iPSC Karyotyping and Pluripotency Evaluation

IPSCs were karyotyped in metaphase by the Shivanand R. Patil Cytogenetics and Molecular Laboratory at the University of Iowa using Leica Microsystems metaphase scanning platform and CytoVision version 7.7 software. Cells were grown in vitro and arrested at metaphase with colcemid. Chromosomes were stained by the G-banding method, counted and structurally evaluated for the presence or absence of detectable rearrangements. At least 20 cells were analyzed for each iPSC line. For TaqMan hPSC ScoreCard™ analysis, total RNA was isolated using the NucleoSpin RNA purification kit (Takara Bio, San Jose, CA). cDNA was generated from 1 µg of RNA using VILO cDNA synthesis kit (Thermo Fisher Scientific). cDNA was added to a TaqMan hPSC scorecard plate (Thermo Fisher Scientific) and amplified using a QuantStudio 6 Flex real-time PCR system. Results were analyzed using Thermo Fisher’s cloud-based analysis suite.