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

PLoS One. 2015 Jun 15;10(6):e0129242. doi: 10.1371/journal.pone.0129242. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amyloid Precursor Protein Secretases / antagonists & inhibitors*
  • Animals
  • Cell Culture Techniques*
  • Cell Self Renewal / drug effects*
  • Enzyme Inhibitors / pharmacology*
  • Forkhead Transcription Factors / genetics
  • Forkhead Transcription Factors / metabolism
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Mice
  • Mice, Transgenic
  • Nephrons / cytology*
  • Stem Cells / cytology*
  • Stem Cells / drug effects*
  • Stem Cells / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • Enzyme Inhibitors
  • Forkhead Transcription Factors
  • Foxd1 protein, mouse
  • Homeodomain Proteins
  • Hoxb7 protein, mouse
  • Six2 protein, mouse
  • Transcription Factors
  • Amyloid Precursor Protein Secretases

Grant support

This study was supported by generous funds provided by the Chau-Li Foundation (to NY).