-Response after 24 Hours – A difference in GFP

expression is observed as the doxycycline concentration is increased. Transfected cells reach an upper

plateau of GFP expression at 24 hours with 500ng/mL of doxycycline.

Cell-Construct

0 n

g/m

L

50 n

g/m

L

100 n

g/m

L

250 n

g/m

L

500 n

g/m

L

1000

ng/m

L

Contro

l*

RBMC-pRTS-1-BMP2

RBMC-pRTS-1-SP7

ROS-pRTS-1-BMP2

ROS-pRTS-1-RUNX2

ROS-pRTS-1-SP7

RBMC-pRTS-1-RUNX2

* Cell lysates obtained

from the same clones

of each respective

constructs. Control

cells have been

continuously exposed

to 500ng/mL of dox

since transfection.

77

4.10 Induction Kinetics

The induction kinetics of the pRTS-1-X transfected RBMC and ROS cells were studied

using the conditioned media and the cell lysates at each of the six time points. Visible GFP

expression is achieved 8 hours post-induction with 500ng/mL Dox and a continuous increase in

GFP expression is observed up to 72 hours (figure 4.10.1).

78

0

20

40

60

80

100

0 Hour 1 Hour 2 Hours 4 Hours 8 Hours 24Hours

72Hours

144Hours

Pro

tein

Exp

ress

ion

re

lati

ve t

o 1

44

ho

urs

(%

)

Number of Hours Post Doxycycline Exposure

RBMC Induction Kinetics (500ng/mL)

BMP2

RUNX2

SP7

Poly. (Series4)

0

20

40

60

80

100

0 Hour 1 Hour 2 Hours 4 Hours 8 Hours 24Hours

72Hours

144Hours

Pro

tein

Exp

ress

ion

re

lati

ve t

o 1

44

ho

urs

(%

)

Number of Hours Post Doxycycline Exposure

ROS Induction Kinetics (500ng/mL)

BMP2

RUNX2

SP7

Poly. (Series4)

Figure 4.10.1 pRTS-1-x Transfected RBMC and ROS Cells Induction Kinetics (500ng/mL dox)

0 h

our

1 h

our

2 h

ours

4 h

ours

24 h

ours

72 h

ours

14

4 h

ou

rs

RBMC-pRTS-1-BMP2

RBMC-pRTS-1-RUNX2

ROS-pRTS-1-BMP2

ROS-pRTS-1-RUNX2

Cell-Construct

ROS-pRTS-1-SP7

8 h

ours

RBMC-pRTS-1-SP7

79

A preliminary study indicated that the GFP expression of pRTS-1-X transfected RBMCs

reached a basal level approximately 18 days (figure 4.10.2 [C]) after doxycycline was removed

from the culture media and 16 days for ROS cells. Once basal level was reached, cells were

exposed to 500ng/mL of doxycycline and stable clones began expressing GFP again within 24

hours (figure 4.10.2 [D]), indicating that GFP expression could be turned “off” and repeatedly

turned “on”. This was completed three times during the course of this thesis.

4.11 Alkaline Phosphatase Assay

Alkaline phosphatase assay was completed to check for increased osteogenic activity.

When the conditioned media (CM) was transferred from the culture flasks of pRTS-1-X

transfected ROS or RBMC cells to a C2C12 plate and incubated for three days, minimal ALP

activity was observed (figure 4.11.1). BMP2, RUNX2, and SP7 conditioned media generated

three or four small patches of cells where ALP activity was shown (figure 4.11.1[A-C]). The

controls – CM of pRTS-1-Luciferase and CM of non-transfected RBMC cells did not induce

ALP activity while the rhBMP-2 as positive control produced a clear ALP activity (figure 4.11.1

[D-F]).

Figure 4.10.2 pRTS-1 Transfected RBMC Doxycycline Kinetics – RBMC-pRTS-1 clone was exposed

to 500ng/mL to doxycycline to induce GFP expression within 12 hours [A&D]. Doxycycline was

removed from the culture media and GFP expression was lost in 18 days [C].

