Evaluation of Undifferentiated State of Human iPS Cells Using C2MAP™ Cell Culture Media Analysis Platform

Significance of the Topic

The reliable evaluation of human induced pluripotent stem cell (iPSC) status is essential for advancing regenerative medicine. Traditional invasive assays interrupt cell integrity and are limited to endpoint analysis. A non-invasive metabolite profiling approach enables continuous monitoring of cell health and differentiation, improving scalability and quality control for therapeutic applications.

Objectives and Study Overview

This study leveraged the C2MAP™ cell culture media analysis platform combined with liquid chromatography–tandem mass spectrometry (LC-MS/MS) to identify secreted biomarkers in iPSC culture supernatants. Key aims included:

• Detecting metabolites that distinguish undifferentiated iPSCs from cells induced toward each germ layer.
• Investigating the biological role of these metabolites in maintaining or altering cell state.

Methodology and Instrumentation

Human iPSCs (PFX#9 line) were maintained in TeSR™-E8™ medium on vitronectin-N–coated six-well plates at 1×105 cells per well. Culture supernatants were completely exchanged and collected every 24 hours over a six-day period. Samples were processed automatically on the C2MAP platform for simultaneous LC-MS/MS quantification of 95 target compounds spanning sugars, amino acids, vitamins, nucleic acid derivatives and other small metabolites. Data analysis employed C2MAP TRENDS for visualization and a Multi-omics Analysis Gadget Package for statistical comparisons.

Main Results and Discussion

Out of 95 analytes, 55 were consistently detected in culture supernatants. Volcano plot analysis at day 3 identified two key biomarkers:

• Kynurenine, elevated specifically in undifferentiated iPSCs.
• 2-Aminoadipic acid, elevated in cells undergoing ectodermal differentiation.

Time-course profiling revealed:

• In undifferentiated cultures, progressive consumption of tryptophan accompanied by increasing kynurenine secretion.
• In ectodermally differentiated cultures, a gradual rise in 2-aminoadipic acid levels.

Inhibition studies provided mechanistic insights:

• Adding an IDO1 inhibitor reduced kynurenine production and suppressed iPSC proliferation, confirming the role of tryptophan metabolism in self-renewal.
• Blocking the aryl hydrocarbon receptor (AhR) decreased both cell growth and kynurenine secretion, indicating that the Kyn-AhR complex drives transcription of pluripotency factors (POU5F1, NANOG, EP300) and reinforces its own pathway.
• In ectodermal differentiation, inhibiting KAT2 limited 2-aminoadipic acid formation and partially inhibited differentiation, suggesting that kynurenine catabolism shifts cell fate toward ectoderm.

Benefits and Practical Applications

This non-invasive LC-MS/MS–based workflow enables real-time monitoring of iPSC status without cell disruption. It supports:

• Quality assurance in cell manufacturing processes.
• Early detection of unwanted differentiation or loss of pluripotency.
• High-throughput screening for media optimization and biomarker discovery.

Used Instrumentation

• C2MAP™ cell culture media analysis platform for automated sample preparation.
• Liquid chromatography–tandem mass spectrometer (LC-MS/MS).
• C2MAP TRENDS viewer software and Multi-omics Analysis Gadget Package for data processing and statistical analysis.

Future Trends and Opportunities

Expanding this approach may include:

• Integration with real-time online monitoring systems for continuous culture surveillance.
• Extension to additional differentiation lineages and primary cell types.
• Combination with transcriptomic and proteomic data for comprehensive multi-omics cell state mapping.

Conclusion

The study demonstrates that C2MAP LC-MS/MS profiling of culture supernatants can non-invasively distinguish undifferentiated iPSCs from ectodermally differentiated cells via specific metabolite biomarkers. Kynurenine and 2-aminoadipic acid not only report cell state but also play functional roles in self-renewal and lineage commitment. This platform offers a scalable strategy for quality control in stem cell production.

References

1. Yamamoto T, Hatabayashi K, Arita M, et al. Kynurenine signaling through the aryl hydrocarbon receptor maintains the undifferentiated state of human embryonic stem cells. Sci Signal. 2019;12(594):eaaw3306.