Optimized, Efficient Measurement of the Expression of Undifferentiated Stem Cell Markers in Human Induced Pluripotent Stem Cells (iPSCs) by Flow Cytometry

Induced pluripotent stem cells (iPSCs) have revolutionized the fields of regenerative medicine, disease modeling, and drug discovery. However, the usage of iPSCs for various applications has been hampered by the observed line-to-line variability in their differentiation capacity. Therefore, it is important to verify the pluripotent status of iPSCs. A very effective way to define the pluripotent state of iPSCs is by evaluating the expression of established undifferentiated stem cell markers. A bona fide iPSC must have high, homogeneous expression of these markers. Here, we present a cost-effective platform that can be readily utilized by researchers to define the pluripotency status of iPSCs by measuring the expression of surface and intracellular markers by flow cytometry. © 2025 Wiley Periodicals LLC. Basic Protocol 1: iPSC culture and collection for flow cytometry analysis Basic Protocol 2: Staining of iPSCs for extracellular and intracellular undifferentiated stem cell markers Basic Protocol 3: Flow cytometry acquisition Basic Protocol 4: Flow cytometry data analysis.