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

Stem Cell Rev Rep. 2022 Dec;18(8):2995-3007. doi: 10.1007/s12015-022-10402-3. Epub 2022 Jun 3.

Abstract

For safe regenerative medicines, contaminated or remaining tumorigenic undifferentiated cells in cell-derived products must be rigorously assessed through sensitive assays. Although in vitro nucleic acid tests offer particularly sensitive tumorigenicity-associated assays, the human pluripotent stem cell (hPSC) detectability is partly constrained by the small input amount of RNA per test. To overcome this limitation, we developed reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays that are highly gene specific and robust against interfering materials. LAMP could readily assay microgram order of input sample per test and detected an equivalent model of 0.00002% hiPSC contamination in a simple one-pot reaction. For the evaluation of cell-derived total RNA, RT-LAMP detected spiked-in hPSCs among hPSC-derived trilineage cells utilizing multiple pluripotency RNAs. We also developed multiplex RT-LAMP assays and further applied for in situ cell imaging, achieving specific co-staining of pluripotency proteins and RNAs. Our attempts uncovered the utility of RT-LAMP approaches for tumorigenicity-associated assays, supporting practical applications of regenerative medicine.

Keywords: Loop-mediated isothermal amplification (LAMP); Pluripotent stem cell; Regenerative medicine; Regulatory science.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Humans
  • Nucleic Acid Amplification Techniques* / methods
  • Pluripotent Stem Cells*
  • RNA
  • Sensitivity and Specificity

Substances

  • RNA

Supplementary concepts

  • LAMP assay