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. 2015 Nov 26:5:17258.
doi: 10.1038/srep17258.

The Molecular Karyotype of 25 Clinical-Grade Human Embryonic Stem Cell Lines

Maurice A Canham  1 , Amy Van Deusen  1 , Daniel R Brison  2 , Paul A De Sousa  3   4 , Janet Downie  3 , Liani Devito  5 , Zoe A Hewitt  6 , Dusko Ilic  5 , Susan J Kimber  7 , Harry D Moore  6 , Helen Murray  3 , Tilo Kunath  1
Affiliations

The Molecular Karyotype of 25 Clinical-Grade Human Embryonic Stem Cell Lines

Maurice A Canham et al. Sci Rep. .

Abstract

The application of human embryonic stem cell (hESC) derivatives to regenerative medicine is now becoming a reality. Although the vast majority of hESC lines have been derived for research purposes only, about 50 lines have been established under Good Manufacturing Practice (GMP) conditions. Cell types differentiated from these designated lines may be used as a cell therapy to treat macular degeneration, Parkinson's, Huntington's, diabetes, osteoarthritis and other degenerative conditions. It is essential to know the genetic stability of the hESC lines before progressing to clinical trials. We evaluated the molecular karyotype of 25 clinical-grade hESC lines by whole-genome single nucleotide polymorphism (SNP) array analysis. A total of 15 unique copy number variations (CNVs) greater than 100 kb were detected, most of which were found to be naturally occurring in the human population and none were associated with culture adaptation. In addition, three copy-neutral loss of heterozygosity (CN-LOH) regions greater than 1 Mb were observed and all were relatively small and interstitial suggesting they did not arise in culture. The large number of available clinical-grade hESC lines with defined molecular karyotypes provides a substantial starting platform from which the development of pre-clinical and clinical trials in regenerative medicine can be realised.

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Figures

Figure 1
Figure 1. Culture of 3 clinical-grade hESC lines.
(A) Man11, Man12, and Shef6 hESCs were cultured in Laminin-521 in Essential 8 medium prior to collection of genomic DNA. (B) Human ESC lines maintained expression of the pluripotent marker, NANOG, during expansion. Scale bar, 90 μm.
Figure 2
Figure 2. Duplications found in hESC lines that are present on the DGV.
(A) Chromosome 6 ideograms from SNP array analysis of MasterShef3 revealed a 267 kb duplication near the telomere, which contained 3 genes, MLLT4, KIF25, and FRMD1. Duplications of this size, or greater, have been reported and annotated on the DGV with an estimated frequency of 2.82% in the human population. (B) A 144 kb duplication was observed on chromosome 12p13.31 of RC17 hESCs. This region contained two genes, SCL2A14 and SLC2A3, and is represented on the DGV (3.9% frequency in humans).
Figure 3
Figure 3. A common deletion observed in two unrelated hESC lines.
Chromosome 8 ideograms from SNP array analysis of KCL031 and RC9 hESC lines revealed a 120 kb deletion on chromosome 8q24.23 (red arrow). This deletion is relatively common in the human population (3.85%), and does not contain any protein-coding genes.
Figure 4
Figure 4. An identical duplication found in sibling KCL hESC lines, but not present on the DGV.
Chromosome 5 ideograms from SNP array analysis of KCL032 and KCL033 hESC lines with the common CNV indicated by blue arrows. This 2.4 Mb duplication on chromosome 5p14.3 included the CDH18 gene. A duplication of this size was not present on the DGV, but a smaller duplication (nsv597424) covering most of the CDH18 exons was present, and a number of smaller deletions have been observed in this region. Only published CNVs greater than 100 kb are represented here.
Figure 5
Figure 5. A novel duplication observed in MasterShef7.
A 516 kb duplication on chromosome 16p11.2 was detected in MasterShef7 by SNP array analysis. This region contained 26 protein-coding genes, and a duplication of this size has not been reported to date. Smaller duplications have been observed in this region, including a duplication (esv2758642) spanning 7 of the genes. Only published CNVs greater than 10 kb are represented here.
Figure 6
Figure 6. Interstitial CN-LOH regions detected in clinical-grade hESC lines.
Chromosome ideograms from SNP array analysis showing CN-LOH regions indicated by green arrows: (A) A 3.6 Mb region on chromosome 2p16.2-16.1 in RC11. (B) A 3.5 Mb region on chromosome 2q11.1-11.2 in KCL040. (C) A 3.4 Mb region around chromosome 12q21.31-21.33 in MasterShef5.
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