Evaluating Reprogramming Efficiency and Pluripotency of the Established Human iPSCS by Pluripotency Markers
| Autor: | Ruowen Zhang, Kejin Hu, Ricardo Raúl Cevallos, Emon Hossain |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Homeobox protein NANOG education.field_of_study medicine.diagnostic_test Population Cell Biology LIN28 Cell biology Flow cytometry 03 medical and health sciences 030104 developmental biology 0302 clinical medicine medicine.anatomical_structure SOX2 embryonic structures medicine education Induced pluripotent stem cell Reprogramming reproductive and urinary physiology 030217 neurology & neurosurgery |
| Zdroj: | Methods in Molecular Biology ISBN: 9781071610831 |
| DOI: | 10.1007/978-1-0716-1084-8_15 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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