STEM CELL-BASED THERAPY FOR TREATMENT OF

CANCER

BY
SANI AHMAD

EDUCATION
SCHOOL OF DISTANCE LEARNING
UNIVERSITI SAINS MALAYSIA

MAY 2016
STEM CELL-BASED THERAPY FOR TREATMENT OF
CANCER

BY
SANI AHMAD

A THESIS SUBMITTED IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR BACHELOR OF
SCIENCE

MAY 2016

ii
“I declare that this report entitle ‘Stem Cell-Based Therapy for Treatment of Cancer’
is the result of my own research except cited in the references The report has not been
accepted for any degree and is not concurrently submitted in candidature of other
degree”

Signature : …………………………….

Name of Student : SANI BIN AHMAD

Date : 18 MAY 2016

iii
 DEDICATION

It gives me great pleasure to convey a special dedication to my beloved family

members, my respected supervisor, my lectures and my friends.

My utmost gratitude goes to all of you for your support and encouragement.

iv
 ACKNOWLEDGEMENT

In the name of Allah SWT, the most gracious and the Most Merciful, I offer
my humble gratitude to Allah for giving me the strength to complete this thesis.
First and foremost, my sincere gratitude and appreciation goes to my
supervisor, Dr. Siti Razila Binti Abdul Razak for all the ideas, critics, guidance and
patience throughout preparing this thesis.
I also would like to extend my gratitude and affection to Universiti Sains
Malaysia library, Universiti Teknologi Malaysia Library and Perpustakaan Awam
Johor because provide me facilities in order to complete this thesis.
Last but not least, to all my family and friends. Thank you for providing me
the support with your patience, love, encouragement and inspiration that has greatly
facilitated the completion of this challenging effort.

v
 STEM CELL-BASED THERAPY FOR TREATMENT OF
CANCER

ABSTRACT

Stem cells (SCs) are subset of cell, which have the unique ability to preserve itself by
self-renewal and have a potential to differentiate to different types of mature cells.
There are three major types of SCs, which are embryonic stem cells (ESCs), adult
stem cells (ADSCs) and induced pluripotent stem cells (iPSCs). Bone marrow
transplantation (BMT) is one of the established stem cell-based therapies mostly for
treatment of blood-associated cancers such as leukaemia, lymphoma and myeloma. It
can be delivered to patient either by autologous, allogeneic or syngeneic
transplantation. Besides BMT, there is also some research, which consider future
application that still undergoes development and clinical trial. One of the examples is
stem cell for therapeutic delivery by using MSCs. It combines between genetically
engineered vehicles stem cell therapy and suicide gene therapy. These methods were
best to treat metastatic brain cancer. The other future application of the stem cell-
based therapy is by creating anticancer stem cells agent known as OH14. This OH14
will act as inhibitor of c-FLIP (cellular FLICE-like inhibitory protein) which capable
targeting tumor’s forming cell. These methods were best to treat breast, colon,
pancreas and prostate cancer. It also has capabilities to prevent cancer from regrowth.
Stem cell also can be genetically modified to target specific tumor types and secrete
anticancer proteins such as interferons  and , interleukins 2 and 12 or chemokine
(signaling protein). These methods were best to treat brain tumor and other
malignancies such as leukimia and Hodgkin’s lymphoma. Stem cell can also
genetically modified to induce cancer cell death by acting as a suicide gene. This
method is believes to treat various tumor cancers with minimal side effect. With all
the ongoing studies on stem cell-based therapy, has brought in a lot of optimistic hope
amongst researchers, doctors, and not to forget the patients who are the chief
beneficiary of this innovation

vi
 TERAPI BERASASKAN STEM SEL BAGI MERAWAT
KANSER

ABSTRAK

Sel stem (SCs) adalah subset sel, yang mempunyai keupayaan unik untuk memelihara
dirinya dengan pembaharuan diri dan mempunyai potensi untuk membezakan kepada
pelbagai jenis sel matang. Terdapat tiga jenis utama SCs, iaitu sel stem embrio, sel
stem dewasa (ADSCs) dan merangsang sel stem pluripotent (iPSCs). Pemindahan
sum-sum tulang (BMT) adalah salah satu terapi berasaskan sel stem yang sedia ada
untuk merawat pelbagai jenis kanser berkaitan darah seperti leukemia, limfoma dan
myeloma. Ia boleh dipindahkan kepada pesakit sama ada dengan cara autologous,
allogeneic atau pemindahan syngeneic. Selain BMT, terdapat juga beberapa kajian,
yang masih menjalani pembangunan dan percubaan klinikal. Salah satu contohnya
ialah sel stem untuk penghantaran terapeutik dengan menggunakan MSC. Ia
menggabungkan antara pengangkutan kejuruteraan genetik terapi sel stem dan gen
terapi membunuh diri. Kaedah-kaedah ini adalah terbaik untuk merawat kanser otak
metastatik. Antara contoh terapi berasaskan sel stem yang lain adalah dengan
mewujudkan sel stem agen anti-kanser yang dikenali sebagai OH14. Sebatian OH14
ini akan bertindak sebagai perencat c-FLIP (selular FLICE seperti protein yg
menghalang) yang mensasarkan pada tumor sel yang terbentuk. Kaedah ini dikatakan
sesuai untuk merawat kanser payudara, kolon, pankreas dan prostat. Ia juga
mempunyai keupayaan untuk mencegah kanser dari pertumbuhan semula. Stem sel
juga boleh diubahsuai secara genetik untuk menyasarkan jenis tumor tertentu dan
merembeskan protein anti-kanser seperti Interferon α dan β, interleukin 2 dan 12 atau
chemokine (isyarat protein). Kaedah boleh digunakan untuk merawat tumor otak,
leukimia dan limfoma Hodgkin. Sel stem juga boleh diubahsuai secara genetik untuk
mendorong kematian sel kanser dengan bertindak sebagai gen bunuh diri. Kaedah ini
percaya untuk merawat pelbagai jenis kanser tumor dengan kesan sampingan yang
minimum. Kajian berterusan ke atas terapi berasaskan sel stem, memberi harapan
optimis di kalangan penyelidik, doktor dan pesakit.

vii
 TABLE OF CONTENTS
CHAPTER TITLE PAGE
AUTHOR’S DECLARATION iii
DEDICATION iv
ACKNOWLEDGMENT v
ABSTRACT vi
ABSTRAK vii
TABLE OF CONTENT viii
LIST OF FIGURES xi
LIST OF TABLES xii
LIST OF ABBREVIATIONS xiii

1 INTRODUCTION 1
1.1 Backgrounds and History 1

2 STEM CELL 4
2.1 What is stem cell? 4
2.2 Basic Characteristic of Stem Cell 5
2.2.1 Self Renewal 5
2.2.2 Potency 5
2.2.2.1 Totipotent 6
2.2.2.2 Pluripotent 6
2.2.2.3 Multipotent 6
2.2.2.4 Oligopotent 7
2.2.2.5 Unipotent 7
2.2.3 Clonality 8
2.3 Types of Stem Cell 8
2.3.1 Embryonic Stem Cell (ESCs) 8
2.3.1.1 Characteristic of ESCs 10
2.3.1.2 Sources of ESCs 11
2.3.1.2.1 Spare or special purpose
embryos 11
2.3.1.2.2 Clone Embryos 11

viii
 2.3.1.2.3 Aborted Fetuses 12
2.3.1.2.4 Amniotic Fluid Stem Cells 12
2.3.2 Adult Stem Cell (ADSCs) 13
2.3.2.1 Characteristic of ADSCs 14
2.3.2.2 Type of ADCs 14
2.3.2.2.1 Hematopoietic Stem Cells
(HSCs) 14
2.3.2.2.2 Mesenchymal Stem Cells
(MSCs) 15
2.3.2.3 Sources of Adult Stem Cells 17
2.3.2.3.1 Cadavers 17
2.3.2.3.2 Umbilical Cord 17
2.3.2.3.3 Bone Marrow 18
2.3.2.3.4 Skin Fibroblast 18
2.3.3 Induced Pluripotent Stem Cells (IPSCs) 19
2.3.3.1 Characteristic of iPSCs 20
2.3.3.2 Generation of iPSCs 20

