TY - JOUR
T1 - Development of selective cytotoxic viral vectors for concentration of undifferentiated cells in cardiomyocytes derived from human induced pluripotent stem cells
AU - Kono, Ken
AU - Sawada, Rumi
AU - Kuroda, Takuya
AU - Yasuda, Satoshi
AU - Matsuyama, Satoko
AU - Matsuyama, Akifumi
AU - Mizuguchi, Hiroyuki
AU - Sato, Yoji
N1 - Publisher Copyright:
© 2019, The Author(s).
PY - 2019/12/1
Y1 - 2019/12/1
N2 - Cell-processed therapeutic products (CTPs) derived from human pluripotent stem cells (hPSCs) have innovative applications in regenerative medicine. However, undifferentiated hPSCs possess tumorigenic potential; thus, sensitive methods for the detection of residual undifferentiated hPSCs are essential for the clinical use of hPSC-derived CTPs. The detection limit of the methods currently available is 1/10 5 (0.001%, undifferentiated hPSCs/differentiated cells) or more, which could be insufficient for the detection of residual hPSCs when CTPs contain more than 1 × 10 5 cells. In this study, we developed a novel approach to overcome this challenge, using adenovirus and adeno-associated virus (AdV and AAV)-based selective cytotoxic vectors. We constructed AdV and AAV vectors that possess a suicide gene, iCaspase 9 (iCasp9), regulated by the CMV promoter, which is dormant in hPSCs, for the selective expression of iCasp9 in differentiated cells. As expected, AdV/CMV-iCasp9 and AAV/CMV-iCasp9 exhibited cytotoxicity in cardiomyocytes but not in human induced pluripotent stem cells (hiPSCs). The vectors also induced apoptosis in hiPSC-derived cardiomyocytes, and the surviving cells exhibited higher levels of hPSC marker expression. These results indicate that the AdV- and AAV-based cytotoxic vectors concentrate cells expressing the undifferentiated cell markers in hiPSC-derived products and are promising biological tools for verifying the quality of CTPs.
AB - Cell-processed therapeutic products (CTPs) derived from human pluripotent stem cells (hPSCs) have innovative applications in regenerative medicine. However, undifferentiated hPSCs possess tumorigenic potential; thus, sensitive methods for the detection of residual undifferentiated hPSCs are essential for the clinical use of hPSC-derived CTPs. The detection limit of the methods currently available is 1/10 5 (0.001%, undifferentiated hPSCs/differentiated cells) or more, which could be insufficient for the detection of residual hPSCs when CTPs contain more than 1 × 10 5 cells. In this study, we developed a novel approach to overcome this challenge, using adenovirus and adeno-associated virus (AdV and AAV)-based selective cytotoxic vectors. We constructed AdV and AAV vectors that possess a suicide gene, iCaspase 9 (iCasp9), regulated by the CMV promoter, which is dormant in hPSCs, for the selective expression of iCasp9 in differentiated cells. As expected, AdV/CMV-iCasp9 and AAV/CMV-iCasp9 exhibited cytotoxicity in cardiomyocytes but not in human induced pluripotent stem cells (hiPSCs). The vectors also induced apoptosis in hiPSC-derived cardiomyocytes, and the surviving cells exhibited higher levels of hPSC marker expression. These results indicate that the AdV- and AAV-based cytotoxic vectors concentrate cells expressing the undifferentiated cell markers in hiPSC-derived products and are promising biological tools for verifying the quality of CTPs.
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UR - http://www.scopus.com/inward/citedby.url?scp=85062587201&partnerID=8YFLogxK
U2 - 10.1038/s41598-018-36848-5
DO - 10.1038/s41598-018-36848-5
M3 - Article
C2 - 30842516
AN - SCOPUS:85062587201
SN - 2045-2322
VL - 9
JO - Scientific reports
JF - Scientific reports
IS - 1
M1 - 3630
ER -
