Pluripotency Assessment by Flow Cytometry

Pluripotency is the ability of stem cells to differentiate into all somatic cell types, a functional attribute that must be experimentally demonstrated¹. To classify a cell line as pluripotent, it must be able to differentiate into cells from all three embryonic germ layers—endoderm, mesoderm, and ectoderm—without any genetic or epigenetic manipulation.  

Evidence of differentiation should include quantitative measurements showing an upregulation of germ layer markers, along with a simultaneous downregulation of markers indicative of the undifferentiated state1. While xenograft (teratoma) assays have been used traditionally to demonstrate pluripotency, in vitro differentiation offers a reliable alternative, providing adequate evidence of pluripotency without animal welfare or regulatory concerns^1,2.  

WiCell’s Pluripotency Assay offers a straightforward and reliable way to assess the pluripotency of your stem cell lines to ensure the integrity of your research.

To assess the pluripotency of a stem cell line, WiCell first induces differentiation to the three embryonic germ layers in vitro using STEMDiff™ Trilineage Differentiation Kit from STEMCELL™ Technologies. With this kit, undifferentiated cells are divided into three separate cultures, where each culture is exposed to lineage-specific media that promotes differentiation. Our trilineage differentiation protocol is optimized for cell lines maintained in mTeSR1/mTeSR Plus on Matrigel/Cultrex. 

Following differentiation, marker expression for the differentiated cultures and the undifferentiated control are measured and compared using the BD Biosciences Accuri™ C6 Plus Flow Cytometer to confirm pluripotency. The specific markers used for flow cytometry are listed in the table below: 

Flow cytometry data analysis is performed using FCS Express™ from De Novo Software. First, non-viable cells are excluded from analysis using the LIVE/DEAD™ Green Dead Cell Stain Kit (Invitrogen™). Compensation matrices are computationally generated from single-color controls and applied to all sample data files. All data for samples and compensation controls are acquired on the same day on the same instrument to ensure consistency. Subsequent gating for undifferentiated markers Oct4 and Nanog is set based on the dominant dual positive population in undifferentiated cells. This gate placement is then maintained for cells from each lineage culture to quantify the downregulation of undifferentiation markers. 

Figure 1. Gating for Undifferentiated Oct 4 and Nanog. Quadrant gates for dual positive population is set using the undifferentiated culture data. Each lineage type is then analyzed using this dual positive gate placement.

Gating for lineage specific markers is set based on the dominant dual positive population of cells from each specific lineage culture. The placement is then maintained for cells from the undifferentiated culture to show potential upregulation of those markers from undifferentiated to differentiated cells. 

Figure 2. Gating for Endoderm markers Sox17 and FoxA2. Quadrant gates were defined using endoderm culture data to identify the dual-positive population for Sox17 and FoxA2. In this example, 72% of the endoderm culture is dual-positive for Sox17 and FoxA2, while the undifferentiated culture (using the same gate) showed 100% double-negative cells.

Please see our sample Pluripotency Gating Scheme for a more detailed explanation.

It is important to note that because no single gene is uniquely expressed by a single cell type or lineage, conclusions regarding pluripotency should rely on the patterns of dual expression for both markers of each cell type. 

Samples for flow cytometric pluripotency assessment are accepted as cryopreserved cells from cultures maintained in mTeSR1 or mTeSR Plus media grown on Matrigel or Cultrex matrices. Detailed sample submission instructions are available. Please email [email protected] for pricing and additional sample submission information.

WiCell is committed to delivering accurate and reliable characterization services that safeguards your stem cell research. After assay completion, the results for your cell line will be provided in an Assessment of Pluripotency Report. 

• View a sample report.
• Expected values are provided within the report as a guide for interpretation since biases in generating each lineage can occur in pluripotent cells. The context of the cell line’s intended applications should determine the degree of stringency required.

Additionally, WiCell provides flow cytometry testing services to assess the undifferentiated status of your stem cell lines to ensure thorough characterization. 

Contact us for inquiries about transfer of cell lines to our required culture format (mTeSR1/mTeSR Plus on Matrigel/Cultrex), assay customization, live culture sample submission, or any other questions.