Epithelial Regeneration After Doxorubicin Arises Primarily From Early Progeny of Active Intestinal Stem Cells

BACKGROUND & AIMS: aISCs (aISCs) are sensitive to acute insults including chemotherapy and irradiation. Regeneration after aISC depletion has primarily been explored in irradiation (IR). However, the cellular origin of epithelial regeneration after doxorubicin (DXR), a common chemotherapeutic, is poorly understood. METHODS: We monitored DXR’s effect on aISCs by enumerating Lgr5-eGFP(+) and Olfm4(+) crypts, cleaved caspase-3 (CASP3(+)) immunofluorescence, and time-lapse organoid imaging. Lineage tracing from previously identified regenerative cell populations (Bmi1(+), Hopx(+), Dll1(+), and Defa6(+)) was performed with DXR damage. Lineage tracing from aISCs was compared with lineage tracing from early progeny cells (transit-amplifying cells arising from aISCs 1 day predamage) in the context of DXR and IR. We compared stem cell and DNA damage response (DDR) transcripts in isolated aISCs and early progeny cells 6 and 24 hours after DXR. RESULTS: Epithelial regeneration after DXR primarily arose from early progeny cells generated by aISCs. Early progeny cells upregulated stem cell gene expression and lacked apoptosis induction (6 hours DXR: 2.5% of CASP3(+) cells, p<0.0001). aISCs downregulated stem cell gene expression and underwent rapid apoptosis (6 hours DXR: 63.4% of CASP3(+) cells). There was minimal regenerative contribution from Bmi1(+), Hopx(+), Dll1(+), and Defa6(+)-expressing populations. In homeostasis, 48.4% of early progeny cells were BrdU(+), and expressed low levels of DDR transcripts. CONCLUSIONS: We show that DXR effectively depleted aISCs in the small intestine and subsequent epithelial regeneration depended on nonquiescent early progeny cells of aISCs. The chemoresistant phenotype of the early progeny cells may rely on a dampened DDR in contrast to aISCs’ robust DDR, which facilitates expeditious apoptosis.