MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells.
The efficiency of pluripotent stem cell differentiation is highly variable, often resulting in heterogeneous populations that contain undifferentiated cells. Here we developed a sensitive, target-specific, and general method for removing undesired cells before transplantation. MicroRNA-302a-5p (miR-302a) is highly and specifically expressed in human pluripotent stem cells and gradually decreases to basal levels during differentiation. We synthesized a new RNA tool, miR-switch, as a live-cell reporter mRNA for miR-302a activity that can specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 0.05% of hiPSCs in a heterogeneous population and can prevent teratoma formation in an in vivo tumorigenicity assay. Automated and selective hiPSC-elimination was achieved by controlling puromycin resistance using the miR-302a switch. Our system uniquely provides sensitive detection of pluripotent stem cells and partially differentiated cells. In addition to its ability to eliminate undifferentiated cells, miR-302a switch also holds great potential in investigating the dynamics of differentiation and/or reprograming of live-cells based on intracellular information.
Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
Callum J C
CJ
Parr
Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
Shota
S
Katayama
Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
Kenji
K
Miki
Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
Yi
Y
Kuang
Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
Yoshinori
Y
Yoshida
Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
Asuka
A
Morizane
Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
Jun
J
Takahashi
Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.|Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA.
Shinya
S
Yamanaka
Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
Hirohide
H
Saito
D002454:Cell Differentiation / Q000235:genetics*; D019070:Cell Lineage / Q000235:genetics; D065150:Cellular Reprogramming / Q000235:genetics*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism* / Q000637:transplantation; D035683:MicroRNAs / Q000235:genetics*; D033581:Stem Cell Transplantation / Q000379:methods
C532169:MIRN302A microRNA, human; D035683:MicroRNAs