Stem Cell Reviews and Reports

, Volume 10, Issue 4, pp 472–479 | Cite as

Aberrant Patterns of X Chromosome Inactivation in a New Line of Human Embryonic Stem Cells Established in Physiological Oxygen Concentrations

  • Juliana Andrea de Oliveira Georges
  • Naja Vergani
  • Simone Aparecida Siqueira Fonseca
  • Ana Maria Fraga
  • Joana Carvalho Moreira de Mello
  • Maria Cecília R. Maciel Albuquerque
  • Litsuko Shimabukuro Fujihara
  • Lygia Veiga Pereira
Article

Abstract

One of the differences between murine and human embryonic stem cells (ESCs) is the epigenetic state of the X chromosomes in female lines. Murine ESCs (mESCs) present two transcriptionally active Xs that will undergo the dosage compensation process of XCI upon differentiation, whereas most human ESCs (hESCs) spontaneously inactivate one X while keeping their pluripotency. Whether this reflects differences in embryonic development of mice and humans, or distinct culture requirements for the two kinds of pluripotent cells is not known. Recently it has been shown that hESCs established in physiological oxygen levels are in a stable pre-XCI state equivalent to that of mESCs, suggesting that culture in low oxygen concentration is enough to preserve that epigenetic state of the X chromosomes. Here we describe the establishment of two new lines of hESCs under physiological oxygen level and the characterization of the XCI state in the 46,XX line BR-5. We show that a fraction of undifferentiated cells present XIST RNA accumulation and single H3K27me foci, characteristic of the inactive X. Moreover, analysis of allele specific gene expression suggests that pluripotent BR-5 cells present completely skewed XCI. Our data indicate that physiological levels of oxygen are not sufficient for the stabilization of the pre-XCI state in hESCs.

Keywords

Human embryonic stem cells X chromosome inactivation Hypoxia Epigenetics 

Notes

Acknowledgments

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico/Departamento de Ciência e Tecnologia doMinistério da Saúde (CNPq/MS/DECIT), Banco Nacional de Desenvolvimento Econômico e Social (BNDES), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grant CEPID 2013/08135-2) and Financiadora de Estudos e Projetos (FINEP). AMF and JM have fellowships from FAPESP; NJ and SASF have fellowships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Conflict of Interest

The authors declare no conflicts of interest.

Supplementary material

12015_2014_9505_Fig5_ESM.jpg (64 kb)
Supplemental Figure 1

In situ analysis of XCI in additional hESCs. Undifferentiated and differentiated HUES1, HUES9, HUES14 and HEK293 (positive control) cells analyzed by XIST-RNA FISH (red). Below, undifferentiated BR-5 cells cultured in 20 % O2, HUES9 and HEK293 (positive control) analyzed by XIST-RNA FISH (green) over-exposed. Nuclei stained by DAPI (blue). (JPEG 63 kb)

12015_2014_9505_MOESM1_ESM.tif (2.3 mb)
High resolution image (TIFF 2356 kb)

