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. 2011 Jan 7;8(1):106-18.
doi: 10.1016/j.stem.2010.12.003.

Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture

Louise C Laurent  1 , Igor Ulitsky, Ileana Slavin, Ha Tran, Andrew Schork, Robert Morey, Candace Lynch, Julie V Harness, Sunray Lee, Maria J Barrero, Sherman Ku, Marina Martynova, Ruslan Semechkin, Vasiliy Galat, Joel Gottesfeld, Juan Carlos Izpisua Belmonte, Chuck Murry, Hans S Keirstead, Hyun-Sook Park, Uli Schmidt, Andrew L Laslett, Franz-Josef Muller, Caroline M Nievergelt, Ron Shamir, Jeanne F Loring
Affiliations

Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture

Louise C Laurent et al. Cell Stem Cell. .

Abstract

Genomic stability is critical for the clinical use of human embryonic and induced pluripotent stem cells. We performed high-resolution SNP (single-nucleotide polymorphism) analysis on 186 pluripotent and 119 nonpluripotent samples. We report a higher frequency of subchromosomal copy number variations in pluripotent samples compared to nonpluripotent samples, with variations enriched in specific genomic regions. The distribution of these variations differed between hESCs and hiPSCs, characterized by large numbers of duplications found in a few hESC samples and moderate numbers of deletions distributed across many hiPSC samples. For hiPSCs, the reprogramming process was associated with deletions of tumor-suppressor genes, whereas time in culture was associated with duplications of oncogenic genes. We also observed duplications that arose during a differentiation protocol. Our results illustrate the dynamic nature of genomic abnormalities in pluripotent stem cells and the need for frequent genomic monitoring to assure phenotypic stability and clinical safety.

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Figures

Figure 1
Figure 1
Duplications and large deletions identified by CNVPartition mapped onto the genome, for all samples. The number and extent of regions of CNV regions are shown. Duplicated regions (3 or 4 copies) are shown in the dark bars, deleted regions (0 or 1 copy) are shown in the light bars, and copy neutral LOH regions are place on the ideograms of the chromosomes. Where 5 or more samples of the same cell type have aberrations at the same region, the number of samples affected is indicated (e.g. “×5). Regions for hESC samples are shown in red, regions for hiPSC samples are shown in blue, and regions for non-PSC samples are shown in green. Some aneuploidies had been identified prior to hESC derivation, and are indicated as “known from PGS”. Regions where the CNV is present in only a subpopulation of the cells in a sample are denoted “(sub).” The 3 regions of duplication on chromosome 20 that arose in a subpopulation of the cells during differentiation of the WA07P96CMD7 sample are indicated. CNVs that overlap with the common CNVs observed in 450 HapMap samples (Conrad et al., 2010) are indicated by an asterisk. See also Figure S1, and Tables S1, S2, S3, and S4).
Figure 2
Figure 2
Details of regions of CNV on chromosome 12 (A) and chromosome 20 (B). Areas of duplication are shown in red bars for hESC samples and blue for hiPSC samples. Areas of overlap between the hPSC samples are highlighted in pink. The pluripotency-associated genes NANOG and DNMT3B are highlighted by red ovals. The dashed vertical blue lines in B indicate the boundaries of the DNMT3B gene. The lower panel of B shows the BAF plots demonstrating that a duplication on chromosome 20 arose during long-term passage of the hESC line CM14. See also Figures S2 and S3, and Table S5.
Figure 3
Figure 3
Number of regions of duplication and deletion, as identified by CNVPartition. Cumulative distribution function plots of the numbers of 0-copy (total allelic loss), 1-copy, and 3-copy, and total CNVs for each sample type (hESCs, hiPSCs, and non-PSCs) are shown in A-C. The tables list the average number per sample (D) of each type of CNV for the hESC, hiPSC, and non-PSC samples. Error bars indicate the standard deviation. Wilcoxon Rank Sum p-values are listed in (E) for each type of CNV, comparing hESC vs. non-pluripotent and hiPSC vs. non-pluripotent. Significant p-values (<0.05) are highlighted in red. See also Figure S4.
Figure 4
Figure 4
Dynamic copy number changes over long-term passage. (A) BAF and LRR plots of chromosome 15 for early and late passage samples of the MIZ4 hESC line. The early passage plots show normal autosomal BAF and LRR distributions, while the late passage plots indicate that a subpopulation of the cells have a duplication of chromosome 15. (B) BAF and LRR plots of the X chromosome for early and late passage samples of the UC06 hESC line. There is a subtle widening of the band of heterozygous SNPs in the BAF plot for the early passage sample, which has separated into two distinct bands in the BAF plot for the late passage sample, indicating that the small subpopulation of cells carrying a duplication of the X chromosome in the early passage population has outcompeted the cells without the duplication over long-term passage.
Figure 5
Figure 5
Duplications on chromosome 20 arising over a five day period during directed differentiation of hESCs to cardiomyocytes. (A) The top two panels show the BAF and LRR plots at Day 2 of the differentiation protocol; the bottom two panels show the plots at Day 7. Three segments showing different degrees of separation of the “cloud” of BAF values for heterozygous SNPs are labeled 1, 2, and 3. (B) The BAF distance for heterozygous SNPs are shown for regions duplicated on chromosome 20. The BAF distance for mixtures of known ratios of HDF51 cells (which have no duplication on chromosome 20) and HDF51IPS11P33 cells (which have a duplication of the proximal portion of the q-arm) are shown on the left (BAF and LRR plots are shown in Figure S4A). The BAF distances for the three partially duplicated segments (corresponding to the segments labeled 1, 2, and 3 in A) are shown on the right, and have been used to estimate the percent of the population carrying the duplication. See also Figure S5.
Figure 6
Figure 6
Regions of duplication (identified by CNVPartition) and deletion (identified by both CNVPartition and replicate error analysis), in the HDF51 fibroblast line (green) and the HDF51IPS lines (blue). Duplicated regions (3 or 4 copies) are shown in the dark bars above the ideogram of the chromosomes, and deleted regions (0 or 1 copy) are shown in the light bars above the ideograms of the chromosomes. The line number and passage number of the HDF51IPS line are shown adjacent to each bar, with regions where the CNV is present in only a subpopulation of the cells in a sample are denoted “(sub).” The names of genes overlapping the regions of CNV are indicated. See also Figure S6 and Table S6.
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Comment in

  • iPSCs: induced back to controversy.
    Panopoulos AD, Ruiz S, Izpisua Belmonte JC. Panopoulos AD, et al. Cell Stem Cell. 2011 Apr 8;8(4):347-8. doi: 10.1016/j.stem.2011.03.003. Cell Stem Cell. 2011. PMID: 21474093

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