Abstract 13750: A Promising Highly Sensitive and Quantitative Assays of Tumorigenicity Essential for Facilitating Safety Studies of Human Induced Pluripotent Stem Cells -derived Cardiomyocytes for Future Therapeutic Application
Background: Although transplantation of cardiomyocytes derived from induced pluripotent stem cells (iPSCs) into the heart is a promising approach for heart disease, the presence of residual undifferentiated iPSCs may limit safety of this treatment. Transplantation of human iPSCs (hiPSCs) into immune-deficient mice leads to the formation of differentiated tumors comprising all three germ layers, resembling spontaneous human teratomas. Teratoma assays are considered to be a standard for demonstrating the differentiation potential of hiPSCs and hold promise as a standard for assessing the safety of hiPSC-derived cardiomyocytes intended for therapeutic applications.
Methods and Results: We examined three in vitro tumorigenicity assays (soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction [qRT-PCR] assay) and in vivo assay to detect residual undifferentiated cells. Soft agar colony formation assay was unable to detect hiPSCs. Next, we tied to detect residual undifferentiated cells by flow cytometry using six antibodies which recognize stem cell marker antigens, (Oct3/4, Nanog, SSEA-3, SSEA-4, TRA1-60, and TRA1-81). The anti-TRA1-60 antibody clearly distinguished hiPSCs from cardiomyocytes; it deteced 0.1% residual undifferentiated hiPSCs that were spiked in primary cardiomyocytes. Furthermore, to identify highly selective markers for undifferentiated hiPSCs, we compared mRNA levels of Oct3/4, Nanog, Sox2, Lin28 and Rex1 in hiPSCs and primary cardiomyocytes by qRT-PCR. Lin28 mRNA was not detected in primary cardiomyocytes, whereas the qRT-PCR assay detected 0.01% residual undifferentiated hiPSCs that were spiked in primary cardiomyocytes. In vivo study, the potency of teratoma formation was tested in immune-deficient mice. Among iPSC-derived cardiomyocytes, cells that contain more than 2% Lin28 positive fraction formed tumors. However, cell population that contained a Lin28 fraction less than 2% did not form tumors.
Conclusion: We established core parameters for detecting residual tumor-forming cells. Our results provide highly sensitive and quantitative assays essential for facilitating safety studies of hiPSC-derived cardiomyocytes for future therapeutic application.
Author Disclosures: E. Ito: None. S. Miyagawa: None. A. Saito: None. S. Fukushima: None. S. Masuda: None. T. Kawamura: None. Y. Imanishi: None. N. Sougawa: None. M. Shiozaki: None. H. Iseoka: None. A. Harada: None. M. Takeda: None. Y. Sato: None. Y. Sawa: None.
- © 2014 by American Heart Association, Inc.