Use of IPs cell derived neural stem cells as a cellular vehicle for glioma and breast cancer therapy

USE OF iPS CELL-DERIVED NEURAL STEM CELL AS A CELLULAR VEHICLE FOR GLIOMA AND BREAST CANCER THERAPY ESTHER LEE XING WEI B,Biomed Sci., University of Melbourne A THESIS SUBMITTED FOR TH

USE OF iPS CELL-DERIVED NEURAL STEM CELL AS A CELLULAR VEHICLE FOR GLIOMA AND BREAST

CANCER THERAPY

ESTHER LEE XING WEI (B,Biomed Sci., University of Melbourne)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILISOPHY

DEPARTMENT OF BIOLOGICAL SCIENCES

NATIONAL UNIVERSITY OF SINGAPORE

&

INSTITUTE OF BIOENGINEERING AND NANOTECHNOLOGY (A*STAR)

2011

ACKNOWLEDGEMENTS

First and foremost I would like to thank A/P Wang Shu for his scientific support and guidance The members of our lab: Ghayathri Balasundaram, Xiaoying Bak, Yukti Choudhury, Timothy Kwang, Dang Hoang Lam, Jiakai Lin, Seong Loong Lo, Yovita Ida Purwanti, Chrishan Julian Alles Ramachandra, Mohammad Shahbazi, Chunxiao Wu, Kai Ye, Ying Zhao, Jieming Zeng and Detu Zhu have been invaluable resources, not only for their technical help, tips and advices but also the camaraderie

I am grateful for my family, especially my husband and son who have been with me through the tough times They have been nothing but supportive in all my endevaours Finally and most importantly,I would also like to thank God for His countless grace

LIST OF PUBLICATIONS

E X W Lee, D.H Lam and S.Wang iPS-cell derived Neural Stem Cell for Glioma Therapy Poster presentation World Stem Cell Summit, Detriot, USA,

2010

X Y Bak, D H Lam, J Yang, K Ye, E X W Lee, S K Lim and S Wang,

"Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells as Cellular Delivery Vehicles for Prodrug Gene Therapy of Glioblastoma," Human Gene Therapy, (2011)

E X W Lee, D.H Lam, C.X Wu, J Yang, C.K Tham, W.H Ng and S Wang

Glioma Gene Therapy Using Induced Pluripotent Stem Cell Derived Neural Stem Cells Molecular Pharmaceutics , Oct 3;8(5):1515-24Jul 22,2011

E X W Lee, Y Kai, G Balasundaram, F Tay, S.Goh, S Wang

Comparison of three suicide gene/prodrug systems in cancer gene therapy mediated by baculovirus-transduced neural stem cells derived from human pluripotent stem cells Poster presentation Stem Cell Society Singapore Symposium 2011

TABLE OF CONTENTS

1 Introduction

1.1 Glioblastoma 13

1.2 Breast cancer 13

1.3 Gene therapy: the methods of gene transfer 14

1.3.1 Viral vectors 14

1.3.1.1 Adenovirus 15

1.3.1.2 Retrovirus 16

1.3.1.3 Baculovirus 16

1.3.2 Synthetic vectors 18

1.3.3 Molecular approach for cancer gene therapy 18

1.3.3.1 Prodrug converting/suicide gene therapy 18

1.3.3.2 Expression of molecules that affect angiogenesis, tumor invasion and metastasi 20

1.3.3.3 RNA interference (RNAi) targeting approach 21

1.3.4 Immunomodulation approach to cancer gene therapy 21

1.3.5 Current status of gene therapy for glioma and breast cancer 22

1.4 Stem cells 23

1.4.1 Adult stem cells 24

1.4.1.1 Neural stem cells 24

1.4.2 Induced pluripotent stem cells 25

1.5 Stem cell as a vehicle for cancer gene therapy 26

1.5.1 Stem cell based cancer gene therap 26

1.5.2 Cancer tropism of stem cells 27

1.5.3 Route of administration of stem cell 28

1.5.4 Clinical trials with stem cell based cancer gene therapy 28

1.5.5 Future of stem cell based cancer gene therapy 30

2 Aims and objective 31

3 Material and methods 33

3.1 Cell culture 33

3.2 Mouse iPSC-derived NSC for glioma therapy 34

3.2.1 Lentivirus preparation, reprogramming and maintenance of mouse iPSC 34

3.2.2 Alkaline phosphatase staining 36

3.2.3 Generation of mouse iPSC-derived NSC (msiPSC-NSC) 36

3.2.4 Characterization of msiPSC-NSC 37

3.2.5 RT2 profiler PCR array 38

3.2.6 Baculoviral vector construction and transduction 40

3.2.7 Cytotoxicity assay 40

3.2.8 In vitro migration assay 3.2.9 In vitro sensitivity of TK expressing-neural stem cells (msiPSC-NSCtk) to GCV 42

3.2.10 In vitro tumoricidal effect in glioma cells 42

3.2.11 Animal study design: the use of msiPSC-derived NSC for glioma therapy 42

3.3 Comparison of different delivery methods of stem cells in breast cancer

mouse model 43

3.3.1 Derivation, maintenance of iPSC-NSC 43

3.3.2 Characterization of iPSC-NSC 44

3.3.3 Fluorescence labelling and biocompatibility analysis 45

3.3.4 Animal study design: comparison of different delivery methods 45

3.4 Comparison of 3 suicide gene systems: HSVtk, Fcy and CodA 46

3.4.1 Baculovirus preparation and cell transduction 46

3.4.2 In vitro sensitivity of HSVtk, Fcy and CodA-expressing iPSC-NSC 47

3.4.3 Animal study design: comparison of 3 prodrug systems in 4T1 breast cancer mouse models 49

4 Results 4.1 Use of msiPSC-NSC in glioma therapy 50

4.1.1 Aim 50

4.1.2 Reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells and the derivation of neural stem cells 50

4.1.3 msiPSC-NSC display migratory behavior to glioma 57

4.1.4 msiPSC-NSC can be successfully transduced with baculovirus 59

4.1.5 msiPSC-NSCtk generated by baculoviral transduction display bystander killing effects on glioma cells in vitro 65

