Fluorescence imaging of in vivo miR-124a-induced neurogenesis of neuronal progenitor cells using neuron-specific reporters.
BACKGROUND
Facilitation of the differentiation of the stem cells toward neuronal lineage is crucial for enhancing the differentiation efficacy of grafted stem cells for the possible treatment of neurodegenerative disorders. MicroRNA124a (miR-124a) has been considered as a neuronal lineage regulator, possessing the capability to activate neuronal differentiation. In this study, using a neuronal promoter-based reporter and live-cell fluorescence imaging, we visualized in vitro and in vivo the enhanced neuronal differentiation of neuronal progenitor cells with miR-124a overproduction.


METHODS
The neuron specific alpha1 tubulin promoter-driven RFP reporter (pTa1-RFP) was used to trace the miR-124a-induced neuronal differentiation in live cell condition. MiR-124a or miR-scramble in 10 % glucose buffer was mixed with in vivo-jetPEITM and in vivo fluorescence images were obtained daily using Maestro spectral fluorescent imager.


RESULTS
Neurite outgrowth was clearly seen in F11 cells after miR-124a transfection, and immunofluorescence staining showed increase of Tuj1 and NF at 48 hours. When pTa1-RFP-transfected F11 cells were implanted simultaneously with miR-124a into the nude mice, gradually increasing reporter signals and morphological changes indicated neuronal differentiation for 48 hours in live cells in vitro. The miR-124a-treated F11 cells showed higher reporter signals on in vivo fluorescence imaging than miR-scramble-treated cells, which were verified by ex vivo confirmation of Tuj1 and NF expression.


CONCLUSIONS
These results indicated that neuronal reporter-based neurogenesis imaging can be used for monitoring miR-124a acting as neuronal activator when miRNA was injected in in vivo PEI-coated form for miRNA-mediated regenerative therapy.
Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, South Korea.|Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, and College of Medicine or College of Pharmacy, Seoul National University, Seoul, South Korea.
Jaeho
J
Jang
Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, South Korea.
Song
S
Lee
Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, South Korea.|Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, and College of Medicine or College of Pharmacy, Seoul National University, Seoul, South Korea.
Hyun Jeong
HJ
Oh
Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, South Korea.
Yoori
Y
Choi
Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, South Korea.
Jae Hyouk
JH
Choi
Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, South Korea. hdw6592@daum.net.|Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, and College of Medicine or College of Pharmacy, Seoul National University, Seoul, South Korea. hdw6592@daum.net.
Do Won
DW
Hwang
Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, South Korea. dsl@plaza.snu.ac.kr.|Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, and College of Medicine or College of Pharmacy, Seoul National University, Seoul, South Korea. dsl@plaza.snu.ac.kr.
Dong Soo
DS
Lee
Activation of neuronal differentiation; In vivo fluorescence imaging; Neuron-specific reporter gene; miR-124a