Abstract: FR-OR100

Multi-Segmented Kidney Organoids Derived from Human ES Cells by Stepwise Transcription Factor Administration

Session Information

Category: Developmental Biology and Inherited Kidney Diseases

  • 402 Stem Cells

Authors

  • Hiratsuka, Ken, Keio University School of Medicine, Tokyo, Japan
  • Monkawa, Toshiaki, Keio University School of Medicine, Tokyo, Japan
  • Yamaguchi, Shintaro, Keio University School of Medicine, Tokyo, Japan
  • Morizane, Ryuji, Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Ko, Shigeru B.h., Keio University School of Medicine, Tokyo, Japan
  • Itoh, Hiroshi, Keio University School of Medicine, Tokyo, Japan
  • Ko, Minoru S.h., Keio University School of Medicine, Tokyo, Japan
Background

We have recently reported a method to differentiate renal tubule-like cells from human Embryonic Stem Cells(hESCs) by a combinatorial administration of defined transcription factor(TF) mRNAs. However, a critical problem of this protocol, referred as direct reprogramming, is that it generates only proximal and distal tubular cells, but not other cell types. Here, we identified TFs that can induce the formation of SIX2+ metanephric mesenchyme/renal vesicles, which can be further differentiated into all other renal linages.

Methods

We utilized the human ES lines with doxycycline-inducible transcription factors (~700 genes), analyzed correlation of gene expression response to the induction of human TFs with tissue-specific gene expression in silico, and identified candidate TFs for differentiating towards renal lineages. Modified mRNAs for TFs were synthesized by in vitro transcription. hESCs were transfected with several combinations of synthetic mRNAs for four days by lipofection and cultured for up to 14 days to form kidney organoids, mimicking renal developmental stage. hESCs-derived kidney organoids were used for RNA sequencing analysis(RNA-Seq) and were tested functional assays to model kidney injury.

Results

Based on in-silico analysis, we identified a set of four TFs that induce SIX2+ metanephric mesenchyme and another set of four TFs that help differentiate toward renal vesicles. Two days after the transfection of the first set of four TFs together into hESCs, metanephric mesenchyme cell populations (SIX2+, SALL1+) were induced efficiently (~95%). Subsequent administration of the second set of four TFs transfection induced differentiation into renal vesicles(PAX8+LHX1+). On day 14, epithelial characteristic changes and multi-segmented kidney structures containing podocyte(PNA+PODXL+), proximal tubules(AQP1+KSP+), and distal tubules(CDH1+BRN1+) were observed in both 2D and 3D cultures. RNA-Seq analysis also showed the global expression profile closely resembled kidney profile in renal developmental stages. Moreover, treatment with gentamicin, nephrotoxic agent, induced KIM-1 expression.

Conclusion

We have identified specific TFs for the differentiation of hESCs toward renal lineage via SIX2+ metanephric mesenchyme, and generated multi-segmented functional kidney organoids.