A total of endogenous genes

A total of 508 endogenous genes were differentially expressed in LU07+Dox brain metabolism when compared with LU07, H9, and H9+Dox cells including several pluripotency- and/or cancer-related genes (Figure 5D). However, the fact that the Pluripotency Scores of LU07+Dox cells and LU07 cells were indistinguishable indicates that the PluriTest algorithm trained on a dataset from bona fide PSCs considered the global gene expression profile of LU07+Dox cells to be within the range of normal hPSCs. By contrast, in hECs, a total of more than 5,500 genes were different (Figure 5C). This higher degree of deregulation in global gene expression resulted in Pluripotency Scores at or below the threshold of normal hPSCs. Similar to LU07+Dox cells, a number of pluripotency-associated genes were upregulated in hECs; in general their induction was higher than in LU07+Dox cells (Figure 5D). It seems likely that the differentiation deficiency of LU07+Dox cells was caused by upregulation of the endogenous pluripotency-associated genes in combination with the reactivated transgenes. The overlap of approximately 400 deregulated genes shared between hEC and LU07+Dox cells suggest the possibility that given an externally validated dataset, additional models and scores within the PluriTest-framework could be trained to reliably detect other potentially differentiation-defective hPSC cell lines with properties similar to those of LU07+Dox cells. In conclusion, the PluriTest provides quantitative information regarding whether a given cell line resembles normal hPSCs at a global molecular level. Higher-resolution RNA-sequencing data and the addition of epigenetic and microRNA profiles are likely to further improve the quality control of undifferentiated hPSCs. Short-term in vitro differentiation can reveal differences in functional pluripotency which are similar to the in vivo data. We therefore propose PluriTest in combination with the hPSC ScoreCard for routine characterization of hPSCs used for in vitro disease modeling and drug testing. Given the large numbers of hiPSC lines expected to be generated in the future, this would lead to a significant reduction of animal experiments and contribute to implementation of the “3Rs” policy (Replacement, Reduction, Refinement). Remarkably, the Teratoma assay is the only one of the three methods able to reveal malignant potential; this is a critical exclusion criterion for future hPSC clinical application. Indeed, the Teratoma assay has been proposed as a readout for tumorigenicity, although a quantitative analysis is currently lacking (Bulic-Jakus et al., 2016). However, the usefulness of a xenograft model for the prediction of malignancy in autologous clinical applications needs to be investigated much more in detail. In this respect mice with a humanized immune system may represent a further advance. Ideally, markers indicating potential malignancy could already be identified in undifferentiated cells.
Experimental Procedures Full details are provided in Supplemental Experimental Procedures.
Author Contributions
Acknowledgments We thank Dr. M. Zenke (RWTH Aachen University) and Dr. P.W. Andrews (University of Sheffield) for providing H9Hyb and 2102Ep hECs, respectively, and Dr. C. Dambrot for assistance in generating LUMC007iCTRL01 cells. We are grateful to Dr. I. Damjanov (Kansas University) and Dr. J.W. Oosterhuis (Erasmus Medical Center) for help with histology data analysis. We thank Dr. X. Yu (Amsterdam Medical Center) for his help with experiments in the initial phase of the project. S. Maas and S. Silvestri (LUMC) are thanked for preparation of cryosections, and J.A. Stoop (Erasmus Medical Center) for performing the CD30 immunohistochemistry staining. We are also grateful to Dr. K. Szuhai and D. de Jong for karyotype analysis and to Dr. R. Tsonaka for support with statistics (all LUMC). Special thanks to the LUMC hiPSC core facility staff for support. R. van der Straaten and J.J.M. Drabbels (LUMC) are thanked for providing access to the Viia7 instrument. Dr. Jan-Bas Prins (LUMC) is thanked for support and critical review of the animal experiments, and Dr. F.J. Müller (University Hospital Kiel) for critical reading of the manuscript. This work is part of the research program “Meer Kennis met Minder Dieren” (More Knowledge with Fewer Animals) with project number 40-42600-98-028, which is financed by the Netherlands Organization for Scientific Research (NWO).