3 STEM CELL-BASED THERAPY FOR TREATMENT 22

OF CANCER
3.1 Stem Cell Therapies 22
3.2 Stem Cell Transplantation 22
3.2.1. Autologous Stem Cell Transplant 22
3.2.2. Allogeneic Stem Cell Transplant 23
3.2.3. Syngeneic Stem Cell Transplant 24
3.3 Established Stem Cell Therapies 24
3.3.1. Bone Marrow Transplantation 25
3.4 Therapeutic Potential of Stem Cell Therapy 28
3.4.1. Stem Cell for Therapeutic Delivery 28
3.4.2. Creating Anticancer Stem Cells 29
3.4.3. Genetic Modification of Stem Cells to 29
Secrete Anticancer Proteins
3.4.4. Genetic Modification of Stem Cells to 30

ix
 Induce Cancer Cell Death

4 DISCUSSION 31
4.1 Limitation of Stem Cell-Based Therapy 31
4.1.1. Mismatched transplant 31
4.1.2. Cancer recurrence 32
4.1.3. Side Effects 32
4.1.4. Financial challenges 33
4.2 Controversy and Etiquette Issue 33
4.3 Legislation on Human Embryonic Stem Cell 34

5 CONCLUSION 35

REFERENCES 37

x
 LIST OF FIGURES

FIGURE NO. TITLE PAGE

1.1 Stages of tumor development 2

2.1 Blood stem cell give arise into blood related cells 7
2.2 Human blastocyst 5 days old 9
2.3 Isolation and differentiation of embryonic stem cells 9
2.4 Human embryonic stem cells 10
2.5 Nuclear transfer procedure using transfer pipette 12
2.6 Newborn baby in fully intact amniotic sac 13
2.7 MSCs observed under fluorescent microscope 16
2.8 Location of somatic stem cell in the human body 16
2.9 Number of registered clinical trials of mesenchymal stem 17
cell-based therapy
2.10 Umbilical cord blood 18
2.11 A colony of induced pluripotent stem cells 19
2.12 Procedure for generation of induced pluripotent stem cells 21
(iPSCs)
3.1 Bone marrow transplant step by step 26
3.2 Bone marrow aspiration and biopsy 27

xi
 LIST OF TABLES

TABLE NO. TITLE PAGE

3.1 Stem Cell Based Therapies and Cancer Types 25

xii
 LIST OF ABBREVIATIONS

ADCs - Adult Stem Cells

BMT - Bone Marrow Transfer
CML - Chronic Myelogenous Leukemia
DNA - Deoxyribonucleic Acid
ESCs - Embryonic Stem Cells
GVC - Ganciclovir

GVHD - Graft-versus-host-disease
hESCs - Human Embryonic Stem Cells
HLA - Human Leukocyte Antigen
HSCs - Hematopoietic Stem Cells
ICM - Inner Cell Mass
IVF - In Vitro Fertilization
iPSCs - Induce Pluripotent Stem Cells
MS - Multiple Sclerosis
MSCs - Mesenchymal Stem Cells
Nanog - Homeobox Transcription Factor
NSCs - Neural Stem Cells

Oct-4 - Octamer-binding Transcription Factor 4

PE- - Pseudomonas Exotoxin
SAA - Severe Aplastic Anemia
SCs - Stem Cells
SCNT - Somatic Nuclear Transfer
Sox2 - Sex determining region Y-box 2
SSEA-4 - Stage Specific Embryonic Antigen 4
Tra-1-81 - Tumor Resistance Antigen-1-81
T/NK - T-Cell/Natural Killer

xiii
 CHAPTER 1

INTRODUCTION

1.1 Backgrounds and History

Cancer is known as most common cause of mortality in Peninsular Malaysia with a

total of 21,773 cancer cases diagnosed among Malaysian in 2006 and 18,219 new cancer
cases were registered in Malaysia at the National Cancer Registry (National Cancer
Registry 2006, 2011). Even with latest advances in the treatments of cancer, the clinical
outcome is still yet far from necessity.
According to National Institutes of Health (2007), cancer is referred to a group of
diseases, which may consist of more than 100 diseases. It is develop from time to time, and
involving uncontrolled division of the body’s cells. Cancer has the ability to develop in
virtually any of body’s tissue which each has a unique features, but most of them were
produce by same basic processes (Figure 1.1). It starts with genetic mutation within cell,
which may vary in number base on their type of tumor. This genetic mutant cells then will
continue to hyperplasia which is divided and grow too rapidly. At certain times, one cells
from this group of cells experiences another mutation, which increase their tendency to
keep divided rapidly that form an obviously abnormal look which known as dysplasia. All
these cells will remain within its original tissue which known as in situ cancer. It may
contain indefinitely or may metastases to other neighbor tissue and establish new tumor.

1
 Figure 1.1: Stages of tumor development (Winslow, T. 2014)

There are a few options of treatments available for cancer patient to cure their
disease. One of the example is through surgery. Surgery works the best for patient who
suffered from solid tumor. The treatment involve cutting or removal of cancer cells using
cutting tools such as knives, scalpels and other sharp tools. It can also be performed by
cryosurgery, which involve the use of liquid nitrogen or argon gas to kill the abnormal
tissue. Anyhow, this cryosurgery’s methods are only suitable for an early stage cancer.
Besides cryotherapy, laser is one of option that is often used to treat tumors. It can be used
to treat tumor on the surface or inside lining of the organs. It used a powerful beam light to
cut through the tissue. It also can be used to shrink or even destroy the tumor and its
growth. Another method that make used of light to treat a cancer is photodynamic therapy.
This treatment used certain drugs that will response to certain type of light. Whenever the
tumor is exposed to the light, it will activate the drug that will kill the nearby cancer cells.
Normally, this kind of therapy is available for treatment of skin cancer, mycosis fungoid
and non-small lung cancer. There is also a surgery method that only used the high
temperature instead of sharp tools. The cancer tissues were exposed to the high temperature
that will kill or make the cancerous cells sensitive to radiation or chemo drugs.
Radiofrequency is one of the examples of hyperthermia. It makes used of high energy to
create an adequate heat to kill the cancerous cell. However this kind of therapy is still under
clinical trial (National Cancer Institute, 2015a).
Radiation therapy is also available for treatment of cancer. There are two kind of
radiation therapies. One is external beam radiation therapy, by using external beam that

2
navigate and spread the beam around specific targeted body part. While secondly, internal
radiation therapy that use solid or liquid, that is placed in our body or nearby the cancer
cells. There is also drug-based type of therapy such as chemotherapy, hormone therapies,
Bisphosphonates and targeted cancer therapy that make used of drug or substances to attack
the cancer cells while sparing the normal healthy cells (National Cancer Institute, 2015b).
Stem cell-based therapy is one of the treatments that is available for curing cancer.
It is one of the exciting and controversial areas of medical sciences since the first successful
bone marrow transplant in 1956 (Thomas et al., 1957) to isolation of embryonic stem cell
from mice in 1980s until the great finding in curing cancer by generate genome-wide
mapping of DNA modification called 5-hydroxymethhylcytosine (5mhmC) in embryonic
stem cell which were founded by researcher at Universiti of California, Los Angeles
(Stroud et al., 2011).
Stem cell based therapies are emerging as a promising strategy to combat cancer.
Multiple stem cell types shows a positive tropism towards tumor. This stem cell was
engineered to response as a therapeutic agent, which acts as an effective pathotropic
delivery vehicle to the target sites of malignancy. This kind of therapies is more effective
than conventional treatment as it has an ability to target cancerous cells while sparing a
healthy cell or tissue (Stuckey & Shah 2014).
There a few other ways available for treatment of cancer using modified stem cell.
The modified stem cells will include technologies related to genetic modification that
involve secretion of anti-cancer protein, induction of cancer cell death, stem cell as nano-
particle carriers and stem cell loaded with oncolytic virus. The technologies will be further
discussed in the next chapter.

