Figure 6

All-mESC organoids form nephrons connected to a ureteric bud/ collecting duct tree. (a) Bright field images of all-mESC-derived organoids, the control being an aggregate of iNP and iSP cells with no eUB: the iNP + iSP + eUB organoids grew (n = 15/18) while the controls did not (n = 0/3). Scale bar, 200 µm. (b) Early nephrogenesis around eUB branch tips at day 3 [an S shaped body is marked by a dotted line in the inset image] (n = 4/4), which grew and formed (c) a single connected ureteric bud-nephron tree structure by day 7 (n = 4/4). Scale bar: (b) 100 µm, (c) 200 µm. (d) Full-length nephron formation with podocytes, proximal tubules and distal tubules (n = 4/4); and (e) no uroplakin-positive ureter formation (n = 0/6) at day 10. Scale bar, 100 µm. (f) Uroplakin-positive ureter-like structure formation in the organoids induced by BMP4-soaked beads (5 µg /ml). Collecting duct-nephron tree is stained for ECAD. (1) Merged and (2) uroplakin channel images are shown. n = 2/6. Scale bar, 100 µm. (g) The lower concentration of BMP4 (1 µg/ml) did not induce uroplakin-positive structures (n = 4/4). Abbreviations: POD: podocalyxin (podocyte marker), JAG: jagged1 (proximal tubule marker), NKCC2: Na–K–Cl cotransporter 2 (distal tubular marker), ECAD: E cadherin (ureteric bud/distal tubular marker), CD31: platelet endothelial cell adhesion molecule (endothelial cells marker), URO: uroplakin (ureter marker), CK8: cytokeratin 8 (ureteric bud marker), WT1: Wilms Tumour 1 (podocyte/cap mesenchyme marker).