The goals of this study were to document the proliferative response of intestinal stem cells (ISCs) during regeneration after damage from doxorubicin (DXR) and to characterize the signals responsible for ISC activation. To this end, jejuni from DXR-treated mice were harvested for histology, assessment of ISC numbers and proliferation by flow cytometry, crypt culture, and RNA analyses. Histology showed that crypt depth and width were increased 4 days after DXR. At this time point, flow cytometry on tissue collected 1 hour after EdU administration revealed increased numbers of CD24loUEA− ISCs and increased percentage of ISCs cycling. In culture, crypts harvested from DXR-treated mice were equally proliferative as those of control mice. Addition of subepithelial intestinal tissue (SET) collected 4 days after DXR elicited increased budding (1.4 ± 0.3 vs. 5.1 ± 1.0 buds per enteroid). Microarray analysis of SET collected 4 days after DXR revealed 1,030 differentially expressed transcripts. Cross comparison of Gene Ontology terms considered relevant to ISC activation pointed to 10 candidate genes. Of these the epidermal growth factor (EGF) family member amphiregulin and the BMP antagonist chordin-like 2 were chosen for further study. In crypt culture, amphiregulin alone did not elicit significant budding, but amphiregulin in combination with BMP antagonism showed marked synergism (yielding 6.3 ± 0.5 buds per enteroid). These data suggest a critical role for underlying tissue in regulating ISC behavior after damage, and point to synergism between amphiregulin and chordin-like 2 as factors which may account for activation of ISCs in the regenerative phase.
Objective: Characterize signals required for epithelial regeneration after damage from Doxorubicin (Doxo). We hypothesize these signals arise from underlying tissue. Methods: Jejuni from Doxo‐treated mice were harvested for histology, assessment of ISC proliferation by flow cytometry, crypt culture, and microarray. Results: Crypt depth increases 4 days after Doxo; flow data confirm increased ISC proliferation at this time: 14±5 % of CD24lo cells are EdU+ in controls vs. 37±4%, 4d after Doxo (p=0.02). Interestingly, Lgr5‐EGFPhi cells are 7‐fold decreased at this time. In culture, crypts harvested from Doxo‐treated mice are equally proliferative as those of control mice. Addition of de‐epithelialized intestinal tissue (DEIT) from mice 4d after Doxo yields an increased average bud number per enteroid [1.45±0.31 buds vs. 5.10±0.96 buds (p <0.05)]; it also induces proliferation of crypts from treated animals [1.16±0.30 buds vs. 4.83±0.29 buds (p<0.0001)]. Microarray analysis of DEIT shows differentially expressed genes and pathways, potentially driving this observed effect. Conclusions: These data suggest a critical role of underlying tissue in regulating ISC behavior after damage, and warrant further investigation of mechanisms.
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