(A)P

hase

-Contr

ast

(B)F

luore

scent

12 Hours Post-Transfection

Dox Induced Stable Clone

Dox Removed Stable Clone

Dox Re-induced Stable Clone

B C D A

80

To test the autocrine and paracrine effects of transfected cells, C2C12 cells were co-

cultured with transfected RBMCs and ROS cells. In the co-culture ALP assays, RBMC-RUNX2,

RBMC-SP7, and RBMC-pRTS-1-Luc did not produce any visible ALP activity but RBMC-

pRTS-1-BMP2 co-cultured with C2C12 cells produced high ALP activity over the controls. The

ROS co-culture experiments presented strong ALP activity over all plates with no significant

distinction between the different pRTS-1-X constructs (figure 4.11.2).

Figure 4.11.1 Alkaline Phosphatase Assay (Conditioned Media):

(A) C2C12 cells exposed to RBMC-pRTS-1-BMP2 conditioned media

(B) C2C12 cells exposed to RBMC-pRTS-1-RUNX2 conditioned media

(C) C2C12 cells exposed to RBMC-pRTS-1-SP7 conditioned media

(D) C2C12 cells exposed to RBMC-pRTS-1-Luc conditioned media

(E) C2C12 cells exposed to untransfected RBMC conditioned media

(F) C2C12 cells exposed to rhBMP-2

A

D

B

E

C

F

81

Figure 4.11.2 Alkaline Phosphatase Assay (Co-Culture) – All ROS co-cultures produced strong ALP activity

with no difference from the controls. RBMC-pRTS-1-BMP2 co-culture produced high ALP activity in comparison

to all other constructs and controls.

RBMC ROS

Untransfected Cells

Co-Culture

pRTS-1-Luc

pRTS-1-BMP2

pRTS-1-RUNX2

pRTS-1-SP7

82

Chapter 5 – Discussion

Bone regeneration induced through direct delivery of rhBMP-2 remains problematic

because of the wasteful burst release from the carrier that creates the need for super-

physiological doses to treat non-union bone defects. Furthermore, as a result of such high doses,

there is an increased risk of complications and increased costs associated with this treatment.

Thus, it is clear that there is a need to improve the current inefficient use of expensive rhBMP-2.

By genetically engineering cells and engrafting them, a continuous expression of

osteogenic factors can be sustained.118

Furthermore, by implementing a tightly regulated control

mechanism to gene therapy, this thesis aimed to control the quantity and duration of osteogenic

factors present during bone regeneration and thus reduce the need for super-physiological doses.

Current literature has shown engraftment of genetically engineered mesenchymal stem

cell through viral transfections to be successful, although with certain limitations. Twenty six

research articles reviewed by Van Damme et al. presented transient (days) and long term (weeks

to months) expression of a transgene through viral transfections. There are five main groups of

viral vectors used for engineering: adenovirus, lentivirus, retrovirus, adeno-associated viruses

(AAV), and herpes simplex virus-1 (HSV-1).111,118

However, despite their potential for

successful gene therapy treatment, there are serious concerns related to immune response, lack of

viral vector specificity leading to dissemination of the vector, and insertional mutagenesis.111

Furthermore, viral vector preparation is a time-consuming process that requires high level of

safety.31

These issues make viral vector transfections an unsafe method of gene transfer for

engineering cells.

Alternatively, several groups attempted to address the super-physiological dose

requirement by engineering cells with the TET-On25

or TET-Off 59,82

regulation system to control

83

the expression of BMP-2 in vivo. Although all three papers have shown bone regeneration, two

TET-induced/repressed methods of transfection were completed through adenoviruses and

adeno-associated two vector viruses and suffer the same issues described previously.25,82

Moutsatsos et al. used non-viral multi-vector TET-Off system and transfected C3H10T1/2 MSC

cells using Lipofectamine to control the expression of BMP-2 in vitro and in vivo.79

The latter

demonstrated controlled expression of BMP-2 with MSC cells and showed bone regeneration but

this system contains two issues that can be improved: multi-vector transfection and high

background activity.

In this thesis, a new vector was utilized to control gene expression. The pRTS-1 vector is

a one-vector system that contains both the response and reporter elements of the TET-On

system.4 Furthermore, the inclusion of a TET-repressor lowers the background gene expression

and thus allows for a tighter regulation.

During the course of the current research, five different transfection methods, both

chemical and electrical, were tested on three different cell types. While some degree of

transfection efficiency was achieved by combining specific parameters with RBMCs and ROS

cells, the project has also exposed a number of problems. Detailed explanation and the beneficial

implications of this new system will be discussed below in the context of the experimental

results.

5.1 Development of Stable pRTS-1-BMP-2 Cell System is Only Achieved with

Electroporation Combined with RBMCs and ROS cells

Three cell types were selected for incorporating the pRTS-1-BMP-2 inducible system:

two primary cell types, HUCPVC and RBMC, and an immortalized cell line, ROS. The inducible

system was incorporated into cells using chemical and electrical transfection methods and the

84

results from these experiments present significant variations. These variations are dependent on

the cell type and the transfection method and they affect cell viability, cell properties, and

transfection efficiencies. Following is a summary (Table 5.1) of the transfection experiments and

their respective results:

Table 5.1 Summary of All Transfection Experiments in Chronological Order HUCPVC RBMC ROS

Amaxa

Nucleofector

II

Low viability Unexpected low viability and proliferation

problem Low transfection

efficiency

Proliferation delayed

(>2 week delay

before proliferation

resumes) or stopped

Loss of GFP

expression over

prolonged expansion

Invitrogen

Lipofectamine

2000

High viability Chemical transfection methods tested to improve

transfection efficiency No transfection

achieved

No viable cells after

selection

Qiagen

Superfect

High viability RBMC introduced to see if HUCVPC is the cause of low transfection efficiency

Low transfection

efficiency

High transfection

efficiency

Transient transfection (7 to 10 days)

No viable cells after selection

Clonetech

Xfect

High viability

Low transfection efficiency


(>3 week delay


resumes)


(>2 week delay


resumes)

Tested immortalized

cell line to see if

proliferation problems

could be overcome

Proliferation

unaffected by

transfection

Long-term transient transfection (~5months)

Bio-Rad Gene

Pulser MXcell

Electroporator

No viable cells

through all

parameters post-

selection

Low viability

Low transfection efficiency

Rate of expansion unaffected

Stable clones generated

Loss of expression over prolonged expansion

85

These results suggest that with the current available transfection methods, it is difficult to

implement the pRTS-1-BMP-2 inducible plasmid into cells and we suggest that these variables

are mainly associated with the large size of the plasmid (18.5+kbp). The following sections

discuss in detail the chemical and electrical transfection methods and the variable results found

in the experiments.

5.1.1 Chemical Transfection Methods

Reported in this thesis are three chemical transfection methods from Clonetech,

Invitrogen, and Qiagen. Protocols achieve transfection using different mechanisms and they

produce high cell viability but low transfection efficiency. This thesis has reported that chemical

transfections achieve a transient-only state with major negative changes to the cells.

The first significant observation was the low transfection efficiency. In comparison to a

near 100% transfection efficiency with the small 3kbp control GFP plasmid (pmaxGFP plasmid,

Amaxa Nucleofection Optimization Kit), the transfection efficiencies with the pRTS-1 based

plasmids were 0.01% for HUCPVCs and between 10-50% for RBMCs, and ROS cells. Initially,

no cellular disruptions were observed but the overall number of transfected cells decreased after

a hygromycin b selection pressure was applied. These data suggests an improper transfection or a

transient-only transfection with a period shorter than that of the selection period.