4.1.6 Gene therapy using HSVtk-expressing NSCs in glioma mouse model successfully reduces tumor growth rate 67

4.2 Comparison of different stem cell delivery methods in breast cancer mouse

model 67

4.2.1 Aim 67

4.2.2 DiR labeled iPSC-NSC as a tool for tracking stem cell migration in

vivo 67

4.2.3 iPSC-NSC injected intravenously exhibits observable but limited

migration to mammary fatpad tumor in vivo 69

4.2.4 Baculovirus successfully transduces iPSC-NSC iPSCNSCtk/GCV

exhibits breast cancer therapeutic effect 73

4.3 Comparison of 3 suicide gene systems: HSVtk, Fcy and CodA 76

4.3.1 Aim 76

4.3.2 iPSC-NSC transduced with baculovirus carrying herpes simplex

virus thymidine kinase or cytoskine deaminase are sensitive to GCV

and 5’-FC respectively 76 4.3.3 A small population of suicide gene carrying cells is sufficient to elicit

bystander mediated killing effect 80

4.3.4 CD/5-FC system shows slightly better therapeutic effect in 4T1

breast cancer mouse in vivo 82

5 Discussion 86

5.1 Use of msiPSC-NSC in glioma therapy 86

5.1.1 NSCs as a suitable gene delivery vehicle for glioma therapy 86

5.1.2 The population size of NSC is a limiting factor for gene delivery 87

5.1.3 Using baculoviral vector for gene transfer 89

5.1.4 The immunomodulatory effects of NSCs 90

5.1.5 The suitability of U87 glioblastoma model as a tool for studying glioma therapy in vivo 92

5.2 Comparison of different stem cell delivery methods in breast cancer mouse model 92

5.2.1 Issues regarding the different methods of stem cell administration 93

5.2.2 DiR as a suitable label for in vivo tracking of iPSC-NSC 94

5.3 Comparison of 3 suicide gene systems: HSVtk, Fcy and CodA 95

5.3.1 HSVtk/GCV and CD/5-FC mediates bystander effect by different methods 96

5.3.2 Tumor growth rates were reduced but no complete eradication of cancer was observed 98

5.3.3 Possible strategies to improve therapeutic efficiency 98

5.3.4 Possible cytotoxic side effects from prodrug treatment 100

6 Conclusion 101

7 Future Direction 106

Summary

Using neural stem cells with tumor tropic migratory capacity to deliver therapeutic genes is an attractive strategy in eliminating metastatic or disseminated tumor Different methods have been studied and developed to isolate or generate NSCs, but it has not been assessed whether induced pluripotent stem cells (iPSCS), a type or pluripotent stem cells that hold great potential for regenerative medicine, can be used as a source for derivation of NSCs with tumor tropism In this study,

we used a conventional lentivirus transduction method to derive both mouse and human iPSCs from embryonic fibroblasts and then generated NSCs from these iPSCs To investigate whether the iPSC-derived NSCs can be used in the treatment of disseminated brain tumor and metastatic breast cancer, the cells were transduced with baculoviral vector containing the herpes simplex virus thymidine kinase or the cytosine deaminase suicide gene In the glioma study, the mouse iPSC-NSCtk were injected contralaterally to tumor inoculation site in a mouse intracranial human glioma xenograft model In the breast cancer study, the human iPSC-NSCtk, iPSC-NSCFcy, iPSC-NSCCodA were injected either intravenously or directly into the tumor site We observed that NSCs expressing the suicide gene were, in the presence of respective prodrugs ganciclovir or 5-fluorocytosine, effective with varying extents in inhibiting the cancer development

of the glioma xenograft and breast cancer metastatic model and prolonging the survival of tumor-bearing mice Our findings provide evidence for the feasibility of using iPSC derived NSCs as a vehicle for targeted anticancer gene therapy

LIST OF TABLES

Table 1 RT-PCR primer sequences for pluripotent stem cell “stemness”-specific and neural stem cell-specific markers 57 Table 2 Changes in expression of embryonic stem cell-specific markers and embryonic stem cell differentiation/lineage marker genes between msiPSCs and MEFs 58

LIST OF FIGURES

Figure 1 Generation of iPS cells from mouse embryonic fibroblasts (MEFs) 55

Figure 2 Generation of neural stem cells from mouse iPS cells and characterization of derived NSCs 56

Figure 3 in vivo and in vitro migratory behaviour of msiPSC-NSC to glioma 60

Figure 4 Mouse iPS cell-derived NSCs can be used to deliver HSVtk suicide gene using a baculoviral vector 64

Figure 5 in vitro bystander killing effect of msiPSC-NSCtk 65

Figure 6.HSVtk/GCV gene therapy for glioma mediated by msiSPC-NSC 68

Figure 7 Characterization of iPSC-NSC labeled with DiR 70

Figure 8 Biodistribution of intravenous administered iPSC-NSC in breast cancer 73

Figure 9 Confocal Imaging of DiR labelled iPSC-NSC ex vivo 74

Figure 10 in vivo tumor monitoring of breast cancer development, and comparison of the therapeutic effect of different administration methods of stem cells 77

Figure 11 iPSC-NSC transduced with baculovirus expressing HSVtk, Fcy and CodA 80

Figure 12 Dose-dependent response of suicide gene-transduced iPSC-NSC to prodrugs 81

Figure 13 Sensitivity of bystander mediated killing effects of HSVtk/GCV, Fcy/5-FC and CodA/5-Fcy/5-FC in co-culture experiments 83

Figure 14 HSVtk/GCV and CD/5-FC for breast cancer therapy mediated by iPSC-NSC 86 Figure 15 Survival analysis of breast cancer receiving HSVtk/GCV or CD/5-FC treatment mediated by iPSC-NSC 87

DMEM Dulbecco's Modified Eagle Medium

EGF Epidermal Growth Factor

FBS Fetal Bovine Serum

FCS Fetal calf serum

GBM Glioblastoma Multiforme

GCV Ganciclovir

hESC Human embryonic stem cell

hESC-NSC Human embryonic stem cell-derived neural stem cell

HSVtk Herpes simplex virus thymidine kinase

IL Interleukin

IP Intraperitoneal

iPSC human induced pluripotent stem cell

iPSC-NSC human induced pluripotent stem cell derived-neural stem cell iPSC-

LIF Leukemia inhibitory factor

MEF Mouse embryonic fibroblasts

miRNA Micro RNA

MMLV Moloney murine leukemia virus

MSC Mesenchymal stem cell

msiPSC Mouse induced pluripotent stem cell

1.1 Glioblastoma

Malignant brain tumors such as glioblastoma multiforme (GBM) and childhood brain cancer, medulloblastoma, have remained virtually untreatable and lethal The median survival is 1 year or less for GBM (Black PM and Loeffler J, 2005; Packer et al., 1999) Current treatment involving radical surgical resection, followed by radiation and chemotherapy substantially improves the survival rate

in some patients, however it remains incurable in a large proportion of patients due to its exceptional ability in infiltrating into surrounding neural tissues distinct from primary tumor localization These microsatellites then act as seeds for recurrent tumor growth (Claes A et al., 2007; Jeon JY et al., 2008)

