Reduced Cytotoxicity by Repetitive mRNA Transfection in Differentiated Neurons

Seung Hwan Ko; Jin Sun Kang; Sang-Mi Kim; Eun-Hye Lee; Chang-Hwan Park

doi:10.15283/ijsc22125

Reduced Cytotoxicity by Repetitive mRNA Transfection in Differentiated Neurons

Int J Stem Cells. 2023 Feb 28;16(1):117-122. doi: 10.15283/ijsc22125. Epub 2022 Dec 31.

Authors

Seung Hwan Ko¹, Jin Sun Kang¹, Sang-Mi Kim², Eun-Hye Lee^{3

4}, Chang-Hwan Park^{1

2

5}

Affiliations

¹ Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Korea.
² Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea.
³ Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
⁴ Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
⁵ Department of Microbiology, College of Medicine, Hanyang University, Seoul, Korea.

Abstract

Background and objectives: mRNA-based protein expression technology has been used to express functional proteins. We have previously generated dopamine neurons from rat-embryo derived neural precursor cells (NPCs) through repeated transfection of synthetic transcription factor mRNA encoding dopamine-inducible genes. However, NPCs began to die approximately 10 d post-transfection. In this study, we examined a long-term transfection protocol that did not affect cell viability.

Methods and results: Experiments were performed in eight groups sorted according to the start date of mRNA transfection. mRNA was transfected into NPCs daily for 21 d and live cell images of each group were recorded. NPCs which were differentiated for more than five days showed sustained gene expression and appreciable viability despite daily mRNA transfection for 21 d.

Conclusions: Repeated mRNA transfection requires cells with a sufficient differentiation period.

Keywords: Delayed initiation; Neuron survival; Neuronal differentiation; mRNA transfection.

Grants and funding

Acknowledgments This work was supported by the Individual Basic Science and Engineering Research Program (2019R1A2C2005681) of the National Research Foundation of the Ministry of Science and ICT in Korea.