Highly Sensitive Detection of Human Pluripotent Stem Cells by Loop-Mediated Isothermal Amplification

Area of Science:

• Biotechnology
• Regenerative Medicine
• Molecular Biology

Background:

• Ensuring the safety of regenerative medicines requires rigorous detection of tumorigenic undifferentiated cells in cell-derived products.
• Current in vitro nucleic acid tests for tumorigenicity are sensitive but limited by the small RNA input per test for human pluripotent stem cells (hPSCs).

Purpose of the Study:

• To develop a highly sensitive and robust assay for detecting human pluripotent stem cell (hPSC) contamination in cell-derived products.
• To overcome the limitations of small RNA input in existing nucleic acid detection methods.

Main Methods:

• Development of gene-specific and robust reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays.
• Assay optimization to handle microgram quantities of input sample.
• Application of multiplex RT-LAMP assays and in situ cell imaging for co-staining of pluripotency proteins and RNAs.

Main Results:

• The RT-LAMP assay detected as little as 0.00002% induced hPSC contamination in a single reaction.
• RT-LAMP successfully identified spiked-in hPSCs within hPSC-derived trilineage cells using multiple pluripotency RNA markers.
• Multiplex RT-LAMP assays enabled simultaneous detection and imaging of pluripotency markers.

Conclusions:

• RT-LAMP assays offer a sensitive and practical method for detecting tumorigenicity-associated contamination in regenerative medicine products.
• This approach supports the safe clinical application of regenerative medicine by enhancing quality control of cell-derived therapeutics.