Differentiation-Defective Human Induced Pluripotent Stem Cells Reveal Strengths and Limitations of the Teratoma Assay and In Vitro Pluripotency Assays

Summary The ability to form teratomas in vivo containing multiple somatic cell types is regarded as functional evidence of pluripotency for human pluripotent stem cells (hPSCs). Since the Teratoma assay is animal dependent, laborious, and only qualitative, the PluriTest and the hPSC ScoreCard assay have been developed as in vitro alternatives. Here we compared normal hPSCs, induced hPSCs (hiPSCs) with reactivated reprogramming transgenes, and human embryonal carcinoma cells (hECs) in these assays. While normal hPSCs gave rise to typical teratomas, the xenografts of the hECs and the hiPSCs with reactivated reprogramming transgenes were largely undifferentiated and malignant. The hPSC ScoreCard assay confirmed the line-specific differentiation propensities in vitro. However, when undifferentiated cells were analyzed by the PluriTest, only hECs were identified as abnormal whereas all other cell lines were indistinguishable and resembled normal hPSCs. Our results indicate that pluripotency assays are best selected on the basis of intended downstream applications.
Highlights • Side-by-side comparison of teratomas/TeratoScore, hPSC ScoreCard, and PluriTest • hiPSCs with reactivated transgenes form embryonal carcinomas in vivo • hiPSCs with reactivated transgenes show impaired differentiation capacity in vitro • PluriTest does not distinguish hiPSCs with reactivated transgenes from normal hPSCs
Salvatori, Freund and colleagues performed a side-by-side comparison of Teratoma assay, ScoreCard, and PluriTest using normal hPSCs, hiPSCs with reactivated reprogramming transgenes, and embryonal carcinoma cells. For assessment of pluripotency the Teratoma assay can be replaced by a combination of ScoreCard and PluriTest. Notably, only the Teratoma assay is able to reveal the potential malignancy of hPSCs.