As a result, to enrich the number of transfected cells, fluorescence-activated cell sorting

(FACS) was conducted at every passage level to isolate and enrich the number of transfected

cells. HUCPVCs transfected with Lipofectamine 2000 or Superfect ceased to proliferate; thus,

only Xfect transfections were isolated using FACS. Despite continuous attempts to isolate and

expand the transfected cells, results from FACS show a decrease in GFP expression over each

86

passage level. These data suggest a loss of pRTS-1 plasmid from the cells and again confirm the

transient nature of chemical transfections.

The second significant observation was the diminished rate of proliferation of cells post-

transfection. Results indicated a delay or loss of proliferation for HUCPVCs while RBMCs

exhibited recovery after 14 days. This diminished rate was observed in all experimental repeats

and clones of RBMC and ROS. Furthermore, clones with resumed proliferation exhibited a

gradual loss of GFP expression over time.

Similar results were found in the literature. Hunt et al. transfected Human Umbilical Vein

Endothelial Cells (HUVEC), which are similar to HUCPVCs, with a 4.7kbp GFP-encoded

plasmid using nine different chemical transfection methods.44

Their results were similar to

HUCPVCs where the cell viability and the rate of proliferation diminished post transfection. The

authors suggested that the toxicity of the chemical reagents may alter the strength of the cell

membrane by generating reactive oxygen species, thus resulting in mass cell death.44

Upon

review, all three chemical transfections used in this thesis research contain proprietary chemical

reagents which alter cell properties for the purpose of gene insertion and an example of a change

to the cell membrane can be visualized from Xfect transfections. As seen in figure 4.4.4.3,

adjacent cells located close to one another forms large complexes. At higher magnification, these

cells appear to be disintegrated. This effect is observed when Xfect transfection is completed on

high density population of cells. The changes observed here and elsewhere44

suggest reagent

toxicity as a possible cause for low cell viability and diminished rate of proliferation.

With the chemical transfection methods currently available, stable transfections of

primary cells seem unlikely. Transient transfections may be achieved but the extremely low cell

survival makes this an inefficient approach. Furthermore, due to the transient nature of

87

transfected primary cells, even if sufficient transfection efficiency is observed at 24hours, the

signal rapidly decays when assayed at 48 hours. This was observed in HUVEC transfections44

and also in HUCPVC, RBMC, and ROS cells in this research. At the present time, regardless of

the cell type, chemically induced transfections are able to achieve only transient transfections

with the large pRTS-1 plasmid.

5.1.2 Electrical Transfection Methods

Two electrical transfection methods were used: Nucleofection and Electroporation. The

results of the methods showed variations. Cell type, plasmid concentration, and transfection

conditions were the key factors that impacted the overall cell viability and efficiency of

transfection.

5.1.2.1 Nucleofection

HUCPVCs, RBMC, and ROS were transfected with the large pRTS-1 plasmid (19+kbp)

and the small control plasmid (pmaxGFP) using eleven different programs of Amaxa‟s

Nucleofector II. Two specific transfection kits were used: Cell Line Optimization Nucleofector

Kit and the Human Mesenchymal Stem Cell Kit. Both of these protocols utilize a unique buffer

to aid in transfection but showed no significant difference in results.

In this study, all three cell types showed diminished proliferation rate post transfection

with the pRTS-1 plasmid but showed no significant difference with the control plasmid. After

four days of selection and induction process, only a small percentage of HUCPVCs survived the

hygromycin B selection process and expressed GFP (<0.01%), thus making nucleofection a non-

viable method of transfection.

88

Surviving HUCPVCs resumed proliferation after three weeks of inactivity and matched

the proliferation rate of a non-transfected HUCPVC. At passage number two, 6% of the cells

expressed GFP and were sorted using FACS to enrich the quantity of transfected cells.

