Flow Cytometric Characterization of Pluripotent Cell Protein Markers in Naïve, Formative, and Primed Pluripotent Stem Cells

Joshua G Dierolf; Kristin Chadwick; Courtney R Brooks; Andrew J Watson; Dean H Betts

doi:10.1007/978-1-0716-2281-0_8

Flow Cytometric Characterization of Pluripotent Cell Protein Markers in Naïve, Formative, and Primed Pluripotent Stem Cells

Methods Mol Biol. 2022:2490:81-92. doi: 10.1007/978-1-0716-2281-0_8.

Authors

Joshua G Dierolf¹, Kristin Chadwick², Courtney R Brooks¹, Andrew J Watson^#^{1

3

4}, Dean H Betts^#^{5

6

7}

Affiliations

¹ Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, ON, Canada.
² Robarts Research Institute, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, ON, Canada.
³ Department of Obstetrics and Gynecology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, ON, Canada.
⁴ The Children's Health Research Institute (CHRI), Lawson Health Research Institute, London, ON, Canada.
⁵ Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, ON, Canada. dean.betts@schulich.uwo.ca.
⁶ Department of Obstetrics and Gynecology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, ON, Canada. dean.betts@schulich.uwo.ca.
⁷ The Children's Health Research Institute (CHRI), Lawson Health Research Institute, London, ON, Canada. dean.betts@schulich.uwo.ca.

^# Contributed equally.

PMID: 35486241
DOI: 10.1007/978-1-0716-2281-0_8

Abstract

Here we describe methodologies to characterize, delineate, and quantify pluripotent cells between naïve, formative, and primed pluripotent state mouse embryonic stem cell (mESCs) populations using flow cytometric analysis. This methodology can validate pluripotent states, sort individual cells of interest, and determine the efficiency of transitioning naïve mESCs to a primed-like state as mouse epiblast-like cells (mEpiLCs) and onto fully primed mouse epiblast stem cells (mEpiSCs). Quantification of the cell surface markers; SSEA1(CD15) and CD24 introduces an effective method of distinguishing individual cells from a population by their respective positioning in the pluripotent spectrum. Additionally, this protocol can be used to demarcate and sort cells via fluorescently activated cell sorting for downstream applications. Flow cytometric analysis within mESCs, mEpiLCs, and mEpiSCs can be efficiently completed using these optimized protocols.

Keywords: Embryonic stem cells; Flow cytometry; Formative pluripotency; Naïve pluripotency; Pluripotent stem cells; Primed pluripotency; mEpiSC.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Differentiation
Germ Layers
Mice
Mouse Embryonic Stem Cells
Pluripotent Stem Cells*

Grants and funding

CIHR/Canada