The Evaluation of Tumorigenicity and Characterization of Colonies in a Soft Agar Colony Formation Assay Using Polymerase Chain Reaction

In regenerative medicine, the tumorigenic potency of cells in cellular therapy products (CTPs) is a major concern for their application to patients. This study presents a method-the soft agar colony formation assay using polymerase chain reaction (PCR)-to evaluate tumorigenicity. MRC-5 cells, contaminated with HeLa cells, were cultured for up to 4 weeks in soft agar medium. Cell-proliferation-related mRNAs, Ki-67 and cyclin B, could be detected in 0.01% of HeLa cells after 5 days of culture, whereas cyclin-dependent kinase 1 (CDK1) could be detected after 2 weeks. On the other hand, CDK2, proliferating cell nuclear antigen (PCNA), and minichromosome maintenance protein 7 (MCM7) were not useful to detect HeLa cells even after 4 weeks of culture. The cancer stem cell (CSC) markers, aldehyde dehydrogenase 1 (ALDH1) and CD133 in 0.01% of HeLa cells, could be detected 2 and 4 weeks after culture, respectively. However, another CSC marker CD44 was not useful because its expression was also detected in MRC-5 cells alone. This study suggests that the application of the PCR method to the soft agar colony formation assay could evaluate not only the tumorigenic potency in the short-term but also characterize the colonies, eventually improving the safety of CTPs.