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Characterization of X‐Chromosome Inactivation Status in Human Pluripotent Stem Cells

Jennifer A. Erwin1,2,3,  Jeannie T. Lee1,2,3

1Department of Genetics, Harvard Medical School, Boston, Massachusetts
2Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts
3Howard Hughes Medical Institute, Boston, Massachusetts



Publication Name: 
Current Protocols in Stem Cell Biology
Unit Number: 
UNIT 1B.6
DOI: 
10.1002/9780470151808.sc01b06s12
Online Posting Date: 
March, 2010
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Abstract

This unit describes a method of performing fluorescent in situ hybridization (FISH) of XIST and Cot-1 RNA in human pluripotent stem cells (hPSC) to characterize the epigenetic status of X-chromosome inactivation (XCI). hPSC laboratories commonly practice karyotypic analysis to monitor genetic stability; however, epigenetic stability is often overlooked. Several laboratories have recently shown that markers of XCI can be used as one effective screen to monitor the epigenetic status of hPSCs. Human embryonic stem cells (HESC) fall into three classes of XCI states: upregulating XIST upon differentiation, always expressing XIST in the undifferentiated and differentiated states, and never expressing XIST in the undifferentiated and differentiated states. Failure to express XIST represents an especially concerning state in hESC, as this state does not occur in healthy female cells but is often seen in malignancies. Herein, methods of carrying out XIST RNA and Cot-1 RNA FISH are described. FISH analysis of XIST RNA, unlike general expression analysis such as RT-PCR, allows for the classification of XCI on a single-cell level, enabling a quantitative determination of the degree of epigenetic change across the population. The complementary Cot-1 analysis measures the extent of repeat element expression throughout the nucleus and therefore enables determination, at a cytological level, of the extent to which the X chromosome is silent. Because the different steps of XCI are some of the first epigenetic changes to take place in differentiating hESC, analysis of the XCI state provides a first indication of an hESC culture's overall health. Curr. Protoc. Stem Cell Biol. 12:1B.6.1-1B.6.11. © 2010 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; epigenetics; X-chromosome inactivation; XIST

     
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation, Slide Preparation, and Fixation of Human Pluripotent Stem Cells
  • Support Protocol 1: XIST DNA Probe Preparation by Nick Translation
  • Support Protocol 2: Cot-1 Probe Labeling by Random Priming
  • Support Protocol 3: DNA Precipitation of Labeled Probes for Analysis of X-Chromosome Inactivation
  • Basic Protocol 2: X-Chromosome Inactivation Detected by RNA Fluorescent In Situ Hybridization
  • Alternate Protocol: DNA Fluorescent In Situ Hybridization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Topics
    • Stem Cells
    • Cell Culture
     
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Materials

Basic Protocol 1: Isolation, Slide Preparation, and Fixation of Human Pluripotent Stem Cells

 Materials
  • hPSCs cultures (starting with at least 1 × 105 cells per hybridization spot)
  • Phosphate buffered saline without CaCl2 and without MgCl2 (CMF-PBS), room temperature and ice cold
  • 0.05% (w/v) trypsin/EDTA
  • MEF medium (see recipe)
  • CSK-T solution (see recipe), ice cold
  • 4% paraformaldehyde in 1× CMF-PBS, pH 7.2 (4% PFA)
  • 70% ethanol, ice cold
  • 37°C incubator
  • 5-ml serological pipets
  • 15-ml conical tubes (Falcon)
  • Cytospin centrifuge
  • Positively charged slides (e.g., Fisherbrand Superfrost/Plus)
  • Coplin jars

Support Protocol 1: XIST DNA Probe Preparation by Nick Translation

 Materials
  • XIST exon1 DNA (GenBank U80460: 61251–69449)
  • Nick translation kit (Roche, cat. no. 10976776001) containing:
    • dATP
    • dGTP
    • dCTP
    • 10× buffer
    • Enzyme mix
  • Cy3-dUTP (Amersham, cat. no. PA53022)
  • PCR thermal cycler

Support Protocol 2: Cot-1 Probe Labeling by Random Priming

 Materials
  • Cot-1 DNA (Invitrogen, cat. no. 15279-011)
  • Prime-It Fluor with FITC-dUTP kit (Stratagene, cat. no. 300380) containing:
    • Random 9-mers
    • 5× nucleotide buffer
    • Fluor-12-dUTP
    • Klenow
    • Stop buffer
  • PCR thermal cycler

Support Protocol 3: DNA Precipitation of Labeled Probes for Analysis of X-Chromosome Inactivation

 Materials
  • Cy3-dUTP-labeled XIST DNA probe reaction mix (see Support Protocol 1)
  • Fluor-12-dUTP-labeled Cot-1 DNA reaction mix (see Support Protocol 2)
  • Herring sperm DNA (Promega, cat. no. D1811)
  • 3 M sodium acetate, pH 5.0
  • 70% and 100% ethanol
  • Hybridization buffer (see recipe)

Basic Protocol 2: X-Chromosome Inactivation Detected by RNA Fluorescent In Situ Hybridization

