Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Embryonic and induced pluripotent stem cell staining and sorting with the live-cell fluorescence imaging probe CDy1

Nature Protocols volume 6, pages 1044–1052 (2011)Cite this article

Subjects

Abstract

Detecting and isolating specific types of cells is crucial to understanding a variety of biological processes, including development, aging, regeneration and pathogenesis; this understanding, in turn, allows the use of cells for therapeutic purposes, for which stem cells have emerged recently as invaluable materials. The current methods of isolation and characterization of stem cells depend on cell morphology in culture or on immunostaining of specific markers. These methods are, however, time consuming and involve the use of antibodies that may often make the cells unsuitable for further study. We recently developed a fluorescent small molecule named CDy1 (compound of designation yellow 1) that selectively stains live embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). This protocol describes detailed procedures for staining ESC and iPSC in live conditions and for fluorescence-activated cell sorting (FACS) of ESC using CDy1. Cell staining, image acquisition and FACS can be done within 6 h.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Chemical structure and report of CDy1.
Figure 2
Figure 3: Isolating stem cells by CDy1 staining and FACS sorting.
Figure 4: Selective staining of mESC with CDy1.
Figure 5: hESC-BG01V characterization and staining with CDy1.
Figure 6: iPSC characterization and staining with CDy1.
Figure 7: Isolation of mESCs by FACS using CDy1.

References

  1. Hanna, J.H., Saha, K. & Jaenisch, R. Pluripotency and cellular reprogramming: facts, hypotheses, unresolved issues. Cell 143, 508–525 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Wobus, A.M. & Boheler, K.R. Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol. Rev. 85, 635–678 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Dimos, J.T. et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321, 1218–1221 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Park, I.H. et al. Disease-specific induced pluripotent stem cells. Cell 134, 877–886 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Nelson, T.J. et al. Repair of acute myocardial infarction by human stemness factors induced pluripotent stem cells. Circulation 120, 408–416 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Laflamme, M.A. et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat. Biotechnol. 25, 1015–1024 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Amariglio, N. et al. Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS Med. 6, e1000029 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Pera, M.F., Filipczyk, A.A., Hawes, S.M. & Laslett, A.L. Isolation, characterization, and differentiation of human embryonic stem cells. Methods Enzymol. 365, 429–446 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. O'Connor, M.D. et al. Alkaline phosphatase-positive colony formation is a sensitive, specific, and quantitative indicator of undifferentiated human embryonic stem cells. Stem Cells 26, 1109–1116 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Yamagata, K., Ueda, J., Mizutani, E., Saitou, M. & Wakayama, T. Survival and death of epiblast cells during embryonic stem cell derivation revealed by long-term live-cell imaging with an Oct4 reporter system. Dev. Biol. 346, 90–101 (2010).

    Article  CAS  PubMed  Google Scholar 

  11. Lee, J.S., Kim, Y.K., Vendrell, M. & Chang, Y.T. Diversity-oriented fluorescence library approach for the discovery of sensors and probes. Mol. Biosyst. 5, 411–421 (2009).

    Article  CAS  PubMed  Google Scholar 

  12. Lee, J.S. et al. Synthesis of a BODIPY library and its application to the development of live cell glucagon imaging probe. J. Am. Chem. Soc. 131, 10077–10082 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. Kim, Y.K. et al. Control of muscle differentiation by a mitochondria-targeted fluorophore. J. Am. Chem. Soc. 132, 576–579 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. Oliveira, F.F. et al. On the use of 2,1,3-benzothiadiazole derivatives as selective live cell fluorescence imaging probes. Bioorg. Med. Chem. Lett. 20, 6001–6007 (2010).

    Article  CAS  PubMed  Google Scholar 

  15. Yamagata, K. DNA methylation profiling using live-cell imaging. Methods 52, 259–266 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Storms, R.W. et al. Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity. Proc. Natl. Acad. Sci. USA 96, 9118–9123 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Moreb, J.S., Zucali, J.R., Ostmark, B. & Benson, N.A. Heterogeneity of aldehyde dehydrogenase expression in lung cancer cell lines is revealed by Aldefluor flow cytometry-based assay. Cytometry B Clin. Cytom. 72, 281–289 (2007).

    Article  PubMed  Google Scholar 

  18. Im, C.N. et al. A fluorescent rosamine compound selectively stains pluripotent stem cells. Angew. Chem. Int. Ed. Engl. 49, 7497–7500 (2010).

    Article  CAS  PubMed  Google Scholar 

  19. Ahn, Y.H., Lee, J.S. & Chang, Y.T. Combinatorial rosamine library and application to in vivo glutathione probe. J. Am. Chem. Soc. 129, 4510–4511 (2007).

    Article  CAS  PubMed  Google Scholar 

  20. Evans, M.J. & Kaufman, M.H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

    Article  CAS  PubMed  Google Scholar 

  21. Lin, S. & Talbot, P. Methods for culturing mouse and human embryonic stem cells. Methods Mol. Biol. 690, 31–56 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. Takahashi, K., Okita, K., Nakagawa, M. & Yamanaka, S. Induction of pluripotent stem cells from fibroblast cultures. Nat. Protoc. 2, 3081–3089 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported by intramural funding from A*STAR (Agency for Science, Technology and Research, Singapore) Biomedical Research Council and a Young Investigator Award R-143-000-353-101 granted to Y.-T.C. from the National University of Singapore.

Author information

Authors and Affiliations

  1. Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore

    Nam-Young Kang, Seong-Wook Yun, Sung-Jin Park & Young-Tae Chang

  2. College of Pharmacy, Sunchon National University, Sunchon, Korea

    Hyung-Ho Ha

  3. Department of Chemistry, National University of Singapore, Singapore, Republic of Singapore

    Hyung-Ho Ha & Young-Tae Chang

Authors
  1. Nam-Young Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Seong-Wook Yun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hyung-Ho Ha
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sung-Jin Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Young-Tae Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.-Y.K. and S.-J.P. performed the experiments and processed the data. N.-Y.K. and S.-W.Y. wrote the manuscript. H.-H.H. synthesized CDy1 and analyzed its chemical properties. Y.-T.C. conceived, directed and supervised the project.

Corresponding author

Correspondence to Young-Tae Chang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kang, NY., Yun, SW., Ha, HH. et al. Embryonic and induced pluripotent stem cell staining and sorting with the live-cell fluorescence imaging probe CDy1. Nat Protoc 6, 1044–1052 (2011). https://doi.org/10.1038/nprot.2011.350

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.350

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing