TY - CHAP
T1 - Evaluating Reprogramming Efficiency and Pluripotency of the Established Human iPSCS by Pluripotency Markers
AU - Cevallos, Ricardo Raúl
AU - Hossain, Md Emon
AU - Zhang, Ruowen
AU - Hu, Kejin
N1 - Funding Information:
The research in Hu laboratory is supported by an NIH 1R01GM127411-01A1 and the American Heart Association (17GRNT33670780).
Publisher Copyright:
© 2021, Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - Human induced pluripotent stem cells
KW - Human iPSCs
KW - Immunophenotyping
KW - iPSC reprogramming
KW - Pluripotency
KW - Pluripotency factors
KW - Pluripotency surface markers
UR - http://www.scopus.com/inward/record.url?scp=85096756961&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-1084-8_15
DO - 10.1007/978-1-0716-1084-8_15
M3 - Chapter
C2 - 33226623
AN - SCOPUS:85096756961
T3 - Methods in Molecular Biology
SP - 235
EP - 249
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -