Maintenance of Mouse Nephron Progenitor Cells in Aggregates with Gamma-Secretase Inhibitor.

Shunsuke Yuri,Norimoto Yanagawa,Naomi Yanagawa,Masaki Nishikawa,Oak D Jo,Atsushi Asakura

doi:10.1371/journal.pone.0129242

Shunsuke Yuri, Norimoto Yanagawa + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0129242

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Journal: PloS one	Publication Date: Jun 15, 2015
Citations: 14	License type: CC BY 4.0

Affiliation: University of California, Los Angeles

Abstract

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.

Highlights

The development of mammalian kidney mainly involves three types of cells, i.e., metanephric mesenchyme (MM), ureteric bud (UB) and stromal mesenchyme (SM) [1]
When aggregates were reconstituted without UB cells by manually separating Venus+-UB from the surrounding Venus--mesenchyme in E11.5 Hoxb7-Venus mouse embryonic kidneys, no Six2+-nephron progenitor cells (NPC) could be detected after 7 days in culture (Fig 1A)
Since the combination of Fgf9 and Bmp7 was suggested to be the niche signal capable of maintaining NPC for up to 5 days [17], we tested the effect of adding Fgf9, Bmp7 and heparin to the aggregates without UB cells

Summary

Introduction

The development of mammalian kidney mainly involves three types of cells, i.e., metanephric mesenchyme (MM), ureteric bud (UB) and stromal mesenchyme (SM) [1]. UB is first induced by MM to extend from the nephric duct and undergoes elaborate branching morphogenesis to give rise to the collecting duct system [2]. MM is induced by UB to form the remaining nephron structures [3,4]. The SM cells play important roles in UB branching morphogenesis [5], MM differentiation [6,7] and the formation of vasculature in the kidney [8,9].

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