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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.

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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.


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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




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.

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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)

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

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