Potential use of human pluripotency-related gene expression reporter cell line for screening small molecules to enhance induction of pluripotency
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The reprogramming of somatic cells into induced pluripotentstem cells (iPSCs) is a crucial development in regenerativemedicine, providing patient-specific cells for therapeutic uses. Traditional methods often utilize viral vectors and transcriptionfactors that pose tumorigenic risks, rendering them unsuitablefor clinical applications. This study explored the use of chemicalsas a non-tumorigenic alternative for cell reprogramming. Utilizing CRISPR/Cas9 technology, we previously created iPSCsexpressing OCT4-EGFP and NANOG-tdTomato, and derivedOCT4-EGFP and NANOG-tdTomato fibroblastic cells (ON-FCs). These cells were reprogrammed using episomal vectors, andtheir pluripotency was validated by fluorescence and FACSanalyses. High-content screening was employed to assess smallmolecules that improve reprogramming efficiency, confirmingthe usefulness of ON-FCs as a dual reporter cell line for identifyingsmall molecules effective in generating human iPSCs. This study underscores the utility of a dual reporter system andhigh-content screening in identifying effective reprogrammingchemicals, establishing a scalable platform for high-throughputscreening. Discovering new chemicals that can reprogram iPSCswould provide a non-tumorigenic method to advance the fieldof regenerative medicine.
Abstract
The reprogramming of somatic cells into induced pluripotentstem cells (iPSCs) is a crucial development in regenerativemedicine, providing patient-specific cells for therapeutic uses. Traditional methods often utilize viral vectors and transcriptionfactors that pose tumorigenic risks, rendering them unsuitablefor clinical applications. This study explored the use of chemicalsas a non-tumorigenic alternative for cell reprogramming. Utilizing CRISPR/Cas9 technology, we previously created iPSCsexpressing OCT4-EGFP and NANOG-tdTomato, and derivedOCT4-EGFP and NANOG-tdTomato fibroblastic cells (ON-FCs). These cells were reprogrammed using episomal vectors, andtheir pluripotency was validated by fluorescence and FACSanalyses. High-content screening was employed to assess smallmolecules that improve reprogramming efficiency, confirmingthe usefulness of ON-FCs as a dual reporter cell line for identifyingsmall molecules effective in generating human iPSCs. This study underscores the utility of a dual reporter system andhigh-content screening in identifying effective reprogrammingchemicals, establishing a scalable platform for high-throughputscreening. Discovering new chemicals that can reprogram iPSCswould provide a non-tumorigenic method to advance the fieldof regenerative medicine.