Esther Wolfs1, Bryan Holvoet1, Laura Ordovas2,3, Natacha Breuls1,4, Nicky Helsen2,3, Matthias Schönberger5, Susanna Raitano2,3, Tom Struys6, Bert Vanbilloen1, Cindy Casteels1, Maurilio Sampaolesi4, Koen Van Laere1, Ivo Lambrichts6, Catherine M Verfaillie2,3, Christophe M Deroose7. 1. Molecular Small Animal Imaging Centre, Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium. 2. Stem Cell Institute, KU Leuven, Leuven, Belgium. 3. Stem Cell Biology and Embryology, Department of Development and Regeneration, KU Leuven, Leuven, Belgium. 4. Translational Cardiomyology Laboratory, Department of Development and Regeneration, KU Leuven, Leuven, Belgium. 5. Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium; and. 6. Laboratory of Histology, Morphology Research Group, Biomedical Research Institute, Universiteit Hasselt, Diepenbeek, Belgium. 7. Molecular Small Animal Imaging Centre, Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium christophe.deroose@uzleuven.be.
Abstract
Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging.
Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging.
Authors: Nourhan Shalaby; John Kelly; Francisco Martinez; Mathew Fox; Qi Qi; Jonathan Thiessen; Justin Hicks; Timothy J Scholl; John A Ronald Journal: Mol Imaging Biol Date: 2022-02-10 Impact factor: 3.484
Authors: Candice Ashmore-Harris; Samuel Ji Blackford; Benjamin Grimsdell; Ewelina Kurtys; Marlies C Glatz; Tamir S Rashid; Gilbert O Fruhwirth Journal: Stem Cell Res Date: 2019-10-15 Impact factor: 2.020