References

  1. 1.
    Evans, M., & Kaufman, M. (1981). Establishment in culture of pluripotent cells from mouse embryos. Nature, 292(5819), 154–156.PubMedCrossRefGoogle Scholar
  2. 2.
    Payer, B., & Lee, J. T. (2008). X chromosome dosage compensation: how mammals keep the balance. Annual Review of Genetics, 42, 733–772.PubMedCrossRefGoogle Scholar
  3. 3.
    Nesbitt, M. N. (1971). X chromosome inactivation mosaicism in the mouse. Developmental Biology, 26, 252–263.CrossRefGoogle Scholar
  4. 4.
    McMahon, A., & Monk, M. (1983). X-chromosome activity in female mouse embryos heterozygous for pgk-1 and Searle’s translocation, T(X; 16) 16H. Genetics Research, 41, 69–83.CrossRefGoogle Scholar
  5. 5.
    Morey, C., & Avner, P. (2011). The demoiselle of X-inactivation: 50 years old and as trendy and mesmerising as ever. PLoS Genetics, 7(7), e1002212. Available: http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1002212. Accessed 06 May 2013.PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147.PubMedCrossRefGoogle Scholar
  7. 7.
    Dhara, S. K., & Benvenisty, N. (2004). Gene trap as a tool for genome annotation and analysis of X chromosome inactivation in human embryonic stem cells. Nucleic Acids Research, 32(13), 3995–4002.PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Reubinoff, B. E., Pera, M. F., & Fong, C. Y. (2000). Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature Biotechnology, 18, 399–404.PubMedCrossRefGoogle Scholar
  9. 9.
    Amps, K., Andrews, P. W., Anyfantis, G., Armstrong, L., Avery, S., et al. (2011). Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage. Nature Biotechnology, 29, 1132–1144.PubMedCrossRefGoogle Scholar
  10. 10.
    Silva, S. S., Rowntree, R. K., Mekhoubad, S., & Lee, J. T. (2008). X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America, 105, 4820–4825.PubMedCentralPubMedGoogle Scholar
  11. 11.
    Shen, Y., Matsuno, Y., Fouse, S. D., Rao, N., Root, S., et al. (2008). X-inactivation in female human embryonic stem cells is in a nonrandom pattern and prone to epigenetic alterations. Proceedings of the National Academy of Sciences of the United States of America, 105, 4709–4714.PubMedCentralPubMedGoogle Scholar
  12. 12.
    Brons, G. M., Smithers, L. E., Trotter, M. W. B., Rugg-Gunn, P., Sun, B., et al. (2007). Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature, 448, 191–195.PubMedCrossRefGoogle Scholar
  13. 13.
    Tesar, P. J., Chenoweth, J. G., Brook, F. A., Davies, T. J., Evans, E. P., et al. (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature, 448, 196–199.PubMedCrossRefGoogle Scholar
  14. 14.
    van den Berg, I. M., Galjaard, R. J., Laven, J. S. E., & van Doorninck, J. H. (2011). XCI in preimplantation mouse and human embryos: first there is remodelling…. Human Genetics, 130(2), 203–215.PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Lengner, C. J., Gimelbrant, A. A., Erwin, J. A., Cheng, A. W., Guenther, M. G., et al. (2010). Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations. Cell, 141(5), 872–883.PubMedCrossRefGoogle Scholar
  16. 16.
    Mitalipova, M., & Palmarini, G. (2006). Isolation and characterization of human embryonic stem cells. Methods in Molecular Biology, 331, 55–76.PubMedGoogle Scholar
  17. 17.
    Gosden, C., Davdson, C., & Robertson, M. (1992). Lymphocyte culture. In D. E. Rooney & B. H. Czepulkowsky (Eds.), Human cytogenetics (pp. 37–47). Oxford: Oxford University Press.Google Scholar
  18. 18.
    Prokhorova, T. A., Harkness, L. M., Frandsen, U., Ditzel, N., Schroder, H. D., et al. (2009). Teratoma formation by human embryonic stem cells is site-dependent and enhanced by the presence of matrigel. Stem Cells and Development, 18(1), 47–54.PubMedCrossRefGoogle Scholar
  19. 19.
    Teklenburg, G., Weimar, C. H. E., Fauser, B. C. J. M., Macklon, N., Geijsen, N., et al. (2012). Cell lineage specific distribution of H3K27 trimethylation accumulation in an in vitro model for human implantation. PLoS One, 7(3), e32701.PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Cowan, C. A., Klimanskaya, I., McMahon, J., Atienza, J., Witmyer, J., et al. (2004). Derivation of embryonic stem-cell lines from human blastocysts. New England Journal of Medicine, 350, 1353–1356.PubMedCrossRefGoogle Scholar
  21. 21.
    Fraga, A. M., Sukoyan, M., Rajan, P., Braga, D. P., Iaconelli, A., Jr., et al. (2011). Establishment of a Brazilian line of human embryonic stem cells in defined medium: implications for cell therapy in an ethnically diverse population. Cell Transplantation, 20(3), 431–440.PubMedCrossRefGoogle Scholar
