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Abstract: FR-OR039

Manipulation of Nephron Patterning Signals Enables Selective Induction of Podocytes from Human Pluripotent Stem Cells

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 501 Development, Stem Cells, and Regenerative Medicine: Basic

Authors

  • Yoshimura, Yasuhiro, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan
  • Taguchi, Atsuhiro, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan
  • Mukoyama, Masashi, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
  • Nishinakamura, Ryuichi, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan
Background

Podocytes, which play an important filtration role in the kidney, are derived from nephron progenitor cells (NPCs). Recently, we and others have established the induction methods of kidney organoids via NPCs from human induced pluripotent stem cells (hiPSCs). However, it remains a challenge to control the patterning process of nephron development and selectively induce a specific nephron segment.

Methods

We first developed a method to purify NPCs from mouse embryonic kidneys and cultured the NPCs ex vivo to investigate the required signals for podocyte induction. We then applied the findings obtained from mouse NPCs to hiPSC-derived NPCs to establish a method to selectively induce human podocytes.

Results

Mouse NPCs differentiation experiments identified that 1) optimal duration and strength of Wnt signaling was essential for mesenchymal-to-epithelial transition and podocyte differentiation, 2) inhibition of TGF-β signaling supported the expansion of proximal segment of renal vesicle, and 3) inhibition of TGF-β signaling in the later step enriched podocyte fraction by suppressing the differentiation of other nephron segments. The resultant protocol was successfully applied to the induction of Nephrin/Podocalyxin-positive podocytes from hiPSCs with more than 90% of efficiency. Importantly, the expression levels of Nephrin and Podocin genes in the induced podocytes were 105 to 106 times higher than that in the immortalized podocyte cell line and comparable to the sorted human adult podocytes. RNA-seq analysis further confirmed characteristic similarities between induced podocytes and human adult podocytes. Electron microscopy revealed foot process-like protrusions with Nephrin protein expression on the cell surface. Furthermore, exposure of induced podocytes to puromycin aminonucleoside reduced the expression of foot process-associated proteins, suggesting their functional responsiveness.

Conclusion

We elucidated the signals required for podocyte differentiation from NPCs and established a novel method to selectively induce human podocytes from hiPSCs. Induced podocytes exhibited gene expression profiles resembling their counterparts in vivo as well as morphological and functional properties of podocytes and will serve as new resources for disease modeling, nephrotoxicity testing, and regenerative medicine.

Funding

  • Government Support - Non-U.S.