Ferritin as a Transgenic MRI Reporter in Mouse Embryonic Stem Cells Público

Abstract

Ferritin as a Transgenic MRI Reporter in Mouse Embryonic Stem Cells

By Jun Liu

Embryonic stem cells hold great promise for regenerative medicine. To facilitate their translation into clinical practice, new methods to monitor stem cell transplant in vivo using transgenic MRI reporter are explored in the current study.

Among potential choices for MRI reporter genes, we focused on two critical players in iron homeostasis: ferritin heavy chain (FTH) and transferrin receptor (Tfrc), and began our study with parallel comparisons of the function and safety of FTH, Tfrc and their co-expression in clonal transgenic 293HEK and C6 glioma cell lines. It was discovered that un-regulated co-expression of FTH and Tfrc was associated with insignificant functional improvement but severe toxicity, so this combination was eliminated as a strategy. When expressed individually, FTH and Tfrc each were shown to be safe and effective MRI reporters. Closer examination of the underlying mechanisms of their reporter function suggested FTH was potentially the safer option between the two, being able to achieve the same level of MRI contrast with less intracellular iron accumulation, while retaining the capacity for effective regulation of iron uptake. FTH was subsequently chosen for introduction into mouse embryonic stem cells (mESCs).

In mESCs, moderate levels of FTH expression did not impair cell growth, nor were these levels disruptive to ESC pluripotency. When transplanted and monitored in vivo, FTH transgenes induced significant MRI contrast comparable to those achieved in other cell types. These findings suggest that FTH can function as a safe and effective molecular reporter in mESCs without external contrast agent. This has opened up new possibilities for stem cell imaging include longitudinal monitoring of cell transplants and, at the molecular level, potential to monitor differentiation status of stem cells and to detect expression of therapeutic genes in stem cell based gene therapies. Further efforts are needed to provide an accurate guideline of applicability and to improve on detection sensitivity of the transgenic reporter, before the full benefit of a MRI reporter-ESC combination can be realized.

Table of Contents

TABLE OF CONTENTS CHAPTER I: GENERAL INTRODUCTION

1 Embryonic Stem Cells 2 Brief review of imaging modalities 6 Choice of MRI reporter genes 10 CHAPTER II: Seeking the Right Reporter 14 Introduction 15 Materials and Methods 23 Results 30 Discussion 50 CHAPTER III: Monitoring mESCs with FTH Transgenic Reporter 89 Introduction 90 Materials and Methods 92 Results 99 Discussion 109 CHAPTER IV: Conclusions and Future Directions 130 Conclusions 131 Future Directions 137 REFERENCES 146

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Palabra Clave	Reporter gene MRI Embryonic Stem cells
Committee Chair / Thesis Advisor	Chan, Anthony, Emory University
Committee Members	Walker, Lary, Emory University Csete, Marie E, Emory University Smith, Yoland, Emory University Mao, Hui, Emory University

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