Chief National Institute of Health Sciences Kawasaki, Kanagawa, Japan
Abstract: Human pluripotent stem cells (hPSCs) have infinite self-renewal capacity and can differentiate to many cell types in vitro, thus　making them an appropriate choice as a raw material for cell-based therapeutic products (CTPs). In order to ensure the safety of CTPs, assessment and appropriate management of tumorigenicity of the final product are important issues. Genomic instability is a potential hazard related to the risk of tumorigenicity because it is expected to increase the probability of karyotypic abnormalities or genomic mutations and to thereby increase the probability of cellular transformation. In particular, CTPs derived from human induced pluripotent stem cells (hiPSC) are predicted to accumulate genetic mutations and increase genomic instability because they require long-term culture for somatic cell reprograming, cloning of cell lines, cell banking, maintenance and expansion, and cell differentiation into desired cell type. Currently, it is recommended that hiPSC-derived CTPs be checked for karyotypic abnormalities and SNV/Indel or structural abnormalities in cancer-related genes. However, the safety of cells with genomic mutations, which are seen in vitro, has not been concluded. In this study, we generated three TP53 mutant hiPSC lines by homozygous mutation of TP53 high frequency mutations (R175H, R248Q, and R273H) by genome editing and analyzed the effects of TP53 mutations on genomic instability and tumorigenicity of hiPSC derived CTPs. The results of this study showed that (1) TP53 mutants have an increased proliferation rate compared to the parental line, (2) TP53 mutants show resistance to cell differentiation, (3) analysis by exome sequencing shows no increase in SNVs common to TP53 mutations, and (4) in vivo tumorigenicity study of cardiomyocytes derived from TP53 mutated hiPSCs, immature teratomas were observed in the TP53 mutant groups. These results are important findings showing the association between TP53 mutations and genomic instability/tumorigenicity, and are expected to lead to the establishment of scientific evidence-based genomic instability evaluation criteria.
Funding Source: This work was partly supported by the Japan Agency for Medical Research and Development (AMED) under grant number 24bk0104157j0203.
