UNIVERSITI PUTRA MALAYSIA

NON-VIRAL TRANSFECTION OF BONE MARROW-DERIVED MESENCHYMAL STEM CELL WITH HUMAN INTERFERON-GAMMA GENE AND IN VITRO

EFFICIENCY AGAINST CHRONIC MYELOID LEUKAEMIA CELLS

LIEW LEE CHUEN

FPSK(m) 2013 22

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NON-VIRAL TRANSFECTION OF BONE MARROW-DERIVED

MESENCHYMAL STEM CELL WITH HUMAN INTERFERON-GAMMA

GENE AND IN VITRO EFFICIENCY AGAINST CHRONIC MYELOID

LEUKAEMIA CELLS

By

LIEW LEE CHUEN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the Degree of Master of Science

June 2013

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COPYRIGHT

All material contained within the thesis, including without limitation text, logos,

icons, photographs and all other artwork, is copyright material of University Putra

Malaysia unless otherwise stated. Use may be made of any material contained within

the thesis for non-commercial purposes from the copyright holder. Commercial use

of material may only be mad with the express, prior, written permission of University

Putra Malaysia.

Copyright © University Putra Malaysia

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Master of Science

NON-VIRAL TRANSFECTION OF BONE MARROW-DERIVED

MESENCHYMAL STEM CELL WITH HUMAN INTERFERON-GAMMA

GENE AND IN VITRO EFFICIENCY AGAINST CHRONIC MYELOID

LEUKAEMIA CELLS

By

LIEW LEE CHUEN

June 2013

Chairman : Maha Abdullah, PhD

Faculty : Medicine and Health Sciences

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder caused by

the BCR/ABL gene rearrangement, known as the Philadelphia (Ph) chromosome. To

date, the only curative therapy for CML is allogeneic stem cell transplantation.

However, significant morbidity and mortality are associated with the procedure and

the need for a matched donor makes this option not available to the majority of the

patients. Currently, various studies have been carried out to develop an alternative

approach for CML treatment, for example targeted gene delivery of therapeutic

cytokines. In this study, the feasibility of using bone marrow-derived mesenchymal

stem cell (BM-MSC) in delivering human interferon-gamma (hIFN-γ) gene for

targeted CML therapy was explored. Mesenchymal stem cells (MSC) were

successfully isolated from human bone marrow aspirates and their biological

properties were similar to those of MSC reported. Expanded BM-MSC were

transfected with plasmid containing hIFN-γ gene (pORF-hIFN-γ) via nucleofection.

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Gene transfer efficiency was determined based on intracellular hIFN-γ expression via

flow cytometry and was found to be at 54.28±11.34%. The in vitro expression of

hIFN-γ mRNA and protein in BM-MSC were also analysed at intervals of 24 h, up to

5 days post nucleofection, via real-time PCR and ELISA, respectively. Real-Time

PCR data analysis showed significant up-regulation of hIFN-γ mRNA in

nucleofected BM-MSC when compared to non-transfected BM-MSC (P=0.043).

BM-MSC harbouring pORF-hIFN-γ could express hIFN-γ protein in vitro. This

cytokine production was achieved as high as 3.47±1.03 ng/ml after 72 hours of

nucleofection. The effect of hIFN-γ produced in nucleofected BM-MSC on the

proliferation of CML cell line (K562) in vitro was also investigated. K562 growth

was inhibited at 61.12±16.38% after seven days of co-culture with BM-MSC

expressing hIFN-γ (P=0.006). In conclusion, findings in the current study indicated

that hIFN-γ produced by genetically engineered BM-MSC successfully inhibited the

proliferation of K562 cells in vitro. Thus, MSC as cellular vehicle in hIFN-γ gene

delivery could be further explored as a promising treatment option for CML patients.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

TRANSFEKSI GEN INTERFERON-GAMMA KE DALAM SEL STEM

MESENKIMIA DARIPADA SUMSUM TULANG SECARA BUKAN VIRUS

DAN KEBERKESANAN MENENTANG SEL LEUKEMIA MIELOGENUS

KRONIK IN VITRO

Oleh

LIEW LEE CHUEN

Jun 2013

Pengerusi : Maha Abdullah, PhD

Fakulti : Perubatan dan Sains Kesihatan

Leukemia mielogenus kronik (CML) merupakan sejenis leukemia kronik yang

dicirikan oleh peningkatan penghasilan klon sel-sel mieloid secara tidak terkawal

