Search

Summary: Intestinal epithelial cells have a defined hierarchy with stem cells located at the bottom of the crypt and differentiated cells more at the top. Epithelial cell renewal and differentiation are strictly controlled by various regulatory signals provided by epithelial as well as surrounding cells. Although there is evidence that stromal cells contribute to the intestinal stem cell niche, their markers and the soluble signals they produce have been incompletely defined. Using a number of established stromal cell markers, we phenotypically and functionally examined fibroblast populations in the colon. CD90+ fibroblasts located in close proximity to stem cells in vivo support organoid growth in vitro and express crucial stem cell growth factors, such as Grem1, Wnt2b, and R-spondin3. Moreover, we found that CD90+ fibroblasts express a family of proteins—class 3 semaphorins (Sema3)—that are required for the supportive effect of CD90+ fibroblasts on organoid growth. : Stem cell proliferation is dependent on regulatory signals from surrounding cells, including stromal cells. Karpus et al. identify a membrane marker, CD90, that specifically marks stem cell niche fibroblasts in the colon. CD90+ fibroblasts produce class 3 semaphorins that promote stem cell proliferation via the Nrp2 receptor. Keywords: colon, intestine, stem cell, niche, fibroblast, stroma, Gli1, CD90, semaphorin, Nrp2

Upon intestinal epithelial damage a complex wound healing response is initiated to restore epithelial integrity and defend against pathogenic invasion. Epithelium-derived Indian Hedgehog (Ihh) functions as a critical sensor in this process. Signaling occurs in a paracrine manner because the receptor for Ihh is expressed only in the mesenchyme, but the exact Hedgehog target cell has remained elusive. The aim of this study was to elucidate further the nature of this target cell in the context of intestinal inflammation. Methods: Hedgehog activity was modulated genetically in both cell type–specific and body-wide models and the resulting animals were analyzed for gene expression profiles and sensitivity for dextran sodium sulfate (DSS) colitis. To characterize the Hedgehog target cell, Gli1-CreERT2-Rosa26-ZsGreen animals were generated, which express ZsGreen in all Hedgehog-responsive cells. These cells were characterized using flow cytometry and immunofluorescence. Results: Loss of Indian Hedgehog from the intestinal epithelium resulted in a rapid increase in expression of inflammation-related genes, accompanied by increased influx of immune cells. Animals with epithelium-specific deletion of Ihh or lacking the Hedgehog receptor Smoothened from Hedgehog target cells were more sensitive to DSS colitis. In contrast, specific deletion of Smoothened in the myeloid compartment did not alter the response to DSS. This suggests that Hedgehog signaling does not repress intestinal immunity through an effect on myeloid cells. Indeed, we found that Hedgehog-responsive cells expressed gp38, smooth muscle actin, and desmin, indicating a fibroblastic nature. Ihh signaling inhibited expression of C-X-C motif chemokine ligand 12 (CXCL12) in fibroblasts in vitro and in vivo, thereby impairing the recruitment of immune cells. Conclusions: We show that epithelium-derived Indian Hedgehog signals exclusively to fibroblasts in the intestine. Loss of Ihh leads to a rapid immune response with up-regulation of fibroblast-derived CXCL12, and migration of immune cells into the lamina propria.