Knowledge on how to maintain and expand nephron progenitor cells (NPC) in vitro is important to provide a potentially valuable source for kidney replacement therapies. In our present study, we examined the possibility of optimizing NPC maintenance in the "re-aggregate" system. We found that Six2-expressing (Six2(+))-NPC could be maintained in aggregates reconstituted with dispersed cells from E12.5 mouse embryonic kidneys for at least up to 21 days in culture. The maintenance of Six2(+)-NPC required the presence of ureteric bud cells. The number of Six2(+)-NPC increased by more than 20-fold at day 21, but plateaued after day 14. In an attempt to further sustain NPC proliferation by passage subculture, we found that the new (P1) aggregates reconstituted from the original (P0) aggregates failed to maintain NPC. However, based on the similarity between P1 aggregates and aggregates derived from E15.5 embryonic kidneys, we suspected that the differentiated NPC in P1 aggregates may interfere with NPC maintenance. In support of this notion, we found that preventing NPC differentiation by DAPT, a γ-secretase inhibitor that inhibits Notch signaling pathway, was effective to maintain and expand Six2(+)-NPC in P1 aggregates by up to 65-fold. The Six2(+)-NPC in P1 aggregates retained their potential to epithelialize upon exposure to Wnt signal. In conclusion, we demonstrated in our present study that the "re-aggregation" system can be useful for in vitro maintenance of NPC when combined with γ-secretase inhibitor.

A method to maintain and rebuild ureteric bud (UB)-like structures from UB cells in vitro could provide a useful tool for kidney regeneration. We aimed in our present study to establish a serum-free culture system that enables the expansion of UB progenitor cells, i.e., UB tip cells, and reconstruction of UB-like structures. We found that fibroblast growth factors or retinoic acid (RA) was sufficient for the survival of UB cells in serum-free condition, while the proliferation and maintenance of UB tip cells required glial cell-derived neurotrophic factor together with signaling from either WNT-β-catenin pathway or RA. The activation of WNT-β-catenin signaling in UB cells by endogenous WNT proteins required R-spondins. Together with Rho kinase inhibitor, our culture system facilitated the expansion of UB tip cells to form UB-like structures from dispersed single cells. The UB-like structures thus formed retained the original UB characteristics and integrated into the native embryonic kidneys.