Fig. 2

Cell seeding and whole-organ culture of decellularized rat intestine. a Fluorescence micrographs of GFP-positive human iPSC-derived mid-hindgut spheroids budding out from 2-dimensional culture of definitive endoderm on a tissue culture plate (left), a spheroid consisting of CDX2-positive cells (middle), a spheroid stained with E-cadherin and vimentin demonstrating cells of both epithelial and mesodermal fate (right). Scale bars, 1 mm (left) and 100 µm (middle/right). b Schematic of seeding mid-hindgut spheroids into the intestinal lumen of a rat intestinal scaffold. c Whole-mount fluorescence micrographs showing GFP-positive mid-hindgut spheroids proliferating to cover regions of the intestinal wall over the culture period. Scale bars, 2 mm. d Quantification of scaffold luminal coverage using visualization of GFP-positive signal at 1 day and 14 days of spheroid culture (n = 3). Error bars, mean ± s.d. of experimental values. **P < 0.01, Student’s t test. e Hematoxylin and eosin staining (left) and fluorescence micrographs of the intestinal epithelium engrafted on rat intestinal scaffold at day 28 of culture in vitro. E-cadherin-positive epithelial cells form a monolayer on the luminal surface of the intestine (middle) and express CDX2 (right), indicating intestinal lineage. Scale bars, 100 µm. f Transmission electron micrograph of regenerated intestinal epithelium on an intestinal scaffold. Presence of microvilli and cellular junctions (white arrowheads) are identified. g Characterization of iPSC-derived epithelium shows that villin is predominantly expressed on the apical surface of the epithelium, indicating enterocytes (left), a subset of cells express chromogranin A, indicating enteroendocrine cells (middle left), a supportive mesenchyme containing smooth muscle cells stains positive staining for vimentin and SMA (middle right) and a proliferative cell population is indicated by positive Ki67 staining (right). Scale bars, 100 µm; 10 µm (insets)