3
 CHAPTER 2

STEM CELL

2.1 What Are Stem Cells?

Stem cell is a special cell, which has an ability to preserve itself through
self-renewal to produce the exactly same copy of itself. Stem cells also have abilities to
generate a mature cell, which later can transform into other specialised cell type in a body
(Noor, et al., 2014). Stem cells are found in all multicellular organisms which can either
mitotically divided into self-renewal and produce more stem cell or differentiate into a
mature cells of some particular tissue (Bose & Sudheer 2014).
The ability of stem cell to self-renew and differentiate into specialised cell types
give us hopes, as it has a potential to replace or to repair any damaged tissue. It can also be
used as an organ transplant and treating diseases in human.
Stem cell has an ability to differentiate into several cell types. It can be either a
pluripotent stem cell that has abilities to giving a rise to every cell of the organism (except
trophoblast of the placenta) or multipotent stem cell, which can give rise to all cell types in
organ where the multipotent resides (Sanberg, & Sanberg, 2006).
Stem cell research has become a centre of attention to researcher because of their
potential of self-renewal and differentiation ability. Researchers are finding new ways to
use stem cell in many kind of therapy including rebuild broken tissue or organ and treating
various diseases including cancer. Besides that, stem cells also help researcher to
understand more of how some diseases occur by observing how stem cells developed and
working in bones, heart muscles and other related organ or tissue. Stem cells also can be
serving as a testing platform to test for new drugs to look for safety and effectiveness
before it can be tested on human.

4
2.2 Basic Characteristics of Stem Cell

Basically, there are three important characteristics of stem cells, which are self-
renewal, potency and clonality.

2.2.1 Self Renewal

Stem cell has the abilities of self-renewal to make more stem cell through cell
division while maintains its undifferentiated state. The stem cells can also generate one
daughter stem cell and another cell which is a differentiated cells. Anyhow this application
is differ from adult progenitor cells that reduce the abilities of self-renewal, which produce
only one lineage of differentiated cells (Weiner, 2008).
The stem cell divides either asymmetrically or symmetrically to form another one or
two daughter stem cell, which has the same potential as the mother cell. This is an essential
process to increase their number during development and to maintain their numbers during
adult tissue and to help restore the stem cell pool after the injury. These self-renewal
process were controlled to ensure their potency during replication by regulating the proto-
oncogenes, a gene code for protein that regulate the self-renewal, the gatekeeper tumor
suppressor gene which responsible for limiting the self-renewal and the caretaker tumor
suppressors gene, which is responsible for maintaining the integrity of its genomic (He et
al., 2009)

2.2.2 Potency

Potency can be defined as a potential to differentiate. Various stem cells have the
ability to differentiate up to 210 tissues that constitute the human body. The term potency
were used to provide hierarchy for organizing cell types and its “value” (Lynch, 2011). In
stem cell, potency is one of the important characteristics of stem cells. Potency can be
divided into five categories based on their degree of differentiation which are totipotent,
pluripotent, multipotent, oligopotent and unipotent.

5
2.2.2.1 Totipotent

Totipotent stem cells are the most flexible stem cells. It has the total potential to
develop into any kind of cells that are found in our body. In early cell division in
embryonic development, totipotent cells will divide and replicate into unlimited number of
cells without even losing their potency. It can produce more and more totipotent cells
within approximately four days. After that, the totipotent cells start to specialise into
pluripotent cells.
Because of these properties, totipotent stem cell has become an ideal choice for a
stem cell-based therapy for curing disease. These cells also give tremendous opportunity to
researcher not only to study about the disease cells but also to find ways to stop, cure and
prevent the production of the disease cells. However, all researcher still bind to law and
ethical concern to used totipotent stem cells in therapies (Murnaghan, 2015).

2.2.2.2 Pluripotent

Pluripotent stem cell is a second phase after the totipotent stem cell in early cell
division of embryonic development. It also has the abilities almost the same as totipotent
stem cells except for creating an entire organism. The inner cell mass (ICM) in blastocyst
are the example of pluripotent stem cells that will develop into other specialised cell types
in human body. Anyhow, it lacks an ability to differentiate into some part of tissue that are
vital for fetal development such as placenta. The abilities to differentiate into specialised
cell make this kind of stem cells available to be use as replacement of broken or infected
cells and tissues (Murnaghan, 2016a).

2.2.2.3 Multipotent

Multipotent stem cells have limited ability to differentiate. It can only give rise into
limited range of cells and only within related tissue type. For example, a multipotent blood
stem can only give rise into a blood-related cell such as red blood, white blood or platelets
(Figure 2.1) (Murnaghan, 2016b).

6
 Figure 2.1: Blood stem cell ggive arise intto blood relaated cells (W
Winslow, 20008)

2.22.2.4 Oligoppotent

Oligoppotent stem
m cells have limited abillity to differrentiate intoo specific typpes of cell
suuch as endodderm, ectodeerm and meesoderm. It oonly can diffferentiate innto a few tyypes of cell
me any of tthe blood ccell, which found in
for example; lymphoid stem cell can becom
mphatic sysstem such aas T cells, B cells and plasma cellls. Howeverr this oligoppotent cell
lym
aree not able too differentiaate to other kkind of bloood cell such as platelet or red bloodd cell.
Accorrding to Majjo et al. (20008), oligopootent stem ccells inside corneal epitthelium of
moouse has thhe abilities to generatee goblet cell if provideed with connjuctival ennvironment
whhile in ocullar surface of pig show
w that, oliggopotent steem cell hass abilities too generate
inddividual collonies of corrneal and coonjuctival ceells

2.22.2.5 Unipootent

mpare to othher type of stem cells

Unipootent stem cells has a veery limited abilities com
pootency. Their abilities to differenttiated are very
v limitedd. Howeverr the abilitiees to self-
rennewal placee unipotentt stem cellss as valuablle potentiall for therappeutic used to treat a
dissease.
Unipootent stem ccells are arissing from m
multipotent stem cells. Later, it coontinues to
diffferentiate aand give risse to certainn type of steem cells that later will form a tissuue specific

7
cell, which provide function and structure to our organ or body.
One example of unipotent cell is epithelium which have an ability to form skin
cells. It can be developed by using skin stem cells and used for transplant to replace
damage skin. However, due to limitation in technology, this reconstruct and development
of unipotent are almost impossible and extremely slow (Murnaghan, 2014).

2.2.3 Clonality

Stem cells clonality can be defined as stem cells that are genetically identical to a
parent cell by sharing a common ancestry with the common properties (Glauche et al.,
2013). The easier way to understand fundamental of clonality is look at tumour as an
example. The tumour cells were formed originally from a single transform cell which are
known as cell of origin (Carlson, 2010).

2.3 Type of Stem Cells

Stem cells can be divided into 3 major types known as embryonic stem cells
(ESCs), adult stem cells (ADSCs) and induced pluripotent stem cells (iPSCs) (Turksen,
2014).

