Longitudinal Monitoring of Stem Cell Grafts In Vivo Using Magnetic Resonance Imaging with Inducible Maga as a Genetic Reporter

Abstract

Purpose: The ability to longitudinally monitor cell grafts and assess their condition is critical for the clinical translation of stem cell therapy in regenerative medicine. Developing an inducible genetic magnetic resonance imaging (MRI) reporter will enable non-invasive and longitudinal monitoring of stem cell grafts in vivo. Methods: MagA, a bacterial gene involved in the formation of iron oxide nanocrystals, was genetically modified for in vivo monitoring of cell grafts by MRI. Inducible expression of MagA was regulated by a Tet-On (Tet) switch. A mouse embryonic stem cell-line carrying Tet-MagA (mESC-MagA) was established by lentivirus transduction. The impact of expressing MagA in mESCs was evaluated via proliferation assay, cytotoxicity assay, teratoma formation, MRI, and inductively coupled plasma atomic emission spectroscopy (ICP-OES). Mice were grafted with mESCs with and without MagA (mESC-MagA and mESC-WT). The condition of cell grafts with induced “ON” and non-induced “OFF” expression of MagA was longitudinally monitored in vivo using a 7T MRI scanner. After imaging, whole brain samples were harvested for histological assessment. Results: Expression of MagA in mESCs resulted in significant changes in the transverse relaxation rate (R2 or 1/T2) and susceptibility weighted MRI contrast. The pluripotency of mESCs carrying MagA was not affected in vitro or in vivo. Intracranial mESC-MagA grafts generated sufficient T2 and susceptibility weighted contrast at 7T. The mESC-MagA grafts can be monitored by MRI longitudinally upon induced expression of MagA by administering doxycycline (Dox) via diet. Conclusion: Our results demonstrate MagA could be used to monitor cell grafts noninvasively, longitudinally, and repetitively, enabling the assessment of cell graft conditions in vivo.

New imaging probes to track cell fate: reporter genes in stem cell research

“…However, using these nanoparticles with reporter genes brings about several limitations. For long-term imaging, residual iron oxide nanoparticles left over from previous imaging sessions may interfere with the signal, leading to inferior contrast-to-noise ratio [15,16]. On the other hand, if we use techniques that utilize reporter genes without the administration of iron-oxide nanoparticles, the resolution obtained by the MRI scanner is low [15].…”

“…For long-term imaging, residual iron oxide nanoparticles left over from previous imaging sessions may interfere with the signal, leading to inferior contrast-to-noise ratio [15,16]. On the other hand, if we use techniques that utilize reporter genes without the administration of iron-oxide nanoparticles, the resolution obtained by the MRI scanner is low [15]. The rise in iron stores poses another issue—augmenting Fenton reaction, leading to toxicity [14, 17].…”

“…Thus, the chronic overexpression of ferritin as a reporter gene could have cytotoxic effects. A study showed that one way to address this issue is through the use of an inducible reporter gene system, such as MagA with the Tet-on expression system [20]. The function of the gene is transportation of iron and intracellular formation of magnetite crystals causing a significant reduction in signal on T2 weighted MRI images [17].…”

“…Another benefit of utilizing this approach is that levels of MagA can be controlled via administration of a tetracycline antibiotic. Having an inducible reporter system leads to a reduced accumulation of MagA and iron limiting cytotoxicity and thus permits longitudinal imaging [20]. Although this technique can potentially overcome the limitation that ferritin overexpression induces, MagA is a non-human gene, which could lead to adverse immune responses.…”

“…Cerenkov luminescence imaging (CLI) utilizes Cerenkov radiation, a form of electromagnetic radiation emitted when a particle exceeds the speed of light while traveling in a medium. As the moving particle displaces electrons in the medium, they eventually return to their ground state and emit photons that can be detected by a CCD camera [15]. A major limitation of this method is that the wavelength of light emitted peaks around 450 nm and the signal emitted is weak requiring long exposure times to obtain a quality image [9], which is not ideal for in vivo experiments…”