MagA as a genetic MRI reporter for longitudinal in vivo stem cell monitoring Open Access

Cho, In (2014)

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The ability to longitudinally monitor cell grafts and assess their condition is critical for the clinical translation of stem cell therapy in regenerative medicine. Here, we investigate feasibility of using MagA as a genetic MRI reporter for longitudinal stem cell graft in vivo. MagA is a bacterial gene involved in forming iron oxide nanocrystals. MagA expression was regulated by the Tet-On switch, hence reducing cytotoxicity and allowing inducible monitoring by supplementing doxycycline (Dox). We established a mouse embryonic stem cell-line carrying Tet-MagA (mESC-MagA) by lentivirus transduction. Expression of MagA in mESCs resulted in significant changes in transverse relaxation rate (R2 or 1/T2) in vitro. mESC with and without MagA (mESC-MagA and mESC-WT) were grafted to striatum of mice brains and longitudinally monitored in vivo using MRI with "ON" (Dox+) and "OFF" (Dox-) conditions. Intracranial mESC-MagA grafts generated sufficient T2 and susceptibility weighted contrast at 7T, allowing for visualization of the graft by MRI longitudinally in controlled "ON" and "OFF" fashion upon induced expression of MagA by administrating Dox in diet. Our results suggest MagA can be used to monitor cell grafts noninvasively and longitudinally by repeated induction, enabling the assessment of cell graft conditions.

Table of Contents

Chapter 1: Introduction

1.1 General introduction 2

1.2 Criteria of an ideal imaging reporter 6

1.3 Introduction to available imaging modalities 8

1.3.1 Single-photon emission computed tomography (SPECT) 9

1.3.2 Positron emission tomography (PET) 10

1.3.3 Computed tomography (CT) 11

1.3.4 Ultrasound (US) 12

1.3.5 Bioluminescence imaging (BLI) 12

1.3.6 Fluorescence light imaging (FLI) 13

1.3.7 Magnetic resonance imaging (MRI) 14 MRI basics 15 Labeling methods 16 Exogenous MRI reporter 18 Endogenous MRI reporter 20

1.3.8 Chemical exchange saturation transfer (CEST) 24

1.3.9 Multimodal imaging methods 25

1.4 Magnetosome as an endogenous MRI genetic reporter 25

1.4.1 Use of magnetosome related genes in MRI 27

1.4.2 MagA as a candidate for in vivo monitoring using MRI 28

1.5 Discussion 30

1.6 Study proposal 31

Chapter 2: In vitro evaluation of MagA as a genetic MRI reporter for stem cell monitoring

2.1 Abstract 49

2.2 Introduction 49

2.3 Methods 52

2.4 Results

2.4.1 Establishment of a mESC-MagA clonal cell line 60

2.4.2 Expression profiling 61

2.4.3 Evaluation of pluripotency 62

2.4.4 Impact of MagA expression in cellular functions 62

2.4.5 In vitro evaluation of MagA as MRI reporter 63

2.4.6 In vitro spontaneous differentiation 64

2.5 Discussion 66

Chapter 3: In vivo monitoring of stem cell graft using MagA as a genetic MRI reporter

3.1 Abstract 89

3.2 Introduction 89

3.3 Methods 92

3.4 Results

3.4.1 In vivo induction of MagA 97

3.4.2 Longitudinal repetitive induced expression of MagA in vivo 98

3.5 Discussion 101

Chapter 4: Conclusions and future direction

4.1 Discussion 122

4.2 Summary 122

4.3 Limitations and future directions 124

4.3.1 Improving sensitivity 127

4.3.2 Longitudinal monitoring 129

4.3.3 Other applications of MagA 129

4.4 Conclusion 130

References 132

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