1.2 Breast cancer

Breast cancer is the most common female malignancy diagnosed in women in developed nations, with an estimated 208,000 women diagnosed with this disease in the United States alone in 2010 (Jemal A et al., 2010) Breast cancer

is a heterogeneous entity with distinct subsets characterised by differences in tumor biology and response to therapy The majority of breast cancers are estrogen receptor -and/or progesterone-positive, and approximately one in five breast cancers is human epidermal growth factor receptor 2/erb-B2 (HER2)-positive breast cancers Hormone receptor-positive and HER2-positive breast cancers have highly effective targeted treatments available Triple-negative breast cancer (TNBC) accounts for 15% to 20% of breast cancers (Konecny G

et al., 2003) Therapy for TNBC is empiric and not based on tumor biology, thus increasing the risk of toxicity without potential benefit As these cancers have a high risk of relapse, irrespective of grade and stage, they account for a large proportion of metastatic breast cancers (Irvin WJ and Carey 2008) 4T1 mammary carcinoma cell line is a suitable experimental animal model for human mammary cancer When introduced orthotopically, it is capable of metastasis to several organs affected in breast cancer including lungs, liver, brain and bone (Aslakson CJ and Miller FR 1992; Yoneda T et al., 2000; Eckhardt BL et al., 2005).The progressive spread to the organs and draining lymph nodes is very similar to that of human mammary cancer (Pulaski BA and Ostrand-Rosenberg S 1998)

1.3 Gene therapy: the methods for gene transfer

Gene therapy is the transfer of genetic material into individuals for therapeutic purposes by altering cellular functions or structure at the molecular level Gene therapy can be carried out by three main routes: gene addition, gene correction/alteration and gene knockdown of which gene addition is the most commonly attempted (Mark A Kay 2011) Two general ways to perform gene

therapy: (1) direct in vivo method and (2) an indirect ex vivo method Gene

delivery methods can be divided into viral methods (lentivirus, adenovirus and retrovirus) and non viral methods (lipoplexes, naked DNA, RNA)

1.3.1 Viral methods

Although non-viral approaches have become more common, two-thirds of gene therapy clinical trials performed used viral vectors due to the poor transfection efficiency and relatively low long term transfection rates of non-viral methods (Edelstein ML et al., 2007)

1.3.1.1 Adenovirus

Adenovirus (AV) is a non-enveloped double stranded DNA viral vector and currently the most commonly used vector in gene therapy trials (Horne et al., 1975; Edelstein ML et al., 2007).It can be used in either replication deficient or replicating forms (Chiocca et al., 2003) Replicating viruses are oncolytic, selectively lysing and dividing tumor cells, and there have been ways to engineer replicating oncolytic virus to achieve tumor selectivity Onyx-015 and Ad5-Delta24 are 2 widely studied oncolytic AV

Onyx015 has deletion in E1B55K gene that allows its replication in p53 defective tumor cells specifically, and its tumor-specific lysis has been enhanced

in clinical trials when combined with chemotherapy in brain, neck and gastrointestinal cancers (Heise et al., 1997; Jiang G et al, 2011) AV mutated in E1A and E1B showed significant antitumor effect in intracranial glioma xenografts with increase tumor selectivity (Gomes-Mazano et al., 2004)

The main advantage of adenoviral vectors are their high efficiency of transduction, ability to infect dividing and non-dividing cells without risk of insertional mutagenesis as they remain as an episomal element after infection, larger DNA load than retrovirus and high level of gene expression, though this is

transient and declines fairly rapidly (Immonen et al, 2004; Edelstein ML et al., 2007) A major disadvantage of adenoviral vectors lies in the activation of both

the innate and adaptive parts of the recipient's immune system when applied in

vivo This immune response may provide additional antitumor effects in glioma

treatment (Sandamair et al., 2000).However, the inflammatory responses can be very strong and can be fatal in patients treated with these AV constructs (Ritler T

et al., 2002)

1.3.1.2 Retrovirus

Retroviruses are RNA viruses that carry a gene for reverse transcriptase that transcribe viral gene material into double-stranded DNA intermediate This DNA intermediate can stably integrate into host DNA, passing to all daughter cells derived from transfected cell (Goff and Lobel, 1987; Oligino et al.,2000) There are two main types of retrovirus employed: lentivirus and Moloney murine leukemia virus (MMLV), with the MMLV more utilised in clinical trials (Marshal 2002) However there is no guarantee that integrated DNA sequences will not cause mutations or malignancies, and its use is also limited by the vector’s inability to infect non-dividing cells and low transduction efficiency (Rainov and Ren 2003)

1.3.1.3 Baculovirus

The insect baculovirus Autographa californica multiple nucelopolyhedrovirus

(acMNPV)-based vector with a circular double-stranded DNA genome has

recently been introduced as a delivery vehicle for transgene expression in mammalian cells, It is capable of transducing MSCs, MSC-derived adipogenic, osteogenic and chondrogenic progenitor cells, hESCs and primary neural cells with varying extents of transduction efficiency Baculovirus mediated transduction had no significant effect on the characteristics of the cells (Ho Y-C et al., 2005;

Ho Y-C et al., 2006; Zeng J et al,2007) Commonly used gene therapy viral vectors such as retrovirus, lentivirus, adenovirus and adeno-associated virus (AAV) in nature have evolved efficient pathways for cellular uptake and viral gene expression (Mark A Kay 2011) However, retroviral and lentiviral vectors randomly integrate into the host genome and has resulted in malignant development (Hacein-Bey-Abina S et al., 2003) The immune response elicited

by adenovirus has hindered its development as a suitable vehicle for gene therapy (Ritler T et al., 2002) and although AAV confers long-term expression as compared to baculovirus’ transient expression, it has limited 2.5-4kb cloning capacity and inconvenient scale up production Furthermore, the majority of human population has been exposed to adenovirus and AAV infections, thus pre-existing immunity might limit its application (Bangari DS and Mittal SK, 2006; Zaiss AK and Muruve DA, 2005) As compared to adenovirus and AAV, baculovirus is an insect virus and humans do not appear to possess pre-existing antibody and T-cell specifically against baculovirus Baculovirus does not integrate into the genome, replicate or is toxic in transduced cells In addition, the construction, propagation and scaling up of baculovirus can be performed easily

