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Adaptation to culture of human embryonic stem cells and oncogenesis in vivo

Abstract

The application of human embryonic stem cells (HESCs) to provide differentiated cells for regenerative medicine will require the continuous maintenance of the undifferentiated stem cells for long periods in culture. However, chromosomal stability during extended passaging cannot be guaranteed, as recent cytogenetic studies of HESCs have shown karyotypic aberrations. The observed karyotypic aberrations probably reflect the progressive adaptation of self-renewing cells to their culture conditions. Genetic change that increases the capacity of cells to proliferate has obvious parallels with malignant transformation, and we propose that the changes observed in HESCs in culture reflect tumorigenic events that occur in vivo, particularly in testicular germ cell tumors. Further supporting a link between culture adaptation and malignancy, we have observed the formation of a chromosomal homogeneous staining region in one HESC line, a genetic feature almost a hallmark of cancer cells. Identifying the genes critical for culture adaptation may thus reveal key players for both stem cell maintenance in vitro and germ cell tumorigenesis in vivo.

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Figure 1: Representative cytogenetic data from a HESC line maintained in Sheffield.
Figure 2: Ideogram of all reported chromosome abnormalities in HESCs.
Figure 3: Interphase FISH analysis of H14.s3 cell line hybridized with an iso17q probe (Kreatech Biotechnology) specific for the p53 (17p13) and MPO (17q23) genes.
Figure 4: Evidence of a minimal region of amplification on chromosome 17q in culture-adapted HESCs.
Figure 5: Characterization of the HSR in the abnormal HESC line H14.

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References

  1. O'Neill, L.P., Vermilyea, M.D. & Turner, B.M. Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations. Nat. Genet. 38, 835–841 (2006).

    Article  CAS  Google Scholar 

  2. Bradley, A., Evans, M., Kaufman, M.H. & Robertson, E. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309, 255–256 (1984).

    Article  CAS  Google Scholar 

  3. Hoffman, L.M. & Carpenter, M.K. Characterization and culture of human embryonic stem cells. Nat. Biotechnol. 23, 699–708 (2005).

    Article  CAS  Google Scholar 

  4. Enver, T. et al. Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells. Hum. Mol. Genet. 14, 3129–3140 (2005).

    Article  CAS  Google Scholar 

  5. Herszfeld, D. et al. CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells. Nat. Biotechnol. 24, 351–357 (2006).

    Article  CAS  Google Scholar 

  6. Andrews, P.W. et al. Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin. Biochem. Soc. Trans. 33, 1526–1530 (2005).

    Article  CAS  Google Scholar 

  7. Liu, Y. et al. Genome wide profiling of human embryonic stem cells (hESCs), their derivatives and embryonal carcinoma cells to develop base profiles of U.S. Federal government approved hESC lines. BMC Dev. Biol. 6, 20 (2006).

    Article  CAS  Google Scholar 

  8. Boveri, T. The origin of malignant tumours (Williams and Walkin, Baltimore, 1914).

    Google Scholar 

  9. Oosterhuis, J.W. & Looijenga, L.H. Testicular germ-cell tumours in a broader perspective. Nat. Rev. Cancer 5, 210–222 (2005).

    Article  CAS  Google Scholar 

  10. Damjanov, I. Teratocarcinoma stem cells. Cancer Surv. 9, 303–319 (1990).

    CAS  PubMed  Google Scholar 

  11. Moller, H. Clues to the aetiology of testicular germ cell tumours from descriptive epidemiology. Eur. Urol. 23, 8–13; discussion 14–15 (1993).

    Article  CAS  Google Scholar 

  12. Damjanov, I. Pathogenesis of testicular germ cell tumours. Eur. Urol. 23, 2–5; discussion 6–7 (1993).

    Article  Google Scholar 

  13. Andrews, P.W. From teratocarcinomas to embryonic stem cells. Phil. Trans. R. Soc. Lond. B 357, 405–417 (2002).

    Article  Google Scholar 

  14. Draper, J.S. et al. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat. Biotechnol. 22, 53–54 (2004).

    Article  CAS  Google Scholar 

  15. Mitalipova, M.M. et al. Preserving the genetic integrity of human embryonic stem cells. Nat. Biotechnol. 23, 19–20 (2005).

    Article  CAS  Google Scholar 

  16. Brimble, S.N. et al. Karyotypic stability, genotyping, differentiation, feeder-free maintenance, and gene expression sampling in three human embryonic stem cell lines derived prior to August 9, 2001. Stem Cells Dev. 13, 585–597 (2004).

    Article  CAS  Google Scholar 

  17. Buzzard, J.J., Gough, N.M., Crook, J.M. & Colman, A. Karyotype of human ES cells during extended culture. Nat Biotechnol. 22, 381–382; author reply 382 (2004).

