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In vivo bioluminescence reporter gene imaging for the activation of neuronal differentiation induced by the neuronal activator neurogenin 1 (Ngn1) in neuronal precursor cells

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European Journal of Nuclear Medicine and Molecular Imaging Aims and scope Submit manuscript

Abstract

Purpose

Facilitation of the ability of neuronal lineages derived from transplanted stem cells to differentiate is essential to improve the low efficacy of neuronal differentiation in stem cell therapy in vivo. Neurogenin 1 (Ngn1), a basic helix-loop-helix factor, has been used as an activator of neuronal differentiation. In this study, we monitored the in vivo activation of neuronal differentiation by Ngn1 in neuronal precursor cells using neuron-specific promoter-based optical reporters.

Methods

The NeuroD promoter coupled with the firefly luciferase reporter system (pNeuroD-Fluc) was used to monitor differentiation in F11 neuronal precursor cells. In vitro luciferase activity was measured and normalized by protein content. The in vivo-jetPEITM system was used for in vivo transgene delivery. The IVIS 100 imaging system was used to monitor in vivo luciferase activity.

Results

The Ngn1-induced neuronal differentiation of F11 cells generated neurite outgrowth within 2 days of Ngn1 induction. Immunofluorescence staining demonstrated that early and late neuronal marker expression (βIII-tubulin, NeuroD, MAP2, NF-M, and NeuN) was significantly increased at 3 days after treatment with Ngn1. When Ngn1 and the pNeuroD-Fluc vector were cotransfected into F11 cells, we observed an approximately 11-fold increase in the luciferase signal. An in vivo study showed that bioluminescence signals were gradually increased in Ngn1-treated F11 cells for up to 3 days.

Conclusion

In this study, we examined the in vivo tracking of neuronal differentiation induced by Ngn1 using an optical reporter system. This reporter system could be used effectively to monitor the activation efficiency of neuronal differentiation in grafted stem cells treated with Ngn1 for stem cell therapy.

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Acknowledgments

This work was supported by the Nano-Biotechnology Project (Regenomics, 20100002086), the WCU project of the MEST and the National Research Foundation of Korea (NRF) (R31-2008-000-10103-0), the future-based technology development program of the NRF funded by the MEST (20100028755), and the Basic Science Research Program through the NRF funded by the Ministry of Education, Science and Technology (2012R1A1A2008799) and a grant (kiom-2010-2) from the Inter-Institutional Collaboration Research Program provided by the Korea Research Council of Fundamental Science & Technology.

Conflict of Interest

None.

Author information

Authors and Affiliations

  1. Department of Nuclear Medicine, College of Medicine, Seoul National University, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, Korea

    Hyun Jeong Oh, Do Won Hwang, Hyewon Youn & Dong Soo Lee

  2. Institute of Radiation Medicine, Medical Research Center, Seoul National University, Seoul, Korea

    Do Won Hwang

  3. Cancer Imaging Center, Seoul National University Cancer Hospital, Seoul National University, #207-4, Samsung Cancer Research Bldg. 103 Daehak-ro, Jongno-gu, 110-799, Seoul, Korea

    Hyewon Youn

  4. Laboratory of Molecular Imaging & Therapy, Cancer Research Institute, Seoul National University, Seoul, Korea

    Hyewon Youn

  5. Department of Molecular Medicine and Biopharmaceutical Science, WCU Graduate School of Convergence Science and Technology, Seoul National University, Seoul, Korea

    Hyun Jeong Oh & Dong Soo Lee

Authors
  1. Hyun Jeong Oh
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  2. Do Won Hwang
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Corresponding authors

Correspondence to Hyewon Youn or Dong Soo Lee.

Additional information

Hyun Jeong Oh and Do Won Hwang contributed equally to this work.

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Supplementary Fig. 1

Quantitative immunohistochemical analysis of neuronal differentiation of F11 cells isolated from the subcutaneous region. The implanted F11 cells were isolated from each thigh and prepared as paraffin slides. Quantitative immunohistochemical analysis for the immature neuronal marker Tuj-1, as well as the mature neuronal markers NeuroD, MAP2, NF-M and NeuN was performed in Mock-treated and Ngn1-treated groups using TissueFAXS2.0. (JPEG 52 kb)

ESM 1

High resolution image (TIFF 974 kb)

Supplementary Fig. 2

TissueFAXS2.0 analysis for ex vivo validation of neuronal differentiation in an orthotopic animal model with Ngn1-induced F11 cells. The brains were removed and serial (10 μm) thick cryosections were prepared. TissueFAXS2.0 was used to quantitatively analyse neuron-specific antibodies against the immature neuronal marker Tuj-1, and the mature neuronal markers NeuroD, MAP2, NF-M and NeuN in the brain area (JPEG 53 kb)

ESM 2

High resolution image (TIFF 974 kb)

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Oh, H.J., Hwang, D.W., Youn, H. et al. In vivo bioluminescence reporter gene imaging for the activation of neuronal differentiation induced by the neuronal activator neurogenin 1 (Ngn1) in neuronal precursor cells. Eur J Nucl Med Mol Imaging 40, 1607–1617 (2013). https://doi.org/10.1007/s00259-013-2457-0

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  • DOI: https://doi.org/10.1007/s00259-013-2457-0

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