2.3.1 Embryonic Stem Cells (ESCs)

Our life begins with fertilization of an egg and sperms which produce zygote.
Zygote then undergoes a cell division and cleavage to increase the number of the cells. The
zygote then reach the morula stage which contain about 4 to 16 cells. After the morula
arrived at uterus via fallopian tube, it later will form a fluid filled cavity which known as
blastocoel. The development of the embryo known as blastocyst then continued with the
number of cell range from 40 to 150 cells. The cells, which in the inner blastocyst then
forms the inner cell mass (ICM) or also known as embryoblast. Naturally, this embryoblast
has pluripotent properties. This inner blastocyst, which 5-7 days old is the best source of
the embryonic stem cells (Figure 2.2 and 2.3) (Sant 2008; Kumar 2008).

8
 Figure 2.2: Human blastocyst 5 days old (Zhang et al. 2009).

An embryonic stem cell is a “special cells” that have the ability to fully differentiate
into any type of cells such as skin, skeletal and neural cells (Figure 2.3). According to
recent study by Biswas, & Hutchins, (2007) shows that the embryonic stem cell can be
directed toward specific lineage differentiation programs in vitro. However all these cells
(in vitro) need a special requirement such as feeder cell, serum, cytokines and such to make
sure it maintain their undifferentiated state in culture before we manipulated their growth
towards any lineage-specific differentiation program. With recent advance in culturing and
successful in exploited their pluripotent properties, it gives us a hope to used it as a stem
cell based therapy in future.

Figure 2.3: Isolation and differentiation of embryonic stem cells (Genetic science learning
center).

9
2.3.1.1 Characteristics of ESCs

There are few basic characteristics of embryonic stem cells such as pluripotent
properties. ESCs also on certain condition can propagate themselves indefinitely in
undifferentiated state and can start to differentiate after feeding with appropriate signal
(Axell, 2009). Basically, size of human embryonic stem cells (Figure 2.4) derived from
early embryos (blastocyst) is approximately four times diameter of human hair (Blair,
2015). These ESCs have the abilities to grow and differentiate into more than 220 cell types
of the body and can give rise to all three embryonic germ ectoderm, mesoderm and
endoderm even after being cultured for a long period.

Figure 2.4: Human embryonic stem cells (hESCs) (Russo, 2005)

Ectoderm germ layers can give rise to organ such as brain, hair, skin, teeth, spinal
cord, eyes and mouth. Mesoderm has abilities to give rise to muscle, connective tissue,
blood vessel and heart while Endoderm layer can give a rise to gut such as pancreas, liver,
and stomach, lung, germ cell (sperm or egg) and the bladder.
However ESCs also possess ability induce a cancerous tumor. One case was
reported in 2009 where the 17 years old Israeli boy who undergoes experimental treatment
to treat his neurodegenerative disease known as ataxia telangiectasia (AT). The ESCs were
injected into his brain and spinal cord when he was nine, and additional injection when he
was at 10 and 12 years old. The boy later suffered with 2 tumors, one in his spinal cord and
another one appeared in his brains. The researchers believe that injected tissue was the
factor that spark the tumors in this boy (Cernansky, 2009).

10
2.3.1.2 Sources of ESCs

According to Lauren Pecorino (2001), ESCs can be harvested from various sources
such as spares or special purpose embryos, cloned embryos, aborted fetus and amniotic
fluid.

2.3.1.2.1 Spare or special purpose embryos

The spare embryos may come from leftover or no longer needed embryo by couples
to have children, which stored at fertility clinics. Millions of unused human embryos which
created from IVF pregnancies been thrown away each year. IVF is the process of complex
series of procedures used to treat fertility or genetic problems and assist with the
conception of a child. During IVF, mature eggs are retrieved from ovary and fertilized by
sperm.
For every woman who conceives a child through IVF, half (7-8 embryos) of the
embryos, that poor in quality will discarded as a medical waste. While the healthy unused
embryos will remain in freezer until it expired. And from the record, almost 1.7 million
spare embryos were discarded since 21 years ago (Dought, 2012).
Therefore, instead of keeping them in freezer until expired, these embryos can be
used in stem cell base therapies. The recent study also found that all these poor quality of
embryo actually possible to derive a stem cell (Lerou et al., 2008).

2.3.1.2.2 Cloned Embryos

Same as spare or special embryo, a cloned embryo also is one of an alternative

source available for embryonic stem cell. Cloning is involved making duplicates of
biological material. Cloning an embryo is using cloning technique, known as somatic cell
nuclear transfer (Figure 2.5). This technique was used to create the first cloned mammal
(sheep) which known as dolly in 1996. The same technique later was used by a group of
scientist. The egg cell’s (oocyte) nucleus were removed and replaced with nucleus from
other cell. The oocyte can review a nucleus without chromosomal damage (Campbell et al.,

11
1993).

Figure 2.5: Nuclear transfer procedure using transfer pipette (Niclos, J., 2012).

2.3.1.2.3 Aborted Fetuses

Fetal tissue is the richest primordial stem cells with some properties, which is very
useful for transplantation. Fetal tissue that can be harvest from the aborted fetuses has the
abilities of proliferating faster and rapid than a mature cell. This fetus cells are very
responsive to the environment and condition with the capabilities to grow, migrate,
elongate and establish a functional connection with other cells. Due to the low level of
histocompatibility antigens, fetal tissue will not easily rejected by the recipient (patient
body) (Bhattacharya, 2004).
However, as we know, certain country has a straight law regarding abortion. In
Malaysia for examples, a medical practitioner or researcher are bind to Act 574 of Penal
Code (revised 1997), Section 312 and Section 314 as stated in Guideline On Termination
Of Pregnancy (TOP) For Hospitals. Any violation to the any related law will be penalized
accordingly (Ministry of Health, 2012).

2.3.3.4 Amniotic Fluid Stem Cells

Amniotic fluid stem cells are a cell that has been isolated from amniotic fluid,
which contain in amniotic sac (Figure 2.6) of a pregnant woman. This amniotic fluid has a
significantly potential for regenerative medicine. These multipotent cells have ability to

12
differentiate into cell types from each embryonic germ layer (Carro, et al. 2008).

Figure 2.6: Newborn baby in fully intact amniotic sac (Siddique, 2013)

2.3.2 Adult Stem Cells (ADSCs)

Adult stem cells or also known as a somatic stem cells are undifferentiated cells that
can be found in children and adult body. This adult stem cell serves as replenish and
regenerate the damage tissue.
Research on adult stem cells begin was begun in 1950s where researcher discover
that bone marrow containing at least two kinds of stem cell which is hematopoietic stem
cells (blood cells) and bone marrow stromal stem cell which also known as mesenchymal
stem cells (National Institute of Health, Dept. of Health and Human Services. 2015).
The usage of the adult stem cells is less controversial compare to embryonic cells as
adult stem cells can be harvest from adult body tissue while ESCs have triggered enormous
debate due to the usage and destruction of an embryo. ADSCs share the same ability as
ESCs, which is, can differentiate into more than one type of cell, however they’re often
restricted to only certain lineages. Each different type of stem cell has different capabilities
to trans-differentiation (become another lineage). Anyhow, until then, there is no exact
proof of this claim (Murnaghan, 2016c).

