Kidney Week

Abstract: FR-PO763

Vascularized Kidney Organoids for Modeling PKD

Session Information

• Development and Organoid Models
  November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

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

Ken Hiratsuka,

• Miyoshi, Tomoya, Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States

Tomoya Miyoshi,

• Lin, Neil, Wyss Institute/Harvard University, Boston, Massachusetts, United States

Neil Lin,

• Van breugel, Koen, Wyss Institute/Harvard University, Boston, Massachusetts, United States

Koen Van breugel,

• Gupta, Navin R., Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States

Navin R. Gupta,

• Valerius, M. Todd, Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States

M. Todd Valerius,

• Matsumoto, Takuya, Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States

Takuya Matsumoto,

• Lewis, Jennifer A., Wyss Institute/Harvard University, Boston, Massachusetts, United States

Jennifer A. Lewis,

• Morizane, Ryuji, Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts, United States

Ryuji Morizane,

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 Metacore^TMwere 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 –