Modeling M-Cell Differentiation in Intestinal Organoids

Proximal small intestine was incubated with 2 mM EDTA/PBS for 30 min on ice. Then, intestinal crypts were isolated by vigorous pipetting and embedded in Matrigel (Corning). Crypts were cultured in Advanced DMEM/F12 (Thermo Fisher Scientific) containing 10 ng/ml EGF (PeproTech), R-spondin1 conditioning medium (provided by C.J. Kuo; Ootani et al., 2009 (link)), 100 ng/ml Noggin (PeproTech), B27 supplement (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), and 1 mM N-acetylcysteine (Sigma-Aldrich). Recombinant glutathione S-transferase–mouse RANKL fusion protein (GST-RANKL; 500 ng/ml; Knoop et al., 2009 (link)) and LT α₁β₂ (1 µg/ml; R&D Systems) were used for the stimulation of organoids. Recombinant mouse IL-22 (100 ng/ml; PeproTech) was added into RANKL-stimulated organoids to evaluate the effect of IL-22 signaling on M cell differentiation.

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Jinnohara T., Kanaya T., Hase K., Sakakibara S., Kato T., Tachibana N., Sasaki T., Hashimoto Y., Sato T., Watarai H., Kunisawa J., Shibata N., Williams I.R., Kiyono H, & Ohno H. (2017). IL-22BP dictates characteristics of Peyer’s patch follicle-associated epithelium for antigen uptake. The Journal of Experimental Medicine, 214(6), 1607-1618.

Publication 2017

Matrigel Advanced DMEM/F12 EGF Noggin B27 supplement N2 supplement N-acetylcysteine LT α1β2 Recombinant mouse IL-22

Acetylcysteine Conditioning medium Crypts Edta Glutathione s transferase Il 22 Intestinal M cell Matrigel Mouse Noggin Organoids Rankl Recombinant fusion protein Small intestine Supplement

Corresponding Organization : Yokohama City University

Other organizations : Keio University, The University of Tokyo, Emory University, Japan Science and Technology Agency

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Variable analysis

independent variables

Incubation with 2 mM EDTA/PBS for 30 min on ice
Stimulation of organoids with recombinant glutathione S-transferase–mouse RANKL fusion protein (GST-RANKL; 500 ng/ml)
Stimulation of organoids with LT α1β2 (1 µg/ml)
Addition of recombinant mouse IL-22 (100 ng/ml) to RANKL-stimulated organoids

dependent variables

Isolation of intestinal crypts
Differentiation of M cells in organoids

control variables

Culturing of crypts in Advanced DMEM/F12 containing 10 ng/ml EGF, R-spondin1 conditioning medium, 100 ng/ml Noggin, B27 supplement, N2 supplement, and 1 mM N-acetylcysteine

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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