Fig. 3. BMP gradient in colon assembloids and its effect on colonic crypt formation.

a Dot plot showing expression of BMP signaling molecules in the identified stromal cell clusters from assembloids. b UMAP expression plots of BMP ligand genes (Bmp2, Bmp5, and Bmp7) and BMP antagonist genes (Grem1, Grem2, and Mgp) for stromal cells in assembloids. c Single-molecule in situ hybridization (sm-ISH) using serial sections showing localization of Bmp2 and Grem1 in assembloids and colon tissue; quantification for Bmp2 and Grem1 signal area divided into top and bottom in the crypt (n = 3 biological replicates per group). d Duplex sm-ISH showing co-localization of Pdgfra and Bmp2 in assembloids. e Hematoxylin and eosin (H&E) staining of an assembloid derived from a wild-type mouse (WT) and an assembloid comprised of epithelial cells from an Axin2CreErt2/Bmpr1a^fl/fl mouse and WT mesenchymal cells from a WT mouse (Bmpr1a^ΔEPI). f, g) Immunofluorescence images and quantification of WT and Bmpr1a^ΔEPI assembloids stained for KI67, KRT20, and MUC2 (n = 3 biological replicates per group). h sm-ISH images and quantification of Bmp2 expression in WT and Bmpr1a^ΔEPI assembloids (n = 3 biological replicates per group). i qPCR for expression of the stem cell marker Lgr5, the colonocyte marker Krt20, the BMP ligand Bmp2, and the BMP target Id1 from organoids cultured with different concentrations of BMP2 (n = 3 biological replicates per group). j qPCR for expression of Id1 and Bmp2 from organoids cultured in full medium (FM) or in medium without sWnt, R-spondin 1, CHIR99021, or noggin (-WCRN) (n = 3 biological replicates per group). Scale bars: 100 µm. Data are presented as mean ± SEM. Statistical analyses were performed using Student’s t-test (two-tailed) for (c, g, h), and (j). Fib Fibroblast, MF Myofibroblast. scRNA-seq data are from two biological replicates per group. Images are representative of at least three biological replicates. Source data are provided as a Source Data file.