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


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.


Human embryonic stem cells X chromosome inactivation Hypoxia Epigenetics 



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)


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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

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