(Hu YC 2008)

1.3.2 Synthetic vectors

The limitations of viral vectors, their relatively small capacity for therapeutic DNA and in particular the safety concerns have prompted the development of synthetic vectors not based on viral systems The popularity of this method was reflected in the slight increase of its use in clinical trials (Edelstein ML et al., 2007) This system consists of the ‘naked’ DNA with the delivery constituent To create delivery platforms, DNA has been complexed to many different types of macromolecules eg polysaccharides, poly-lysine, poly-ethylenimine, but the delivery efficiency has remained poor and expression does not persist for sufficiently long periods Hence this approach can only prove useful if the problems associated with delivery efficacy can be overcome (Kay MA 2011)

1.3.3 Molecular approaches for cancer gene therapy

The molecular approach for cancer gene therapy is divided mainly into the several groups Firstly, suicide gene therapy which targets cancer cells at molecular levels Secondly, genes involved in cancer development, tumor invasion and metastasis and thirdly, RNAi that employs oligonucleotides that can bind to specific RNA or DNA sequences

1.3.3.1 Prodrug converting/suicide genes

Suicide gene therapy involves delivery of a specific enzyme that can produce cell death through conversion of an inactive non-toxic prodrug into a cytotoxic drug

metabolite Prodrug-activating enzymes are normally absent or poorly expressed

in mammalian cells The tumor targeting of gene therapy uses specific delivery vehicles that restricts enzyme expression to the transduced tumor cells and adjacent surrounding tumor cells through diffusion or formation of gap junctions

of drug metabolite to generate a bystander effect Two systems studied extensively are the cytosine deaminase/5-fluorocytosine (CD/5-FC) and herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system

CD has been identified in two forms: yeast CD (Fcy) and E.coli CD (CodA)

The CD/5-FC enzyme/prodrug system utilises the diffusion method It converts the antifungal agent, 5-FC, to diffusible, highly cytotoxic compound 5-fluorouracil (5-FU) Intracellular 5-FU is then converted to active metabolites 2-fluoro-2-dUMP (FdUMP) which is an inhibitor of thymidylate synthase and hence of DNA synthesis by deprivation of deoxy-thymidine triphosphate (dTTP) 5-FU although effective in treatment of brain metastasis, cannot penetrate the blood brain barrier Hence stem cell driven prodrug cancer therapy is a much more efficient delivery method This is further elaborated in section 3.1

The cytotoxicity towards glioma cells is enhanced by a bystander effect when NSCs are armed with suicide genes CD and HSVtk (Aboody K.S et al., 2000; Li S et al., 2005; Uhl M et al., 2005) HSVtk mediates GCV activation in

competes with dGTP for incorporation into DNA in internucleotide linkages (Rubsam LZ et al., 1998; Cheng YC et al., 1983) In animal studies, the treated group display significantly extended lifespan and reduction in tumour mass In the HSVtk/GCV system, this cytotoxic bystander effect is based on phosphorylated GCV as a cytotoxic metabolite transferred from cell to cell via gap junctions (Uhl M et al., 2005)

1.3.3.2 Expression of molecules that affect angiogenesis, tumor invasion and metastasis

Angiogenesis is a good target for cancer gene therapy as solid tumors are limited

in size without sufficient oxygen and nutrition Commonly used anti-angiogenic genes are vascular endothelial growth factor (VEGF) receptors and angiostatin Ribozymes or antisense strands can be used against angiogenic gene products

of tumor cells (Folkman J 2002) Genes involved in invasion and metastasis are either deficient (eg TIMP-1) or are overexpressed (eg metalloproteinase

Adapted from: www.invivogen.com

be potentially used to block the invasion and metastasis of cancer Antisense oligonucleotides against mRNA of Bcl-2, c-myc and ras family of oncogenes has resulted in tumor growth inhibition in solid tumors and leukemia (Marcucci et al., 2003; Scharovsky et al., 2000)

1.3.3.3 RNA interference (RNAi) targeting

RNAi is a RNA dependent, gene silencing process that exerts its gene silencing effect through chromatin remodelling, blocking protein synthesis and cleaving specifically targeting mRNA, either via the engagement of small inhibitory or interfering RNA (siRNA) or microRNAs (miRNA) Studies that compare normal versus cancer cells have discovered miRNAs that are implicated in growth control, angiogenesis and metastasis of cancer The use of RNAi in probing the functions of various genes in several tumors has also facilitated the systemic search for new drug targets (Eis et al., 2005; Liu et al., 2003) The ability of RNAi

to silence disease-associated genes in animal models has shown much promise for RNAi-based reagents for clinical applications to treat cancer siRNAs are readily synthesized with low production costs compared to protein or antibody therapies However, their stability in blood and delivery methods are challenges that must be solved for developing effective RNAi reagents for cancer therapy (Kota J et al., 2009)

1.3.4 Immunomodulation approach to cancer gene therapy

The failure of normal immune surveillance mechanisms, leading to an inability to recognize cancer cells as “foreign cells”, is integral in tumor development Hence vectors that express genes that activate the host immune system or bypass some of these defects have been utilized The genes found to alter the local immune microenvironment eg interleukin(IL)-2, IL-4, IL-12, tumor necrosis factor, interferon-γ, granulocyte-macrophage colony stimulating factor(GM-CSF); and genes that have immuno-stimulatory activity or T-cell co-stimulatory molecules are some of them (Rosenberg SA 2004)

1.3.5 Current status of gene therapy for glioma and breast cancer

The first gene therapy clinical trial was conducted in melanoma patients (Rosenberg et al., 1989) followed by treatment of 2 children with severe combined immunodeficiency (ADA-SCID) using retrovirus encoding adenosine deaminase gene (Anderson et al., 1999) Thus far, the majority of clinical trials in gene therapy have been aimed at treatment of various immunodeficiences, cancers including lung, skin, gynaecological and neurological diseases (Edelstein