    Article  CAS  Google Scholar 

  18. Imreh, M.P. et al. In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells. J. Cell. Biochem. 99, 508–516 (2006).

    Article  CAS  Google Scholar 

  19. Maitra, A. et al. Genomic alterations in cultured human embryonic stem cells. Nat. Genet. 37, 1099–1103 (2005).

    Article  CAS  Google Scholar 

  20. Josephson, R. et al. A molecular scheme for improved characterization of human embryonic stem cell lines. BMC Biol. 4, 28 (2006).

    Article  Google Scholar 

  21. Reuter, V.E. Origins and molecular biology of testicular germ cell tumors. Mod. Pathol. 18 (suppl. 2), S51–S60 (2005).

    Article  CAS  Google Scholar 

  22. Samaniego, F. et al. Cytogenetic and molecular analysis of human male germ cell tumors: chromosome 12 abnormalities and gene amplification. Genes Chromosom. Cancer 1, 289–300 (1990).

    Article  CAS  Google Scholar 

  23. Kraggerud, S.M. et al. Genome profiles of familial/bilateral and sporadic testicular germ cell tumors. Genes Chromosom. Cancer 34, 168–174 (2002).

    Article  CAS  Google Scholar 

  24. Summersgill, B.M. et al. Definition of chromosome aberrations in testicular germ cell tumor cell lines by 24-color karyotyping and complementary molecular cytogenetic analyses. Cancer Genet. Cytogenet. 128, 120–129 (2001).

    Article  CAS  Google Scholar 

  25. von Eyben, F.E. Chromosomes, genes, and development of testicular germ cell tumors. Cancer Genet. Cytogenet. 151, 93–138 (2004).

    Article  CAS  Google Scholar 

  26. Sugawara, A., Goto, K., Sotomaru, Y., Sofuni, T. & Ito, T. Current status of chromosomal abnormalities in mouse embryonic stem cell lines used in Japan. Comp. Med. 56, 31–34 (2006).

    CAS  PubMed  Google Scholar 

  27. Lastowska, M. et al. Breakpoint position on 17q identifies the most aggressive neuroblastoma tumors. Genes Chromosom. Cancer 34, 428–436 (2002).

    Article  CAS  Google Scholar 

  28. Tirkkonen, M. et al. Molecular cytogenetics of primary breast cancer by CGH. Genes Chromosom. Cancer 21, 177–184 (1998).

    Article  CAS  Google Scholar 

  29. Azuhata, T. et al. The inhibitor of apoptosis protein survivin is associated with high-risk behavior of neuroblastoma. J. Pediatr. Surg. 36, 1785–1791 (2001).

    Article  CAS  Google Scholar 

  30. Cowan, C.A. et al. Derivation of embryonic stem-cell lines from human blastocysts. N. Engl. J. Med. 350, 1353–1356 (2004).

    Article  CAS  Google Scholar 

  31. Korkola, J.E. et al. Down-regulation of stem cell genes, including those in a 200-kb gene cluster at 12p13.31, is associated with in vivo differentiation of human male germ cell tumors. Cancer Res. 66, 820–827 (2006).

    Article  CAS  Google Scholar 

  32. Skotheim, R.I. et al. Novel genomic aberrations in testicular germ cell tumors by array-CGH, and associated gene expression changes. Cell. Oncol. 28, 315–326 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Roelofs, H. et al. Restricted 12p amplification and RAS mutation in human germ cell tumors of the adult testis. Am. J. Pathol. 157, 1155–1166 (2000).

    Article  CAS  Google Scholar 

  34. Zafarana, G. et al. 12p-amplicon structure analysis in testicular germ cell tumors of adolescents and adults by array CGH. Oncogene 22, 7695–7701 (2003).

    Article  CAS  Google Scholar 

  35. Ludwig, T.E. et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185–187 (2006).

    Article  CAS  Google Scholar 

  36. Inzunza, J. et al. Comparative genomic hybridization and karyotyping of human embryonic stem cells reveals the occurrence of an isodicentric X chromosome after long-term cultivation. Mol. Hum. Reprod. 10, 461–466 (2004).

    Article  CAS  Google Scholar 

  37. Sperger, J.M. et al. Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc. Natl. Acad. Sci. USA 100, 13350–13355 (2003).

    Article  CAS  Google Scholar 

  38. Dhara, S.K. & Benvenisty, N. Gene trap as a tool for genome annotation and analysis of X chromosome inactivation in human embryonic stem cells. Nucleic Acids Res. 32, 3995–4002 (2004).

    Article  CAS  Google Scholar 

  39. Sandberg, A.A., Meloni, A.M. & Suijkerbuijk, R.F. Reviews of chromosome studies in urological tumors. III. Cytogenetics and genes in testicular tumors. J. Urol. 155, 1531–1556 (1996).