However, by passage number eight, the number of GFP expressing cells decreased to 1.3% and

despite the continuous cell enrichment process, the expression level diminished rapidly after

each passage. These data suggest that nucleofection is unable to transfect our large plasmid to

produce stable cell clones.

As seen in Table 5.1, results of nucleofection show low viability and low transfection

efficiency. The results exhibited significant variation on cell viability in regards to the size of the

plasmid but a large difference was observed between programs. Transfection efficiencies of the

surviving cells were less than 1% with the pRTS-1 plasmid but near 100% with the control

plasmid. Furthermore, transfected cells exhibited a similar diminished proliferation rate as

observed with chemical transfection and could be explained again with Hunt et al.‟s suggestion

of chemical toxicity.44

As stated at the start of this section, two kits were used to transfect the

cells and they both contain proprietary chemical reagents which are known to be toxic to cells

upon prolonged exposure. These buffers are known to have detrimental effect on the cells and

transfection is suggested to be completed within 15 minutes of suspension in this solution.

Alternatively, because electroporation forces plasmids into cells via an electrical charge,

the strength of the charge or the size of the plasmid may damage the cell membrane and cause

the low initial viability. Upon review of the literature, the size of plasmid affects transfection

efficiencies and it has been suggested that a smaller DNA plasmid will improve cell

transfection.134

Despite nucleofection‟s enhancement of gene integration for long term

transfections, our results indicate that a stable transfection of pRTS-1 is unlikely and only a

89

transient transfection is possible and only under a strict set of conditions such as those used in

this research.

5.1.2.2 Electroporation

Electroporation is an alternate transfection method with an open-ended optimization

process. An electroporator differs by allowing the user to alter the type of pulse, duration,

voltage, and resistance of each transfection. Optimization of these parameters affected the cell

viability and transfection efficiencies of pRTS-1 transfections. We found that too low voltage,

duration, or plasmid concentration all resulted in high viability but low transfection efficiency

whereas parameters that were too high resulted in massive cell death.

The results of the pRTS-1 transfections varied depending on the plasmid as well as the

cell type. HUCPVCs transfection results were identical to nucleofection. Cell viability was less

than 0.01% with diminished proliferation post-transfection. For RBMC and ROS cells, the

viability was greater than 50% and the rate of proliferation was unaffected. These data suggested

a major difference between nucleofection and electroporation.

When the protocols are compared, the main difference is the reagent the cells are

suspended in prior to and during transfection. Electroporation is carried out in a relatively inert

OPTI-MEM whereas Nucleofection is conducted in a proprietary buffer created by Amaxa. This

implies that the buffer may have a toxic effect on the cell and if so, Hunt et al.‟s suggestion of

reagent toxicity44

and its effect on cell proliferation may be true.

The second variance of electroporation was the transfection efficiency. Twenty-four

hours post-transfection and exposure to dox, GFP was expressed in RBMC and ROS cells. After

72 hours of exposure to dox and selection, GFP expression was seen in the majority of the

90

surviving cells. This high expression level could be explained by the unaffected proliferation rate

and hygromycin B selection process. The selection process destroyed non-transfected cells while

the transfected cells proliferated with the resistance gene. Together, these two processes greatly

increased the number of transfected cells in 72 hours. Furthermore, the high level of GFP

expression reduced the need for FACS. Transfected cells sustained their GFP expression for 7

months before significant decrease in expression level was observed for all clones.

5.2 pRTS-1-BMP-2 Presents Low Background Expression and Doxycycline Dose-

Dependent Activation

The protein expression pattern observed in this research agreed with the data published

by Bornkamm et al.4 In the absence of dox, there is minimal background activity observed as a

result of the transcriptional silencer (tTS) and there is a doxycycline dose-dependent expression

level as observed in figure 4.9. These two observations highlight the hallmark benefits of the

pRTS-1 system, low background activity and control. In figure 4.9, the BMP-2 expression at 0

ng/mL of dox is minimal but as the concentration increases towards 1 µg/mL, an increase is

observed in all clones. This curve continues until the upper plateau is reached, where a maximum

expression level is achieved. These data coincides with those found previously.4, 30

