Multiparameter flow cytometry for the characterization of human embryonic stem cells

Area of Science:

• Stem Cell Biology
• Immunology
• Biotechnology

Background:

• Investigating human embryonic stem cell (HESC) pluripotency relies on accurate marker expression analysis.
• Flow cytometry is a key technique for multiparameter analysis of cell surface markers.

Purpose of the Study:

• To evaluate the impact of adjusting flow cytometry gating strategies on identifying HESC populations.
• To address antibody cross-reactivity issues affecting marker expression analysis in HESC cultures.

Main Methods:

• Utilized multiparameter staining and flow cytometry on HUES7 human embryonic stem cell cultures.
• Investigated marker co-expression patterns, specifically focusing on SSEA-1 and SSEA-4 antibodies.
• Compared gating strategies at 95% and 99% confidence levels using isotype controls.

Main Results:

• A multidimensional approach identified distinct cell populations but revealed cross-reactivity between SSEA-4 and SSEA-1 antibodies, creating false positives for SSEA-1.
• Increasing the confidence level of control gates to 99% significantly reduced SSEA-1 false positives.
• The 99% gating strategy also decreased positive expression signals for SSEA-4 and Tra-1-60.

Conclusions:

• A 99% confidence gating strategy can mitigate antibody cross-reactivity in flow cytometry analysis of HESCs.
• Further optimization of fluorophore/antibody combinations and selection of strongly expressing markers are necessary for routine implementation of 99% gating.
• Careful gating strategy optimization is crucial for accurate pluripotency marker assessment in stem cell research.