Abstract B50: Supercooling under magnetic field can control the risk for malignant transformations of human pluripotent stem (iPS) cells
Authors: Moriauchi Hisashi , Mihara Makoto , Chung Raymond , Zhang Yue , Sato Chifumi 
Published: June 2012

Background: The solution in order to control the risk for malignant transformations of human pluripotent stem (iPS) cells are still unclear. Therefore, to seek the solution in order to control the tumorigenicity of human iPS cells is needed in the clinical applications of human iPS cells.

Methods: By using the technology of supercooling under magnetic field (2 kHz), integration-free human iPS cell lines from adult human dermal fibroblasts were supercooled and stored until the use at −80° C at a fixed −1°C/min with ROCK inhibitor with serum replacement medium and 10% DMSO. As for the control groups, the conventional cryopreservation method group (Nature Protocols, 2010) and the conventional cultivation method group (Takahashi K et al, Cell, 2007) were settled. Furthermore, mRNA expression levels of Oct 3/4 and Nanog in the human iPS cell lines after thawing or during the conventional cultivation were investigated by using real-time quantitative PCR (Notes: mRNA expression levels of Oct3/4 and Nanog in the human iPS cell lines of prefreezing were defined as 1.0 [arbitrary units.]). Moreover, the occurrences of mutant p53 as a risk factor for malignant transformations of mouse or human iPS cells (Sarig R et al, J Exp Med, 2010, and Mizuno H et al, PNAS, 2010) were evaluated. In addition, all integration-free human iPS cell lines have been compared at the same timepoint/passage number after generation.

Results: The survival rates of the human iPS cell lines for the supercooling under magnetic field group in 1 passage after thawing was higher than the conventional cryopreservation method group (96% in the supercooling under magnetic field group and 80% in the conventional cryopreservation method group: p&lt;0.0001, t-test). Furthermore, mRNA expression levels of Oct 3/4 and Nanog in the human iPS cell lines of 40 passages after thawing were higher in the conventional cryopreservation method group compared with the supercooling under magnetic field group (p&lt;0.0001, t-test). Moreover, in 40 passages after thawing, the human iPS cell lines which carry mutant p53 were observed in the conventional cryopreservation method group more than the supercooling under magnetic field group (11/24 in the conventional cryopreservation method group, 0/24 in the supercooling under magnetic field group; p&lt;0.0001, Mann-Whitney U test). Also, in 40 passages of the human iPS cell lines in the conventional cultivation method group, the human iPS cell lines which carry mutant p53 were 17.4 % (4/23). Therefore, the human iPS cell lines which carry mutant p53 were observed in the conventional cultivation method group more than the supercooling under magnetic field group (p=0.035, Mann-Whitney U test). In addition, the cases of the human iPS cell lines which carry mutant p53 were observed when mRNA expression levels of Oct 3/4 was 2.3 (arbitrary units) and more or mRNA expression levels of Nanog was 2.0 (arbitrary units) and more in the human iPS cell lines of 40 passages.

Discussion and Conclusion: In the current study, we have found that the supercooling under magnetic field can avoid the occurrences of mutant p53 as a risk factor for malignant transformations of human iPS cells by controlling the overexpressions of Oct 3/4 and/or Nanog.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B50.