13
2.3.2.1 Characteristics of ADSCs

ADSCs can be characterised by it size which apparently small with highly nucleus-
cytoplasm ratio. It also did not have the specific lineage marker and give a poor staining
result with vital dyes such as Hoechst 33342. It express various surface markers such as
CD34, CD90, CD133 and CD 117 (Gonzalez, & Bernad, 2012).
Basically ADSCs has less potential than ESCs. However, ADSCs has the ability to
make an identical copy of itself for such a long period. It also can give rise of mature cell,
which have the same characteristic and morphology in term of shape and functions.
According to National Institute of Health, Department of Health and Human
Services (2001a), ADSCs has no such definitive mean of characterisation different from
ESCs, which can be defined by their origin, ICM of blastocyst. There is no way to know
the origin of adult stem cells in any mature tissue. The definition of ADSCs may vary in
some scientific literature from a simple definition to a rigid experimental criteria which
must be fulfilled before particular ADSCs is characterise. It is almost impossible to design
an experiment in organisms as complex as human to identify and track ADCs in vivo. The
majority of the researchers claim that identification of ADSCs actually relay on two
characteristics; first is the appropriate ADSCs morphology and second is by using surface
marker that identify them as belonging to the tissue. Anyhow, the fundamental of ADCs is
the cell must capable to self-renew for the lifetime of that organism.

2.3.2.2 Types of ADCs

Basically, there are two types of ADCs, which are Hematopoietic Stem Cells
(HSCs) and Mesenchymal Stem Cells (MSCs). It was classified by its properties of blood
forming capabilities.

2.3.2.2.1 Hematopoietic Stem Cells (HSCs)

Hematopoietic stem cells (HSCs) are stem cells that responsible of forming blood
and immune cells. These HSCs can be isolated from our blood or harvested from a bone

14
marrow.
Anyhow, over the years, the terms that relate between hematopoiesis and stem cell
may lead to some confusion. Although they (at an early stage of development) have the
abilities to differentiate along with multiple lineages and posses extensive proliferative
potential however, this HSCs are lack in abilities of self-renewal in vivo. HSCs also low in
frequencies in normal hematopoietic tissues (Keller, 1992).
According to National Institutes of Health, U.S Department of Health and Human
Services (2011b), HSCs basically face two major problems in order to expend their abilities
beyond just only as the replacement of blood and immune system. First, the lack of abilities
to replicate it selves and differentiate to other specialised cell type in vitro and secondly,
there is no accurate method to distinguish stem cells from others cells that recovered from
the blood or bone marrow. This is due to their characteristic and behavior that just exactly
like a normal white blood. There is almost impossible to distinguish between these two cell
unless through the cell surface protein which act as a markers of the white blood cells.

2.3.2.2.2 Mesenchymal Stem Cells (MSCs)

Mesenchymal stem cell or also known as mesenchymal progenitor cells or subset of

non-hematopoietic cells are exist within the bone marrow stroma (Minguell et al., 2001).
MSCs can be characterise by it morphology (Figure 2.7). It posses a small cell body
with some cell process which usually thin and long and widely dispersed and matrix
extracellular. It populated by some reticular fibrils. It cell body contain large and round
nucleus that surrounded by finely disperse chromatin particles which give the nuclear a
clear appearance (Banerjee, 2014) This adult stem cell can be found in almost any of the
tissue in the body of fully developed organisms (Figure 2.8). Sometimes, multiple types of
adult stem cells with the vast potential to differentiate into many other types of cell can be
found and isolated from the same tissue. (Turksen, 2014).

15
 Figure 2.77: MSCs observed undeer fluorescennt microscoope (Stem Cell
C Institutee, 2016)

Figure 2.8: Locations of

F o somatic stem cells inn the human body (Geneetic science learning
centeer)

w done inn 1974, by a scientist

The fiirst isolationn and charaacterisation of MSCs was
naame Friedennstein and hhis colleagues. Since thhen, researchh in MSCs are rapidlyy increased
evvery year w
with a numbeers of cliniccal trial figuures were rregistered (F
Figure 2.9) with high
ennthusiasm inn many counntries. This is due to theeir propertiees that free from ethicaal concerns
annd low risk oof teratoma formation, tthe (Wei et al., 2013).

16
 Figure 2.9: Number of registered clinical trials of mesenchymal stem cells-based therapy
(Wei et al., 2013).

2.3.2.3 Sources of Adult Stem Cells

Besides our own tissue or organ in own body, somatic stem cells for stem cell
therapies can also be obtained from cadavers and umbilical cord.

2.3.2.3.1 Cadavers

Cadavers or dead bodies do not only provide spare organs for transplants but also
can serve as pool of a source of stem cells. Huge numbers of stem cells can be obtained
form bone marrow, five days after death. It could possibly be used in such of variety of
treatment. Recently, scientist has successfully isolate cell from postmortem arterial segment
and kept in tissue banking facilities up to 5 years. This successful could represent as an
innovative and the unlimited reservoir of different stem cell bank for future used (Valente
et al., 2014).

2.3.2.3.2 Umbilical Cord

Umbilical cord (Figure 2.10) blood that rich in cell is one of good source of
hematopoietic (HSCs) stem cells, which can give rise to all other blood cells trough
haematopoiesis process.

17
 Figure 2.10: Umbilical cord blood (Qiao’s Pathology)

The first scientist’s groups that introduce the use of umbilical cord blood as a
transferable hematopoetic stem cell are Boyse, Bard, J., and Bronxmeyer 1980s (Lopez
2010). Besides hematopoietic stem cell, umbilical cord also known as rich with pluripotent
mesenchymal cells. It holds big medical potential to cure many disease and blood cancers
such as acute lymphocytic and myeloid leukemia (Moise, 2005).

2.3.2.3.3 Bone Marrow

Bone marrow stem cells are one of the sources of the adult stem cell. It represents
approximately 1 per 10.000 cells. Because of their abilities of mobilization from bone
marrow to bloodstream, ADSCs also can be found in peripheral blood (Gonzalez, &
Bernad, 2012).
Bone marrow has vast abilities to populate a numerous body tissues such as bone,
liver, cardiac muscle, colon and skin. In normal body of adult with range of 70 KG has
functional hematopoietic marrow volume at least 1.7 liter and it will increase accordingly
due to infection or hemorrhage up to six times, which approximately 10.2 liter (Hassan, &
El-Sheemy, 2004).

2.3.2.3.4 Skin Fibroblast

Skin fibroblast or also known as dermal fibroblast a most numerous cells dermal

18
layer of the skin. Besides it main function of fibroblast which preserve the structural
integrity of skins it also can serve as a stem cell sources (Bai, 2013).
Recent study discovered that multipotent dermal stem cells isolated from a human
neonatal foreskins has abilities to differentiated into multiple cell linage which including
pigmented melanocytes (Li et al., 2012)

2.3.3 Induced Pluripotent Stem Cells (IPSCs)

Induced pluripotent stem cells or also known as iPSCs (Figure 2.11) is an embryo-
like stem cells than can be derived from patient’s own cells. It is a mature cells that
undergoes chemically engineered of genetic by reverting adult state to an immature state
that have the same fetal or embryonic stem cells properties. The iPSCs also carries the
same genes as non-engineered cell thus it will not be rejected by the body. The iPSCs also
serve as good alternative to avoid ethical dilemma and controversy that may arise for
certain stem cell that derived and required killing of the embryos.

Figure 2.11: A colony of induced pluripotent stem cells (Salk Institute for Biological
Studies, 2012).

19
2.3.3.1 Characteristic of iPSCs

According to Tiscornia et al., (2011), there are a few basic characteristics of iPSCs.
For example, iPSCs has the ESC-like morphology such as forming a tight colonies with
high nucleus to cytoplasm ratio. The cell have different shape and the appearance depend
on culture. Beside that, iPSCs and ESCs also give positive staining for alkaline
phosphatase. The presence and activity of reprogramming transgenes also detected and can
be tested using PCR or Southern blot. iPSCs also give a positivity for pluripotency marker
such as Oct4, Nanog, Sox2, Tra-1-81, SSEA4, Rex and telomerase. iPSCs also act as
promoter methylation analysis of key pluripotency genes, usually Oct4 and Nanog. It also
has ability to differentiate into three germ layers, which are endoderm, mesoderm and
ectoderm.

