Potential use of human pluripotency-related gene expression reporter cell line for screening small molecules to enhance induction of pluripotency.

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is a crucial development in regenerative medicine, providing patient-specific cells for therapeutic uses. Traditional methods often utilize viral vectors and transcription factors that pose tumorigenic risks, rendering them unsuitable for clinical applications. This study explored the use of chemicals as a non-tumorigenic alternative for cell reprogramming. Utilizing CRISPR/Cas9 technology, we previously created iPSCs expressing OCT4-EGFP and NANOG-tdTomato, and derived OCT4-EGFP and NANOG-tdTomato fibroblastic cells (ON-FCs). These cells were reprogrammed using episomal vectors, and their pluripotency was validated by fluorescence and FACS analyses. High-content screening was employed to assess small molecules that improve reprogramming efficiency, confirming the usefulness of ON-FCs as a dual reporter cell line for identifying small molecules effective in generating human iPSCs. This study underscores the utility of a dual reporter system and high-content screening in identifying effective reprogramming chemicals, establishing a scalable platform for high-throughput screening. Discovering new chemicals that can reprogram iPSCs would provide a non-tumorigenic method to advance the field of regenerative medicine. [BMB Reports 2025; 58(4): 183-189].