 Materials
  • XIST/Cot-1-labeled probe (see Support Protocols 1 through 3)
  • Fixed slides in 70% ethanol (see Basic Protocol 1)
  • 80%, 90%, and 100% ethanol, ice cold
  • 2× SSC/50% (v/v) formamide (see recipe for 20× SSC)
  • 2× SSC (see recipe for 20× SSC)
  • Vectashield with DAPI (Vector)
  • Clear nail polish
  • 42°C and 80°C heating blocks or water baths
  • Humidified chamber at 37°C (e.g., pipet tip container with water in the bottom)
  • Glass coverslips
  • Coplin jars
  • 45°C incubator with agitator
  • Upright epifluorescence microscope with 60× oil immersion lens or confocal microscope equipped with filters (compatible for imaging with DAPI, Cy3, Cy5, and FITC)

Alternate Protocol: DNA Fluorescent In Situ Hybridization

 Additional Materials (also see Basic Protocol 2)
  • Unlabeled Cot-1 DNA
  • Hybridization buffer (Cambio)
  • Concentrated whole X-chromosome paint (Cambio)
  • 0.2% (v/v) Tween in CMF-PBS
  • 2% (w/v) paraformaldehyde (PFA) in CMF-PBS/0.2% (v/v) Tween
  • RNase A (400 µg/ml in CMF-PBS)
  • CMF-PBS
  • 0.2 N HCl/0.2% (v/v) Tween
  • 70% (v/v) formamide/2× SSC (see recipe for 20× SSC)
  • Fixed slides in 70% ethanol (see Basic Protocol 2)
  • Rubber cement
Looking for materials?  Suppliers Guide
     
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Literature Cited

Literature Cited
    Andrews, P.W., Matin, M.M., Bahrami, A.R., Damjanov, I., Gokhale, P., and Draper, J.S. 2005. Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: Opposite sides of the same coin. Biochem. Soc. Trans. 33:1526-1530.
    Baker, D.E., Harrison, N.J., Maltby, E., Smith, K., Moore, H.D., Shaw, P.J., Heath, P.R., Holden, H., and Andrews, P.W. 2007. Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat. Biotechnol. 25:207-215.
    Donohoe, M.E., Silva, S.S., Pinter, S.F., Xu, N., and Lee, J.T. 2009. The pluripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting. Nature 460:128-132.
    Guo, G., Yang, J., Nichols, J., Hall, J.S., Eyres, I., Mansfield, W., and Smith, A. 2009. Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 136:1063-1069.
    Hall, L.L., Byron, M., Butler, J., Becker, K.A., Nelson, A., Amit, M., Itskovitz-Eldor, J., Stein, J., Stein, G., Ware, C., and Lawrence, J.B. 2008. X-inactivation reveals epigenetic anomalies in most HESC but identifies sublines that initiate as expected. J. Cell Physiol. 216:445-452.
    Hoffman, L.M., Hall, L., Batten, J.L., Young, H., Pardasani, D., Baetge, E.E., Lawrence, J., and Carpenter, M.K. 2005. X-inactivation status varies in human embryonic stem cell lines. Stem Cells 23:1468-1478.
    Maherali, N., Sridharan, R., Xie, W., Utikal, J., Eminli, S., Arnold, K., Stadtfeld, M., Yachechko, R., Tchieu, J., Jaenisch, R., Plath, K., and Hochedlinger, K. 2007. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell. 1:55-70.
    Navarro, P., Chambers, I., Karwacki-Neisius, V., Chureau, C., Morey, C., Rougeulle, C., and Avner, P. 2008. Molecular coupling of XIST regulation and pluripotency. Science 321:1693-1695.
    Pageau, G., Hall, L., Ganesan, S., Livingston, D., and Lawrence, J. 2007. The disappearing Barr body in breast and ovarian cancers. Nat. Rev. Cancer 7:628-633.
    Payer, B. and Lee, J.T. 2008. X chromosome dosage compensation: How mammals keep the balance. Annu. Rev. Genet. 42:733-772.
    Shen, Y., Matsuno, Y., Fouse, S.D., Rao, N., Root, S., Xu, R., Pellegrini, M., Riggs, A.D., and Fan, G. 2008. X-inactivation in female human embryonic stem cells is in a nonrandom pattern and prone to epigenetic alterations. Proc. Natl. Acad. Sci. U.S.A. 105:4709-4714.
    Silva, S.S., Rowntree, R.K., Mekhoubad, S., and Lee, J.T. 2008. X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 105:4820-4825.
    Sirchia, S.M., Ramoscelli, L., Grati, F.R., Barbera, F., Coradini, D., Rossella, F., Porta, G., Lesma, E., Ruggeri, A., Radice, P., Simoni, G., and Miozzo, M. 2005. Loss of the inactive X chromosome and replication of the active X in BRCA1-defective and wild-type breast cancer cells. Cancer Res. 65:2139-2146.
    Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861-872.
    Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282:1145-1147.
    Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, II, and Thomson, J.A. 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917-1920.
     
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