ML et al., 2007) Combining adenoviral vectors expressing macrophage colony stimulating factor (GMCSF) and a means to restrict vector replication to tumor cells has proven successful in several different cancer trials (Koski A et al., 2010; Burke J.M et al., 2010) T-cells engineered to co-express tumor-specific receptors have also shown persistent antitumor activity in neuroblastoma patients (Pule M.A et al., 2008)

granulocyte-Although gene therapy holds much promise, there have been unfortunate adverse cases with inevitable effects on progress in this area Alain Fischer’s group reported successful treatment of children with X-linked SCID with gene delivered using retroviral vectors (Cavazzana-Calvo M et al., 2000) 2 of the 10 patients developed leukemia within 3 years of gene therapy treatment due to retroviral insertion into proximity of the LMO-2 proto-oncogene promoter (Hacein-Bey-Abina S et al., 2003)

Current gene therapy trials have shown differing extents of anti-tumor efficiency in clinical studies for malignant glioma These include the use of interleukins, interferons in immunotherapy, toxins that target specifically to surface receptors over-expressed on tumor cells and HSV mutant vectors and oncolytic conditionally replicating adenoviruses (Rainov NG et al., 2008; Chiocca

EA et al., 2008; Immonen A et al., 2004; Germano IM et al., 2003; Rainov NG 2000) The therapeutic potential for encoding anti-GBM specific siRNA and miRNA is also being explored (Graner et al., 2009)

A variety of gene therapy approaches have been evaluated for treatment

of breast carcinoma, with majority of clinical trials focusing on p53, and combining the replacement of p53 function with chemotherapy and radiation therapy However varying success have been reported in these clinical trials (Adis R&D Profile, 2003; Lebedeva S et al., 2001; Nielsen LL et al., 1998) With respect to suicide gene therapy, a variety of enzyme/prodrug systems have been investigated in preclinical studies, but those focusing on transducing tumors to express HSVtk, with systemic administration of GCV have demonstrated varying

efficacy as well (Braybrooke JP et al., 2005; Vlachaki MT et al., 2001) Stem cells

as delivery vehicles have been shown to enhance gene delivery efficiency (elaborated in section 3.1)

1.4 Stem cells

1.4.1 Adult stem cells (ASCs)

Adult stem cells (ASCs), also known as somatic stem cells, are multipotent undifferentiated stem cells found in a tissue or organ that can renew itself and can differentiate to yield some or all of the major specialized cell types of the tissue or organ Unlike the pluripotent ESCs or iPSCs, ASC proliferation is limited, making generation of large quantities of stem cells difficult One of the most-well studied is the adult hematopoietic stem cells, which give rise to all the blood cells Hematopoietic stem cells are in clinical trials for genetic diseases such as sickle cell, β-thalassemia and multiple sclerosis (Trounson A et al., 2011) Autologous adipose stem cells and the stromal vascular fractions are being used for soft tissue engineering in breast augmentation, fistulas in Crohn’s disease (Sullivan

KM et al., 2010)

1.4.1.1 Neural stem cells (NSCs)

NSCs are multipotent cells with the ability to self renew and generate mature cells in three fundamental neural lineages (astrocytes, oligodendrocytes and neurons) throughout the nervous system NSCs for can be derived directly from the neuroectodermal structures (central nervous system, CNS) or from ES cells

when subjected to appropriate stimuli in vitro (Conti L et al., 2005) They display migratory ability and innate tropism towards intracranial pathologies and have the ability to populate a developing region and/or repopulate an ablated or degenerated region of nervous system and because NSCs share a variety of similarities with brain tumor cells including molecular signature, hence some believe that endogenous NSCs are involved in development of brain tumors (Sutter R et al., 2007) NSCs have proven to be able to improve motor function in animal models of spinal cord injury (Ronaghi M et al., 2009) NSCs can successfully be used to derive dopaminergic neurons that when transplanted into rat Parkinson models, form dopaminergic connections able to produce functional improvement and behavioral recovery (Wijeyekoon R et al., 2009) Hence NSCs are ideal therapeutic agents in a variety of neurological diseases (Parker M.A et al., 2005; Yip S et al., 2006)

1.4.2 Induced pluripotent stem cells (iPSC)

Embryonic stem (ES) cells can be expanded to virtually unlimited numbers and have the potential to generate all types of cells in culture Hence ESC are an attractive donor source for transplantation and hold much promise to revolutionise regenerative medicine (Lerou P.H and Daley G.Q., 2005) ES cell therapy is however complicated by immune rejection due to immunological incompatibility between patient and donor ES cells

in vitro reprogramming of mature mouse fibroblasts into pluripotent stem

cells (induced pluripotent stem cells, iPS cells) was first achieved using retroviral

transduction of four transcription factors Oct4, sox2, c-Myc and Klf4 (Takahashi K

et al., 2007) Direct reprogramming of mouse cells has been extended to Rhesus macaque and human (Huangfu D et al., 2008; Park I.H et al.,2008) Subsequently, to avoid concerns regarding the oncogenicity of factors used in reprogramming and potential for insertional mutagenesis caused by integrating retroviral gene transfer vectors, several groups reported successful alternative virus-free reprogramming approaches such as the use of small-molecule chemicals in combination with genetic factors (Huangfu D et al., 2008 ) protein transduction for direct delivery of transcription factors (Bosnali M and Edenhofer F., 2008) and delivery of reprogramming genes using non-integrating gene transfer systems (Okita K et al., 2008; Kaji K et al., 2009).iPS cells might be an ideal cell source for cell therapy given that they can be derived from the patient to

be treated and hence are genetically identical cells that might avoid immune rejection

1.5 Stem cells as a vehicle for cancer gene therapy

1.5.1 Stem cell based cancer gene therapy

Gene directed enzyme prodrug therapy or suicide gene therapy did not show clinically significant effect in the past, perhaps caused by the missing tumor specificity of this approach Stem cell based enzyme-prodrug therapy represents

a more specific, less toxic and tailored approach to treating invasive cancers MSCs and NSCs have been studied in great detail for stem cell based cancer gene therapy MSCs are easily isolated from a variety of tissues such as the

bone marrow, adipose tissues, umbilical cord and amniotic fluid (Zvaifler NJ et al., 2000; Zuk PA et al., 2001; Erices A et al., 2000; Igura K et al., 2004), MSCs have also shown tropism to tumors such as glioma, breast, lung among many other tumors as well as injured ischemic tissues (Chamberlain G et al., 2007; Karnoub

