Abstract
Purpose :
iPSC derived RPE transplants potentially offer a treatment for macular degeneration. The clinical use of iPSC-derived RPE cells requires testing of the final product for the presence of contaminating iPSCs. While LIN28A has been suggested as a marker of iPSC contamination in RPE, it is likely that regulatory bodies will require redundant testing with additional markers. To accomplish this we sought to identify and validate a new marker of pluripotency that could be used to identify contaminating iPSCs in a preparation of iPSC-derived RPE cells.
Methods :
We performed RNASeq on iPSC’s and IPSC-derived RPE from the same donor and screened for mRNAs that were in the iPSC dataset and not the RPE dataset. To confirm the RNASeq data we performed SybrGreen reverse transcription PCR on ZSCAN10 a candidate gene identified from the RNASeq dataset. qPCR was used to determine relative levels of expression of ZSCAN10 across iPSC clones from different donors and from clones isolated from the same donor. Publicly available databases were screened to determine expression of ZSCAN10 in tissue.
Results :
Of the top 10 genes identified by our RNASeq screen, ZSCAN10 was the only gene identified that is a known marker of pluripotency. Using RT-PCR we observed ZSCAN10 expression in 15 of 15 iPSC cell clones from 6 different donors, but not in RPE cultures derived from those donors. Using qPCR the ZSCAN10 signal was observed in similar amounts in all iPSC tested regardless of donor. ZSCAN10 was only observed in the iPSC cells and was absent from all iPSC-RPE preparations tested. Screening of publicly available databases identified very low levels of expression in human tissue, predominantly in brain and kidney.
Conclusions :
ZSCAN10 is a strong marker of undifferentiated iPSC impurities in a population of iPSC-derived RPE.
This is a 2021 ARVO Annual Meeting abstract.