ASN's Mission

ASN leads the fight to prevent, treat, and cure kidney diseases throughout the world by educating health professionals and scientists, advancing research and innovation, communicating new knowledge, and advocating for the highest quality care for patients.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on Twitter

Kidney Week

Abstract: FR-PO763

Vascularized Kidney Organoids for Modeling PKD

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Hiratsuka, Ken, Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Miyoshi, Tomoya, Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Lin, Neil, Wyss Institute/Harvard University, Boston, Massachusetts, United States
  • Van breugel, Koen, Wyss Institute/Harvard University, Boston, Massachusetts, United States
  • Gupta, Navin R., Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Valerius, M. Todd, Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Matsumoto, Takuya, Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Lewis, Jennifer A., Wyss Institute/Harvard University, Boston, Massachusetts, United States
  • Morizane, Ryuji, Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States
Background

Various models of 3D culture have been developed to model PKD cystogenesis. While recent studies demonstrated the utility of hPSC-derived kidney organoids for modeling ADPKD, even control organoids exhibited cyst formation when forskolin was administered. Moreover, those organoid models displayed cyst formation in both proximal and distal nephrons, which may not fully recapitulate cyst pathogenesis of PKD patients. We postulate that vascularized kidney organoids developed in vitro under flow may provide a better model for in vivo cystogenesis via activation of ciliary signals without the need for forskolin.

Methods

PKHD1-mutant hPSCs were generated by CRISPR/Cas9 genome editing. Hetero and homozygous mutants with frameshift mutations were selected with deep-seq. Kidney organoids were generated following our reported protocol and cultured in vitro under static or flow conditions. Forskolin was tested for cyst formation. cAMP activation was evaluated by ELISA. Control and cystic organoids were characterized by immunostaining and transcriptome analyses. To evaluate differential gene expression profiles, microarray (3D-Gene®) and MetacoreTMwere used.

Results

CRISPR-mutant kidney organoids (static culture) with homozygous mutations in PKHD1 exhibited cyst formation in both proximal and distal nephrons when treated with forskolin, while a heterozygous mutant did not form cysts. Forskolin significantly increased cell proliferation marked by Ki67 in both tubular cells and interstitial cells in homozygous mutant organoids. Further, microarray analysis revealed differential gene expression induced by forskolin and/or PKHD1 homozygous mutations, which was associated with >50 signal pathways. Some signal pathways have been implicated in PKD cystogenesis, yet others, apparently altered by forskolin, appeared to be non-specific to PKD cystogenesis. By contrast, CRISPR-mutant, vascularized kidney organoids cultured under flow exhibited cyst formation and Na, K-ATPase mislocalization solely in distal nephrons without addition of forskolin, which are consistent with clinical findings.

Conclusion

The fluidic chip model of PKD organoids demonstrated clinically relevant phenotypes of PKD patients, which would complement current PKD models to better understand PKD pathomechanisms for new therapeutic development.

Funding

  • NIDDK Support