Peyer’s Patch M cells derive from Lgr5+ stem cells require SpiB and are induced by RankL in cultured ‘organoids’. Mus musculus

Peyer’s Patches consist of domains of specialized intestinal epithelium overlying Gut-Associated Lymphoid Tissue (GALT). Luminal antigens reach the GALT by translocation through epithelial gatekeeper cells, so-called M cells. We have recently demonstrated that all epithelial cells required for the digestive functions of the intestine are generated from Lgr5-expressing stem cells. Here, we show that M cells also derive from these crypt-based Lgr5 stem cells. The Ets family transcription factor Spi-B, known to control effector functions of bone marrow-derived immune cells, is specifically expressed in M cells. In Spi-B-/- mice, M cells are entirely absent, which occurs in a cell-autonomous fashion. It has been shown that Tnfsf11 (RankL) can induce M cell development in vivo. In intestinal organoid (‘mini-gut’) culture, we show that stimulation with RankL induces SpiB expression within 24hrs and subsequently of other M cell markers. We conclude that RankL-induced expression of Spi-B is essential for Lgr5 stem cell-derived epithelial precursors to develop into M cells. Overall design: Small intestinal organoids were derived from wildtype (WT) mice. Recombinant mouse RankL (BioLegend) was added to the organoid culture medium in concentrations of 50-200ng/ml and fresh medium was added at day 2 and day 5. At the indicated time points, organoids were harvested for RNA isolation and microarray analysis to look for gene expression changes in reponse to RanKL.