Stem Cell Reviews and Reports

, Volume 7, Issue 4, pp 969–975 | Cite as

DNA Methylation Assay for X-Chromosome Inactivation in Female Human iPS Cells

  • Lesli A. Kiedrowski
  • Gordana Raca
  • Jennifer J. Laffin
  • Benjamin S. Nisler
  • Kimberly Leonhard
  • Erik McIntire
  • Karen Dyer Mongomery
Article

Abstract

Remarkable interest in the epigenetic status of human induced pluripotent stem (iPS) cells inspired numerous studies of their X-inactivation patterns. However, both the presence and the absence of X-inactivation have been described to date in undifferentiated iPS cells. The reasons for the discordant results between different studies are unclear, and further X-inactivation testing is warranted for all female human iPS cell lines. Some of the inconsistency in the current data most likely results from the use of different X-inactivation assays by different authors. We provide a detailed protocol for a simple, reliable and affordable X-inactivation assay based on promoter methylation and CAG-repeat polymorphism in the human androgen receptor (AR) gene at Xq11.2. This assay is commonly used in clinical genetic laboratories and we propose that it could be ideal for routine assessment and monitoring of the X-inactivation status in female human iPS cell lines.

Keywords

iPS cells X-inactivation AR methylation IMR90 

References

  1. 1.
    Maherali, N., Sridharan, R., Xie, W., et al. (2007). Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell, 1(1), 55–70.PubMedCrossRefGoogle Scholar
  2. 2.
    Navarro, P., & Avner, P. (2009). When X-inactivation meets pluripotency: an intimate rendezvous. FEBS Letters, 583, 1721–1727.PubMedCrossRefGoogle Scholar
  3. 3.
    Wutz, A., & Gribnau, J. (2007). X inactivation Xplained. Current Opinion in Genetics & Development, 17(5), 387–393.CrossRefGoogle Scholar
  4. 4.
    Tchieu, J., Kuoy, E., Chin, M. H., et al. (2010). Female human iPSCs retain an inactive X chromosome. Cell Stem Cell, 7(3), 329–342.PubMedCrossRefGoogle Scholar
  5. 5.
    Marchetto, M. C., Carromeu, C., Acab, A., et al. (2010). A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell, 143(4), 527–539.PubMedCrossRefGoogle Scholar
  6. 6.
    Shen, Y., Matsuno, Y., Fouse, S. D., 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(12), 4709–4714.PubMedCrossRefGoogle Scholar
  7. 7.
    Hall, L. L., Byron, M., Butler, J., et al. (2008). X-inactivation reveals epigenetic anomalies in most hESC but identifies sublines that initiate as expected. Journal of Cellular Physiology, 216(2), 445–452.PubMedCrossRefGoogle Scholar
  8. 8.
    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.PubMedCrossRefGoogle Scholar
  9. 9.
    Lengner, C. J., Gimelbrant, A. A., Erwin, J. A., et al. (2010). Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations. Cell, 141(5), 872–883.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(12), 4820–4825.PubMedCrossRefGoogle Scholar
  11. 11.
    Allen, R. C., Zoghbi, H. Y., Moseley, A. B., Rosenblatt, H. M., & Belmont, J. W. (1992). Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation. American Journal of Human Genetics, 51(6), 1229–1239.PubMedGoogle Scholar
  12. 12.
    Thouin, M. M., Giron, J. M., & Hoffman, E. P. (2003). Detection of nonrandom X chromosome inactivation. Curr Protoc Hum Genet, Chapter 9:Unit 9 7.Google Scholar
  13. 13.
    Talebizadeh, Z., Bittel, D. C., Veatch, O. J., Kibiryeva, N., & Butler, M. G. (2005). Brief report: non-random X chromosome inactivation in females with autism. Journal of Autism and Developmental Disorders, 35(5), 675–681.PubMedCrossRefGoogle Scholar
  14. 14.
    Yu, J., Vodyanik, M. A., Smuga-Otto, K., et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science, 318(5858), 1917–1920.PubMedCrossRefGoogle Scholar
  15. 15.
    Edwards, A., Hammond, H. A., Jin, L., Caskey, C. T., & Chakraborty, R. (2005). Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics, 12(2), 241–253.CrossRefGoogle Scholar
  16. 16.
    Basehore, M. J., & Friez, M. J. (2009). Molecular analysis of Fragile X syndrome. Curr Protoc Hum Genet, Chapter 9:Unit 9 5.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Lesli A. Kiedrowski
    • 1
  • Gordana Raca
    • 1
    • 2
  • Jennifer J. Laffin
    • 1
    • 3
  • Benjamin S. Nisler
    • 4
  • Kimberly Leonhard
    • 4
  • Erik McIntire
    • 4
  • Karen Dyer Mongomery
    • 4
  1. 1.Clinical Genetic Laboratories, Wisconsin State Laboratory of HygieneUniversity of Wisconsin-Madison School of Medicine and Public HealthMadisonUSA
  2. 2.Department of Pathology and Laboratory MedicineUniversity of Wisconsin-Madison School of Medicine and Public HealthMadisonUSA
  3. 3.Department of PediatricsUniversity of Wisconsin-Madison School of Medicine and Public HealthMadisonUSA
  4. 4.Cytogenetics LaboratoryWiCell Research InstituteMadisonUSA

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