akibat daripada translokasi gen ABL dan gen BCR, umumnya dikenali sebagai

kromosom Philadelphia. Sehingga kini, transplantasi sumsum tulang merupakan

satu-satunya terapi kuratif yang berpotensi untuk CML. Walau bagaimanapun,

morbiditi and mortaliti yang dikaitkan dengan prosedur transplantasi and keperluan

penderma yang sesuai telah menyebabkan terapi tersebut tidak dapat disediakan

kepada kebanyakan pesakit. Oleh yang demikian, pelbagai penyelidikan telah

dijalankan pada masa kini dengan tujuan untuk memperkembangkan satu rawatan

novel untuk CML, contohnya penghantaran sitokin terapeutik ke sasaran. Dalam

kajian ini, penggunaan sel stem mesenkima yang diperoleh daripada sumsum tulang

dalam penghantaran gen interferon-gamma manusia (hIFN-γ) sebagai terapi sasaran

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bagi CML telah dikaji. Sel stem mesenkima (MSC) telah berjaya dipencilkan

daripada aspirasi sumsum tulang manusia dan sifat-sifat biologi sel ini adalah serupa

dengan MSC yang dilaporkan. MSC sumsum tulang manusia (BM-MSC) ini

kemudian ditransfeksi dengan plasmid mengekod gen hIFN-γ dengan menggunakan

teknologi nukleofeksi. Keberkesanan transfeksi telah ditentu berdasarkan ekspresi

hIFN-γ intraselular dengan menggunakan sitometri aliran. Keberkesan transfeksi

sebanyak 54.28±11.34% telah tercapai selepas 24 jam nukleofeksi. Ekspresi mRNA

dan protein hIFN-γ di dalam BM-MSC secara in vitro juga dikaji setiap 24 jam

selepas nukleofeksi, selama 5 hari dengan menggunakan tindak balas rantai

polimerase masa nyata (real-time PCR) dan ELISA masing-masing. Keputusan

tindak balas rantai polimerase masa nyata menunjukan peningkatan ekspresi mRNA

hIFN-γ yang ketara dalam sampel nukleofeksi BM-MSC berbanding dengan BM-

MSC yang tidak dinukleofeks (P=0.043). Di samping itu, BM-MSC yang telah

dinukleofeks dengan plasmid pORF-hIFN-γ juga berupaya menghasilkan protein

hIFN-γ secara in vitro. Produksi protein hIFN-γ yang paling tinggi ialah 72 jam

selepas nukleofeksi, dimana sebanyak 3.47±1.03 ng/ml protein hIFN-γ telah dikesan.

Selain itu, kesan hIFN-γ yang dihasilkan oleh nukleofeksi BM-MSC terhadap

proliferasi sel CML (K562) juga turut dikaji. Proliferasi sel K562 telah ditindas

sebanyak 61.12±16.38% apabila kultur bersama nukleofeksi BM-MSC selepas 7 hari

(P=0.006). Secara kesimpulannya, penemuan dalam kajian semasa menunjukkan

bahawa hIFN-γ yang dihasilkan secara modifikasi genetik BM-MSC telah berjaya

menindas proliferasi sel K562 in vitro. Oleh itu, MSC sebagai pengangkutan selular

dalam penghantaran gen hIFN-γ boleh terus diterokai sebagai rawatan alternatif yang

berkesan untuk pesakit CML.

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ACKNOWLEDGEMENTS

First and foremost, I would like to express utmost gratitude to my supervisor, Assoc.

Prof. Dr. Maha Abdullah, for her patience to guide me along the research with her

immense knowledge and motivation. Her valuable guidance has helped me

throughout the research and completion of this thesis.

It gives me great pleasure in acknowledging my co-superviors, Emeritus Prof. Dr.

Cheong Soon Keng, Assoc. Prof. Dr. Leong Chooi Fun and Assoc. Prof. Dr. Chong

Pei Pei, for their timely support and insightful comments. My sincere thanks also go

to Dato’ Mohd Farid Ariffin, the Founder and President of National Cancer Council

Malaysia (MAKNA), for his moral support in research activities and generosity in

providing grant to enable the research to be carried out. I would also like to share the

credit of my work with my seniors and fellow labmates – Wei Yi, Chee Yin, Erica,

Yuen Fen and especially Hoon Koon and Rachel, for their guidance and discussion

that has polished up both my research and thesis writing.

Last but not least, I would like to thank my family and friends for their love and

unconditional support, letting me focus on my study and help me to make it through

hurdles that I faced throughout the study.

