Purpose: Mesenchymal stem cells (MSCs) have been of great interest for cell-replacement therapy in neurodegenerative disease due to substantial merit capable of directly using patient own cells. Developing the imaging system for tracking dynamic chang...
Purpose: Mesenchymal stem cells (MSCs) have been of great interest for cell-replacement therapy in neurodegenerative disease due to substantial merit capable of directly using patient own cells. Developing the imaging system for tracking dynamic change of the neuronal differentiation process from MSCs is important to evaluate the transdifferentiation efficacy of grafted MSCs. In this study, we aimed to assess real-time live cell imaging for detection of differentiation process by the small molecule known as “compound 1” (C18H17N3O3) to facilitate neuronal transdifferentiation induced from MSCs using a neuronal promoter-driven fluorescence imaging system. Materials and methods: Rat bone marrow-derived MSCs were treated with 20 μM compound 1 to induce differentiation of MSCs into neuronal cells. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were performed for evaluation of neuronal marker expression. Plasmid vector containing red fluorescence reporter genes under the control of the tubulin α1 (Tα1) promoter (Tα1p-DsRed2) were transfected into MSCs to examine the change of fluorescence signal during neuronal differentiation. Static microscopic imaging and time-lapse live cell imaging were performed for monitoring of neuronal differentiation progress from MSCs by compound 1 using confocal microscopy and fluorescence live cell microscope imaging device, respectively. Results: Two days after treatment with compound 1, MSC cells showed changes of neuron-like phenotype changes, as well as increased expression of neuron-specific markers such as neuron specific enolase (NSE), class III β-tubulin (Tuj 1), and synaptophysin. Immunofluorescence staining results revealed that Tuj 1 expression level was increased after induction of neuronal differentiation by compound 1. When Tα1p-DsRed2 reporter vector was transfected into MSCs, immunostaining data displayed that the enhanced fluorescence signals were detected in the cytoplasm region of MSCs after treatment of compound 1. In vitro monitoring in MSC transfected with Tα1p-DsRed2 reporters showed progressively increased fluorescence signals by 30 h after treatment of compound 1, corresponding with progressive neuronal differentiation from MSCs. Conclusions: We examined an efficient neuronal differentiation pattern by compound 1, and monitored neuronal differentiation process by neuron specific promoter-based fluorescence reporter system in cellular level. These findings will be helpful in understanding the efficacy of neuronal differentiation derived from MSCs for stem cell-based therapy.