AD et al., 2007; Loebinger MR et al., 2009; Xin H et al., 2009; Bak et al., 2010; Goldstein RH et al., 2010) In these models, MSCs have successfully homed to tumors from a large variety of administration routes including the carotid artery, femur, tail vein, tibia and trachea Intracranial or intravenous injected NSCs have been shown to migrate towards CNS locus with injury or pathology Besides GBM and medulloblastoma (Aboody KS et al., 2000; Seung SK et al., 2006) , NSCs stably expressing therapeutic genes can be designed to treat animal models of neurological disorders including Parkinson’s Huntington’s, ALS, stroke and lysosomal storage disease (Kim SU et al., 2006; Lee ST et al., 2005; Hwang

DH et al., 2009; Lee HJ et al., 2007)

1.5.2 Cancer tropism of stem cells

NSCs are attracted to various brain lesions such as cancers and areas of neurodegeneration Brain tumor targeting behavior of NSCs is believed to be mediated by chemo-attractant molecules and their respective receptors including stem cell factor (SCF)/c-kit (Sun L et al., 2004), stomal cell-derived factor (SDF-1), CXC chemokine receptor 4(CXCR4) and vascular endothelial growth factor(VEGF)/VEGF receptor (VEGFR)-1 and VEGFR2 (Ehtesham M et al., 2004; Schmidt NO et al., 2005) The fate of NSCs in the presence of lesions are not

well understood as most preclinical studies use only surrogate markers (Benedetti S et al., 2000; Li S et al., Cancer Gene therapy 2005)

Commonly used vectors have little or no migratory potential or specific tropism, hence gene therapy is quite limited in glioma cells that have infiltrated normal brain parenchyma There are many reports documenting the ability of transplanted NSCs to migrate not only to a tumour mass but also towards infiltrative “satellite” tumour cells in glioma animal models This makes NSCs suitable vehicles for gene delivery in treating highly invasive intracranial tumours (Aboody K.S et al., 2000; Glass R et al., 2000)

1.5.3 Route of administration of stem cells

One limiting variable in development of stem cell therapeutics is the identification

of a delivery method that is effective and minimally invasive Intravenous administration offers the easiest access to circulation with possible distribution throughout multiple tissues including lung, liver, kidney, spleen, bone marrow tissues The drawback is the large proportion of first-pass pulmonary sequestration (Barbash et al., 2003; Tolar et al., 2006) Intra-arterial administration offers an alternative method to further localise the placement of stem cells whilst bypassing the high pulmonary first pass effect However a recent investigation into this method showed a significant amount of variability in cell delivery following intracarotid infusion of MSCs immediately after induced ischemic stroke (Walczak et al., 2008) Direct implantation of stem cells will maximise stem cell load at site of disease/injury but invasiveness and possibility

of further tissue damage during cell transplantation must be considered The ideal stem cell delivery vehicle would combine the accessibility of intravenous administration with the ability to focus progenitor cell proliferation

1.5.4 Current clinical trials with stem cell based gene therapy

Recently a clinical trial based largely on Aboody K.S‘s work using human immortalised NSC, HB1.F3 as vehicles for cytosine deaminase has been launched (clinicaltrials.gov identifier: NCT 01172964) (Aboody K.S et al., 2000) The application of primary NSC in clinical glioma therapy is limited by practical and ethical problems Since it is difficult to isolate NSCs in a sufficient number, immortalised NSC lines were prepared and used in several preclinical models of prodrug cancer gene therapy (Kim SK et al., 2006; Aboody KS et al., 2006)

MSCs can be isolated from blood, adipose tissue, umbilical cord blood and placenta (Zvaifler NJ et al., 2000; Zuk PA et al., 2001; Erices A et al., 2000; Igura K et al., 2004) Due to this convenient isolation, lack of significant immunogenicity and feasibility for allogeneic transplantation, MSCs are widely used in ongoing clinical trials involving ischemic stroke, multiple sclerosis, acute leukemia among others (www.clinicaltrial.gov) However, several challenges have hindered the clinical applications of MSCs in breast cancer mouse models, such as the possibility of MSCs causing enhanced metastasis (Karnoub et al., 2007; Goldstein RH et al.,2010) Therapeutic benefit is highly questionable in models of experimental autoimmune encephalitis (EAE) where decreased methylation and inflammation had occurred in MSC-transplanted EAE mice,

indicating an immunosuppressive effect (Zappia et al, 2005) It has been suggested that administration of MSCs can contribute to angiogenesis in the pathological brain and a subset of primary GBM tumors and their derived tumor cell lines express cellular and molecular markers that are associated with MSCs (Chen J et al., 2003; Tso CL et al., 2006) In view of MSC as a cellular carrier, it

is important to carefully address their biosafety

1.5.5 Future of stem cell based cancer gene therapy

The failures of most suicide gene therapies were mainly caused by the inability of vectors carrying the suicide gene to reach invasive tumor cells distant from the tumor bulk as well as inefficient spread of the vectors within the tumor With the discovery of the tumor tropic nature of MSCs and NSCs (Aboody K et al., 2008; Bak et al., 2010; Goldstein RH et al., 2010), these stem cells hold great potential in enhancing the efficiency of gene therapy for cancer As of June 2011, the public clinical trials database http://clinicaltrials.gov shows that NSCs have been used for clinical trails involving lysosomal storage diseases, Parkinson’s and stroke and only a single study involving the use of NSCs for glioma (Aboody

et al., 2008)

Sourcing and safety of stem cells are two of the most important challenges The main objective for sourcing is to find autologous, non immunogenic, readily available stem cells that can be obtained with minimal ethical objections and that

display relative ease of manipulation in vitro without requiring immortlization

Ethical considerations of sourcing embryonic stem cells would argue for the use

of adult sources The experimental work on NSCs is promising but the derivation

of NSCs from adult brains is not possible Hence the derivation of NSCs from iPSCs is hoped to be able to overcome these hurdles, although there is a clear need to deremine the biological significance of the immunogenicity and epigenicity of iPSCs and iPSC-derived cells before they can be taken to clinical trials

2 Aims and objectives

In the first section we would like to investigate whether tumor tropic mouse derived NSC can be used in glioblastoma therapy, by first using a conventional lentivirus transduction method to derive iPSCs from primary MEF and then generating NSCs from these iPSCs To investigate whether the iPSC-derived NSCs can be used in the treatment of disseminated brain tumors, the iPSC-NSCs were transduced with a baculoviral vector containing the HSVtk contralateral to a tumor inoculation site in a mouse intracranial human glioma xenograft model Due to the availability of valid murine tumor models, murine NSC lines are often used to investigate the migratory capacity of tumor tropic NSCs and predict their preclinical therapeutic efficacy (Aboody K et al., 2000; Ehtesham M et al., 2000; Lorico A et al., 2008) Pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be expanded indefinitely

iPSC-in culture and have the potential to generate all types of cells iPSC-in vitro Hence they are attractive cell sources to derive differentiated cells, including NSCs for multiple purposes (Murray CE and Keller G, 2009; Amabile G and Meissner A,

