New Abstractt -10-28-20




Adam J. Hirst 1, Alicia Zhang 1, Mark Hills 1, Allen C. Eaves 1, 2, Sharon A. Louis 1 and Arwen L. Hunter 1.

1. STEMCELL Technologies Inc., Vancouver, Canada.

2. Terry Fox Laboratory, BC Cancer Agency, Vancouver, Canada.

Chromosomal aberrations, including numerical aneuploidy, chromosomal rearrangements and sub-microscopic changes, have been widely reported in cultured human pluripotent stem cells (hPSCs). Genetic variants can affect hPSC growth rates, cell survival and differentiation potential. Recurrent genetic abnormalities observed in hPSCs are also observed in human cancers, an observation that raises concerns for downstream clinical applications. The hPSC Genetic Analysis Kit employs a qPCR-based method designed to rapidly detect the most common genetic abnormalities observed in hPSC cultures. Specifically, primer-probe assays were optimized to detect the minimal critical regions on chromosomes 1q, 8q, 10p, 12p, 17q 18q, 20q and Xp, as well as a control region on chromosome 4p. These regions represent approximately 70% of all reported abnormalities in hPSC cultures. Amplification efficiencies for all primer-probe sets were measured at ≥ 90% (n = 2). Abnormalities were detected in 4 different hPSC lines each containing a 1q duplication, 10p deletion, 12 trisomy or 20q duplication (p < 0.001), with no other genetic abnormalities detected in other regions (p > 0.1). Duplication of 20q11.21 is a submicroscopic abnormality often missed when using G-banding karyotyping. As a case study, we analyzed the WLS-4D1 human induced pluripotent stem cell (hiPSC) line using G-banding, fluorescent in situ hybridization (FISH), and the hPSC Genetic Analysis Kit. Although this hiPSC line was found to be karyotypically normal by G-banding, duplication of 20q11.21 was only detected by using the hPSC Genetic Analysis Kit and confirmed by FISH. To determine assay sensitivity, fluorescently-labelled hPSC lines known to be abnormal for 10p, 12p and 20q were mixed with unlabeled normal diploid hPSC at varying ratios. Results indicate that our qPCR-based approach was able to detect genetically abnormal hPSCs when present at a minimal frequency of 30% (n = 3; p xtagstartz 0.05). In summary, the hPSC Genetic Analysis Kit offers researchers a reliable, fast and cost-effective tool to routinely monitor and pre-screen the hPSC lines in their laboratory for recurrent genetic abnormalities.

Credits

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