Skip to main content

Thank you for visiting 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.

Characterization of pluripotent stem cells


Characterization of pluripotent stem cells is required for the registration of stem cell lines and allows for an impartial and objective comparison of the results obtained when generating multiple lines. It is therefore crucial to establish specific, fast and reliable protocols to detect the hallmarks of pluripotency. Such protocols should include immunocytochemistry (takes 2 d), identification of the three germ layers in in vitro–derived embryoid bodies by immunocytochemistry (immunodetection takes 3 d) and detection of differentiation markers in in vivo–generated teratomas by immunohistochemistry (differentiation marker detection takes 4 d). Standardization of the immunodetection protocols used ensures minimum variations owing to the source, the animal species, the endogenous fluorescence or the inability to collect large amounts of cells, thereby yielding results as fast as possible without loss of quality. This protocol provides a description of all the immunodetection procedures necessary to characterize mouse and human stem cell lines in different circumstances.

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

Get just this article for as long as you need it


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

Figure 1: Possible cross-links.
Figure 2: Background as a result of using mouse antibodies in mouse samples.
Figure 3: Isoforms of Oct4.
Figure 4: The use of SlideFlasks in immunodetection.
Figure 5: Quick pluripotency assays.
Figure 6: Antigen retrieval process over cultured stem cells.
Figure 7: Characterization of a pluripotent stem cell line.
Figure 8: AP staining.
Figure 9: Pluripotency detection in hESCs.
Figure 10: Pluripotency detection in mESCs.
Figure 11: Differentiation test in vitro.
Figure 12: Differentiation test in vivo.
Figure 13: Analysis of teratomas.


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

    Article  CAS  Google Scholar 

  2. Thomson, J.A. et al. Embryonic stem cells lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    Article  CAS  Google Scholar 

  3. 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  Google Scholar 

  4. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblast by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  Google Scholar 

  5. Carpenter, M.K. & Bhatia, M. Characterization of human embryonic stem cells. in Handbook of Stem Cells, Vol 1: Embryonic stem cells (eds. Lanza, R. et al.) 407–411 (Elsevier/Academic Press, 2004).

  6. Ohnuki, M., Takahashi, K. & Yamanaka, S. Generation and characterization of human induced Pluripotent stem cells. Curr. Protoc. Stem Cell Biol. 9, 4A.2.1–4A.2.25 (2009).

    Google Scholar 

  7. Crook, J.M., Hei, D. & Stacey, G. International Stem Cell Banking Initiative: raising standards to bank on. In Vitro Cell Dev. Biol. Anim. 46, 169–172 (2010).

    Article  Google Scholar 

  8. Adewumi, O. et al. Characterization of human embryonic stem cell lines by the international stem cell initiative. Nat. Biotechnol. 25, 803–815 (2007).

    Article  CAS  Google Scholar 

  9. Neale, F., Clubb, J.S., Hotchks, D. & Posen, S. Heat stability of human placental alkaline phosphatase. J. Clin. Pathol. 18, 359–363 (1965).

    Article  CAS  Google Scholar 

  10. Henthorn, P., Zervos, P., Raducha, M., Harris, H. & Kadesch, T. Expression of a human placental alkaline phosphatase gene in transfected cells: use as a reporter for studies of gene expression. Proc. Natl. Acad. Sci. USA 85, 6342–6346 (1988).

    Article  CAS  Google Scholar 

  11. Maatman, R. et al. Aggregation of embryos and embryonic stem cells. Transgenic mouse. Methods Mol. Biol. 209, 201–230 (2002).

    Google Scholar 

  12. Wang, J., Levasseur, D.N. & Orkin, S.H. Requirement of Nanog dimerization for stem cell self-renewal and pluripotency. Proc. Natl. Acad. Sci. USA 105, 6326–6331.

  13. Das, S., Jena, S. & Levasseur, D.N. Alternative splicing produces Nanog protein variants with different capacities for self-renewal and pluripotency in embryonic stem cells. J. Biol. Chem. 286, 42690–42703 (2011).

    Article  CAS  Google Scholar 

  14. Cauffman, G., Van de Velde, H., Liebaers, I. & Van Steirteghem, A. Oct-4 mPNA and protein expression during human preimplantation development. Mol. Hum. Reprod. 11, 173–181 (2005).

    Article  CAS  Google Scholar 

  15. Cauffman, G., Liebaers, I., Van Steirteghem, A. & Van de Velde, H. POU5F1 isoforms show different expression patterns in human embryonic stem cells and preimplantation embryos. Stem Cells 24, 2685–2691 (2006).

    Article  CAS  Google Scholar 

  16. Müller, F.J. et al. A bioinformatic assay for pluripotency in human cells. Nat. Methods 8, 315–317 (2011).

    Article  Google Scholar 

  17. Hentze, H. et al. Teratoma formation by human embryonic stem cells: evaluation of essential parameters for future safety studies. Stem Cell Res. 3, 198–210 (2009).

    Article  Google Scholar 

  18. Itskovitz-Eldor, J. et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. 6, 88–95 (2000).

    Article  CAS  Google Scholar 

  19. Aasen, T. et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat. Biotechnol. 26, 1276–1284 (2008).

    Article  CAS  Google Scholar 

  20. Aran, B. et al. Derivation of human embryonic stem cells at the Center of Regenerative Medicine in Barcelona. In vitro Cell Dev. Biol. Anim. 46, 356–366 (2010).

