Tissue underlying the intestinal epithelium elicits proliferation of intestinal stem cells following cytotoxic damage

Abstract

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.

Epithelial WNT Ligands Are Essential Drivers of Intestinal Stem Cell Activation

“…Chemotherapy treatments, such as doxorubicin, elicit injury patterns similar to radiation treatment, with loss of LGR5 + populations within the crypt. Activation and increased proliferation of surface glycoprotein CD24-labeled ISCs following injury can reconstitute the damaged epithelium (Dekaney et al, 2009; Seiler et al, 2015). Genetic mouse models resulting in the direct ablation of LGR5-labeled CBCs have also shown the importance of the reserve ISCs as well as the newly characterized secretory and absorptive progenitors after LGR5 + cell loss (Tetteh et al, 2016; van Es et al, 2012; Buczacki et al, 2013).…”

Regulation and plasticity of intestinal stem cells during homeostasis and regeneration

“…In the Drosophila midgut, BMP is induced upon injury and acts directly on ISCs to limit their expansion (Guo et al, 2013). In the murine intestine, the BMP inhibitor chordin-like 2 is upregulated together with EGF ligand amphiregulin in sub-epithelial tissues after treatment with the chemotherapeutic doxorubicin (Seiler et al, 2015). This upregulation occurs in concert with an increase in ISC proliferation.…”

Functional transcriptomics in diverse intestinal epithelial cell types reveals robust gut microbial sensitivity of microRNAs in intestinal stem cells

“…We achieved a robust ~700-fold knockdown of miR-375-3p at day 8 using a LNA inhibitor complementary to miR-375-3p (LNA-375; Figure 4c). At both day 4 and 8, LNA-375 treated enteroids exhibited dramatically increased budding (Figure 4d & 4e, Supplemental Figure 6ac), a marker of IESC proliferative capacity (35,36), relative to Mock and LNA-Scramble treated enteroids. Consistent with this finding, whole mount staining of the enteroids also showed increased Ki67 upon knockdown of miR-375-3p (Figure 4f), though no difference in enteroid size (Supplemental Figure 6d) nor passage efficiency was observed (data not shown).…”