High-resolution DNA analysis of human embryonic stem cell lines reveals culture-induced copy number changes and loss of heterozygosity

Journal name:
Nature Biotechnology
Volume:
28,
Pages:
371–377
Year published:
DOI:
doi:10.1038/nbt.1615
Received
Accepted
Published online

Abstract

Prolonged culture of human embryonic stem cells (hESCs) can lead to adaptation and the acquisition of chromosomal abnormalities, underscoring the need for rigorous genetic analysis of these cells. Here we report the highest-resolution study of hESCs to date using an Affymetrix SNP 6.0 array containing 906,600 probes for single nucleotide polymorphisms (SNPs) and 946,000 probes for copy number variations (CNVs). Analysis of 17 different hESC lines maintained in different laboratories identified 843 CNVs of 50 kb–3 Mb in size. We identified, on average, 24% of the loss of heterozygosity (LOH) sites and 66% of the CNVs changed in culture between early and late passages of the same lines. Thirty percent of the genes detected within CNV sites had altered expression compared to samples with normal copy number states, of which >44% were functionally linked to cancer. Furthermore, LOH of the q arm of chromosome 16, which has not been observed previously in hESCs, was detected.

At a glance

Figures

  1. Amplifications contribute to majority of total genomic size affected by CNV in hESCs.
    Figure 1: Amplifications contribute to majority of total genomic size affected by CNV in hESCs.

    (a,b) Average chromosomal distribution of 50 kb–3 Mb size CNVs in hESCs (a) and in Caucasian HapMap population (b). The majority (72%) of the total genomic size affected by CNVs found in hESCs corresponded to amplifications, whereas gains and losses were equally distributed in the HapMap samples. Chromosomal distribution differences between hESCs and HapMap were most prominent in chromosomes 10, 14, 20, X and Y.

  2. LOH and CNV regions change in culture.
    Figure 2: LOH and CNV regions change in culture.

    (a) The number of LOH, CNV and passages between sample collections in sample pairs (H9 P25/P34, CCTL-14 P38/P49, I3 P41/P55, HS293 P26/P60, H7 P30/P91). CNVs that remained stable during the culture are marked with dashed line. (b) The percentage of total genomic area changed plotted against the passages in culture shows clear correlation within chromosomes 1 (78%), 10 (89%), 17 (84%), 20 (90%) and X (88%) in H7 sample series, all P < 0.05. All seven samples are from the same hESC line H7 (P30, P38, P128, P132, P230 and P237). Large chromosomal changes in addition to CNVs were included in the analysis.

  3. Chromosomal abnormalities detected.
    Figure 3: Chromosomal abnormalities detected.

    (a) The array karyotype of the sample H7 (s6) P237 shows deletions of extra abnormal chromosome 1 in 1p35 and in 1p terminus, as well as gains of 9p13–p21.2 and 10p11.2–p15, which were not seen by conventional karyotyping. (b) Mosaic karyotype of FES61, having an extra copy of chromosomes 3, 5, 11, 16, 17 and 20 and two extra copies of chromosome 12 in half of the cell population, was seen on the array karyoview as multiple CNVs in the chromosomes of the extra copy and total gain in the case of chromosome 12. (c) Summary of the large karyotype abnormalities detected. Gain, blue (↑); loss, red (↓). Each individual CNV is marked with a symbol: , gain, , loss.

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Referenced accessions

Gene Expression Omnibus

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Author information

Affiliations

  1. Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.

    • Elisa Närvä,
    • Reija Autio,
    • Nelly Rahkonen,
    • Omid Rasool &
    • Riitta Lahesmaa
  2. Department of Signal Processing, Tampere University of Technology, Tampere, Finland.

    • Reija Autio,
    • Lingjia Kong &
    • Olli Yli-Harja
  3. Centre for Stem Cell Biology and the Department of Biomedical Science, University of Sheffield, Sheffield, UK.

    • Neil Harrison &
    • Peter W Andrews
  4. Stem Cell Technologies Ltd., Jerusalem, Israel.

    • Danny Kitsberg
  5. Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn and Hertie Foundation, Bonn, Germany.

    • Lodovica Borghese &
    • Oliver Brüstle
  6. Faculty of Medicine, Technion-Israel Institute of Technology and Department of Obstetrics and Gynecology, Rambam Health Care Campus, Haifa, Israel.

    • Joseph Itskovitz-Eldor
  7. Department of Biology, Faculty of Medicine, Masaryk University & Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Brno, Czech Republic.

    • Petr Dvorak
  8. Department CLINTEC, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.

    • Outi Hovatta
  9. Program of Molecular Neurology, Biomedicum Stem Cell Center, University of Helsinki, Helsinki, Finland.

    • Timo Otonkoski &
    • Timo Tuuri
  10. Children's Hospital, University of Helsinki, Helsinki, Finland.

    • Timo Otonkoski
  11. Institute of Reproductive and Developmental Biology, Faculty of Medicine, Imperial College London, Hammersmith Campus, London, UK.

    • Wei Cui
  12. Sheffield Diagnostic Genetic Services, Sheffield Children's NHS Trust, Sheffield, UK.

    • Duncan Baker &
    • Edna Maltby
  13. Centre for Stem Cell Biology and the Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK.

    • Harry D Moore
  14. Stem Cell Unit, Department of Genetics, The Institute of Life Sciences, The Hebrew University, Jerusalem, Israel.

    • Nissim Benvenisty
  15. Institute for Systems Biology, Seattle, Washington, USA.

    • Olli Yli-Harja

Contributions

E.N., R.A., N.B., P.W.A., O.Y.-H. and R.L. designed the experiments, E.N. and R.L. were responsible for the coordination of the project and microarray experiments. R.A., E.N. and O.Y.-H. were responsible for data analysis, integration and statistical analysis. N.R. performed RNA extractions. L.K. built the gene annotation list of genes overlapping CNVs. D.B. performed conventional karyotyping. E.N. and N.R. performed copy-number state validations with RT-PCR. J.I.-E. provided I3 and I6 lines for the study. P.D., O.H., T.O., T.T., N.B., W.C., O.B., E.M., H.D.M., P.W.A., O.Y.-H. and R.L. provided the samples and coordinated the project in their groups. E.N., R.A., N.R., L.K., N.H., D.K., L.B., J.I.-E., O.R., P.D., O.H., T.O., T.T., N.B., W.C., O.B., D.B., E.M., H.D.M., P.W.A., O.Y.-H. and R.L. contributed to writing the paper.

Competing financial interests

D.K. is affiliated with Stem Cell Technologies, Ltd. (However, the study was not supported by the company.)

Corresponding authors

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Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (304K)

    Supplementary Figs. 1–4 and Supplementary Tables 4,5,6,12

Excel files

  1. Supplementary Table 1. (92K)

    SNP profiles and Hapmap codes.xls

  2. Supplementary Table 2. (956K)

    CNV region list

  3. Supplementary Table 3. (2.4K)

    HapMap CNV region list

  4. Supplementary Table 7. (1M)

    Genes affected by CNVs HapMap

  5. Supplementary Table 8. (236K)

    Genes affected by CNVs

  6. Supplementary Table 9. (60K)

    genes changed by adaptation

  7. Supplementary Table 10a. (172K)

    integrated analysis, losses

  8. Supplementary Table 10b. (3.9K)

    integrated analysis, gains

  9. Supplementary Table 11. (20K)

    Culture conditions

Additional data