For optimal use of human induced pluripotent stem cells (hiPSCs) it is essential to identify lines that are fully reprogrammed and of high quality with proven pluripotency in terms of differentiation. The ability to form teratomas in vivo is regarded as functional evidence of pluripotency for human pluripotent stem cells (hPSCs). Since the Teratoma assay is animal-dependent, laborious and only qualitative, there is an ongoing debate whether it is an acceptable tool. This has led to the development of an assay for quantitative analysis of teratomas (TeratoScore) as well as in vitro alternatives such as PluriTest and the hPSC Scorecard. We compared normal hPSCs, hiPSCs with reactivated reprogramming transgenes and nullipotent human embryonal carcinoma (hEC) cells in these assays. Cells were cultured on Vitronectin in TESR-E8 media (hPSCs) or in DMEM/10% FCS (hEC cells). The quality of undifferentiated cells was analysed by FACS for OCT3/4 as well as karyotyping. As assessed by immunohistochemistry and immunofluorescent staining the normal hPSCs gave rise to typical teratomas whereas the xenografts of the hEC cells and the hiPSCs with the reactivated reprogramming transgenes were largely undifferentiated and displayed a malignant phenotype.

TeratoScore confirmed typical teratomas and tumors lacking differentiation but was unable to identify partially differentiated tumors. The hPSC Scorecard assay confirmed the line-specific differentiation propensities in vitro. However, when undifferentiated cells were analysed with PluriTest only hEC cells were identified as distinct. All other undifferentiated cells lines including hiPSCs with reactivated transgenes were undistinguishable and resembled normal hPSCs. We propose a combination of PluriTest and hPSC Scorecard for quantitation of pluripotency status and function of hPSCs used for in vitro purposes. By contrast, the Teratoma assay is the only method which is able to determine whether hPSCs could potentially develop a malignant phenotype, resulting in exclusion for any clinical application.