This dissertation would not have been possible without the help and guidance of all

the individuals mentioned above, and to whom that I did not mention but

nevertheless contributed in one way or another to the success of this thesis, a big

THANK YOU to you all.

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I certify that a Thesis Examination Committee has met on 7 June 2013 to conduct the

final examination of Liew Lee Chuen on her thesis entitled “Non-viral Transfection

of Bone Marrow-Derived Mesenchymal Stem Cell woth Human Interferon-gamma

Gene and In Vitro Efficiency Against Chronic Myeloid Leukaemia Cells” in

accordance with the Universities and University Colleges Act 1971 and the

Constitution of the University Putra Malaysia [P.U.(A) 106] 15 March 1998. The

Committee recommends that the student be awarded the Master of Science.

Members of Thesis Examination Committee were as follows:

Mohamad Aziz bin Dollah, PhD

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Sharmili Vidyadaran, PhD

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Internal Examiner)

Noorjahan Banu binti Mohamad Alitheen, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular

Universiti Putra Malaysia

(Internal Examiner)

Ainoon Othman

Professor

Faculty of Medicine and Health Sciences

Universiti Sains Islam Malaysia

(External Examiner)

____________________________

NORITAH OMAR, PhD

Assoc. Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: 2 August 2013

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Maha Abdullah, PhD

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Chong Pei Pei, PhD

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Member)

Cheong Soon Keng

Professor and Dean

Faculty of Medicine and Health Sciences

Universiti Tunku Abdul Rahman

(Member)

Leong Chooi Fun

Associate Professor

Department of Pathology

Universiti Kebangsaan Malaysia Medical Centre

(Member)

____________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is my original work except for the quotations and citations

which have been duly acknowledged. I also declare that it has not been previously,

and is not concurrently, submitted for any other degree at Universiti Putra Malaysia

or at any other institution.

__________________

LIEW LEE CHUEN

Date: 7th June 2013

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRACK iv

ACKNOWLEDGEMENTS vi

APPROVAL vii

DECLARATION ix

LIST OF TABLES xiv

LIST OF FIGURES xv

LIST OF ABBREVIATIONS xvii

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 6

2.1 Chronic Myeloid Leukaemia

2.1.1 Introduction

2.1.2 Treatment Outcome

6

6

7

2.2 An Overview of Stem Cell 9

2.3 Mesenchymal Stem Cell (MSC) 11

2.3.1 Historical Overview and Nomenclature of

MSC

11

2.3.2 Properties of MSC 14

2.3.2.1 Morphology and Immunophenotype 14

2.3.2.2 Differentiation Capacity 16

2.3.2.3 Immunimodulatory Properties

2.3.2.4 Homing Ability

2.3.3 Clinical Potential of Mesenchymal Stem Cells

2.3.3.1 MSC for the Treatment of Graft-

Versus-Host Disease

2.3.3.2 The Use of MSC in Regenerative

Medicine

2.3.3.3 MSC as Potential Vehicle for

Therapeutic Genes Delivery

17

19

21

22

26

29

2.4 Gene Delivery Systems 33

2.4.1 Viral-based Gene Delivery System

2.4.1.1 Integrating Viral Vector

2.4.1.2 Non-integrating Viral Vector

34

35

39

2.4.2 Non-viral Gene Delivery System

2.4.2.1 Chemical Methods

2.4.2.2 Physical Methods

40

41

44

2.4.3 Nucleofection 47

2.5 The Interferons (IFNs) 49

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3 MATERIALS AND METHODS 54

3.1 Experimental Design 54

3.2 Isolation of Mononuclear Cells From Bone Marrow

Aspirate

56

3.3 Cell Count 57

3.4 Cell Culture 58

3.4.1 Culture of Bone Marrow-Derived Adherent

Cells

58

3.4.2 Culture of Chronic Myeloid Leukaemia Cells

Line (K562)

59

3.5 Characterisation of Bone Marrow-Derived Adherent

Cells

59

3.5.1 Morphology Study under Microscopic

Observation

59

3.5.2 Immunophenotyping 60

3.5.3 Differentiation Assays

3.5.3.1 Adipogenic Differentiation

3.5.3.2 Osteogenic Differentiation

3.5.3.3 Chondrogenic Differentiation

61

61

62

64

3.6 Preparation of Plasmid pORF-hIFN-γ for Transfection

3.6.1 Plasmid pORF-hIFN-γ

3.6.2 E. coli Culture Media and Antibiotic

3.6.2.1 Liquid Media

3.6.2.2 Solid Media

3.6.2.3 Antibiotic

3.6.3 Growth and Storage of pORF-transformed

Bacteria

3.6.4 Verification of Plasmid pORF-hIFN-γ from

Transformed E. coli Clones

3.6.4.1 Colony PCR (Polymerase Chain

Reaction)