2.3.3.2 Generation of iPSCs

iPSCs are pluripotent are adult cells that have been genetically reprogrammed to an
embryonic stem cell-like state by induce the ESC-gene expression. This iPSCs generation
share the same properties as pluripotent stem cell or embryonic stem cells such ability to
express of pluripotency-related genes, DNAs methylation patterns, self-renewal, embryoid
body formation, teratoma formation and viable chimera formation. Anyhow, iPSCs and
ESCs are not identical in terms of differentiation capacity and epigenetic features. There are
many various approached can be used to generate this clinical-compatible iPSCs, for
example by using non-integrating, non-viral and non-genetic method (Sohn et al., 2012).
iPSCs can be obtained by reprogramming somatic cell into a pluripotent state.
Basically creating iPS cells can be divided into five step (Figure 2.12). First of all, cell from
patient either skins of fibroblast were isolated and grown on a dish. After that, cell was
treated with reprogramming factors. The culture then leaves for rest for a few weeks and
pluripotent stem cell was produced. The conditions of stem cell in dish were changed
accordingly to stimulate cell to differentiate into a variety of cell types.

20
Figure 2.12: Proceduree for generattion of inducced pluripottent stem ceells (iPSCs) (Genetic
F
sciience learninng center).

21
 CHAPTER 3

STEM CELL-BASED THERAPY FOR TREATMENT OF CANCER

3.1 Stem Cell Therapies

Stem cell based therapy is rely on the tumor-tropic properties of cancer cells. The
ultimate goal of cancer therapy strategies is to develop an antitumor agent that can robustly
target all cancerous cells while sparing all normal and healthy cells. (Kim et al., 2014).
In this chapter, we will look further on type of stem cells-based therapy for treating
cancer and how to manipulate stem cell as an anticancer agent and some genetic engineered
stem cell to stop and kill the cancer cells. We will also look on some established treatment
and some examples of future treatments that are still under clinical trial using stem cell to
cure cancer.

3.2 Stem Cell Transplant

There are three basic types of transplant for curing cancer (named according who
supply the stem cells), which is autologous (derived from patient own body), allogeneic
(from other donor either it closely related or not) and syngeneic (from identical twin or
triplet).

3.2.1 Autologous Stem Cell Transplants

Autologous stem cell transplant is a transplant that used the stem cells that derived
from patient own body. Healthy stem cell was harvested before the patient undergoes
chemotherapy and radiation therapy before the treatment destroys the cells. This healthy
stem cell was obtained from bone marrow or blood which then keeping frozen for the later
use. After chemo and or radiation done, the stock healthy stem cells were transplant back to
the patient. The advantage by using this method is, there is low-to-no risk of graft-versus-
host-disease (GVHD) or a new infection from other person. This kind of transplant usually

22
used to treat a various diseases such as testicular, neuroblastoma and certain cancer in
children.
One of the example of autologous stem cell application for curing cancer is
autologous bone marrow transplantation for primary nasal T/NK cell lymphoma that was
reported by Liang et al. (1997). These primary nasal T/NK cell lymphoma is uncommon in
western world compare to Orientals. These tumors normally attack and grow on mid-line of
nasal cavity. Before the application on autologous stem cell transplant, only 50% of stage
one patient’s are durable remission and some of them face fatal if the disease are
disseminated. However in 1992, three Chinese patient that suffered primary nasal T/NK
cell lymphoma undergo high-dose chemotherapy and autologous bone marrow rescue
resulting two of them successfully remission completed while another third patient
unfortunately died due to progressive disease. This report as one of many evidences (Chim,
et al., 2004; Au, et al., 2003) that high-dose chemotherapy and autologous marrow or stem
cell transplant can be effective treatment for relapsed primary nasal lymphoma.

3.2.2 Allogeneic Stem Cell Transplants

Allogeneic stem cell transplantation is a stem cell transplantation derived from a

donor (maybe relative or complete stranger), which involve harvesting adequate number of
the stem cells from histocompatible donor (compatibility tissue with the almost the same
HLA) to infusion into patient body after high-intensity chemotherapy or radiation (Bosi et
al., 2005).
In Malaysia, allogeneic stem cell transplant is predominant with 70.7% compare to
29.3% are autologous with majority to treat malignant disorders (Gan et al., 2008).
Allogeneic stem cell transplantation might be a high-risk procedure with possible adverse
effect such as patient’s immune system rejects the donated stem cell (Host-versus-graft
effect) and immune reaction by the donor cells against the patient’s tissues (graft-versus-
host-disease: GVHD).
There are few examples of allogeneic bone marrow transplant for curing some
disease, for example for curing chronic myelogenous leukemia in a patient with multiple
sclerosis which reported by McAllister et al. (1997) and aplastic anemia, which reported by

23
Storb et al. (1976)

3.2.3 Syngeneic stem cell transplants

Syngeneic stem cell transplant involve harvesting stem cells from donor, which
from patient’s identical twin or triplet and infusing back them into the patient. Identical
twins, which have identical genetic types, were considerate as a perfect match. The first
attempt syngeneic transplant is in 1960s to treat aplastic anemia when they found that
hematopoietic system in humans could be replaced with identical genetic donors (Whedon
& Wujcik, 1997).
Based on case report shown by Hwang et al. (2000), there are two cases (woman
21-year-old and 35 year-old man) of severe aplastic anemia (SAA) that successfully treated
by syngeneic peripheral blood stem cell transplantation using immunosuppression and
without high-dose chemotherapy or irradiation for conditioning. The women patient
receives BMT from her identical twin sister, while the man from identical twin brother.
After the transplant, the patients show no rejection up to 22-month post peripheral blood
stem cell transplantation. The successful of these two cases as one of many evidences that
syngeneic transplant can be used as therapy for the cancer treatment.

3.3 Established Stem Cell Therapies

There a few well-established diseases that successfully treated using stem cell
method such as acute myelogeneous leukemia, acute lymphoblastic leukemia, chronic
lymphocytic leukemia, chronic myelogeneous leukemia and some other type of leukemias
and lymphomas (Table 3.1).
Beside that, stem cell therapies were successfully treating some diseases that affect
bone marrow such as severe aplastic anemia, fanconi anemia, pure red cell aplasia and
several bone marrow failure syndrome (Dokal & Vulliamy, 2010).
Beta thalassemia major and sickle cell disease are the example of
hemoglobinopathies, the diseases that have poor red blood cells. This disease has been
successfully treated with stem cells therapies. Stem cell therapies also successfully treated

24
some inherited metabolic disorders such as krabbe disease, Hurler syndrome,
adrenoleukodystophy and metachromatic leukodystrophy
According to Luca (2015) since 1970s, skin stem cells were used to treat life-
threatening patient who suffered from severe burns on their body. There are also some
applications of stem cells (Holoclar® System) to treat damaged cornea due to injury caused
by chemical. This therapy is already gained an approval in Europe.

Stem Cell-Based Therapies Cancer Type

Bone Marrow Transplant
Myelogeneous leukemia, acute lymphoblastic
leukemia, chronic lymphocytic leukemia, chronic
myelogenous leukemia, hemoglobinopathies, krabbe
disease, hurler syndrome, adrenoleukodystophy,
metachromatic leukodystrophy

Stem Cell for Therapeutic Brain cancer, metastatic tumour, multiple tumour
Delivery lesion

Anticancer stem cells Breast cancer, colon cancer, pancreas cancer and
prostate cancer.