2009) iPSCs might be a more powerful option for animal experimentation compared to ESCs since they can be easily generated from mature cells The differentiated iPSC can be fully syngeneic in immunocompetent hosts, thus circumventing problems of immunological incompatibility

In the second section, we want to determine a suitable administration method for stem cells in breast cancer therapy by observing the distribution of intravenous and intratumor administered iPSC-NSC in breast cancer mice These human iPSC-NSCs were labeled with a near-infrared (NIR) lipophilic carbocyanine dye: 1,1’dioctacedcyl-3,3,3’3’-tetramethylindotricarbocyanine iodide (DiR) It has been used to safely and directly label the membranes of several cell types with absorption and fluorescence maxima at 750 and 782 nm respectively (Kalchenko V et al., 2006)

In this study, we compared different routes for administering NSCs derived from iPSCs in a 4T1 mammary fatpad breast cancer mouse model A major factor that has been limiting the development of stem cell therapeutics is a effective minimally invasive administration method Intravenous administration offers easy access to the circulation but large proportion of cells are lost due to first-pass pulmonary sequestration The direct implantation of stem cells to the tumor site would maximize the stem cell load at the site but invasiveness of the

approach must be considered The questions we asked were whether the

presence of breast cancer influence the distribution of administered iPSC-NSC, and how the route of administration of transplanted cells affects this distribution

In the final section, we compared the therapeutic efficacy of 3 different suicide gene/prodrug systems with the use of iSPC-NSC in the 4T1 breast cancer mouse model iPSC-NSC were transduced with a baculoviral vector

carrying HSVtk, the yeast cytosine deaminase (Fcy) or Escherichia coli cytosine

deaminase (CodA) HSVtk converts the antiviral drug, GCV, to a toxic triphosphate form In the other system, the cytosine deaminase (CD) gene converts 5-FC into the toxic anabolite 5-fluorouracil (5-FU) Both HSVtk/GCV and CD/5-FC has been shown to have varying efficacies in cancer treatments (Trinh

QT et al., 1995; Kuriyama S et al., 1999 ; Corban-Wilhem H et al., 2003) In this study, we want to compare the strength of the bystander effect with the use of iPSC-NSC as a vehicle The success of gene therapy against cancer with suicide gene/prodrug system hinges not only on the strength of the bystander effect but also the efficiency of baculoviral-mediated gene expression

3 Material and methods

3.1 Cell culture

Primary mouse embryonic fibroblasts (MEF, CF-01) were obtained from forebrain

of embryonic mice strain CF1 (Biopolis Shared Facilities, Singapore) C17.2 cells were kindly provided by Prof E.Arenas (Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden), maintained in DMEM supplemented with 15% FCS, 5% horse serum, penicillin-streptomycin, and NEAA Human embryonic stem cell line H1-derived MSCs and were generated and maintained using a method previously described (Hwang et al., 2008, Bak

XY et al.,2011 ) MSC1 were maintained in Dulbecco's Modified Eagle Medium (DMEM), 10% FBS, Pen-strep, 5mM L-glutamine and non-essential amino acids (NEAA, Invitrogen, Carlsbad, CA) H1 embryonic stem cells were obtain from WiCell Institute (Madison, WI) and cultured in mTeSR-1 medium (Stem Cell Technologies, Inc, Singapore) on hESC-qualified matrigel (BD Biosciences, USA) H1-derived Neural stem cells (NSC1) were generated and maintained using a monoculture adherent method previously described (Swistowski A et al.,2009) NSC1 were maintained in Neurobasal media containing 1 x NEAA, L-Glutamine (2 mM), 1 X B27, 20 ng/ml FGF2 and LIF (Invitrogen) Human glioma cell line, U87-MG luciferase (U87-luc) and murine breast cancer cell line, 4T1-luciferase (4T1-luc2) was obtained from Caliper Life Sciences (Hopkinton, MA) Lentiviral packaging cell line, HEK293FT was purchased from Invitrogen, C6 rat glioma cell lines were purchased from American Type Culture Collection (ATCC) and C17.2 cells were kindly provided by Prof E.Arenas (Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden) CF01, U87MG-luci and C6 were maintained in DMEM supplemented with 10% FBS, Penicillin-Streptomycin and 5mM L-glutamine (Invitrogen) 4T1-luci was maintained in RPMI supplemented with 10% FBS, Penicillin-Streptomycin and 5

mM L-glutamine (Invitrogen)

3.2 Mouse iPSC –derived NSCs for glioma therapy

3.2.1 Lentivirus preparation, reprogramming and maintenance of mouse iPS (msiPSC)

The single lentiviral vector expressing four transcription factors, Oct4, Sox2, Klf4 and cMyc driven by the constitutive EF1α promoter (STEMCCA-EF1α) was kindly provided by Gustavo Mostoslavsky (Department of Medicine, Boston

University School of Medicine) Lentivirus was prepared using 293FT packaging

cells and ViraPower Lentiviral Expression Systems (Invitrogen) as per manufacturer’s instruction Supernatants were collected every 12 hours over two consecutive days starting 48 hours after transfection Viral particles were concentrated by centrifugation at 16,500 rpm at 4ºC Lentiviral titering was measured using HT1080, by transducing HT1080 cells with 10-fold serial dilutions of STEMCCA- EF1α lentivirus After 10-12 days of antibiotic selection, antibiotic-resistant colonies can be detected using crystal violet staining Blue-stained colonies were counted and used to determine lentiviral titre Multiplicity of infectivity (MOI) 5 was used for reprogramming 1 x 105 CF01 were seeded in 35mm dishes and infected with 10 µl (MOI 5) of concentrated virus, in the presence of 5µg/ml polybrene in MEF media After 16 hours, MEF media was replaced with mouse ES cell media (DMEM supplemented with 15% FBS, L-Glutamine, NEAA , β-mercaptoethanol , Invitrogen, and 1000 U/ml leukemia inhibitory factor (LIF), Peprotech, Rocky Hill, NJ), and changed everyday (Detailed Takahashi et al., 2006) Colonies were picked 14 days post-transduction based on morphology and clonally expanded on Mitomycin C (Sigma-Aldrich, St Louis, MO) treated MEFs in mouse ES cell media Derived mouse induced pluripotent stem cells (msiPS) were cultured in mouse ES culture media and routinely expanded in 1:5-1:8 ratio (detailed Takashi et al.2006)

msiPS cells were assessed using alkaline phosphatase (AP) staining with the AP

substrate kit (Sigma Aldrich) in accordance to manufacturer’s instruction For in

vitro differentiation of msiPSC to embryoid bodies, msiPSCs were trypsinized

and transferred to bacterial culture dishes without ES cell culture media without LIF, After 3 days, aggregates were collected and plated on gelatin coated dishes, Total RNA was isolated from plated embryoid bodies on day 6 for RT-PCR analysis

