Fig 1: Characterization of hiPSC-derived macrophage-integrated gut organoids. (A) Enhanced green fluorescent protein (EGFP)–human-induced pluripotent stem cells (hiPSCs), which constitutively expressed EGFP under a cytomegalovirus promoter, were cultured under feeder-free conditions in StemFlex medium (1) and differentiated into monocyte-like cells (pMCs) (2). Scale bars: black, 300 µm; white, 100 µm (2) (B) Immunofluorescence staining for ionized calcium-binding adapter molecule 1 (IBA1) in human intestine. Scale bar, 100 µm. (C) Immunofluorescence staining for IBA1 and CX3CR1 in MC-XF-HIOs derived from a non-EGFP hiPSC line (Edom-iPSCs). Scale bar: 50 µm. (D) Immunostaining for caudal type homeobox 2 (CDX2), villin, zonula occludens-1 (ZO-1), E-cadherin (ECAD), glycoprotein 2 (GP2), mucin 2 (MUC2), defensin alpha 6 (DEFA6), protein gene product 9.5 (PGP9.5), and smooth muscle actin (SMA). PGP9.5-positive enteric neuronal cells were surrounded by SMA-positive mesenteric tissue in MC-XF-HIOs. Cell nuclei were counterstained with 4', 6-diamidino-2-phenylindole, dihydrochloride (DAPI). Scale bars, 100 µm. Anti-CDX2 (1:1000, ab76541; Abcam), anti-villin (1:50, sc-7672; Santa Cruz Biotechnology, Dallas, TX), anti-GP2 (1:1000, HPA016668; Sigma-Aldrich), anti-MUC2 (1:50, sc-7314; Santa Cruz Biotechnology), anti-PGP9.5 (1:10, ab8189; Abcam), anti-SMA (1:400, A2547; Sigma-Aldrich), anti-ECAD (1:50, 610181; BD Pharmingen, San Diego, CA), anti–ZO-1 (1:100, 40-2200; Invitrogen), and anti-DEFA6 (1:500, HPA019462; Sigma-Aldrich) were used as primary antibodies.