5.3 pRTS-1 Controlled Kinetics

As discussed earlier in this chapter and by Bornkamm et al., clones exhibited variations

in the level and duration of inducibility. Clones of ROS-pRTS-1 and RBMC-pRTS-1 expressed

maximum GFP within 72 hours of exposure to doxycycline and lost its GFP expression upon

removal of doxycycline after 16-18 days. The relatively long deactivation period of GFP is due

to its long half-life and does not correspond to the expression kinetics of BMP-2.4 BMP-2 has a

91

much shorter half-life in vivo.54

Thus, even if the two genes are expressed at an equivalent level

through the bidirectional promoter36

, the effects of the BMP-2 gene will not last the same

duration as the GFP.

A preliminary study on re-inducibility was tested. The sustained inducibility over time

varied between different cells and clones4 but in the absence of dox, the gene of interest was not

expressed. These non-induced clones were then re-exposed to dox and again, cells achieved

expression in 72 hours and were suppressed in 16-18 days.

Subsequently, clones were continuously cultured in an activation-deactivation cycle for

eight months. These clones exhibited continuous but diminished activity near the end of this

period where the GFP expressing number of cells diminished rapidly. Current literature cannot

explain this limited stability; thus, additional work is required to better understand this

phenomenon.

5.4 pRTS-1 Bioactivity

Bioactivity of the pRTS-1 systems were verified through an alkaline phosphatase (ALP)

activity assay. ALP assay was used to study the characteristic changes to osteogenic protein

expression in C2C12 cells as a result of the inducible system.

As expected, the positive controls showed increased ALP activity. Activated RBMC-

pRTS-1-BMP-2 generated a significant increase in ALP activity over the negative control. The

ALP activity of both negative controls (RBMC and RBMC-pRTS-1-Luc) were minimal,

suggesting that dox-exposed RBMC-pRTS-1-BMP-2 could induce osteogenesis.

In contrast, high levels of ALP activity were observed from all dox-exposed and, non-

exposed ROS-pRTS-1-X cells, as well as the negative control. Kartsogiannis et al. states that

92

ROS generates a large amount of ALP activity due to its osteogenic nature57

and as a result, may

mask the effects of the dox-induced ROS-pRTS-1.

5.5 Experimental Limitations

Throughout this research thesis, our goal was to enhance gene therapy by developing a

tightly regulated, doxycycline dose-dependent cellular expression system that addressed two

major aspects of the tissue engineering triad. This system utilizes the strength of rhBMP-2in

conjunction with a novel source of stem cells, HUCPVCs, to control the expression at will. The

concept was groundbreaking and the potential for this system could be extended beyond

osteogenic gene therapy but several difficulties were faced through the course of this research

and as a result, the project turned into a proof of concept rather than a viable application for

human gene therapy.

The first major experimental limitation was the transfection. The pRTS-1 plasmid is a

large 18.5kbp plasmid which contains numerous benefits over the two-vector based Tet-On

system. However, the current methods of transfection have not been optimized to transfect large

plasmids and as a result, proved to be a major hurdle in this research. In the case of electrical

transfection methods, the cell viability was low due to cell shearing or reagent toxicity whereas

chemical transfections, proved to be only transient with low efficiencies were achieved. The

data from this research shows a clear distinction between the large (18.5+kbp) pRTS-1 plasmid

and the small (3kbp) control GFP plasmid. When all other parameters are kept equal,

transfections with the control plasmid shows minimal disturbance to cell viability and high

transfection efficiency while pRTS-1 transfections presented massive cell death post-transfection.

Furthermore, large quantities of cells that survive transfection were lost upon selection.