    Article  CAS  Google Scholar 

  40. Kawakami, T. et al. The roles of supernumerical X chromosomes and XIST expression in testicular germ cell tumors. J. Urol. 169, 1546–1552 (2003).

    Article  Google Scholar 

  41. Yang, S.H., Jaffray, E., Hay, R.T. & Sharrocks, A.D. Dynamic interplay of the SUMO and ERK pathways in regulating Elk-1 transcriptional activity. Mol. Cell 12, 63–74 (2003).

    Article  CAS  Google Scholar 

  42. Wu, X., Noh, S.J., Zhou, G., Dixon, J.E. & Guan, K.L. Selective activation of MEK1 but not MEK2 by A-Raf from epidermal growth factor-stimulated HeLa cells. J. Biol. Chem. 271, 3265–3271 (1996).

    Article  CAS  Google Scholar 

  43. Ishitani, K. et al. p54nrb acts as a transcriptional coactivator for activation function 1 of the human androgen receptor. Biochem. Biophys. Res. Commun. 306, 660–665 (2003).

    Article  CAS  Google Scholar 

  44. Bai, V.U., Cifuentes, E., Menon, M., Barrack, E.R. & Reddy, G.P. Androgen receptor regulates Cdc6 in synchronized LNCaP cells progressing from G1 to S phase. J. Cell. Physiol. 204, 381–387 (2005).

    Article  CAS  Google Scholar 

  45. Rapley, E.A. et al. Localization to Xq27 of a susceptibility gene for testicular germ-cell tumours. Nat. Genet. 24, 197–200 (2000).

    Article  CAS  Google Scholar 

  46. Daley, G.Q. & Baltimore, D. Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific P210bcr/abl protein. Proc. Natl. Acad. Sci. USA 85, 9312–9316 (1988).

    Article  CAS  Google Scholar 

  47. Wang, N., Trend, B., Bronson, D.L. & Fraley, E.E. Nonrandom abnormalities in chromosome 1 in human testicular cancers. Cancer Res. 40, 796–802 (1980).

    CAS  PubMed  Google Scholar 

  48. Looijenga, L.H. et al. Comparative genomic hybridization of microdissected samples from different stages in the development of a seminoma and a non-seminoma. J. Pathol. 191, 187–192 (2000).

    Article  CAS  Google Scholar 

  49. Biedler, J.L. & Spengler, B.A. A novel chromosome abnormality in human neuroblastoma and antifolate-resistant Chinese hamster cell lives in culture. J. Natl. Cancer Inst. 57, 683–695 (1976).

    Article  CAS  Google Scholar 

  50. Benner, S.E., Wahl, G.M. & Von Hoff, D.D. Double minute chromosomes and homogeneously staining regions in tumors taken directly from patients versus in human tumor cell lines. Anticancer Drugs 2, 11–25 (1991).

    Article  CAS  Google Scholar 

  51. Albrecht, S., Armstrong, D.L., Mahoney, D.H., Cheek, W.R. & Cooley, L.D. Cytogenetic demonstration of gene amplification in a primary intracranial germ cell tumor. Genes Chromosom. Cancer 6, 61–63 (1993).

    Article  CAS  Google Scholar 

  52. van Dartel, M. & Hulsebos, T.J. Amplification and overexpression of genes in 17p11.2 p12 in osteosarcoma. Cancer Genet. Cytogenet. 153, 77–80 (2004).

    Article  CAS  Google Scholar 

  53. Skotheim, R.I. et al. Novel genomic aberrations in testicular germ cell tumours by array-CGH, and associated gene expression changes. Cell. Oncol. 28, 315–326 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Feng, G.S. et al. Grap is a novel SH3-SH2-SH3 adaptor protein that couples tyrosine kinases to the Ras pathway. J. Biol. Chem. 271, 12129–12132 (1996).

    Article  CAS  Google Scholar 

  55. Burdon, T., Smith, A. & Savatier, P. Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol. 12, 432–438 (2002).

    Article  CAS  Google Scholar 

  56. Kang, H.B. et al. Basic fibroblast growth factor activates ERK and induces c-fos in human embryonic stem cell line MizhES1. Stem Cells Dev. 14, 395–401 (2005).

    Article  CAS  Google Scholar 

  57. Cervantes, R.B., Stringer, J.R., Shao, C., Tischfield, J.A. & Stambrook, P.J. Embryonic stem cells and somatic cells differ in mutation frequency and type. Proc. Natl. Acad. Sci. USA 99, 3586–3590 (2002).

    Article  CAS  Google Scholar 

  58. Hong, Y. & Stambrook, P.J. Restoration of an absent G1 arrest and protection from apoptosis in embryonic stem cells after ionizing radiation. Proc. Natl. Acad. Sci. USA 101, 14443–14448 (2004).

