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 - Funding Information:
We would like to thank Dr. Naoya Koizumi and Dr. Takamasa Hirai for technical advice on AdV infection. This research was supported by Japan Agency for Medical Research and Development under Grant Number JP17mk0104044 and JP18mk0104118.
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.
UR - http://www.scopus.com/inward/record.url?scp=85062587201&partnerID=8YFLogxK
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
VL - 9
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
IS - 1
M1 - 3630
ER -