Monitoring and visualizing microRNA dynamics during live cell differentiation using microRNA-responsive non-viral reporter vectors.
MicroRNA (miRNA) activity differs with cell type, suggesting it can be used as a cell marker. In this study, we developed novel miRNA-responsive non-viral reporter vectors to continuously monitor and visualize miRNA dynamics during differentiation and to efficiently purify target living cells. Each vector codes miRNA-responsive and reference reporter genes in a single mRNA. These two genes are independent modules but transcribed by a single promoter, which enables us to distinguish miRNA-mediated post-transcriptional repression from transcriptional repression. We generated stable, miRNA-responsive vector-containing human induced pluripotent stem cells (hiPSCs) using the piggyBac transposon or episomal vectors. We could continuously monitor the differentiation status of living hiPSCs by detecting the activity of hiPSC-specific miRNA (miR-302a*). In addition, we could selectively sort hiPSC-derived cardiomyocytes using cardiomyocyte-specific miRNA (miR-208a or miR-1)-reporter vectors. Our miRNA reporter system provides a simple way to quantitatively and continuously monitor and visualize changes in the cellular state and should facilitate a broad range of studies that depend on cellular changes including drug discovery and cell-fate conversion.
Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
Hideyuki
H
Nakanishi
Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
Kenji
K
Miki
Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
Kaoru R
KR
Komatsu
Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
Masayuki
M
Umeda
Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
Megumi
M
Mochizuki
Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
Azusa
A
Inagaki
Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
Yoshinori
Y
Yoshida
Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. Electronic address: hirohide.saito@cira.kyoto-u.ac.jp.
Hirohide
H
Saito
D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics; D005434:Flow Cytometry; D018628:Gene Dosage; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000378:metabolism*; D016678:Genome; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic
C532169:MIRN302A microRNA, human; D035683:MicroRNAs
Cell sorting; Differentiation; Live cell imaging; Non-viral vector; Pluripotent stem cell; microRNA