Genetic Modification stem Malignancies such as leukemia and Hodgkin’s

cell to secrete anticancer lymphoma.
protein
Genetic modification of stem Prostate cancer, metastatic tumour
cell to induce cancer cell
death

Table 3.1: Stem Cell Based Therapies and Cancer Types

3.3.1 Bone Marrow Transplantation

Bone Marrow Transplant (BMT) is the most successful method for treatment of
blood related disorders such as leukemia or lymphoma. It involves transplantation of
multipotent stem cell involving either autologous bone marrow transplant, allogeneic bone
marrow transplant or syngeneic bone marrow transplant.
In Malaysia, the first BMT was successfully performed in 1987 on pediatric patient
at University Malaya Medical Center (UMMC) while the first adult BMT was performed in

25
1993 also at the same hospital. Since then, BMT research center has rapidly growing with 8
BMT centers with a total of 1155 total transplantation performed until 2006 with range
from 100 to 150 of transplantation per year (Gan et al., 2008).
The procedure for BMT can be summarised in the Figure 3.1. Basically, the
procedure will start with biopsy and aspiration (Figure 3.2) of the bone marrow from
patient or donor pelvis (hip bone). Around 1 liter of bone marrow will be harvested and
processed with antibodies in the lab to purify and concentrate the stem cells. Then stem
cells will be cryopreserved until the blood transfusion took place. After that, a patient will
undergoes high dose of chemotherapy or radiation to kill all cancerous cell. Chemotherapy
or sometimes referred to simply as “chemo” is the use of a drug or combination of drugs to
treat cancer by stopping or slowing the growth of cancer cells. There are more that 100
drugs available to treat cancer with variety of purposes while radiation therapy is uses high-
energy radiation to shrink tumors and kill cancer cell. The radiation delivered by a machine
outside the body known as external-beam radiation therapy or internal radiation therapy by
placed radioactive material in the body near the cancer cells. After chemotherapy and
radiation took place, thawed stem cells will be infused back into patient blood stream using
central venous catheter (Antin & Raley, 2013; Chu & DeVita, 2006; National Cancer
Institute, 2015a; b).

Figure 3.1: Bone marrow transplant step by step (The Editors of Encyclopedia

26
 Britannica, 2014)

Figure 3.2: Bone marrow aspiration and biopsy (Winslow, 2007).

In typical stem cell transplant for curing cancer, chemotherapy has been introduced
to patient along with a radiation therapy to prevent cancer cells from multiplying, invading,
metastasizing and ultimately killing the patient. However, chemotherapy and radiation
therapy will kill both cancerous and healthy cells including stem cells in the bone marrow.
Soon after the “killing” processes were done, patients were infuse with the healthy stem
cells thru vein to replace the entire destroyed cell. This engraftment process happen from
time to time until it settles in bone marrow and begun to grow and make a healthy blood.
These procedures a more or less likely, a normal blood transfusion (Cancer Research UK,
2015; Skeel & Khleif, 2011).
According to research done by Attal, et al. (1996), high-dose chemotherapy
combined with BMT improves the response rate, event-free survival, and overall survival
up to 81 percent in patient with myeloma. Matthay et al. (1999), also found that, event-free
survival rate was significantly better for patient who suffer high-risk neuroblastoma receive
chemotherapy and bone marrow transplant compare to patient who undergo13-cis-retinoic
acid treatment or continuation chemotherapy without transplant.

27
3.4 Therapeutic Potential of Stem Cell Therapy

Besides traditional therapies using stem cell, there also some research make used
the stem cell properties to stop and prevent the cancer cell from emerge and growing.
Anyhow, this research consider of the future application, which still undergoes
development and clinical trial.
Clinical study over last ten years suggest that stem cell transplantation has a huge
potential as therapy for some diseases such as neurodegenerative diseases, Parkinson’s
disease and Huntington’s disease. It also offers tremendous potential to treat many other
human diseases and treat such as damage tissue, injury, ageing, cancer, diabetes and
blindness (Watt & Driskell, 2009).
Stem cell therapy also can serve as therapeutic option to some injury and disease
condition such as musculoskeletal myriad, which presently have a very limited therapeutic
option (Bahney, & Miclau, 2012)

3.4.1 Stem cell for therapeutic delivery

Targeted delivery by the anti-cancer agents is one of the promising fields in stem
cell as therapeutic delivery. Mesenchymal stem cell, also known as MSCs said have the
abilities as a tumor-tropic and migratory properties thus have a huge potential to serve as a
vehicle for targeted drugs delivery system for isolated tumor and metastatic disease (Gao et
al., 2013).
MSCs also have abilities to localise and integrate into tumor stroma and deliver
anti-cancer agent such as oncolytic viruses that preferentially infects and kill cancer cells.
(Gjorgieva et al., 2013).
With all these properties, it became promising future especially in cancer
treatments. Anyhow, these systemic deliveries in gene therapy face some problem due to
some factor such as immune reaction of the patient, poor tissue permeation and non-
specific accumulation in a normal tissue. Recent study was done by Serakinci et al. (2011)
has found that the best way to used this therapeutic delivery approach by combines between
genetically engineered vehicles stem cell therapy and suicide gene therapy. This can reduce

28
the risk of secondary tumor and improved the targeting therapy.
A study was conduct by Bagci-Onder et al. (2015) Discover the efficacy of
therapeutic stem cell for metastatic brain cancer which also known as secondary brain
cancer by using mouse model in vivo. They also successfully explore the ability of
engineered adult stem cell, which implanted or injected efficiently home to metastatic
tumour deposit in brain. These tumour-seeking stem cell-based therapies might successfully
treat multiple metastatic tumour lesions in the brain. They also successfully establish a
novel therapeutic approach by using engineered neural and mesenchymal stem cell for
primary brain neoplasm to express tumour-specific biomolecules. They also claim has
successfully demonstrate the efficient of stem cell-based for anti-tumour therapies both in
nodular and invasive glioblastoma.

3.4.2 Creating anticancer stem cells.

Many of the cancer patients only survive within a short period before the cancer
start to recurrence. This is because conventional cancer drugs only shrink the tumors
instead of killing the cancer stem cells (CSCs). Therefore researchers start to find ways on
how to stop the cancer growth of cells by creating anti-cancer stem cells.
One of the latest finding on anti-cancer stem cell agent is known as OH14. OH14 is
an inhibitor of c-FLIP (cellular FLICE-like inhibitory protein) was discovered by a group
of scientist from Cardiff University. According to the latest news from Cardiff University
(2015), that compound are capable targeting aggressive tumor forming cell which common
in breast, colon, pancreas and prostate cancer. It will target the c-FLIP with the OH14
compound that activated it self-defense mechanism against the immune system. This
compound also will prevent cancer from regrowth. Anyhow, according to them, this
compound is still under development phase and once completed, then it will continue to the
clinical trial, licensed by Tiziana Life Sciences.

3.4.3 Genetic modification of stem cells to secrete anticancer proteins

Stem cell also can be genetically modified to target specific tumor types and secrete

29
therapeutic protein such as interferons (signaling protein to responds the presence of tumor
cells)  and , interleukins 2 and 12 or chemokine (signaling protein) CX3CL1 (Gjorgieva
et al., 2013).
One sample of clinical research is Engineering Toxin-Resistance Therapeutic Stem
Cells to Treat Brain Tumors that were conducted by Stuckey et al. (2014). They
successfully construct a stem cell genetically that resistance of toxin and at the same time
secrete PE-cytotoxin. PE-based cytotoxins have been used with the great success to treat
much kind of malignancies such as leukemia and Hodgkin’s lymphoma.

