Fig. 2

Serial culture of epithelial cells derived from small intestinal organoids A Phase-contrast micrograph of intestinal epithelial cells at passage 2 on mouse embryonic fibroblasts (MEF) (left) and immunohistochemistry of intestinal epithelial cells with an antibody to CDX2 (right). Immunohistochemistry was performed on cells in iPGell. Scale bars: 200 (left) and 50 (right) µm. B Immunocytochemistry of intestinal epithelial cells at passage 2 on MEF with an antibody to CDX2. Nuclei were stained with DAPI. Scale bar: 100 µm. C Phase-contrast micrograph of and immunohistochemistry of mesenchymal stromal cells. Mesenchymal stromal cells exhibit spindle-shaped morphology. Mesenchymal stromal cells were positive for Vimentin and α-SMA, but negative for Desmin. Nuclei were stained with DAPI. Scale bars: 100 (white) and 50 (yellow) µm. D Expression heat map of gene expression in intestinal epithelial cells and mesenchymal stromal cells. E Immunocytochemistry of mesenchymal stromal cells. Mesenchymal stromal cells were positive for trophocyte markers PDGFRA and CD81. Nuclei were stained with DAPI. Scale bar: 50 µm. F Phase-contrast micrograph mesenchymal stromal cells. Days after a passage are shown. Scale bars: 200 µm. G Trophic factor gene expression of intestinal epithelial cells and mesenchymal stromal cells. H Boxplot of representative Rspo, WNT, and BMP inhibitor families. Expression levels were calculated from the results of independent (biological) triplicate experiments (intestinal epithelial cells and mesenchymal stromal cells) and single experiments (normal adult intestine tissue). Boxplots are expressed as mean ± SD. I Phase-contrast micrograph and immunohistochemistry of intestinal epithelial cells on organoid-derived mesenchymal stromal cells with an antibody to CDX2. Immunohistochemistry was performed on cells in iPGell. Scale bars: 200 (left) and 50 (right) µm. J Immunocytochemistry of intestinal epithelial cells on organoid-derived mesenchymal stromal cells with an antibody to CDX2. Nuclei were stained with DAPI. Scale bars: 100 µm. K Schematic of successful maintenance and culture of intestinal epithelial cells on organoid-derived mesenchymal stromal cells. Epithelial cells and mesenchymal stromal cells were derived from the same organoids