3.2.2 Alkaline phosphatase staining

msiPS cells were evaluated for alkaline phosphatase (AP) activity using the AP substrate kit (Sigma Aldrich) in accordance to manufacturer’s instruction

3.2.3 Generation of mouse iPSC-derived neural stem cells (msiPSC-NSC)

Neural differentiation of mouse ES cells is detailed here (Ying QL Smith AG et al., 2003) Using this method, mouse ES cells can be efficiently converted to Sox1 expressing NSCs Briefly msiPSCs were cultured under feeder-free conditions in ESC media for not more than 5 passages They are resuspended in N2B27 media and plated onto 0.1% gelatin coated 100mm dish at 1.2 x 106 density Culture medium is changed everyday, in the process removing dead or detached cells Under these conditions approximately 60-80% of cells will undergo neural lineage specification within 4-5 days and with overt neuronal differentiation detectable from day 5 onwards On day 7 differentiated cultures are trypsinized and 2-3 x 106 cells are re-plated on 100mm bacteriological dishes in NS

expansion media Within 2-3 days, the dish will contain many floating aggregates that can be harvested at 90 x g for 5min This step removes debris and dead cells, enriching for NSC founders, and ensures complete media exchange Cells can then be re-plated in fresh NS expansion medium onto gelatin coated dishes NSCs can be re-plated as single cells in 1:3-1:5 splitting ratio

3.2.4 Characterization of msiPSC-NSCs

Immunostaining and western blot assay was performed with the following primary antibodies: anti-Sox2, anti-Oct3, anti-KLF4, anti-Nanog (R&D systems, Minneapolis, MN), anti-cMyc, anti-Nestin, anti-GFAP, anti-thymidine kinase (TK), anti-SSEA1, anti-III-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-actin (Sigma-Aldrich) For immunostaining, antibody was used 1:200 and in western blot analysis was 1:2000 Secondary horseradish peroxidase-linked antibodies were purchased from Upstate, Millipore (Billerica, MA) Secondary fluorescent Alexa488, Alexa546 conjugated anti-mouse, anti-rabbit were purchased from Invitrogen The samples were counterstained with Hoechst

33422 (Invitrogen) before observation

To examine differentiation of NSCs to GFAP positive astrocytes, NSCs were exposed to 1% FCS in NS expansion media with N2 supplement, without EGF/FGF on gelatin coated dishes For neuronal differentiation, NSCs were harvested and re-plated on laminin coated 6-well plates in NS expansion media with FGF (5 ng/ml), modified N2 and B27 (Gibco, Invitrogen) Media is half-

changed every 2-3 days and after 7 days replaced with NS expansion media plus B27, without EGF or FGF-2

In vitro migration assay of NSCs toward glioma cells was examined using Boyden chamber assays A migration kit from BD Falcon with 24 well cell culture plates was used Well plates were separated into 2 chambers by an insert membrane with 8 µm pore A day before assay, 2.5 x 105

mouse embryonic fibroblasts (MEF), C6 and U87 glioma cells were seeded into each lower chamber, NSCs were labeled with Calcein-AM (Invitrogen) and serum starved overnight Cell culture media in lower chamber was removed the next day and replaced with optiMEM 0.8 x 105 Calcein-AM labeled-NSCs were seeded into inserts and incubated overnight at 37 ºC in optiMEM After 24 hours and 48 hours incubation, migrating cells found at insert bottoms can be quantified by measuring fluorescence intensity with fluorescence plate reader (GENios Pro, Tecan, Switzerland) Values are expressed as the mean±SD in percentage with migration towards optiMEM alone as basal migration rate Statistical analysis was done using Student’s T-test

Total RNA isolated with Trizol (Invitrogen), and further cleaned up using Turbo DNase (Applied Biosytems, Life Technologies, Singapore) Superscript III First Strand Synthesis System (Invitrogen) was used to produce cDNA from RNA by reverse transcription with oligo (dT) priming Reverse-Transcription (RT) PCR amplification of cDNA was carried out using Taq polymerase (Fermentas, Glen Burnie, Maryland ) RT-PCR primer sequences are listed in Table 1

3.2.5 RT 2 Profiler PCR array

Mouse embryonic stem cell and neural stem cell array ( PAMM-081A and PAMM-404A respectively , SABiosciences) was used to screen the expression of stem cell differentiation markers, RNA was isolated with Trizol (Invitrogen) and cleaned up with Turbo DNase (Applied Biosystems), mentioned above RT2 First Strand Kit (SABiosciences C-03) and 2 x RT2 SYBR Green/Fluorescein qPCR Master Mix (SABioscences PA-011-24) was used to prepare first strand cDNA for Real-Time PCR Real Time PCR was performed with BioRad iCycler with the following thermal cycling condition: 10 minutes at 95 ºC, followed by 40 cycles at

95 ºC for 15 seconds and 60 ºC for 1min with fluorescence detection The initial

10 minutes at 95 ºC is needed to activate the polymerase, and subsequent steps for detecting the melt curve Three independent replicates from each group: MEF, msiPS and msiPS-NSCs were used for analysis The threshold cycle (Ct) values are then exported to Excel spreadsheet for use with the SABiosciences PCR Array Data Analysis Web Portal:

http://www.SABiosciences.com/pcrarraydataanalysis.php

The PCR Array Data Analysis examines the threshold cycle of the genomic DNA control, reverse transcription control and positive PCR control before calculating the ∆ Ct for each pathway-focused gene in each plate

∆ Ct = CtGOI

- CtAVG HKG (GOI:Gene of interest, HKG: Housekeeping Gene)