Kidney Week

Abstract: FR-PO346

A Novel Organoid Model for Autosomal Dominant Polycystic Kidney Disease Based on Mouse Nephron Progenitor Cells (NPCs)

Session Information

• Genetic Diseases: Models, Mechanisms, Treatments
  November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1101 Genetic Diseases of the Kidneys: Cystic

Authors

• Ishimoto, Yu, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States

Yu Ishimoto,

• Araoka, Toshikazu, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Kyoto, Japan

Toshikazu Araoka,

• Menezes, Luis F., National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States

Luis F. Menezes,

• Shimizu, Tatsuya, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Kyoto, Japan

Tatsuya Shimizu,

• Mae, Shin-ichi, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Kyoto, Japan

Shin-ichi Mae,

• Osafune, Kenji, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Kyoto, Japan

Kenji Osafune,

• Germino, Gregory G., National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States

Gregory G. Germino,

Background

An in vitro cell culture system that accurately recapitulates ADPKD cystogenesis is needed, and a human iPS cell-derived nephron organoid system is one of the options. However, the process takes >20 days, differentiation efficiency varies between cell lines, and long-term methods of induction of differentiation result in higher inter-experiment variability. Moreover, the lack of a mouse-based system means that the results of in vitro studies cannot be easily assessed in a whole organism. To overcome these problems, we have established a mouse nephron organoid system from primary NPCs.

Methods

We sorted NPCs from mouse E13.5 embryonic kidneys using antibodies that detect kidney lineage differentiation markers. Sorted NPCs were maintained in 3D expansion culture and evaluated for their nephrogenic potential. We modified conditions to assess organoid differentiation using both air-fluid and suspension culture methods. Finally, we used established differentiation protocols to evaluate cystogenesis in nephron organoids derived from NPCs of ADPKD mouse models.

Results

Robo2^high/Pdgfrb^--/Podocalyxin^-- cells (NPCs) sorted from E13.5 embryonic WT, Pkd1^KO/KO,, Pkd2^KO/KO, and Pkd1^cko/cko;tamoxifen Cre kidneys could be cultured for >40 passages and maintain nephrogenic potential, and they could be differentiated into nephron organoids using either air-fluid or suspension culture methods in <10 days. NPCs established from ADPKD models spontaneously formed cyst-like structures without inducers like forskolin in suspension culture but not in air-fluid culture.

Conclusion

Mouse primary NPCs could be maintained long term with nephrogenic potential. ADPKD mutants spontaneously make cyst-like structures when grown in suspension. This is the first mouse NPC-based organoid system that appears to make cyst-like structures spontaneously and will be a useful tool to reveal mechanisms of cystogenesis in ADPKD.

Funding

• NIDDK Support