Assessing the developmental and malignant potential of human pluripotent stem cells by RNA-seq analysis of Teratomas

The International Stem Cell Initiative compared three common approaches for assessing pluripotent stem cells (PSC). The formation of teratomas in vivo, or embryoid bodies (EB) in vitro, provide direct tests of differentiation, whereas PluriTest predicts pluripotency through bioinformatic analysis of transcriptomes of undifferentiated cells. We studied the teratomas by histology and TeratoScore, which analyzes gene expression in each tumor. For the EB assay, we assessed differentiation under neutral conditions and under conditions promoting differentiation to ectoderm, mesoderm, or endoderm lineages. Comparison of each method showed that all assays predict pluripotency, but they have different endpoints that provide different insights. For example, tumors from a subset of lines also contained undifferentiated cells and yolk sac elements that indicate possible malignant potential. This characteristic was not detected by the other two methods. Our results highlight the need for multiple assays to assess both pluripotency and potential malignancy in clinical applications of PSCs. RNA-seq analysis of human pluripotent stem cell-derived teratomas obtained from 15 human pluripotent stem cells lines from 4 laboratories with 2-3 replicates each, 37 samples in total.