    Article  CAS  Google Scholar 

  59. Fluckiger, A.C. et al. Cell cycle features of primate embryonic stem cells. Stem Cells 24, 547–556 (2006).

    Article  CAS  Google Scholar 

  60. Downes, C.S. et al. A topoisomerase II-dependent G2 cycle checkpoint in mammalian cells. Nature 372, 467–470 (1994).

    Article  CAS  Google Scholar 

  61. Damelin, M., Sun, Y.E., Sodja, V.B. & Bestor, T.H. Decatenation checkpoint deficiency in stem and progenitor cells. Cancer Cell 8, 479–484 (2005).

    Article  CAS  Google Scholar 

  62. Amon, A. The spindle checkpoint. Curr. Opin. Genet. Dev. 9, 69–75 (1999).

    Article  CAS  Google Scholar 

  63. Wilton, L. Preimplantation genetic diagnosis for aneuploidy screening in early human embryos: a review. Prenat. Diagn. 22, 512–518 (2002).

    Article  Google Scholar 

  64. Hardy, K. Cell death in the mammalian blastocyst. Mol. Hum. Reprod. 3, 919–925 (1997).

    Article  CAS  Google Scholar 

  65. von Zglinicki, T., Pilger, R. & Sitte, N. Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts. Free Radic. Biol. Med. 28, 64–74 (2000).

    Article  CAS  Google Scholar 

  66. Andrews, P.W. et al. Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro. Lab. Invest. 50, 147–162 (1984).

    CAS  PubMed  Google Scholar 

  67. Andrews, P.W., Goodfellow, P.N., Shevinsky, L.H., Bronson, D.L. & Knowles, B.B. Cell-surface antigens of a clonal human embryonal carcinoma cell line: morphological and antigenic differentiation in culture. Int. J. Cancer 29, 523–531 (1982).

    Article  CAS  Google Scholar 

  68. Amit, M. et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271–278 (2000).

    Article  CAS  Google Scholar 

  69. Pyle, A.D., Lock, L.F. & Donovan, P.J. Neurotrophins mediate human embryonic stem cell survival. Nat. Biotechnol. 24, 344–350 (2006).

    Article  CAS  Google Scholar 

  70. Liu, X. et al. Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission. Dev. Dyn. 209, 85–91 (1997).

    Article  CAS  Google Scholar 

  71. Longo, L., Bygrave, A., Grosveld, F.G. & Pandolfi, P.P. The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism. Transgenic Res. 6, 321–328 (1997).

    Article  CAS  Google Scholar 

  72. Draper, J.S., Moore, H.D., Ruban, L.N., Gokhale, P.J. & Andrews, P.W. Culture and characterization of human embryonic stem cells. Stem Cells Dev. 13, 325–336 (2004).

    Article  CAS  Google Scholar 

  73. Caisander, G. et al. Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture. Chromosome Res. 14, 131–137 (2006).

    Article  CAS  Google Scholar 

  74. Rosler, E.S. et al. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev. Dyn. 229, 259–274 (2004).

    Article  CAS  Google Scholar 

  75. Xiao, L., Yuan, X. & Sharkis, S.J. Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells. Stem Cells 24, 1476–1486 (2006).

    Article  CAS  Google Scholar 

  76. Kim, S.J. et al. Efficient derivation of new human embryonic stem cell lines. Mol. Cells 19, 46–53 (2005).

    CAS  PubMed  Google Scholar 

  77. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    Article  CAS  Google Scholar 

  78. Smith, A.C. et al. Interstitial deletion of (17)(p11.2p11.2) in nine patients. Am. J. Med. Genet. 24, 393–414 (1986).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to C. Cowan and D. Melton for providing the HUES1-17 HESC lines, and to J. Thomson for the H1, H7 and H14 HESC lines. We are grateful to our colleagues, especially J. Jackson, K. Amps, G. Bray and G. Bingham for culture of the HESCs. In addition, we would like to acknowledge B. Aflatoonian and L. Ruban for their assistance in the derivation and proliferation of the Shef cell lines. This work was supported by grants from the Medical Research Council, Yorkshire Cancer Research, The Engineering and Physical Sciences Research Council and The Juvenile Diabetes Research Foundation.

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Correspondence to Peter W Andrews.

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Supplementary Figure 1

FISH on the Shef5 cell line with the SMS and MDS probes specific for the critical regions of Smith Magenis syndrome and Miller Dieker syndrome at 17p11.2 and 17p13.3, respectively. (PDF 457 kb)

Supplementary Methods (PDF 132 kb)

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Baker, D., Harrison, N., Maltby, E. et al. Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat Biotechnol 25, 207–215 (2007). https://doi.org/10.1038/nbt1285

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