  22. 22.
    Mello, J. C. M., Araújo, E. S. S., Stabellini, R., Fraga, A. M., Souza, J. E. S., et al. (2010). Random X inactivation and extensive mosaicism in human placenta revealed by analysis of allele-specific gene expression along the X chromosome. PLoS ONE, 5, e10947.CrossRefGoogle Scholar
  23. 23.
    Ge, B., Gurd, S., Gaudin, T., Dore, C., Lepage, P., et al. (2005). Survey of allelic expression using EST mining. Genome Research, 15(11), 1584–1591.PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Kay, G. F., Penny, G. D., Patel, D., Ashworth, A., Brockdorff, N., & Rastan, S. (1993). Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation. Cell, 72, 171–182.PubMedCrossRefGoogle Scholar
  25. 25.
    Carrel, L., & Willard, H. F. (2005). X-inactivation profile reveals extensive variability in X-linked gene expression in females. Nature, 434, 400–404.PubMedCrossRefGoogle Scholar
  26. 26.
    Hoffman, L. M., Hall, L., Batten, J. L., Young, H., Pardasani, D., et al. (2005). X-Inactivation status varies in human embryonic stem cell lines. Stem Cells, 23, 1468–1478.PubMedCrossRefGoogle Scholar
  27. 27.
    Dvash, T., Lavon, N., & Fan, G. (2010). Variations of X chromosome inactivation occur in early passages of female human embryonic stem cells. PLoS ONE, 5(6), e11330. doi:10.1371/journal.pone.0011330.PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Mak, W., Nesterova, T. B., de Napoles, M., Appanah, R., Yamanaka, S., et al. (2004). Reactivation of the paternal X chromosome in early mouse embryos. Science, 303, 666–669.PubMedCrossRefGoogle Scholar
  29. 29.
    Okamoto, I., Otte, A. P., Allis, C. D., Reinberg, D., & Heard, E. (2004). Epigenetic dynamics of imprinted X inactivation during early mouse development. Science, 303, 644–649.PubMedCrossRefGoogle Scholar
  30. 30.
    van den Berg, I. M., Laven, J. S., Stevens, M., Jonkers, I., Galjaard, R. J., et al. (2009). X chromosome inactivation is initiated in human preimplantation embryos. American Journal of Human Genetics, 84, 771–779.PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Okamoto, I., Patrat, C., Thépot, D., Peynot, N., Fauque, P., et al. (2011). Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development. Nature, 472, 370–374.PubMedCrossRefGoogle Scholar
  32. 32.
    Bruck, T., & Benvenisty, N. (2011). Meta-analysis of the heterogeneity of X chromosome inactivation in human pluripotent stem cells. Stem Cell Research (Amsterdam), 6, 187–193.CrossRefGoogle Scholar
  33. 33.
    Nazor, K. L., Altun, G., Lynch, C., Tran, H., Harness, J. V., Slavin, I., et al. (2012). Recurrent variations in DNA methylation in human pluripotent stem cells and their differentiated derivatives. Cell Stem Cell, 10(5), 620–634.PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Mekhoubad, S., Bock, C., de Boer, A. S., Kiskinis, E., Meissner, A., & Eggan, K. (2012). Erosion of dosage compensation impacts human iPSC disease modeling. Cell Stem Cell, 10(5), 595–609.PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Bruck, T., Yanuka, O., & Benvenisty, N. (2013). Human pluripotent stem cells with distinct X inactivation status show molecular and cellular differences controlled by the X-linked ELK-1 gene. Cell Reports, 4, 262–270.PubMedCrossRefGoogle Scholar
  36. 36.
    Chan, Y. S., Goke, J., Ng, J. H., Lu, X., Gonzales, K. A. U., Tan, C. P., et al. (2013). Induction of a human pluripotent state with distinct regulatory circuitry that resembles preimplantation epiblast. Cell Stem Cell, 13, 663–675.PubMedCrossRefGoogle Scholar
  37. 37.
    Gafni, O., Weinberger, L., Mansour, A. A., Manor, Y. S., Chomsky, E., Ben-Yosef, D., et al. (2013). Derivation of novel human ground state naïve pluripotent stem cells. Nature. doi:10.1038/nature12745.Google Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Juliana Andrea de Oliveira Georges
    • 1
    • 2
  • Naja Vergani
    • 1
    • 2
  • Simone Aparecida Siqueira Fonseca
    • 1
    • 2
    • 4
  • Ana Maria Fraga
    • 1
    • 2
  • Joana Carvalho Moreira de Mello
    • 1
    • 2
  • Maria Cecília R. Maciel Albuquerque
    • 3
  • Litsuko Shimabukuro Fujihara
    • 3
  • Lygia Veiga Pereira
    • 1
    • 2
    • 4
  1. 1.Laboratory of Molecular GeneticsUniversity of São PauloSão PauloBrazil
  2. 2.National Laboratory of Embryonic Stem Cell (LaNCE), Department of Genetics and Evolutionary BiologyUniversity of São PauloSão PauloBrazil
  3. 3.Fertivitro - Center of Human ReproductionSão PauloBrazil
  4. 4.Center for Cell-based TherapySão Paulo Research Foundation (FAPESP)São PauloBrazil

Personalised recommendations