93

An alternative approach to resolving the low transfection efficiency issue is to re-

engineer the beneficial portions of the pRTS-1 vector (TET-repressor and the response/reporter

elements of TET-On) into viral vectors. If the benefits of removing super-physiological doses of

BMP-2 are higher than the risks, genetically engineering pRTS-1 into MSCs through viral

transfections may be considered. As reviewed by Thomas et al., adenovirus may package up to

30kbp depending on the helper agent but the high inflammatory potential makes it less desirable.

Alternatively, viral vectors such as retrovirus or lentivirus can package approximately 8kbp.111

The two enhancements of the pRTS-1 can be excised down to roughly 11kbp but this can be

reduced further if the reporter gene of the bidirectional promoter is excised (figure 5.5). This

would still allow a tight regulation while having both elements of the TET-On system

incorporated into a viral vector with low inflammatory potential.

The second experimental limitation was associated with the cell type. HUCPVC, which

has not been fully characterized, proved to be a difficult target for large plasmid transfections.

Figure 5.5 pRTS-1 Vector – The red area indicates the regions that include the response and

reporter element as well as the TET-repressor responsible for low background activity.

94

HUCPVCs were unable to proliferate when a large plasmid was incorporated and as a result,

alternate cell types were used in this research thesis.

The third experimental limitation was the verification of the VEGF-A construct. The

purpose of this gene was to induce angiogenesis at the site of transplant. As explained in the

introduction, vascularization of transplanted tissue quickened the regeneration process.80

The

difficulty in this thesis was associated with the development of the pRTS-1-VEGF-A system.

Despite several attempts to confirm the orientation of the VEGF-A gene insert, the quality of the

sequencing data was always below the acceptable threshold. We carried on with cell

transfections to see if we can detect the expression at a protein level, however, we were unable to

detect VEGF-A from any clones.

The final experimental limitation was associated with RUNX2 and OSX constructs.

RUNX2 and OSX are key genes necessary during osteoblastogenesis. Literature indicates that in

the absence of these genes, osteoblasts are not formed and as a result, no bone is formed.8 The

mechanism and signaling pathway is yet to be fully understood but due to their importance in

osteoblastogenesis, we created the pRTS-1-RUNX2 and pRTS-1-OSX constructs to learn the

effects of up regulating the genes above physiological levels. We were able to control the

expression level but failed to detect any enhancement to osteogenesis. Unfortunately, the current

understanding of RUNX2 and OSX could not explain the lack of osteogenic effect found in our

research.

95

Conclusions

Five conclusions can be drawn from this research.

1. Stable clones of RBMCs and ROS cells transfected with the pRTS-1-BMP-2 plasmids

can be generated by electroporation.

2. Stable clones can be induced and un-induced, in a dose-dependent manner, with

doxycycline.

3. Stable clones can re-achieve near-maximum gene expression within 72 hours using

500ng/mL doxycycline.

4. Xfect transfected RBMCs and ROS cells rapidly lose gene expression upon removal of

the selection pressure.

5. Amaxa Nucleofection, Clontech Xfect, Invitrogen Lipofectamine, and Qiagen SuperFect

did not generate stable clones.

96

Future Directions

The aim of this research was to study a controlled one-vector expression system through

non-viral transfection methods with MSCs. This research work implies that by controlling the

quantity and duration of gene expression during bone regeneration, two issues of the current

treatment can be addressed: the burst release of rhBMP-2, and the need for super-physiological

dose. But as discussed in section 5.5, several aspects of this research warrant further study.

1. in vivo engraftment of pRTS-1-X stable clones into animal models for ectopic bone

formation followed by bone regeneration in non-union bone defects.

2. Optimization and increasing transfection efficiency for the large pRTS-1 vector.

3. In addition, understanding the minor differences found between transfection methods and

attempting to explain the low transfection efficiency associated with large vectors would

also warrant further research.

4. Finally, it may be possible to re-engineer the beneficial portions of the pRTS-1 vector

(TET-repressor and the response/reporter elements of TET-On) into a viral vector to

study the potential to regulate gene expression with low background activity.

97

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