WS6 enables scalable ex vivo expansion and gene editing of epithelial basal stem cells

Orr, J. C.; Haughey, E. K.; Farr, A. S.; Pearce, D. R.; McCarthy, N. A.; Reddy, S. K.; Rouhani, M. J.; Percival, C.; Rose, I.; Straatman-Iwanowska, A.; Dale, R.; Guthrie, M.; Benedetti, G.; Pape, O. R.; Ocampo-Godinez, J. M.; Maughan, E. F.; Butler, C. R.; Moulding, D. A.; Kreins, A. Y.; Giobbe, G. G.; De Coppi, P.; Grey, W.; Dost, A. F. M.; Hirst, R. A.; Baines, D. L.; Ishii, Y.; O'Callaghan, C.; Janes, S. M.; Hynds, R. E.

doi:10.1101/2025.09.15.675861

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bioRxiv · 10.1101/2025.09.15.675861

WS6 enables scalable ex vivo expansion and gene editing of epithelial basal stem cells

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Abstract

Modeling human epithelial diseases and developing cell-based therapies require robust methods to expand and manipulate epithelial stem and progenitor cells in vitro. Basal stem/progenitor cells from stratified epithelia can be expanded in 3T3-J2 fibroblast feeder cell co-culture systems, and the addition of the ROCK inhibitor Y-27632 enhances proliferation and culture longevity, a phenomenon described as conditional reprogramming. Here, we present a method incorporating the small molecule WS6 to further improve the proliferation and lifespan of cultured epithelial cells from multiple tissues, including airway, skin, and thymus. Cells maintained in this medium ( EpMED; FAD+Y+WS6) retain basal stem/progenitor cell identity and function, including the capacity to differentiate. We demonstrate their capacity to engraft in vivo in a tracheal transplantation model. In a second application, we generate clonal CRISPR-Cas9 genome edited nasal cultures, introducing targeted knockouts of DNAH5 or DNAI2 to create primary ciliary dyskinesia disease models. We anticipate that our method will have broad applications in epithelial cell biology, disease modeling, and regenerative medicine, while reducing reliance on immortalized or cancer cell lines and animal experimentation.

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Orr, J. C., Haughey, E. K., Farr, A. S., Pearce, D. R., McCarthy, N. A., Reddy, S. K., Rouhani, M. J., Percival, C., Rose, I., Straatman-Iwanowska, A., Dale, R., Guthrie, M., Benedetti, G., Pape, O. R., Ocampo-Godinez, J. M., Maughan, E. F., Butler, C. R., Moulding, D. A., Kreins, A. Y., Giobbe, G. G., De Coppi, P., Grey, W., Dost, A. F. M., Hirst, R. A., Baines, D. L., Ishii, Y., O'Callaghan, C., Janes, S. M., Hynds, R. E.. 2025-09-15. WS6 enables scalable ex vivo expansion and gene editing of epithelial basal stem cells. https://doi.org/10.1101/2025.09.15.675861

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