June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Identification and validation of ZSCAN10 as a marker of iPSC impurities in cultures of iPSC-derived RPE.
Author Affiliations & Notes
  • Alan D Marmorstein
    Ophthalmology, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Matthew Hill
    Ophthalmology, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Cynthia Andrews-Pfannkoch
    Ophthalmology, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Jose Pulido
    Ophthalmology, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Footnotes
    Commercial Relationships   Alan Marmorstein, LAgen Laboratories LLC (I), LAgen Laboratories LLC (E), Seeing Medicines Inc (S); Matthew Hill, None; Cynthia Andrews-Pfannkoch, None; Jose Pulido, LAgen Laboratories LLC (I)
  • Footnotes
    Support  NIH U01-EY030547
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 3312. doi:
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      Alan D Marmorstein, Matthew Hill, Cynthia Andrews-Pfannkoch, Jose Pulido; Identification and validation of ZSCAN10 as a marker of iPSC impurities in cultures of iPSC-derived RPE.. Invest. Ophthalmol. Vis. Sci. 2021;62(8):3312.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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