ABSTRACT
Progenitors and mature cells can maintain the intestinal epithelium by dedifferentiation and facultative intestinal stem cell (fISC) function when active ISCs (aISCs) are lost to damage. Here, we modeled fISC activation in mouse intestinal organoids with doxorubicin (DXR) treatment, a chemotherapeutic known to ablate Lgr5+ aISCs in vivo. Similar fISC gene activation was observed between organoids treated with low versus high DXR, despite significantly decreased survival at the higher dose. aISCs exhibited dose-dependent loss after DXR treatment but survived at doses compatible with organoid survival. We ablated residual aISCs after DXR treatment using a Lgr52A-DTR allele and observed that aISC survival of the initial genotoxic insult is required for organoid survival following DXR treatment. These results suggest that although typical fISC genes are activated by DXR-induced injury in organoids, functional stemness remains dependent on the aISC pool. Finally, we show that human intestinal organoids require higher doses of DXR to induce loss of survival and downregulation of LGR5. Our data establish a reproducible model of DXR-induced injury in intestinal organoids and reveal differences in in vitro responses to an established in vivo damage modality.
Footnotes
Author contributions
Conceptualization: A.G., A.D.G.; Methodology: J.L., A.G.; Formal analysis: J.L., A.G., N.V.J., L.L.A., S.S., F.T., T.H., S.F.W., A.D.G.; Investigation: J.L., A.G., N.V.J., L.L.A., S.S., F.T., T.H., S.F.W., A.D.G.; Resources: N.B., A.D.G.; Data curation: J.L., L.L.A., S.S., F.T., T.H., S.F.W., A.D.G.; Writing - original draft: J.L., A.G., A.D.G.; Writing - review & editing: J.L., A.G., N.V.J., L.L.A., S.S., F.T., T.H., S.F.W., N.B., A.D.G.; Visualization: J.L., A.G., N.V.J., L.L.A., F.T., T.H., S.F.W., A.D.G.; Supervision: A.D.G.; Project administration: A.D.G.; Funding acquisition: A.D.G.
Funding
This work was funded by the National Institutes of Health (R35GM142503 and R35GM142503-01S1 to A.D.G.; F31DK136254 to N.V.J.). L.L.A. was supported by the Undergraduate Research Training Initiative for Student Enhancement (U-RISE) (T34) funded by the National Institute of General Medical Sciences (GM145428; University of New Mexico). Deposited in PMC for release after 12 months.
Data availability
All relevant data can be found within the article and its supplementary information.
