Fig. 3

Extensive proliferation of organoid-derived intestinal epithelial cells on a stromal cell feeder layer A Schematic of organoid-derived intestinal epithelial cell culture system. Intestinal epithelial and mesenchymal stromal cells can be cultured, passaged and frozen, allowing for large stocks of cells. Intestinal epithelial and mesenchymal stromal cells are designated as RYU and LONG, respectively, as code names. B Phase-contrast micrographs of RYU growing on LONG. RYU formed colonies and proliferated. Scale bars: 100 µm. C Immunocytochemistry of RYU P6 on a dish with the proliferating cell marker Ki67 and the epithelial cell-specific cadherin (ECAD (E-Cadherin/Cadherin-1/CD324)). Nuclei were stained with DAPI. Scale bar: 50 µm. D Growth curve of RYU. Each point indicates the timing of passages. RYU were passaged into 4 dishes at each passage. E Histology and immunohistochemistry of RYU at passage 4 in iPGell. Immunohistochemistry was performed with antibodies to CDX2 and Villin. Scale bars: 50 µm. F Histology and immunohistochemistry of RYU at passage 6 in iPGell. Immunohistochemistry was performed with antibodies to CDX2 and Villin. Scale bars: 50 µm. G Histology and immunohistochemistry of RYU at passage 9 in iPGell. Immunohistochemistry was performed with antibodies to CDX2 and Villin. Scale bars: 50 µm. H Histology and immunohistochemistry of RYU at passage 12 in iPGell. Immunohistochemistry was performed with antibodies to CDX2 and Villin. Scale bars: 50 µm. I Karyotypic analysis revealed that RYU at passage 13 have normal karyotypes. J Immunohistochemistry of RYU and LONG at the subrenal capsule of the kidney (n = 3, respectively), using human-specific Lamin A/C antibody. Scale bars of zoomed sections represent 100 µm