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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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.
The authors declare no competing financial interests.
Alkaline phosphatase staining. Flow chart showing steps 1 to 9 of the protocol. Timing: 30 min. (PDF 295 kb)
Pluripotency detection. Flow chart showing Steps 10 to 37 of the protocol. Timing: 2 days. (PDF 315 kb)
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)
Differentiation detection in EBs over SlideFlask (OPTION B). Flow chart showing steps 38 B/ I to XVIII. Timing: 3 days. (PDF 325 kb)
Differentiation detection in teratomas (OPTION A). Flow chart showing steps 44 A/ I to XXV of the protocol. Timing: 5 days. (PDF 328 kb)
Cell proliferation in teratomas (OPTION B). Flow chart showing steps 44 B/ I to XXVII. Timing: 6-8 days. (PDF 376 kb)
Proliferation versus apoptosis in teratomas (OPTION C). Flow chart showing steps 44C /I to XXIX. Timing: 3 days. (PDF 178 kb)
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)
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). https://doi.org/10.1038/nprot.2012.154
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