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Long term non-invasive imaging of embryonic stem cells using reporter genes

Abstract

Development of non-invasive and accurate methods to track cell fate after delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. In this protocol, we describe the in vivo monitoring of stem cell survival, proliferation and migration using reporter genes. We established stable ES cell lines constitutively expressing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein, firefly luciferase and herpes simplex virus thymidine kinase (HSVtk)) reporter genes using lentiviral transduction. We used fluorescence-activated cell sorting to purify these populations in vitro, bioluminescence imaging and positron emission tomography (PET) imaging to track them in vivo and fluorescence immunostaining to confirm the results ex vivo. Unlike other methods of cell tracking, such as iron particle and radionuclide labeling, reporter genes are inherited genetically and can be used to monitor cell proliferation and survival for the lifetime of transplanted cells and their progeny.

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Figure 1: Schematic diagram of the double fusion (DF) and triple fusion (TF) lentiviral constructs, as well as the underlying mechanism of each imaging modality.
Figure 2: Comparison of physical labeling versus reporter gene labeling for tracking fate of transplanted human embryonic stem (hES) cells and human embryonic stem cell-derived endothelial cells (hESC-ECs).
Figure 3: Bioluminescence imaging of transplanted embryonic stem (ES) cells.
Figure 4: Small animal micro positron emission tomography (PET) imaging of transplanted embryonic stem (ES) cells.

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Acknowledgements

We thank funding support from AHA 0970394N, NIH HL089027 and NIH DP2OD004437 (J.C.W.).

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N.S. and A.L. contributed equally to the preparation of this manuscript. J.C.W. provided advice and proofread the manuscript.

Corresponding author

Correspondence to Joseph C Wu.

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Sun, N., Lee, A. & Wu, J. Long term non-invasive imaging of embryonic stem cells using reporter genes. Nat Protoc 4, 1192–1201 (2009). https://doi.org/10.1038/nprot.2009.100

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