Published papers

Currently, we can label the certain cells by transducing specific genes, called reporter genes, and distinguish them from other cells. For example, fluorescent protein such as green fluorescence protein (GFP) is commonly used for cell labeling. However, fluorescent protein is difficult to observe in living animals. We can observe the reporter signals of the luciferin-luciferase system from the outside of living animals using in vivo imaging systems, although the resolution of this system is low. Therefore, in this study, we examined the reporter genes, which allowed the MRI-mediated observation of labeled cells in living animals. As a preliminary stage of animal study, we transduced some groups of plasmids that coded the protein that could take and store metal ions to the cell culture, added metal ions solutions, and measured their T1 or T2 relaxation values. Finally, we specified the best reporter gene combination for MRI, which was the combination of transferrin receptor, DMT1, and Ferritin-M6A for T1WI, and Ferritin-M6A for T2WI.

Introduction: Cell transplantation finds broad applications in medical science, with applications ranging from stem cell therapies to cancer research. Despite its widespread use, inherent risks such as tumor formation and immune rejection necessitate a comprehensive understanding of transplanted cell dynamics. Thus, tracing cellular behavior is a critical aspect of medical research, particularly in the context of cell transplantation. The capacity to precisely monitor and evaluate the behavior of transplanted cells over time is essential for evaluating therapeutic effectiveness, safety profiles, and long-term consequences. Traditional imaging approaches, like Z-stack and overlay images, present challenges due to limitations in sample size, determining cell location and migration, and only observing the one moment of the therapeutical application. However, recent advancements in imaging technologies have significantly improved our ability to trace cellular behavior in vivo. Bioluminescence imaging (BLI) has emerged as a powerful tool for non-invasive, real-time monitoring of cell survival, proliferation, and distribution in animal models. The in vivo imaging system (IVIS) for instance, focuses on its non-invasive nature and versatile applications in real-time investigations. Genetically modified cells express luciferase, allowing for the detection of light emission when luciferin is administered. BLI offers high sensitivity and the ability to track cells over extended periods, providing crucial information about cell engraftment and persistence. Method: Transfecting human adipose mesenchymal stem cells (adMSCs) with a lentiviral vector encoding firefly luciferase under the CAG promoter (CAG-ffLuc-cp156), which allows to establish a comprehensive understanding of adMSC behavior, distribution, and therapeutic safety, addressing a critical obstacle in the clinical evaluation of stem cell applications. The study tracked transfected adMSCs over seven days, with subsequent analysis of human DNA distribution by Alu-PCR. Result: Data indicates adMSCs disappear from the recipient by day 7, corroborated by the absence of human DNA in tested organs. The primary objective is to present a methodology for subconjunctival delivery, investigating the biodistribution and migration of adMSCs post-injection, with potential implications for various cell therapies. Conclusion: This study provides a valuable methodology for investigating cell behavior post-injection, contributing to the optimization of cell therapies for clinical applications. Furthermore, it highlights the safety of applying adMSCs with relatively low potential of tumorgenicity.

Neuronal Elav-like (nElavl or neuronal Hu) proteins are RNA-binding proteins that regulate RNA stability and alternative splicing, which are associated with axonal and synaptic structures. nElavl proteins promote the differentiation and maturation of neurons via their regulation of RNA. The functions of nElavl in mature neurons are not fully understood, although Elavl3 is highly expressed in the adult brain. Furthermore, possible associations between nElavl genes and several neurodegenerative diseases have been reported. We investigated the relationship between nElavl functions and neuronal degeneration using Elavl3−/− mice. Elavl3−/− mice exhibited slowly progressive motor deficits leading to severe cerebellar ataxia, and axons of Elavl3−/− Purkinje cells were swollen (spheroid formation), followed by the disruption of synaptic formation of axonal terminals. Deficit in axonal transport and abnormalities in neuronal polarity was observed in Elavl3−/− Purkinje cells. These results suggest that nElavl proteins are crucial for the maintenance of axonal homeostasis in mature neurons. Moreover, Elavl3−/− mice are unique animal models that constantly develop slowly progressive axonal degeneration. Therefore, studies of Elavl3−/− mice will provide new insight regarding axonal degenerative processes.