Evaluating Reprogramming Efficiency and Pluripotency of the Established Human iPSCS by Pluripotency Markers

Ricardo Raúl Cevallos, Md Emon Hossain, Ruowen Zhang, Kejin Hu

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

4 Scopus citations


The pluripotency of human induced pluripotent stem cells (HiPSCs) cannot be tested strictly in a similar way as we can do for the mouse ones because of ethical restrictions. One common and initial approach to prove the pluripotency of an established human iPSC line is to demonstrate expression of a set of established surface and intracellular pluripotency markers. This chapter provides procedures of immunocytochemistry of the established HiPSC lines for a set of the signature intracellular pluripotency proteins, OCT4, SOX2, NANOG, and LIN28. We also describe cell phenotyping by flow cytometry for the five established human pluripotency surface markers, SSEA3, SSEA4, TRA-1-60, TRA-1-81, and TRA2-49 (ALP). Numbers of ALP+ and TRA-1-60+ colonies are the most widely used parameters for evaluation of human iPSC reprogramming efficiency. Therefore, this chapter also provides detailed steps for substrate colorimetric reaction of the ALP activity, as well as the TRA-1-60 staining, of the iPSC colonies in the reprogramming population.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages15
StatePublished - 2021

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Human induced pluripotent stem cells
  • Human iPSCs
  • Immunophenotyping
  • iPSC reprogramming
  • Pluripotency
  • Pluripotency factors
  • Pluripotency surface markers


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