A Simplified In Vitro Teratoma Assay for Pluripotent Stem Cells Injected Into Rodent Fetal Organs

Teratoma formation assays are established methods for evaluating the pluripotency of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. Teratoma formation in immunodeficient mice takes approximately 2 months. Here, we have developed a novel assay system for developing teratomas in vitro from ES cells and iPS cells in a short period. In vitro culture of ES, iPS, and mesenchymal stem cells (MSCs) in fetal rat metanephroi for 1 week resulted in distinct cell-dependent distribution patterns: Pluripotent cells (ES and iPS cells) formed aggregated masses, whereas MSCs showed disseminated distribution. The aggregated masses that had developed from ES cells and iPS cells after 2 weeks of culture comprised teratomas, though they were largely composed of immature components. Furthermore, in vitro organ culture for 1 week followed by relay transplantation into immunodeficient mice resulted in considerably rapid growing teratomas (teratomas developed in 4 weeks) having similar pathological features as of the teratomas developed using conventional 7-week in vivo teratoma formation assays. In addition, the initial cell number required in the in vitro assay was 1 × 10³ cells, which was about 1% of the number of cells required in the conventional in vivo teratoma formation assays. These results suggest that the in vitro teratoma assay is a rapid and convenient screening system and might be an alternative method for developing teratomas for investigating the pluripotency of ES cells and iPS cells.