Abstract
Transgenic mouse with a stably integrated reporter gene(s) can be a valuable resource for obtaining uniformly labeled stem cells, tissues, and organs for various applications. We have generated a transgenic mouse model that ubiquitously expresses a tri-fusion reporter gene (fluc2-tdTomato-ttk) driven by a constitutive chicken β-actin promoter. This “Tri-Modality Reporter Mouse” system allows one to isolate most cells from this donor mouse and image them for bioluminescent (fluc2), fluorescent (tdTomato), and positron emission tomography (PET) (ttk) modalities. Transgenic colonies with different levels of tri-fusion reporter gene expression showed a linear correlation between all three-reporter proteins (R2=0.89 for TdTomato vs Fluc, R2=0.94 for Fluc vs TTK, R2=0.89 for TdTomato vs TTK) in vitro from tissue lysates and in vivo by optical and PET imaging. Mesenchymal stem cells (MSCs) isolated from this transgenics showed high level of reporter gene expression, which linearly correlated with the cell numbers (R2=0.99 for bioluminescence imaging (BLI)). Both BLI (R2=0.93) and micro-PET (R2=0.94) imaging of the subcutaneous implants of Tri-Modality Reporter Mouse derived MSCs in nude mice showed linear correlation with the cell numbers and across different imaging modalities (R2=0.97). Serial imaging of MSCs transplanted to mice with acute myocardial infarction (MI) by intramyocardial injection exhibited significantly higher signals in MI heart at days 2, 3, 4, and 7 (p<0.01). MSCs transplanted to the ischemic hindlimb of nude mice showed significantly higher BLI and PET signals in the first 2 weeks that dropped by 4th week due to poor cell survival. However, laser Doppler perfusion imaging revealed that blood circulation in the ischemic limb was significantly improved in the MSCs transplantation group compared with the control group. In summary, this mouse can be used as a source of donor cells and organs in various research areas such as stem cell research, tissue engineering research, and organ transplantation.
Highlights
Over the last decade, regenerative medicine with the use of stem cells has appeared as a potential therapeutic alternative for diseases in different organs/systems, such as endocrine, musculoskeletal, and the cardiovascular system [1,2,3,4]
Micro-positron emission tomography (PET) scanning was performed one hour after the injection, and we found that a minimum of 2.5x105 Mesenchymal stem cells (MSCs) can be visualized by PET imaging (Figure 6C)
The multimodality fusion reporter genes offers possibility to monitor molecular events ranging from a single live cell (fluorescence imaging, Fluorescence activated cell sorting (FACS)) to the multicellular environment of living mice with higher sensitivity, and with tomographic information from deep tissues
Summary
Regenerative medicine with the use of stem cells has appeared as a potential therapeutic alternative for diseases in different organs/systems, such as endocrine, musculoskeletal, and the cardiovascular system [1,2,3,4]. Recent advances in molecular biology and imaging have allowed for the successful non-invasive monitoring of transplanted stem cells in the living subject by labeling transplanted cells. Direct labeling strategies appear to be a good imaging method for detection of cells shortly after transplantation, providing a good signal-to-noise ratio, but less suited for long-term monitoring of stem cell viability [5]. The development of reporter gene strategies made it possible to accurately study the biology of stem cells based on the physiologic activity of transplanted cells [6,7,8,9,10]. In particular the ability of transducing reporter transgenes in non-dividing cells, especially lentivirus was successfully used for reporter gene delivery to the stem cells in many previous studies [11]. Current methods of introducing reporter genes are not satisfactory for transplantation studies
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