3.6.4.2 Gel Electrophoresis

3.6.4.3 Plasmid Extraction and Sequencing

65

65

67

67

68

68

69

69

69

71

72

3.7 Nucleofection of Bone Marrow-Derived

Mesenchymal Stem Cells

3.7.1 Nucleofection Protocol

3.7.2 Gene Transfection Efficiency and Viability

of Cells Post Nucleofection

3.7.2.1 Transfection Efficiency of pmaxGFP

into BM-MSC

3.7.2.2 Transfection Efficiency of pORF-

hIFN-γ into BM-MSC

3.7.2.3 Viability of Cells after

Nucleofection

3.7.3 Morphology, Immunophenotyping and

Differentiation Potential of Nucleofected

BM-MSC

74

74

75

75

76

77

78

3.8 Functional Status of Human Interferon Gamma Gene

in Nucleofected BM-MSC

3.8.1 Quantitative Real-Time PCR Analysis of

78

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hIFN-γ mRNA Expression in Nucleofected

BM-MSC

3.8.1.1 Total Ribonucleic Acid (RNA)

Extraction

3.8.1.2 DNase I Treatment of RNA

3.8.1.3 Determination of RNA Integrity

3.8.1.4 Reverse Transcription of RNA to

cDNA

3.8.1.5 Quantitative Real-Time PCR

Quantification

3.8.2 Protein Expression of hIFN-γ in

Nucleofected BM-MSC via ELISA

79

80

81

82

83

85

3.9 K562 Cell Proliferation Assay

3.9.1 Establishment of Growth Profile for K562

cells

3.9.2 Inhibition Effect of Nucleofected BM-MSC

on K562 Cell Proliferation

86

86

87

3.10 Statistical Analysis

89

4 RESULTS 90

4.1 Characterisation of Isolated Bone Marrow-Derived

Adherent Cells

90

4.1.1 Morphology Study under Microscopic

Observation

90

4.1.2 Immunophenotyping 92

4.1.3 Differentiation Assays

4.1.3.1 Adipogenic Differentiation

4.1.3.2 Osteogenic Differentiation

4.1.3.3 Chondrogenic Differentiation

94

94

96

98

4.2 Verification of Plasmid pORF-hIFN-γ from

Transfected E. coli Colonies

4.2.1 Colony PCR (Polymerase Chain Reaction)

4.2.2 Plasmid Purification and Verification

100

100

102

4.3 Transfection Efficiency and Viability of Cell Post

Nucleofection

103

4.3.1 Nucleofection of BM-MSC with plasmid

pmaxGFP

4.3.2 Nucleofection of BM-MSC with plasmid

pORF-hIFN-γ

103

106

4.4 Morphology, Immunophenotypes and Differentiation

Potential of Nucleofected BM-MSC

108

4.5 mRNA Expression of hIFN-γ in Nucleofected BM-

MSC

110

4.5.1 RNA Purity and Integrity

4.5.2 Real-Time PCR Quantification

110

111

4.5.2.1 Validation Experiment 111

4.5.2.2 mRNA Expression of hIFN-γ in

nucleofected BM-MSC

115

4.6 Protein Expression of hIFN-γ in Nucleofected BM-

MSC

117

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4.7 K562 Cell Proliferation Assay 119

4.7.1 Establishment of Growth Profile for K562

Cells

4.7.2 Inhibition Effect of Nucleofection BM-MSC

on K562 Cell Proliferation

119

120

5 DISCUSSION 122

5.1 Characterisation of Isolated Bone Marrow-Derived

Adherent Cells

123

5.2 Transfection Efficiency and Viability of BM-MSC

Post Nucleofection

126

5.3 In vitro Expression of Human Inteferon-gamma in

Nucleofected BM-MSC and Their Inhibition Effect on

K562 Cell Proliferation

129

6 CONCLUSION AND FUTURE RECOMMENDATIONS

135

6.1 Conclusion 135

6.2 Future recommendations

137

REFERENCES 139

APPENDIX A – Information Sheets 163

APPENDIX B – Data Sets 169

BIODATA OF STUDENT 186

PUBLICATION 187