Background: Although transplantation of human induced pluripotent stem cells derived cardiomyocytes (hiPSCs-CMs) have a possibility of treatment for heart failure, potential tumorigenicity is major concern in clinical application. It has been suggested that residual undifferentiated hiPSCs and malignant transformed cells may have responsibility for the tumor formation post-transplantation. Whereas, a highly sensitive tumorigenecity assay for the detection of these cells in hiPSCs-CMs has not been fully investigated. We herein aimed to establish the tumorigenecity assay in vitro and in vivo which verify the safety of hiPSCs-CMs.
Methods: In vitro, malignant transformed cells in hiPSCs-CMs was evaluated by soft agar colony formation assay. Further, undifferentiated hiPSCs in hiPSCs-CMs was examined by FACS and qRT-PCR. In addition, we transplanted hiPSCs-CMs to T-cell deficient nude rats to examine the possibility of teratoma formation.
Results: In soft agar colony formation assay, cervical cancer cells formed colony but hiPSCs-CMs did not form colony in vitro. In karyotype test, no abnormality occurred during the hiPSCs culture and cardiomyocyte differentiation. Measurement of TRA 1-60 by FACS and Lin 28 by qRT-PCR showed the highest sensitivity to detect undifferentiated hiPSC compared with other stem cell markers because no expression in terminally differentiated CMs and 100% expression in immature hiPS cells. QRT-PCR (limitation in detection; 0.01%) was more sensitive than FACS (0.1%). In vivo tumorigenicity test, in order to investigate the microenvironment of the transplantation site, hiPSCs-CMs sheets were transplanted into the heart and subcutaneous in NOG mice of the same individual. It was shown that the heart was more sensitivity to tumor formation than subcutaneous (heart vs subcutaneous; 44/47 vs. 30/47). ROC curve analysis revealed that no tumor formation was detected in the hiPSCs-CMs containing less than 0.33% Lin28-positive fraction (AUC=0.75, P<0.03) in vivo study.
Conclusion: it was suggested that combination of in vitro quantification of tumorigenic cells by measuring Lin28 mRNA expression and in vivo tumorigenecity assays can verify the safety of hiPSCs-CMs for cell transplantation therapy.