Genomic abnormalities

Principles of stem cell biology and cancer: future applications and therapeutics. Edited by T. Regad, T. J. Sayers and R. C. Rees. John Wiley & Sons (2015)

1. Isolation and characterization of human embryonic stem cells and future applications in tissue engineering therapies

1.3. Stem cell quality and culture adaptation with reference to cancer

Genomic abnormalities that have been observed in pluripotent stem cell cultures range from large chromosomal changes to single-nucleotide mutations. Chromosomal aberrations

The study of large chromosomal aberrations has been possible since chromosomal banding methods were established in the late 1960s. ‘Karyotyping’, in which metaphase chromosomes are stained with either quinacrine mustard (q-banding) (Caspersson et al., 1970) or Giemsa (g-banding) (Sumner et al., 1971) to give a characteristic banding pattern to each chromosome, is now a routine method. Depending on the chromosomal region, a resolution of 5 – 10 megabases can be achieved. The detection of aneuploidy in patient cells can be an indicator or marker for disease; for example, trisomy 21 is found in Down syndrome.

Initial studies revealed that hESC lines could maintain a normal diploid set of chromosomes during extended periods in culture (>6 months) (Thomson et al., 1998). However, follow-up studies soon revealed that hESC lines could also acquire chromosomal changes (Draper et al., 2003) and thereby emphasized the need for genome monitoring.

Recurrent large aberrations in hESCs after extended culture are mostly gains of regions in chromosomes 1, 12, 17 and X. Interestingly, the most frequent gain of human chromosome 17 (Figure 1.6) is also syntenic to the distal part of mouse chromosome 11, which is most often gained in mESCs (Ben-David and Benvenisty, 2012). Such changes are nonrandom gains that seem to be selected for by in vitro culture systems, and have been seen to occur at a rate of 10 – 20%. However, the general frequency of changes, including subchromosomal changes, is at a rate of 30 – 35%; this includes aberrations that are selected against during culture and those that are introduced at derivation or come from the embryo (Amps et al., 2011). The observed frequency of chromosomal abnormalities clearly reiterates the need to monitor cells over time, with karyotyping being the most commonly used method.

 Principles of Stem Cell Biology and Cancer 1.6

Figure 1.6. Illustration of karyotype with an extra chromosome 17. Trisomy 17 is one of the most common chromosome changes acquired during hESC culture. Copy-number variations

While karyotyping initially identified large chromosomal changes, recent application of higher-resolution technologies has both confirmed such large deviations and revealed additional changes on a subchromosomal level. Several studies using single-nucleotide polymorphism (SNP) data have established that all hESC lines exhibit copy-number variations (CNVs) of various sizes, many of which are specific to hESCs (Figure 1.7). At a higher resolution, changes that naturally exist in the human population must be differentiated from changes that have been acquired during in vitro culture. Analysis conducted on early and late passage cell populations revealed several regions with gain or loss of heterozygosity (Narva et al., 2010; Hanahan and Weinberg, 2011; Avery et al., 2013). In particular, a minimal amplicon in chromosome 20q11.21 was found in more than 20% of cell lines (Werbowetski-Ogilvie et al., 2009; Amps et al., 2011). Furthermore, it was revealed that the gain of this minimal amplicon introduces a resistance to apoptosis, most likely caused by one specific gene, BCL2L1. A simple genomic quantitative polymerase chain reaction (qPCR)-based approach or fluorescence in situ hybridization (FISH) on karyotyping slides should be a good measure to verify that this region has not changed in a particular set of hESC cultures (Avery et al., 2013).

Principles of Stem Cell Biology and Cancer 1.7

Figure 1.7. Illustration of a possible CNV in hESs. Single-nucleotide variations

With the advent of whole-genome sequencing, a few studies on iPSCs have been able to increase their resolution to the single base pair level and have thus identified single-nucleotide variations (SNVs) (Gore et al., 2011). An average of five to six mutations in coding regions have been reported, but many of these likely derive from the parental cell lines. It is important to bear in mind that only a few complete human genomes have been sequenced to date, so the extent of normal variation amongst our population is unclear. However, over time, and with further advanced sequencing technology, bigger data sets will reveal more answers with regards to genome stability. More scientific studies using whole-genome sequencing on hESC lines will be very interesting and may reveal significant SNVs that cannot be detected with other methods and which impact the quality of hESC lines.


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