3.4.4 Genetic modification of stem cells to induce cancer cell death

Mesenchymal stem cells or neural stem cell can be genetically engineered to act as a
suicide gene therapy to treat various malignant. This genetic modified stem cell has
advantage of being safe where the stem cell is being eliminated after the therapeutic effect
thus there is no long-term fate concern in future (Stuckey & Shah, 2014).
This genetically engineered suicide gene therapy approach are involving the NSCs
are modified to express gene coding for an enzyme (enzyme-prodrug suicide gene therapy
system) which convert systemically administered inactive prodrug into toxic metabolites at
a tumor location. There two systems can be used to engineer the NSCs to archive this
purpose. One is by using HSV-thymidine kinase (HASV-tk) that responsible to convert the
inactive prodrug ganciclovir (GCV) to toxic metabolite GCV-triphosphate and second
system by using cytosine deaminase (CD) gene that converts inactive 5-fluorocytosine (5-
FC) to 5-fluorouracil (5-FU) (Ahmed et al., 2010).

30
 CHAPTER 4

DISCUSSION

4.1. Limitation of Stem Cell Based Therapy

Even though stem cell-base therapy is the best option for treatment of cancer, there
is consequences may have side impact or risk to take. Most of the stem-cell base therapy is
still in the preliminary research phase. Some of the researches for example held by China
do not even have a proper medical standard documentation! And there is a record that in
certain country that does not fully supervise used sharks and sheep stem cell to treating
human patient! (SCL Support, 2013).
Even for some center that has legal approval from government. It is still then raising
a concern either that stem cell-based therapy will 100% work to cure on all patients without
any life-threaten side effect.
In this last chapter, we will look for some risk and danger that may arise, challenges
and some controversy and etiquette issue related to stem base therapy, especially to treat
cancer.

4.1.1 Mismatched transplant

There were risks where when the transplant if not really a perfect match or
mismatch. When someone has a mismatched transplant, they will more likely have a
reaction after the transplant known as GVHD. GVHD is an immunological disorder, which
may occur after a bone marrow, or stem cell transplant whenever someone who receives a
bone marrow tissue or cells from a donor that not really matched. This new transplanted
cells regard the recipient’s body as foreign, thus the cell will attack the recipient’s body.
GVHD may affect some organ system such as gastrointestinal tract, which can
caused diarrhea, vomiting, anorexia and or abdominal pain. GVHD also can cause Liver

31
disease such as liver dysfunction after BMT such as veno-occlusive disease, drug toxicity,
viral infection, sepsis or iron overload. While skin, which effected with GVHD cause
maculopapular rash others possible diseases. The patients require to continued treatment
with immunosuppressive drugs that increases their risks for serious infections and other
complications (Ferrara et al., 2009).

4.1.2 Cancer recurrence

Some stem-based therapy may be failure and cause recurrence of the cancer. For
example in autologous transplant, the cancer cells may be picked up along with the healthy
stem cells when it was first time harvest from the patient and later on were transplant back
to patient body. This will cause cancer recurrence (American Cancer Society, 2013).
It might be recurrence because of low in immune system as this method does not
improve a patient immune system when stem cells were engraft thus will give ability to
cancer start to grow again. Anti-cancer drugs or to treat stem cell in other method in order
to reduce a number of cancel cell does not promise fully successful as it has not yet been
proven to reduce the rick of cancer recurrence. This method also at the same time will
purging the healthy stem cell and can causing serious problem due to longer time to
produce normal blood and white blood cells, which increase a risk of infections or bleeding
problem (Campbell, 2006)

4.1.3 Side Effects

As we discuss briefly before in topic type of stem cells transplant, the bad impact of
chemo and radiation that not only kill the cancerous cell but also kill the health cells. There
are also concerns on side effect that may occur due to chemo and radiation. When the
patient treat with chemotherapies which one of the process in stem cell therapy (bone
marrow transplant), the drugs that been used to destroy the cell in the body will also cause
hair lost, nauseous, rashers and in some cases lost feeling in fingertips and toes (Adams,
2004).

32
4.1.4 Financial Challenges

One of the biggest challenges to patient is the high cost for stem cell-based therapy
for treatment of cancer. According to Gan et al. (2008), in Malaysia the cost for
transplantation ranging from 500 MYR to 100,000 MYR. Even though there are two
government hospitals (Ampang Hospital and University Malaya Medical Centre) provided
the transplant with subsidized cost, but the waiting list is way too long while other private
medical centre such as Subang Jaya Medical Centre may offer the treatment but with highly
cost involve.

4.2. Controversy and Etiquette Issue

Even though the abilities of stem cell that want to be used in therapies can be tested
using animal, such as mice before tested to human, it is not really sufficient to know the
real potential of that stem cell to developed tissue replacement. This is because animal has a
different development substantial biological and different stem cell compare to human, thus
will not fully represent the actual result when apply to human (Committee on the Biological
and Biomedical (National Research Council, 2005).
Stem cell research also bound by the laws and regulation set by the religious
council. For example in Malaysia, Majlis Fatwa is Islamic religious authorities has set the
guideline in Convention of The Committee of National Council for Islamic Religious Affairs
in Malaysia (67th assembly) over the issue therapeutic cloning and stem cell research in
2005 has decided any commercial research and has nothing to do with the health of mother
or fetus are prohibited. Any of research on embryo must grant permission from both
husband and wife. And according to Majlis Fatwa, the pre-embryo from the research are
absolutely prohibited to be implanted into other women or co-wife. SCNT also prohibited
(Haram) in Islam based on method sad al-zaraie which mean, “blocking the means to evil”
(National Fatwa Council, 2005).

33
4.3 Legislation on Human Embryonic Stem Cell

The also some country has a strict policies regarding human embryonic stem cell
(hESC) for example in certain country like Austria, France, Germany, Ireland, Italy,
Netherland and Norway are prohibited on research of human embryonic stem cell while
Australia, Belgium, China, Denmark, Finland, Hungary, India, Israel, Japan, Singapore,
Spain, South Korea, Sweden and United Kingdom were regulated by law (Ministry of
Health Malaysia, 2009).
Malaysia has release a guidelines for stem cell research therapy by Ministry of
Health in 2009 where Institutional Review Board (IRB) and or Institutional Ethic
Committee (IEB) must review all stem cell research and application for approval (Ministry
of Health Malaysia, 2009).

34
 CHAPTER 5

CONCLUSION

Stem cell-based therapy has the potential to cure many human diseases. The ability to self-
renewal and differentiate give us hopes, as it has a potential to replace or to repair any
damage tissue. It also can be an effective treatment for people with certain form of cancer
such as leukemia, lymphoma, multiple myeloma and neuroblastoma. Anyhow, at the
meantime, only hematopoietic stem cell is available and the best option with high success
rate while other potential therapy still undergoes clinical trial and some of them still lack in
documentation, therefore, it is important to establish an entity who responsible for
reviewing all the related research to make sure it complies with the discipline of study and
documented it. In addition, we also need to solve the biggest issue, which is searching for
matching donor. Searching for the right donor is a crucial issue, as un-matching donor will
cause side effect to the patient and may causing death. Therefore, besides focusing on the
research itself, it is important we find the solution on how we can solve the demand of stem
cell itself. As conclusion, stem cell-based therapy do look promising for a cancer treatment,
however there’s a lots of limitation and challenge that we need to consider. I believe, if all
the limitation and challenge that been discuss in previous chapter has been solve, stem cell-
based therapy will become a main solution to the various type of medical potential to cure
many disease including all type of cancer.

35
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