    Article  Google Scholar 

  21. Keller, G.M. In vitro differentiation of embryonic stem cells. Curr. Opin. Cell Biol. 7, 862–869 (1995).

    Article  CAS  Google Scholar 

  22. Cerdan, C., Hong, S.H. & Bhatia, M. Formation and hematopoietic differentiation of human embryoid bodies by suspension and hanging drop cultures. Curr. Protoc. Stem Cell Biol. 3, 1D.2.1–1D.2.16 (2007).

    Google Scholar 

  23. Sundström, J. et al. Characterization of the model for experimental testicular teratoma in 129/SvJ-mice. Br. J. Cancer 80, 149–160 (1999).

    Article  Google Scholar 

  24. Gertow, K. et al. Isolation of human embryonic stem cell–derived teratomas for the assessment of pluripotency. Curr. Protoc. Stem Cell Biol. 3, 1B.4.1–1B.4.29 (2007).

    Google Scholar 

  25. Campos, P.B., Sartore, R.C., Abdalla, S.N. & Rehen, S.K. Chromosomal spread preparation of human embryonic stem cells for karyotyping. J. Vis. Exp. 31, e1512 (2009).

    Google Scholar 

  26. Schröck, E. et al. Multicolor spectral karyotyping of human chromosomes. Science 273, 494–497 (1996).

    Article  Google Scholar 

  27. Gonzalez, F. et al. Generation of mouse induced pluripotent stem cells by transient expression of a single non-viral polycistronic vector. Proc. Natl. Acad. Sci. USA 106, 8918–8922 (2009).

    Article  CAS  Google Scholar 

  28. Raya, A. et al. Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Nature 460, 53–59 (2009).

    Article  CAS  Google Scholar 

  29. Kawamura, T. et al. Linking the p53 tumor suppressor pathway to somatic cell reprogramming. Nature 460, 1140–1144 (2009).

    Article  CAS  Google Scholar 

  30. Giorgetti, A. et al. Generation of induced pluripotent stem cells from cord blood using OCT4 and SOX2. Cell Stem Cell 5, 353–357 (2009).

    Article  CAS  Google Scholar 

  31. Montserrat, N. et al. Generation of feeder free pig induced pluripotent stem cells without Pou5f1. Cell Transplant. 21, 815–825 (2011).

    Article  Google Scholar 

Download references


We are grateful to all the researchers who provided us their samples to be analyzed, with special mention to I. Rodriguez, A. Giorgietti, A. Consiglio, R. Vassena, N. Montserrat, C. Eguizabal and S. Menendez. Work in the laboratory of J.C.I.B. was supported by grants from TERCEL-ISCII-MINECO, CIBER, Fundacion Cellex, Sanofi, The Leona M. and Harry B. Helmsley Charitable Trust and the G. Harold and Leila Y. Mathers Charitable Foundation.

Author information

Authors and Affiliations



All authors contributed equally to this work. M.M. designed the protocols, analyzed the data and wrote the paper; L.M. and C.P. performed and optimized the protocols; C.M. contributed to the microscope observations, live-cell staining and flow cytometry analysis; M.C. performed cell cultures and EB formation, and helped with the manuscript; L.L.-R. helped with the manuscript and gave conceptual advice; C.R.E. performed and optimized the protocols; and J.C.I.B. supervised the project and wrote the manuscript.

Corresponding author

Correspondence to Juan Carlos Izpisua Belmonte.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Alkaline phosphatase staining. Flow chart showing steps 1 to 9 of the protocol. Timing: 30 min. (PDF 295 kb)

Supplementary Figure 2

Pluripotency detection. Flow chart showing Steps 10 to 37 of the protocol. Timing: 2 days. (PDF 315 kb)

Supplementary Figure 3

Differentiation detection in EBs in suspension (OPTION A). Flow chart showing steps 38 A/I to XVI of the protocol. Timing: 5 days. (PDF 299 kb)

Supplementary Figure 4

Differentiation detection in EBs over SlideFlask (OPTION B). Flow chart showing steps 38 B/ I to XVIII. Timing: 3 days. (PDF 325 kb)

Supplementary Figure 5

Differentiation detection in teratomas (OPTION A). Flow chart showing steps 44 A/ I to XXV of the protocol. Timing: 5 days. (PDF 328 kb)

Supplementary Figure 6

Cell proliferation in teratomas (OPTION B). Flow chart showing steps 44 B/ I to XXVII. Timing: 6-8 days. (PDF 376 kb)

Supplementary Figure 7

Proliferation versus apoptosis in teratomas (OPTION C). Flow chart showing steps 44C /I to XXIX. Timing: 3 days. (PDF 178 kb)

Supplementary Video 1

Time lapse obtained from 15 h of a colony staining with Tra-1-81. Merge images of the green fluorescence and the transmitted light image. Image information: Inverted microscope: Leica DMI4000. Image pixels: 1024 x 1024. Resolution: 8 bits. Image: xyt series. Scale Bar: 75 μm. (MPG 2600 kb)

Supplementary Video 2

Time lapse obtained from 15 h of a colony staining with Tra-1-81, showing only the green fluorescence of the previous video. Image information: Inverted microscope: Leica DMI4000. Image pixels: 1024 x 1024. Resolution: 8 bits. Image: xyt series. Scale Bar: 75 μm. (MPG 706 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Martí, M., Mulero, L., Pardo, C. et al. Characterization of pluripotent stem cells. Nat Protoc 8, 223–253 (2013).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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.


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