Figure 2
Regeneration of nephrons derived from transplanted rat NPCs in an adult NOG mouse using the nephron progenitor cell replacement system. (a) Schematic of the experimental protocol. (b) We observed Six2-GFP fluorescence in E13.5 MNs of the Six2-iDTR mice (scale bars, 500 µm). (c) DT and rat GFP-RPCs were injected under the renal capsule. The RPCs showed strong green fluorescent protein (GFP) expression (CAG promoter); therefore, we were unable to observe Six2-GFP (native promoter) under a fluorescence stereoscopic microscope alongside GFP-RPCs after transplantation (scale bars, 500 µm). (d) MNB was isolated from the Six2-iDTR mouse embryo (scale bar, 500 µm). (e) MNB-containing rat RPCs and DT were transplanted into the vicinity of the aorta of an adult NOG mouse. The arrow indicates MNB. (f) Fourteen days after transplantation, GFP expression was evaluated and identified (scale bars, 1 mm). (g) Frozen sections were created from the collected MNB. We observed GFP-positive glomerular (arrow head) and tubular structures (arrow) (left column; scale bars, 100 µm). HE staining demonstrated glomeruli (arrow head) and tubules (arrow) regenerated from the transplanted rat NPCs (right column; scale bar, 100 µm). (h) Frozen sections were analysed by immunostaining. We identified neo-glomeruli (lower column) and tubules (upper column) that originated from transplanted rat GFP-NPCs. Glomeruli had nephrin-positive epithelial cells lining CD31-positive vascular endothelial cells (right lower). All the cells originating in the NPCs were GFP-positive. Nephrin is a podocyte marker, megalin is a proximal tubular marker, E-cadherin is a distal tubular marker, and CD31 is an endothelial marker (scale bars, left upper: 100 µm, left lower; 100 µm, right upper: 25 µm, right lower: 10 µm). Representative images of five specimens from three independent transplantation experiments are indicated.
