Like any other method karyotyping cannot prove

Like any other method, karyotyping cannot prove the absence of variant cells. Nonetheless, mosaicism can be excluded to a certain degree by analyzing an appropriate number of metaphases (Hook, 1977). The exact number of metaphases scored may depend on the ultimate application of cells. For example, for routine culturing of hPSCs scoring of 20 or 30 metaphases may be sufficient, as this allows exclusion of 14% or 10% of mosaicism at 95% confidence, respectively. However, detection of very low levels of mosaicism (<1%) would require testing of more than 500 metaphases, which is impractical for routine testing of cultures but may be warranted if the nuciferine are being used in clinical applications. Given this dependence of the sensitivity of karyotyping on the numbers of metaphases analyzed, any karyotyping report and/or published data should clearly indicate the numbers of metaphases scored. One drawback to karyotyping in the context of routine laboratory practice is the need for expert cytogenetics analyses, which can hamper the frequency of the testing. Yet variant hPSCs with common genetic changes rapidly outcompete normal cells in culture, making early detection and frequent testing for abnormalities essential (Olariu et al., 2010). With this in mind, we designed a qPCR assay for detecting common genetic changes in hPSCs. When tested on a panel of cell lines, our assay accurately reflected the data obtained by karyotyping and/or FISH. The simplicity of the method allows for the data to be obtained within the same day, making it an ideal approach for routine screening of the common changes that occur in hPSC lines. A disadvantage of the qPCR-based assessment (which is also true for FISH) is that the assays can only detect changes at predetermined loci rather than assess the whole genome, and so they do not negate the need for detailed genetic analysis to detect other changes. Nonetheless, due to the non-random changes observed in hPSCs short-day plants should be possible to create a panel of primers that would detect the majority of the changes. We estimated that the panel of primers used in this study would detect about 45% of the reported genetic changes summarized in Figure 1. The flexibility of the method allows for further primer pairs to be designed to cover additional regions in the genome, although this would increase the overall cost of screening. Unlike G-banding, which generally may not be able to detect changes smaller than 5 Mb, qPCR and FISH are useful for detecting small amplifications and deletions. A case in point is the gain of chromosome 20q11.21. The gain of 20q11.21 is a particularly insidious genetic aberration as it occurs in many cell lines with no overt karyotypic changes (Amps et al., 2011). The 20q11.21 gain appears relatively frequently and, once acquired, confers selective advantage to variant cells, which rapidly overtake the culture (Amps et al., 2011; Avery et al., 2013). Thus, hPSC cultures should be regularly tested for the chromosome 20q CNV. Whereas FISH and karyotyping typically require specialist staff and often must be outsourced at costs of up to US$850, the qPCR can usually be performed in-house for the cost of reagents. The sensitivity of 5%–10% obtained by PCR-based methods and FISH may be suitable for routine culturing of cells, but the applications of hPSCs in the clinic will likely require a more rigorous sensitivity. Some of the alternative methods for detection of genetic aberrations in hPSCs that were not included in our study include comparative genomic hybridization (CGH) or SNP array platforms, e-karyotyping, and next-generation sequencing. Previous studies on the sensitivity of CGH and SNP arrays in detecting mosaicism in various clinical samples estimated the sensitivity of 8%–20% (Ballif et al., 2006; Cross et al., 2007; Valli et al., 2011), whereas an estimated sensitivity of e-karyotyping is around 30% (Ben-David et al., 2013).